HK1253363A1 - Nucleic acids for treatment of allergies - Google Patents

Abstract

The present invention relates to nucleic acids for treatment of allergies, partifularly, provides DNA vaccines for the treatment of allergies. The vaccines comprise the coding sequence for one or more allergenic epitopes, and preferably the full protein sequence, of the allergenic protein from which the epitope(s) is derived, fused inframe with the lumenal domain of the lysosomal associated membrane protein (LAMP) and the targeting sequence of LAMP. The vaccines allow for presentation of properly configured three dimensional epitopes for production of an immune response. The vaccines can be multivalent molecules, and/or can be provided as part of a multivalent vaccine containing two or more DNA constructs.

Description

Nucleic acids for the treatment of allergies

The application is a divisional application of international application No. 6/15/2012/042552 on international application No. 2012/6/2012/2015 to chinese national stage on 2/6/2015, No. 201280075163.5, title "nucleic acid for treating allergy". Cross reference to related patent applications

This application is dependent on and claims the disclosure of U.S. provisional patent application No. 61/496,866, filed 6/14 2011, the entire disclosure of which is hereby incorporated by reference herein.

Technical Field

The present invention relates to the fields of molecular biology and medicine. More particularly, the present invention relates to nucleic acids useful as DNA vaccines and methods for their use in treating subjects suffering from or susceptible to allergy.

Background

Allergy is a hypersensitivity disease characterized by the production of IgE antibodies to anti-allergens (or allergy-causing molecules). Allergies affect more than 25% of the population. Allergies can enter the human body by a number of routes including respiratory tract, skin contact, ingestion, insect bites or drug infusion.

Allergy disease management includes diagnosis and treatment. Allergy scientists use a variety of techniques such as skin prick tests, techniques based on the adsorption of radioallergens, ELISA or challenge tests to demonstrate allergen specific IgE and to identify the source of the allergen to diagnose allergy. The treatment of allergies is most commonly divided into two categories: avoidance and administration of antihistamines. The third alternative is allergy immunotherapy, which requires that patients receive weekly injections consisting of small amounts of aggressive allergens to help the immune system retrain (reuducete) their response to allergens.

The use and production of allergen fusion proteins is well known in the art. For example, U.S. patent No.7,566,456 teaches fusion proteins having IgE and IgG binding domains and encoding an allergen. In addition, WO 97/07218 teaches allergen-anti-CD 32 fusion proteins for use in allergy immunotherapy. However, neither of these documents teaches how their respective fusion proteins interact with T cells by antigen presentation to induce or alter Th1 responses. Furthermore, there is no theoretical link between directing a vaccine containing anti-CD 32 to dendritic cells to achieve positive induction of Th1 cells. Both of these documents teach a composition that therapeutically introduces an allergen such that the allergen may be present in serum as an allergen fusion protein.

It has been determined by Toda et al (2002) that T cell epitopes from allergens (in this case the Cry J2 epitope at amino acids 247-. Toda et al (2002) describe the use of a specific composition that is a DNA vaccine encoding the major CD4T cell epitope (at amino acids 247-258) of Cry J2 attached to a class II related constant chain peptide (CLIP). CLIP contains lysosomal/endosomal trafficking sequences and contains a domain that binds to the peptide binding groove of MHC II. Toda et al (2002) showed that immunization with the Cry J2 peptide/CLIP DNA vaccine resulted in mice initiating a major Th1 response characterized by higher IFN-. gamma.and IgG2a production. However, Toda et al do not teach intracellular targeting of the entire protein coding sequence of an allergen that can be used for allergy-specific immunotherapy.

U.S. Pat. No.6,982,326 and U.S. Pat. No.6,090,386 describe nucleic acid sequences encoding the Japanese cedar (Cryptomeria japonica) major pollen allergens Cry J1, Cry J2, Jun I and Jun v I, as well as fragments or peptides thereof. The invention also provides purified Cry J1, Cry J2, Jun s I and Jun v I and at least one fragment thereof, and synthetically prepared Cry J1, Cry J2, Jun s I or Jun v I fragments, produced in a host cell transformed with a nucleic acid sequence encoding Cry J1, Cry J2, Jun s I and Jun v I or at least one fragment thereof, and Cry J1, Cry J2, Jun s I or Jun v I fragments or at least one fragment thereof. Cry J1, Cry J2, Jun s I and Jun v I and fragments thereof are disclosed as useful for diagnosing, treating and preventing Japanese cedar pollinosis. The invention also provides isolated peptides of CryJ1 and Cry J2. Peptides within the scope of this invention comprise at least one T cell epitope, or preferably at least two T cell epitopes, of Cry J1 or Cry J2. The invention also relates to modified peptides having similar or enhanced therapeutic properties as the corresponding naturally occurring allergen or portion thereof but with reduced side effects. Also provided are methods of treating or diagnosing sensitivity to japanese cedar pollen in an individual, as well as therapeutic compositions and multiple peptide formulations (polypeptide formulations) comprising one or more of the peptides of the invention. The invention does not teach how to incorporate epitopes or allergens into DNA vaccines with immunostimulatory properties.

U.S. Pat. No.7,547,440 and U.S. Pat. No.7,112,329 identified T cell epitope sites on Japanese cypress pollen allergen molecules by stimulating T cell lines established from patients suffering from Japanese cypress pollen allergy with overlapping peptides encompassing the primary structure of Japanese cypress (hinoki) pollen allergen. The peptides are useful in peptide-based immunotherapy for patients suffering from spring tree pollinosis, including Japanese cypress pollinosis patients having cross-reactivity with Japanese cypress pollen. The peptide can also be used for diagnosing spring tree pollinosis. The invention is limited to diagnostics and epitope polypeptide delivery.

DNA vaccines have evolved as an alternative to traditional whole cell or whole virus vaccines. Generally, a DNA vaccine is an engineered nucleic acid comprising a sequence encoding one or more epitopes. The nucleic acid is delivered to a cell, typically an Antigen Presenting Cell (APC), the nucleic acid is expressed, epitopes present on the expressed protein are processed in the endosomal/lysosomal compartment and eventually presented on the surface of the cell. U.S. patent No.5,633,234 to August et al discloses and characterizes endosomal/lysosomal targeting sequences for Lysosomal Associated Membrane Proteins (LAMP). This patent identifies key residues in the C-terminal region of the protein that are necessary for targeting of the protein to the endosomal/lysosomal compartment. The patent discloses that fusion of an antigenic peptide with a C-terminal LAMP targeting sequence provides enhanced epitope processing and presentation for the generation of an immune response.

In addition, U.S. patent application publication No.2004/0157307 to Harris et al discloses the use of the LAMP lumen domain (luminal domain) as a "transport domain" to direct chimeric proteins expressed by DNA vaccines across one or more cellular compartments/organelles, such as across the lysosomal vesicular pathway. The chimeric protein includes the luminal domain of the LAMP polypeptide, an antigenic domain comprising a peptide epitope sequence previously identified and selected from an antigenic protein, a transmembrane domain, and an endosomal/lysosomal targeting sequence.

DNA vaccines have been proposed as a treatment for allergic diseases (Raz et al, 1996; Hartl et al, 2004; Hsu et al, 1996; Crameri 2007; Weiss et al, 2006). The rationale is that the allergen protein encoded by the DNA vaccine will preferentially activate allergen specific Th1 cell responses accompanied by interferon production by APC, natural killer cells (NK) and T cells, rather than activating characteristic Th2 type responses such as secretion of IL-4, IL-5 and IL-13, and IgE formation by B lymphocytes and maturation and recruitment of eosinophils in late phase reactions. However, the underlying mechanisms of differential induction of the Th1 and Th2T cell phenotypes appear to involve a number of factors, such as the unique properties of bacterial DNA of vaccine formulations (e.g., unmethylated and CpG DNA residues), the cytokine environment caused by innate immunity, and the cell trafficking characteristics of allergens (Chen et al, 2001; Kaech et al, 2002). There has been no invention or method that successfully addresses the uncertainty of allergy treatment by delivery of allergen-encoding nucleic acids. Thus, such an allergy treatment has not been achieved to date. Furthermore, the administration of DNA vaccines for the treatment of allergic diseases has resulted in the secretion of allergen peptides into the extracellular environment, possibly leading to the unexpected induction of allergic responses by activation of IgE.

Disclosure of Invention

The present invention provides nucleic acids (also referred to herein as "constructs") encoding allergenic proteins, allergenic polypeptides, and allergenic peptides. The nucleic acids are designed for delivery to immune cells and production of allergenic proteins, polypeptides and peptides in those cells. The encoded proteins, polypeptides and peptides have targeting sequences for targeting the protein to the MHC-II compartment for processing and display of one or more epitopes, resulting in an immune response to said epitopes. In general, the nucleic acid comprises the following domains, which are associated with the respective domains of the encoded protein: a signal sequence field; an intra-organelle stabilizing domain; an allergen domain; a transmembrane domain; and a cytoplasmic lysosomal/endosomal targeting domain.

In the context of an encoded protein, a signal sequence is provided to direct the encoded protein to the endoplasmic reticulum or lysosome. The intra-organelle stabilizing domain is a sequence designed to be resistant to proteolysis and designed to protect the rest of the protein (especially the allergen domain) from degradation prior to processing for epitope presentation by the cell. In an exemplary embodiment, the intra-organelle stabilizing domain is the luminal domain of LAMP-1. The allergen domain comprises a sequence of one or more allergenic epitopes that can be used to elicit an immune response in the animal in which the epitope is presented. Typically, the allergen domain comprises one or more allergen proteins, but in some embodiments, immunogenic polypeptides or peptide fragments of the allergen proteins may be used. In the exemplary embodiments discussed below, the epitopes are epitopes of plant allergens. In the encoded proteins of the invention, the allergen domain does not include a signal peptide, such as a signal peptide that naturally occurs as part of an allergen protein. The allergen domain may comprise a single allergenic protein, polypeptide, or peptide, or may comprise two or more allergenic proteins, polypeptides, or peptides. If two or more allergens are present, each allergen may be from the same species/source or one or more may be from one or more different sources. If two or more allergens are present, they may be expressed synergistically to provide equal copy numbers of each coding region in the expressed protein. The transmembrane domain may be any sequence suitable for directing the insertion and transport of proteins into and through the membrane. Many such sequences are known in the art or can be readily designed. The lysosomal/endosomal targeting domain can be any sequence capable of directing a peptide to the lysosome or endosome. Such sequences are known in the art and are exemplified herein by the cytoplasmic tail sequence of LAMP-1.

As mentioned above, in preferred embodiments, the nucleic acid comprises an allergen domain that includes the entire allergenic coding sequence of an allergenic protein, but lacks the coding sequence of the signal sequence of the allergen. In some embodiments, the nucleic acid of the invention does not comprise the entire allergenic coding sequence, but only a sufficient amount of the coding sequence to enable the encoded polypeptide to fold upon expression to achieve the native three-dimensional structure of at least one epitope present on the polypeptide. If less than the entire coding sequence is present, as in a construct comprising the entire allergen coding sequence, the nucleic acid construct also lacks the coding sequence for the naturally occurring signal peptide of the allergenic polypeptide or peptide.

In preferred embodiments, the nucleic acid construct comprises a plurality of coding sequences for allergenic proteins, polypeptides and/or peptides in the allergen domain. Each allergen present may be from the same source, each from a different source, or any combination thereof.

The nucleic acids of the invention, and thus the encoded proteins, polypeptides and peptides, are useful in methods of treating subjects, particularly animal subjects, suffering from or potentially developing an allergy. In general, the methods of treatment according to the invention comprise administering a nucleic acid of the invention to a subject in an amount sufficient to deliver the nucleic acid to one or more immune cells of the immune system, and preferably to one or more Antigen Presenting Cells (APCs). Once delivered, the nucleic acid is expressed, the encoded protein is processed within the cell, and the epitope is displayed on the surface of the cell. The therapeutic methods can be thought of as methods of using nucleic acids and proteins to provide a therapeutic or prophylactic immune response.

Drawings

Fig. 1 is a schematic illustration of a nucleic acid according to one embodiment of the invention, wherein a single antigen comprising a single epitope is provided in the allergen domain.

FIG. 2 shows a vector map of a nucleic acid according to the invention in which the allergen domain comprises CryJ2 allergen (allergen from Cryptomeria japonica) inserted between the human LAMP N-terminal sequence (SS and ISOD) and the human LAMP C-terminal sequence (TM and TG), but no signal sequence.

FIG. 3 is a schematic representation of a nucleic acid according to an alternative embodiment of the invention, wherein multiple epitope sequences of a single allergen are provided in the allergen domain.

FIG. 4 is a schematic representation of a nucleic acid according to an alternative embodiment of the invention, wherein a plurality of different allergen sequences are provided in the allergen domain.

Figure 5 shows a vector map of a nucleic acid according to the invention, wherein the allergen domain comprises the allergen sequences (no signal peptide) of the allergen CryJ1 (allergen from cryptomeria japonica) and the allergen CryJ2 (allergen from cryptomeria japonica).

Figure 6A shows a vector map of a nucleic acid comprising three peanut allergens (AraH1, AraH2, and AraH3, all lacking a signal sequence) in the allergen domain.

FIG. 6B shows a schematic of the protein encoded by the nucleic acid of FIG. 6A.

FIG. 7 shows a vector map of a nucleic acid according to the invention, depicting a naturally occurring signal sequence lacking the CryJ1 allergen sequence. This particular construct was used in experiments described in detail below to show the importance of removing the native signal sequence of the allergen sequence.

Figure 8 shows a vector map of a nucleic acid construct not encompassed by the present invention in which the CryJ2 allergen is encoded on the plasmid backbone but lacks the SS, IOS, TM and TG domains. This construct was used as a comparative control in the experiments described in detail below.

FIG. 9 shows a Western blot depicting the expression of constructs according to the invention in 293 cells. Panel a shows the expression of the CryJ1-CryJ2 combination allergen (see figure 5) and the CryJ2 allergen alone (see figure 2) in the constructs according to the invention, when assayed with anti-CryJ 2 antibody. Panel B shows the expression of a combination of allergen CryJ1-CryJ2 and CryJ1 allergen (lacking its native signal sequence; see FIG. 7) when assayed with anti-CryJ 1 antibody. Panel B also shows that expression of CryJ1 allergen was not detectable in constructs (vector map not shown) in which the native signal sequence of CryJ1 allergen was not removed.

FIG. 10 shows a line graph depicting the potency of a nucleic acid construct according to the invention compared to other constructs comprising a allergen sequence. Panel A (FIG. 10A) shows that a significant increase in IgG1 production and assay values was seen as a result of administration of the CryJ2-LAMP construct of the invention (see FIG. 2) compared to a construct comprising a plasmid backbone fused to a CryJ2 coding sequence (see FIG. 8). Panel B (fig. 10B) shows that a significant increase in IgG2a production and assay values was seen due to administration of the CryJ2-LAMP construct of the invention (according to fig. 10A) compared to the construct comprising a plasmid backbone fused to a CryJ2 coding sequence (according to fig. 10A).

Figure 11 depicts a bar graph showing the dose effect of the CryJ2-LAMP construct in mice. Panel A (FIG. 11A) plots IgG2a detection values at 21 and 28 days after injection of DNA vaccine at various amounts ranging from 10 μ g to 100 μ g, compared to injection of vector DNA alone. Panel B (FIG. 11B) plots IgG1 detection values at 21 and 28 days after injection of DNA vaccine at various amounts ranging from 10 μ g to 100 μ g, compared to injection of vector DNA alone.

FIG. 12 depicts a bar graph showing the induction of IL-4 and IFN- γ in spleen cultures of mice treated with the CryJ2-LAMP construct of the invention compared to the vector alone. Panel A (FIG. 12A) shows the effect of IL-4. Panel B (FIG. 12B) shows the effect of IFN- γ.

FIG. 13 depicts a line graph showing the efficacy of immunization of previously sensitized mice with CryJ2-LAMP DNA vaccine. Panel a (fig. 13A) shows IgG1 titer over time. Panel B (fig. 13B) shows IgG2a titers over time.

FIG. 14 depicts a bar graph showing the induction of IFN-g (Panel A (FIG. 14A)) and IL-4 (Panel B (FIG. 14B)) in mouse spleen cell cultures.

Figure 15 depicts a bar graph showing quantification of circulating CryJ2 protein in immunized mice.

Fig. 16 plots a bar graph of guinea pig data showing IgG1 (panel a (fig. 16A)) and IgG2 (panel B (fig. 16B)) detected in guinea pigs immunized with CryJ2-LAMP construct and challenged with recombinant CryJ 2.

Figure 17 depicts a bar graph showing anti-CryJ 2 responses in new zealand white rabbits immunized with CryJ2-lampd dna vaccine during 85 days of toxicology GLP safety studies.

FIG. 18 depicts a Western blot showing the co-expression of peanut allergens H1, H2 and H3 from the constructs according to the invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention. It should be understood that the following discussion of exemplary embodiments is not intended to limit the invention, but is instead broadly disclosed herein. Rather, the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the present invention. The practice of the present invention employs, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are well explained in the literature known to those of ordinary skill in these arts and need not be described in detail herein. Likewise, practice of the invention with respect to medical treatment follows standard protocols known in the art, and those protocols need not be described in detail herein.

Before the embodiments of the invention are described in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, if a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Every smaller range between any stated value or every intervening value in a stated range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where one, both, or neither of the limits is included in the smaller range is also encompassed within the invention, subject to any specifically excluded limit in the stated range. If a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. It will thus be appreciated that where a range of values is provided, each value within the range, and each range falling within the range, is also inherently recited, and the specific recitation of each value and each possible range of values is not intended to exclude those values and ranges, but is done so for convenience of the reader and for brevity of this disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. In the event that the disclosure conflicts with any incorporated publication, the disclosure controls.

As used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a allergen" includes a plurality of such allergens, and reference to "the sample" includes reference to one or more samples and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the use of terms that may be described using equivalent terms includes the use of those equivalent terms. Thus, for example, use of the term "subject" should be understood to include the terms "animal," "human," and other terms used in the art to refer to a subject undergoing medical treatment.

As used herein, the term "comprising" is intended to mean that the constructs, compositions, and methods include the elements and/or steps recited, but do not exclude other elements and/or steps. "consisting essentially of …" when used to define constructs, compositions, and methods is meant to exclude any other elements or steps that are significant to the constructs, compositions, and methods. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from the isolation and purification process and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "consisting of …" means that other ingredients than the balance of elements are excluded and the method steps are based on the application of the composition of the invention. Embodiments defined by each of these transition terms are within the scope of the invention.

A "chimeric DNA" is an identifiable segment of DNA in a larger DNA molecule that does not naturally occur in association with the larger molecule. Thus, when a chimeric DNA encodes a protein segment, the coding sequence of the segment will be flanked by DNA that does not flank the coding sequence in any naturally occurring genome. In the case where the flanking DNA encodes a polypeptide sequence, the encoded protein is referred to as a "chimeric protein" (i.e., a protein having non-naturally occurring amino acid sequences fused together). Allelic variations or naturally occurring mutational events do not result in a chimeric DNA or chimeric protein as defined herein.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to a polymeric form of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides can have any three-dimensional structure and can perform any known or unknown function. The term "polynucleotide" includes, for example, single-, double-and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may also comprise modified nucleic acid molecules (e.g., comprising modified bases, sugars, and/or internucleotide linkages).

As used herein, the term "peptide" refers to a compound that is two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds or by other bonds (e.g., as esters, ethers, etc.). The term "peptide" is used herein generically to refer to peptides (i.e., polyamino acids of 2 to about 20 residues), polypeptides (i.e., peptides of about 20 residues to about 100 residues), and proteins (i.e., peptides having about 100 residues or more).

As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including both glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics. Peptides of three or more amino acids are often referred to as oligopeptides if the peptide chain is short. Although the term "protein" encompasses the term "polypeptide," a "polypeptide" can be a protein that is shorter than its full length.

The term "allergen" refers to any naturally occurring protein or mixture of proteins that have been reported to induce an allergic response, i.e., an IgE-mediated response, upon their repeated exposure to an individual. An allergen is any compound, substance or material capable of causing an allergic reaction. An allergen is generally understood as a subclass of antigens, which are compounds, substances or materials capable of eliciting an immune response. For the practice of the present invention, the allergen may be selected from the group consisting of natural or native allergens, modified natural allergens, synthetic allergens, recombinant allergens, allergoids, and mixtures or combinations thereof, among others. Of particular interest are allergens capable of causing IgE-mediated immediate hypersensitivity reactions.

Examples of naturally occurring allergens include pollen allergens (such as tree, weed, herb and grass pollen allergens), mite allergens (from e.g. house dust mites and storage mites), insect allergens (such as allergens of inhalant, saliva and venom origin), animal allergens from e.g. saliva, hair and dander of animals (such as dogs, cats, horses, rats, mice, etc.), fungal allergens and food allergens. The allergen may be in the form of: allergen extract, purified allergen, modified allergen or recombinant mutant allergen, allergen fragment of more than 30 amino acids or any combination thereof.

By their chemical or biochemical properties, the allergens may represent natural or recombinant proteins or peptides, fragments or truncated forms of natural or recombinant proteins or peptides, fusion proteins, synthetic compounds (chemical allergens), synthetic compounds mimicking allergens, or allergens altered by chemical or physical methods such as allergens modified by heat denaturation.

Allergens can be classified as major allergens by several tests. An allergen is generally classified as a major allergen if at least 25% of the patients show strong IgE binding (score 3) and at least 50% of the patients show moderate binding (score 2), as determined by CRIE (cross Radio immuno electrophoresis) (CRIE strong binding, i.e. IgE binding visible on X-ray film after one day; CRIE moderate binding, i.e. binding occurring after 3 days; CRIE weak binding, i.e. binding occurring after 10 days). At least 10% of patients classify allergens as moderate allergens for strong IgE binding, and less than 10% classify allergens as minor allergens for significant specific binding. Other methods may also be used to determine IgE binding, such as IgE blotting.

An "epitope" is a structure, usually composed of short peptide sequences or oligosaccharides, that is specifically recognized or specifically bound by a component of the immune system. T cell epitopes have been shown collectively as linear oligopeptides. Two epitopes correspond to each other if they can be specifically bound by the same antibody. Two epitopes correspond to each other if both epitopes are capable of binding the same B cell receptor or binding the same T cell receptor and binding of one antibody to its epitope substantially prevents binding of the other epitope (e.g., less than about 30%, preferably less than about 20%, and more preferably less than about 10%, 5%, 1%, or about 0.1% of the other epitope).

As used herein, two nucleic acid coding sequences "correspond to" each other if the two nucleic acid coding sequences or their complements encode the same amino acid sequence.

As used herein, a polynucleotide or polynucleotide region (or polypeptide region) that has a certain percentage (at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%) of "sequence identity" to another sequence means that when optimally aligned, either manually or using software programs routine in the art, the percentage of bases (or amino acids) in the two sequences being compared are identical.

Two nucleotide sequences are "substantially homologous" or "substantially similar" when at least about 50%, at least about 60%, at least about 70%, at least about 75%, and preferably at least about 80%, and most preferably at least about 90 or 95% of the nucleotides match over a DNA sequence of defined length. Similarly, two polypeptide sequences are "substantially homologous" or "substantially similar" when at least about 40%, at least about 50%, at least about 60%, at least about 66%, at least about 70%, at least about 75%, and preferably at least about 80%, and most preferably at least about 90 or 95% or 98% of the amino acid residues of the polypeptides match over a polypeptide sequence of defined length. Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence databases (sequence databank). Substantially homologous nucleic acid sequences can also be identified in DNA hybridization experiments, for example, under stringent conditions, which are defined for a particular system. Defining appropriate hybridization conditions is within the skill of the art. For example, stringent conditions may be: hybridization was performed with 5XSSC and 50% formamide at 42 ℃ and washed with 0.1XSSC and 0.1% sodium dodecyl sulfate at 60 ℃.

"conservatively modified variants" of the domain sequences may also be provided. With respect to particular nucleic acid sequences, the term "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or if the nucleic acids do not encode an amino acid sequence, to essentially identical sequences. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed-base and/or deoxyinosine residues (Batzer et al, 1991, Nucleic Acid Res. ("Nucleic acids research") 19: 5081; Ohtsuka et al, 1985, J.biol.chem. ("J.Biol.Chem.) 260: 2605. Amp. 2608; Rossolini et al, 1994, mol.cell.Probes (molecular and cellular probes) 8: 91-98).

The terms "biologically active fragment," "biologically active form," "biologically active equivalent" and "functional derivative" of a wild-type protein refer to a substance that has a biological activity that is at least substantially the same as (e.g., not significantly different from) the biological activity of the wild-type protein when measured using an assay suitable for detecting activity. For example, a biologically active fragment comprising a trafficking domain is a fragment that can be co-localized to the same compartment as a full-length polypeptide comprising the trafficking domain.

A cell has been "transformed", "transduced" or "transfected" by an exogenous or heterologous nucleic acid when the nucleic acid has been introduced into the cell. The transforming DNA may or may not be integrated (covalently linked) with the chromosomal DNA that makes up the genome of the cell. In prokaryotes, yeast and mammalian cells, for example, the transforming DNA may be maintained on an episomal element such as a plasmid. In eukaryotic cells, a stably transformed cell is one in which the transforming DNA has integrated into the chromosome such that it is inherited by daughter cells through chromosome replication. The stability is demonstrated by the ability of the eukaryotic cell to establish a cell line or clone consisting of a population of progeny cells containing the transformed DNA. A "clone" is a population of cells derived by mitosis from a single cell or a common ancestor. A "cell line" is a clone of primary cells that is capable of stable growth in vitro for many generations (e.g., at least about 10 generations).

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that serves as an autonomous unit of DNA replication in vivo.

As used herein, "viral vector" refers to a virus or viral particle comprising a polynucleotide to be delivered into a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. In aspects where gene transfer is mediated by an adenoviral vector, a vector construct refers to a polynucleotide comprising an adenoviral genome or portion thereof and selected non-adenoviral genes associated with adenoviral capsid proteins.

As used herein, a "nucleic acid delivery vector" is a nucleic acid molecule that can transport a polynucleotide of interest into a cell. Preferably, such vectors comprise a coding sequence operably linked to an expression control sequence. However, the polynucleotide sequence of interest does not necessarily comprise a coding sequence. For example, in one aspect, the polynucleotide sequence of interest is an aptamer that binds to a target molecule. In another aspect, the sequence of interest is a complement of a regulatory sequence that binds to the regulatory sequence to inhibit regulation of the regulatory sequence. In yet another aspect, the sequence of interest is itself a regulatory sequence (e.g., for titrating out a regulatory factor in a cell).

As used herein, a "nucleic acid delivery vehicle" is defined as any molecule or group of molecules or macromolecules (e.g., genes or gene fragments, antisense molecules, ribozymes, aptamers, etc.) that can carry an inserted polynucleotide into a host cell and occur in association with a nucleic acid delivery vector as described above.

As used herein, "nucleic acid delivery" or "nucleic acid transfer" refers to the introduction of an exogenous polynucleotide (e.g., a transgene) into a host cell, regardless of the method used for the introduction. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotide either contain an origin of replication compatible with the host cell or that the replicon, e.g., an extrachromosomal replicon (e.g., plasmid) or nuclear or mitochondrial chromosome, be integrated into the host cell.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA transcribed from the genomic DNA.

As used herein, "under transcriptional control" or "operably linked" refers to the expression (e.g., transcription or translation) of a polynucleotide sequence, which expression is controlled by appropriate juxtaposition of an expression control element and the coding sequence. In one aspect, when an expression control sequence controls and regulates the transcription of a DNA sequence, then the DNA sequence is "operably linked" to the expression control sequence.

As used herein, a "coding sequence" is a sequence that is transcribed and translated into a polypeptide when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence are determined by an initiation codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Polyadenylation signals and transcription termination sequences will usually be located 3' to the coding sequence.

As used herein, "genetic modification" refers to any addition or deletion or disruption to the normal nucleotide sequence of a cell. Any method that can effect genetic modification of an APC is within the spirit and scope of the present invention. Methods recognized in the art include virus-mediated gene transfer, liposome-mediated transfer, transformation, transfection, and transduction, for example, the use of virus-mediated gene transfer such as DNA virus-based vectors (e.g., adenovirus, adeno-associated virus, and herpes virus) and retroviral-based vectors.

As used herein, "lysosomal/endosomal compartment" refers to membrane-bound acidic vesicles containing LAMP molecules in the membrane, hydrolases that function in antigen processing, and MHC class II molecules for antigen recognition and presentation.

This compartment serves as a degradation site for foreign material internalized from the cell surface by any of a variety of mechanisms, including endocytosis, phagocytosis, and pinocytosis, as well as intracellular material delivered to this compartment by specialized autolysis phenomena (see, e.g., de Duve, eur.j. Biochem. (european journal of biochemistry) 137:391,1983). The term "endosome" as used herein encompasses lysosomes.

As used herein, "lysosome-associated organelle" refers to any organelle comprising lysozyme and includes, but is not limited to, MIIC, CUV, melanosome, secretory granules, lytic granules, compact granules of platelets, basophilic granules, burbeck granules, phagolysosomes, secretory lysosomes, and the like. Preferably, such an organelle lacks the mannose 6-phosphate receptor and comprises a LAMP, but may or may not comprise an MHC class II molecule. For Reviews, see, e.g., Blott and Griffiths, Nature Reviews, Molecular Cell Biology (Nature review, Molecular Cell Biology), 2002; dell' Angelica et al, The FASEB Journal (J. FASEB) 14: 1265-.

As used herein, "LAMP polypeptide" refers to LAMP-1, LAMP-2, CD63/LAMP-3, DC-LAMP, or any lysosomal-associated membrane protein or homolog, ortholog, variant (e.g., allelic variant), and modified form (e.g., comprising one or more naturally occurring or engineered mutations). In one aspect, the LAMP polypeptide is a mammalian lysosomal associated membrane protein, such as a human or mouse lysosomal associated membrane protein. More generally, an "lysosomal membrane protein" refers to any protein that comprises a domain present in the membrane of an endosomal/lysosomal compartment or lysosomal-associated organelle and that also comprises an intraluminal domain.

As used herein, "targeting" refers to directing the chimeric proteins of the invention to a preferred site, such as a polypeptide sequence of an organelle or compartment where antigen processing and binding to MHC II occurs. As used herein, "targeting domain" refers to a series of amino acids required for delivery to a cellular compartment/organelle. Preferably, the targeting domain is a sequence that binds to a linker protein or an AP protein (e.g., AP1, AP2, or AP3 protein). Exemplary targeting domain sequences are described, for example, in Dell' Angelica, 2000.

As used herein, in vivo nucleic acid delivery, nucleic acid transfer, nucleic acid therapy, and the like, refers to the direct introduction of a vector comprising an exogenous polynucleotide into the body of an organism (such as a human or non-human mammal), thereby introducing the exogenous polynucleotide into the cells of such organism in vivo.

As used herein, the term in situ refers to a type of in vivo nucleic acid delivery in which the nucleic acid is brought into proximity with a target cell (e.g., without systemic administration of the nucleic acid). For example, in situ delivery methods include, but are not limited to, injecting the nucleic acid directly into the site (e.g., into a tissue, such as a tumor or heart muscle), contacting the nucleic acid with a cell or tissue through an open surgical field, or delivering the nucleic acid to the site using a medical access device, such as a catheter.

As used herein, the terms "isolated" and "purified" are sometimes used interchangeably to refer to separation from components (cells and other components) to which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are typically associated in nature. For example, for a polynucleotide, an isolated polynucleotide is a polynucleotide that is separated from the 5 'and 3' sequences with which the polynucleotide is normally associated in the chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof need not be "isolated" to distinguish it from its naturally occurring counterpart. Furthermore, the terms "isolated" and "purified" do not imply complete isolation and complete purification. These terms are used to denote partial and complete purification from some or all of the other materials naturally associated with the polynucleotide or the like. Thus, these terms may mean isolated or purified from one of the naturally-associated substances (e.g., DNA isolated or purified from RNA), from other substances of the same general class of molecules (e.g., a particular protein exhibits 20% purity compared to all proteins in a sample), or any combination. Isolation and purification can mean any level of about 1% to about 100% (including 100%). As such, an "isolated" or "purified" cell population is substantially free of cells and materials with which it is associated in nature. By substantially free of impurities or substantially purified APCs is meant that at least 50% of the cell population is APC, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of non-APC cells with which it is associated in nature. Of course, those skilled in the art will recognize that all specific values, including fractions of values, are encompassed within these ranges without necessarily requiring each specific value to be listed herein. Values are not specifically disclosed for the sake of brevity; however, the reader should understand that each specific value is inherently disclosed and encompassed by the invention.

As used herein, "target cell" or "recipient cell" refers to an individual cell or a cell that is desired to be or has become the recipient of an exogenous nucleic acid molecule, polynucleotide, and/or protein. The term is also intended to include progeny of a single cell, and due to natural, accidental, or deliberate mutation, the progeny may not necessarily be identical (either morphologically or in genomic or total DNA complement) to the original parent cell. The target cell may be in contact with other cells (e.g., as in a tissue) or may circulate in the body of an organism.

The term "antigen presenting cell" or "APC" as used herein means any cell that presents on its surface an antigen associated with a major histocompatibility complex molecule or a part thereof, or, one or more atypical MHC molecules or parts thereof. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells.

As used herein, "engineered antigen presenting cell" refers to an antigen presenting cell that has a non-native molecular moiety on its surface. For example, such a cell may naturally have no co-stimulatory molecules on its surface or may have additional artificial co-stimulatory molecules on its surface in addition to the natural co-stimulatory molecules, or may express non-natural class II molecules on its surface.

As used herein, the term "immune effector cell" refers to a cell that is capable of binding an antigen and mediating an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and Cytotoxic T Lymphocytes (CTLs), such as CTL lines, CTL clones, and CTLs from tumors, inflammation or other infiltrates.

As used herein, the terms "subject" and "patient" are used interchangeably to refer to an animal to which the present invention is directed. The term animal should be understood to include both human and non-human animals; the terms human and/or non-human animal are used if a distinction between the two is desired. In embodiments, the subject or patient is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals (e.g., cattle, sheep, pigs), sport animals (e.g., horses), and pets (e.g., dogs and cats).

Clinical allergy symptoms are known to those skilled in the art and do not require an exhaustive list to be given herein. Non-limiting examples include rhinitis, conjunctivitis, asthma, urticaria, eczema, which includes reactions in the skin, eyes, nose, upper and lower respiratory tract, where common symptoms are e.g. redness and itching of eyes and nose, itching and runny nose, cough, asthma, shortness of breath, itching and tissue swelling.

Examples of "immunological in vivo tests" are Skin Prick Tests (SPT), conjunctival challenge tests (CPT), bronchial attacks with allergens (BCA) and various clinical tests in which one or more allergy symptoms are monitored. See, for example, Haugaard et al, J Allergy Clin Immunol (J. Allergy clinical immunology), Vol 91, No. 3, p. 709 & 722, p. 3 1993.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier known in the art, such as phosphate buffered saline solutions, water and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see martin remington's pharm.sei. ("remton's pharmaceutical science"), 15 th edition (mark publishing company (Mack PubL co., Easton) (1975)).

As used herein, "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, correct and/or normalize an abnormal physiological response. In one aspect, a "therapeutically effective amount" is an amount sufficient to reduce a clinically important pathological feature (e.g., tumor volume size, antibody production, cytokine production, fever or white blood cell count, or histamine level) by at least about 30%, more preferably by at least 50%, and most preferably by at least 90%.

An "antibody" is any immunoglobulin that binds a specific epitope, including antibodies and fragments thereof. The term encompasses polyclonal antibodies, monoclonal antibodies, and chimeric antibodies (e.g., bispecific antibodies). An "antibody binding site" is the structural portion of an antibody molecule consisting of the variable and hypervariable regions of the heavy and light chains, which specifically binds to an antigen. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of immunoglobulin molecules that contain paratopes, including Fab portions, Fab 'portions, F (ab')₂And part f (v), which are preferably used in the treatment methods described herein.

The term "oromucosal administration" refers to a route of administration in which a dosage form is placed under the tongue or elsewhere in the mouth to bring an active ingredient into contact with the mucosa of the oral cavity or pharynx of a patient in order to obtain a local or systemic effect of the active ingredient. An example of a route of oromucosal administration is sublingual administration. The term "sublingual administration" refers to a route of administration in which the dosage form is placed on the base of the tongue in order to obtain a local or systemic effect of the active ingredient. As used herein, the term "intradermal delivery" means the delivery of a vaccine to the dermis in the skin. However, the vaccine will not necessarily be located only in the dermis. The dermis is a layer of skin in the human skin that is located between about 1.0 and about 2.0mm from the surface, but there is a certain amount of variation between individuals and in different parts of the body. Typically, access to the dermis is expected by penetration 1.5mm below the skin surface. The dermis is located between the stratum corneum and epidermis of the surface and the underlying subcutaneous layer. Depending on the mode of delivery, the vaccine may ultimately reside only or primarily within the dermal layer, or it may ultimately be distributed within the epidermis or dermis.

As used herein, the term "prevention" in the context of allergy immunotherapy, allergy treatment or other terms describing interventions designed for allergy patients means prevention of IgE response in at least 20% of all patients. The term "prevention" does not mean complete prevention of development of IgE-mediated disease in all patients, and such a definition is not within the scope of the present invention for treating allergy by a mechanism that reduces symptoms of allergy, and is inconsistent with the use of this term in the art. It is well known to those skilled in the art of allergy immunotherapy that allergy treatment is not 100% effective in 100% of patients and thus the absolute definition of "prevention" is not applicable in the context of the present invention. The present invention contemplates the art-recognized concept of prevention.

The invention provides polynucleotides, polyamines, and methods of treating a subject in need thereof. In a broad sense, the polynucleic acid may be considered a nucleic acid (e.g., DNA, RNA) vaccine for intracellular production of an allergenic sequence (polyamino acid) that elicits a protective immune response in a subject to which the polynucleic acid is administered. The polynucleic acids, when administered, preferentially elicit a cell-mediated immune response via the MHC-II pathway and the production of IgG antibodies by activating an allergen-specific helper T cell type 1 (Th1) cellular response (with interferon production by APC, NK cells and T cells) rather than a Th2 type response, which involves the production of IgE antibodies, granulocytes (e.g. eosinophils) and other substances. To some extent, both MHC-II and MHC-I responses can be generated. However, the present invention provides a response which is predominantly or substantially MHC-II. Preferably, the nucleic acid does not encode an antibiotic resistance gene.

The present invention is based, at least in part, on the recognition that: the combination of certain structural (and thus functional) elements may provide advantageous properties for nucleic acid vaccines and encoded allergens, thereby enabling allergy treatment approaches to meet unmet needs in the art. In various embodiments of the invention (which are intended to be understood as embodiments that exist alone as independent embodiments and as embodiments that combine two or more features of independent embodiments), the combination includes the use of a lysosomal trafficking domain to direct an allergen amino acid sequence to a lysosome having an MHC II protein. This is done so that an IgG response occurs predominantly to the allergen sequence rather than an IgE response. In addition, an independent embodiment or a combination of embodiments provides a construct comprising a nucleic acid sequence of sufficient length to encode an amino acid sequence that provides a naturally occurring three-dimensional structure of an epitope. In preferred embodiments, the nucleic acid sequence provides/encodes the full-length allergen coding sequence, but lacks any naturally occurring signal peptide sequence associated with the allergen sequence. In other embodiments, the nucleic acid sequence encodes at least one allergenic region of an allergen, rather than a full-length allergen protein (and also lacks a signal sequence if the signal sequence is naturally present). Although the art recognizes that an immune response can be generated against the primary sequence of an epitope, the present invention recognizes that a nucleic acid vaccine for generating an MHC-II immune response against an encoded epitope preferably uses a nucleic acid construct that: which encodes sufficient sequence data to produce the correct three-dimensional peptide structure in the region containing the allergenic epitope, at least when the allergenic sequence is delivered to lysosomes for processing. While not being limited to any particular molecular theory, it is believed that delivery of a protein, polypeptide, or peptide that is correctly three-dimensionally folded into an endosome can improve processing and presentation of allergenic epitopes for immune response.

As another example of an embodiment that can be performed alone or as part of a combination of embodiments, expression of multiple allergens from a single construct is provided. To date, there has been no indication that nucleic acid vaccines against allergens can be efficiently prepared and used. The present invention provides not only an effective nucleic acid vaccine against allergens but also an effective nucleic acid vaccine against multiple allergens simultaneously. The allergen may be an allergen from the same source (e.g., a single plant), or may be an allergen from two or more sources (e.g., trees, flowers, food, etc.). As noted above, full-length allergen sequences (lacking any naturally associated signal sequence of the allergen) may be used, or allergenic moieties may be used. In constructs comprising multiple allergen sequences, any mixture of full-length or truncated allergen sequences may be used. In addition, for other embodiments, it is preferred to remove the naturally occurring signal sequence of each allergen sequence (i.e., the naturally occurring signal sequence of each allergen sequence is not present in the construct).

Although the use of signal sequences for independent allergenic sequences within the allergenic domain has been found to be detrimental to the function of the nucleic acid construct, the use of signal sequence regions or domains within the nucleic acid vaccine construct has been found to be an important feature. As such, in embodiments, the nucleic acid vaccine includes at least one signal sequence within the signal sequence domain to direct the encoded peptide to and through the membrane. Although the amino acid sequence of the signal sequence may vary from construct to construct, and any known signal sequence may be selected, it has been found that in preferred embodiments, the signal sequence is present and is provided in frame with the coding sequence of the allergen sequence. The use of a single signal sequence is sufficient to direct the entire encoded chimeric protein to and across the membrane. As such, it is not necessary that a signal sequence be used for each allergen sequence, and in fact, has been found to be detrimental to the correct positioning, processing and expression of the allergen epitope on the surface of immune cells.

Furthermore, in particular embodiments and in combinations of embodiments, it has been found that chelation or physical protection of allergen sequences during transfer of the polypeptide from the cytoplasm to the endosome, including in the endosome, before cleavage of the polypeptide into units for presentation on the cell surface, may be an important factor in providing a useful nucleic acid vaccine according to the invention. As such, in general, the invention includes constructs comprising an intra-organelle stabilizing domain (IOSD) to protect the allergen sequence.

The nucleic acids of the invention comprise at least the following domains: a signal sequence domain; an intra-organelle stabilizing domain; an allergen domain (which may comprise a single allergen or two or more allergens, each comprising one or more allergenic epitopes); a transmembrane domain; and a cytoplasmic lysosomal/endosomal targeting domain. Each domain is present on a single chimeric or engineered nucleic acid. The domains may be combined in any linear order using techniques known in the art and widely practiced. In preferred embodiments, the domains are combined and arranged such that they constitute a single open reading frame encoding the chimeric protein, operably linked to transcription elements sufficient for expression of the chimeric protein. The nucleic acid may thus be an expression vector, such as a plasmid, phagemid, viral vector or the like. Preferably, the nucleic acid comprises a transcription element suitable for expression in a mammalian cell, such as a human cell. Such expression vector elements and expression vectors are known in the art and are widely used, as exemplified by U.S. patent application publication No.2004/0157307, which is incorporated herein by reference. A non-limiting example of a plasmid backbone used to generate the nucleic acid construct according to the present invention is sometimes referred to herein as the "pITI" plasmid, the sequence of which is provided in SEQ ID NO 1.

Three exemplary configurations of the nucleic acids of the invention are depicted schematically in FIGS. 1, 3 and 4, respectively. Figure 1 shows the sequential arrangement of domains in which a single allergen comprising a single epitope is included in the encoded chimeric protein. Figure 3 shows a sequential arrangement of domains in which multiple different epitopes of a single allergen are included within the allergen domain of the encoded chimeric protein. These two epitopes are arranged so that they are in the same reading frame and thus both are produced as part of a chimeric protein. One skilled in the art will immediately recognize that three or more epitopes may be provided within an epitope domain in the same reading frame using standard molecular biology techniques. Figure 4 shows a sequential arrangement of domains in which two different allergens are present in the allergen domain. Of course, the skilled person will recognise that each allergen sequence may contain one or more allergenic epitopes. From these three schematic representations of an example of a nucleic acid of the invention, the reader will immediately recognize that any number of allergens from any number of sources and containing any number of epitopes can be included within an allergen domain and linked in frame using standard molecular biology techniques.

FIG. 2 depicts a vector map of a nucleic acid according to one embodiment of the invention ("pITI-CRY J2-LAMP"; also sometimes referred to herein as "CRYJ 2-LAMP") that is generally relevant to the embodiment of the invention depicted schematically in FIG. 1. The vector or delivery vehicle comprises a plasmid backbone having a pUC origin of replication and various transcription and expression elements for the production of the encoded protein. More specifically, it includes the sequence of the pITI backbone (SEQ ID NO: 1). It should be noted that according to a preferred embodiment of the invention, the nucleic acid construct does not comprise an antibiotic resistance gene. The nucleic acid also comprises a sequence of the encoded protein that comprises an N-terminal region of the human LAMP protein, which includes a signal sequence and an intra-organelle stabilizing domain. The nucleic acid also provides the sequence of the encoded protein, which comprises the CryJ2 allergen sequence (lacking its signal sequence) fused in frame to the N-terminal region of the LAMP protein. The nucleic acid also includes a sequence encoding a portion of the C-terminal region of the human LAMP protein, which includes a transmembrane region and a targeting region. The coding region of the CRY J2-LAMP chimeric protein sequence is provided as SEQ ID NO 2. The amino acid sequence of the CRY J2-LAMP chimeric protein is provided as SEQ ID NO 3.

In exemplary embodiments, the invention also relates to nucleic acid constructs for the delivery and expression of other allergens of cryptomeria japonica (including CryJ1 allergen). Using the same plasmid backbone, the pITI-CRYJ1-LAMP construct has been generated. The chimeric protein can elicit an MHC class II immune response. The coding region of the pITI-CRYJ1-LAMP construct is given as SEQ ID NO. 4. The amino acid sequence of the CRY J1-LAMP chimeric protein is provided as SEQ ID NO 5.

As shown in fig. 3 and 4, the allergen domain may comprise a allergen having a plurality of allergenic epitopes, or may comprise a plurality of allergens (each having one or more allergenic epitopes). FIG. 5 depicts a vector map of one particular exemplary embodiment of a nucleic acid construct, wherein the allergenic domain includes two allergenic sequences. In this exemplary embodiment, the allergen domain contains the CryJ1 and CryJ2 allergens of cryptomeria japonica (each lacking its native signal sequence) fused in frame to the LAMP signal sequence domain and the intra-organelle stabilizing domain and at the N-terminus. The CryJ1-CryJ2 sequence is also fused at the C-terminus to the LAMP transmembrane and targeting domains. The entire nucleotide sequence of the coding region of the chimeric protein is given in SEQ ID NO 6. The entire amino acid sequence of the encoded chimeric protein is given in SEQ ID NO 7, wherein: residues 1-27 represent the signal sequence of the chimeric protein; residues 28-380 represent the intra-organelle stabilizing domain (taken from the sequence of human LAMP); residues 381 and 382 represent a linker; residue 383-735 represents the coding region of CryJ1 (without its signal sequence); residue 736-741 represents a linker region; residue 742-1232 represents the coding region for the CryJ2 allergen; residues 1233-1234 represent the linker region; residues 1235-1258 represent the transmembrane and targeting domains; and residues 1259-1270 represent an additional C-terminal residue.

The nucleic acid constructs of the invention are essentially unlimited in the number of allergens that can be synergistically produced. As such, two, three, four, five, six, ten, twenty or more different allergens (from the same source or a mixture of different sources) may be included in the nucleic acid construct of the present invention. FIG. 6A sets forth a vector map of another exemplary nucleic acid according to embodiments of the present invention. The vector or delivery vehicle includes a plasmid backbone having a pUC origin of replication and various transcription and expression elements for production of the encoded protein. The scaffold may be, but need not be, the pITI scaffold of SEQ ID NO: 1. The nucleic acid also comprises a sequence of the encoded protein comprising an N-terminal region of the human LAMP protein, the N-terminal region including a signal sequence domain and an intra-organelle stabilizing domain. The nucleic acid also provides the sequence of the encoded chimeric protein comprising the peanut allergen polyprotein AraH1/AraH2/AraH 3. The nucleic acid also includes a sequence encoding a portion of the C-terminal region of the human LAMP protein, which includes a transmembrane region and a targeting region. The nucleotide sequence of the coding region of the chimeric protein is provided as SEQ ID NO 8. The chimeric proteins encoded by the vectors of FIG. 6A are given schematically in FIG. 6B (and as SEQ ID NO: 9).

The domains present in the nucleic acids of the invention are described in more detail below with respect to the functions provided by the encoded chimeric proteins. It will be appreciated that the practice of the invention is not dependent on or limited to any particular nucleic acid or protein sequence, but rather, it is a combination of elements and domains that provide advantages and specificity to the construct. It will also be understood that when discussed in the context of physical and functional properties of the encoded protein, the description is with respect to the domains of the nucleic acid construct, and vice versa. It is sufficient to inform the person skilled in the art about the physical and functional properties of the nucleic acid or protein. It is a simple matter to exploit the degeneracy of the computer and genetic code to obtain all possible nucleic acid molecules encoding known protein sequences and to obtain the protein encoded by the nucleic acid. Thus, reference to a physical or functional property of a particular protein sequence immediately discloses to the skilled person all possible nucleic acid sequences which are related to that physical or functional property and vice versa.

The design and combination of two or more nucleic acid molecules or sequences to obtain a sequence encoding a chimeric protein according to the invention is also well within the skill of the person skilled in the art. Likewise, the selection and combination of transcriptional and translational control elements to express coding sequences and chimeric proteins in vivo or in vitro as desired is well within the purview of the skilled artisan. Thus, these commonly used techniques need not be discussed in detail herein to enable practice of the present invention.

The nucleic acids of the invention comprise a signal sequence domain. The signal sequence domain contains a signal sequence for insertion of the encoded chimeric protein into a biological membrane defining a boundary between an external environment and an internal environment. The signal sequence also directs the transfer of the protein from the external environment to the internal environment. The general structure of signal sequences is well known in the art, as there are many examples of specific signal sequences. The practitioner can choose any suitable signal sequence at will, depending on various selection parameters for each embodiment that fall within the scope of the invention. In an exemplary embodiment, the signal sequence is one that directs the chimeric protein to the endoplasmic reticulum. It is worth noting at this point that the signal sequence domain is only that portion of the chimeric protein that contains the signal sequence. As such, the naturally occurring signal sequence of the allergen present in the allergen domain has been removed prior to inclusion of the allergen sequence in the construct. It has been found that removal of these individual signal sequences improves the overall performance of the construct in vivo.

The nucleic acids of the invention comprise an intra-organelle stabilizing domain (IOSD). The IOSD comprises a sequence encoding an amino acid sequence that binds to one or more sequences in the allergen domain via a chemical bond and protects those sequences from degradation (e.g., proteolysis) before the chimeric protein reaches the endosomal/lysosomal compartment. In essence, IOSD can be envisioned as protective caps for allergen domain sequences, shielding those sequences, especially allergenic epitope sequences, from proteolytic enzymes, low pH and other protein destabilizing substances and conditions. The IOSD may be any of a number of known or engineered sequences, including but not limited to LAMP polypeptide lumenal domain and macrosialin/CD 68 protein, macrosialin/CD 68 protein is a highly glycosylated transmembrane protein expressed as a late endosomal protein in macrophages and macrophage-like cells. A key feature of IOSD is the ability of IOSD to bind to the allergen domain and protect it from proteolysis until release of MHC class II molecules from invariant peptides. In this way, the three-dimensional structure of the allergenic epitope is maintained until active MHC class II molecules are available for interaction. In preferred embodiments, the IOSD comprises all or part of the sequence of the lysosomal protein. In some embodiments, the IOSD is a protein or polypeptide other than the LAMP polypeptide luminal domain, such as, but not limited to, ambroxol/CD 68.

The nucleic acid construct of the invention comprises an allergen domain. The allergen domain comprises one or more sequences encoding an allergen protein, polypeptide or peptide comprising one or more allergenic epitopes. The allergen domain does not include a signal sequence from the allergen present. Many proteinaceous allergens are known in the art, and any one or combination of allergens and/or allergenic epitopes may be used according to the present invention. If less than the full length allergenic sequence is used, preferably one or more surfaces of the full length allergenic proteins are provided in their natural positional context within the allergenic protein. More specifically, the present invention provides improved nucleic acid vaccines wherein the vaccine encodes chimeric proteins that retain or substantially retain their three-dimensional structure until an MHC class II molecule is capable of binding an epitope on the chimeric protein. In this way, an improved immune response may be elicited compared to delivering short peptides to MHC class II molecules, which will typically lack the appropriate three-dimensional structure. Thus, it is preferred that the allergen domain encodes a relatively long amino acid sequence comprising one or more epitopes (if originally present on the allergen protein).

The allergen domain may comprise two or more allergens, each containing one or more allergenic epitopes. It is well known that certain allergenic proteins contain two or more epitopes. Because preferred embodiments of the present invention use the entire allergenic coding region (i.e., the coding region lacking the signal sequence) of the allergenic protein, or a substantial portion thereof, certain allergen domains will include two or more epitopes in their naturally occurring relationship. Alternatively, two or more known epitopes may be fused into one coding region. Again, in exemplary embodiments, two or more allergenic proteins, or allergenic regions thereof, are present in the allergenic domain. If two or more epitopes are engineered to be present in a single epitope domain, the epitopes may be from the same antigenic protein. Alternatively, they may be from two different proteins of the same species. Again, they may be from the same protein of two different species. Furthermore, they may be from two or more different proteins of two or more different species. Essentially, any combination of epitopes of the same or different proteins from the same or different species is contemplated by the present invention. Likewise, the order of the individual allergens and epitopes may be varied in any way that is imaginable. The mixing of allergenic proteins and/or allergenic peptides from multiple species enables the production of robust nucleic acid vaccines that can provide treatment for allergies against organisms of a single origin (e.g., trees of a particular species) based on multiple allergens, and can provide treatment for allergies against organisms of multiple origin (e.g., multiple plants that release spores during the same season of the year). The ability to combat multiple allergens by a single nucleic acid vaccine has not been demonstrated to date.

The nucleic acid construct of the invention further comprises a transmembrane domain. Transmembrane domains are well-known and well-characterized physical and functional elements of proteins, part of which are present on both sides of biological membranes. In essence, a transmembrane domain is a linear sequence of amino acids that are generally hydrophobic or lipophilic in nature and serve to anchor proteins at biological membranes. Generally, such sequences are 20-25 residues in length. Such sequences will be well known to those skilled in the art and suitable transmembrane sequences can be readily obtained or engineered for use in the present invention.

In addition to the elements discussed above, the nucleic acids of the invention also comprise a targeting domain. A targeting domain is a sequence encoding an amino acid sequence for targeting the encoded chimeric protein to an endosomal/lysosomal compartment. Although not so limited in their identity, in preferred embodiments, the targeting domain comprises a C-terminal cytoplasmic targeting sequence of the LAMP polypeptide, DC-LAMP, LAMP2, LAMP-3, LIMP II, ENDOLYN, or megasialin/CD 68.

In embodiments, the nucleic acids of the invention comprise as part of the allergen domain the sequence of SEQ ID NO:2 (i.e., the Cry J2 nucleotide sequence lacking its signal sequence) or another sequence encoding SEQ ID NO:3 (i.e., the Cry J2 protein sequence lacking its signal sequence). SEQ ID NO:2 consists of nucleotides encoding the entire protein coding sequence of Cry J2, with the exception of the signal sequence (i.e., SEQ ID NO:2), Cry J2 is a pectate lyase protein present in Japanese cedar pollen. It is well known in the art that Cry J2 is associated with seasonal and persistent allergic reactions in areas where cedar pollen is present. Cry J2-specific IgE is commonly present in allergy patients in areas close to cedrol. It should be noted that in the sequence listing provided as part of the present disclosure, the signal sequence (if present) of each allergen is annotated. It will be appreciated that in the context of the constructs of the invention, these signal sequences are not present.

In other embodiments, the nucleic acid comprises the sequence of SEQ ID NO:4 (i.e., the Cry J1 nucleotide sequence, lacking its signal sequence) or another sequence encoding SEQ ID NO:5 (i.e., the Cry J2 protein sequence, lacking its signal sequence). In other embodiments, the nucleic acid comprises the sequences of both SEQ ID NO 2 and SEQ ID NO 4 or other sequences encoding SEQ ID NO 3 and SEQ ID NO 5, respectively. In embodiments, the nucleic acid comprises one or more of the other sequences disclosed herein, such as those encoding any of the following allergens: cry J3(Cry J3.8; Cryptomeria japonica; SEQ ID NO: 10; signal sequence residues 1-26), CJP-4 (Cryptomeria japonica; SEQ ID NO:11), CJP-6 (Cryptomeria japonica; SEQ ID NO:12), CJP-8 (Cryptomeria japonica; SEQ ID NO: 13; signal sequence residues 1-35), CPA63 (Cryptomeria japonica; SEQ ID NO: 14; signal sequence residues 1-20), CJP38 (Cryptomeria japonica; SEQ ID NO: 15; signal sequence residues 1-28), Cha O1 (Chamaecyparis obtusa (C.obtusea.); SEQ ID NO: 16; signal sequence residues 1-21), Juna 1 (Juniperus procumbens (J.ashei); ID NO: 17; signal sequence residues 1-21), Junv 1 (J.virginiana); SEQ ID NO: 18; signal sequence residues 1-21), Cup a 1 (Hipponach saxara (H.arizonica); SEQ ID NO: 19; signal sequence residues 1-21), Juno 1 (Juniperus tamariscina (J.Oxycedrus); SEQ ID NO: 20; signal sequence residues 1-21), Cup s1 (Sempervirens (C.); SEQ ID NO: 21; signal sequence residues 1-21), Chao 2 (Thuja Nipponica; SEQ ID NO: 22; signal sequence residues 1-22), Jun a2 (Juniperus albuginella; SEQ ID NO: 23; signal sequence residues 1-22), Cup a2 (Hipponach saxara; SEQ ID NO:24), Jun a 3 (Junicosa laevigata; SEQ ID NO: 25; signal sequence residues 1-16), Jun r 3 (Juniperus communis (J.rigida); signal residues 1-26; signal sequence residues 26-26; Juniperus communis 1-26), Cup s 3 (Selaginella moellendorfii; SEQ ID NO: 27; signal sequences from residues 1 to 26), Cup a 3 (Arizona sonehz sala; SEQ ID NO:28), Ch4A (P.montiola, Calif.), SEQ ID NO: 29; signal sequences from residues 1 to 25), Ch4-1 (Douglas fir (P.menziesii); SEQ ID NO: 30; signal sequences from residues 1 to 26), PT-1 (P.taeda; SEQ ID NO:31) and LTP (P.Abies); SEQ ID NO: 32; signal sequences from residues 1 to 25). Nucleic acid and amino acid sequences not listed with reference to SEQ ID NO are also publicly available. The protein sequence according to the invention can be obtained from the nucleic acid sequence by mere implementation of a computer program. Of course, sequences that are biochemically homologous to these protein sequences are also encompassed by these examples. For example, sequences that exhibit 30% or greater identity to the disclosed sequences, e.g., 40% or greater, 50% or greater, 75% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater identity are encompassed by these embodiments. It is understood that this concept applies not only to the particular sequences of the allergens disclosed herein, but also to all protein and nucleic acid sequences provided herein. In addition, as noted above, each value within the disclosed ranges is to be understood to be specifically encompassed by the present disclosure.

In a specific example of the invention, a DNA vaccine comprising SEQ ID NO 2 or another sequence encoding SEQ ID NO 3 in the allergen domain is provided. When such a vaccine is administered to a patient presenting with evidence of equivalent Japanese cedar allergy, the vaccine results in de novo synthesis of a fusion or chimeric (these two terms are used interchangeably herein) protein comprising the allergen Cry J2 (given in SEQ ID NO: 3). Due to the combination of domains present on the chimeric protein, the protein is directed from the endoplasmic reticulum into the endocytolysosomal pathway, resulting in processing of the fusion protein into epitopes in MHC vesicles, some of which bind to MHC class II molecules, resulting in an enhanced humoral immune response.

In another example of the invention, a DNA vaccine is provided comprising the sequence of SEQ ID NO. 4 or another sequence encoding SEQ ID NO.5 in the allergenic domain. When such a vaccine is administered to a patient presenting with evidence of equivalent Japanese cedar allergy, the vaccine results in the de novo synthesis of a fusion or chimeric (these two terms are used interchangeably herein) protein comprising the allergen Cry J1 (given in the sequence of SEQ ID NO: 4). Due to the combination of domains present on the chimeric protein, the protein is directed from the endoplasmic reticulum into the endocytolysosomal pathway, resulting in processing of the fusion protein into epitopes in MHC vesicles, some of which bind to MHC class II molecules, resulting in an enhanced humoral immune response.

In another example of the present invention, a DNA vaccine comprising SEQ ID NO 6 in the allergen domain is provided. When such a vaccine is administered to a patient presenting with evidence of equivalent cryptomeria japonica allergy, the vaccine results in de novo synthesis of a fusion or chimeric (the terms are used interchangeably herein) protein comprising the allergens CryJ1 and Cry J2 (SEQ ID NO: 7). Due to the combination of domains present on the chimeric protein, the protein is directed from the endoplasmic reticulum into the endocytolysosomal pathway, resulting in processing of the fusion protein into epitopes in MHC vesicles, some of which bind to MHC class II molecules, resulting in an enhanced humoral immune response.

In another example of the present invention, a nucleic acid encoding the entire protein-encoding sequence of Jun a 1, Jun a 1 being a pectate lyase belonging to the genus Aspercus, is provided in the allergen domain. Jun a 1 displays a high degree of sequence identity to Cry J1 and retains enzyme activity similar to Cry J1 and has a high degree of similarity among known epitopes.

It is well known that other polypeptides have cross-reactivity with Cry J1 and that this cross-reactivity is due to sharing epitopes associated with the enzymatic activity of pectate lyase family polypeptides. This family includes the major allergens of hinoki (Ch o 1)) and includes the allergens from: aoshi juniper (Jun a 1), Thuja occidentalis (Jun V1), Arizona asiatica (Cuppress arizonica) (Cup a 1), Juniperus Oxyphylla (Jun o 1), and Juniperus communis (Cups 1). It has been observed in the literature that there is strong cross-reactivity to pollen from cedarwood (Cupressus) in allergy patients. Table I below depicts a table showing the levels of cross-reactivity between related proteins. Although the invention is described in detail with respect to Cry J1 and Cry J2, it is to be understood that one or more allergens disclosed herein and in particular in table I can be used in addition to or as an alternative to Cry J1 and Cry J2 sequences.

Table I: cross-reactivity of Cryptomeria japonica with other allergens

It is well known in the art that certain sites (i.e., fusion sites) for inserting the nucleotide sequence of a coding region into the nucleotide sequence of a different coding region are more advantageous than others. The positions of the allergen sequences as taught, for example, in fig. 1-5 are taught as being advantageous for use of the compositions taught in the present invention. It is within the scope of the invention to move the location of the allergen sequence to other locations, such as the luminal domain of the LAMP polypeptide or other intra-organelle stabilizing domains. However, it is preferred that the allergen is not located within the coding region of the transmembrane or cytoplasmic domain. In a preferred example of the present invention, the allergen sequence is inserted into the lumen domain of the LAMP polypeptide within 5 amino acids from the junction point with the transmembrane domain and up to 20 amino acids on the 5' N terminal side of the lumen domain of the LAMP polypeptide.

The nucleic acids of the invention may be provided as purified or isolated molecules. The nucleic acid may also be provided as part of a composition. The composition may consist essentially of the nucleic acid, meaning that the nucleic acid is the only nucleic acid in the composition suitable for expression of the coding sequence. Alternatively, the composition may comprise a nucleic acid of the invention. In an exemplary embodiment, the composition is a pharmaceutical composition comprising a nucleic acid of the invention and one or more pharmaceutically acceptable substances or carriers. In some embodiments, the composition comprises a substance that promotes uptake of nucleic acids by a cell. In some embodiments, the composition comprises a targeting molecule that facilitates delivery of the nucleic acid to a particular cell type, such as an immune cell (e.g., APC). In embodiments, the nucleic acid is part of a delivery vehicle or delivery vector for delivering the nucleic acid to a cell or tissue.

In a particular example of the invention, the composition comprises a mixture of two DNA vaccines, wherein one vaccine comprises the sequence of one allergen and wherein the other vaccine comprises the sequence of the other allergen. The two vaccine constructs can be mixed together in a ratio of 1:1, 1:2, 1:3, 1:4, in order up to 1: 10. The preferred ratio is 1: 1.

In particular examples of the invention, the nucleic acids of Cry J1, Cry J2, and/or Jun a2 are present in a nucleic acid delivery vector. In a preferred embodiment of the invention, the nucleic acid delivery vector does not contain an antibiotic resistance gene, such as the nucleic acid delivery vector taught in U.S. patent application publication No.2008/006554 or the vector disclosed in or derived from U.S. patent application publication No. 2006/003148. In a particular example of the invention, the nucleic acid is a viral vector, such as an adenoviral vector.

The nucleic acids and compositions are novel and are useful as agents for reducing allergy in a patient. For example, the nucleic acids and compositions may be used to reduce pollinosis in patients with proven allergies associated with Japanese cedar pollen or caused by homologous pollen or allergens. As another non-limiting example, the nucleic acid may be used to reduce food allergies, such as to peanuts or other nuts. Nucleic acids and compositions are delivered to treat pollinosis caused by japanese cedar pollen, such that the nucleic acids and compositions transfect antigen presenting cells resulting in increased serum levels of immunoglobulin g (igg) specific for epitopes contained within Cry J1 and/or Cry J2. The response can be used to alleviate symptoms of allergy. Allergens that deliver other allergies (including ragweed, other tree pollen and food) also result in increased serum levels of IgG.

The invention also provides methods of treating a subject in need thereof. The method is a method of prophylactically or therapeutically treating a subject suffering from or at risk of developing an allergy to one or more allergens. The method comprises administering a DNA vaccine according to the invention to a subject in an amount sufficient to cause the APC to take in and express the DNA vaccine. Expression of the DNA vaccine results in presentation of the encoded allergenic epitope on APC and development of an IgG immune response.

In a particular example of the invention, SEQ ID NO 2, SEQ ID NO 4 and/or another allergen coding sequence is administered to a cell. In a preferred embodiment, the cell is an antigen-presenting cell, such as a dendritic cell. Preferably, the dendritic cells are human dendritic cells. The present invention may be administered by methods known in the art as effective delivery methods for nucleic acid vaccines, including intramuscular injection, subcutaneous injection, electroporation, gene gun immunization, or liposome-mediated transfer.

The present invention provides a preparation useful for treating pollinosis associated with Japanese cedar. It has been previously determined that delivery of a DNA plasmid encoding the protein coding sequence of an allergen to an animal increases IFN- γ production and decreases IL-4 production, and that it is useful in treating animals that are allergic to specific allergens. The present invention provides improved DNA vaccine compositions for treating patients with allergy associated with japanese cedar pollen. The fusion proteins of the invention have a specific intracellular trafficking pattern that intersects MHC class II vesicles and results in enhanced presentation of allergen epitopes to the immune system, resulting in, inter alia, an enhanced antibody response. The nucleic acids and compositions provided herein are useful for performing allergy immunotherapy.

The present invention provides a formulation that results in an increased specific antibody response when administered to a cell. The increased antibody response to allergens is useful in the treatment of IgE-mediated allergic diseases. IgE has certain properties related to its cellular confinement and intracellular signaling upon binding of homologous allergens. IgE against allergens is produced when B cells receive IL-4 secreted by Th2 cells. This helps direct B cells to produce IgE class antibodies. Upon secretion by B cells, IgE binds to Fc-eRI (its high affinity receptor expressed by mast cells and eosinophils), causing these cells and the animal to become susceptible to subsequent allergen exposure. Thus, allergic symptoms can be triggered upon ingestion, inhalation, or mucosal contact with allergens. Due to the binding properties of antibodies, it has been proposed that one way to alleviate the symptoms of allergy is by chelating free allergens to which IgE can bind by competition with other antibody types. In particular, allergy preparations that increase IgG have been proposed as a way to alleviate allergic diseases. The invention described herein induces enhanced IgG production, thereby causing a reduction in the ratio of IgE to IgG in a clinically significant manner. Results of the studies that have been performed indicate that on day 98, the IgG levels induced by the Cry J2-LAMP construct are greater than those induced by delivery of the nucleotide encoding the unmodified Cry J2.

In another example of the invention, a method is taught for selecting pectate lyase polypeptides present in cedar pollen to determine the degree of sequence homology to the amino acid or nucleic acid sequence of Cry J1 (a pectate lyase) such that a novel composition of matter similar to Cry J1 can be produced, and such that administration of the homologous composition of matter to a patient will produce a therapeutic result useful in treating allergy associated with cedar pollen.

Examples of the invention

The present invention will now be described with reference to exemplary embodiments thereof. The following examples are intended to give the reader a better understanding of the construction and activity of the constructs of the invention and should not be construed as limiting the scope of the invention.

Example 1: general materials and methods

Immunization and serum Collection

Female BALB/c mice, six to eight weeks old, were purchased from Harlan Laboratories, Frederick, Maryland, Fraderrick, Maryland and maintained in our animal facility in Rockville, Maryland. DNA immunization was performed intramuscularly or intradermally with 50. mu.g plasmid DNA in sterile PBS in a volume of 100. mu.l. Serum was obtained by orbital bleeding and stored at-20 ℃ for later analysis. For sensitization, mice were injected with 5. mu.g/ml recombinant CRYJ2(rCRYJ2) or recombinant CRYJ1(rCYRJ1) and 100. mu.l alum (2mg/ml) in a total volume of 200. mu.l. Mice were bled weekly and sera were analyzed by ELISA for CRY J-specific antibodies.

Guinea pig

Female guinea pigs were purchased and housed at the Spring Valley laboratory (Woodline, MD) of the forest crown line, maryland. DNA immunization was performed intramuscularly with 100. mu.g plasmid DNA in a volume of 200. mu.l sterile saline. Serum was obtained by cardiac blood sampling and stored at 20 ℃ for later analysis.

Detection of CYRJ 2-specific immunoglobulin response

Nunc Maxisorp immunoassay plates were coated with rCRYJ2 in PBS at a concentration of 5. mu.g/ml at 4 ℃. After blocking with 1% BSA in PBS, the serum was diluted in PBS containing 0.05% tween 20 (PBS-T), added and incubated for 1 hour. IgG, IgG1, or IgG2a bound to CRY J2 immobilized on the wells was detected using peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2a antibodies (Jackson Laboratories). TMB substrate (KPL) was added and the enzyme activity was stopped with TMB stop solution. Plates were read at 450 nm. In some cases, plates were read using Sure Stop Solution (KPL) and at 650 nm.

Preparation of splenocytes for cytokine measurement

Spleens were aseptically removed and picked to prepare single cell suspensions. To study primary responses, splenocytes were plated in 24-well plates (4 × 10)⁵Individual cells/well) in the presence or absence of 10. mu.g/ml, 5. mu.g/ml or 2.5. mu.g/ml rCRYJ2 for 72 hours.

Cytokine assay

Supernatants were assayed for the presence of IFN-. gamma.and IL-4 by ELISA. The matched antibody pairs were used for IFN-. gamma.and IL-4 and were performed according to the manufacturer's instructions. Standard curves were generated using mouse recombinant IFN-. gamma.and IL-4. All antibodies and cytokines were purchased from Invitrogen, Carlsbad, CA, calsbad, california. The detection limits of the IFN-. gamma.and IL-4 assays were 20 and 10pg/ml, respectively.

Example 2: expression of allergens from constructs

To show that the nucleic acid constructs of the invention can be used to express one or more allergens in transformed cells, human 293 cells were transfected with CryJ2-LAMP plasmid, CryJ1+ J2-LAMP plasmid (FIG. 4), CryJ1-LAMP plasmid, CryJ 1-plasmid (lacking CryJ1 signal sequence; FIG. 7) and a base plasmid vector alone (negative control; SEQ ID NO: 1). The results of the experiment are shown in fig. 9.

FIG. 9A shows the results of the transfection reaction, detected with anti-CryJ 2 antibody. Briefly, thirty micrograms of cell lysate was electrophoresed and then transferred to a membrane for immunoblot analysis. Proteins were detected by immunoblotting with the CryJ2 monoclonal antibody followed by chemiluminescence. As can be seen from the figure, constructs comprising the CryJ2 allergen alone and the CryJ1+ CryJ2 allergen were detected (lanes 2 and 3), while no other allergens were detected. In this experiment, the naturally occurring signal sequences of the CryJ1 and CryJ2 allergens were removed prior to the experiment, except for the constructs in lane 5. These results show that not only the construct of the present invention is suitable for expressing an allergen, but also a plurality of allergens can be co-expressed.

FIG. 9B shows the results of the transfection reaction, detected with anti-CryJ 1 antibody. Briefly, thirty micrograms of cell lysate was electrophoresed and then transferred to a membrane for immunoblot analysis. Proteins were detected by immunoblotting with the CryJ1 monoclonal antibody followed by chemiluminescence. As can be seen from the figure, the construct comprising the CryJ1+ CryJ2 allergen (lacking the natural signal sequence) was detected (lane 3), as was the construct comprising the CryJ1 allergen from which the naturally occurring signal sequence had been removed (lane 5). However, constructs in which the Cryl allergen included its native signal sequence were not detected. These results show that the constructs of the invention are suitable for the expression and detection of a variety of allergens, and that removal of the naturally occurring signal sequence is important in the expression and detection of the product.

Example 3: data supporting the MHC II processing pathway of the constructs

To determine whether the chimeric proteins produced by the constructs of the invention were processed by the MHC II pathway, a set of experiments were performed to compare the immune response to the CryJ2 protein when administered as coding region on a plasmid or as allergen domain on a construct according to the invention. The results are given in panels a and B of fig. 10.

More specifically, the figure shows the CryJ2 specific response after four DNA immunizations and sensitization with crude pollen extract. Groups of mice (n-5) were immunized subcutaneously with either CRYJ2-LAMP plasmid DNA or CRYJ2 plasmid (see fig. 8) DNA on days 0, 7, 14 and 21. Six weeks after the last DNA immunization (day 77), mice were sensitized with crude pollen extract in alum and given booster doses three weeks later (day 91). Data show the values generated from pooled sera at each time point. IgG1 (panel a) and IgG2a (panel B) responses in mice receiving CRYJ2-LAMP DNA remained elevated throughout 112 days and were much higher than those mice receiving CRYJ2 plasmid DNA that did not include LAMP. Delivery of the allergen cause via the construct according to the invention provides a superior MHC II response than delivery of the allergen without the background of the construct of the invention.

Example 4: theoretical basis of dose-comparison of immune responses against constructs at different doses and against individual vectors

Figure 11 shows CryJ 2-specific responses for IgG2a production and IgG1 production after four DNA immunizations at different dose levels. Groups of mice (n ═ 5) were given 10 μ g, 50 μ g, or 100 μ g of CRYJ2-LAMP plasmid DNA or vector DNA intramuscularly on days 0, 7, 14, and 21. Three weeks after the last DNA immunization, mice were sacrificed and spleens removed for cytokine induction assays.

The data show the values generated from pooled sera for each vaccine dose. All three concentrations of CRYJ2-LAMP plasmid DNA caused IgG1 and IgG2a responses, with a 50 μ g dose showing that the highest antibody response had been caused. Any concentration of vector alone did not induce any antibody response. These data show that there is a dose-dependent response to elicit an immune response, and that the immune response is at least in part an MHC class II response.

Example 5: additional data illustrating immune response via the MHC II pathway

In this set of experiments, cytokine secretion in the supernatant of stimulated splenocytes was determined using IL-4 and IFN- γ as markers. Specifically, splenocytes from mice (n-3) were collected on day 42 and cultured in the presence of 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, or no rCRYJ 2. From untreated mice (For spleen cells of micro)Negative control was performed. IL-4 and IFN- γ levels in pg/ml were measured by ELISA on splenocytes stimulated with rCRYJ 2.

The data are given in panels a and B of fig. 12. The data show that mice receiving 50 μ g of CRYJ2-LAMP plasmid DNA have significantly higher expression of IFN- γ (a definitive biomarker of MHC II immune response pathway activation) than those receiving lower doses of plasmid DNA. Minimal IL-4 level responses were seen in any of the groups, IL-4 being a definitive biomarker for the MHC I pathway. There was also minimal, if any, response to IL-5 (data not shown). These results indicate that Cry J2-LAMP DNA immunization induced recruitment of Th1 memory cells but not Th2 cells, as indicated by production of IFN- γ rather than IL-4 following stimulation with recombinant Cry J2 protein.

Example 6: study of therapeutic Effect of immunization with CryJ2-LAMP DNA vaccine in mice previously sensitized with CryJ2

To study the therapeutic effect of the DNA-LAMP-CryJ2 vaccine, a group of mice (n ═ 5) was sensitized with three 5 μ g injections of CryJ2 recombinant protein and, four weeks later, treated with four injections of CryJ2-LAMP plasmid DNA, which were given at weekly (7 day) intervals. This DNA immunization induced a boosting effect of IgG2a and a temporary increase of IgG1 antibodies, indicating a Th 1-mediated regulatory effect of the DNA vaccine. Two additional DNA immunizations at day 167 and day 174 boosted a CRYJ 2-specific IgG2a response and little change in IgG1 response. Visual examination of the mice showed no physical discomfort or skin reaction. The appetite also did not change and they did not appear to be asleep. The effect on IgG1 and IgG2a titers are shown in panels a and B of fig. 13, respectively.

Example 7: induction of IFN-gamma and IL-4 in mouse splenocyte cultures

The therapeutic effect of cytokine induction on CryJ2-LAMP DNA vaccine was also investigated. Splenocytes from mice (n-3) were harvested on day 183 and stimulated with various concentrations of rCRYJ 2. Splenocytes from untreated mice were used as negative controls. IL-4 and IFN- γ levels in pg/ml were measured by ELISA on splenocytes stimulated with rCRYJ 2. Significantly increased IFN- γ expression was detected in the CRYJ2-LAMP immunized group compared to IFN- γ expression in the vector group. However, IL-4 expression did not appear to differ from the vector set. The increase in IFN- γ by Cry 12-LAMP DNA immunization is presumably related to the recruitment of antigen-specific Th1 cells and the production of a Th1 cytokine environment. The data obtained from this experiment are given in panels a and B of fig. 14.

Example 8: detection of circulating CryJ2 protein in serum

Mice were immunized with Cry J2 protein, pDNA-Cry J2 (without LAMP) and Cry 12-LAMP-vax. Serum samples were taken on days 0, 1, 2,3, 4 and 7 and evaluated for the presence of free Cry J2 protein in a sensitive sandwich immunoassay. Free Cry J2 was detected in the protein and non-LAMP immunization. However, no free allergen was detected at any time point in any experiment performed with Cry J2-LAMP-vax immunized mice (lowest detectable level was 2 ng/ml). Data supporting this argument is given in fig. 12.

The LAMP vaccine according to the invention will be the only formulation that can treat allergy without introducing free allergen systemically into the patient. This is unlike traditional immunotherapy, which sometimes can lead to allergic reactions due to systemic introduction of allergens. This experiment shows that mice receiving the Cry J2-LAMP DNA plasmid do not have free Cry J2 protein and thus are not released into the systemic circulation, as is seen for mice given protein alone or no LAMP of Cry J2 DNA.

Example 9: efficacy of DNA vaccines in guinea pigs

To expand the scientific understanding of the function of the nucleic acid construct in other animals, studies were conducted in female guinea pigs immunized with the CryJ2-LAMPDNA vaccine and then challenged with recombinant CryJ2 protein. The results of the study are shown in panels a and B of fig. 16.

Specifically, female guinea pigs received 100 μ g of CRYJ2-LAMP DNA vaccine or intramuscular injection of vector alone on days 0, 7, and 14. Four weeks after the last DNA vaccine immunization on day 14, guinea pigs were given subcutaneous injections of 10. mu.g/ml rCRYJ2 protein/alum on days 42 and 49. Serum samples were obtained from guinea pigs on days 0, 21, 35, 63 and 77. The data show that for IgG2, the average absorbance values of guinea pigs receiving CRYJ2-LAMP DNA increased up to day 35 with little or no IgG1 response. The increase in IgG2a was consistent with that typically seen in Th1 biased responses.

Example 10: additional investigations in other mammals-toxicological data to demonstrate safety

New Zealand white rabbits received an intramuscular injection of 4.128mg of CRYJ2-LAMP DNA. Age and sex matched control rabbits received saline alone. Rabbits were immunized on days 1, 14, 28, 42, and 56. Serum samples were obtained from rabbits on days 1, 14, 28, 42, 56, 58, and 85. The average absorbance values of rabbit sera at 1:100 after multiple intramuscular injections of CryJ2-LAMP plasmid or saline are shown in figure 17. As can be seen from the figure, the data show that the average absorbance values for saline-receiving rabbits are less than 0.100. The absorbance values of rabbits in the group treated with CRYJ2-LAMP DNA generally increased up to day 42, and in some cases up to day 85.

Example 11: applicability to food allergen

Over the past 25 years, 8 important peanut allergens have been identified based on sensitization in peanut allergy sufferers. Three major peanut allergens are most commonly recognized by IgE of peanut allergic individuals: 65-100% recognize Arah1, which is a 63.5kDa seed storage pea globulin (vicilin) family protein; 71-100% recognize Ara h2, which is a 17kDa seed storage lupin conglutin family protein; and 45-95% recognize Ara h3, a 14kDa seed storage glycinin family protein. In addition to being a common causative factor in peanut-dependent allergies and allergies, these three proteins also appear to promote stronger allergies. Targeting these allergens has the potential to provide the broadest protection against strong allergies among diverse populations of peanut allergies as a basis for immunotherapy of peanut allergies. Phase I clinical trials are currently underway which use hypoallergenic forms of the three major allergens and heat inactivated bacterial adjuvants as allergy immunotherapy. This trial is ongoing, but the ultimate commercialization of such therapies would be a challenge due to the highly complex manufacturing processes.

In order to solve the increased incidence of food allergies, in particular peanut allergies, a nucleic acid construct according to the invention is produced. This construct is depicted in fig. 6A, and a schematic of the encoded chimeric protein is depicted in fig. 6B, as discussed above. The constructs are useful for generating a primary MHC II response in a subject administered the construct. The presence of the three most common peanut allergens in a single chimeric protein provides a broad immune effect that will treat the vast majority of peanut allergies in a population.

This construct was expressed and the results are shown in figure 18. FIG. 18 shows that all three of the allergens can be expressed and detected as single polymeric proteins on Western blots.

It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the invention and in the construction of nucleic acid constructs without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Additional sequences of the sequence Listing

In addition to the sequences provided in the official sequence listing provided as part of this application, the following sequences form part of this disclosure:

the nucleotide sequence of the coding region of the Cryl-Cry2-LAMP chimeric construct is as follows:

SEQ ID NO:6-Cry J1+J2-LAMP

nucleic acid sequence of the coding region of Ara H1/H2/H3 polyprotein:

SEQ ID NO:8

the amino acid sequence of the coding region of the Ara H1/H2/H3 polyprotein chimeric construct is as follows:

SEQ ID NO:9-AraH-LAMP

sequence listing

<110> immunotherapy Co., Ltd (IMMUNOMIC THERAPEUTICS, INC.)

<120> NUCLEIC acid FOR treating allergy (NUCLEIC ACIDS FOR TREATMENT OF ALLERGIES)

<130>SPI175586-11

<150>PCT/US2012/042552

<151>2012-06-15

<150>61/496,866

<151>2011-06-14

<160>32

<170>PatentIn version 3.5

<210>1

<211>2898

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence: Synthetic

pITI plasmid polylnucleotide

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ccgcctaatg agcgggcttt tttttcttag gccttcttcc gcttcctcgc tcactgactc 60

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 120

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 180

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 240

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 300

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 360

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 420

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 480

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 540

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 600

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 660

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 720

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 780

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 840

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 900

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 960

aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 1020

atttcgttca tccatagttg cctgactcct gcaaaccacg ttgtggtaga attggtaaag 1080

agagtcgtgt aaaatatcga gttcgcacat cttgttgtct gattattgat ttttggcgaa 1140

accatttgat catatgacaa gatgtgtatc taccttaact taatgatttt gataaaaatc 1200

attaggtacc ccggctctag atggcatgac attaacctat aaaaataggc gtatcacgag 1260

gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 1320

ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 1380

gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 1440

actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 1500

catcagattg gctattggcc attgcatacg ttgtatccat atcataatat gtacatttat 1560

attggctcat gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag 1620

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 1680

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 1740

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 1800

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 1860

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 1920

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 1980

ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 2040

caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 2100

tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 2160

tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 2220

tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggctcgca tctctccttc 2280

acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc 2340

tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc 2400

gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac 2460

gctttgcctg accctgcttg ctcaactcta gttctctcgt taacttaatg agacagatag 2520

aaactggtct tgtagaaaca gagtagtcgc ctgcttttct gccaggtgct gacttctctc 2580

ccctgggctt ttttcttttt ctcaggttga aaagaagaag acgaagaaga cgaagaagac 2640

aaagccgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 2700

gcagtcttcg tttcgcccag cggtaccgga tccgtcgacg gggggagatc tttttccctc 2760

tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag 2820

gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac 2880

ataagggcgg ccgctagc 2898

<210>2

<211>4157

<212>DNA

<213>Cryptomeria japonica

<400>2

ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac 60

tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag 120

ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt 180

gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc 240

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 300

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 360

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 420

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 480

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 540

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 600

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 660

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 720

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 780

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 840

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 900

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 960

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200

tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260

tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320

tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380

ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680

aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740

atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820

aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880

gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940

ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000

gaactacgac accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060

agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120

tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180

cgttcagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240

ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300

atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360

tgccaccatc caggcgtacc tttccaacag cagcttcagc aggggagaga cacgctgtga 3420

acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480

cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540

gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600

gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660

cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720

gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780

cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840

cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900

caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960

ggaggagtgt ctgctggacg agaacagcct cgaggatcag tcagcgcaga tcatgctgga 4020

tagcgtggtg gagaagtacc tgaggagtaa caggtcactg cgcaaggttg agcattccag 4080

acacgacgct atcaacatct tcaacgtgga gaagtacggt gctgtcggag acgggaagca 4140

cgactgcacc gaagcct 4157

<210>3

<211>911

<212>PRT

<213>Cryptomeria japonica

<400>3

Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val

20 25 30

Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala

35 40 45

Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu

50 55 60

Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly

65 70 75 80

Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly

85 90 95

His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val

100 105 110

Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro

115 120 125

Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile

130 135 140

Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val

145 150 155 160

His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala

165 170 175

Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln

180 185 190

Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser

195 200 205

Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser

210 215 220

Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn

225230 235 240

Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn

245 250 255

Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu

260 265 270

Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe Gln

275 280 285

Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln

290 295 300

Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala

305 310 315 320

Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys

325 330 335

Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn

340 345 350

Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe

355 360 365

Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Asp Gln

370 375 380

Ser Ala Gln Ile Met Leu Asp Ser Val Val Glu Lys Tyr Leu Arg Ser

385 390 395 400

Asn Arg Ser Leu Arg Lys Val Glu His Ser Arg His Asp Ala Ile Asn

405 410 415

Ile Phe Asn Val Glu Lys Tyr Gly Ala Val Gly Asp Gly Lys His Asp

420 425 430

Cys Thr Glu Ala Phe Ser Thr Ala Trp Gln Ala Ala Cys Lys Asn Pro

435 440 445

Ser Ala Met Leu Leu Val Pro Gly Ser Lys Lys Phe Val Val Asn Asn

450 455 460

Leu Phe Phe Asn Gly Pro Cys Gln Pro His Phe Thr Phe Lys Val Asp

465 470 475 480

Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Asn Asn Arg

485 490 495

Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Lys

500 505 510

GlyVal Ile Asp Gly Gln Gly Lys Gln Trp Trp Ala Gly Gln Cys Lys

515 520 525

Trp Val Asn Gly Arg Glu Ile Cys Asn Asp Arg Asp Arg Pro Thr Ala

530 535 540

Ile Lys Phe Asp Phe Ser Thr Gly Leu Ile Ile Gln Gly Leu Lys Leu

545 550 555 560

Met Asn Ser Pro Glu Phe His Leu Val Phe Gly Asn Cys Glu Gly Val

565 570 575

Lys Ile Ile Gly Ile Ser Ile Thr Ala Pro Arg Asp Ser Pro Asn Thr

580 585 590

Asp Gly Ile Asp Ile Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn

595 600 605

Thr Ile Gly Thr Gly Asp Asp Cys Val Ala Ile Gly Thr Gly Ser Ser

610 615 620

Asn Ile Val Ile Glu Asp Leu Ile Cys Gly Pro Gly His Gly Ile Ser

625 630 635 640

Ile Gly Ser Leu Gly Arg Glu Asn Ser Arg Ala Glu Val Ser Tyr Val

645 650 655

His Val Asn Gly Ala Lys Phe Ile Asp Thr Gln Asn Gly Leu Arg Ile

660 665 670

Lys Thr Trp Gln Gly Gly Ser Gly Met Ala Ser His Ile Ile Tyr Glu

675 680 685

Asn Val Glu Met Ile Asn Ser Glu Asn Pro Ile Leu Ile Asn Gln Phe

690 695 700

Tyr Cys Thr Ser Ala Ser Ala Cys Gln Asn Gln Arg Ser Ala Val Gln

705 710 715 720

Ile Gln Asp Val Thr Tyr Lys Asn Ile Arg Gly Thr Ser Ala Thr Ala

725 730 735

Ala Ala Ile Gln Leu Lys Cys Ser Asp Ser Met Pro Cys Lys Asp Ile

740 745 750

Lys Leu Ser Asp Ile Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala Ser

755 760 765

Cys Leu Asn Asp Asn Ala Asn Gly Tyr Phe Ser Gly His Val Ile Pro

770 775 780

Ala Cys Lys Asn Leu Ser Pro Ser Ala Lys Arg Lys Glu Ser Lys Ser

785790 795 800

His Lys His Pro Lys Thr Val Met Val Glu Asn Met Arg Ala Tyr Asp

805 810 815

Lys Gly Asn Arg Thr Arg Ile Leu Leu Gly Ser Arg Pro Pro Asn Cys

820 825 830

Thr Asn Lys Cys His Gly Cys Ser Pro Cys Lys Ala Lys Leu Val Ile

835 840 845

Val His Arg Ile Met Pro Gln Glu Tyr Tyr Pro Gln Arg Trp Ile Cys

850 855 860

Ser Cys His Gly Lys Ile Tyr His Pro Glu Phe Thr Leu Ile Pro Ile

865 870 875 880

Ala Val Gly Gly Ala Leu Ala Gly Leu Val Leu Ile Val Leu Ile Ala

885 890 895

Tyr Leu Val Gly Arg Lys Arg Ser His Ala Gly Tyr Gln Thr Ile

900 905 910

<210>4

<211>5326

<212>DNA

<213>Cryptomeria japonica

<400>4

ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac 60

tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag 120

ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt 180

gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc 240

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 300

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 360

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 420

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 480

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 540

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 600

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 660

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 720

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 780

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 840

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 900

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 960

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200

tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260

tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320

tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380

ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680

aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740

atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820

aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880

gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940

ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000

gaactacgac accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060

agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120

tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180

cgttcagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240

ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300

atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360

tgccaccatc caggcgtacc tttccaacag cagcttcagc aggggagaga cacgctgtga 3420

acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480

cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540

gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600

gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660

cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720

gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780

cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840

cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900

caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960

ggaggagtgt ctgctggacg agaacagcct cgaggacaat cctattgatt cctgctggcg 4020

tggagattct aactgggcac agaaccggat gaaactggct gactgtgccg tgggctttgg 4080

ctcttccact atgggaggga agggaggcga cctgtacact gttacaaaca gcgacgacga 4140

ccctgtcaat ccagcacccg gaaccttgag atatggtgca acgcgagacc gaccactttg 4200

gatcatcttt agcggaaaca tgaacatcaa gttgaagatg cctatgtaca tagctgggta 4260

caaaaccttc gacggcagag gagcccaagt gtacattggc aacggaggtc cctgcgtgtt 4320

catcaagcgt gttagtaatg tgatcattca cggtctgcac ctctatggct gttcaacaag 4380

cgtgctgggg aatgtgctga tcaatgagtc attcggtgtt gaacccgtgc acccacagga 4440

cggtgatgcg ttgacactga ggacagccac caatatctgg attgaccata acagtttctc 4500

taacagctca gatggcctgg tggatgtcac cttgagtagc acaggggtca caatcagcaa 4560

caatctgttc ttcaaccatc ataaggtgat gctgctgggc cacgacgatg cgtattccga 4620

cgataagagc atgaaagtga cggtggcctt taaccagttt ggtcctaact gtggacagcg 4680

gatgcctaga gccaggtacg gactggtgca cgtggccaac aacaactatg atccgtggac 4740

tatctatgca attggcggtt cttccaaccc gacgatactg agtgaaggga actcctttac 4800

cgctcccaat gagagctaca agaagcaggt caccatccgc ataggctgca aaactagttc 4860

atcctgtagc aactgggtgt ggcagtccac tcaagatgtc ttctacaacg gagcttactt 4920

cgttagcagt gggaaatacg aaggtggcaa catatacaca aagaaagagg ctttcaatgt 4980

ggagaatggc aatgccactc cccagctcac caagaatgca ggggtgctca cctgctccct 5040

gagcaaacgg tgcgaattca cgctgatccc catcgctgtg ggtggtgccc tggcggggct 5100

ggtcctcatc gtcctcatcg cctacctcgt cggcaggaag aggagtcacg caggctacca 5160

gactatctag taaagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc 5220

ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga 5280

attttttgtg tctctcactc ggaaggacat aagggcggcc gctagc 5326

<210>5

<211>773

<212>PRT

<213>Cryptomeria japonica

<400>5

Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu

1 5 1015

Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val

20 25 30

Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala

35 40 45

Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu

50 55 60

Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly

65 70 75 80

Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly

85 90 95

His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val

100 105 110

Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro

115 120 125

Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile

130 135 140

Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val

145 150 155 160

His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala

165 170 175

Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln

180 185 190

Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser

195 200 205

Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser

210 215 220

Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn

225 230 235 240

Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn

245 250 255

Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu

260 265 270

Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe Gln

275 280 285

Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln

290 295 300

Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala

305 310 315 320

Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys

325 330 335

Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn

340 345 350

Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe

355 360 365

Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Asp Asn

370 375 380

Pro Ile Asp Ser Cys Trp Arg Gly Asp Ser Asn Trp Ala Gln Asn Arg

385 390 395 400

Met Lys Leu Ala Asp Cys Ala Val Gly Phe Gly Ser Ser Thr Met Gly

405 410 415

Gly Lys Gly Gly Asp Leu Tyr Thr Val Thr Asn Ser Asp Asp Asp Pro

420 425 430

Val Asn Pro Ala Pro Gly Thr Leu Arg Tyr Gly Ala Thr Arg Asp Arg

435 440 445

Pro Leu Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys Leu Lys Met

450 455 460

Pro Met Tyr Ile Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly Ala Gln

465 470 475 480

Val Tyr Ile Gly Asn Gly Gly Pro Cys Val Phe Ile Lys Arg Val Ser

485 490 495

Asn Val Ile Ile His Gly Leu His Leu Tyr Gly Cys Ser Thr Ser Val

500 505 510

Leu Gly Asn Val Leu Ile Asn Glu Ser Phe Gly Val Glu Pro Val His

515 520 525

Pro Gln Asp Gly Asp Ala Leu Thr Leu Arg Thr Ala Thr Asn Ile Trp

530 535 540

Ile Asp His Asn Ser Phe Ser Asn Ser Ser Asp Gly Leu Val Asp Val

545 550 555 560

Thr Leu Ser Ser Thr Gly Val Thr Ile Ser Asn Asn Leu Phe Phe Asn

565 570575

His His Lys Val Met Leu Leu Gly His Asp Asp Ala Tyr Ser Asp Asp

580 585 590

Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asn Cys

595 600 605

Gly Gln Arg Met Pro Arg Ala Arg Tyr Gly Leu Val His Val Ala Asn

610 615 620

Asn Asn Tyr Asp Pro Trp Thr Ile Tyr Ala Ile Gly Gly Ser Ser Asn

625 630 635 640

Pro Thr Ile Leu Ser Glu Gly Asn Ser Phe Thr Ala Pro Asn Glu Ser

645 650 655

Tyr Lys Lys Gln Val Thr Ile Arg Ile Gly Cys Lys Thr Ser Ser Ser

660 665 670

Cys Ser Asn Trp Val Trp Gln Ser Thr Gln Asp Val Phe Tyr Asn Gly

675 680 685

Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr

690 695 700

Lys Lys Glu Ala Phe Asn Val Glu Asn Gly Asn Ala Thr Pro Gln Leu

705 710 715 720

Thr Lys Asn Ala Gly Val Leu Thr Cys Ser Leu Ser Lys Arg Cys Glu

725 730 735

Phe Thr Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val

740 745 750

Leu Ile Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser His Ala

755 760 765

Gly Tyr Gln Thr Ile

770

<210>6

<211>6817

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence: Synthetic

Cry J1&J2-LAMP polynucleotide

<400>6

ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac 60

tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag 120

ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt 180

gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc 240

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 300

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 360

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 420

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 480

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 540

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 600

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 660

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 720

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 780

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 840

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 900

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 960

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200

tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260

tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320

tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380

ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680

aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740

atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820

aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880

gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940

ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000

gaactacgacaccaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060

agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120

tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180

cgtccagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240

ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300

atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360

tgccaccatc caggcgtacc tttccaacag cagcttcagc cggggagaga cacgctgtga 3420

acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480

cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540

gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600

gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660

cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720

gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780

cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840

cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900

caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960

ggaggagtgt ctgctggacg agaacagcct cgaggacaat cctattgatt cctgctggcg 4020

tggagattct aactgggcac agaaccggat gaaactggct gactgtgccg tgggctttgg 4080

ctcttccact atgggaggga agggaggcga cctgtacact gttacaaaca gcgacgacga 4140

ccctgtcaat ccagcacccg gaaccttgag atatggtgca acgcgagacc gaccactttg 4200

gatcatcttt agcggaaaca tgaacatcaa gttgaagatg cctatgtaca tagctgggta 4260

caaaaccttc gacggcagag gagcccaagt gtacattggc aacggaggtc cctgcgtgtt 4320

catcaagcgt gttagtaatg tgatcattca cggtctgcac ctctatggct gttcaacaag 4380

cgtgctgggg aatgtgctga tcaatgagtc attcggtgtt gaacccgtgc acccacagga 4440

cggtgatgcg ttgacactga ggacagccac caatatctgg attgaccata acagtttctc 4500

taacagctca gatggcctgg tggatgtcac cttgagtagc acaggggtca caatcagcaa 4560

caatctgttc ttcaaccatc ataaggtgat gctgctgggc cacgacgatg cgtattccga 4620

cgataagagc atgaaagtga cggtggcctt taaccagttt ggtcctaact gtggacagcg 4680

gatgcctaga gccaggtacg gactggtgca cgtggccaac aacaactatg atccgtggac 4740

tatctatgca attggcggtt cttccaaccc gacgatactg agtgaaggga actcctttac 4800

cgctcccaat gagagctaca agaagcaggt caccatccgc ataggctgca aaactagttc 4860

atcctgtagc aactgggtgt ggcagtccac tcaagatgtc ttctacaacg gagcttactt 4920

cgttagcagt gggaaatacg aaggtggcaa catatacaca aagaaagagg ctttcaatgt 4980

ggagaatggc aatgccactc cccagctcac caagaatgca ggggtgctca cctgctccct 5040

gagcaaacgg tgcggcggtg gtggcctcga ggatcagtca gcgcagatca tgctggatag 5100

cgtggtggag aagtacctga ggagtaacag gtcactgcgc aaggttgagc attccagaca 5160

cgacgctatc aacatcttca acgtggagaa gtacggtgct gtcggagacg ggaagcacga 5220

ctgcaccgaa gccttttcta cagcctggca agctgcctgc aagaatccct cagccatgct 5280

cctcgtgcct gggtctaaga agtttgtcgt gaataacctt ttcttcaatg gaccctgcca 5340

gccacacttt accttcaaag ttgatgggat catcgcagcc tatcagaacc cagctagctg 5400

gaagaacaat cggatctggt tgcagtttgc caaactgaca ggattcaccc tgatggggaa 5460

aggcgtgatc gacggacagg gcaaacagtg gtgggcaggg cagtgcaagt gggtcaatgg 5520

tagggagatt tgcaatgaca gggaccgtcc taccgctatc aagtttgatt tcagcacagg 5580

actgattatt caggggttga agctgatgaa tagtccagag tttcaccttg tgtttggcaa 5640

ttgtgaaggt gtgaagatca taggcattag cattacagca cctcgcgatt ctcccaatac 5700

ggacggcatt gacatcttcg cctccaagaa ctttcacctg caaaagaata ccattggcac 5760

aggcgacgac tgcgtggcca ttggcactgg cagcagcaat atcgttatcg aagatttgat 5820

atgtggtcct gggcatggca taagcattgg aagcctgggt agagaaaact caagagctga 5880

agtcagctat gttcacgtta acggagcgaa gttcattgat acccagaacg gactgcgaat 5940

caaaacttgg caagggggaa gtggcatggc atctcacatc atctacgaga acgtcgagat 6000

gatcaattcc gagaacccca tactgattaa ccaattctat tgtacttccg cctctgcctg 6060

ccagaatcag agatcagccg tgcagattca ggacgtgaca tacaagaata tccgagggac 6120

gagcgctacc gctgccgcaa tacagctcaa atgttccgat agcatgccct gcaaagatat 6180

caagcttagt gatatctccc tcaaactgac tagcggaaag atagcgtcct gtctcaatga 6240

taacgcaaat ggctacttct cagggcatgt gatccctgca tgcaaaaacc ttagcccgag 6300

tgcgaaacgc aaagaatcca aatcccataa gcatccgaag actgtgatgg tcgagaacat 6360

gagagcctac gacaaaggga accggacgag gattctgctg ggctctcgac cgccaaactg 6420

taccaacaaa tgtcacggtt gttctccatg caaagctaaa ctggtgatag tgcatcgcat 6480

catgcctcaa gagtactatc cccagcgttg gatttgtagt tgccatggca agatctatca 6540

cccagaattc acgctgatcc ccatcgctgt gggtggtgcc ctggcggggc tggtcctcat 6600

cgtcctcatc gcctacctcg tcggcaggaa gaggagtcac gcaggctacc agactatcta 6660

gtaaggatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc 6720

tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt 6780

gtctctcact cggaaggaca taagggcggc cgctagc 6817

<210>7

<211>1270

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence: Synthetic

chimeric polypeptide of Cry J1 and CryJ2 allergens of

c. japonica

<400>7

Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val

20 25 30

Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala

35 40 45

Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu

50 55 60

Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly

65 70 75 80

Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly

85 90 95

His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val

100 105 110

Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro

115 120 125

Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile

130 135 140

Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val

145 150 155 160

His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala

165 170 175

Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln

180 185 190

Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser

195 200 205

Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser

210 215 220

Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn

225 230 235 240

Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn

245 250 255

Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu

260 265 270

Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe Gln

275 280 285

Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln

290 295 300

Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala

305 310 315 320

Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys

325 330 335

Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn

340 345 350

Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe

355 360 365

Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Asp Asn

370 375 380

Pro Ile Asp Ser Cys Trp Arg Gly Asp Ser Asn Trp Ala Gln Asn Arg

385 390 395 400

Met Lys Leu Ala Asp Cys Ala Val Gly Phe Gly Ser Ser Thr Met Gly

405 410 415

Gly Lys Gly Gly Asp Leu Tyr Thr Val Thr Asn Ser Asp Asp Asp Pro

420 425 430

Val Asn Pro Ala Pro Gly Thr Leu Arg Tyr Gly Ala Thr Arg Asp Arg

435 440 445

Pro Leu Trp Ile Ile Phe Ser Gly Asn Met Asn Ile Lys Leu Lys Met

450 455 460

Pro Met Tyr Ile Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly Ala Gln

465 470 475 480

Val Tyr Ile Gly Asn Gly Gly Pro Cys Val Phe Ile Lys Arg Val Ser

485 490 495

Asn Val Ile Ile His Gly Leu His Leu Tyr Gly Cys Ser Thr Ser Val

500 505 510

Leu Gly Asn Val Leu Ile Asn Glu Ser Phe Gly Val Glu Pro Val His

515 520 525

Pro Gln Asp Gly Asp Ala Leu Thr Leu Arg Thr Ala Thr Asn Ile Trp

530 535 540

Ile Asp His Asn Ser Phe Ser Asn Ser Ser Asp Gly Leu Val Asp Val

545 550 555 560

Thr Leu Ser Ser Thr Gly Val Thr Ile Ser Asn Asn Leu Phe Phe Asn

565 570 575

His His Lys Val Met Leu Leu Gly His Asp Asp Ala Tyr Ser Asp Asp

580 585 590

Lys Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asn Cys

595 600 605

Gly Gln Arg Met Pro Arg Ala Arg Tyr Gly Leu Val His Val Ala Asn

610 615 620

Asn Asn Tyr Asp Pro Trp Thr Ile Tyr Ala Ile Gly Gly Ser Ser Asn

625 630 635 640

Pro Thr Ile Leu Ser Glu Gly Asn Ser Phe Thr Ala Pro Asn Glu Ser

645 650 655

Tyr Lys Lys Gln Val Thr Ile Arg Ile Gly Cys Lys Thr Ser Ser Ser

660 665 670

Cys Ser Asn Trp Val Trp Gln Ser Thr Gln Asp Val Phe Tyr Asn Gly

675 680 685

Ala Tyr Phe Val Ser Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr

690 695 700

Lys Lys Glu Ala Phe Asn Val Glu Asn Gly Asn Ala Thr Pro Gln Leu

705 710 715 720

Thr Lys Asn Ala Gly Val Leu Thr Cys Ser Leu Ser Lys Arg Cys Gly

725 730 735

Gly Gly Gly Leu Glu Asp Gln Ser Ala Gln Ile Met Leu Asp Ser Val

740 745 750

Val Glu Lys Tyr Leu Arg Ser Asn Arg Ser Leu Arg Lys Val Glu His

755 760 765

Ser Arg His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala

770 775 780

Val Gly Asp Gly Lys His Asp Cys Thr Glu Ala Phe Ser Thr Ala Trp

785 790 795 800

Gln Ala Ala Cys Lys Asn Pro Ser Ala Met Leu Leu Val Pro Gly Ser

805 810 815

Lys Lys Phe Val Val Asn Asn Leu Phe Phe Asn Gly Pro Cys Gln Pro

820 825 830

His Phe Thr Phe Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro

835 840 845

Ala Ser Trp Lys Asn Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr

850 855 860

Gly Phe Thr Leu Met Gly Lys Gly Val Ile Asp Gly Gln Gly Lys Gln

865 870 875 880

Trp Trp Ala Gly Gln Cys Lys Trp Val Asn Gly Arg Glu Ile Cys Asn

885 890 895

Asp Arg Asp Arg Pro Thr Ala Ile Lys Phe Asp Phe Ser Thr Gly Leu

900 905 910

Ile Ile Gln Gly Leu Lys Leu Met Asn Ser Pro Glu Phe His Leu Val

915 920 925

Phe Gly Asn Cys Glu Gly Val Lys Ile Ile Gly Ile Ser Ile Thr Ala

930 935 940

Pro Arg Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Ala Ser Lys

945 950 955 960

Asn Phe His Leu Gln Lys Asn Thr Ile Gly Thr Gly Asp Asp Cys Val

965 970 975

Ala Ile Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu Ile Cys

980 985 990

Gly Pro Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Glu Asn Ser

995 1000 1005

Arg Ala Glu Val Ser Tyr Val His Val Asn Gly Ala Lys Phe Ile

1010 1015 1020

Asp Thr Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser

1025 1030 1035

Gly Met Ala Ser His Ile Ile Tyr Glu Asn Val Glu Met Ile Asn

1040 1045 1050

Ser Glu Asn Pro Ile Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala

1055 1060 1065

Ser Ala Cys Gln Asn Gln Arg Ser Ala Val Gln Ile Gln Asp Val

1070 1075 1080

Thr Tyr Lys Asn Ile Arg Gly Thr Ser Ala Thr Ala Ala Ala Ile

1085 1090 1095

Gln Leu Lys Cys Ser Asp Ser Met Pro Cys Lys Asp Ile Lys Leu

1100 1105 1110

Ser Asp Ile Ser Leu Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys

1115 1120 1125

Leu Asn Asp Asn Ala Asn Gly Tyr Phe Ser Gly His Val Ile Pro

1130 1135 1140

Ala Cys Lys Asn Leu Ser Pro Ser Ala Lys Arg Lys Glu Ser Lys

1145 1150 1155

Ser His Lys His Pro Lys Thr Val Met Val Glu Asn Met Arg Ala

1160 1165 1170

Tyr Asp Lys Gly Asn Arg Thr Arg Ile Leu Leu Gly Ser Arg Pro

1175 1180 1185

Pro Asn Cys Thr Asn Lys Cys His Gly Cys Ser Pro Cys Lys Ala

1190 1195 1200

Lys Leu Val Ile Val His Arg Ile Met Pro Gln Glu Tyr Tyr Pro

1205 1210 1215

Gln Arg Trp Ile Cys Ser Cys His Gly Lys Ile Tyr His Pro Glu

1220 1225 1230

Phe Thr Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu

1235 1240 1245

Val Leu Ile Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser

1250 1255 1260

His Ala Gly Tyr Gln Thr Ile

1265 1270

<210>8

<211>8023

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence: Synthetic

polynucleotide coding region for the Ara H1/H2/H3 polyprotein

<400>8

ccgcctaatg agcgggcttt tttttcttag ggtgcaaaag gagagcctgt aagcgggcac 60

tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag 120

ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt 180

gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccccttc ttccgcttcc 240

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 300

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 360

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 420

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 480

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 540

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 600

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 660

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 720

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 780

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 840

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 900

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 960

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1020

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1080

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1140

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1200

tcagcgatct gtctatttcg ttcatccata gttgcctgac tcctgcaaac cacgttgtgg 1260

tagaattggt aaagagagtc gtgtaaaata tcgagttcgc acatcttgtt gtctgattat 1320

tgatttttgg cgaaaccatt tgatcatatg acaagatgtg tatctacctt aacttaatga 1380

ttttgataaa aatcattagg taccccggct ctagatggca tgacattaac ctataaaaat 1440

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 1500

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1560

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 1620

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 1680

aggagaaaat accgcatcag attggctatt ggccattgca tacgttgtat ccatatcata 1740

atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 1800

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 1860

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 1920

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 1980

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2040

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2100

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2160

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2220

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2280

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 2340

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 2400

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 2460

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 2520

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 2580

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 2640

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 2700

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 2760

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 2820

aagacgaaga agacaaaccg tcgtcgacat ggcgccccgc agcgcccggc gacccctgct 2880

gctgctactg ctgttgctgc tgctcggcct catgcattgt gcgtcagcag caatgtttat 2940

ggtgaaaaat ggcaacggga ccgcgtgcat aatggccaac ttctctgctg ccttctcagt 3000

gaactacgac accaagagtg gccctaagaa catgaccctt gacctgccat cagatgccac 3060

agtggtgctc aaccgcagct cctgtggaaa agagaacact tctgacccca gtctcgtgat 3120

tgcttttgga agaggacata cactcactct caatttcacg agaaatgcaa cacgttacag 3180

cgttcagctc atgagttttg tttataactt gtcagacaca caccttttcc ccaatgcgag 3240

ctccaaagaa atcaagactg tggaatctat aactgacatc agggcagata tagataaaaa 3300

atacagatgt gttagtggca cccaggtcca catgaacaac gtgaccgtaa cgctccatga 3360

tgccaccatc caggcgtacc tttccaacag cagcttcagc aggggagaga cacgctgtga 3420

acaagacagg ccttccccaa ccacagcgcc ccctgcgcca cccagcccct cgccctcacc 3480

cgtgcccaag agcccctctg tggacaagta caacgtgagc ggcaccaacg ggacctgcct 3540

gctggccagc atggggctgc agctgaacct cacctatgag aggaaggaca acacgacggt 3600

gacaaggctt ctcaacatca accccaacaa gacctcggcc agcgggagct gcggcgccca 3660

cctggtgact ctggagctgc acagcgaggg caccaccgtc ctgctcttcc agttcgggat 3720

gaatgcaagt tctagccggt ttttcctaca aggaatccag ttgaatacaa ttcttcctga 3780

cgccagagac cctgccttta aagctgccaa cggctccctg cgagcgctgc aggccacagt 3840

cggcaattcc tacaagtgca acgcggagga gcacgtccgt gtcacgaagg cgttttcagt 3900

caatatattc aaagtgtggg tccaggcttt caaggtggaa ggtggccagt ttggctctgt 3960

ggaggagtgt ctgctggacg agaacagcct cgagaagtcc agcccctacc agaagaaaac 4020

cgagaacccc tgcgcccagc ggtgcctgca gtcttgtcag caggaacccg acgacctgaa 4080

gcagaaggcc tgcgagagcc ggtgcaccaa gctggaatac gaccccagat gcgtgtacga 4140

ccctagaggc cacaccggca ccaccaacca gagaagccct ccaggcgagc ggaccagagg 4200

cagacagcct ggcgactacg acgacgacag acggcagccc agaagagaag agggcggcag 4260

atggggacct gccggcccta gagagagaga acgcgaggaa gattggagac agcccagaga 4320

ggactggcgg aggccttctc accagcagcc ccggaagatc agacccgagg gcagagaagg 4380

cgagcaggaa tggggcacac ctggctctca cgtgcgcgag gaaaccagcc ggaacaaccc 4440

cttctacttc ccctcccggc ggttcagcac cagatacggc aaccagaacg gccggatcag 4500

agtgctgcag agattcgacc agcggagccg gcagttccag aacctgcaga accaccggat 4560

cgtgcagatc gaggccaagc ccaacaccct ggtgctgccc aaacacgccg acgccgacaa 4620

catcctcgtg atccagcagg gccaggccac cgtgacagtg gccaacggca acaacagaaa 4680

gagcttcaac ctggacgagg gccacgccct gagaatcccc agcggcttca tcagctacat 4740

cctgaacaga cacgacaatc agaacctgag ggtggccaag atcagcatgc ccgtgaacac 4800

ccctggccag ttcgaggact tcttccccgc atcctcccgg gaccagagca gctacctgca 4860

gggcttcagc cggaataccc tggaagccgc cttcaacgcc gagttcaacg agatcagacg 4920

ggtgctgctg gaagagaacg ctggcggaga gcaggaagaa cggggccaga gaagatggtc 4980

caccagaagc agcgagaaca acgagggcgt gatcgtgaag gtgtccaaag aacacgtgga 5040

agaactgacc aagcacgcca agagcgtgtc caagaagggc tccgaggaag agggggacat 5100

caccaacccc atcaatctga gagagggcga gcccgacctg agcaacaact tcggcaagct 5160

gttcgaagtg aagcccgaca agaagaaccc ccagctgcag gacctggaca tgatgctgac 5220

ctgcgtggaa atcaaagagg gggccctgat gctgccacac ttcaactcca aagccatggt 5280

catcgtggtc gtgaacaagg gcaccggcaa cctggaactg gtggccgtgc ggaaagagca 5340

gcagcagaga ggccgcagag aggaagaaga ggacgaggac gaagaagaag agggatccaa 5400

ccgggaagtg cggcggtaca ccgccagact gaaagaaggc gacgtgttca tcatgcctgc 5460

cgcccacccc gtggccatca atgcctctag cgagctgcat ctgctgggct tcggcattaa 5520

cgccgagaac aatcaccgga tctttctggc cggcgacaaa gacaacgtga tcgaccagat 5580

cgagaagcag gccaaggacc tggcctttcc cggctctggc gaacaagtgg aaaagctgat 5640

caagaaccag aaagaaagcc acttcgtgtc cgccagaccc cagagccagt ctcagagccc 5700

tagctccccc gagaaagagt ctcctgagaa agaggaccag gaagaggaaa accagggcgg 5760

caagggccct ctgctgagca tcctgaaggc cttcaatggc ggcggaggca ggcagcagtg 5820

ggaactgcag ggcgacagaa gatgccagtc ccagctggaa cgggccaacc tgaggccttg 5880

cgagcagcac ctgatgcaga aaatccagcg cgacgaggac agctacggcc gggatcctta 5940

cagccccagc caggaccctt actcccctag ccaggatccc gacagaaggg acccctacag 6000

ccctagcccc tacgatagaa gaggcgccgg aagcagccag caccaggaaa gatgctgcaa 6060

cgagctgaac gagtttgaga acaaccagcg ctgcatgtgc gaggccctgc agcagatcat 6120

ggaaaatcag agcgaccggc tgcagggacg gcagcaggaa cagcagttca agagagagct 6180

gcggaacctg ccccagcagt gtggactgag agccccccag agatgcgacc tggaagtgga 6240

aagcggcggc agagatcggt acggcggagg gggcgtgacc ttcagacagg gcggagaaga 6300

gaatgagtgc cagtttcagc ggctgaacgc ccagaggccc gacaacagaa tcgagagcga 6360

gggcggctac atcgagacat ggaaccccaa caaccaggaa tttcagtgcg ctggggtggc 6420

cctgagcagg accgtgctga gaagaaatgc cctgaggcgg cccttctaca gcaacgcccc 6480

cctggaaatc tacgtgcagc agggcagcgg ctacttcggc ctgatctttc ccggatgccc 6540

ctccacctat gaggaacccg ctcaggaagg cagacggtat cagagccaga agcctagcag 6600

acggttccaa gtgggccagg acgatcccag ccaacagcag caggactctc accagaaggt 6660

gcaccgcttc gacgagggcg acctgatcgc tgtgccaacc ggcgtggcct tctggatgta 6720

caacgacgag gataccgacg tcgtgaccgt gaccctgagc gacaccagct ccatccacaa 6780

ccagctggac cagttcccca ggcggtttta cctggccggc aatcaggaac aggaatttct 6840

gagataccag cagcagcagg gctccagacc ccactacaga cagatcagcc ctagagtgcg 6900

gggcgacgaa caggaaaatg agggcagcaa catcttctcc ggctttgccc aggaatttct 6960

gcagcacgcc ttccaggtgg accggcagac cgtggaaaac ctgagaggcg agaacgagag 7020

agaggaacag ggcgccatcg tgactgtgaa gggcggcctg aggatcctga gccccgacga 7080

agaggatgag tcctctagaa gcccccccaa ccgccgggaa gagttcgatg aggaccgcag 7140

cagacctcag cagcggggga agtacgacga gaacaggcgg ggctacaaga acggcatcga 7200

ggaaacaatc tgcagcgcca gcgtgaagaa gaatctgggc cggtccagca accccgacat 7260

ctacaatcca caggccggca gcctgcggag cgtgaacgaa ctggatctgc ccatcctggg 7320

atggctgggc ctgtctgccc agcacggcac catctaccgg aacgccatgt tcgtgcctca 7380

ctacaccctg aatgcccaca ccatcgtggt ggctctgaac ggccgcgccc acgtccaagt 7440

ggtggacagc aacggcaatc gggtgtacga tgaagaactg caggaaggac acgtcctggt 7500

ggtgccccag aattttgccg tggccgccaa ggcccagtcc gagaactatg agtatctggc 7560

cttcaagacc gacagccggc cctctatcgc caatcaagcc ggcgagaaca gcatcatcga 7620

caacctgccc gaggaagtgg tggccaacag ctaccggctg cctagagagc aggcccggca 7680

gctgaagaac aacaaccctt tcaagttctt cgtgccccca ttcgaccacc agagcatgag 7740

agaggtggcc gaattcacgc tgatccccat cgctgtgggt ggtgccctgg cggggctggt 7800

cctcatcgtc ctcatcgcct acctcgtcgg caggaagagg agtcacgcag gctaccagac 7860

tatctagtaa ggatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 7920

agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 7980

ttttgtgtct ctcactcgga aggacataag ggcggccgct agc 8023

<210>9

<211>1672

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence: Synthetic

chimeric polypeptide of AraH1,2 and 3 allergens

<220>

<221>misc_feature

<222>(1)..(27)

<223>Signal

<220>

<221>misc_feature

<222>(28)..(380)

<223>N-lamp

<220>

<221>misc_feature

<222>(383)..(983)

<223>AraH1

<220>

<221>misc_feature

<222>(988)..(1138)

<223>AraH2

<220>

<221>misc_feature

<222>(1143)..(1634)

<223>AraH3

<220>

<221>misc_feature

<222>(1637)..(1672)

<223>TM/CYTO

<400>9

Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met Val

20 25 30

Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala

35 40 45

Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu

50 55 60

Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly

65 70 75 80

Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly Arg Gly

85 90 95

His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val

100 105 110

Gln Leu Met Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro

115 120 125

Asn Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile

130 135 140

Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val

145 150 155 160

His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala

165 170175

Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln

180 185 190

Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser

195 200 205

Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser

210 215 220

Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn

225 230 235 240

Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn

245 250 255

Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu

260 265 270

Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe Gln

275 280 285

Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln

290 295 300

Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala

305 310 315 320

Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys

325 330 335

Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val Asn

340 345 350

Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe

355 360 365

Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Lys Ser

370 375 380

Ser Pro Tyr Gln Lys Lys Thr Glu Asn Pro Cys Ala Gln Arg Cys Leu

385 390 395 400

Gln Ser Cys Gln Gln Glu Pro Asp Asp Leu Lys Gln Lys Ala Cys Glu

405 410 415

Ser Arg Cys Thr Lys Leu Glu Tyr Asp Pro Arg Cys Val Tyr Asp Pro

420 425 430

Arg Gly His Thr Gly Thr Thr Asn Gln Arg Ser Pro Pro Gly Glu Arg

435 440 445

Thr Arg Gly Arg Gln Pro Gly Asp Tyr Asp Asp Asp ArgArg Gln Pro

450 455 460

Arg Arg Glu Glu Gly Gly Arg Trp Gly Pro Ala Gly Pro Arg Glu Arg

465 470 475 480

Glu Arg Glu Glu Asp Trp Arg Gln Pro Arg Glu Asp Trp Arg Arg Pro

485 490 495

Ser His Gln Gln Pro Arg Lys Ile Arg Pro Glu Gly Arg Glu Gly Glu

500 505 510

Gln Glu Trp Gly Thr Pro Gly Ser His Val Arg Glu Glu Thr Ser Arg

515 520 525

Asn Asn Pro Phe Tyr Phe Pro Ser Arg Arg Phe Ser Thr Arg Tyr Gly

530 535 540

Asn Gln Asn Gly Arg Ile Arg Val Leu Gln Arg Phe Asp Gln Arg Ser

545 550 555 560

Arg Gln Phe Gln Asn Leu Gln Asn His Arg Ile Val Gln Ile Glu Ala

565 570 575

Lys Pro Asn Thr Leu Val Leu Pro Lys His Ala Asp Ala Asp Asn Ile

580 585 590

Leu Val Ile Gln Gln Gly Gln Ala Thr Val Thr Val Ala Asn Gly Asn

595 600 605

Asn Arg Lys Ser Phe Asn Leu Asp Glu Gly His Ala Leu Arg Ile Pro

610 615 620

Ser Gly Phe Ile Ser Tyr Ile Leu Asn Arg His Asp Asn Gln Asn Leu

625 630 635 640

Arg Val Ala Lys Ile Ser Met Pro Val Asn Thr Pro Gly Gln Phe Glu

645 650 655

Asp Phe Phe Pro Ala Ser Ser Arg Asp Gln Ser Ser Tyr Leu Gln Gly

660 665 670

Phe Ser Arg Asn Thr Leu Glu Ala Ala Phe Asn Ala Glu Phe Asn Glu

675 680 685

Ile Arg Arg Val Leu Leu Glu Glu Asn Ala Gly Gly Glu Gln Glu Glu

690 695 700

Arg Gly Gln Arg Arg Trp Ser Thr Arg Ser Ser Glu Asn Asn Glu Gly

705 710 715 720

Val Ile Val Lys Val Ser Lys Glu His Val Glu Glu Leu Thr Lys His

725 730735

Ala Lys Ser Val Ser Lys Lys Gly Ser Glu Glu Glu Gly Asp Ile Thr

740 745 750

Asn Pro Ile Asn Leu Arg Glu Gly Glu Pro Asp Leu Ser Asn Asn Phe

755 760 765

Gly Lys Leu Phe Glu Val Lys Pro Asp Lys Lys Asn Pro Gln Leu Gln

770 775 780

Asp Leu Asp Met Met Leu Thr Cys Val Glu Ile Lys Glu Gly Ala Leu

785 790 795 800

Met Leu Pro His Phe Asn Ser Lys Ala Met Val Ile Val Val Val Asn

805 810 815

Lys Gly Thr Gly Asn Leu Glu Leu Val Ala Val Arg Lys Glu Gln Gln

820 825 830

Gln Arg Gly Arg Arg Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu

835 840 845

Gly Ser Asn Arg Glu Val Arg Arg Tyr Thr Ala Arg Leu Lys Glu Gly

850 855 860

Asp Val Phe Ile Met Pro Ala Ala His Pro Val Ala Ile Asn Ala Ser

865 870 875 880

Ser Glu Leu His Leu Leu Gly Phe Gly Ile Asn Ala Glu Asn Asn His

885 890 895

Arg Ile Phe Leu Ala Gly Asp Lys Asp Asn Val Ile Asp Gln Ile Glu

900 905 910

Lys Gln Ala Lys Asp Leu Ala Phe Pro Gly Ser Gly Glu Gln Val Glu

915 920 925

Lys Leu Ile Lys Asn Gln Lys Glu Ser His Phe Val Ser Ala Arg Pro

930 935 940

Gln Ser Gln Ser Gln Ser Pro Ser Ser Pro Glu Lys Glu Ser Pro Glu

945 950 955 960

Lys Glu Asp Gln Glu Glu Glu Asn Gln Gly Gly Lys Gly Pro Leu Leu

965 970 975

Ser Ile Leu Lys Ala Phe Asn Gly Gly Gly Gly Arg Gln Gln Trp Glu

980 985 990

Leu Gln Gly Asp Arg Arg Cys Gln Ser Gln Leu Glu Arg Ala Asn Leu

995 1000 1005

Arg Pro Cys Glu Gln His Leu Met Gln Lys IleGln Arg Asp Glu

1010 1015 1020

Asp Ser Tyr Gly Arg Asp Pro Tyr Ser Pro Ser Gln Asp Pro Tyr

1025 1030 1035

Ser Pro Ser Gln Asp Pro Asp Arg Arg Asp Pro Tyr Ser Pro Ser

1040 1045 1050

Pro Tyr Asp Arg Arg Gly Ala Gly Ser Ser Gln His Gln Glu Arg

1055 1060 1065

Cys Cys Asn Glu Leu Asn Glu Phe Glu Asn Asn Gln Arg Cys Met

1070 1075 1080

Cys Glu Ala Leu Gln Gln Ile Met Glu Asn Gln Ser Asp Arg Leu

1085 1090 1095

Gln Gly Arg Gln Gln Glu Gln Gln Phe Lys Arg Glu Leu Arg Asn

1100 1105 1110

Leu Pro Gln Gln Cys Gly Leu Arg Ala Pro Gln Arg Cys Asp Leu

1115 1120 1125

Glu Val Glu Ser Gly Gly Arg Asp Arg Tyr Gly Gly Gly Gly Val

1130 1135 1140

Thr Phe Arg Gln Gly Gly Glu Glu Asn GluCys Gln Phe Gln Arg

1145 1150 1155

Leu Asn Ala Gln Arg Pro Asp Asn Arg Ile Glu Ser Glu Gly Gly

1160 1165 1170

Tyr Ile Glu Thr Trp Asn Pro Asn Asn Gln Glu Phe Gln Cys Ala

1175 1180 1185

Gly Val Ala Leu Ser Arg Thr Val Leu Arg Arg Asn Ala Leu Arg

1190 1195 1200

Arg Pro Phe Tyr Ser Asn Ala Pro Leu Glu Ile Tyr Val Gln Gln

1205 1210 1215

Gly Ser Gly Tyr Phe Gly Leu Ile Phe Pro Gly Cys Pro Ser Thr

1220 1225 1230

Tyr Glu Glu Pro Ala Gln Glu Gly Arg Arg Tyr Gln Ser Gln Lys

1235 1240 1245

Pro Ser Arg Arg Phe Gln Val Gly Gln Asp Asp Pro Ser Gln Gln

1250 1255 1260

Gln Gln Asp Ser His Gln Lys Val His Arg Phe Asp Glu Gly Asp

1265 1270 1275

Leu Ile Ala Val Pro Thr Gly Val AlaPhe Trp Met Tyr Asn Asp

1280 1285 1290

Glu Asp Thr Asp Val Val Thr Val Thr Leu Ser Asp Thr Ser Ser

1295 1300 1305

Ile His Asn Gln Leu Asp Gln Phe Pro Arg Arg Phe Tyr Leu Ala

1310 1315 1320

Gly Asn Gln Glu Gln Glu Phe Leu Arg Tyr Gln Gln Gln Gln Gly

1325 1330 1335

Ser Arg Pro His Tyr Arg Gln Ile Ser Pro Arg Val Arg Gly Asp

1340 1345 1350

Glu Gln Glu Asn Glu Gly Ser Asn Ile Phe Ser Gly Phe Ala Gln

1355 1360 1365

Glu Phe Leu Gln His Ala Phe Gln Val Asp Arg Gln Thr Val Glu

1370 1375 1380

Asn Leu Arg Gly Glu Asn Glu Arg Glu Glu Gln Gly Ala Ile Val

1385 1390 1395

Thr Val Lys Gly Gly Leu Arg Ile Leu Ser Pro Asp Glu Glu Asp

1400 1405 1410

Glu Ser Ser Arg Ser Pro Pro AsnArg Arg Glu Glu Phe Asp Glu

1415 1420 1425

Asp Arg Ser Arg Pro Gln Gln Arg Gly Lys Tyr Asp Glu Asn Arg

1430 1435 1440

Arg Gly Tyr Lys Asn Gly Ile Glu Glu Thr Ile Cys Ser Ala Ser

1445 1450 1455

Val Lys Lys Asn Leu Gly Arg Ser Ser Asn Pro Asp Ile Tyr Asn

1460 1465 1470

Pro Gln Ala Gly Ser Leu Arg Ser Val Asn Glu Leu Asp Leu Pro

1475 1480 1485

Ile Leu Gly Trp Leu Gly Leu Ser Ala Gln His Gly Thr Ile Tyr

1490 1495 1500

Arg Asn Ala Met Phe Val Pro His Tyr Thr Leu Asn Ala His Thr

1505 1510 1515

Ile Val Val Ala Leu Asn Gly Arg Ala His Val Gln Val Val Asp

1520 1525 1530

Ser Asn Gly Asn Arg Val Tyr Asp Glu Glu Leu Gln Glu Gly His

1535 1540 1545

Val Leu Val Val Pro Gln AsnPhe Ala Val Ala Ala Lys Ala Gln

1550 1555 1560

Ser Glu Asn Tyr Glu Tyr Leu Ala Phe Lys Thr Asp Ser Arg Pro

1565 1570 1575

Ser Ile Ala Asn Gln Ala Gly Glu Asn Ser Ile Ile Asp Asn Leu

1580 1585 1590

Pro Glu Glu Val Val Ala Asn Ser Tyr Arg Leu Pro Arg Glu Gln

1595 1600 1605

Ala Arg Gln Leu Lys Asn Asn Asn Pro Phe Lys Phe Phe Val Pro

1610 1615 1620

Pro Phe Asp His Gln Ser Met Arg Glu Val Ala Glu Phe Thr Leu

1625 1630 1635

Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val Leu Ile

1640 1645 1650

Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg Ser His Ala Gly

1655 1660 1665

Tyr Gln Thr Ile

1670

<210>10

<211>225

<212>PRT

<213>Cryptomeria japonica

<400>10

Met Ala Lys Val Ser Asp Leu Ala Leu Leu Leu Val Ala Gly Met Ala

1 5 10 15

Ile Ser Leu Tyr Ile Gln Glu Thr Gly Ala Val Lys Phe Asp Ile Lys

20 25 30

Asn Gln Cys Gly Tyr Thr Val Trp Ala Ala Gly Leu Pro Gly Gly Gly

35 40 45

Gln Gln Leu Thr Gln Gly Gln Thr Trp Thr Val Asn Leu Ala Ala Gly

50 55 60

Thr Gln Ser Ala Arg Phe Trp Gly Arg Thr Gly Cys Ser Phe Asp Ala

65 70 75 80

Ser Gly Lys Gly Thr Cys Gln Thr Gly Asp Cys Gly Gly Gln Leu Ser

85 90 95

Cys Thr Val Ser Gly Ala Val Pro Ala Thr Leu Ala Glu Tyr Thr Gln

100 105 110

Ser Asp Gln Asp Tyr Tyr Asp Val Ser Leu Val Asp Gly Phe Asn Ile

115120 125

Pro Leu Ser Ile Asn Pro Thr Asn Ala Gln Cys Thr Ala Pro Ala Cys

130 135 140

Lys Ala Asp Val Asn Ala Val Cys Pro Ala Glu Leu Lys Val Asp Gly

145 150 155 160

Gly Cys Lys Ser Ala Cys Ala Ala Phe Gln Thr Asp Gln Tyr Cys Cys

165 170 175

Thr Gly Thr Tyr Ala Asn Ser Cys Pro Ala Thr Asn Tyr Ser Met Ile

180 185 190

Phe Lys Asn Gln Cys Pro Gln Ala Tyr Ser Tyr Pro Lys Asp Asp Thr

195 200 205

Ala Thr Phe Ala Cys Pro Ser Gly Thr Asp Tyr Ser Ile Val Phe Cys

210 215 220

Pro

225

<210>11

<211>281

<212>PRT

<213>Cryptomeria japonica

<400>11

Met Gly Ile Met Ala Thr Gln Asn Ser Lys Ser Asn Ile Phe Trp Ser

1 5 10 15

Ser Ser Ala Ser Val Val Leu Val Leu Leu Leu Leu Val Asp Val Gly

20 25 30

Val Cys Gln Asn Cys Gly Cys Asn Gly Leu Cys Cys Ser Gln Tyr Gly

35 40 45

Tyr Cys Gly Ser Gly Glu Ala Tyr Cys Gly Ala Gly Cys Lys Glu Gly

50 55 60

Pro Cys Ser Ser Ser Ser Pro Pro Ser Thr Gly Thr Gly Val Gly Ser

65 70 75 80

Ile Val Ser Ser Asp Val Phe Asn Ser Ile Val Gly Gly Ala Ala Ser

85 90 95

Gly Cys Ala Gly Asn Gly Phe Tyr Thr Tyr Asp Ser Phe Ile Ser Ala

100 105 110

Ala Asn Ala Phe Asn Gly Phe Gly Thr Ser Gly Ser Ser Asp Val Asn

115 120 125

Lys Arg Glu Ile Ala Ala Phe Phe Ala Asn Ala Ala His Glu Thr Gly

130 135 140

Gly Phe Cys Tyr Ile Glu Glu Gln Asn Pro Thr Ser Ile Tyr Cys Asp

145 150 155 160

Ala Ser Asn Thr Gln Tyr Pro Cys Ala Ser Gly Lys Thr Tyr His Gly

165 170 175

Arg Gly Pro Leu Gln Leu Ser Trp Asn Tyr Asn Tyr Gly Ala Ala Gly

180 185 190

Ser Tyr Ile Gln Phe Asp Gly Leu Asn Asn Pro Glu Ile Val Gly Thr

195 200 205

Asp Ser Thr Ile Ser Phe Lys Thr Ala Val Trp Phe Trp Met Val Asn

210 215 220

Ser Asn Cys His Thr Ala Ile Thr Ser Gly Gln Gly Phe Gly Ala Thr

225 230 235 240

Ile Arg Ala Ile Asn Ser Met Glu Cys Asp Gly Gly Asn Ala Ala Thr

245 250 255

Val Ala Ser Arg Val Asn Tyr Tyr Gln Lys Phe Cys Gln Gln Leu Asn

260 265 270

Val Asp Thr Gly Ser Ala Leu Gln Cys

275 280

<210>12

<211>306

<212>PRT

<213>Cryptomeria japonica

<400>12

Met Gly Gly Ser Arg Val Leu Ile Ile Gly Gly Thr Gly Tyr Ile Gly

1 5 10 15

Arg His Val Thr Asn Ala Ser Leu Ala Gln Gly His Pro Thr Phe Leu

20 25 30

Leu Val Arg Glu Ile Thr Pro Ser Asn Pro Glu Lys Ala Gln Leu Leu

35 40 45

Glu Ser Phe Thr Ser Lys Gly Ala Thr Leu Val Gln Gly Ser Ile Asp

50 55 60

Asp His Ala Ser Leu Val Ala Ala Leu Lys Lys Val Asp Val Val Ile

65 70 75 80

Ser Thr Leu Gly Ala Pro Gln Ile Ala Asp Gln Phe Asn Leu Ile Lys

85 90 95

Ala Ile Lys Glu Val Gly Thr Ile Lys Arg Phe Phe Pro Ser Glu Phe

100 105 110

Gly Asn Asp Val Asp Lys His HisAla Val Glu Pro Met Lys Ser Met

115 120 125

Phe Asp Leu Lys Ile Lys Leu Arg Arg Thr Ile Glu Ala Glu Gly Ile

130 135 140

Pro His Thr Tyr Val Val Pro His Cys Phe Ala Gly Tyr Phe Leu Thr

145 150 155 160

Asn Leu Ala Gln Leu Gly Leu Ala Ala Pro Pro Arg Asp Lys Ile Val

165 170 175

Ile Tyr Gly Asp Gly Thr Thr Lys Ala Val Tyr Met Lys Glu Glu Asp

180 185 190

Ile Gly Thr Phe Thr Ile Lys Ala Val Asp Asp Pro Arg Thr Leu Asn

195 200 205

Lys Thr Leu Tyr Leu Lys Pro Pro Ala Asn Thr Ile Ser Thr Asn Asp

210 215 220

Leu Val Ala Leu Trp Glu Ala Lys Ile Gly Lys Thr Leu Glu Lys Val

225 230 235 240

Tyr Leu Ser Glu Glu Gln Val Leu Lys Leu Leu Gln Asp Thr Pro Phe

245 250 255

Pro Gly Thr Phe Met Val Ser Ile Phe His Thr Ile Tyr Val Lys Gly

260 265 270

Asp Gln Thr Asn Phe Gln Ile Gly Pro Asp Gly Val Glu Ala Ser Ala

275 280 285

Leu Tyr Pro Asp Val Lys Tyr Thr Thr Val Glu Glu Tyr Ile Ser Ala

290 295 300

Phe Val

305

<210>13

<211>165

<212>PRT

<213>Cryptomeria japonica

<400>13

Met Ala Met Arg Met Lys Ser Ser Ser Met Ser Ser Tyr Arg Phe Ser

1 5 10 15

Tyr Cys Gln Met Met Leu Val Leu Met Val Met Thr Leu Val Gln Ile

20 25 30

Gly Ala Ala Gln Ser Asp Thr Asn Ser Cys Val Asn Ser Leu Val Pro

35 40 45

Cys Ala Ser Tyr Leu Asn Ala Thr Thr Lys Pro Pro Asp Ser Cys Cys

50 55 60

Val Pro Leu Leu Asn Val Ile Gln Thr Gln Gln Gln Cys Leu Cys Asn

65 70 75 80

Leu Leu Asn Ser Ser Ile Val Lys Gln Ser Ser Ile Asn Ile Thr Gln

85 90 95

Ala Leu Asn Ile Pro Arg Leu Cys Gly Asp Thr Asn Val Ser Thr Asp

100 105 110

Ala Cys Ser Thr Asn Ala Thr Ala Asn Ala Pro Ser Ala Ser Thr Thr

115 120 125

Pro Ser Val Pro Ala Asp Thr Gly Asp Ser Ser Gly Ile Gly Ala Thr

130 135 140

Ser Leu Gln Ile Phe Leu Pro Leu Leu Ala Val Phe Phe Leu Gly Val

145 150 155 160

Phe Lys Ser Phe Pro

165

<210>14

<211>472

<212>PRT

<213>Cryptomeria japonica

<400>14

Met Ala Arg Arg Leu Cys Ser Phe Leu Leu Ser Phe Leu Ile Ile Val

1 5 10 15

Ser Val Trp Ala Glu Asn Ser Lys Phe Ala Arg Leu Asn Leu Ala Ser

20 25 30

Phe Thr Trp Lys Asp Ala Glu Asp Asn Lys Asn Cys Ser Ala Gly Glu

35 40 45

Leu Glu Thr Ser Ser Leu Ser Val Met His Ile Gln Gly Lys Cys Ser

50 55 60

Pro Phe Arg Leu Leu Asn Ser Ser Trp Trp Thr Ala Val Ser Glu Ser

65 70 75 80

Ile Lys Gly Asp Thr Ala Arg Tyr Arg Ala Met Val Lys Gly Gly Trp

85 90 95

Ser Ala Gly Lys Thr Met Val Asn Pro Gln Glu Asp Ala Asp Ile Pro

100 105 110

Leu Ala Ser Gly Gln Ala Glu Ser Ser Ser Asn Tyr Ile Ile Lys Leu

115 120 125

Gly Phe Gly Thr Pro Pro Gln Ser Phe Tyr Thr Val Leu Asp Thr Gly

130 135 140

Ser Asn Ile Ala Trp Ile Pro Cys Asn Pro Cys Ser Gly Cys Ser Ser

145 150 155 160

Lys Gln Gln Pro Phe Glu Pro Ser Lys Ser Ser Thr Tyr Asn Tyr Leu

165 170 175

Thr Cys Ala Ser Gln Gln Cys Gln Leu Leu Arg Val Cys Thr Lys Ser

180 185 190

Asp Asn Ser Val Asn Cys Ser Leu Thr Gln Arg Tyr Gly Asp Gln Ser

195 200 205

Glu Val Asp Glu Ile Leu Ser Ser Glu Thr Leu Ser Val Gly Ser Gln

210 215 220

Gln Val Glu Asn Phe Val Phe Gly Cys Ser Asn Ala Ala Arg Gly Leu

225 230 235 240

Ile Gln Arg Thr Pro Ser Leu Val Gly Phe Gly Arg Asn Pro Leu Ser

245 250 255

Phe Val Ser Gln Thr Ala Thr Leu Tyr Asp Ser Thr Phe Ser Tyr Cys

260 265 270

Leu Pro Ser Leu Phe Ser Ser Ala Phe Thr Gly Ser Leu Leu Leu Gly

275 280 285

Lys Glu Ala Leu Ser Ala Gln Gly Leu Lys Phe Thr Pro Leu Leu Ser

290 295 300

Asn Ser Arg Tyr Pro Ser Phe Tyr Tyr Val Gly Leu Asn Gly Ile Ser

305 310 315 320

Val Gly Glu Glu Leu Val Ser Ile Pro Ala Gly Thr Leu Ser Leu Asp

325 330 335

Glu Ser Thr Gly Arg Gly Thr Ile Ile Asp Ser Gly Thr Val Ile Thr

340 345 350

Arg Leu Val Glu Pro Ala Tyr Asn Ala Met Arg Asp Ser Phe Arg Ser

355 360 365

Gln Leu Ser Asn Leu Thr Met Ala Ser Pro Thr Asp Leu Phe Asp Thr

370 375 380

Cys Thr Asn Arg Pro Ser Gly Asp Val Glu Phe Pro Leu Ile Thr Leu

385 390 395 400

His Phe Asp Asp Asn Leu Asp Leu Thr Leu Pro Leu Asp Asn Ile Leu

405 410 415

Tyr Pro Gly Asn Asp Asp Gly Ser Val Leu Cys Leu Ala Phe GlyLeu

420 425 430

Pro Pro Gly Gly Gly Asp Asp Val Leu Ser Thr Phe Gly Asn Tyr Gln

435 440 445

Gln Gln Lys Leu Arg Ile Val His Asp Val Ala Glu Ser Arg Leu Gly

450 455 460

Ile Ala Ser Gly Asn Cys Asp Gly

465 470

<210>15

<211>348

<212>PRT

<213>Cryptomeria japonica

<400>15

Met Glu Leu Leu Lys Gln His Arg Tyr Met Phe Leu Leu Ile Ser Cys

1 5 10 15

Ile Val Ile Leu Leu Asn Ser Met His Ala Asp Cys Glu Gln Ile Gly

20 25 30

Val Asn Tyr Gly Met Asp Gly Asn Asn Leu Pro Ser Ala Gly Asp Val

35 40 45

Val Ser Leu Met Lys Lys Asn Asn Ile Gly Lys Met Arg Ile Phe Gly

50 55 60

Pro Asn Ala Asp Val Leu Arg Ala Phe Ala Asn Ser Arg Ile Glu Val

65 70 75 80

Ile Val Gly Val Glu Asn Lys Gly Leu Glu Ala Val Ala Ser Ser Gln

85 90 95

Asp Ser Ala Asn Gly Trp Val Asn Asp Asn Ile Lys Pro Phe Tyr Pro

100 105 110

Ser Thr Asn Ile Lys Tyr Ile Ala Val Gly Asn Glu Val Leu Glu Met

115 120 125

Pro Asp Asn Ala Gln Tyr Val Ser Phe Leu Val Pro Ala Ile Lys Asn

130 135 140

Ile Gln Thr Ala Leu Glu Asn Ala Asn Leu Gln Asn Asn Ile Lys Val

145 150 155 160

Ser Thr Ala His Ala Met Thr Val Ile Gly Thr Ser Ser Pro Pro Ser

165 170 175

Lys Gly Thr Phe Lys Asp Ala Val Lys Asp Ser Met Ser Ser Ile Leu

180 185 190

Gln Phe Leu Gln Asp His Gly Ser Pro Phe Met Ala Asn Val Tyr Pro

195 200 205

Tyr Phe Ser Tyr Asp Gly Asp Arg Ser Ile Lys Leu Asp Tyr Ala Leu

210 215 220

Phe Asn Pro Thr Pro Pro Val Val Asp Glu Gly Leu Ser Tyr Thr Asn

225 230 235 240

Leu Phe Asp Ala Met Val Asp Ala Val Leu Ser Ala Met Glu Ser Leu

245 250 255

Gly His Pro Asn Ile Pro Ile Val Ile Thr Glu Ser Gly Trp Pro Ser

260 265 270

Ala Gly Lys Ser Val Ala Thr Ile Glu Asn Ala Gln Thr Tyr Asn Asn

275 280 285

Asn Leu Ile Lys His Val Leu Ser Asn Ala Gly Thr Pro Lys Arg Pro

290 295 300

Gly Ser Ser Ile Glu Thr Tyr Ile Phe Ala Leu Phe Asn Glu Asn Leu

305 310 315 320

Lys Gly Pro Ala Glu Val Glu Lys His Phe Gly Leu Phe Asn Pro Asp

325 330 335

Glu Gln Pro Val Tyr Pro Val Lys Phe Ser Leu Asn

340 345

<210>16

<211>375

<212>PRT

<213>Chamaecyparis obtusa

<400>16

Met Ala Ser Cys Thr Leu Leu Ala Val Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Phe Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ala Asn Trp Asp Gln Asn Arg Met Lys Leu Ala Asp Cys Ala Val Gly

35 40 45

Phe Gly Ser Ser Ala Met Gly Gly Lys Gly Gly Ala Phe Tyr Thr Val

50 55 60

Thr Ser Ser Asp Asp Asp Pro Val Asn Pro Ala Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Arg Ser Leu Trp Ile Ile Phe Ser Lys Asn

85 90 95

Leu Asn Ile Lys Leu Asn Met Pro Leu Tyr Ile Ala Gly Asn Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Glu Val His Ile Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Thr Val Ser His Val Ile Leu His Gly Leu Asn Ile

130 135 140

His Gly Cys Asn Thr Ser Val Ser Gly Asn Val Leu Ile Ser Glu Ala

145 150 155 160

Ser Gly Val Val Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met

165 170 175

Arg Asn Val Thr Asp Val Trp Ile Asp His Asn Ser Leu Ser Asp Ser

180 185 190

Ser Asp Gly Leu Val Asp Val Thr Leu Ala Ser Thr Gly Val Thr Ile

195 200 205

Ser Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His

210 215 220

Ser Asp Ile Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe

225 230 235 240

Asn Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr

245 250 255

Gly Leu Ile His Val Ala Asn Asn Asn Tyr Asp Pro Trp Ser Ile Tyr

260 265 270

Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser

275 280 285

Phe Thr Ala Pro Asn Asp Ser Asp Lys Lys Glu Val Thr Arg Arg Val

290 295 300

Gly Cys Glu Ser Pro Ser Thr Cys Ala Asn Trp Val Trp Arg Ser Thr

305 310 315 320

Gln Asp Ser Phe Asn Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Asn

325 330 335

Glu Gly Thr Asn Ile Tyr Asn Asn Asn Glu Ala Phe Lys Val Glu Asn

340 345 350

Gly Ser Ala Ala Pro Gln Leu Thr Lys Asn Ala Gly Val Leu Thr Cys

355 360 365

Ile Leu Ser Lys Pro Cys Ser

370 375

<210>17

<211>367

<212>PRT

<213>Juniperus ashei

<400>17

Met Ala Ser Pro Cys Leu Ile Ala Val Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Tyr Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ser Asn Trp Asp Gln Asn Arg Met Lys Leu Ala Asp Cys Ala Val Gly

35 40 45

Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Asp Phe Tyr Thr Val

50 55 60

Thr Ser Thr Asp Asp Asn Pro Val Asn Pro Thr Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Lys Ala Leu Trp Ile Ile Phe Ser Gln Asn

85 90 95

Met Asn Ile Lys Leu Lys Met Pro Leu Tyr Val Ala Gly His Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Asp Val His Leu Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Lys Val Ser His Val Ile Leu His Ser Leu His Ile

130 135 140

His Gly Cys Asn Thr Ser Val Leu Gly Asp Val Leu Val Ser Glu Ser

145 150 155 160

Ile Gly Val Glu Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met

165 170 175

Arg Asn Val Thr Asn Ala Trp Ile Asp His Asn Ser Leu Ser Asp Cys

180 185 190

Ser Asp Gly Leu Ile Asp Val Thr Leu Gly Ser Thr Gly Ile Thr Ile

195 200 205

Ser Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His

210 215 220

Asp Asp Thr Tyr Asp Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe

225 230 235 240

Asn Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr

245 250 255

Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp ProTrp Asn Ile Tyr

260 265 270

Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser

275 280 285

Phe Thr Ala Pro Ser Glu Ser Tyr Lys Lys Glu Val Thr Lys Arg Ile

290 295 300

Gly Cys Glu Ser Pro Ser Ala Cys Ala Asn Trp Val Trp Arg Ser Thr

305 310 315 320

Arg Asp Ala Phe Ile Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Thr

325 330 335

Glu Glu Thr Asn Ile Tyr Asn Ser Asn Glu Ala Phe Lys Val Glu Asn

340 345 350

Gly Asn Ala Ala Pro Gln Leu Thr Lys Asn Ala Gly Val Val Thr

355 360 365

<210>18

<211>367

<212>PRT

<213>Juniperus virginiana

<400>18

Met Ala Ser Pro Cys Leu Ile Ala Phe Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Cys Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ser Asn Trp Gly Gln Asn Arg Met Lys Leu Ala Asp Cys Ala Val Gly

35 40 45

Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Asp Phe Tyr Thr Val

50 55 60

Thr Ser Ala Asp Asp Asn Pro Val Asn Pro Thr Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Lys Thr Leu Trp Ile Ile Phe Ser Gln Asn

85 90 95

Met Asn Ile Lys Leu Lys Met Pro Leu Tyr Val Ala Gly His Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Asp Val His Leu Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Lys Val Ser His Val Ile Leu His Gly Leu His Ile

130 135 140

His Gly Cys Asn Thr Ser Val Leu Gly Asp Val Leu Val Ser Glu Ser

145 150 155 160

Ile Gly Val Val Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met

165 170 175

Arg Asn Val Thr Asn Ala Trp Ile Asp His Asn Ser Leu Ser Asp Cys

180 185 190

Ser Asp Gly Leu Ile Asp Val Thr Leu Gly Ser Thr Gly Ile Thr Ile

195 200 205

Phe Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His

210 215 220

Asp Asp Thr Tyr Asp Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe

225 230 235 240

Asn Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr

245 250 255

Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp Asn Ile Tyr

260 265 270

Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser

275 280 285

Phe Thr Ala Pro Asn Glu Asn Tyr Lys Tyr Glu Val Thr Lys Arg Ile

290 295 300

Gly Cys Glu Ser Thr Ser Ala Cys Ala Asn Trp Val Trp Arg Ser Thr

305 310 315 320

Arg Asp Ala Phe Ser Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Ile

325 330 335

Glu Glu Thr Asn Ile Tyr Asn Ser Asn Glu Ala Phe Lys Val Glu Asn

340 345 350

Gly Asn Ala Ala Pro Gln Leu Thr Lys Asn Ala Gly Val Val Ala

355 360 365

<210>19

<211>367

<212>PRT

<213>Hexalectris arizonica

<400>19

Met Ala Ser Pro Cys Leu Val Ala Val Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Tyr Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ser Asn Trp Asp Gln Asn Arg Met Lys Leu Ala Asp Cys Val Val Gly

35 40 45

Phe Gly Ser Leu Thr Met Gly Gly Lys Gly Gly Glu Ile Tyr Thr Val

50 55 60

Thr Ser Ser Asp Asp Asn Pro Val Asn Pro Thr Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Lys Ala Leu Trp Ile Ile Phe Ser Gln Asn

85 90 95

Met Asn Ile Lys Leu Gln Met Pro Leu Tyr Val Ala Gly Tyr Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Asp Val His Leu Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Thr Ala Ser His Val Ile Leu His Gly Leu His Ile

130 135 140

His Gly Cys Asn Thr Ser Val Leu Gly Asp Val Leu Val Ser Glu Ser

145 150 155 160

Ile Gly Val Glu Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met

165 170 175

Arg Asn Val Thr Asn Ala Trp Ile Asp His Asn Ser Leu Ser Asp Cys

180 185 190

Ser Asp Gly Leu Ile Asp Val Thr Leu Gly Ser Thr Gly Ile Thr Ile

195 200 205

Ser Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His

210 215 220

Asp Asp Thr Tyr Asp Asp Asp Ile Ser Met Lys Val Thr Val Ala Phe

225 230 235 240

Asn Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr

245 250 255

Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Gln Trp Asn Ile Tyr

260 265 270

Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser

275 280 285

Pro Thr Ala Pro Ser Glu Ser Tyr Lys Lys Glu Val Thr Lys Arg Ile

290 295 300

Gly Cys Glu Ser Thr Ser Ala Cys Ala Asn Trp Val Trp Arg Phe Thr

305310 315 320

Arg Asp Ala Phe Thr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Ala

325 330 335

Glu Glu Thr Asn Ile Tyr Asn Ser Asn Glu Ala Phe Lys Val Glu Asn

340 345 350

Gly Asn Ala Ala Pro Gln Leu Thr Gln Asn Ala Gly Val Val Thr

355 360 365

<210>20

<211>366

<212>PRT

<213>Juniperus oxycedrus

<400>20

Met Ala Ser Pro Cys Leu Arg Ala Val Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Tyr Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ser Asn Trp Gly Gln Asn Arg Met Lys Leu Ala Asp Cys Val Val Gly

35 40 45

Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Glu Phe Tyr Thr Val

50 5560

Thr Ser Ala Glu Asp Asn Pro Val Asn Pro Thr Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Lys Ala Leu Trp Ile Ile Phe Ser Gln Asn

85 90 95

Met Asn Ile Lys Leu Lys Met Pro Leu Tyr Val Ala Gly His Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Asp Val His Leu Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Lys Val Ser His Val Ile Leu His Gly Leu His Ile

130 135 140

Gly Cys Asn Thr Ser Val Leu Gly Asp Val Leu Val Ser Glu Ser Ile

145 150 155 160

Gly Val Glu Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met Arg

165 170 175

Asn Val Thr Asn Ala Trp Ile Asp His Asn Ser Leu Ser Asp Cys Ser

180 185 190

Asp Gly Leu Ile Asp Val Thr Leu Gly Ser Thr Gly Ile Thr Ile Ser

195 200 205

Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His Asp

210 215 220

Asp Thr Tyr Asp Asn Asp Lys Ser Met Lys Val Thr Val Ala Phe Asn

225 230 235 240

Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr Gly

245 250 255

Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp Asn Ile Tyr Ala

260 265 270

Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser Phe

275 280 285

Thr Ala Pro Ser Glu Ser Tyr Lys Lys Glu Val Thr Lys Arg Ile Gly

290 295 300

Cys Glu Ser Thr Ser Ala Cys Ala Asn Trp Val Trp Arg Ser Thr Arg

305 310 315 320

Asp Ala Phe Thr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Ile Glu

325 330 335

Glu Thr Asn Ile Tyr Asn Ser Asn Glu Ala Phe Lys Val Glu Asn Gly

340 345 350

Asn Ala Ala Pro Gln Leu Thr Lys Asn Ala Gly Val Val Thr

355 360 365

<210>21

<211>367

<212>PRT

<213>Cupressus sempervirens

<400>21

Met Asp Ser Pro Cys Leu Ile Ala Val Leu Val Phe Leu Cys Ala Ile

1 5 10 15

Val Ser Cys Tyr Ser Asp Asn Pro Ile Asp Ser Cys Trp Arg Gly Asp

20 25 30

Ser Asn Trp Asp Gln Asn Arg Met Lys Leu Ala Asp Cys Ala Val Gly

35 40 45

Phe Gly Ser Ser Thr Met Gly Gly Lys Gly Gly Asp Ile Tyr Thr Val

50 55 60

Thr Ser Ala Glu Asp Asn Pro Val Asn Pro Thr Pro Gly Thr Leu Arg

65 70 75 80

Tyr Gly Ala Thr Arg Glu Lys Ala Leu Trp Ile Ile Phe Ser Gln Asn

85 90 95

Met Asn Ile Lys Leu Lys Met Pro Leu Tyr Val Ala Gly His Lys Thr

100 105 110

Ile Asp Gly Arg Gly Ala Asp Val His Leu Gly Asn Gly Gly Pro Cys

115 120 125

Leu Phe Met Arg Lys Val Ser His Val Ile Leu His Gly Leu His Ile

130 135 140

His Gly Cys Asn Thr Ser Val Leu Gly Asn Val Leu Val Ser Glu Ser

145 150 155 160

Ile Gly Val Glu Pro Val His Ala Gln Asp Gly Asp Ala Ile Thr Met

165 170 175

Arg Asn Val Thr Asn Ala Trp Ile Asp His Asn Ser Leu Ser Asp Cys

180 185 190

Ser Asp Gly Leu Ile Asp Val Thr Leu Ser Ser Thr Gly Ile Thr Ile

195 200 205

Ser Asn Asn His Phe Phe Asn His His Lys Val Met Leu Leu Gly His

210 215 220

Asp Asp Thr Tyr Asp Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe

225 230 235 240

Asn Gln Phe Gly Pro Asn Ala Gly Gln Arg Met Pro Arg Ala Arg Tyr

245 250 255

Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Gln Trp Asn Ile Tyr

260 265 270

Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly Asn Ser

275 280 285

Phe Ala Ala Pro Asn Glu Asn Tyr Lys Lys Glu Val Thr Lys Arg Ile

290 295 300

Gly Cys Val Ser Thr Ser Ala Cys Ala Asn Trp Val Trp Arg Ser Thr

305 310 315 320

Arg Asp Ala Phe Ser Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Thr

325 330 335

Glu Glu Thr Asn Ile Tyr Thr Ser Asn Glu Ala Phe Lys Val Glu Asn

340 345 350

Gly Asn Leu Ala Pro Gln Leu Thr Lys Asn Ala Gly Val Val Ala

355 360 365

<210>22

<211>514

<212>PRT

<213>Chamaecyparis obtusa

<400>22

Met Gly Met Lys Phe Met Ala Ala Val Ala Phe Leu Ala Leu Gln Leu

1 5 10 15

Ile Val Met Ala Ala Ala Glu Asp Gln Ser Ala Gln Ile Met Leu Asp

20 25 30

Ser Asp Ile Glu Glu Tyr Leu Arg Ser Asn Arg Ser Leu Lys Lys Leu

35 40 45

Val His Ser Arg His Asp Ala Ala Thr Val Phe Asn Val Glu Gln Tyr

50 55 60

Gly Ala Val Gly Asp Gly Lys His Asp Ser Thr Glu Ala Phe Ala Thr

65 70 75 80

Thr Trp Asn Ala Ala Cys Lys Lys Ala Ser Ala Val Leu Leu Val Pro

85 90 95

Ala Asn Lys Lys Phe Phe Val Asn Asn Leu Val Phe Arg Gly Pro Cys

100 105 110

Gln ProHis Leu Ser Phe Lys Val Asp Gly Thr Ile Val Ala Gln Pro

115 120 125

Asp Pro Ala Arg Trp Lys Asn Ser Lys Ile Trp Leu Gln Phe Ala Gln

130 135 140

Leu Thr Asp Phe Asn Leu Met Gly Thr Phe Val Ile Asp Gly Gln Gly

145 150 155 160

Gln Gln Trp Trp Ala Gly Gln Cys Lys Val Val Asn Gly Arg Thr Val

165 170 175

Cys Asn Asp Arg Asn Arg Pro Thr Ala Ile Lys Ile Asp Tyr Ser Lys

180 185 190

Ser Val Thr Val Lys Glu Leu Thr Leu Met Asn Ser Pro Glu Phe His

195 200 205

Leu Val Phe Gly Glu Cys Glu Gly Val Lys Ile Gln Gly Leu Lys Ile

210 215 220

Lys Ala Pro Arg Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Ala

225 230 235 240

Ser Lys Arg Phe His Ile Glu Lys Cys Val Ile Gly Thr Gly Asp Asp

245 250 255

Cys Ile Ala Ile Gly Thr Gly Ser Ser Asn Ile Thr Ile Lys Asp Leu

260 265 270

Ile Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Asp

275 280 285

Asn Ser Arg Ala Glu Val Ser His Val His Val Asn Arg Ala Lys Phe

290 295 300

Ile Asp Thr Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser

305 310 315 320

Gly Leu Ala Ser Tyr Ile Thr Tyr Glu Asn Val Glu Met Ile Asn Ser

325 330 335

Glu Asn Pro Ile Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ser Ala

340 345 350

Cys Gln Asn Gln Arg Ser Ala Val Gln Ile Gln Gly Val Thr Tyr Lys

355 360 365

Asn Ile His Gly Thr Ser Ala Thr Ala Ala Ala Ile Gln Leu Met Cys

370 375 380

Ser Asp Ser Val Pro Cys Thr Gly Ile Gln Leu Ser Asn Val Ser Leu

385390 395 400

Lys Leu Thr Ser Gly Lys Pro Ala Ser Cys Val Asp Lys Asn Ala Arg

405 410 415

Gly Phe Tyr Ser Gly Arg Leu Ile Pro Thr Cys Lys Asn Leu Arg Pro

420 425 430

Gly Pro Ser Pro Lys Glu Phe Glu Leu Gln Gln Gln Pro Thr Thr Val

435 440 445

Met Asp Glu Asn Lys Gly Ala Cys Ala Lys Gly Asp Ser Thr Cys Ile

450 455 460

Ser Leu Ser Ser Ser Pro Pro Asn Cys Lys Asn Lys Cys Lys Gly Cys

465 470 475 480

Gln Pro Cys Lys Pro Lys Leu Ile Ile Val His Pro Asn Lys Pro Gln

485 490 495

Asp Tyr Tyr Pro Gln Lys Trp Val Cys Ser Cys His Asn Lys Ile Tyr

500 505 510

Asn Pro

<210>23

<211>507

<212>PRT

<213>Juniperus ashei

<400>23

Met Ser Met Lys Phe Met Ala Ala Leu Ala Phe Leu Ala Leu Gln Leu

1 5 10 15

Ile Val Met Ala Ala Gly Glu Asp Gln Ser Ala Gln Ile Met Leu Asp

20 25 30

Ser Asp Thr Lys Gln Tyr His Arg Ser Ser Arg Asn Leu Arg Lys Ala

35 40 45

Val His His Ala Arg His Asp Val Ala Ile Val Phe Asn Val Glu His

50 55 60

Tyr Gly Ala Val Gly Asp Gly Lys His Asp Ser Thr Asp Ala Phe Glu

65 70 75 80

Lys Thr Trp Asn Ala Ala Cys Asn Lys Leu Ser Ala Val Phe Leu Val

85 90 95

Pro Ala Asn Lys Lys Phe Val Val Asn Asn Leu Val Phe Tyr Gly Pro

100 105 110

Cys Gln Pro His Phe Ser Phe Lys Val Asp Gly Thr Ile Ala Ala Tyr

115 120 125

Pro Asp Pro Ala Lys Trp Leu Asn Ser Lys Ile Trp Met His Phe Ala

130 135 140

Arg Leu Thr Asp Phe Asn Leu Met Gly Thr Gly Val Ile Asp Gly Gln

145 150 155 160

Gly Asn Arg Trp Trp Ser Asp Gln Cys Lys Thr Ile Asn Gly Arg Thr

165 170 175

Val Cys Asn Asp Lys Gly Arg Pro Thr Ala Ile Lys Ile Asp Phe Ser

180 185 190

Lys Ser Val Thr Val Lys Glu Leu Thr Leu Thr Asn Ser Pro Glu Phe

195 200 205

His Leu Val Phe Gly Glu Cys Asp Gly Val Lys Ile Gln Gly Ile Lys

210 215 220

Ile Lys Ala Pro Arg Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe

225 230 235 240

Ala Ser Lys Arg Phe Glu Ile Glu Lys Cys Thr Ile Gly Thr Gly Asp

245 250 255

Asp Cys Val Ala Val Gly Thr Gly Ser Ser Asn Ile Thr Ile Lys Asp

260 265 270

Leu Thr Cys Gly Pro Gly His Gly Met Ser Ile Gly Ser Leu Gly Lys

275 280 285

Gly Asn Ser Arg Ser Glu Val Ser Phe Val His Leu Asp Gly Ala Lys

290 295 300

Phe Ile Asp Thr Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly

305 310 315 320

Ser Gly Leu Ala Ser His Ile Thr Tyr Glu Asn Val Glu Met Ile Asn

325 330 335

Ala Glu Asn Pro Ile Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ala

340 345 350

Ala Cys Lys Asn Gln Arg Ser Ala Val Lys Ile Gln Asp Val Thr Phe

355 360 365

Lys Asn Ile His Gly Thr Ser Ala Thr Thr Ala Ala Ile Gln Leu Met

370 375 380

Cys Ser Asp Ser Val Pro Cys Ser Asn Ile Lys Leu Ser Asn Val Phe

385 390 395 400

Leu Lys Leu Thr Ser Gly Lys Val Ala Thr Cys Val Asn Lys Asn Ala

405410 415

Asn Gly Tyr Tyr Thr Asn Pro Leu Asn Pro Ser Cys Lys Ser Leu His

420 425 430

Pro Gly Arg Thr Pro Lys Glu Leu Glu Leu His Gln Lys Pro Thr Thr

435 440 445

Leu Leu Met Asp Glu Lys Met Gly Ala Ser Leu Asn Ser Ser Pro Pro

450 455 460

Asn Cys Lys Asn Lys Cys Lys Gly Cys Gln Pro Cys Lys Pro Lys Leu

465 470 475 480

Ile Ile Val His Pro Asn Gln Pro Glu Asp Tyr Tyr Pro Gln Arg Trp

485 490 495

Val Cys Ser Cys His Asn Lys Ile Tyr Asn Pro

500 505

<210>24

<211>384

<212>PRT

<213>Hexalectris arizonica

<400>24

His Asp Val Ala Ile Val Phe Asn Val Glu His His Gly Ala Val Gly

1 5 10 15

Asp Gly Asn His Asp Ser Thr Asp Ala Phe Glu Lys Thr Trp Asn Glu

20 25 30

Ala Cys Lys Thr Leu Ser Ala Val Phe Leu Val Pro Ala Asn Lys Lys

35 40 45

Phe Val Val Val Asn Asn Leu Val Phe Tyr Gly Pro Cys Gln Pro His

50 55 60

Phe Ser Pro Lys Val Asp Gly Ile Ile Ala Ala Tyr Pro Asp Pro Val

65 70 75 80

Lys Trp Lys Asn Ser Lys Ile Trp Met His Phe Ala Arg Leu Thr Asp

85 90 95

Phe Asn Leu Met Gly Thr Gly Val Ile Asp Gly Gln Gly Ser Lys Trp

100 105 110

Trp Ser Asp Gln Cys Lys Thr Val Asn Gly Arg Thr Val Cys Asn Asp

115 120 125

Lys Gly Arg Pro Thr Ala Ile Lys Ile Asp Phe Ser Lys Ser Val Thr

130 135 140

Val Lys Glu Leu Thr Leu Met Asn Ser Pro Glu Phe His Leu Val Phe

145 150 155160

Gly Glu Cys Asp Gly Val Lys Ile Gln Gly Ile Lys Ile Lys Ala Pro

165 170 175

Lys Glu Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Gly Ser Lys Arg

180 185 190

Phe Glu Ile Glu Lys Cys Ile Ile Gly Thr Gly Asp Asp Cys Val Ala

195 200 205

Ile Gly Thr Gly Ser Ser Asn Ile Thr Ile Thr Asp Leu Thr Cys Gly

210 215 220

Pro Gly His Gly Met Ser Ile Gly Ser Leu Gly Lys Gly Asn Ser Arg

225 230 235 240

Ser Glu Val Ser Phe Val His Leu Asp Gly Ala Lys Phe Ile Asp Thr

245 250 255

Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser Gly Leu Ala

260 265 270

Ser His Ile Thr Tyr Glu Asn Val Glu Met Val Asn Ala Glu Asn Pro

275 280 285

Ile Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ala Cys Glu Asn Gln

290 295 300

Arg Ser Ala Val Lys Ile Glu Asp Val Trp Phe Lys Asn Ile His Gly

305 310 315 320

Thr Ser Ala Thr Ala Ala Ala Ile Gln Leu Met Cys Ser Asp Ser Val

325 330 335

Pro Cys Ser Asn Ile Lys Leu Ser Asn Val Val Leu Lys Leu Ser Ser

340 345 350

Gly Lys Val Ala Ala Cys Val Asn Lys Asn Ala Asn Gly Tyr Tyr Thr

355 360 365

Asn Pro Leu Asn Pro Pro Cys Lys Ser Leu His Pro Gly Pro Thr Pro

370 375 380

<210>25

<211>225

<212>PRT

<213>Juniperus ashei

<400>25

Met Ala Arg Val Ser Glu Leu Ala Phe Leu Leu Ala Ala Thr Leu Ala

1 5 10 15

Ile Ser Leu His Met Gln Glu Ala Gly Val Val Lys Phe Asp Ile Lys

2025 30

Asn Gln Cys Gly Tyr Thr Val Trp Ala Ala Gly Leu Pro Gly Gly Gly

35 40 45

Lys Arg Leu Asp Gln Gly Gln Thr Trp Thr Val Asn Leu Ala Ala Gly

50 55 60

Thr Ala Ser Ala Arg Phe Trp Gly Arg Thr Gly Cys Thr Phe Asp Ala

65 70 75 80

Ser Gly Lys Gly Ser Cys Gln Thr Gly Asp Cys Gly Gly Gln Leu Ser

85 90 95

Cys Thr Val Ser Gly Ala Val Pro Ala Thr Leu Ala Glu Tyr Thr Gln

100 105 110

Ser Asp Gln Asp Tyr Tyr Asp Val Ser Leu Val Asp Gly Phe Asn Ile

115 120 125

Pro Leu Ala Ile Asn Pro Thr Asn Ala Gln Cys Thr Ala Pro Ala Cys

130 135 140

Lys Ala Asp Ile Asn Ala Val Cys Pro Ser Glu Leu Lys Val Asp Gly

145 150 155 160

Gly Cys Asn Ser Ala Cys Asn Val Phe Lys Thr Asp Gln Tyr Cys Cys

165 170 175

Arg Asn Ala Tyr Val Asp Asn Cys Pro Ala Thr Asn Tyr Ser Lys Ile

180 185 190

Phe Lys Asn Gln Cys Pro Gln Ala Tyr Ser Tyr Ala Lys Asp Asp Thr

195 200 205

Ala Thr Phe Ala Cys Ala Ser Gly Thr Asp Tyr Ser Ile Val Phe Cys

210 215 220

Pro

225

<210>26

<211>225

<212>PRT

<213>Juniperus rigida

<400>26

Met Ala Arg Val Ser Glu Leu Ala Leu Leu Leu Val Ala Thr Leu Ala

1 5 10 15

Ile Ser Leu His Met Gln Glu Ala Gly Ala Val Lys Phe Asp Ile Lys

20 25 30

Asn Gln Cys Gly Tyr Thr Val Trp Ala Ala Gly Leu Pro Gly Gly Gly

35 40 45

Lys Arg Leu Asp GlnGly Gln Thr Trp Thr Leu Asn Leu Ala Ala Gly

50 55 60

Thr Ala Ser Ala Arg Phe Trp Gly Arg Thr Gly Cys Thr Phe Asp Ala

65 70 75 80

Ser Gly Lys Gly Ser Cys Lys Thr Gly Asp Cys Gly Gly Gln Leu Ser

85 90 95

Cys Thr Val Ser Gly Ala Val Pro Ala Thr Leu Ala Glu Tyr Thr Gln

100 105 110

Ser Asp Gln Asp Tyr Tyr Asp Val Ser Leu Val Asp Gly Phe Asn Ile

115 120 125

Pro Leu Ala Ile Asn Pro Thr Asn Ala Gln Cys Thr Ala Pro Ala Cys

130 135 140

Lys Ala Asp Ile Asn Ala Val Cys Pro Ser Glu Leu Lys Val Glu Gly

145 150 155 160

Gly Cys Asn Ser Ala Cys Asn Val Phe Gln Thr Asp Gln Tyr Cys Cys

165 170 175

Arg Asn Ala Tyr Val Asp Asn Cys Pro Ala Thr Asn Tyr Ser Lys Ile

180 185 190

Phe Lys Asn Gln Cys Pro Gln Ala Tyr Ser Tyr Ala Lys Asp Asp Thr

195 200 205

Ala Thr Phe Ala Cys Ala Ser Gly Thr Asp Tyr Ser Ile Val Phe Cys

210 215 220

Pro

225

<210>27

<211>224

<212>PRT

<213>Cupressus sempervirens

<400>27

Met Ala Arg Val Ser Glu Leu Ala Leu Leu Leu Val Ala Thr Leu Ala

1 5 10 15

Ile Ser Leu His Met Gln Glu Ala Gly Ala Val Lys Phe Asp Ile Lys

20 25 30

Asn Gln Cys Gly Tyr Thr Val Trp Ala Ala Gly Leu Pro Gly Gly Gly

35 40 45

Lys Arg Leu Asp Gln Gly Gln Thr Trp Thr Val Asn Leu Ala Ala Gly

50 55 60

Thr Ala Ser Ala Arg Phe Trp Gly Arg Thr Gly Cys Thr Phe Asp Ala

6570 75 80

Ser Gly Lys Gly Ser Cys Arg Ser Gly Asp Cys Gly Gly Gln Leu Ser

85 90 95

Cys Thr Val Ser Gly Ala Val Pro Ala Thr Leu Ala Glu Tyr Thr Gln

100 105 110

Ser Asp Lys Asp Tyr Tyr Asp Val Ser Leu Val Asp Gly Phe Asn Ile

115 120 125

Pro Leu Ala Ile Asn Pro Thr Asn Thr Lys Cys Thr Ala Pro Ala Cys

130 135 140

Lys Ala Asp Ile Asn Ala Val Cys Pro Ser Glu Leu Lys Val Asp Gly

145 150 155 160

Gly Cys Asn Ser Ala Cys Asn Val Leu Gln Thr Asp Gln Tyr Cys Cys

165 170 175

Arg Asn Ala Tyr Val Asp Asn Cys Pro Ala Thr Asn Tyr Ser Lys Ile

180 185 190

Phe Lys Asn Gln Cys Pro Gln Ala Tyr Ser Tyr Ala Lys Asp Asp Thr

195 200 205

Ala Thr Phe Ala Cys Ala Ser Gly Thr Asp Tyr Ser Ile Val PheCys

210 215 220

<210>28

<211>199

<212>PRT

<213>Hexalectris arizonica

<400>28

Val Lys Phe Asp Ile Lys Asn Gln Cys Gly Tyr Thr Val Trp Ala Ala

1 5 10 15

Gly Leu Pro Gly Gly Gly Lys Glu Phe Asp Gln Gly Gln Thr Trp Thr

20 25 30

Val Asn Leu Ala Ala Gly Thr Ala Ser Ala Arg Phe Trp Gly Arg Thr

35 40 45

Gly Cys Thr Phe Asp Ala Ser Gly Lys Gly Ser Cys Arg Ser Gly Asp

50 55 60

Cys Gly Gly Gln Leu Ser Cys Thr Val Ser Gly Ala Val Pro Ala Thr

65 70 75 80

Leu Ala Glu Tyr Thr Gln Ser Asp Gln Asp Tyr Tyr Asp Val Ser Leu

85 90 95

Val Asp Gly Phe Asn Ile Pro Leu Ala Ile Asn Pro Thr Asn Thr Lys

100 105 110

Cys Thr Ala Pro Ala Cys Lys Ala Asp Ile Asn Ala Val Cys Pro Ser

115 120 125

Glu Leu Lys Val Asp Gly Gly Cys Asn Ser Ala Cys Asn Val Leu Gln

130 135 140

Thr Asp Gln Tyr Cys Cys Arg Asn Ala Tyr Val Asn Asn Cys Pro Ala

145 150 155 160

Thr Asn Tyr Ser Lys Ile Phe Lys Asn Gln Cys Pro Gln Ala Tyr Ser

165 170 175

Tyr Ala Lys Asp Asp Thr Ala Thr Phe Ala Cys Ala Ser Gly Thr Asp

180 185 190

Tyr Ser Ile Val Phe Cys Pro

195

<210>29

<211>274

<212>PRT

<213>Pinus monticola

<400>29

Met Gly Asn Ser Ser Gly Asn Ser Leu Met Val Leu Leu Leu Val Leu

1 5 10 15

Leu Leu Val Gly Val Thr Val Asn Ala Gln Asn Cys Gly Cys Ala Ser

20 25 30

Gly Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Ser Ser Ser Ala Tyr

35 40 45

Cys Gly Ala Gly Cys Lys Ser Gly Pro Cys Ser Gly Gly Gly Ser Pro

50 55 60

Ser Gly Gly Gly Gly Ser Val Gly Thr Ile Ile Ser Gln Ser Phe Phe

65 70 75 80

Asn Gly Leu Ala Gly Gly Ala Ala Ser Ser Cys Glu Gly Lys Gly Phe

85 90 95

Tyr Thr Tyr Asn Ala Phe Ile Ala Ala Ala Asn Ala Tyr Ser Gly Phe

100 105 110

Gly Thr Thr Gly Ser Ala Asp Val Thr Lys Arg Glu Leu Ala Ala Phe

115 120 125

Leu Ala Asn Val Met His Gly Thr Gly Gly Met Cys Tyr Ile Asn Glu

130 135 140

Arg Thr Pro Pro Met Ile Tyr Cys Met Ser Ser Ala Thr Trp Pro Cys

145 150 155160

Ala Ser Gly Lys Ser Tyr His Gly Arg Gly Pro Leu Gln Leu Ser Trp

165 170 175

Asn Tyr Asn Tyr Gly Ala Ala Gly Gln Ser Ile Gly Phe Asp Gly Val

180 185 190

Asn Asn Pro Glu Lys Val Gly Gln Asp Ser Thr Ile Ser Phe Lys Thr

195 200 205

Ala Val Trp Phe Trp Met Lys Asn Ser Asn Cys His Ser Ala Ile Thr

210 215 220

Ser Gly Gln Gly Phe Gly Gly Thr Ile Lys Ala Ile Asn Ser Gln Glu

225 230 235 240

Cys Asn Gly Gly Asn Ser Gly Glu Val Asn Ser Arg Val Asn Tyr Tyr

245 250 255

Lys Asn Ile Cys Ser Gln Leu Gly Val Asp Pro Gly Ala Asn Leu Ser

260 265 270

Cys His

<210>30

<211>277

<212>PRT

<213>Pseudotsuga menziesii

<400>30

Met Gly Lys Thr Gly Gly Glu Lys Trp Val Met Ala Leu Val Leu Val

1 5 10 15

Leu Leu Leu Leu Gly Val Ser Val Asn Ala Gln Asn Cys Gly Cys Ala

20 25 30

Ser Gly Leu Cys Cys Ser Lys Tyr Gly Tyr Cys Gly Thr Thr Ser Ala

35 40 45

Tyr Cys Gly Thr Gly Cys Arg Ser Gly Pro Cys Ser Ser Asn Ser Gly

50 55 60

Gly Gly Ser Pro Ser Gly Gly Gly Gly Ser Val Gly Thr Ile Ile Ser

65 70 75 80

Gln Ser Ile Phe Asn Gly Leu Ala Gly Gly Ala Ala Ser Ser Cys Glu

85 90 95

Gly Lys Gly Phe Tyr Thr Tyr Thr Ala Phe Ile Lys Ala Ala Ser Ala

100 105 110

Tyr Ser Gly Phe Gly Thr Thr Gly Ser Asn Asp Val Lys Lys Arg Glu

115 120 125

Leu Ala Ala Phe Phe Ala Asn Val Met His Glu Thr Gly Gly Leu Cys

130 135 140

Tyr Ile Asn Glu Arg Asn Pro Pro Met Ile Tyr Cys Asn Ser Ser Ser

145 150 155 160

Thr Trp Pro Cys Ala Ser Gly Lys Ser Tyr His Gly Arg Gly Pro Leu

165 170 175

Gln Leu Ser Trp Asn Tyr Asn Tyr Gly Ala Ala Gly Lys Ser Ile Gly

180 185 190

Phe Asp Gly Leu Asn Asn Pro Glu Lys Val Gly Gln Asp Ala Thr Ile

195 200 205

Ser Phe Lys Thr Ala Val Trp Phe Trp Met Asn Asn Ser Asn Cys His

210 215 220

Ser Ala Ile Thr Gly Gly Gln Gly Phe Gly Ala Thr Ile Lys Ala Ile

225 230 235 240

Asn Ser Gly Glu Cys Asn Gly Gly Asn Ser Gly Glu Val Ser Ser Arg

245 250 255

Val Asn Tyr Tyr Arg Lys Ile Cys Ser Gln Leu Gly Val Asp Pro Gly

260 265 270

AlaAsn Val Ser Cys

275

<210>31

<211>308

<212>PRT

<213>Pinus taeda

<400>31

Met Gly Ser Arg Ser Arg Ile Leu Leu Ile Gly Ala Thr Gly Tyr Ile

1 5 10 15

Gly Arg His Val Ala Lys Ala Ser Leu Asp Leu Gly His Pro Thr Phe

20 25 30

Leu Leu Val Arg Glu Ser Thr Ala Ser Ser Asn Ser Glu Lys Ala Gln

35 40 45

Leu Leu Glu Ser Phe Lys Ala Ser Gly Ala Asn Ile Val His Gly Ser

50 55 60

Ile Asp Asp His Ala Ser Leu Val Glu Ala Val Lys Asn Val Asp Val

65 70 75 80

Val Ile Ser Thr Val Gly Ser Leu Gln Ile Glu Ser Gln Val Asn Ile

85 90 95

Ile Lys Ala Ile Lys Glu Val Gly Thr Val Lys Arg Phe Phe Pro Ser

100 105 110

Glu Phe Gly Asn Asp Val Asp Asn Val His Ala Val Glu Pro Ala Lys

115 120 125

Ser Val Phe Glu Val Lys Ala Lys Val Arg Arg Ala Ile Glu Ala Glu

130 135 140

Gly Ile Pro Tyr Thr Tyr Val Ser Ser Asn Cys Phe Ala Gly Tyr Phe

145 150 155 160

Leu Arg Ser Leu Ala Gln Ala Gly Leu Thr Ala Pro Pro Arg Asp Lys

165 170 175

Val Val Ile Leu Gly Asp Gly Asn Ala Arg Val Val Phe Val Lys Glu

180 185 190

Glu Asp Ile Gly Thr Phe Thr Ile Lys Ala Val Asp Asp Pro Arg Thr

195 200 205

Leu Asn Lys Thr Leu Tyr Leu Arg Leu Pro Ala Asn Thr Leu Ser Leu

210 215 220

Asn Glu Leu Val Ala Leu Trp Glu Lys Lys Ile Asp Lys Thr Leu Glu

225 230 235 240

Lys Ala Tyr Val Pro Glu Glu Glu Val Leu Lys Leu Ile Ala Asp Thr

245 250 255

Pro Phe Pro Ala Asn Ile Ser Ile Ala Ile Ser His Ser Ile Phe Val

260 265 270

Lys Gly Asp Gln Thr Asn Phe Glu Ile Gly Pro Ala Gly Val Glu Ala

275 280 285

Ser Gln Leu Tyr Pro Asp Val Lys Tyr Thr Thr Val Asp Glu Tyr Leu

290 295 300

Ser Asn Phe Val

305

<210>32

<211>173

<212>PRT

<213>Picea abies

<400>32

Met Asp Ser Arg Arg Leu Lys Arg Ser Gly Ile Val Cys Met Val Leu

1 5 10 15

Met Ser Met Leu Met Leu Val Val Cys Glu Asp Ser Asp Asn Thr Ala

20 25 30

Cys Leu Ser Ser Leu Ser Ser Cys Ala Pro Tyr Leu Asn Ala Thr Thr

35 40 45

Lys Pro Asp Ser Ser Cys Cys Ser Ala Leu Ile Ser Val Ile Asp Lys

50 55 60

Asp Ser Gln Cys Leu Cys Asn Leu Leu Asn Ser Asp Thr Val Lys Gln

65 70 75 80

Leu Gly Val Asn Val Thr Gln Ala Met Lys Met Pro Ala Glu Cys Gly

85 90 95

Lys Asn Val Ser Ala Thr Gln Cys Asn Lys Thr Ala Thr Ser Gly Gly

100 105 110

Ser Ser Val Gly Lys Thr Pro Thr Ser Thr Pro Pro Pro Ser Ser Ala

115 120 125

Thr Pro Ser Thr Thr Thr Ile Thr Lys Ser Asn Ser Asn Ala Ala Ala

130 135 140

Ser Val Ser Val Lys Met Phe Pro Val Ala Ala Leu Val Phe Val Ala

145 150 155 160

Val Ala Ser Val Leu Gly Leu Lys Gly Pro Cys Leu Arg

165 170

Claims (18)

1. A nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein consists of an amino acid sequence having at least 80% identity to SEQ ID No.5, SEQ ID No. 3, and SEQ ID No. 9.

2. The nucleic acid molecule of claim 1, wherein the chimeric protein consists of an amino acid sequence having at least 80% identity to SEQ ID NO 5.

3. The nucleic acid molecule of claim 1, wherein the chimeric protein consists of an amino acid sequence having at least 80% identity to SEQ ID NO 3.

4. The nucleic acid molecule of claim 1, wherein the chimeric protein consists of an amino acid sequence having at least 80% identity to SEQ ID NO 9.

5. The nucleic acid molecule of claim 2 or 3, wherein the nucleic acid molecule comprises DNA.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A pharmaceutical composition comprising the expression vector of claim 6.

8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier.

9. Use of an expression vector according to claim 6 for the preparation of a pharmaceutical composition for the treatment of pollen allergy.

10. A nucleic acid vaccine comprising the pharmaceutical composition of claim 7 or 8.

11. Use of a pharmaceutical composition according to claim 7 or 8 for the preparation of a nucleic acid vaccine for the treatment of pollen allergy.

12. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule comprises DNA.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. A pharmaceutical composition comprising the expression vector of claim 13.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. Use of an expression vector according to claim 13 for the preparation of a pharmaceutical composition for the treatment of peanut allergy.

17. A nucleic acid vaccine comprising the pharmaceutical composition of claim 14 or 15.

18. Use of a pharmaceutical composition according to claim 14 or 15 for the preparation of a nucleic acid vaccine for the treatment of peanut allergy.