CN100460509C - Trichinella paramyosin gene and its application - Google Patents

Abstract

The present invention relates to paramyosin gene of new trichina antigen, constitution of the vector containing the gene, prokaryon expression vector containing the gene and the expressed protein. The present invention also relates to the application of the gene and its coded protein. When the recombinant protein of trichina paramyosin gene is used in immunizing animal, high titer antibody and effective immunoprotection against attacting infection will generate.

Description

Trichinella paramyosin gene and its application

field of invention

The invention relates to a trichinella spiralis new antigen gene paramyosin gene, the construction of a vector containing the gene, the prokaryotic expression vector, the expressed protein and the antibody produced by the protein-immunized animal. The present invention also relates to the application of said gene and protein.

Background technique

Trichinella laspiralis (Trichinella spiralis, referred to as Trichinella spiralis), can infect humans and more than 150 kinds of animals. Huge economic losses to animal husbandry. The life history of Trichinella spiralis is briefly described as follows: When a human or animal host eats raw or semi-raw meat (pork, dog meat, etc.) containing Trichinella spiralis cysts, the larvae in the cysts escape under the action of gastric juice and intestinal juice and develop into adults in the small intestine. After mating, the females produce new larvae. The newborn larva invades the lymphatic vessels or veins of the intestinal mucosa, and reaches the striated muscle of the host along with the lymphatic and blood circulation to continue to develop. The pathogenic process of Trichinella spiralis is divided into three stages: invasion stage, larval migration and cyst formation stage, that is, every link in the life cycle of the worm can make the host pathogenic.

The clinical manifestations of trichinellosis are complex and diverse, and it is difficult to make an accurate diagnosis in time only based on clinical symptoms, which makes timely treatment difficult. Therefore, the immunodiagnosis and prevention of the disease have become a top priority. Because pathogens cannot be subcultured in large quantities in vitro, the acquisition of antigens is limited; coupled with the complexity and diversity of Trichinella spiralis antigens, there is currently no good antigen with high sensitivity and high specificity as a candidate antigen for diagnostic or protective vaccines molecular.

Contents of the invention

The purpose of the present invention is to find and clone a new trichinella antigen gene, to study the immunological function of the gene recombinant protein, and to provide new candidate antigen molecules for the immunodiagnosis, immune prevention and treatment of trichinosis.

The invention provides isolated polynucleotide molecules comprising a nucleotide sequence selected from the group consisting of:

(A) at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% homologous nucleotide sequences;

(B) a nucleotide sequence that can hybridize with the sequence shown in SEQ ID NO: 1 or the complementary sequence of the open reading frame in SEQ ID NO: 1 under moderately stringent hybridization conditions, preferably high stringent hybridization conditions;

(C) a nucleotide sequence that encodes the same protein as SEQ ID NO: 1 but differs in sequence due to the degeneracy of the genetic code;

(D) a fragment of the nucleotide sequence described in (A), (B) or (C); and

(E) A nucleotide sequence complementary to the nucleotide sequence described in (A), (B), (C) or (D).

Preferably, the polynucleotide molecule of the invention encodes an immunogenic polypeptide.

In a specific embodiment, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO: 1 or the open reading frame sequence shown in nucleotides 75-2732 of SEQ ID NO: 1.

The present invention also provides an isolated polypeptide comprising an amino acid sequence selected from the following group of amino acid sequences:

(A) an amino acid sequence at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% homologous to the amino acid sequence shown in SEQ ID NO: 2;

(B) due to substitution, deletion or insertion of one or more (eg 1-25, 1-20, 1-15, 1-10, 1-5) amino acid residues with SEQ ID NO: 2 amino acid sequences that differ from the sequence shown; and

(C) An immunogenic fragment of the amino acid sequence described in (A) or (B).

Preferably, the polypeptides of the invention are immunogenic.

In a specific embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

The present invention also provides a recombinant vector comprising the polynucleotide molecule of the present invention. Preferably, the recombinant vector is an expression vector, which contains and is capable of expressing the polynucleotide molecule of the present invention.

The present invention also provides host cells containing the recombinant expression vector of the present invention.

The present invention also provides a method for producing a polypeptide, comprising culturing the host cell of the present invention under conditions capable of expressing the polypeptide and recovering the expressed polypeptide.

The present invention also provides antibodies that specifically bind to the polypeptides of the present invention. The antibodies may be polyclonal or monoclonal.

The present invention also provides a pharmaceutical composition for preventing or treating Trichinella spiralis infection in animals, especially livestock and humans. The pharmaceutical composition contains the polypeptide or polynucleotide or antibody of the present invention.

The present invention also provides a diagnostic kit for diagnosing Trichinella spiralis infection in animals, especially livestock and humans, which contains the polynucleotide molecule of the present invention, the nucleic acid probe capable of detecting the polynucleotide molecule of the present invention, A primer that can specifically amplify the polynucleotide molecule of the present invention, the polypeptide of the present invention, or the antibody of the present invention.

The present invention provides the polynucleotide molecule of the present invention, the nucleic acid probe capable of detecting the polynucleotide molecule of the present invention, the primer capable of specifically amplifying the polynucleotide molecule of the present invention, the polypeptide of the present invention, or Use of the antibody of the present invention in the preparation of diagnostic reagents for diagnosing Trichinella infection in animals, especially livestock and humans.

The present invention also provides the use of the polynucleotide molecules or polypeptides or antibodies of the present invention in the preparation of medicaments for preventing or treating Trichinella spiralis infection in humans and animals.

The present invention also provides oligonucleotides, which contain at least 15 of the nucleotide sequence shown in SEQ ID NO: 1 or the open reading frame sequence shown in nucleotides 75-2732 of SEQ ID NO: 1 or its complementary sequence , preferably at least 25, more preferably at least 50 nucleotides or similar sequences thereof, which can hybridize with the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence under highly stringent hybridization conditions. Preferably, the oligonucleotide is a nucleic acid probe capable of detecting the polynucleotide molecule of the present invention, or a primer capable of specifically amplifying the polynucleotide molecule of the present invention.

The present invention also provides the use of the oligonucleotides of the present invention for diagnosing Trichinella infection in animals, especially livestock and humans.

The present invention also provides a fusion protein comprising the polypeptide of the present invention and another fusion object. In a specific embodiment, the other fusion partner is β-galactosidase, glutathione-S-transferase or polyhistidine, etc.

The nucleotide sequence of the present invention can be obtained from the cDNA gene library of Trichinella spiralis by immune screening. The invention also relates to variants of this sequence, such as deletions, additions or substitutions of one or more bases, which do not alter the function of the encoded protein. Variants may be naturally occurring equivalent variants or non-naturally occurring variants.

The polypeptide of the present invention can be prepared by constructing a prokaryotic or eukaryotic expression vector of the above gene, transforming it into a suitable host cell, and purifying the polypeptide from the culture. The polypeptide of the present invention includes the polypeptide of Sequence 2, and its derivatives, fragments or analogs with equivalent functions.

As a specific example, the inventors of the present invention utilize molecular biology techniques, such as: cDNA library immune screening, molecular cloning, polymerase chain reaction (PCR), DNA sequence determination, Southern hybridization, Western blot hybridization method, clone and identify Trichinella spiralis Antigen new gene paramyosin gene; using gene expression and immunology technology to obtain the gene's recombinant protein and high-titer immune serum; and the protein's cell localization, immunogenicity and immune protection were analyzed.

The technical indicators achieved by the gene are:

1. In the present invention, the full-length cDNA sequence of the gene is obtained by immune screening of a cDNA library. The full length is 2996bp, the start codon ATG is located at the 74th bp of the sequence, TGA is the stop codon, and the encoded protein contains 885 amino acids.

2. The 885 amino acid recombinant protein of Trichinella spiralis paramyosin gene was expressed and purified in Escherichia coli BL21 strain ( FIG. 1 ).

3. Animals immunized with recombinant protein of Trichinella spiralis paramyosin gene obtained high-titer anti-paramyosin immune serum.

4. The recombinant protein of Trichinella spiralis paramyosin gene is recognized by the sera of patients infected with Trichinella spiralis, sick pigs, and sick rabbits. And it reacted with the sera of artificially immunized mice with paramyosin gene recombinant protein (Figure 2).

5. Trichinella spiralis paramyosin gene recombinant protein immunized animals to produce effective immune protection against challenge infection (Table 1).

The present invention discovered the full-length cDNA of the paramyosin antigen gene of Trichinella spiralis; obtained the purified recombinant protein of the paramyosin gene and carried out immunological function research, providing a new protective candidate for the immune prevention of trichinellosis Antigen molecule. And it can also be used to prepare diagnostic reagents.

Description of drawings

Fig. 1 SDS-PAGE electrophoresis: expression and purification of paramyosin gene recombinant protein in Escherichia coli. Among them, each lane represents:

M. Protein Molecular Weight Marker

1. Empty vector whole bacteria protein

2. Whole protein without IPTG induction

3. Whole protein after IPTG induction

4. Purified Paramyosin Gene Recombinant Protein

The arrow points to the recombinant protein band

Figure 2 Western blotting to identify the immunological properties of the paramyosin gene recombinant protein. Among them, each lane represents:

1. Reaction of the purified paramyosin gene recombinant protein with the serum of patients with Trichinella spiralis

2. Reaction of purified paramyosin gene recombinant protein with pig serum infected with Trichinella spiralis

3. Reaction of purified paramyosin gene recombinant protein with rabbit serum infected with Trichinella spiralis

4. The purified paramyosin gene recombinant protein reacted with the recombinant protein artificially immunized mouse serum.

Detailed ways

As used herein, the terms "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "open reading frame (ORF)" and the like refer to single- or double-stranded DNA and RNA molecules, which may contain one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences including exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

Methods of producing and manipulating the polynucleotide molecules and oligonucleotide molecules disclosed herein are known to those skilled in the art and can be performed following recombinant techniques as described (see Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ausubel et al., 1989, Current Techniques in Molecular Biology , Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory Chamber Press, Cold Spring Harbor, New York; Innis et al (eds), 1995, PCR Strategies , Academic Press, Inc., San Diego; and Erlich (eds), 1992, PCR Technology, Oxford University Press, New York).

The present invention provides an isolated polynucleotide molecule encoding an immunogenic polypeptide. In one embodiment, the isolated polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the following members: (1) SEQ ID NO: 1 or the open reading frame (position 75-2732) in SEQ ID NO: 1 The nucleotide sequence shown; (2) at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical to the nucleotide sequence shown in the open reading frame of SEQ ID NO: 1 or SEQ ID NO: 1 , most preferably 95% identical nucleotide sequences; (3) under moderately stringent hybridization conditions (ie in 0.5M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C and bound to Conditions for DNA hybridization of filters and washing in 0.2xSSC/0.1% SDS at 42°C; see Ausubel et al. (eds.), 1989, Current Techniques in Molecular Biology , Vol. 1, GreenPublishing Associntes, Inc., and John Wiley & Sons, Inc., NY, P.2.10.3), preferably highly stringent hybridization conditions (that is, in 0.5M NaHPO ₄ , 7% SDS, 1 mM EDTA, hybridize with DNA bound to the filter at 65 ° C, and at 0.1 x SSC/0.1% SDS at 68 ° C washing conditions; refer to Ausubel et al., 1989, the above-mentioned literature) with the polynucleotide molecular energy with the open reading frame in SEQ ID NO: 1 or SEQ ID NO: 1 or its complementary sequence Hybridized nucleotide sequence; (4) Nucleosides that encode the same sequence as the protein encoded by the open reading frame in SEQ ID NO: 1 or SEQ ID NO: 1, but differ in sequence due to the degeneracy of the genetic code acid sequence; or (5) a nucleotide sequence complementary to any one of the nucleotide sequences in (1)-(4).

In one embodiment, the polynucleotide molecule of the invention comprises a nucleotide sequence encoding Trichinella paramyosin.

In one embodiment, the polynucleotide molecules of the present invention can be used to amplify Trichinella-specific polynucleotide molecules using standard amplification techniques, or as diagnostic reagents to detect Trichinella in animal body fluids or tissue samples infected by Trichinella. Presence of specific polynucleotides.

The invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide homologous to the Trichinella paramyosin or immunogenic polypeptide of the invention. The term "homologous" used herein when referring to a polypeptide homologous to Trichinella paramyosin or an immunogenic polypeptide means that the polypeptide originally has the amino acid sequence of Trichinella spiralis paramyosin or an immunogenic polypeptide, but wherein one or more (e.g. 1-25, 1-20, 1-15, 1-10, 1-5) amino acid residues have been conservatively substituted by a different amino acid residue, and the resulting Polypeptides can be used in the practice of the invention. Conservative amino acid substitutions are known in the art. Rules for making such substitutions include those described by Dayhof, M.D. (1978, National Biomedical Research Foundation, Washington, D.C., Vol. 5, Suppl. 3) and others. More specifically, conservative amino acid substitutions occur within a family of amino acids associated with their acidity, polarity, or side chain size. Genetically encoded amino acids can generally be divided into four groups: (1) acidic amino acids = aspartic acid, glutamic acid; (2) basic amino acids = lysine, arginine, histidine; (3) non- Polar amino acids = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar amino acids = glycine , Asparagine, Glutamine, Cysteine, Serine, Threonine, Tyrosine. Phenylalanine, tryptophan, and tyrosine are also collectively classified as aromatic amino acids. One or more substitutions within any particular group, such as isoleucine or valine for leucine, or glutamic acid for aspartic acid, or serine for threonine, or any other amino acid residue Structurally related amino acid residues, such as amino acid residue substitutions with similar acidity, polarity, side chain size, or similarity in some combination thereof, generally have no effect on the function or immunogenicity of the polypeptide. too much impact.

Substantially homologous proteins and polypeptides are characterized by having one or more (eg, 1-25, 1-20, 1-15, 1-10, 1-5) amino acid substitutions, deletions or additions. These changes are preferably those that affect less, that is, conservative amino acid substitutions (see Table 2) and other substitutions that do not seriously affect the folding and activity of proteins or polypeptides; small deletions, usually small deletions of 1 to about 30 amino acids; and small amino- or carboxy-terminal extensions, such as amino-terminal methionine residues, small connecting peptides up to about 20-25 residues in length, or small extensions (affinity tags) that facilitate purification, such as polyhistidine Bundle, Protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods in Enzymology, 198:3, 1991). DNA encoding affinity tags can be purchased from product suppliers.

As used herein, the polypeptide "can be used to practice the present invention" means that the polypeptide can be used as a diagnostic reagent to detect Trichinella-specific antibodies in the blood or serum samples of animals newly infected by Trichinella spiralis or infected by Trichinella spiralis or can be used as an antigen to produce trichinella-specific antibodies (such as specific binding antibodies, neutralizing antibodies, therapeutic antibodies); or can be used as an immunogen for preventive or therapeutic immunization of animals, or Any use of the polypeptide obtained.

The present invention further provides an oligonucleotide molecule hybridizable to the above-mentioned polynucleotide molecule of the present invention, or to a polynucleotide molecule having a nucleotide sequence that is the complement of the above-mentioned polynucleotide molecule of the present invention. Such oligonucleotide molecules are preferably at least about 15 nt in length, more preferably about 25 nt in length, especially at least 50 nt in length, and can hybridize to one or more of the above polynucleotide molecules under highly stringent conditions. The highly stringent condition is to wash the membrane in 6XSSC/0.5% sodium pyrophosphate, and the washing temperature is about 37°C for those with a length of about 14 bases, about 48°C for those with a length of about 17 bases, and about 20 bases. The base is about 55°C, and the length of about 23 bases is about 60°C. Other hybridization conditions for longer oligonucleotide molecules of the invention can be determined by those skilled in the art following standard techniques. In a preferred embodiment, the oligonucleotide molecule of the invention is complementary to a portion of at least one of the aforementioned polynucleotide molecules of the invention.

The oligonucleotide molecules of the present invention can be used for various purposes, including as primers to amplify Trichinella spiralis-specific polynucleotide molecules for differential diagnosis of diseases, or to encode or be used as antisense molecules for gene regulation. For diagnostic purposes, appropriately designed primers can be used to detect the presence of Trichinella-specific polynucleotide molecules in samples such as animal tissue or body fluids. The production of specific amplification products supports the diagnosis of Trichinella spiralis infection, while the absence of amplification products may indicate the absence of infection. Methods for performing amplification, such as the polymerase chain reaction (PCR) method, are described, for example, in the aforementioned publications edited by Innis et al. (1995, supra) and Erlicch (1992, supra). Other amplification techniques known in the art can also be used, such as ligase chain reaction. Primers for isolating homologous genes from other species or strains of Trichinella spiralis can also be designed using the sequences of the polynucleotide molecules disclosed herein.

The invention further provides cloning vectors comprising the polynucleotide molecules of the invention, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In a preferred embodiment, the present invention provides a recombinant vector comprising a polynucleotide molecule having a nucleotide sequence encoding Trichinella paramyosin or an immunogenic polypeptide.

Preferably, the recombinant vector of the present invention, especially the expression vector, is constructed in such a way that the coding sequence of the polynucleotide molecule of the present invention is operably linked to one or more regulatory elements required for transcription and translation of the coding sequence to produce peptide. The term "regulatory element" as used herein includes, but is not limited to, encoding inducible and non-inducible promoters, enhancers, operators and other elements known in the art that can be used to drive and/or regulate the expression of a polynucleotide coding sequence the nucleotide sequence. In addition, the "operably linked" coding sequence and one or more regulatory elements referred to herein means that the regulatory elements can effectively regulate and allow transcription of the coding sequence or translation of its mRNA, or both functions.

Methods for constructing recombinant vectors containing specific coding sequences operably linked to appropriate regulatory elements are known and can be used to carry out the present invention. These methods include in vitro recombinant techniques, synthetic techniques and in vivo genetic recombination (see eg Maniatis et al. (1989) supra).

Various vectors that can be used to express the Trichinella spiralis paramyosin of the present invention or the immunogenic polypeptide coding sequence of the present invention are known in the art, including recombinant phage DNA, plasmid DNA and cosmid DNA expression containing specific coding sequences carrier. Plasmids that can be engineered to contain polynucleotide molecules of the invention include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, CA), pPL and pKK223 (Pharmacia, Piscataway, NJ), pQE50 (Qiagen, Chatsworth, CA) and pGEM-T EASY (Promega, Madison, WI) and the like. Typical eukaryotic expression vectors that can be engineered to contain polynucleotide molecules of the invention include ecdysone-inducible mammalian expression systems (Invitrogen, Carlsbad, CA), cytomegalovirus promoter-enhancer-based systems (Promega, Madison , WI; Stratagene, La Jolla, CA; Invitrogen), and a baculovirus-based expression system (Promega), among others.

The regulatory elements of these and other vectors can vary in their strength and specificity. Depending on the host/vector system utilized, any of a variety of suitable transcription and translation elements may be used. For example, when cloning in mammalian cell systems, promoters isolated from mammalian cell genomes, such as the mouse metallothionein promoter, or from viruses growing in these cells, such as Vaccinia virus 7.5K promoter or Moloney murine sarcoma virus long terminal repeat. Promoters obtained by recombinant DNA or synthetic techniques may be used to transcribe the inserted sequence. In addition, expression from certain promoters can be enhanced in the presence of specific inducers, such as zinc and cadmium ions for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include β-gal promoter, T7 promoter, TAC promoter, trp and lac promoter, trp-lac fusion promoter, etc. for bacteria; Lyase promoters, such as ADH- and ADH-II promoters, GPK promoters, PGI promoters, TRP promoters, etc.; and SV40 early and late promoters for mammalian cells, adenovirus major late promoters, etc. The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the promoter of the Trichinella spiralis paramyosin gene, which can be used to express the coding sequence of the present invention in Trichinella spiralis.

Specific initiation signals are also required for adequate translation of the inserted coding sequence. These signals typically include the ATG initiation codon and adjacent sequences. In cases where a polynucleotide molecule of the invention comprising its own initiation codon and adjacent sequences is inserted into an appropriate expression vector, it may not be necessary to add additional translational control signals. However, when only a portion of the coding sequence is inserted, exogenous translational control signals including the ATG initiation codon and the like may be required. These exogenous translational control signals and initiation codons can be obtained from a variety of sources, both natural and synthetic. Furthermore, the initiation codon must be in-frame with the coding region to ensure in-frame translation of the entire insert.

Expression vectors can also be constructed that will express a fusion protein comprising a protein or polypeptide of the invention and a fusion partner. Such fusion proteins can be used, for example, to produce antisera against Trichinella proteins, to study the biochemical properties of Trichinella proteins, to engineer Trichinella proteins that exhibit different immunological or functional properties, to help identify or purify recombinantly expressed Trichinella proteins or improve its stability. Possible fusion protein expression vectors include, but are not limited to, inserts encoding β-galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions, and polyhistidine fusions (carrier region ) sequence carrier. Methods that can be used to construct expression vectors encoding these and other fusion proteins are known in the art.

Fusion proteins can be used to aid in the purification of expressed proteins. In a non-limiting embodiment, paramyosin or immunogenic polypeptide-maltose binding fusion protein can be purified using amylose resin and paramyosin or immunogen can be purified using glutathione sepharose beads Sexual polypeptide-glutathione-S-transferase fusion proteins, paramyosin or immunogenic polypeptide-polyhistidine fusion proteins can be purified using divalent nickel resin. Alternatively, the fusion protein can also be purified by affinity chromatography using antibodies against the carrier protein or peptide. For example, a nucleotide sequence encoding a target epitope of a monoclonal antibody can be engineered into an expression vector operably linked to regulatory elements and positioned so that the expressed epitope is fused to the Trichinella protein. In a non-limiting embodiment, standard techniques can be used to insert an epitope tag encoding FLAG ^™ (which is a hydrophilic tag peptide) at a point corresponding to the amino or carboxyl terminus of paramyosin or the immunogenic polypeptide. (Zinternational Biotechnologies Inc.), the expressed paramyosin or immunogenic polypeptide-FLAG ^™ epitope fusion product can then be detected and affinity purified using a commercially available anti-FLAG ^™ antibody.

The expression vector can also be modified to contain a polylinker sequence encoding a specific protease cleavage site, so that the expressed Trichinella protein can be released from the vector region or fusion partner by specific protease treatment. For example, a fusion protein vector may comprise a nucleotide sequence encoding a thrombin or Factor Xa cleavage site.

A signal sequence located upstream of the Trichinella spiralis protein coding sequence and in the same reading frame can be processed into the expression vector using known methods to direct the trafficking and secretion of the expressed protein. Non-limiting examples of signal sequences include those of alpha factors, immunoglobulins, outer membrane proteins, penicillinase, and T cell receptors, among others.

In order to facilitate selection of host cells transformed or transfected with the recombinant vector of the present invention, the vector can be engineered to further include coding sequences for reporter gene products or other selectable markers. Such coding sequences are preferably operably linked to the regulatory elements described above. Reporter genes used in the practice of the present invention are well known in the art and include genes encoding chloramphenicol acetyltransferase (CAT), green fluorescent protein, firefly luciferase, and human growth hormone, among others. Nucleotide sequences encoding selectable markers are known in the art and include those encoding gene products that confer antibiotic resistance or antimetabolite resistance, or provide auxotrophic requirements, and the like. Examples of such sequences include those encoding thymidine kinase activity, or resistance to methotrexate, ampicillin, kanamycin, chloramphenicol, aminoglycosides, or hygromycin, among others.

The present invention further provides transformed host cells comprising the polynucleotide molecules or recombinant vectors of the present invention, and cell lines derived therefrom. Host cells useful in the practice of the present invention may be eukaryotic or prokaryotic. Such transformed host cells include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA vectors, or yeast transformed with recombinant vectors, or animal cells, such as with recombinant viral vectors such as rod-shaped Virus-infected insect cells, or mammalian cells infected with recombinant viral vectors such as adenovirus or vaccinia virus, etc. For example, E. coli strains such as the DH5α strain may be used. Eukaryotic host cells include yeast cells, but mammalian cells such as mouse, hamster, bovine, monkey or human cell lines can also be effectively used. Eukaryotic host cells that can be used to express the recombinant protein of the present invention include Chinese hamster ovary (CHO) cells, NIH/3T3 and the like.

Preferably, a recombinant vector of the invention is transformed or transfected into one or more host cells in a substantially homogeneous culture of cells. Generally, known techniques can be followed, such as protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, infection by contact with recombinant virus, liposome-mediated transfection, DEAE-dextran Techniques such as transfection, transduction, conjugation, or particle bombardment introduce vectors into host cells. Transformants can be selected using standard methods, for example by selecting for cells expressing a selectable marker associated with the recombinant expression vector, such as antibiotic resistance.

Once the expression vector has been introduced into the host cell, standard techniques such as Southern hybridization analysis, restriction analysis, PCR analysis including reverse transcriptase PCR (rt-PCR) can be used to confirm whether the polynucleotide molecule of the present invention is integrated or not. Remain in the host cell genome or exist as episomes, or detect the expected protein product by immunological detection. Host cells containing and/or expressing polynucleotide molecules of the invention can be identified by one of at least four general methods known in the art: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detection of the presence of "marker" gene function; (iii) detection of expression of specific mRNA transcripts in host cells to estimate transcript levels; or (iv) detection by immunoassays known in the art Presence of mature polypeptide product.

Once a polynucleotide molecule of the invention has been stably introduced into a suitable host cell, the transformed host cell can be clonally propagated and the resulting cells cultured under conditions conducive to maximal production of the encoded polypeptide . Such conditions generally involve culturing transformed cells to high densities. When the expression vector contains an inducible promoter, changes in temperature, depletion of nutrients, addition of obligatory inducers (such as carbohydrate analogs, such as isopropyl β-D-thiogalactoside (IPTG), Appropriate induction conditions such as excess metabolic by-product accumulation to induce expression.

When the polypeptide remains within the host cell, the cells should be harvested and lysed, and the lysate extracted from the lysate under extraction conditions known in the art to minimize protein degradation, for example, at 4°C and/or in the presence of protease inhibitors. The product is substantially purified or isolated in . If the polypeptide is secreted from the host cell, the depleted nutrient medium can simply be harvested and the polypeptide substantially purified or isolated therefrom.

When necessary, substantially purify or isolate from cell lysates or culture media using standard methods including, but not limited to, ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density gradient centrifugation, and affinity chromatography. peptide. If the polypeptide lacks biological activity, it can be detected by size, or by reactivity with antibodies specific for the polypeptide, or by the presence of a fusion label. For use in the practice of the present invention, the polypeptide may be secreted into culture fluid or present in a cell lysate in an unpurified state, but preferably has been substantially purified or isolated therefrom. Reference herein to a polypeptide that is "substantially purified" means that the polypeptide constitutes at least about 20% by weight of the protein in a particular preparation. Additionally, reference herein to a polypeptide that is "isolated" means that the polypeptide constitutes at least about 80% by weight of the protein in a particular preparation.

Accordingly, the invention provides substantially purified or isolated Trichinella spiralis paramyosin or immunogenic polypeptides encoded by polynucleotides of the invention. In a preferred embodiment, Trichinella spiralis paramyosin or immunogenic polypeptide has the amino acid sequence of SEQ ID NO:2.

The present invention further provides a polypeptide homologous to said Trichinella spiralis paramyosin or immunogenic polypeptide, wherein the term "homologous" has the meaning defined above for the polypeptide.

The invention further provides a polypeptide consisting of a substantial portion of any one of the above polypeptides of the invention. As used herein - a "substantial portion" or "peptide fragment" of a polypeptide of the invention means less than the entire amino acid sequence of a corresponding full-length polypeptide in composition, but comprising at least about 10%, preferably at least about 20%, of its amino acid sequence, and useful in the practice of the invention ("usable in" has the meaning defined above for polypeptides). Particularly preferred are peptide fragments which are immunogenic (ie capable of inducing an immune response producing antibodies specific against the corresponding full-length Trichinella spiralis polypeptide).

The present invention further provides fusion proteins comprising any one of the above polypeptides fused to carriers or fusion partners known in the art.

The polypeptide of the present invention can be used for various purposes, including as a diagnostic reagent, for example, screening for Trichinella-specific antibodies in animal blood or serum samples by standard detection methods such as ELISA detection; or for producing polyclonal or monoclonal antibodies as described below wherein the antibody can be used as a diagnostic reagent, for example, to screen for Trichinella-specific proteins in animal cell, tissue or body fluid samples by standard techniques such as Western blot detection.

Any polypeptide of the invention can be modified at the protein level to improve or alter its biological or immunological characteristics. Analogs thereof can be prepared by subjecting a polypeptide to one or more chemical modifications using known techniques.

One or more chemical groups or another protein such as serum albumin, keyhole limpet hemocyanin or BSA, or a polyamino acid such as polylysine, or a polysaccharide such as Sepharose may be attached to the molecule. , agarose, or modified or unmodified cellulose) etc., to prepare derivatives of the polypeptide of the present invention. Methods for performing these ligation reactions are known in the art of protein chemistry.

The invention further provides isolated antibodies raised against the polypeptides of the invention. In a preferred embodiment, known methods can be used to generate antibodies against Trichinella paramyosin or immunogenic polypeptides. Trichinella paramyosin or immunogenic polypeptide recombinant protein, or its homologue, fusion protein, substantial part, analog or derivative as described above, can be immunized with partially or substantially purified or isolated Trichinella paramyosin or an immunogenic polypeptide selected from pig, bovine, Various host animals such as horses, rabbits, goats, sheep or mice. Antibody production can be enhanced using adjuvants as described below.

Polyclonal antibodies can be obtained and isolated from the sera of immunized animals and tested for specificity for the antigen using conventional methods. Alternatively, monoclonal antibodies can be prepared and isolated using any technique for the production of antibody molecules from cultured continuous cell lines. These techniques include, but are not limited to, the hybridoma technique previously described by Kohler and Milstein (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al. , 1983, Proceedings of the National Academy of Sciences of the United States of America, 80:2026-2030), and EBV hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-86). Alternatively, single chain antibodies specific for Trichinella spiralis antigens can be produced using techniques described for the production of single chain antibodies (eg, see US Pat. No. 4,946,778).

Antibody fragments containing specific binding sites for polypeptides of the invention are also included within the scope of the invention and can be prepared using known techniques. Such fragments include, but are not limited to, F(ab') ₂ fragments, which can be produced by pepsin digestion of intact antibody molecules, and Fab fragments, which can be produced by reducing the disulfide bridges of F(ab') ₂ fragments. Alternatively, Fab expression libraries can also be constructed (Huse et al., 1989, Science 246: 1275-1281) to rapidly identify Fab fragments with the desired specificity for Trichinella proteins.

Techniques for producing and isolating monoclonal antibodies and antibody fragments are known in the art and are described in more detail in, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory and JW Cooding , 1986, Monoclonal Antibodies: Principles and Practice , Academic Press, London (which is incorporated by reference herein).

The following examples are described by way of example only, and are not intended to limit the scope of the present invention.

Example

Example 1 Cloning and sequence analysis of Trichinella spiralis paramyosin gene

1. Preparation of screening serum (artificially immunized rabbit serum and artificially infected rabbit serum)

Adults of Trichinella spiralis Heilongjiang (International Standard Strain Code ISS533) isolated from pigs were collected, and the adult bodies were placed in a grinder and rapidly ground for 15 minutes under ice bath. Transfer the ground liquid into a 10ml centrifuge tube, and ultrasonicate (on ice) 3 times, 3 minutes each time, with a 1-minute rest in between. Repeated freezing and thawing several times, centrifuging and taking the supernatant to obtain the whole worm soluble antigen. Immunize Japanese big-eared white rabbits (1.6mg/rat), boost once a week, and boost 4 times to obtain artificially immune rabbit serum; experimental rabbits were fed with 4000 Trichinella muscle larvae, and artificially infected rabbit serum was obtained after 4 weeks. As determined by ELISA, the serum titers of the artificially immunized rabbit serum and the artificially infected rabbit serum both reached above 1:4000.

2. Immunoscreening expression library

Trichinella spiralis adult λZAP II cDNA expression library was purchased from Liu Mingyuan (reference "Construction and screening of adult and newborn larvae gene libraries of Chinese Trichinella spiralis isolates", Chinese Journal of Veterinary Medicine, 1998, 18(2): 147-150.).

1 μl Trichinella spiralis adult λZAP II cDNA expression library dilution was added to 600 μl host bacteria XL1-Blue, 37°C, adsorbed for 20 minutes, added to the top layer of agar, and placed upside down in a 42°C incubator to cultivate until phage plaques the size of pinpoints grew. The nitrocellulose membrane (Gelman Company) was spread on the plate and incubated overnight in a 37°C incubator. The next day, mark the membrane at room temperature, remove it from the plate, and immerse it in the primary antibody working solution (artificially immune rabbit serum diluted with TBST at 1:1000) for 30 minutes. Escherichia coli phage lysate (STRTAGENE company). Immerse the membrane in the secondary antibody working solution—1:7500 alkaline phosphatase—goat anti-rabbit IgG (Promega), for 30 minutes. Immerse in color developing solution NBT, BCIP (Promega Company), after color development is sufficient, add ddH ₂ O to terminate. The positive clones obtained were screened with 1:1000 artificial immune rabbit serum, and then re-screened with artificially infected rabbit serum as the primary antibody (diluted with TBST at 1:1000), and 3 positive clones were obtained. After identification by PCR and Southern Blot, the Ts86 clone was selected for DNA sequence determination.

3. Analysis of Ts86cDNA sequence and encoded amino acid

The full length of Ts86cDNA is 2996bp (see SEQ ID NO: 1), with an open reading frame (base 75-2732, 2658b in total), encoding 885 amino acids (see SEQ ID NO: 2), and its theoretical molecular weight is 102KD. After homology analysis of the encoded protein, it was found that the paramyosin homology with Ancylostoma duodenale, Onchocercia volvulus, and Filaria malayi was 82%, 82% and 79%, respectively, and it was inferred that it was Trichinella spiralis paramyosin gene.

Nucleotide sequence (SEQ ID NO: 1) (bases underlined are the starting and ending positions of the open reading frame):

GGCACGAGCAGATCTAAAAGGATCTGTTGTAAATTGATTCTCCATTCGACAATCGAAAAAAC

TACATCAGCACC ATG TCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCAT

GCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCA

ACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGAT

TTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCA

ACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACG

CCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTG

GAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCA

GCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGC

AGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCG

GAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCT

GGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATT

TGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAAGACTCAA

CTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCA

AATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAG

AGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCC

CAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAAGTTGACGATCTGCG

TAAAAAAATGATCCAAAAACAAGCAGAATATGAGGAACAAATTGAAATTATGCTGCAAAAGA

TTTCCCAATTGGAAAAAGCGAAAAGCCGTCTGCAGTCGGAAGTTGAAGTTTTGATAGTTGAT

TTGGAAAAGGCTCAAAGCACCATTGCCATTTTGGAAAGACAAAAAGAACAGCTCGAAAGAAT

GGTCGCCGAGATGAAGACACGCCTGGACGAGGTGACACAAGAGCTCGAAGCTACGCAACGAG

AACTCAGAGCGACGCAAGCGGAATTGCAAAAGATGAAGCATCTTTATGAAAAAGCCGTCGAA

CAGAAAGAAGCTCTGGCTAGGGAGAACAAAAAATTGCAAGACGATTTGCATGAAGCAAAGGA

AGCTTTGGCCGACGCGAACAGAAAATTGCACGAGCTGGATTTGGAGAATGCACGTCTGGCCG

GTGAAATCAGAGAACTGCAAATCGCGCTCAAGGAAGCCGAAGCGGCTAGACGTGACGCGGAA

AGTCGCGCACAGCGTGCAGTAGCTGAATTGCAAGCTCTGCGCGTGGAAATGGAACGTCGTTT

ACAAGAAAAAGAAGAAGAAATGGAAGCATTGCGCAAAAATATGCAGTTCGAAATTGACCGAC

TGGCTGCAGCGTTGGCTGACGCTGAGGCTCGCATGAAGGCGGAAATTTCCCGTCTGAAGAAG

AAATACCAGGCGGAAATTGCCGAACTGGAAATGACCATCGACAATTTGAACCGGGCCAATTT

GGAAGCACAAAAGACGATCAAAAAACAAGCAGACCAACTGAGGGCTTTGCAAAGCAGCTTCG

AAGATTGCCAACGTCAACTGCAACAAACGCTCGACCAGTATGCAATTGCCCAGCGCAAGTTG

TCCGCTTTGTCGGCCGAATTGGAAGACTGCAAGAGTGCCTTGGACACGGCGATCCGGGTGCG

CAAACAAGCCGAAGCGGATCTGGAAGAAGCCCAGGGGAAAATTGCCGATTTGGTCAGCTTGA

ACAACAATTTGACCGCGATCAAGGGCAAATTGGAGACCGACTTGTCGACATGCCAAGCCGAT

TTGGACGAGACGACCAAGAACTGCGAGCCGCCGACGACCGTGCCAACCGAGCTCAAGAGAGA

TGCGGTCCGGGCTATGGAACAACTCCACGAAGAGCAGGAACATTCGATGAAAATCGACGCCA

TGCGCAAAGCGTTGGAAGAGCACGTCAAACAGTTGCAAGTGCAAATCCAAGAAGCCGAAGCG

GCAGCTCTGCTCGGTGGCAAGCGCGTCATTGCCAAACTCGAGACCAGGATTCGAGATTTGGA

AATGGCGTTGGATGAAGAATCGAGACGACACAAAGAAACCCAAGCCCTGCTTGCGCAAAAAGG

ACCGTCGCGTCAAGGAAATGCAAATGCAAGTGGACGAGGAGCACAAAAATTTCGTCATGGCC

CAAGATACTGCCGAACGATTGGCCGAAAAATTGAACATCTACAAGCGACAGTTGACTGAAGC

GGAATCGTTGACAATGCAAAATTTGCAACGTGTCCGTCGTTACCAGCACGAATTGGAAGATG

CCGAAGGTCGAGCCGAACAAGCCGAAAGTAGCTTGCACTTGATCCGTGCCAAGCATCGTTCT

TCCGTTTCCATGGGAAAAAGTCTCCTTCCAAGGTTTACGTGATGGAAGAAGGACATGAATA

T TGA TGAACAATACACTAATAATTAATTGCAGAAATTTATTTTTCTGGTGGAAGAGCAACCA

TGTTTTAGCTTCTTTCTTTCTCAATTTACTACCATAATAATAATAATATTATTAATATATTT

TTAATATACAATGATGATCAATTTTCTTTTTATTCAGAATGTAATCGCCAATTAGCTTTTTT

CTTTTTCTCTCTCAAAACAACCAACCAATCATAATTCTCTTCTTCATTCACGCCAACAAAAAA

AAAAAAAAAAAAAAAAAAAAA

Amino acid sequence within the open reading frame (SEQ ID NO: 2):

MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETE

RELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESED

SLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAA

EKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQ

LEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKS

KFDAEVSLHQEEVDDLRKKMIQKQAEYEEQIEIMLQKISQLEKAKSRLQSEVEVLIVDLEKA

QSTIAILERQKEQLERMVAEMKTRLDEVTQELEATQRELRATQAELQKMKHLYEKAVEQKEA

LARENKKLQDDLHEAKEALADANRKLHELDLENARLAGEIRELQIALKEAEAARRDAESRAQ

RAVAELQALRVEMERRLQEKEEEMEARKNMQFEIDRLAAAALADAEARMKAEISRLKKKYQA

EIAELEMTIDNLNRANLEAQKTIKKQADQLRALQSSFEDCQRQLQQTLDQYAIAQRKLSALS

AELEDCKSALDTAIRVRKQAEADLEEAQGKIADLVSLNNNLTAIKGKLETDLSTCQADLDET

TKELRAADDRANRAQQDAVRAMEQLHEEQEHSMKIDARKALEEHVKQLQVQIQEAEAAALL

GGKRVIAKLETRIRDLEMALDEESRRHKETQACLRKKDRRVKEMQMQVDEEHKNFVMAQDTA

ERLAEKLNIYKRQLTEAESLTMQNLQRVRRYQHELEDAEGRAEQAESSLHLIRAKHRSSVSM

GKSLSSKVYVMEEGHEY

Example 2 Preparation of Paramyosin Gene Recombinant Protein and Artificial Immune Rat Serum

1. Prokaryotic expression and purification of paramyosin gene

According to the Ts86cDNA sequence, a pair of primers were designed, the upstream primer was 5'-CGGGATCCATGTCTCTGTATCGCAGTCCCAGT-3' (SEQ ID NO: 3) (32nt in total, containing a BamHI restriction site), and the downstream primer was 5'-CGGAATTCATATTCATGTCCTTCTCCATCAC-3'( SEQ ID NO: 4) (32nt in total, containing an EcoRI restriction site). Using the Ts86cDNA clone obtained from the immune screening library as a template, PCR was performed, and the product was double-digested with BamHI and EcoRI, and then subcloned into the prokaryotic expression vector PET-28a(+) (Novagen Company). The correct recombinant plasmid was transformed into Escherichia coli BL21 strain (Novagen Company), and induced by 1.0mMIPTG at 150 rpm at 37°C for 3 hours to obtain the recombinant protein of the paramyosin gene (885 amino acids in all open reading frames), and the recombinant protein was passed through His After purification by -binding affinity chromatography column (Novagen Company), it was eluted by an electroeluter (BIO-RAD Company) to make the purity higher (Fig. 1).

2. The purified paramyosin gene recombinant protein was immunized with BALB/c mice (20 μg/only) by multi-point injection through the skin, and thereafter every 14 days with the same dose of antigen plus the same amount of incomplete Freund's adjuvant, boosted twice . After 5 weeks, the eyeballs were removed for blood collection. The antibody titer determined by ELISA is as high as 1:128000 or more.

Example 3 Immune properties of paramyosin gene recombinant protein

Enzyme-linked immunosorbent assay (ELISA) detection

1 μg of paramyosin gene recombinant protein (prepared in Example 2) was used as an antigen to coat a 96-well microtiter plate, the primary antibody was the serum to be tested, and the secondary antibody was the corresponding horseradish peroxidase-labeled (goat anti-human, goat Anti-rabbit or goat anti-pig) secondary antibody, normal human serum, normal rabbit serum, and normal pig serum were used as negative controls. According to the OD value of the serum sample to be tested is greater than 2.1 times of the negative control, it is a positive judgment result. After ELISA detection, the positive rate of trichinellosis patient serum (23 copies) was 86.96%, trichinellosis rabbit serum (2 copies) and trichinellosis pig serum (4 copies) were all positive, indicating that the recombination of the paramyosin gene The protein has specific antigenicity and is a diagnostic reagent with application potential.

2. Western blot analysis

Purified paramyosin gene expression protein (prepared in Example 2) (200ng) was transferred to PVDF membrane after SDS-PAGE electrophoresis, and was detected with ECL chemiluminescence kit (Amersham company), respectively with patient serum 1:200 1: 1:200 of infected pig serum, 1:1000 of infected rabbit serum, and 1:10000 of paramyosin gene recombinant protein artificially immunized mouse serum (prepared in Example 2) were used as the primary antibody. The results showed that there was an obvious Western blot band at about 110KDa (Fig. 2), indicating that the paramyosin gene recombinant protein could be the serum of patients infected with Trichinella spiralis, the serum of pigs infected with Trichinella spiralis, the serum of rabbits infected with Trichinella spiralis and paramyosin. The myosin gene recombinant protein was recognized by the sera of artificially immunized mice.

Embodiment 4 Protection experiment in animal body

Thirty BALB/c mice were randomly divided into 2 groups, 15 in each group. The paramyosin gene recombinant protein immunization group was subcutaneously injected with 20 μg of antigen (prepared in Example 2) plus an equal amount of Freund's complete adjuvant (FCA). Afterwards, the same dose of antigen plus the same amount of incomplete Freund's adjuvant was added every 14 days to boost twice. Ten days after the last injection, each mouse was challenged with 400 infective larvae of Trichinella spiralis. The control group was injected with the same dose of Tris-HCL, and the immune group was challenged with 400 Trichinella infective larvae at the same time. The mice were killed 45 days after the challenge, and the whole muscle was digested with pepsin to collect the larvae and counted. Through one-way analysis of variance, paramyosin gene recombinant protein immune group worm reduction rate (muscle larva reduction rate%=(control group muscle larva number-immune group muscle larva number)/control group muscle larva number * 100%) is 47%, compared with the control group, there was a significant difference (P<0.01) (Table 1).

Since the paramyosin gene recombinant protein in this experiment produced an effective worm reduction rate in animals, it can be used as a protective candidate antigen and applied to the immune prevention of trichinellosis.

Table 1 The protective results of paramyosin gene recombinant protein immunized BALB/c mice

All patents, patent applications and publications listed above are hereby incorporated by reference in their entirety.

It is not intended that the scope of the invention be limited to the particular embodiments described, which are presented as single illustrations of individual aspects of the invention. Functionally equivalent compositions and methods are within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the reading of the foregoing. Such modifications are intended to fall within the scope of the appended claims of the invention.

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Claims (15)

1. isolating polynucleotide molecule, its nucleotide sequence is selected from:

(A) sequence of open reading frame among sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1;

(B) with the protein of SEQ ID NO:1 coding identical sequence but because of the degeneracy of genetic code different nucleotide sequence on sequence; With

(C) with (A) or (B) described nucleotide sequence complementary nucleotide sequence.

2. polynucleotide molecule as claimed in claim 1 is characterised in that to have nucleotide sequence shown in the SEQ ID NO:1.

3. isolated polypeptide, its aminoacid sequence is shown in SEQ ID NO:2.

4. recombinant vectors wherein comprises claim 1 or 2 described polynucleotide molecules.

5. the recombinant vectors of claim 4, it is a recombinant expression vector.

6. the host cell that contains the recombinant vectors of claim 5.

7. produce the method for polypeptide, be included in and cultivate the described host cell of claim 6 under the condition that can make described expression of polypeptides and reclaim expressed polypeptide.

8. antibody, but its specificity is in conjunction with the described polypeptide of claim 3.

9. be used for preventing or treating the pharmaceutical composition of animal or human's trichinzation, contain the described polypeptide of claim 3 in the described pharmaceutical composition.

10. be used for diagnosing the diagnostic kit of animal or human's trichinzation, wherein contain claim 1 or 2 described polynucleotide molecules, the described polypeptide of claim 3 or the described antibody of claim 8.

11. claim 1 or 2 described polynucleotide molecules, the described polypeptide of claim 3 or the described antibody of claim 8 are used for diagnosing purposes in animal or human's the diagnostic reagent of trichinzation in preparation.

12. claim 1 or 2 described polynucleotide molecules or the described polypeptide of claim 3 or the described antibody of claim 8 are used for preventing or treating the purposes of the medicine of animal or human's trichinzation in preparation.

13. fusion rotein wherein comprises the described polypeptide of claim 3 and another merges object.

14. the fusion rotein of claim 13, wherein said fusion is to liking beta-galactosidase enzymes, glutathione-S-transferase or polyhistidine.

15. the purposes of the pharmaceutical composition of claim 9, the diagnostic kit of claim 10 or claim 11, wherein said animal is a livestock.

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