CN111718420A - A kind of fusion protein for gene therapy and its application - Google Patents

Abstract

The present invention relates to a fusion protein for gene therapy and its application. Specifically, the present invention provides an enhanced fusion protein. Compared with the wild-type gene editing protein, the enhanced fusion protein of the present invention can be used for gene therapy, and the effect of gene therapy is better than that of the wild-type gene editing protein.

Description

A kind of fusion protein for gene therapy and its application

technical field

The present invention relates to the field of biotechnology, in particular, to a fusion protein for gene therapy and its application.

Background technique

Gene editing technology is a technology that achieves gene manipulation by artificially breaking double-stranded DNA and using the repair mechanism of double-stranded DNA breaks. The existing gene editing technologies include ZFN, TALEN and CRISPR/Cas9 technology, among which CRISPR/Cas9 technology is the most widely used. CRISPR/Csa9 technology is an acquired immune mechanism derived from bacteria or archaea, which utilizes a single-stranded guide RNA (sgRNA) and Csa9 protein to generate DNA double-strand breaks at specific locations in the genome, and then pass endogenous non-identical DNA double-strand breaks. Source end joining (NHEJ) or homologous recombination (HDR) repair mechanisms to achieve knockout of target genes or insertion of specific genes or fragments.

Although CRISPR/Cas9 technology has extremely powerful functions, it also has its shortcomings, such as: 1. Off-target problem; 2. The limitation of PAM leads to limited target selection; 3. The editing efficiency of some new tools is generally low ( XCas9 and SpCas0-NG), and the current optimization and transformation of gene editing tools are mainly based on the improvement of the accuracy of the tool and the enhancement of the target range, and there is no extensive improvement of various gene editing tools for the gene editing tool itself. Methods.

Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defects and abnormal genes, so as to achieve therapeutic purposes. Traditional gene therapy is based on overexpressing the correct gene to compensate for the missing gene in the patient's body, thereby treating the disease. After the advent of gene editing technology, gene therapy has developed into a precise treatment, which can be precisely corrected from the DNA level. Healing genes to achieve the purpose of fundamentally curing diseases. At present, gene therapy based on gene editing technology is in full swing, and it has been reported that it has achieved promising results in the treatment of many genetic diseases, but for most genetic diseases, the efficiency of gene editing-mediated gene therapy It is still generally low, which greatly limits its wide application. Therefore, based on the newly invented enhanced gene editing tool, the present invention delivers the enhanced gene editing tool into the body of the disease model through a clinically feasible method, in an attempt to improve the efficiency of disease treatment.

Therefore, there is an urgent need in the field to develop a new method of gene therapy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new method of gene therapy.

The first aspect of the present invention provides the use of an active substance for preparing a drug or preparation for gene therapy, wherein the active substance is a fusion protein, or an encoding gene thereof, or an expression vector thereof, wherein the fusion protein The structure is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

Each "-" is independently a linking peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the drug is used to treat diseases that can be treated by gene therapy.

In another preferred embodiment, the diseases include genetic diseases, neurological diseases, cardiac system diseases, ophthalmic diseases, autoimmune diseases, hemophilia, blood diseases, and/or cancer.

In another preferred embodiment, the genetic disease is selected from the group consisting of liver metabolic disease, beta thalassemia, cardiovascular disease, Duchenne muscular dystrophy, hyperoxalate disease, or a combination thereof.

In another preferred embodiment, the liver metabolic disease is selected from the group consisting of familial hypercholesterolemia, hemophilia, phenylketonuria, tyrosinemia, hyperammonemia, or a combination thereof.

In another preferred embodiment, the disease includes a cell proliferative disorder.

In another preferred embodiment, the cell proliferative disorder is selected from the group consisting of lung cancer, colon cancer, rectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma tumor, gastric cancer, ovarian cancer, rheumatoid arthritis, or a combination thereof.

In another preferred example, the disease is a disease for an organ selected from the group consisting of liver, kidney, heart, pancreas, lung, bone, thymus, adrenal gland, oral cavity, pharynx, esophagus, stomach, brain, twelve Duct, jejunum, ileum, large intestine, anus, or a combination thereof.

In another preferred example, when the structure of the fusion protein is shown in formula I', C is none.

In another preferred embodiment, the non-peptide bond includes PEG.

In another preferred embodiment, the gene editing protein is selected from the group consisting of Cas9, Cas9a, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.

In another preferred embodiment, the gene editing protein includes wild-type or mutant gene editing protein.

In another preferred embodiment, the gene editing protein is selected from the group consisting of: Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, or its combination.

In another preferred embodiment, the amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1, 14 or 15.

In another preferred embodiment, the amino acid sequence of the base editor element is shown in SEQ ID NO.: 2 or 12.

In another preferred embodiment, the DNA double-stranded binding domain is a non-sequence-specific DNA double-stranded binding domain.

In another preferred embodiment, the DNA double-stranded binding domain is selected from the group consisting of HMG-D, Sac7d, or a combination thereof.

In another preferred embodiment, the DNA double-stranded binding domain includes a wild-type DNA double-stranded binding domain and a mutant DNA double-stranded binding domain.

In another preferred embodiment, the DNA double-stranded binding domain is derived from Drosophila or Archaea.

In another preferred embodiment, the amino acid sequence of the DNA double-stranded binding domain is shown in SEQ ID NO.: 10 or 11.

In another preferred embodiment, the length of the connecting peptide is 1-100aa, preferably 15-85aa, more preferably 25-70aa.

In another preferred embodiment, the connecting peptide is a sequence shown as Gly-Gly-Ser with n repeats, wherein n is 2-8, preferably n is 3-6.

In another preferred embodiment, the amino acid sequence of the connecting peptide is selected from the following group:

(1) a polypeptide whose amino acid sequence is shown in any of SEQ ID NO.:3-7;

(2) Passing through one or several amino acid sequences shown in any of SEQ ID NO.:3-7, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8 One, more preferably 1-3, most preferably 1 amino acid residues are formed by substitution, deletion or addition, and the amino acid shown in any one of SEQ ID NO.:3-7 having the function of (1) the polypeptide is formed Sequence of polypeptides derived from polypeptides.

In another preferred embodiment, the base editor element includes cytosine deaminase and adenine deaminase.

In another preferred embodiment, the cytosine deaminase includes Apobec1 and Apobec3A.

In another preferred embodiment, the adenine deaminase includes TadA.

In another preferred embodiment, the fusion protein has the amino acid sequence shown in any of SEQ ID NO.:8, 9, and 13.

In another preferred embodiment, the medicine includes:

(a) fusion protein, or its encoding gene, or its expression vector (vector); the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

each "-" is independently a linking peptide or a peptide bond or a non-peptide bond; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of freeze-dried preparation, liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the medicine is an injection dosage form.

In another preferred example, the drug further includes other drugs used for gene therapy.

In another preferred embodiment, the other drugs for gene therapy are selected from the group consisting of antisense nucleotide drugs, EDIT-101 drugs, CTX001, or a combination thereof.

In another preferred embodiment, the drug is a cell preparation.

In another preferred embodiment, the gene therapy is performed by gene editing.

In another preferred embodiment, the gene therapy is performed by gene editing reagents.

In another preferred embodiment, the gene editing reagent comprises a fusion protein, and the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

Each "-" is independently a linking peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the reagent also includes one or more reagents selected from the following group:

(a1) gRNA, crRNA, or a vector for producing said gRNA or crRNA;

(a2) Template for homology-directed repair: single-stranded nucleotide sequence or plasmid vector.

In another preferred embodiment, the gene therapy is carried out by the following method:

Gene editing of cells is performed in the presence of gene editing reagents.

In another preferred embodiment, the cells include human or non-human mammalian cells (such as primates or livestock).

In another preferred example, the cells include cancer cells or normal cells.

In another preferred embodiment, the cells are selected from the group consisting of kidney cells, liver cells, nerve cells, cardiac cells, epithelial cells, muscle cells, somatic cells, bone marrow cells, endothelial cells, or a combination thereof.

In another preferred example, the cells are selected from the group consisting of 293 cells, A549 cells, SW626 cells, HT-3 cells, PA-1 cells, or a combination thereof.

In another preferred embodiment, the cells include HEK293T.

In another preferred embodiment, the gene editing is performed in an in vitro reaction system.

In another preferred embodiment, in the in vitro reaction system, the content of the gene editing reagent is 100ng-700ng, preferably 200ng-600ng, more preferably 300ng-500ng.

In another preferred embodiment, the cells are in vitro cells.

A second aspect of the present invention provides a medicine box, comprising:

(a1) The first container, and the active substance in the first container, or the medicine containing the active substance, wherein the active substance is a fusion protein, or its encoding gene, or its expression vector, wherein the The structure of the fusion protein is shown in the following formula I or I':

C-A-L-B(I)

B-L-A-C(I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

Each "-" is independently a linking peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, the kit further includes:

(a2) A second container, and other drugs for gene therapy located in said second container, or drugs containing other drugs for gene therapy.

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, the medicine in the first container is a unilateral preparation containing the active substance.

In another preferred embodiment, the medicine in the second container is a single preparation containing other medicines for gene therapy.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of freeze-dried preparation, liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the medicine is an injection dosage form.

A third aspect of the present invention provides a pharmaceutical composition, comprising:

(a) active substance, the active substance is a fusion protein, or its encoding gene, or its expression vector (vector), wherein the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

each "-" is independently a linking peptide or a peptide bond or a non-peptide bond; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the expression vector includes a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred embodiment, the vector is selected from the group consisting of lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: freeze-dried preparation, liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection dosage form.

In another preferred embodiment, the pharmaceutical composition further includes other drugs for gene therapy.

In another preferred embodiment, the other drugs for gene therapy are selected from the group consisting of antisense nucleotide drugs, EDIT-101 drugs, CTX001, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition is a cell preparation.

The fourth aspect of the present invention provides a method for treating a disease, the method comprising the steps of: administering an active substance or the kit according to the second aspect of the present invention to an object in need, the active substance being a fusion protein, or Its encoding gene, or its expression vector, wherein the structure of the fusion protein is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

Each "-" is independently a linking peptide or a peptide bond or a non-peptide bond.

In another preferred embodiment, other active substances for treating diseases are used in combination before, at the same time and/or after the administration of the active substances or the kit according to the second aspect of the present invention.

It should be understood that, within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the editing efficiency for two different endogenous targets, indicating that the different connection methods of the DNA double-stranded binding domain and Cas9, and the linker of different lengths have different effects on the efficiency improvement. The HMG-D was selected comprehensively. The domains are preferably linked at the N-terminus of Cas9 by an L4-length linker. Namely: HMG-D-L4-Cas9, where H stands for HMG-D; S stands for Sac7d; L1-L5 stands for linkers of different lengths; mutH stands for mutated HMG-D (V32A and T33A are mutated, reducing binding activity); C stands for Cas9.

Figure 2 shows that the editing efficiency of HMG-D-L4-Cas9 at other endogenous targets can also be improved, and the efficiency is improved by > 20%, preferably > 40%, more preferably > 60% (such as 80%) %), up to 2 times, where H stands for HMG-D.

Figure 3 shows that the double-chain binding domain HMG-D can improve the efficiency of other sources of Cas9 proteins (eg: SaCas9) by more than 20%.

Figure 4 shows that the double-stranded binding domain HMG-D can increase the efficiency of non-Cas9 proteins (eg, AsCas12a) by 10-20%.

Figure 5 shows that the double-stranded binding domain HMG-D can improve the efficiency of epigenetic regulation tools (eg: CRISPR-VPR), and the efficiency can be increased by 2 times.

Figure 6 shows that the double-stranded binding domain HMG-D can improve the efficiency of the single base editing tool ABE, where H stands for HMG-D.

Figure 7 shows the successful targeted knockout of mouse PCSK9 gene by adeno-associated virus (AAV) delivery of SpCas9 fused to HMG-D and sgRNA targeting the therapeutic target of hypercholesterolemia (PCSK9) into mouse liver , the knockout efficiency was significantly higher than that of the SpCas9 group without HMG-D fusion. By monitoring the blood cholesterol levels of the treated mice over a long period of time, we found that the blood cholesterol levels of the treated mice were significantly lower than those of the untreated group, and the treatment group with HMG-D fusion was significantly lower than the treatment group without HMG-D fusion. This indicates that SpCas9 fused to a double-stranded binding protein can significantly help reduce blood cholesterol, which is beneficial for the treatment of cardiovascular disease and familial hypercholesterolemia. Figure 7A shows the editing efficiency of PCSK9 targets in mice , Figure 7B shows the decrease in blood cholesterol levels in the edited mice.

Figure 8 shows that the C-to-T transition of the -114 and -115 sites was achieved by expressing the protein fused to the base editing tool (hA3A-CBE) of the double-stranded binding protein (HMG-D), and electrotransferring sgRNA into hematopoietic stem cells. mutation. The results showed that the editing efficiency of the hA3A-CBE group fused with HMG-D was significantly higher than that of the hA3A-CBE group without HMG-D fusion; at the RNA level, the expression of γ-globin in the hA3A-CBE group fused with HMG-D was also significantly higher. Also at the protein level, the expression of globin in the hA3A-CBE group fused with HMG-D was also significantly higher than that in the hA3A-CBE group without HMG-D fusion; Figure 8A shows the editing efficiency of hematopoietic stem cell targets, Figure 8B The expression of HBG gene mRNA after the edited hematopoietic stem cells is shown, and FIG. 8C shows the expression of γ-globin after the edited hematopoietic stem cells are differentiated.

Detailed ways

After extensive and in-depth research, the present inventors unexpectedly obtained an enhanced fusion protein. Compared with the wild-type gene editing protein, the enhanced fusion protein of the present invention can significantly improve the gene editing efficiency in vivo or in vitro, and the present invention also unexpectedly finds that the gene editing protein and DNA double-stranded binding domain, optional base The fusion protein formed by the base editor element and optional linking peptide can significantly improve the gene editing efficiency (increase ≥20%, such as 80%, even up to 2 times), and the fusion protein of the present invention can be used for gene therapy. On this basis, the present inventors have completed the present invention.

the term

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

The term "about" can refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including (including)" can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

Sequence identity (or homology) is determined by comparison along a predetermined window of comparison (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference ) is determined by comparing the two aligned sequences and determining the number of positions where the same residue occurs. Typically, this is expressed as a percentage. Measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

As used herein, the term "EDIT-101 drug" belongs to a gene therapy drug, which is a class of cells, specifically, EDIT-101 is a drug for the treatment of inherited retinal degeneration diseases (LCA10 disease) using CRISPR gene editing technology, EDIT- 101 is administered by subretinal injection, delivering the gene editing system directly into photoreceptor cells to achieve therapeutic effects.

As used herein, the term "CTX001" belongs to a gene therapy drug, which is a type of cell, specifically, CTX001 is based on CRISPR gene editing technology to achieve therapeutic purposes by cutting the BCL11A gene of β-thalassemia patients.

wild-type gene editing protein

As used herein, "wild-type gene editing protein" refers to a naturally occurring, unmodified gene editing protein whose nucleotides can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR) ), etc., the amino acid sequence of which can be derived from the nucleotide sequence. Sources of the wild-type gene editing protein include (but are not limited to): Streptococcus pyogenes, Staphylococcusaureus, Acidaminococcus sp, Lachnospiraceaebacterium.

In a preferred example of the present invention, the amino acid sequence of the wild-type gene editing protein is shown in SEQ ID NO.: 1 or 14 or 15.

In a preferred embodiment of the present invention, the gene editing proteins include, but are not limited to, Cas9, Cas9a, Cas12, Cas12a, Cas12b, Cas13, and Cas14.

DNA double-strand binding domain

As used herein, the term "DNA double-stranded binding domain" is a DNA double-stranded binding domain that is not sequence specific. Compared with the sequence-specific DNA double-strand binding domain, the non-sequence-specific DNA double-strand binding domain of the present invention is not limited by the DNA sequence, and can theoretically bind to any DNA sequence, therefore, it can be applied to any position binding of DNA.

A preferred DNA double-stranded binding domain sequence is shown in SEQ ID NO.:10 or 11.

base editor

Any of the base editors provided herein are capable of modifying specific nucleotide bases without producing a significant proportion of insertions/deletions. As used herein, "insertion/deletion" refers to the insertion or deletion of nucleotide bases within a nucleic acid. Such insertions or deletions can result in frameshift mutations within the coding region of the gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g., mutate or deaminate) specific nucleotides within a nucleic acid without generating substantial insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of producing a greater proportion of intended modifications (e.g., point mutations or deaminations) relative to insertions/deletions.

Any of the base editors of the present invention are capable of efficiently generating intended mutations, such as point mutations, in nucleic acids (eg, nucleic acids within a genome) without generating large numbers of unintended mutations, such as unintended point mutations.

In the present invention, base editors include cytosine deaminase and adenine deaminase, and other types of base editors are also within the protection scope of the present invention as long as they have the functions of the base editors of the present invention.

In the present invention, the structure after fusion of gene editing protein and base editor is called ABE or CBE, wherein, ABE is the structure after fusion of gene editing protein and adenine deaminase, CBE is gene editing protein and cell Structure of pyrimidine deaminase fusion.

The sequence of a preferred base editor is shown in SEQ ID NO.: 2 or 12.

fusion protein

As used herein, "fusion protein of the present invention", or "polypeptide" refers to the fusion protein of the second aspect of the present invention. The structure of the fusion protein of the present invention is shown in the following formula I or I':

C-A-L-B (I)

B-L-A-C (I’)

In the formula,

A is a gene editing protein,

B is the DNA double-stranded binding domain,

C is an optional base editor element;

L is no or linking peptide,

Each "-" is independently a linking peptide or a peptide bond or a non-peptide bond.

In the present invention, the length of the connecting peptide has an effect on the activity of the fusion protein, and the preferred length of the connecting peptide is 1-100aa, preferably 15-85aa, more preferably 25-70aa.

A preferred linker peptide is shown in SEQ ID NO.: 3-7.

As used herein, the term "fusion protein" also includes variant forms set forth in SEQ ID NO.: 8, 9, or 13 having the above-mentioned activities. These variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (usually 1-2, more preferably 1) amino acids, as well as C-terminal and/or N-terminal additions or One or several (usually within 3, preferably within 2, more preferably within 1) amino acids are deleted. For example, in the art, substitutions with amino acids of similar or similar properties generally do not alter the function of the protein. As another example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as nonlinear polypeptides (eg, cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the fusion proteins of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with a substituent group in each amino acid residue, or (iii) a polypeptide formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide formed by fusing this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretory sequence or a tag sequence such as 6His). Such fragments, derivatives and analogs are well known to those skilled in the art in light of the teachings herein.

A preferred class of active derivatives means that compared with the amino acid sequence of formula I, at most 3, preferably at most 2, more preferably at most 1 amino acid are replaced by amino acids with similar or similar properties to form a polypeptide. These conservatively variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

initial residue representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The present invention also provides analogs of the fusion proteins of the present invention. The differences between these analogs and the polypeptides shown in SEQ ID NO.: 8 or 9 or 13 may be differences in amino acid sequences, or differences in modified forms that do not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of the polypeptide in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, in formula I, A is a gene editing protein, B is HMG-D or Sac7d, C is adenine deaminase or cytosine deaminase or none, L is L1 or L2 or L3 or L4 or L5 or none.

In a preferred embodiment, in formula I, A is a gene editing protein, B is HMG-D, C is either adenine deaminase or cytosine deaminase or none, and L is L4 or L5.

In a preferred embodiment, the fusion protein of the present invention may also include two or more of the A, B, C, and L elements in formula I.

In a preferred embodiment, in formula I, A is a gene editing protein, B is HMG-9, C is none, and L is L4.

In a preferred embodiment, in formula I, A is a gene editing protein, B is HMG-D, C is adenine deaminase, and L is L5.

In a preferred embodiment, in formula I, A is a gene editing protein, B is HMG-D, C is cytosine deaminase, and L is L5.

In a preferred embodiment, the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.: 8, 9 or 13.

adeno-associated virus

Because adeno-associated virus (AAV) is smaller than other viral vectors, has no pathogenicity, and can transfect both dividing and undivided cells, gene therapy methods for genetic diseases based on AAV vectors have been favored. Widespread concern.

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependovirus of the family Parvoviridae, and is a class of single-stranded DNA-deficient viruses with the simplest structure found so far. virus) involved in replication. It encodes the cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in viral replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus, due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and time to express foreign genes in vivo It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have accumulated a lot of data. In medical research, rAAV is used for gene therapy research of various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer carrier, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice, etc.

In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can integrate into the genomes of the cells they infect in a stable and site-specific manner. They are capable of infecting a wide range of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the viral origin of replication. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Adeno-associated virus of any serotype is suitable. Methods for purifying vectors can be found, for example, in US Pat. Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described (see, eg, International Patent Application Publication Nos. WO91/18088 and WO93/09239; US Patent Nos. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by genes of interest, as well as these constructs in vitro (into cultured cells) or in vivo (directly into an organism) ) use to transport the gene of interest. Replication-deficient recombinant AAVs can be prepared by co-transfection of a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeats (ITRs) into a cell line infected with a human helper virus (eg, adenovirus). region, and plasmids carrying the AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is encapsidated into a virion (eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV virions of AAV16). Accordingly, the present disclosure includes recombinant virions (recombinant because they comprise recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and described in U.S. Patent No. 6,596,535.

Expression Vectors and Host Cells

The present invention also relates to vectors comprising the polynucleotides of the present invention, as well as host cells genetically engineered with the vectors of the present invention or fusion protein coding sequences of the present invention, and methods for recombinantly producing the polypeptides of the present invention.

The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally there are the following steps:

(1). Use the polynucleotide (or variant) encoding the fusion protein of the present invention of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Separation and purification of proteins from culture medium or cells.

In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmids and vectors can be used as long as they are replicable and stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, promoter, marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the fusion proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the bacteriophage lambda PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the reverse The LTRs of transcription viruses and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate host cells so that they can express the protein.

Host cells can be prokaryotic cells (such as E. coli), or lower eukaryotic cells, or higher eukaryotic cells, such as yeast cells, plant cells, or mammalian cells (including humans and non-human mammals). Representative examples are: Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, a yeast cell (such as Pichia pastoris, Kluyveromyces, or a combination thereof is selected; preferably, the yeast cells include: Kluyveromyces, more preferably Markus Kluyveromyces, and/or Kluyveromyces lactis) as host cells.

When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on a promoter to enhance transcription of a gene. Illustrative examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers, among others.

It will be clear to those of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention. The medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Gene therapy

Gene therapy for genetic diseases refers to the application of genetic engineering technology to introduce normal genes into patient cells to correct defective genes and cure diseases. The way of correction can be either to repair the defective gene in situ, or to transfer a functional normal gene into a certain part of the cell genome to replace the defective gene to play a role. A gene is a basic functional unit that carries biological genetic information, and is a specific sequence located on a chromosome. The introduction of exogenous genes into biological cells must rely on certain technical methods or vectors. The methods of gene transfer are divided into biological methods, physical methods and chemical methods. Adenoviral vector is one of the most commonly used viral vectors for gene therapy. Gene therapy is mainly to treat diseases that pose a serious threat to human health, including, but not limited to: genetic diseases (such as hemophilia, cystic fibrosis, familial hypercholesterolemia, etc.), malignant tumors, cardiovascular diseases, Infectious diseases (such as AIDS, rheumatoid, etc.). Gene therapy is a high-tech biomedical technology that introduces normal human genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or play a therapeutic role, so as to achieve the purpose of treating diseases. Gene therapy is different from conventional treatments: in general, the treatment of a disease targets various symptoms caused by abnormal genes, while gene therapy targets the root cause of the disease - the abnormal gene itself. Target cells for gene therapy include, but are not limited to, somatic cells, bone marrow cells, hepatocytes, neural cells, endothelial cells, and muscle cells.

In the present invention, efficient gene editing (including gene insertion, replacement, etc.) is performed on the target gene through gene therapy, so as to restore the normal expression of the gene or enhance the expression of the gene, so as to treat related diseases.

genetic disease

In the present invention, hereditary diseases refer to a class of diseases caused by mutations in genomic DNA.

In a preferred embodiment, the genetic diseases of the present invention include, but are not limited to, liver metabolic diseases (such as familial hypercholesterolemia, hemophilia, phenylketonuria, tyrosinemia, hyperammonemia) Symptoms, etc.), beta thalassemia, cardiovascular disease, Duchenne muscular dystrophy, hyperoxalic acidosis.

liver metabolic disease

The liver is the most important metabolic organ in the body, and various substances are metabolized directly or indirectly through the liver. Therefore, hepatocytes express various metabolic enzymes to perform metabolic functions. Once the gene of a certain (or some) enzyme is mutated and affects the expression of the enzyme or its biological function, the upstream metabolites cannot be metabolized in time or the important downstream metabolites cannot be replenished, resulting in irreversible metabolic disorders. , leading to the occurrence of disease.

In addition, the level of cholesterol is often an important indicator for evaluating cardiovascular disease and familial hypercholesterolemia. Cholesterol concentration in the blood increases or accumulates in blood vessels, which will lead to the occurrence of related cardiovascular diseases. The body's metabolism of cholesterol is metabolized by the liver, so the lack or reduction of the ability of cholesterol metabolism in the liver will lead to high blood cholesterol concentration, leading to the occurrence of diseases.

The study of the present invention shows that elevated blood cholesterol is an important factor in the occurrence of cardiovascular disease, and the present invention finds that reducing cholesterol content by drug therapy or gene therapy is the main strategy for the treatment of cardiovascular disease and familial hypercholesterolemia .

beta thalassemia

β-thalassemia is a blood-based genetic disease caused by mutation of the β-globin gene. When the mutation of the β-globin gene results in the inability to express or lack of function of the β-strain protein, it will lead to premature apoptosis of red blood cell precursors and failure to form. . At present, the expression of γ-globin can be reactivated by gene editing strategy to make up for the loss of β-globin, and the production of γ-globin can also form a heterotetramer with α-globin, namely fetal hemoglobin HBF, which is helpful for the treatment of β-globin. Thalassemia. However, γ-globin is mainly expressed in infancy, and then is inhibited by various transcriptional regulators and cannot be expressed. At present, it is increasingly found that certain point mutations can reactivate the expression of γ-globin.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the fusion protein of the present invention can significantly improve the gene editing efficiency in vivo or in vitro.

(2) The present invention finds for the first time that the fusion protein of the present invention can significantly improve the gene editing efficiency in vivo or in vitro, and the improvement range is ≥20%, preferably, >40%, more preferably, >60% (such as 80%) , up to 2 times.

(3) The present invention discovers for the first time the mRNA of the in vitro transcription-enhanced gene editing tool, which improves the success of animal model construction.

(4) The present invention uses the AAV virus packaged with the enhanced gene editing tool, expresses the protein of the enhanced gene editing tool, and improves the treatment effect of the disease.

(5) The present invention utilizes for the first time to improve gene editing efficiency by fusing double-stranded DNA binding domains.

(6) The present invention firstly screened and found a double-stranded DNA binding domain (such as HMG-D) that can effectively improve the efficiency of gene editing, and its very excellent fusion mode.

(7) The present invention finds for the first time that the double-stranded DNA binding domain of the present invention can widely improve the gene editing efficiency of various gene editing tools.

(8) The enhanced gene editing tool of the present invention can improve the success rate of animal model construction and the efficiency of gene therapy.

(9) The enhanced gene editing tool of the present invention can be used for gene therapy.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer the proposed conditions. Percentages and parts are weight percentages and parts unless otherwise specified.

Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

general approach

Method 1: (applicable to Example 1, Example 2, Example 3, Example 4 and Example 6)

1. The density of HEK293T cells in 24-well plate grows to 60-70%. Equimolar amount of plasmid is transfected into cells by transfection reagent PEI, and the medium is changed for 8-10 hours. After cell culture for a period of time (Cas9 system is cultured for 72 hours, The base editing system was cultured for 120 hours), the cells were harvested, and the genome was extracted.

2. Design suitable primers to amplify a sequence of 150-180 bp around the target, perform Hitom library construction, deep sequencing, and analyze and calculate the editing efficiency.

Method 2: (applicable to the animal model construction of Example 7)

1. In vitro transcription-enhanced gene editing tool mRNA and corresponding target sgRNA.

2. The mouse embryos were injected with mRNA and sgRNA, transplanted into surrogate mother mice, F0 generation mice were obtained, the genes were identified, the genotype mutation rate of the mice was counted, and the success rate of model construction was calculated.

Method 3: (applicable to Example 5)

1. The density of HEK293T cells in 24-well plate grows to 60-70%. Equimolar amount of plasmid is transfected into cells by transfection reagent PEI, and the medium is changed for 8-10 hours. After cell culture for a period of time (Cas9 system is cultured for 72 hours, The base editing system was cultured for 120 hours), cells were harvested, RNA was extracted, and cDNA was obtained by reverse transcription.

2. Design appropriate primers for relative quantitative analysis by RT-qPCR to compare the efficiency of target gene transcriptional activation.

Method 4: (applicable to the in vivo gene therapy of Example 8)

1. Adeno-associated virus (AAV) packaging enhanced gene editing tool and sgRNA for therapeutic gene, or homology repair template.

2. By intravenous injection (tail vein) or local injection (intramuscular, etc.) of the packaged AAV to the disease animal model (mice or rat), and simultaneously inject the control virus.

3. Regularly observe, detect the phenotype of the animal model in the treatment group and the animal model in the control group, and evaluate the treatment efficiency.

Method 5: (applicable to the cell gene therapy of Example 9)

1. In vitro expression of proteins of enhanced gene editing tools, and synthesis of sgRNAs of related targets.

2. Electroporation of protein and RNA complexes (RNP) into cells.

3. Genotyping and functional testing.

Example 1 Screening for enhanced gene editing tools

Different kinds of double-stranded DNA-binding domains (HMG-D and Sac7d) were synthesized respectively, and 5 kinds of linkers of different lengths (L1, L2, L3, L4 and L5) were designed and fused to the N- and C-termini of Cas9, The two endogenous targets (VEGF and HBG1/2) were compared, and the enhanced gene editing tools obtained by this series of optimizations were scored according to the statistics of the results (on the basis of good results). (Not good: A; Not good: AA; Good: AAA; Good: AAAA; Very good: AAAAA), the results are as follows: (Note: C means Cas9; L1-L5 means different linkers; H means HMG -D domain; S for sac7d.)

A: S-L1-C, H-L1-C

AA: S-L2-C, S-L3-C, S-L4-C, H-L2-C

AAA: H-L4-C-L4-S, H-L4-C-L5-S

AAAA:H-L3-C,C-L4-S,C-L5-S,C-L4-H,H-L4-C-L4-H,H-L4-C-L5-H,H-L4- H-L4-C, C-L5-H-L5-H

AAAAA:H-L4-C,C-L5-H

Comprehensive statistics, it is found that the effect of HMG-D is very good, and the linker of L4 and L5 length (32 and 64 amino acids) is the optimal linker. By comparing the N-terminal and C-terminal connections, it is found that under the optimal linker conditions, The effect of N-terminal and C-terminal connection is very good, so the inventors obtained an enhanced gene editing tool, that is, HMG-D is fused to the N-terminal or C-terminal of Cas9 through the linker of L4 or L5, which is a very good fusion method. (figure 1).

Similarly, this enhanced gene editing tool that enhances gene editing efficiency through the fusion of double-stranded DNA-binding domains can also be analogized to other types of double-stranded DNA-binding domains, such as widely used zinc finger proteins (ZFPs), other DNA binding domains of transcription factors and HMG-D or Sac7d from other species, etc. The present invention can also improve the gene editing efficiency through these double-stranded DNA binding domains. Therefore, the most important thing of the present invention is to find that the double-stranded DNA binding domain fusion gene editing tool can improve the gene editing efficiency, and the double-stranded DNA is preferably used. DNA binding domain HMG-D.

Comparative example 1 does not work well after fusion with a non-DNA binding domain

In order to further verify that the fusion of the DNA-binding domain can improve the gene editing efficiency, the DNA-binding domain was changed to an unrelated protein such as GFP protein (a non-DNA-binding domain), and it was found that the fusion of the GFP protein could not improve the gene editing efficiency. (figure 1). At the same time, the HMG-D domain was mutated to destroy its DNA binding ability, and a 3 amino acid mutated HMG-D domain (mutHMG-D, mutH for short) was constructed. Through experimental comparison, the mutHMG-D domain also Does not improve gene editing efficiency (Figure 1).

Comparative example 2 DNA binding domains other than HMG-D and Sac7d do not work well

In order to broaden the scope of DNA-binding domains, some single-stranded DNA-binding domains (such as Rad51) were tested, and fusion of Rad51 also failed to improve gene editing efficiency (Figure 1). Therefore, the results show that the above-screened double-stranded DNA binding domains of HMG-D and Sac7d of the present invention are very effective.

Example 2 Improving the gene editing efficiency of SpCas9

The obtained enhanced gene editing tool (that is, the fusion protein of the present invention, such as HMG-D-L4-SpCas9) was further compared in terms of effects on more endogenous targets, and 293T cells were transfected by equimolar ratio. , found that compared with SpCas9 in the compared targets, the editing efficiency of the fusion protein of the present invention was improved by more than 20% (or 60%, or 80%), up to 2 times ( FIG. 2 ).

Example 3 Improving the gene editing efficiency of Cas9 derived from other species (eg SaCas9)

By constructing the HMG-D-L4-SaCas9 expression vector and transfecting 293T cells with an equimolar ratio targeting endogenous targets, it was found that the editing efficiency of SaCas9 after fusion with HMG-D also had a similar effect. (image 3)

Example 4 Improving the gene editing efficiency of other non-Cas9 proteins (such as AsCas12a)

By constructing the HMG-D-L4-AsCas12a expression vector and transfecting 293T cells with an equimolar ratio targeting endogenous targets, it was found that the editing efficiency of AsCas12a after fusion with HMG-D also had a similar effect. (Figure 4)

Example 5 Improving the transcriptional activation efficiency of transcriptional regulatory tools (CRISPR/Cas9a)

By constructing HMG-D-L4-dCas9-VPR expression vector and transfecting 293T cells with equimolar ratio for endogenous targets, it was found that dCas9-VPR after fusion with HMG-D has similar transcriptional activation efficiency to endogenous genes. effect enhancement. (Figure 5)

Example 6 Improving the editing efficiency of base editors

Since the base editor is a fusion of cytosine deaminase (CBE) and adenine deaminase (ABE) at the N-terminus of Cas9, the C-terminus of Cas9 of the base editor passes through L5 linker to fuse HMG-D to develop an enhanced single-base editing tool. Through the comparison of endogenous targets, the editing efficiency of base editors (such as ABE) after fusion with HMG-D has been greatly improved, such as an increase of >1.5-2 times (Figure 6).

The improvement in editing efficiency of fusion proteins fused with other types of base editors was similar or comparable to that of fusion proteins fused with ABE.

Example 7 Improve the success rate of animal model construction

The success rate of animal model construction is increased by in vitro transcription of the mRNA of enhanced gene editing tools.

Example 8 By delivering the enhanced gene editing tool of fusion double-stranded binding protein (HMG-D) to animals, the in vivo editing efficiency of Cas9 can be increased and the efficiency of gene therapy can be improved

Take, for example, a gene therapy approach that lowers blood cholesterol levels in a mouse model.

method:

1. The adeno-associated virus of the AAV8 serotype of SpCas9 fused with HMG-D, and the sgRNA targeting the liver gene PCSK9, were purified by ultracentrifugation, concentrated, and titered by qPCR; 2, 6-8 Mice of appropriate age were injected with the mixture of the two viruses through the tail vein, and the control group was also set up to be injected with the virus mixture of wild-type SpCas9 and sgRNA; 3. Surgery was performed 15 and 35 days after virus injection, and liver tissue was collected under anesthesia. Methods The genome was extracted, and the gene editing efficiency was compared by next-generation sequencing; 4. Starting from 7 days after injection, blood was collected every 7 days, and the total blood cholesterol content was detected by enzyme labeling method; 5. Finally, the wild-type gene editing tools were uniformly compared. and enhanced gene editing tools for efficiency and disease treatment outcomes.

The inventors delivered HMG-D-fused SpCas9 and sgRNA targeting hypercholesterolemia therapeutic target (PCSK9) into mouse liver by adeno-associated virus (AAV), and successfully knocked out mouse PCSK9 gene. The removal efficiency was significantly higher than that of the SpCas9 group without HMG-D fusion (Fig. 7A). By long-term monitoring of the blood cholesterol levels of the treated mice, it was found that the blood cholesterol levels of the treated mice were significantly lower than those of the untreated group, and the treatment group with HMG-D fusion was significantly lower than the treatment group without fusion HMG-D (Figure 7B).

The results show that SpCas9 fused with double-chain binding protein can significantly help to reduce blood cholesterol, which is beneficial to the treatment of cardiovascular disease and familial hypercholesterolemia. At the same time, the delivery of enhanced gene editing tools through AAV significantly enhances the editing efficiency in vivo, which also provides conditions for the treatment of more diseases.

Example 9 The enhanced gene editing tool of double-stranded binding protein (HMG-D) is electrofused into therapeutic cells, which can improve the editing efficiency of therapeutic cells in vitro, which is beneficial to the treatment of diseases

Take hematopoietic stem cell therapy for beta thalassemia as an example.

The inventors used base editing tools to generate therapeutic point mutations in hematopoietic stem cells, thereby activating the expression of gamma globin and treating beta thalassemia. Mutations of C to T at -114 and -115 sites were achieved by expressing a protein fused to a base editing tool (hA3A-CBE) of double-stranded binding protein (HMG-D) and electroporating sgRNA into hematopoietic stem cells.

method:

1. Prokaryotic expression and purification of hA3A-CBE protein fused with HMG-D; 2. Synthesis of sgRNA of target gene; 3. Hematopoietic stem cells were isolated, cultured, incubated with protein and RNA, and electroporated into hematopoietic stem cells to form RNP; 4. Electroporated 72 After hours, collect some cells for genome extraction, PCR, sequencing, analysis and comparison of the mutation efficiency of target genes; 5. The remaining hematopoietic stem cells are differentiated into erythrocytes by conventional methods, and the expression level of γ-globin in erythrocytes is detected by RT-qPCR 6. Isolate the protein in red blood cells and perform chromatography to characterize and quantify the expression of γ-globin; 7. Overall compare the therapeutic effect of the enhanced editing tool on β-thalassemia.

The results showed that the editing efficiency of the hA3A-CBE group fused with HMG-D was significantly higher than that of the hA3A-CBE group without HMG-D fusion (Figure 8A); The expression of HMG-D was also significantly increased (Fig. 8B); also at the protein level, the expression of globin in the hA3A-CBE group fused with HMG-D was also significantly higher than that in the hA3A-CBE group without HMG-D (Fig. 8C). Therefore, the strategy of electrotransforming enhanced gene editing tool proteins can greatly improve the editing efficiency of cells, which is of great significance for various cell therapies (HSC, CART, etc.) in the future.

discuss

In general, the present invention is based on the preclinical development of gene therapy for related diseases based on enhanced gene editing tools. It is implemented by two of the most important gene therapy strategies in the field of gene therapy today, namely: in vivo therapy by in vivo delivery of gene therapy tools and in vitro therapy by isolating patient cells for gene therapy and then transplanting them into patients.

First, most liver metabolic diseases can be treated by virus-targeted delivery of gene therapy drugs. The present invention takes cholesterolemia caused by cholesterol metabolism disorder as an example. Delivery of enhanced gene editing tools through AAV can greatly reduce blood pressure in the body. Cholesterol content is not only beneficial for the treatment of cholesterolemia, but also has a great guiding role for most liver metabolic diseases, and can be extended to general hereditary diseases.

Secondly, the example of the present invention for the treatment of β-thalassemia based on hematopoietic stem cells proves that the enhanced gene editing tool of the present invention can greatly improve the gene editing efficiency of hematopoietic stem cells and improve the effect of the treatment of β-thalassemia. Of course, This can also be extended to more cell therapy, such as immune cells (T cells) for tumor therapy applications, mesenchymal stem cells, etc.

Finally, through the study of two representative cases of in vivo treatment and in vitro treatment, it can be seen that the enhanced gene editing tool of the present invention is superior to the wild type gene editing tool, and shows great prospects in the gene therapy of diseases. And the enhanced gene editing tool of the present invention is not limited to these two diseases, but also includes all diseases that can be treated by the wild-type gene editing tool.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the limited scope of the appended claims of the application.

sequence listing

<110> East China Normal University

Shanghai Bangyao Biotechnology Co., Ltd.

<120> A fusion protein for gene therapy and its application

<130> P2019-2040

<150> CN 201910209634.0

<151> 2019-03-19

<160> 15

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660 665 670

Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys

675 680 685

Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile

785 790 795 800

Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser

805 810 815

Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

820 825 830

Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

835 840 845

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp

850 855 860

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

865 870 875 880

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

885 890 895

Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu

900 905 910

Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala

915 920 925

Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg

930 935 940

Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu

945 950 955 960

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

965 970 975

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

980 985 990

Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp

995 1000 1005

Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His

1010 1015 1020

Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr

1025 1030 1035 1040

Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp

1045 1050 1055

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr

1060 1065 1070

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

1075 1080 1085

Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr

1090 1095 1100

Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala

1105 1110 1115 1120

Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val

1170 1175 1180

Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys

1185 1190 1195 1200

Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1205 1210 1215

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp

1220 1225 1230

Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly

1235 1240 1245

Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu

1250 1255 1260

Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His

1265 1270 1275 1280

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

1285 1290 1295

Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1365 1370 1375

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1380 1385 1390

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys

1395 1400 1405

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1410 1415 1420

<210> 2

<211> 1792

<212> PRT

<213> Artificial sequence

<400> 2

Met Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu

1 5 10 15

Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu

20 25 30

Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile

35 40 45

Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His

50 55 60

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

65 70 75 80

Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met

85 90 95

Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly

100 105 110

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

115 120 125

His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu

130 135 140

Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg

145 150 155 160

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

165 170 175

Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

180 185 190

Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu

195 200 205

Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala

210 215 220

Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val

225 230 235 240

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

245 250 255

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

260 265 270

Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr

275 280 285

Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile

290 295 300

Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly

305 310 315 320

Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu

325 330 335

Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr

340 345 350

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

355 360 365

Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu

370 375 380

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser

385 390 395 400

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

405 410 415

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

420 425 430

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

435 440 445

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

450 455 460

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

465 470 475 480

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

485 490 495

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

500 505 510

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

515 520 525

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

530 535 540

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

545 550 555 560

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

565 570 575

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

580 585 590

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

595 600 605

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

610 615 620

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

625 630 635 640

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

645 650 655

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

660 665 670

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

675 680 685

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

690 695 700

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

705 710 715 720

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

725 730 735

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

740 745 750

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

755 760 765

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

770 775 780

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

785 790 795 800

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

805 810 815

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

820 825 830

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

835 840 845

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

850 855 860

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

865 870 875 880

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

885 890 895

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

900 905 910

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

915 920 925

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

930 935 940

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

945 950 955 960

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

965 970 975

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

980 985 990

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

995 1000 1005

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

1010 1015 1020

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

1025 1030 1035 1040

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

1045 1050 1055

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

1125 1130 1135

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

1140 1145 1150

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

1155 1160 1165

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

1170 1175 1180

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

1185 1190 1195 1200

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

1205 1210 1215

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

1220 1225 1230

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

1235 1240 1245

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1250 1255 1260

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1265 1270 1275 1280

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1285 1290 1295

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1300 1305 1310

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

1315 1320 1325

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1330 1335 1340

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

1345 1350 1355 1360

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1365 1370 1375

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

1380 1385 1390

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1395 1400 1405

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1410 1415 1420

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1425 1430 1435 1440

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1445 1450 1455

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1460 1465 1470

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1475 1480 1485

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1490 1495 1500

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

1505 1510 1515 1520

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1525 1530 1535

Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1540 1545 1550

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1555 1560 1565

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

1570 1575 1580

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1585 1590 1595 1600

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1605 1610 1615

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1620 1625 1630

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1635 1640 1645

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

1650 1655 1660

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1665 1670 1675 1680

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

1685 1690 1695

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1700 1705 1710

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys

1715 1720 1725

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu

1730 1735 1740

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1745 1750 1755 1760

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

1765 1770 1775

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

1780 1785 1790

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<400> 3

Gly Gly Ser Gly Gly Ser Gly Gly Ser

1 5

<210> 4

<211> 18

<212> PRT

<213> Artificial sequence

<400> 4

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 5

<211> 16

<212> PRT

<213> Artificial sequence

<400> 5

Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1 5 10 15

<210> 6

<211> 32

<212> PRT

<213> Artificial sequence

<400> 6

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1 5 10 15

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

<210> 7

<211> 48

<212> PRT

<213> Artificial sequence

<400> 7

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1 5 10 15

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Ser Glu Thr Pro Gly Thr

20 25 30

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

35 40 45

<210> 8

<211> 1567

<212> PRT

<213> Artificial sequence

<400> 8

Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu

1 5 10 15

Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val

20 25 30

Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp

35 40 45

Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp

50 55 60

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

65 70 75 80

Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys

85 90 95

Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu

100 105 110

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

115 120 125

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

130 135 140

Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp

145 150 155 160

Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val

165 170 175

Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu

180 185 190

Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr

195 200 205

Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His

210 215 220

Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu

225 230 235 240

Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr

245 250 255

Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu

260 265 270

Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe

275 280 285

Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn

290 295 300

Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His

305 310 315 320

Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu

325 330 335

Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu

340 345 350

Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe

355 360 365

Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile

370 375 380

Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser

385 390 395 400

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

405 410 415

Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr

420 425 430

Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr

485 490 495

Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His

500 505 510

Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu

515 520 525

Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly

530 535 540

Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys

545 550 555 560

Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu

565 570 575

Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser

580 585 590

Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg

595 600 605

Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu

610 615 620

Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg

625 630 635 640

Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile

645 650 655

Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln

660 665 670

Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu

675 680 685

Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr

690 695 700

Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro

705 710 715 720

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

725 730 735

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

740 745 750

Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp

755 760 765

Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile

770 775 780

Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu

785 790 795 800

Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu

805 810 815

Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys

820 825 830

Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile

930 935 940

Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser

945 950 955 960

Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

965 970 975

Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu

980 985 990

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu

1045 1050 1055

Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala

1060 1065 1070

Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg

1075 1080 1085

Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp

1140 1145 1150

Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His

1155 1160 1165

Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr

1170 1175 1180

Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp

1185 1190 1195 1200

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr

1205 1210 1215

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

1220 1225 1230

Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr

1235 1240 1245

Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala

1250 1255 1260

Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val

1315 1320 1325

Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys

1330 1335 1340

Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1345 1350 1355 1360

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp

1365 1370 1375

Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly

1380 1385 1390

Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu

1395 1400 1405

Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His

1410 1415 1420

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

1425 1430 1435 1440

Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile

1445 1450 1455

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

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1505 1510 1515 1520

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1525 1530 1535

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys

1540 1545 1550

Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1555 1560 1565

<210> 9

<211> 1952

<212> PRT

<213> Artificial sequence

<400> 9

Met Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu

1 5 10 15

Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu

20 25 30

Arg Glu Val Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile

35 40 45

Gly Glu Gly Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His

50 55 60

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

65 70 75 80

Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met

85 90 95

Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly

100 105 110

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

115 120 125

His His Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu

130 135 140

Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg

145 150 155 160

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

165 170 175

Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu

180 185 190

Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu

195 200 205

Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala

210 215 220

Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val

225 230 235 240

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

245 250 255

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

260 265 270

Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr

275 280 285

Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile

290 295 300

Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly

305 310 315 320

Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu

325 330 335

Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr

340 345 350

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

355 360 365

Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu

370 375 380

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser

385 390 395 400

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

405 410 415

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

420 425 430

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

435 440 445

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

450 455 460

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

465 470 475 480

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

485 490 495

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

500 505 510

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

515 520 525

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

530 535 540

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

545 550 555 560

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

565 570 575

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

580 585 590

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

595 600 605

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

610 615 620

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

625 630 635 640

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

645 650 655

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

660 665 670

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

675 680 685

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

690 695 700

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

705 710 715 720

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

725 730 735

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

740 745 750

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

755 760 765

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

770 775 780

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

785 790 795 800

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

805 810 815

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

820 825 830

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

835 840 845

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

850 855 860

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

865 870 875 880

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

885 890 895

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

900 905 910

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

915 920 925

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

930 935 940

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

945 950 955 960

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

965 970 975

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

980 985 990

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

995 1000 1005

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

1010 1015 1020

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

1025 1030 1035 1040

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

1045 1050 1055

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

1125 1130 1135

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

1140 1145 1150

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

1155 1160 1165

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

1170 1175 1180

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

1185 1190 1195 1200

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

1205 1210 1215

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

1220 1225 1230

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

1235 1240 1245

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1250 1255 1260

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1265 1270 1275 1280

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1285 1290 1295

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1300 1305 1310

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

1315 1320 1325

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1330 1335 1340

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

1345 1350 1355 1360

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1365 1370 1375

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

1380 1385 1390

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1395 1400 1405

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1410 1415 1420

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1425 1430 1435 1440

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1445 1450 1455

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1460 1465 1470

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1475 1480 1485

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1490 1495 1500

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

1505 1510 1515 1520

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1525 1530 1535

Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1540 1545 1550

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1555 1560 1565

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

1570 1575 1580

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1585 1590 1595 1600

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1605 1610 1615

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1620 1625 1630

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1635 1640 1645

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

1650 1655 1660

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1665 1670 1675 1680

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

1685 1690 1695

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1700 1705 1710

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys

1715 1720 1725

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu

1730 1735 1740

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1745 1750 1755 1760

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

1765 1770 1775

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

1780 1785 1790

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1795 1800 1805

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Ser Glu Thr Pro Gly Thr

1810 1815 1820

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

1825 1830 1835 1840

Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu

1845 1850 1855

Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val

1860 1865 1870

Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp

1875 1880 1885

Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp

1890 1895 1900

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

1905 1910 1915 1920

Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys

1925 1930 1935

Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu

1940 1945 1950

<210> 10

<211> 112

<212> PRT

<213> Artificial sequence

<400> 10

Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met Leu Trp Leu

1 5 10 15

Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly Ile Lys Val

20 25 30

Thr Glu Val Ala Lys Arg Gly Gly Glu Leu Trp Arg Ala Met Lys Asp

35 40 45

Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp Asp Tyr Asp

50 55 60

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

65 70 75 80

Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys Val Ala Lys

85 90 95

Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp Glu Ser Glu

100 105 110

<210> 11

<211> 66

<212> PRT

<213> Artificial sequence

<400> 11

Met Val Lys Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Thr Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Val Ser Phe

20 25 30

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

35 40 45

Asp Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu

50 55 60

Lys Lys

65

<210> 12

<211> 1667

<212> PRT

<213> Artificial sequence

<400> 12

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

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

20 25 30

Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg

35 40 45

Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly

50 55 60

Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val

65 70 75 80

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

85 90 95

Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly

100 105 110

Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val

115 120 125

Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg

130 135 140

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

145 150 155 160

Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn

165 170 175

Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp

180 185 190

Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro

195 200 205

Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe

210 215 220

Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile

225 230 235 240

Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser

245 250 255

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

260 265 270

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

275 280 285

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

290 295 300

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

305 310 315 320

Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr

325 330 335

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

340 345 350

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

355 360 365

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

370 375 380

Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile

385 390 395 400

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

405 410 415

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

420 425 430

Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile

435 440 445

Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile

450 455 460

Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

595 600 605

Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys

610 615 620

Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr

625 630 635 640

Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro

645 650 655

Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn

660 665 670

Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile

675 680 685

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

690 695 700

Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys

705 710 715 720

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

725 730 735

Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr

740 745 750

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

755 760 765

Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys

770 775 780

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

785 790 795 800

Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys

865 870 875 880

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

885 890 895

Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu

900 905 910

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

915 920 925

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

1010 1015 1020

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

1025 1030 1035 1040

Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg

1045 1050 1055

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln

1060 1065 1070

Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys

1075 1080 1085

Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln

1090 1095 1100

Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val

1105 1110 1115 1120

Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr

1125 1130 1135

Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu

1140 1145 1150

Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe

1235 1240 1245

Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp

1250 1255 1260

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

1265 1270 1275 1280

Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val

1285 1290 1295

Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala

1300 1305 1310

Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile

1315 1320 1325

Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn

1330 1335 1340

Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1345 1350 1355 1360

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Lys Leu Lys Ser Val Lys Glu

1425 1430 1435 1440

Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro

1445 1450 1455

Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu

1460 1465 1470

Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg

1475 1480 1485

Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu

1490 1495 1500

Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr

1505 1510 1515 1520

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

1525 1530 1535

Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser

1540 1545 1550

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

1555 1560 1565

Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala

1570 1575 1580

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

1585 1590 1595 1600

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1605 1610 1615

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

1620 1625 1630

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly

1635 1640 1645

Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys

1650 1655 1660

Arg Lys Val

1665

<210> 13

<211> 1827

<212> PRT

<213> Artificial sequence

<400> 13

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

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

20 25 30

Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg

35 40 45

Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly

50 55 60

Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val

65 70 75 80

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

85 90 95

Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly

100 105 110

Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val

115 120 125

Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg

130 135 140

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

145 150 155 160

Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn

165 170 175

Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp

180 185 190

Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro

195 200 205

Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe

210 215 220

Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile

225 230 235 240

Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser

245 250 255

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

260 265 270

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

275 280 285

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

290 295 300

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

305 310 315 320

Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr

325 330 335

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

340 345 350

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

355 360 365

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

370 375 380

Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile

385 390 395 400

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

405 410 415

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

420 425 430

Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile

435 440 445

Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile

450 455 460

Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

595 600 605

Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys

610 615 620

Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr

625 630 635 640

Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro

645 650 655

Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn

660 665 670

Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile

675 680 685

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

690 695 700

Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys

705 710 715 720

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

725 730 735

Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr

740 745 750

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

755 760 765

Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys

770 775 780

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

785 790 795 800

Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys

865 870 875 880

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

885 890 895

Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu

900 905 910

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

915 920 925

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

1010 1015 1020

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

1025 1030 1035 1040

Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg

1045 1050 1055

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln

1060 1065 1070

Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys

1075 1080 1085

Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln

1090 1095 1100

Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val

1105 1110 1115 1120

Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr

1125 1130 1135

Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu

1140 1145 1150

Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe

1235 1240 1245

Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp

1250 1255 1260

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

1265 1270 1275 1280

Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val

1285 1290 1295

Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala

1300 1305 1310

Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile

1315 1320 1325

Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn

1330 1335 1340

Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1345 1350 1355 1360

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Lys Leu Lys Ser Val Lys Glu

1425 1430 1435 1440

Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro

1445 1450 1455

Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu

1460 1465 1470

Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg

1475 1480 1485

Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu

1490 1495 1500

Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr

1505 1510 1515 1520

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

1525 1530 1535

Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser

1540 1545 1550

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

1555 1560 1565

Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala

1570 1575 1580

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

1585 1590 1595 1600

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1605 1610 1615

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

1620 1625 1630

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly

1635 1640 1645

Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys

1650 1655 1660

Arg Lys Val Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr

1665 1670 1675 1680

Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Ser Glu Thr

1685 1690 1695

Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser

1700 1705 1710

Gly Gly Ser Met Ser Asp Lys Pro Lys Arg Pro Leu Ser Ala Tyr Met

1715 1720 1725

Leu Trp Leu Asn Ser Ala Arg Glu Ser Ile Lys Arg Glu Asn Pro Gly

1730 1735 1740

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

1745 1750 1755 1760

Met Lys Asp Lys Ser Glu Trp Glu Ala Lys Ala Ala Lys Ala Lys Asp

1765 1770 1775

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

1780 1785 1790

Ala Ala Asn Gly Gly Gly Ala Lys Lys Arg Ala Lys Pro Ala Lys Lys

1795 1800 1805

Val Ala Lys Lys Ser Lys Lys Glu Glu Ser Asp Glu Asp Asp Asp Asp

1810 1815 1820

Glu Ser Glu

1825

<210> 14

<211> 1057

<212> PRT

<213> Staphylococcus aureus

<400> 14

Met Glu Ala Ser Met Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly

1 5 10 15

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

20 25 30

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

35 40 45

Glu Gly Arg Arg Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg

50 55 60

Arg His Arg Ile Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu

65 70 75 80

Leu Thr Asp His Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg

85 90 95

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

100 105 110

Leu Leu His Leu Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val

115 120 125

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

130 135 140

Asn Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu

145 150 155 160

Arg Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys

165 170 175

Thr Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys

180 185 190

Ala Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu

195 200 205

Leu Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro

210 215 220

Phe Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His

225 230 235 240

Cys Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn

245 250 255

Ala Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr

260 265 270

Arg Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Lys Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu

340 345 350

Asp Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile

355 360 365

Gln Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile

370 375 380

Glu Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser

385 390 395 400

Leu Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp

405 410 415

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

420 425 430

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

435 440 445

Ile Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val

450 455 460

Ile Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile

465 470 475 480

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

485 490 495

Glu Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile

500 505 510

Ile Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile

515 520 525

Lys Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala

530 535 540

Ile Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp

545 550 555 560

His Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys

565 570 575

Val Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro

580 585 590

Phe Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe

595 600 605

Lys Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys

610 615 620

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

625 630 635 640

Val Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala

645 650 655

Thr Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn

660 665 670

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

675 680 685

Arg Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His

690 695 700

His Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys

705 710 715 720

Glu Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met

725 730 735

Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln

740 745 750

Glu Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys

755 760 765

Asp Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn

770 775 780

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

785 790 795 800

Gly Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp

805 810 815

Asn Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu

820 825 830

Met Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met

835 840 845

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

850 855 860

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

865 870 875 880

Ile Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp

885 890 895

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

900 905 910

Leu Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys

915 920 925

Phe Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr

930 935 940

Glu Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile

945 950 955 960

Ser Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile

965 970 975

Lys Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu

980 985 990

Leu Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr

995 1000 1005

Leu Glu Asn Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile

1010 1015 1020

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

1025 1030 1035 1040

Asn Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys

1045 1050 1055

Gly

<210> 15

<211> 1307

<212> PRT

<213> Acidaminococcus sp

<400> 15

Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr

1 5 10 15

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

20 25 30

Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys

35 40 45

Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln

50 55 60

Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile

65 70 75 80

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

85 90 95

Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly

100 105 110

Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile

115 120 125

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

130 135 140

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

145 150 155 160

Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg

165 170 175

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

180 185 190

Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe

195 200 205

Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn

210 215 220

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

225 230 235 240

Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp

245 250 255

Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu

260 265 270

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

275 280 285

Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro

290 295 300

Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu

305 310 315 320

Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr

325 330 335

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

340 345 350

Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His

355 360 365

Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr

370 375 380

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

385 390 395 400

Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu

405 410 415

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

420 425 430

Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala

435 440 445

Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys

450 455 460

Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu

465 470 475 480

Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe

485 490 495

Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser

500 505 510

Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val

515 520 525

Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp

530 535 540

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

545 550 555 560

Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys

565 570 575

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

580 585 590

Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys

595 600 605

Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr

610 615 620

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

625 630 635 640

Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln

645 650 655

Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala

660 665 670

Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr

675 680 685

Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr

690 695 700

Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His

705 710 715 720

Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu

725 730 735

Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys

740 745 750

Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu

755 760 765

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

770 775 780

Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His

785 790 795 800

Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr

805 810 815

Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His

820 825 830

Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn

835 840 845

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

850 855 860

Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln

865 870 875 880

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

885 890 895

Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg

900 905 910

Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu

915 920 925

Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu

930 935 940

Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val

945 950 955 960

Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile

965 970 975

His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu

980 985 990

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

995 1000 1005

Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn

1010 1015 1020

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

1025 1030 1035 1040

Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly

1045 1050 1055

Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys

1060 1065 1070

Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile

1075 1080 1085

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

1090 1095 1100

His Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn

1105 1110 1115 1120

Arg Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp

1125 1130 1135

Asp Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr

1140 1145 1150

Pro Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg

1155 1160 1165

Phe Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala

1170 1175 1180

Leu Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu

1185 1190 1195 1200

Pro Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val

1205 1210 1215

Ala Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr

1220 1225 1230

Gly Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys

1235 1240 1245

Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala

1250 1255 1260

Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His

1265 1270 1275 1280

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

1285 1290 1295

Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn

1300 1305

Claims (10)

1. Use of an active substance for the manufacture of a medicament or formulation for gene therapy, wherein the active substance is a fusion protein, or a gene encoding it, or an expression vector thereof, wherein the fusion protein has the structure of formula I or I' below:

C-A-L-B (I)

B-L-A-C (I’)

in the formula (I), the compound is shown in the specification,

a is a gene editing protein,

b is a DNA double-stranded binding domain,

c is an optional base editor element;

l is a null or a connecting peptide,

each "-" is independently a linking peptide or non-peptide bond.

2. The use according to claim 1, wherein the medicament is for the treatment of a disease treatable by gene therapy.

3. The use of claim 1, wherein the gene-editing protein is selected from the group consisting of: cas9, Cas9a, Cas12, Cas12a, Cas12b, Cas13, Cas14, or a combination thereof.

4. The use of claim 1, wherein the gene-editing protein is selected from one or more of the following sources: streptococcus pyogenes (Streptococcus pyogenes), Staphylococcus aureus (Staphylococcus aureus), Aminococcus sp, and Lachnospiraceae (Lachnospiraceae bacteria).

5. The use of claim 1, wherein the DNA double-stranded binding domain is a non-sequence specific DNA double-stranded binding domain.

6. The use of claim 1, wherein the DNA double-stranded binding domain is derived from a drosophila or archaea.

7. The use of claim 1, wherein the base editor element comprises a cytosine deaminase, an adenine deaminase.

8. The use of claim 1, wherein the gene therapy is gene therapy by gene editing.

9. The use of claim 1, wherein the gene therapy is gene therapy by a gene editing reagent.

10. The use of claim 9, wherein the gene-editing reagent comprises a fusion protein having the structure of formula I or I' below:

C-A-L-B (I)

B-L-A-C (I’)

in the formula (I), the compound is shown in the specification,

a is a gene editing protein,

b is a DNA double-stranded binding domain,

c is an optional base editor element;

l is a null or a connecting peptide,

each "-" is independently a linking peptide or non-peptide bond.

In another preferred embodiment, the reagent further comprises one or more reagents selected from the group consisting of:

(a1) a gRNA, a crRNA, or a vector for producing the gRNA or crRNA;

(a2) template for homogenous directed repair: a single-stranded nucleotide sequence or a plasmid vector.

Images