A cancer vaccine targeting the epha2 antigen

Abstract

The present application relates to the field of immunotherapy and prevention of cancer, in particular to a tumor vaccine targeting EPHA2. The present application improves the efficiency of EPHA2 protein phagocytosis, processing and presentation by DC cells by fusing EPHA2 protein with DC cell ligand XCL1, and improves the effect of inhibiting tumor growth. Through experiments, it has been verified that the protein expressed by the nucleic acid vaccine of the present application can effectively induce EPHA2 specific T cell response and significantly inhibit the growth of tumors with high expression of EPHA2 in animal tumor models.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to an antigen vaccine targeting EphA2 and a preparation method thereof. BACKGROUND

[0002] Cancer is a major public health problem worldwide, with a rising incidence rate. It is estimated that there will be 4.2 billion new cancer cases worldwide every year by 2025. Despite great progress in the clinical treatment of cancer, millions of people still die from cancer every year, and the improvement of cancer treatment remains a long way to go.

[0003] EphA2 is highly expressed in a variety of cancers, such as prostate cancer, lung cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer and skin cancer, but is relatively low in most normal adult tissues, so EphA2 is an ideal potential therapeutic target for cancer. EphA2 receptor is a 130 kDa transmembrane glycoprotein, and EphA2-ephrin A1 signaling plays an important role in tumorigenesis. In addition, the expression of EphA2 is associated with poor prognosis, increased metastatic potential and reduced survival rate in tumor patients. After more than 30 years of research, the mechanism of EphA2 has gradually become clear, and some drugs targeting EphA2 have been used in clinical research for cancer treatment. Current treatment methods targeting EphA2 include reducing EphA2 expression, promoting EphA2 degradation, blocking endogenous EphA2 activation, EphA2 as a drug delivery target, EphA2-based immunotherapy and EphA2-based combination therapy. Among them, immunotherapy that relies on enhancing the patient's immune defense to fight tumor cells has become a promising new therapy in cancer treatment. For example, after immunizing a colorectal cancer animal model with dendritic cells (DC) loaded with EphA2-derived short peptides, the activity of EphA2-specific cytotoxic T lymphocytes (CTL) and the anti-tumor ability can be improved; adoptive infusion of EphA2-specific T cells can kill EphA2-positive tumor cells and inhibit the occurrence of lung cancer in vivo. Although the above therapies can activate anti-tumor immune responses, their effects are limited and are not sufficient to provide effective anti-tumor protection.

[0004] Most immunotherapy methods rely on the activation of CTLs by mature DCs presenting tumor-associated antigens. Among them, type I conventional DCs (conventional DC1, cDC1) can uptake antigens from dead cells through cross-presentation and present them via MHC-I molecules, thereby activating and expanding specific CTLs. CD103+ DC1 is a very important cDC1 with migration ability, which can carry tumor antigens to the draining lymph nodes for cross-presentation, and expresses XCL1 receptor (XCR1) on the surface, which can be attracted by XCL1 molecules. Therefore, XCL1 can be fused with EphA2 protein to increase the contact between CD103+ DC and EphA2 antigen, thereby enhancing the antigen presentation and immune activation of cDC1 to natural killer cells (NK) and CD8+ CTLs, enhancing the immune response, and providing more effective anti-tumor protection. SUMMARY

[0005] Therefore, the present application provides a new EPHA2-targeted antigen vaccine and a preparation method thereof. Specifically, the present application provides an EPHA2-targeted nucleic acid and protein vaccine, which improves the phagocytosis and presentation efficiency of DCs to EPHA2 protein by fusing and expressing cDC1 ligand XCL1 with EPHA2 protein, enhances EPHA2-specific CD8+ CTL response, and improves the inhibition effect of tumors with high expression of EPHA2.

[0006] The present application provides a fusion protein comprising an amino acid sequence of an extracellular region of EPHA2 capable of inducing specific CD8+ T response or an optimized form of MHC class I molecule binding epitope; a linker; and a human or murine XCL1 protein specifically binding to DC cells with antigen cross-presentation ability;

[0007] wherein:

[0008] (I) the extracellular region of EPHA2 capable of inducing specific CD8+ T response or an optimized form of MHC class I molecule binding epitope has an amino acid sequence as shown in SEQ ID No. 1 or 2; and the human or murine XCL1 specifically binding to DC cells with antigen cross-presentation ability has an amino acid sequence as shown in SEQ ID No. 3 or 4;

[0009] or

[0010] (II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues to the amino acid sequence of (I), and an amino acid sequence functionally identical or similar to the amino acid sequence shown in (I);

[0011] or

[0012] (III), an amino acid sequence having at least 90% sequence identity with the sequence of (I) or (II) and having the same or similar function as the amino acid sequence shown in (I).

[0013] In some embodiments of the present application, the fusion protein comprises an amino acid sequence as shown in SEQ ID No. 9 or SEQ ID No. 10.

[0014] Based on the above, the present application further provides a nucleic acid molecule encoding the fusion protein, wherein the nucleic acid molecule comprises DNA and / or mRNA, and the sequence of the nucleic acid molecule has:

[0015] (I), a nucleotide sequence as shown in any of SEQ ID No. 17~32;

[0016] or

[0017] (II), a complementary nucleotide sequence of the nucleotide sequence as shown in any of SEQ ID No. 15~30; or

[0018] (III), a nucleotide sequence encoding the same protein as the nucleotide sequence of (I) or (II), but different from the nucleotide sequence of (I) or (II) due to the degeneracy of genetic code;

[0019] or

[0020] (IV), a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences of the nucleotide sequence as shown in (I), (II) or (III), and having the same or similar function as the nucleotide sequence as shown in (I), (II) or (III);

[0021] or

[0022] (V), a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of (I), (II), (III) or (IV).

[0023] The present application further provides a recombinant expression vector comprising the vector and the fusion protein or the nucleic acid molecule.

[0024] In some embodiments of the present application, the vector comprises pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP, pVAX1.

[0025] The present application further provides a recombinant strain or cell comprising the fusion protein or the nucleic acid molecule.

[0026] The present invention also provides the use of the fusion protein, the nucleic acid molecule, the recombinant expression vector, or the recombinant strain or cell in the preparation of vaccines for tumors that highly express EPHA2 and / or in the preparation of medicaments for the prevention and / or treatment of tumors that highly express EPHA2.

[0027] In some specific embodiments of the present invention, the metastatic cancer includes tumors that highly express EPHA2, including prostate cancer, lung cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer, and skin cancer.

[0028] This invention also provides a tumor vaccine targeting EPHA2, comprising the fusion protein, the nucleic acid molecule, the recombinant expression vector or the recombinant strain or cell, and pharmaceutically acceptable vectors, excipients and / or adjuvants.

[0029] The present invention also provides medicaments for the prevention and / or treatment of tumors that highly express EPHA2, comprising the fusion protein, the nucleic acid molecule, the recombinant expression vector or the recombinant strain or cell, and pharmaceutically acceptable excipients.

[0030] This invention provides a tumor vaccine targeting EPHA2. The vaccine consists of a human or mouse XCL1 protein specifically binding to dendritic cells (DCs) with antigen cross-presentation capability, fused with the amino acid sequence of the extracellular region of EPHA2 and its N-terminus via a linker. It includes a nucleic acid containing the fusion protein and a vector containing the nucleic acid. This invention relates to the application of the fusion protein, nucleic acid, and vector in the prevention and treatment of tumors highly expressing EPHA2. By fusing the EPHA2 protein with the DC ligand XCL1, this invention improves the efficiency of EPHA2 protein phagocytosis, processing, and presentation by DCs, thereby enhancing its tumor-inhibiting effect. Experimental verification shows that the protein expressed by the nucleic acid vaccine of this invention can effectively induce an EPHA2-specific T-cell response and significantly inhibit the growth of tumors highly expressing EPHA2 in animal tumor models. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0032] Figure 1 The vector map showing the nucleotides encoding the fusion protein is shown; among them Figure 1 A indicates that the CMV promoter is followed by the secretory signal peptide of XCL1, and then by the nucleic acid vaccine coding sequence of the extracellular region of EPHA2; Figure 1 B indicates a sequence for coding nucleic acid vaccines, following the CMV promoter, the XCL1 sequence, and then the extracellular region of EPHA2.

[0033] Figure 2 Figure 1 shows the predicted three-dimensional structure of the fusion protein; the nucleotide sequence encoding the fusion protein was input into http: / / raptorx.uchicago.edu / to predict the three-dimensional structure of the fusion protein, and the predicted spatial structure of the XCL1-EPHA2 fusion protein is shown in the figure;

[0034] Figure 3 Figure 1 shows the predicted three-dimensional structure of the fusion protein; the nucleotide sequence encoding the fusion protein was input into http: / / raptorx.uchicago.edu / to predict the three-dimensional structure of the fusion protein, and the predicted spatial structure of the XCL1-EPHA2 fusion protein is shown in the figure;

[0035] Figure 4 Figure 1 shows the predicted three-dimensional structure of the fusion protein; the nucleotide sequence encoding the fusion protein was input into http: / / raptorx.uchicago.edu / to predict the three-dimensional structure of the fusion protein, and the predicted spatial structure of the XCL1-EPHA2 fusion protein is shown in the figure;

[0036] Figure 5 Figure 1 shows the predicted three-dimensional structure of the fusion protein; the nucleotide sequence encoding the fusion protein was input into http: / / raptorx.uchicago.edu / to predict the three-dimensional structure of the fusion protein, and the predicted spatial structure of the XCL1-EPHA2 fusion protein is shown in the figure; Figure 5 A shows the fusion gene immunization method and the immunization process; Figure 5 B shows the tumor growth curve obtained by measuring the volume of the tumor after inoculation of Hepa1-6. DETAILED DESCRIPTION

[0037] The present application discloses a tumor vaccine targeting EPHA2, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly important to note that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0038] The present application uses the amino acid sequence of the EPHA2 extracellular region as an immunogen to facilitate the purification of soluble protein vaccines. The vaccine of the present application can effectively induce specific T cell immune response against EPHA2 in mice or humans, break the immune tolerance of the original tumor-associated antigen EPHA2, and thus effectively play a role in directly eliminating tumor cells expressing EPHA2, thereby inhibiting tumor growth.

[0039] The present application relates to a fusion protein vaccine for tumors overexpressing EPHA2, which comprises 1) an amino acid sequence of the extracellular region of EPHA2, and 2) a nucleotide sequence of a human or mouse XCL1 protein that specifically binds to DC cells with antigen cross-presentation ability. For example, the sequence shown in SEQ ID No. 1 and SEQ ID No. 2, which specifically binds to DC cells with antigen cross-presentation ability, is fused to the N-terminus of a human or mouse XCL1 protein, and the sequence of the amino acids 1-114 shown in SEQ ID No. 3 and SEQ ID No. 4.

[0040] In some embodiments, the vaccine for tumors overexpressing EPHA2 comprises any of the sequences shown in SEQ ID No. 9 and SEQ ID No. 10.

[0041] The present application relates to a fusion protein vaccine for tumors overexpressing EPHA2, which comprises 1) an amino acid sequence of the extracellular region of EPHA2, and 2) a nucleotide sequence of a human or mouse XCL1 protein that specifically binds to DC cells with antigen cross-presentation ability. For example, the sequence shown in SEQ ID No. 1 and SEQ ID No. 2, which specifically binds to DC cells with antigen cross-presentation ability, is fused to the N-terminus of a human or mouse XCL1 protein, and the sequence of the amino acids 1-114 shown in SEQ ID No. 3 and SEQ ID No. 4.

[0042] In some embodiments, the sequence encoding the amino acid sequence of the extracellular region of EPHA2 and the human or mouse XCL1 protein that specifically binds to DC cells with antigen cross-presentation ability comprises the nucleotide sequences shown in SEQ ID No. 23 and SEQ ID No. 24.

[0043] The present application also provides a recombinant expression vector loaded with the nucleic acid vaccine, which comprises the nucleotide sequence of the fusion protein vaccine and the corresponding nucleic acid vaccine of the present application, and a vector. The vector can be a mammalian cell expression vector or an insect baculovirus expression vector, and specifically, the vector can be pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP, or pVAX1.

[0044] The present application finally provides a vaccine for tumors overexpressing EPHA2, which comprises the fusion protein vaccine, the nucleic acid vaccine, and the recombinant expression vector.

[0045] The vaccine of the present application provides a method for preventing recurrence or treatment of tumors overexpressing EPHA2. The vaccine of the present application can also be used for other indications.

[0046] The application also provides the use of the vaccine, the nucleic acid vaccine and the vector in the preparation of the vaccine for the tumor with high expression of EPHA2.

[0047] The application also provides the use of the vaccine, the nucleic acid vaccine and the vector in the preparation of the vaccine for the tumor with high expression of EPHA2.

[0048] The vaccine or the drug of the application comprises the fusion protein, the nucleic acid, the recombinant expression vector and the optional pharmaceutically acceptable carrier, excipient and / or adjuvant.

[0049] In summary, the application provides the tumor vaccine targeting EPHA2, which is composed of the amino acid sequence of the extracellular region of EPHA2 and the N terminal fused with the human or murine XCL1 protein specifically binding to the DC cells with the antigen cross-presentation ability through the linker, the nucleic acid comprising the fusion protein and the vector where the nucleic acid is located, the use of the fusion protein, the nucleic acid and the vector in the prevention and treatment of the tumor with high expression of EPHA2. The application improves the efficiency of the phagocytosis, processing and presentation of the EPHA2 protein by the DC cells by fusing the EPHA2 protein with the DC cell ligand XCL1, and improves the effect of inhibiting the growth of the tumor. Through the experiment verification, the nucleic acid vaccine of the application can effectively induce the EPHA2 specific T cell response and significantly inhibit the growth of the tumor with high expression of EPHA2 in the tumor animal model.

[0050] The raw materials and reagents involved in the application can be purchased from the market.

[0051] The application is further described below in combination with the examples:

[0052] Example 1 Design scheme of the fusion protein vaccine antigen and construction and preparation of the mammalian cell expression plasmid

[0053] (1) Construction of the fusion gene mammalian cell expression vector:

[0054] According to the amino acid sequences of mouse or human EPHA2 (SEQ ID No. 1 and SEQ ID No. 2) and mouse or human XCL1 (SEQ ID No. 3 and SEQ ID No. 4) proteins, a fusion protein XCL1-EPHA2 was constructed. In order to facilitate the expression of the nucleic acid of the fusion protein to be translated in vivo, the fusion protein can be effectively secreted to the outside of the cell and chemotactic to MHC-II+CD11c+CD8A+antigen cross-presentation DC cells, we retained the secretion signal peptide of the XCL1 protein (SEQ ID No. 5 and SEQ ID No. 6), while selecting the extracellular region of EPHA2 after removing the transmembrane positioning signal peptide (SEQ ID No. 7 and SEQ ID No. 8). The final amino acid sequence of the fusion protein (SEQ ID No. 9 and SEQ ID No. 10). For the EPHA2 protein alone, the secretion signal peptide (SEQ ID No. 11 and SEQ ID No. 12) was retained to connect the EPHA2 extracellular region (SEQ ID No. 7 and SEQ ID No. 8) to ensure that the EPHA2 protein expressed by the EPHA2 expression vector can also be secreted to the outside of the cell. We optimized the nucleotide sequence corresponding to the amino acid sequence for the codon preference of mammalian cells, synthesized by the company, and connected to the pVAX1 expression vector (SEQ ID No. 13 and SEQ ID No. 14).

[0055] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 1 has a nucleotide sequence as shown in SEQ ID No. 15;

[0056] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 2 has a nucleotide sequence as shown in SEQ ID No. 16;

[0057] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 3 has a nucleotide sequence as shown in SEQ ID No. 17;

[0058] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 4 has a nucleotide sequence as shown in SEQ ID No. 18;

[0059] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 5 has a nucleotide sequence as shown in SEQ ID No. 19;

[0060] The nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 6 has a nucleotide sequence as shown in SEQ ID No. 20;

[0061] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 7 has a nucleotide sequence as shown in SEQ ID No. 21;

[0062] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 8 has a nucleotide sequence as shown in SEQ ID No. 22;

[0063] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 9 has a nucleotide sequence as shown in SEQ ID No. 23;

[0064] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 10 has a nucleotide sequence as shown in SEQ ID No. 24;

[0065] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 11 has a nucleotide sequence as shown in SEQ ID No. 25;

[0066] A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 12 has a nucleotide sequence as shown in SEQ ID No. 26;

[0067] Figure 1 A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 12 has a nucleotide sequence as shown in SEQ ID No. 26; Figure 1 A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 12 has a nucleotide sequence as shown in SEQ ID No. 26; Figure 1 A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 12 has a nucleotide sequence as shown in SEQ ID No. 26;

[0068] (2) Amplification of the fusion gene mammalian cell expression vector

[0069] Transformation of bacteria, the -80 °C frozen competent bacteria were placed on ice to melt, when close to complete melting, add 100 ng plasmid, gently mix, ice for 30 mins. The competent was placed in a 42 °C water bath for 60 s, immediately after taking out and placed on ice for 2 mins. Add 500 μL of LB medium without antibiotics into the tube, 37 °C shaking incubator for 1 h. The bacteria were centrifuged at 4000 rpm at room temperature for 2 mins, after discarding part of the supernatant (about 450 μL) resuspend the bacteria, take the appropriate amount of bacterial suspension on the culture dish containing the corresponding antibiotic. The culture dish was placed upside down, 37 °C incubator culture overnight. When the clone size is appropriate (about 16 h) with a gun tip to the clone to add antibiotic LB medium, 37 °C shaking culture to turbidity. Take 15 mL of bacteria cultured to the appropriate concentration, 4000 rpm at room temperature for 5 mins, discard the supernatant. According to the Beijing Tiangen Company's "plasmid extraction kit" goods number (DP103) instructions to extract bacterial DNA. First add 250 μL of RNAse containing cell resuspension P1 to resuspend the bacterial pellet, and transfer the pellet to a 1.5 mL EP tube. Add 250 μL of alkaline cell lysis solution P2, gently invert mix until the liquid is clear. Add 350 μL of neutralization solution P3, invert mix evenly until the appearance of flocculent precipitate. The suspension was centrifuged at 12000 rpm at room temperature for 10 mins. The DNA adsorption column was placed in the recovery tube, 500 μL of equilibration liquid was added to activate the adsorption membrane, and the liquid was discarded after centrifugation at 12000 rpm for 1 min. The supernatant obtained by centrifugation in step 4 was transferred to the DNA adsorption column, and the liquid was discarded after centrifugation at 12000 rpm for 1 min. Add 600 μL of washing solution to the adsorption column, and discard the liquid after centrifugation at 12000 rpm for 1 min. Repeat the washing once. 12000 rpm empty recovery tube 2 mins. Replace the collection tube with a new 1.5 mL EP tube, and let it dry at room temperature for 5 mins. Add 70 μL of 65 °C preheated elution buffer or high pressure purified water, and let it dry at room temperature for 5 mins. Centrifuge at 12000 rpm at room temperature for 3 mins to collect the liquid. Discard the adsorption column, and determine the concentration of the plasmid in the tube and label the plasmid name, concentration, and extraction date.

[0070] Example 2 Prediction of the three-dimensional structure of the fusion protein

[0071] According to the nucleotide sequence of the fusion protein, we use the website http: / / raptorx.uchicago.edu / to predict the three-dimensional structure of XCL1 and EPHA2 fusion protein. XCL1 belongs to chemokines, and its normal chemotactic function requires the preservation of the complete spatial structure. In order to ensure that XCL1 still retains its own spatial structure after fusion with EPHA2, we predict the three-dimensional structure of the amino acid sequence after fusion on the website http: / / raptorx.uchicago.edu / , and the results are as follows Figure 2 It is shown that after XCL1 and EPHA2 fusion, they still retain their original spatial structure and do not affect the chemotactic function of XCL1.

[0072] Example 3 Detection of expression effect of mammalian cell expression vector encoding XCL1-EPHA2 fusion protein

[0073] 24 hours before transfection, 1*10 6 HEK293T cells were inoculated in a 6-well cell culture plate, and when the cell density reached 70%-80%, the transfection experiment was started. When transfecting, the cell culture medium and serum-free Opti-MEM medium were preheated in a 37°C water bath. When transfecting, 5 micrograms of empty vector (Vector), EPHA2 expression vector alone, XCL1-EPHA2 wild-type fusion gene plasmid and 20 μL PEI transfection reagent were added to 200 μL serum-free Opti-MEM, mixed evenly, and then incubated at room temperature for 10 minutes. The cells to be transfected were replaced with fresh medium, and the above transfection system was gently added and shaken gently. After 6 hours of incubation in the cell culture box, the cells were collected and the expression effect of XCL1-EPHA2 fusion gene plasmid in HEK293T cells was detected by Western Blot.

[0074] To facilitate the detection of the expression of the fusion gene, we connect a Flag tag composed of 5 amino acids of DDDDK to the C terminal of the fusion protein, so as to detect the expression of the fusion protein by using Flag tag antibody. The cells are collected, and 60 μL of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor is added. The cells are resuspended, and the cells are lysed at 4°C for 30 minutes. The lysate is centrifuged at 12000 rpm and 4°C for 10 minutes, and the supernatant is collected into a new 1.5 mL EP tube, and the precipitate is discarded. According to the actual volume of the sample, 5x SDS-PAGE protein loading buffer is added, and the sample is heated at 100°C in an air bath for 10 minutes, and then Western blot is performed immediately, and Flag tag antibody (Sigma, F3165) is used for detection, and the results are shown in Figure 3 The empty vector (Vector) has no protein expression, the EPHA2 expression vector alone, and the XCL1-EPHA2 wild type fusion gene plasmid can effectively express and the expression amount is equivalent.

[0075] Example 4, explore whether the immune fusion gene induces stronger cell-specific T cell response

[0076] We extract the EPHA2 and XCL1-EPHA2 wild type plasmid alone, and use the gene gun (GDS-80) of Wealtec company to immunize the mice with plasmid injection. After 14 days, the EPHA2 specific tetramer flow cytometry antibody is used to detect the EPHA2 specific T cell response of the mouse peripheral blood.

[0077] Specifically, first take the mouse peripheral blood inside and outside the canthus, mix after adding heparin sodium anticoagulant, centrifuge at 200g for 10 minutes, discard the supernatant, and use R&D company's red blood cell lysis solution (item number WL2000) to crack the red blood of peripheral blood. Specifically, first dilute lysis solution A with distilled water 10 times to prepare working solution, add 2mL working solution to each peripheral blood sample, resuspend at room temperature for 10 min, during which time neutralizing solution B is diluted 10 times with distilled water to prepare working solution. Ten minutes later, 10mL of neutralizing solution B was added to the lysis solution and neutralized, and then centrifuged at 200g for 10 min. The cell mass was washed once with PBS buffer containing 1% inactivated FBS and subjected to flow cytometry staining: EPHA2-tetramer antibody-FITC, CD8a-PE. After 2 hours, flow cytometry detection was performed. The results are shown in Figure 4 As shown in the figure, the specific T cells of the mouse peripheral blood against EPHA2 after immunization with XCL1-EPHA2 wild type plasmid increased significantly compared with the EPHA2 immunization group alone, indicating that XCL1-EPHA2 wild type plasmid immunization indeed induced specific CD8A cytotoxic T lymphocytes.

[0078] Table 1 Figure 4 Data

[0079]

[0080] Example 5 Intervention effect of fusion gene on the occurrence of CT26 cell transplant tumor overexpressing EPHA2

[0081] We extracted EPHA2 and XCL1-EPHA2 wild type plasmids alone, and immunized mice with plasmid injection using the gene gun (GDS-80) of Wealtec Company. After transplanting hepal-6 cells overexpressing EPHA2, we observed the inhibition of fusion gene immunization on the growth of CT26 tumor cells overexpressing EPHA2.

[0082] We extracted EPHA2 and XCL1-EPHA2 wild type plasmids alone, and immunized mice with plasmid injection using the gene gun (GDS-80) of Wealtec Company. After transplanting hepal-6 cells overexpressing EPHA2, we observed the inhibition of fusion gene immunization on the growth of CT26 tumor cells overexpressing EPHA2. Figure 5 The immunization strategy marked by the time axis of A was used to inject plasmid into mice by gene gun. C57B6 (purchased from Vivotec Laboratories) 6-week-old male mice were divided into three groups: control group, EPHA2 injection group, and XCL1-EPHA2 wild type plasmid injection group, with five mice in each group. Hair removal cream was used to remove hair on the right side of the mice near the inguinal lymph nodes. Then plasmid was injected into the hair removal area by gene gun, 50 μg per mouse, once a week, a total of four times. One week after the last injection, overexpressing EPHA2 CT26 cells were inoculated before the tumorigenic conditions were explored, the time of tumor formation was observed, and the long diameter a and short diameter b of the tumor were measured every two days. The tumor volume was calculated according to a*b*b / 2, and the tumor growth curve was drawn. The results are shown in Figure 5 A, 5B, immunization injection of XCL1-EPHA2 wild type plasmid can effectively delay the occurrence time and growth rate of tumor. It shows that the treatment effect of fusion gene immunization is significantly effective.

[0083] Table 2 Figure 5 B data

[0084]

[0085] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application. SEQUENCE LISTING <110> Beijing Noventor Technology Co., Ltd. <120> A cancer vaccine targeting EphA2 antigen <130> MP21007548 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 977 <212> PRT <213> Artificial Sequence <400> 1 Met Glu Leu Arg Ala Val Gly Phe Cys Leu Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu Asp 20 25 30 Phe Ala Ala Met Lys Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly 35 40 45 Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asp Asp Met Pro Ile Tyr 50 55 60 Met Tyr Ser Val Cys Asn Val Val Ser Gly Asp Gln Asp Asn Trp Leu 65 70 75 80 Arg Thr Asn Trp Val Tyr Arg Glu Glu Ala Glu Arg Ile Phe Ile Glu 85 90 95 Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser 100 105 110 Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Val Asp 115 120 125 Tyr Gly Thr Asn Phe Gin Lys Arg Gin Phe Thr Lys He Asp Thr He 130 135 140 Ala Pro Asp Glu He Thr Val Ser Ser Asp Phe Glu Ala Arg Asn Val 145 150 155 160 Lys Leu Asn Val Glu Glu Arg Met Val Gly Pro Leu Thr Arg Lys Gly 165 170 175 Phe Tyr Leu Ala Phe Gin Asp He Gly Ala Cys Val Ala Leu Leu Ser 180 185 190 Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Met Leu Gin Ser Leu Ala 195 200 205 Arg Phe Pro Glu Thr He Ala Val Ala Val Ser Asp Thr Gin Pro Leu 210 215 220 Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Tyr Gly 225 230 235 240 Gly Glu Gly Pro Leu Met His Cys Thr Val Asp Gly Glu Trp Leu Val 245 250 255 Pro He Gly Gin Cys Leu Cys Gin Glu Gly Tyr Glu Lys Val Glu Asp 260 265 270 Ala Cys Arg Ala Cys Ser Pro Gly Phe Phe Lys Ser Glu Ala Ser Glu 275 280 285 Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Thr Glu Gly 290 295 300 Ala Thr Ser Cys Gln Cys Glu Glu Gly Tyr Phe Arg Ala Pro Glu Asp 305 310 315 320 Pro Leu Ser Met Ser Cys Thr Arg Pro Pro Ser Ala Pro Asn Tyr Leu 325 330 335 Thr Ala Ile Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Ala Pro 340 345 350 Lys Asp Thr Gly Gly Arg Gln Asp Ile Val Tyr Ser Val Thr Cys Glu 355 360 365 Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val 370 375 380 Arg Tyr Ser Glu Pro Pro His Ala Leu Thr Arg Thr Ser Val Thr Val 385 390 395 400 Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Ala Val Glu Ala Arg 405 410 415 Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser 420 425 430 Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Asp Arg 435 440 445 Ser Thr Thr Ser Leu Ser Val Thr Trp Ser Ile Pro Val Ser Gln Gln 450 455 460 Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ala 465 470 475 480 Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp 485 490 495 Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr 500 505 510 Gln Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu 515 520 525 Ser Thr Glu Gly Ser Ala Asn Met Ala Val Ile Gly Gly Val Ala Val 530 535 540 Gly Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Leu Phe Ile His 545 550 555 560 Arg Arg Arg Arg Asn Leu Arg Ala Arg Gln Ser Ser Glu Asp Val Arg 565 570 575 Phe Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro 580 585 590 His Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu 595 600 605 Ile His Pro Ser Cys Val Ala Arg Gin Lys Val He Gly Ala Gly Glu 610 615 620 Phe Gly Glu Val Tyr Lys Gly Thr Leu Lys Ala Ser Ser Gly Lys Lys 625 630 635 640 Glu He Pro Val Ala He Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys 645 650 655 Gln Arg Val Asp Phe Leu Ser Glu Ala Ser He Met Gly Gin Phe Ser 660 665 670 His His Asn He He Arg Leu Glu Gly Val Val Ser Lys Tyr Lys Pro 675 680 685 Met Met He He Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe 690 695 700 Leu Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gin Leu Val Gly Met 705 710 715 720 Leu Arg Gly He Ala Ser Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr 725 730 735 Val His Arg Asp Leu Ala Ala Arg Asn He Leu Val Asn Ser Asn Leu 740 745 750 Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp 755 760 765 Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp 770 775 780 Thr Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp 785 790 795 800 Val Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu 805 810 815 Arg Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn 820 825 830 Asp Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr 835 840 845 Gln Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ser Arg Arg Pro Lys 850 855 860 Phe Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp 865 870 875 880 Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu 885 890 895 Pro Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu 900 905 910 Trp Leu Glu Ser lie Lys Met Gin Gin Tyr Thr Glu His Phe Met Val 915 920 925 Ala Gly Tyr Thr Ala lie Glu Lys Val Val Gin Met Ser Asn Glu Asp 930 935 940 lie Lys Arg lie Gly Val Arg Leu Pro Gly His Gin Lys Arg lie Ala 945 950 955 960 Tyr Ser Leu Leu Gly Leu Lys Asp Gin Val Asn Thr Val Gly lie Pro 965 970 975 lie <210> 2 <211> 976 <212> PRT <213> Artificial Sequence <400> 2 Met Glu Leu Gin Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Ala Gin Gly Lys Glu Val Val Leu Leu 20 25 30 Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr 35 40 45 Gly Lys Gly Trp Asp Leu Met Gin Asn lie Met Asn Asp Met Pro lie 50 55 60 Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp 65 70 75 80 Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile 85 90 95 Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala 100 105 110 Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu 115 120 125 Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr 130 135 140 Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His 145 150 155 160 Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys 165 170 175 Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu 180 185 190 Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu 195 200 205 Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala 210 215 220 Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly 225 230 235 240 Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro 245 250 255 Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala 260 265 270 Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser 275 280 285 Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala 290 295 300 Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro 305 310 315 320 Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr 325 330 335 Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln 340 345 350 Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln 355 360 365 Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg 370 375 380 Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser 385 390 395 400 Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn 405 410 415 Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val 420 425 430 Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser 435 440 445 Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser 450 455 460 Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn 465 470 475 480 Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp 485 490 495 Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln 500 505 510 Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser 515 520 525 Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly 530 535 540 Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg 545 550 555 560 Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe 565 570 575 Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His 580 585 590 Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile 595 600 605 His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe 610 615 620 Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu 625 630 635 640 Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln 645 650 655 Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His 660 665 670 His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met 675 680 685 Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu 690 695 700 Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu 705 710 715 720 Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val 725 730 735 His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val 740 745 750 Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro 755 760 765 Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr 770 775 780 Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 785 790 795 800 Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg 805 810 815 Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp 820 825 830 Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln 835 840 845 Leu Met Met Gin Cys Trp Gin Gin Glu Arg Ala Arg Arg Pro Lys Phe 850 855 860 Ala Asp He Val Ser He Leu Asp Lys Leu He Arg Ala Pro Asp Ser 865 870 875 880 Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser He Arg Leu Pro 885 890 895 Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp 900 905 910 Leu Glu Ser He Lys Met Gin Gin Tyr Thr Glu His Phe Met Ala Ala 915 920 925 Gly Tyr Thr Ala He Glu Lys Val Val Gin Met Thr Asn Asp Asp He 930 935 940 Lys Arg He Gly Val Arg Leu Pro Gly His Gin Lys Arg He Ala Tyr 945 950 955 960 Ser Leu Leu Gly Leu Lys Asp Gin Val Asn Thr Val Gly He Pro He 965 970 975 <210> 3 <211> 115 <212> PRT <213> Artificial Sequence <400> 3 Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro 1 5 10 15 Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys 20 25 30 Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr 35 40 45 Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg 50 55 60 Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala 65 70 75 80 Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu 85 90 95 Thr Val Pro Thr Gly Ala Gln Arg Ser Thr Ser Thr Ala Ile Thr Leu 100 105 110 Thr Gly Gly 115 <210> 4 <211> 114 <212> PRT <213> Artificial Sequence <400> 4 Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala 1 5 10 15 Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys 20 25 30 Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr 35 40 45 Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg 50 55 60 Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val 65 70 75 80 Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln 85 90 95 Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu 100 105 110 Thr Gly <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <400> 5 Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro 1 5 10 15 Trp Val Val Glu Gly 20 <210> 6 <211> 21 <212> PRT <213> Artificial Sequence <400> 6 Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala 1 5 10 15 Tyr Ile Val Glu Gly 20 <210> 7 <211> 513 <212> PRT <213> Artificial Sequence <400> 7 Lys Glu Val Val Leu Leu Asp Phe Ala Ala Met Lys Gly Glu Leu Gly 1 5 10 15 Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile 20 25 30 Met Asp Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Val Ser 35 40 45 Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Glu Glu 50 55 60 Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn 65 70 75 80 Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr 85 90 95 Tyr Ala Glu Ser Asp Val Asp Tyr Gly Thr Asn Phe Gln Lys Arg Gln 100 105 110 Phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser 115 120 125 Asp Phe Glu Ala Arg Asn Val Lys Leu Asn Val Glu Glu Arg Met Val 130 135 140 Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly 145 150 155 160 Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro 165 170 175 Glu Met Leu Gln Ser Leu Ala Arg Phe Pro Glu Thr Ile Ala Val Ala 180 185 190 Val Ser Asp Thr Gln Pro Leu Ala Thr Val Ala Gly Thr Cys Val Asp 195 200 205 His Ala Val Val Pro Tyr Gly Gly Glu Gly Pro Leu Met His Cys Thr 210 215 220 Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Glu 225 230 235 240 Gly Tyr Glu Lys Val Glu Asp Ala Cys Arg Ala Cys Ser Pro Gly Phe 245 250 255 Phe Lys Ser Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His 260 265 270 Thr Leu Pro Ser Thr Glu Gly Ala Thr Ser Cys Gln Cys Glu Glu Gly 275 280 285 Tyr Phe Arg Ala Pro Glu Asp Pro Leu Ser Met Ser Cys Thr Arg Pro 290 295 300 Pro Ser Ala Pro Asn Tyr Leu Thr Ala Ile Gly Met Gly Ala Lys Val 305 310 315 320 Glu Leu Arg Trp Thr Ala Pro Lys Asp Thr Gly Gly Arg Gln Asp Ile 325 330 335 Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys 340 345 350 Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Ala Leu 355 360 365 Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr 370 375 380 Thr Phe Ala Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser 385 390 395 400 Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro 405 410 415 Lys Val Arg Leu Glu Asp Arg Ser Thr Thr Ser Leu Ser Val Thr Trp 420 425 430 Ser Ile Pro Val Ser Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr 435 440 445 Tyr Arg Lys Lys Gly Asp Ala Asn Ser Tyr Asn Val Arg Arg Thr Glu 450 455 460 Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu 465 470 475 480 Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys 485 490 495 Val His Glu Phe Gln Thr Leu Ser Thr Glu Gly Ser Ala Asn Met Ala 500 505 510 Val <210> 8 <211> 514 <212> PRT <213> Artificial Sequence <400> 8 Ala Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly 1 5 10 15 Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met 20 25 30 Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn 35 40 45 Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr 50 55 60 Arg Gly Glu Ala Glu Arg lie Phe lie Glu Leu Lys Phe Thr Val Arg 65 70 75 80 Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe 85 90 95 Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln 100 105 110 Lys Arg Leu Phe Thr Lys lie Asp Thr lie Ala Pro Asp Glu lie Thr 115 120 125 Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu 130 135 140 Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln 145 150 155 160 Asp lie Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys 165 170 175 Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr lie 180 185 190 Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val 195 200 205 Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys 210 215 220 Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gin Cys Leu Cys Gin 225 230 235 240 Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gin Ala Cys Ser Pro Gly 245 250 255 Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu 260 265 270 His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu 275 280 285 Gly Phe Phe Arg Ala Pro Gin Asp Pro Ala Ser Met Pro Cys Thr Arg 290 295 300 Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys 305 310 315 320 Val Glu Leu Arg Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg Glu Asp 325 330 335 Ile Val Tyr Ser Val Thr Cys Glu Gin Cys Trp Pro Glu Ser Gly Glu 340 345 350 Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly 355 360 365 Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn 370 375 380 Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr 385 390 395 400 Ser Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn Gin Thr Glu Pro 405 410 415 Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser 420 425 430 Trp Ser Ile Pro Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr Glu Val 435 440 445 Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr 450 455 460 Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr 465 470 475 480 Leu Val Gin Val Gin Ala Leu Thr Gin Glu Gly Gin Gly Ala Gly Ser 485 490 495 Lys Val His Glu Phe Gin Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu 500 505 510 Ala Val <210> 9 <211> 638 <212> PRT <213> Artificial Sequence <400> 9 Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro 1 5 10 15 Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys 20 25 30 Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr 35 40 45 Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg 50 55 60 Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala 65 70 75 80 Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu 85 90 95 Thr Val Pro Thr Gly Ala Gln Arg Ser Thr Ser Thr Ala Ile Thr Leu 100 105 110 Thr Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Lys Glu Val 115 120 125 Val Leu Leu Asp Phe Ala Ala Met Lys Gly Glu Leu Gly Trp Leu Thr 130 135 140 His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asp Asp 145 150 155 160 Met Pro lie Tyr Met Tyr Ser Val Cys Asn Val Val Ser Gly Asp Gin 165 170 175 Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Glu Glu Ala Glu Arg 180 185 190 lie Phe lie Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro 195 200 205 Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu 210 215 220 Ser Asp Val Asp Tyr Gly Thr Asn Phe Gin Lys Arg Gin Phe Thr Lys 225 230 235 240 lie Asp Thr lie Ala Pro Asp Glu lie Thr Val Ser Ser Asp Phe Glu 245 250 255 Ala Arg Asn Val Lys Leu Asn Val Glu Glu Arg Met Val Gly Pro Leu 260 265 270 Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gin Asp lie Gly Ala Cys Val 275 280 285 Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Met Leu 290 295 300 Gln Ser Leu Ala Arg Phe Pro Glu Thr lie Ala Val Ala Val Ser Asp 305 310 315 320 Thr Gin Pro Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val 325 330 335 Val Pro Tyr Gly Gly Glu Gly Pro Leu Met His Cys Thr Val Asp Gly 340 345 350 Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Glu Gly Tyr Glu 355 360 365 Lys Val Glu Asp Ala Cys Arg Ala Cys Ser Pro Gly Phe Phe Lys Ser 370 375 380 Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro 385 390 395 400 Ser Thr Glu Gly Ala Thr Ser Cys Gin Cys Glu Glu Gly Tyr Phe Arg 405 410 415 Ala Pro Glu Asp Pro Leu Ser Met Ser Cys Thr Arg Pro Pro Ser Ala 420 425 430 Pro Asn Tyr Leu Thr Ala Ile Gly Met Gly Ala Lys Val Glu Leu Arg 435 440 445 Trp Thr Ala Pro Lys Asp Thr Gly Gly Arg Gin Asp Ile Val Tyr Ser 450 455 460 Val Thr Cys Glu Gin Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys 465 470 475 480 Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Ala Leu Thr Arg Thr 485 490 495 Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Ala 500 505 510 Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe 515 520 525 Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg 530 535 540 Leu Glu Asp Arg Ser Thr Thr Ser Leu Ser Val Thr Trp Ser Ile Pro 545 550 555 560 Val Ser Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys 565 570 575 Lys Gly Asp Ala Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser 580 585 590 Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val 595 600 605 Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu 610 615 620 Phe Gln Thr Leu Ser Thr Glu Gly Ser Ala Asn Met Ala Val 625 630 635 <210> 10 <211> 638 <212> PRT <213> Artificial Sequence <400> 10 Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala 1 5 10 15 Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys 20 25 30 Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr 35 40 45 Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg 50 55 60 Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val 65 70 75 80 Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln 85 90 95 Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu 100 105 110 Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ala Gln Gly Lys 115 120 125 Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp 130 135 140 Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met 145 150 155 160 Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly 165 170 175 Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala 180 185 190 Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser 195 200 205 Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr 210 215 220 Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe 225 230 235 240 Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp 245 250 255 Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly 260 265 270 Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala 275 280 285 Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu 290 295 300 Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp 305 310 315 320 Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val 325 330 335 Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp Gly 340 345 350 Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu 355 360 365 Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe 370 375 380 Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro 385 390 395 400 Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg 405 410 415 Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala 420 425 430 Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg 435 440 445 Trp Thr Pro Pro Gin Asp Ser Gly Gly Arg Glu Asp lie Val Tyr Ser 450 455 460 Val Thr Cys Gin Gin Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys 465 470 475 480 Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr 485 490 495 Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr 500 505 510 Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe 515 520 525 Arg Thr Ala Ser Val Ser lie Asn Gin Thr Glu Pro Pro Lys Val Arg 530 535 540 Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser lie Pro 545 550 555 560 Pro Pro Gin Gin Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys 565 570 575 Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser 580 585 590 Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gin Val 595 600 605 Gln Ala Leu Thr Gin Glu Gly Gin Gly Ala Gly Ser Lys Val His Glu 610 615 620 Phe Gin Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu Ala Val 625 630 635 <210> 11 <211> 25 <212> PRT <213> Artificial Sequence <400> 11 Met Glu Leu Arg Ala Val Gly Phe Cys Leu Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Gin Gly 20 25 <210> 12 <211> 24 <212> PRT <213> Artificial Sequence <400> 12 Met Glu Leu Gin Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys 1 5 10 15 Ala Leu Ala Ala Ala Ala Ala Ala 20 <210> 13 <211> 4910 <212> DNA <213> Artificial Sequence <400> 13 gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240 ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420 gcggttttgg cagtacatca atgggcgtgg atagcggtttt gactcacggg gatttccaag 480 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540 aaaatgtcgt aaaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660 attaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720 accgagctcg gatccgaatt cgccaccatg agacttctcc tcctgacttt cctgggagtc 780 tgctgcctca ccccatgggt tgtggaaggt gtggggactg aagtcctaga agagagtagc 840 tgtgtgaact tacaaaccca gcggctgcca gttcaaaaaa tcaagaccta tatcatctgg 900 gagggggcca tgagagctgt aatttttgtc accaaacgag gactaaaaat ttgtgctgat 960 ccagaagcca aatgggtgaa agcagcgatc aagactgtgg atggcagggc cagtaccaga 1020 aagaacatgg ctgaaactgt tcccacagga gcccagaggt ccaccagcac agcgataacc 1080 ctgactgggg gcggaggcgg aggatcaggg ggagggggag gaaaggaagt tgttttgttg 1140 gacttcgcag caatgaaggg agagctcggc tggctcacgc acccctatgg caaagggtgg 1200 gacctgatgc agaacatcat ggacgacatg cctatctaca tgtactcggt gtgcaacgtg 1260 gtatccggcg accaggacaa ctggctccgc accaactggg tgtaccggga ggaggccgag 1320 cgcatcttta ttgagctcaa gttcacggtg cgagactgta acagcttccc gggtggcgcc 1380 agctcatgca aagagacctt caacctctac tatgcagagt cagatgtgga ctatggcacc 1440 aacttccaga agcgccagtt caccaagatt gacaccatcg cccctgacga gatcacggtc 1500 agcagtgact tcgaggctcg caatgtcaag ctgaacgtag aggagcgcat ggtggggccc 1560 cttacccgga agggcttcta cctggccttc caggacatcg gcgcctgcgt ggcgctgctc 1620 tccgttcgcg tctactacaa gaagtgtccc gagatgctgc agagcttggc tcgcttcccc 1680 gagaccattg ctgtcgctgt ctccgataca caacccctgg ccacggtggc cggtacctgc 1740 gtggaccatg ccgtggtgcc ttatgggggc gaggggcctc tcatgcactg cacggtggat 1800 ggcgagtggc tggtgcccat cgggcagtgc ctgtgccagg aaggctacga gaaggtcgag 1860 gatgcctgcc gagcctgttc tccaggattc ttcaagtctg aggcatctga gagcccttgc 1920 ctggagtgtc cagagcatac cctgccatcc acagagggtg ccacctcctg ccagtgtgaa 1980 gaaggctatt tcagggcacc tgaggaccca ctgtccatgt cttgcacacg tccaccctct 2040 gcccccaact acctcacggc cattggcatg ggtgccaaag tagaactgcg ttggacagct 2100 cccaaggaca ctggtggccg ccaggacatt gtctacagtg tcacttgcga acagtgctgg 2160 ccagagtctg gcgagtgtgg gccctgtgag gcgagcgtgc gctattcaga acctcctcac 2220 gccctgaccc gcacgagtgt gacagtcagt gacctggagc cccacatgaa ctataccttc 2280 gctgtcgaag cacgcaatgg tgtctcaggc ctggtgacta gccgaagctt ccggactgcc 2340 agcgtcagta ttaaccaaac agagcccccc aaagtgaggc tggaggaccg aagcaccacc 2400 tccctgagtg tcacctggag catcccggtg tcacagcaga gccgtgtgtg gaagtacgaa 2460 gtcacctacc gcaagaaggg ggatgccaac agctataatg tgcgccgcac ggaaggcttc 2520 tccgtgaccc tggatgacct tgctccggat accacgtacc tggtgcaggt gcaggcgctg 2580 acgcaggagg gccagggagc cggcagcaaa gtgcacgagt tccagacact gtccacggaa 2640 ggatctgcca acatggcggt ggattacaag gatgacgacg ataagtaagc ggccgcctcg 2700 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2760 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2820 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2880 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2940 ctggggatgc ggtgggctct atggcttcta ctgggcggtt ttatggacag caagcgaacc 3000 ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat 3060 ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg 3120 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3180 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3240 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 3300 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt 3360 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 3420 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 3480 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 3540 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 3600 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 3660 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3720 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 3780 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 3840 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 3900 cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt tcctgatgcg 3960 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg 4020 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4080 ctcatgagac aataaccctg ataaatgctt caataatagc acgtgctaaa acttcatttt 4140 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4200 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4260 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4320 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4380 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4440 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4500 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4560 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4620 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4680 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4740 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4800 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4860 gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt 4910 <210> 14 <211> 4910 <212> DNA <213> Artificial Sequence <400> 14 gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240 ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420 gcggttttgg cagtacatca atgggcgtgg atagcggtttt gactcacggg gatttccaag 480 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540 aaaatgtcgt aaaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660 attaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720 accgagctcg gatccgaatt cgccaccatg agacttctca tcctggccct ccttggcatc 780 tgctctctca ctgcatacat tgtggaaggt gtagggagtg aagtctcaga taagaggacc 840 tgtgtgagcc tcactaccca gcgactgccg gttagcagaa tcaagaccta caccatcacg 900 gaaggctcct tgagagcagt aatttttatt accaaacgtg gcctaaaagt ctgtgctgat 960 ccacaagcca catgggtgag agacgtggtc aggagcatgg acaggaaatc caacaccaga 1020 aataacatga tccagaccaa gccaacagga acccagcaat cgaccaatac agctgtgact 1080 ctgactggcg gaggcggagg atcaggggga gggggaggag cgcagggcaa ggaagtggta 1140 ctgctggact ttgctgcagc tggaggggag ctcggctggc tcacacaccc gtatggcaaa 1200 gggtgggacc tgatgcagaa catcatgaat gacatgccga tctacatgta ctccgtgtgc 1260 aacgtgatgt ctggcgacca ggacaactgg ctccgcacca actgggtgta ccgaggagag 1320 gctgagcgta tcttcattga gctcaagttt actgtacgtg actgcaacag cttccctggt 1380 ggcgccagct cctgcaagga gactttcaac ctctactatg ccgagtcgga cctggactac 1440 ggcaccaact tccagaagcg cctgttcacc aagattgaca ccattgcgcc cgatgagatc 1500 accgtcagca gcgacttcga ggcacgccac gtgaagctga acgtggagga gcgctccgtg 1560 gggccgctca cccgcaaagg cttctacctg gccttccagg atatcggtgc ctgtgtggcg 1620 ctgctctccg tccgtgtcta ctacaagaag tgccccgagc tgctgcaggg cctggcccac 1680 ttccctgaga ccatcgccgg ctctgatgca ccttccctgg ccactgtggc cggcacctgt 1740 gtggaccatg ccgtggtgcc accggggggt gaagagcccc gtatgcactg tgcagtggat 1800 ggcgagtggc tggtgcccat tgggcagtgc ctgtgccagg caggctacga gaaggtggag 1860 gatgcctgcc aggcctgctc gcctggattt tttaagtttg aggcatctga gagcccctgc 1920 ttggagtgcc ctgagcacac gctgccatcc cctgagggtg ccacctcctg cgagtgtgag 1980 gaaggcttct tccgggcacc tcaggaccca gcgtcgatgc cttgcacacg acccccctcc 2040 gccccacact acctcacagc cgtgggcatg ggtgccaagg tggagctgcg ctggacgccc 2100 cctcaggaca gcgggggccg cgaggacatt gtctacagcg tcacctgcga acagtgctgg 2160 ccggaggtgg ctgctggagg agcctgggga ggccctgtcc gggcctggaa ggccttcctc 180 ggactgaccc gcaccagtgt gacagtgagc gacctggagc cccacatgaa ctacaccttc 2280 accgtggagg cccgcaatgg cgtctcaggc ctggtaacca gccgcagctt ccgtactgcc 2340 agtgtcagca tcaaccagac agagcccccc aaggtgaggc tggagggccg cagcaccacc 2400 tcgcttagcg tctcctggag catccccccg ccgcagcaga gccgagtgtg gaagtacgag 2460 gtcacttacc gcaagaaggg agactccaac agctacaatg tgcgccgcac cgagggtttc 2520 tccgtgaccc tggacgacct ggccccagac accacctacc tggtccaggt gcaggcactg 2580 acgcaggagg gccagggggc cggcagcaag gtgcacgaat tccagacgct gtccccggag 2640 ggatctggca acttggcggt ggattacaag gatgacgacg ataagtaagc ggccgcctcg 2700 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2760 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2820 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2880 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2940 ctggggatgc ggtgggctct atggcttcta ctgggcggtt ttatggacag caagcgaacc 3000 ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat 3060 ggctttctcg ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg 3120 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3180 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3240 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 3300 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt 3360 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 3420 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 3480 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 3540 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 3600 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 3660 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3720 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 3780 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 3840 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 3900 cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt tcctgatgcg 3960 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacaggtgg cacttttcgg 4020 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4080 ctcatgagac aataaccctg ataaatgctt caataatagc acgtgctaaa acttcatttt 4140 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4200 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4260 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4320 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4380 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4440 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4500 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4560 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4620 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4680 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4740 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4800 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4860 gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt 4910 <210> 15 <211> 2931 <212> DNA <213> Artificial Sequence <400> 15 atggagctcc gggcagtcgg tttctgcctg gcgctgctgt ggggttgcgc gctggcggcc 60 gcggcggcac agggaaagga agttgttttg ttggacttcg cagcaatgaa gggagagctc 120 ggctggctca cgcaccccta tggcaaaggg tgggacctga tgcagaacat catggacgac 180 atgcctatct acatgtactc ggtgtgcaac gtggtatccg gcgaccagga caactggctc 240 cgcaccaact gggtgtaccg ggaggaggcc gagcgcatct ttattgagct caagttcacg 300 gtgcgagact gtaacagctt cccgggtggc gccagctcat gcaaagagac cttcaacctc 360 tactatgcag agtcagatgt ggactatggc accaacttcc agaagcgcca gttcaccaag 420 attgacacca tcgcccctga cgagatcacg gtcagcagtg acttcgaggc tcgcaatgtc 480 aagctgaacg tagaggagcg catggtgggg ccccttaccc ggaagggctt ctacctggcc 540 ttccaggaca tcggcgcctg cgtggcgctg ctctccgttc gcgtctacta caagaagtgt 600 cccgagatgc tgcagagctt ggctcgcttc cccgagacca ttgctgtcgc tgtctccgat 660 acacaacccc tggccacggt ggccggtacc tgcgtggacc atgccgtggt gccttatggg 720 ggcgaggggc ctctcatgca ctgcacggtg gatggcgagt ggctggtgcc catcgggcag 780 tgcctgtgcc aggaaggcta cgagaaggtc gaggatgcct gccgagcctg ttctccagga 840 ttcttcaagt ctgaggcatc tgagagccct tgcctggagt gtccagagca taccctgcca 900 tccacagagg gtgccacctc ctgccagtgt gaagaaggct atttcagggc acctgaggac 960 ccactgtcca tgtcttgcac acgtccaccc tctgccccca actacctcac ggccattggc 1020 atgggtgcca aagtagaact gcgttggaca gctcccaagg acactggtgg ccgccaggac 1080 attgtctaca gtgtcactlg cgaacagtgc tggccagagt ctggcgagtg tgggccctgt 1140 gaggcgagcg tgcgctattc agaacctcct cacgccctga cccgcacgag tgtgacagtc 1200 agtgacctgg agccccacat gaactatacc ttcgctgtcg aagcacgcaa tggtgtctca 1260 ggcctggtga ctagccgaag cttccggact gccagcgtca gtattaacca aacagagccc 1320 cccaaagtga ggctggagga ccgaagcacc acctccctga gtgtcacctg gagcatcccg 1380 gtgtcacagc agagccgtgt gtggaagtac gaagtcacct accgcaagaa gggggatgcc 1440 aacagctata atgtgcgccg cacggaaggc ttctccgtga ccctggatga ccttgctccg 1500 gataccacgt acctggtgca ggtgcaggcg ctgacgcagg agggccaggg agccggcagc 1560 aaagtgcacg agttccagac actgtccacg gaaggatctg ccaacatggc ggtgatcggc 1620 ggtgtggctg taggtgttgt tttgcttctg gtactggcag gagttggcct cttcatccat 1680 cgaaggagga ggaacctgcg ggctcgccag tcctctgagg atgtccgttt ttccaagtca 1740 gaacaactaa agcccctgaa gacctatgtg gatcctcaca cttacgaaga ccccaaccag 1800 gctgtactca agtttaccac cgagatccac ccatcctgtg tggcaaggca gaaggtcatt 1860 ggagcaggag agtttggaga ggtctataaa gggacgctga aggcatcctc ggggaagaag 1920 gagataccgg tggccatcaa gacactgaaa gcgggctaca ctgagaagca gcgggtggac 1980 ttcctgagcg aggccagcat catgggccag tttagccacc acaatatcat ccgcctggag 2040 ggcgtggtct ctaaatacaa acccatgatg attatcacag agtacatgga gaatggagcg 2100 ctagacaagt tccttaggga gaaggatggt gagttcagtg tacttcagtt ggtgggcatg 2160 ctgaggggta tcgcatccgg catgaagtac ctggccaaca tgaactacgt gcaccgggac 2220 ctggccgccc gcaacatcct cgtcaacagc aacctggtgt gcaaggtgtc cgattttggc 2280 ctgtcgcgtg tgctggaaga tgaccccgag gccacctaca ccacaagtgg cggcaagatc 2340 cctattcgat ggacagcccc agaggccatt tcctaccgca agttcacctc agccagcgat 2400 gtgtggagct acggcattgt catgtgggaa gtgatgactt atggcgaacg gccctactgg 2460 gaactgtcaa accacgaggt catgaaagcc atcaacgacg gcttccggct ccctacgccc 2520 atggactgcc cttcagccat ttaccagctc atgatgcagt gctggcagca agagcgctcc 2580 cgccgaccca agtttgccga catcgttagc atcctggaca agctcatccg agcccccgac 2640 tccctcaaga cgctggctga ctttgatccc cgagtgtcca tccggctgcc cagcaccagc 2700 ggctcggagg gagtcccctt ccgtacggtg tccgagtggc tggagagcat caagatgcaa 2760 cagtacacgg aacacttcat ggtggctggc tacacggcca tcgagaaggt ggtacagatg 2820 tccaacgaag acatcaaaag gatcggagtg cgtcttcctg gccaccagaa gcgtattgcc 2880 tacagcctgc tgggactcaa ggaccaggtc aacacagtgg ggattcctat c 2931 <210> 16 <211> 2928 <212> DNA <213> Artificial Sequence <400> 16 atggagctcc aggcagcccg cgcctgcttc gccctgctgt ggggctgtgc gctggccgcg 60 gccgcggcgg cgcagggcaa ggaagtggta ctgctggact ttgctgcagc tggaggggag 120 ctcggctggc tcacacaccc gtatggcaaa gggtgggacc tgatgcagaa catcatgaat 180 gacatgccga tctacatgta ctccgtgtgc aacgtgatgt ctggcgacca ggacaactgg 240 ctccgcacca actgggtgta ccgaggagag gctgagcgta tcttcattga gctcaagttt 300 actgtacgtg actgcaacag cttccctggt ggcgccagct cctgcaagga gactttcaac 360 ctctactatg ccgagtcgga cctggactac ggcaccaact tccagaagcg cctgttcacc 420 aagattgaca ccattgcgcc cgatgagatc accgtcagca gcgacttcga ggcacgccac 480 gtgaagctga acgtggagga gcgctccgtg gggccgctca cccgcaaagg cttctacctg 540 gccttccagg atatcggtgc ctgtgtggcg ctgctctccg tccgtgtcta ctacaagaag 600 tgccccgagc tgctgcaggg cctggcccac ttccctgaga ccatcgccgg ctctgatgca 660 ccttccctgg ccactgtggc cggcacctgt gtggaccatg ccgtggtgcc accggggggt 720 gaagagcccc gtatgcactg tgcagtggat ggcgagtggc tggtgcccat tgggcagtgc 780 ctgtgccagg caggctacga gaaggtggag gatgcctgcc aggcctgctc gcctggattt 840 tttaagtttg aggcatctga gagcccctgc ttggagtgcc ctgagcacac gctgccatcc 900 cctgagggtg ccacctcctg cgagtgtgag gaaggcttct tccgggcacc tcaggaccca 960 gcgtcgatgc cttgcacacg acccccctcc gccccacact acctcacagc cgtgggcatg 1020 ggtgccaagg tggagctgcg ctggacgccc cctcaggaca gcgggggccg cgaggacatt 1080 gtctacagcg tcacctgcga acagtgctgg cccgagtctg gggaatgcgg gccgtgtgag 1140 gccagtgtgc gctactcgga gcctcctcac ggactgaccc gcaccagtgt gacagtgagc 1200 gacctggagc cccacatgaa ctacaccttc accgtggagg cccgcaatgg cgtctcaggc 1260 ctggtaacca gccgcagctt ccgtactgcc agtgtcagca tcaaccagac agagcccccc 1320 aaggtgaggc tggagggccg cagcaccacc tcgcttagcg tctcctggag catccccccg 1380 ccgcagcaga gccgagtgtg gaagtacgag gtcacttacc gcaagaaggg agactccaac 1440 agctacaatg tgcgccgcac cgagggtttc tccgtgaccc tggacgacct ggccccagac 1500 accacctacc tggtccaggt gcaggcactg acgcaggagg gccagggggc cggcagcaag 1560 gtgcacgaat tccagacgct gtccccggag ggatctggca acttggcggt gattggcggc 1620 gtggctgtcg gtgtggtcct gcttctggtg ctggcaggag ttggcttctt tatccaccgc 1680 aggaggaaga accagcgtgc ccgccagtcc ccggaggacg tttacttctc caagtcagaa 1740 caactgaagc ccctgaagac atacgtggac ccccacacat atgaggaccc caaccaggct 1800 gtgttgaagt tcactaccga gatccatcca tcctgtgtca ctcggcagaa ggtgatcgga 1860 gcaggagagt ttggggaggt gtacaagggc atgctgaaga catcctcggg gaagaaggag 1920 gtgccggtgg ccatcaagac gctgaaagcc ggctacacag agaagcagcg agtggacttc 1980 ctcggcgagg ccggcatcat gggccagttc agccaccaca acatcatccg cctagagggc 2040 gtcatctcca aatacaagcc catgatgatc atcactgagt acatggagaa tggggccctg 2100 gacaagttcc ttcgggagaa ggatggcgag ttcagcgtgc tgcagctggt gggcatgctg 2160 cggggcatcg cagctggcat gaagtacctg gccaacatga actatgtgca ccgtgacctg 2220 gctgcccgca acatcctcgt caacagcaac ctggtctgca aggtgtctga ctttggcctg 2280 tcccgcgtgc tggaggacga ccccgaggcc acctacacca ccagtggcgg caagatcccc 2340 atccgctgga ccgccccgga ggccatttcc taccggaagt tcacctctgc cagcgacgtg 2400 tggagctttg gcattgtcat gtgggaggtg atgacctatg gcgagcggcc ctactgggag 2460 ttgtccaacc acgaggtgat gaaagccatc aatgatggct tccggctccc cacacccatg 2520 gactgcccct ccgccatcta ccagctcatg atgcagtgct ggcagcagga gcgtgcccgc 2580 cgccccaagt tcgctgacat cgtcagcatc ctggacaagc tcattcgtgc ccctgactcc 2640 ctcaagaccc tggctgactt tgacccccgc gtgtctatcc ggctccccag cacgagcggc 2700 tcggaggggg tgcccttccg cacggtgtcc gagtggctgg agtccatcaa gatgcagcag 2760 tatacggagc acttcatggc ggccggctac actgccatcg agaaggtggt gcagatgacc 2820 aacgacgaca tcaagaggat tggggtgcgg ctgcccggcc accagaagcg catcgcctac 2880 agcctgctgg gactcaagga ccaggtgaac actgtgggga tccccatc 2928 <210> 17 <211> 345 <212> DNA <213> Artificial Sequence <400> 17 atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60 ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120 ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180 gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240 atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300 ggagcccaga ggtccaccag cacagcgata accctgactg ggggc 345 <210> 18 <211> 342 <212> DNA <213> Artificial Sequence <400> 18 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60 ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120 ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180 attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240 gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300 ggaacccagc aatcgaccaa tacagctgtg actctgactg gc 342 <210> 19 <211> 63 <212> DNA <213> Artificial Sequence <400> 19 atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60 ggt 63 <210> 20 <211> 63 <212> DNA <213> Artificial Sequence <400> 20 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60 ggt 63 <210> 21 <211> 1539 <212> DNA <213> Artificial Sequence <400> 21 aaggaagttg ttttgttgga cttcgcagca atgaagggag agctcggctg gctcacgcac 60 ccctatggca aagggtggga cctgatgcag aacatcatgg acgacatgcc tatctacatg 120 tactcggtgt gcaacgtggt atccggcgac caggacaact ggctccgcac caactgggtg 180 taccgggagg aggccgagcg catctttatt gagctcaagt tcacggtgcg agactgtaac 240 agcttcccgg gtggcgccag ctcatgcaaa gagaccttca acctctacta tgcagagtca 300 gatgtggact atggcaccaa cttccagaag cgccagttca ccaagattga caccatcgcc 360 cctgacgaga tcacggtcag cagtgacttc gaggctcgca atgtcaagct gaacgtagag 420 gagcgcatgg tggggcccct tacccggaag ggcttctacc tggccttcca ggacatcggc 480 gcctgcgtgg cgctgctctc cgttcgcgtc tactacaaga agtgtcccga gatgctgcag 540 agcttggctc gcttccccga gaccattgct gtcgctgtct ccgatacaca acccctggcc 600 acggtggccg gtacctgcgt ggaccatgcc gtggtgcctt atggggcga ggggcctctc 660 atgcactgca cggtggatgg cgagtggctg gtgcccatcg ggcagtgcct gtgccagggaa 720 ggctacgaga aggtcgagga tgcctgccga gcctgttctc caggattctt caagtctgag 780 gcatctgaga gcccttgcct ggagtgtcca gagcataccc tgccatccac agagggtgcc 840 acctcctgcc agtgtgaaga aggctatttc agggcacctg aggacccact gtccatgtct 900 tgcacacgtc caccctctgc ccccaactac ctcacggcca ttggcatggg tgccaaagta 960 gaactgcgtt ggacagctcc caaggacact ggtggccgcc aggacattgt ctacagtgtc 1020 acttgcgaac agtgctggcc agagtctggc gagtgtgggc cctgtgaggc gagcgtgcgc 1080 1140 cacatgaact ataccttcgc tgtcgaagca cgcaatggtg tctcaggcct ggtgactagc 1200 cgaagcttcc ggactgccag cgtcagtatt aaccaaacag agccccccaa agtgaggctg 1260 gagaccgaa gcaccacctc cctgagtgtc acctggagca tcccggtgtc acagcagagc 1320 cgtgtgtgga agtacgaagt cacctaccgc aagaaggggg atgccaacag ctataatgtg 1380 cgccgcacgg aaggcttctc cgtgaccctg gatgaccttg ctccggatac cacgtacctg 1440 gtgcaggtgc aggcgctgac gcaggagggc cagggagccg gcagcaaagt gcacgagttc 1500 cagacactgt ccacggaagg atctgccaac atggcggtg 1539 <210> 22 <211> 1542 <212> DNA <213> Artificial Sequence <400> 22 gcgcagggca aggaagtggt actgctggac tttgctgcag ctggagggga gctcggctgg 60 ctcacacacc cgtatggcaa agggtgggac ctgatgcaga acatcatgaa tgacatgccg 120 atctacatgt actccgtgtg caacgtgatg tctggcgacc aggacaactg gctccgcacc 180 aactgggtgt accgaggaga ggctgagcgt atcttcattg agctcaagtt tactgtacgt 240 gactgcaaca gcttccctgg tggcgccagc tcctgcaagg agactttcaa cctctactat 300 gccgagtcgg acctggacta cggcaccaac ttccagaagc gcctgttcac caagattgac 360 accattgcgc ccgatgagat caccgtcagc agcgacttcg aggcacgcca cgtgaagctg 420 aacgtggagg agcgctccgt ggggccgctc acccgcaaag gcttctacct ggccttccag 480 gatatcggtg cctgtgtggc gctgctctcc gtccgtgtct actacaagaa gtgccccgag 540 ctgctgcagg gcctggccca cttccctgag accatcgccg gctctgatgc accttccctg 600 gccactgtgg ccggcacctg tgtggaccat gccgtggtgc caccgggggg tgaagagccc 660 cgtatgcact gtgcagtgga tggcgagtgg ctggtgccca ttgggcagtg cctgtgccag 720 gcaggctacg agaaggtgga ggatgcctgc caggcctgct cgcctggatt ttttaagttt 780 gaggcatctg agagcccctg cttggagtgc cctgagcaca cgctgccatc ccctgagggt 840 gccacctcct gcgagtgtga ggaaggcttc ttccgggcac ctcaggaccc agcgtcgatg 900 ccttgcacac gacccccctc cgccccacac tacctcacag ccgtgggcat gggtgccaag 960 gtggagctgc gctggacgcc ccctcaggac agcgggggcc gcgaggacat tgtctacagc 1020 gtcacctgcg aacagtgctg gcccgagtct ggggaatgcg ggccgtgtga ggccagtgtg 1080 cgctactcgg agcctcctca cggactgacc cgcaccagtg tgacagtgag cgacctggag 1140 CCCCACATGA ACTACACCTT CACCCTGGAG GCCCGCAATG GCCTCTCAGG CCTGGTAACC 1200 AGCCGCAGCT TCCGTACTGC CAGTGTCAAC ATCAACCAGA CAGAGCCCCC CAAGGTGAGG 1260 CTGGAGGGCC GCAGCACCAC CTCGCTTAGC GTCTCCTGGA GCATCCCCCCC GCAGCAGCA 1320 AGCCGAGTGT GGAAGTACGA GGTCACTTAC CGCAAGAGGG GAGACTCCAA CAGCTACAA 1380 TGTGGCCGCA CCCGAGGGTT CTCCGTGACC CTGGACGACC TGGCCCCAGA CACCACCTAC 1440 CTGGTCCAGG TGCAGGCACG TACGCAGGAG GGCCAGGGGG CCAGCAGCAA GGTGCACGAA 1500 TTCCAGACGC TGTCCCCGGA GGGATCTGGC AACTTGGCGG TG 1542 <210> 23 <211> 1914 <212> DNA <213> Artificial Sequence <400> 23 ATGAGACTTC TCCTCCTGAC TTTCCTGGGA GTCTGCTGCC TCACCCCATG GGTGTGGGAA 60 GTTGTGGGGA CTGAAGTCCT AGAAGAGAGT AGCTGTGTGA ACTTACAAAC CCAGCAGCTG 120 CCAGTTCAAA AAATCAAGAC CTTATATCAT TGGGAGGGGG CCAATGAGAG CTGTAATTTT 180 gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240 atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300 ggagcccaga ggtccaccag cacagcgata accctgactg ggggcggagg cggaggatca 360 gggggagggg gaggaaagga agttgttttg ttggacttcg cagcaatgaa gggagagctc 420 ggctggctca cgcaccccta tggcaaaggg tgggacctga tgcagaacat catggacgac 480 atgcctatct acatgtactc ggtgtgcaac gtggtatccg gcgaccagga caactggctc 540 cgcaccaact gggtgtaccg ggaggaggcc gagcgcatct ttattgagct caagttcacg 600 gtgcgagact gtaacagctt cccgggtggc gccagctcat gcaaagagac cttcaacctc 660 tactatgcag agtcagatgt ggactatggc accaacttcc agaagcgcca gttcaccaag 720 attgacacca tcgcccctga cgagatcacg gtcagcagtg acttcgaggc tcgcaatgtc 780 aagctgaacg tagaggagcg catggtgggg cccttaccc ggaagggctt ctacctggcc 840 ttccaggaca tcggcgcctg cgtggcgctg ctctccgttc gcgtctacta caagaagtgt 900 cccgagatgc tgcagagctt ggctcgcttc cccgagacca ttgctgtcgc tgtctccgat 960 acaacacccc tggccacggt ggccggtacc tgcgtggacc atgccgtggt gccttatggg 1020 ggcgaggggc ctctcatgca ctgcacggtg gatggcgagt ggctggtgcc catcgggcag 1080 tgcctgtgcc aggaaggcta cgagaaggtc gaggatgcct gccgagcctg ttctccagga 1140 ttcttcaagt ctgaggcatc tgagagccct tgcctggagt gtccagagca taccctgcca 1200 tccacagagg gtgccacctc ctgccagtgt gaaaggct atttcagggc acctgaggac 1260 ccactgtcca tgtcttgcac acgtccaccc tctgccccca actacctcac ggccattggc 1320 atgggtgcca aagtagaact gcgttggaca gctcccaagg acactggtgg ccgccaggac 1380 attgtctaca gtgtcacttg cgaacagtgc tggccagagt ctggcgagtg tgggccctgt 1440 gaggcgagcg tgcgctattc agaacctcct cacgccctga cccgcacgag tgtgacagtc 1500 agtgacctgg agccccacat gaactatacc ttcgctgtcg aagcacgcaa tggtgtctca 1560 ggcctggtga ctagccgaag cttccggact gccagcgtca gtattaacca aacagagccc 1620 cccaaagtga ggctggagga ccgaagcacc acctccctga gtgtcacctg gagcatcccg 1680 gtgtcacagc agagccgtgt gtggaagtac gaagtcacct accgcaagaa gggggatgcc 1740 aacagctata atgtgcgccg cacggaaggc ttctccgtga ccctggatga ccttgctccg 1800 gataccacgt acctggtgca ggtgcaggcg ctgacgcagg agggccaggg agccggcagc 1860 aaagtgcacg agttccagac actgtccacg gaaggatctg ccaacatggc ggtg 1914 <210> 24 <211> 1914 <212> DNA <213> Artificial Sequence <400> 24 atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60 ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120 ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180 attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacatgggt gagagacgtg 240 gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300 ggaacccagc aatcgaccaa tacagctgtg actctgactg gcggaggcgg aggatcaggg 360 ggagggggag gagcgcaggg caaggaagtg gtactgctgg actttgctgc agctggaggg 420 gagctcggct ggctcacaca cccgtatggc aaagggtggg acctgatgca gaacatcatg 480 aatgacatgc cgatctacat gtactccgtg tgcaacgtga tgtctggcga ccaggacaac 540 tggctccgca ccaactgggt gtaccgagga gaggctgagc gtatcttcat tgagctcaag 600 tttactgtac gtgactgcaa cagcttccct ggtggcgcca gctcctgcaa ggagactttc 660 aacctctact atgccgagtc ggacctggac tacggcacca acttccagaa gcgcctgttc 720 accaagattg acaccattgc gcccgatgag atcaccgtca gcagcgactt cgaggcacgc 780 cacgtgaagc tgaacgtgga ggagcgctcc gtggggccgc tcacccgcaa aggcttctac 840 ctggccttcc aggatatcgg tgcctgtgtg gcgctgctct ccgtccgtgt ctactacaag 900 aagtgccccg agctgctgca gggcctggcc cacttccctg agaccatcgc cggctctgat 960 gcaccttccc tggccactgt ggccggcacc tgtgtggacc atgccgtggt gccaccgggg 1020 GAGAAGCGTA ATATTAAAGA AGTTTCGGCT GATTCGGCTA ATGCCTGCGG 240 TGCCTGTGCC AGGCAGGCTA CGAGAAGGTG GAGGATGCCT GCCAGGCCTG CTCGCCTGGA 1140 TTTTTTAAAG TTTGAGGCAT CTGAGAGCCC CTGCTTGGAG TZCCTGAGCA CACGCTGCCA 1200 TCCCCTGAGG GTGCCACCTC CTGCGAGTGT GAGGAAGGCT TCTTCCGGGC ACCTCAGGAC 1260 CCAGCGTCGA TGCCTTGCAC ACGACCCCCC TCCGCCCCAC ACTACCTCAC AGCCGTGGGC 1320 ATGGGTGCCA AGGTGGAGCT GCGCTGGACG CCCCCTCAGG ACAGCGGGGC CGCGAGGAC 1380 ATTGTCTACA GCCTCACCTG CAAAGTGCCT GGCCCGAGTC TGGGGAATGC GGGCCGTGT 1440 GAGGCCAGTG TGCCTACTCG GAGCCTCCTC ACGGACTGAC CCACCCAGTG TGACAGTGT 1500 AGCGACCTGG AGCCCCACAT GAACIACACC TTCACCCTGG AGGCCCGCAA TGGCCTCTCA 1560 GGCCTGGTA ACCAGCCGCA GCTTCCGTAC TGCCAGTGTC AGCATCAACC AGACAGAGCC C 1620 CCCAAGGTGA GGCTGGAGGG CCACAGCACCA CCTCGCTTAC GCTCTCCTG GAGCATCCCC 1680 CCGCCGCAGC AGAGCCGAGT GTGGAAGTAC GAGGTCACCT ACCGCAAGAA GGGAGACTCC 1740 aacagctaca atgtgcgccg caccgagggt ttctccgtga ccctggacga cctggcccca 1800 gacaccacct acctggtcca ggtgcaggca ctgacgcagg agggccaggg ggccggcagc 1860 aaggtgcacg aattccagac gctgtccccg gagggatctg gcaacttggc ggtg 1914 <210> 25 <211> 75 <212> DNA <213> Artificial Sequence <400> 25 atggagctcc gggcagtcgg tttctgcctg gcgctgctgt ggggttgcgc gctggcggcc 60 gcggcggcac aggga 75 <210> 26 <211> 69 <212> DNA <213> Artificial Sequence <400> 26 atggagctcc aggcagcccg cgcctgcttc gccctgctgt ggggctgtgc gctggccgcg 60 gccgcggcg 69

Claims

1. A fusion protein, characterized in that, an amino acid sequence of an extracellular region of EPHA2 capable of eliciting specific CD8+ T responses or an optimized version of a MHC class I molecule-binding epitope thereof; a linker; and a human or murine XCL1 protein that specifically binds to DC cells having an antigen cross-presentation ability; the amino acid sequence of the fusion protein is shown as SEQ ID No. 9 or SEQ ID No.

10.

2. A nucleic acid molecule encoding the fusion protein according to claim 1, comprising DNA and / or mRNA, characterized in that, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No. 23 or SEQ ID No.

24.

3. A recombinant expression vector, characterized in that, the nucleic acid molecule of claim 2.

4. The recombinant expression vector of claim 3, wherein, the vector is pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP or pVAX1.

5. A recombinant strain or cell comprising the fusion protein of claim 1 or the nucleic acid molecule of claim 2.

6. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant strain or cell of claim 5 in the preparation of a vaccine for colorectal cancer overexpressing EPHA2 and / or in the preparation of a medicament for preventing and / or treating colorectal cancer overexpressing EPHA2.

7. A vaccine targeting colorectal cancer of EPHA2, characterized in that, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant strain or cell of claim 5, and a pharmaceutically acceptable carrier, excipient and / or adjuvant.

8. A medicament for preventing and / or treating colorectal cancer overexpressing EPHA2, characterized by, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant strain or cell of claim 5, and a pharmaceutically acceptable carrier, excipient and / or adjuvant.

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