CN111440244B - Metastatic cancer vaccine targeting VEGFR2 - Google Patents

Abstract

The present invention relates to the field of immunotherapy and prevention of cancer, and in particular to metastatic cancer vaccines targeting VEGFR2 (KDR). According to the invention, through fusion expression of the VEGFR2 protein for promoting tumor angiogenesis and the DC cell ligand XCL1, the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein expressed by the nucleic acid vaccine can be effectively combined with DC cells, induces VEGFR2 specific T cell response and remarkably inhibits the growth of VEGFR 2-highly-expressed tumors in various models.

Description

Metastatic cancer vaccine targeting VEGFR2

Technical Field

The present invention relates to the field of immunotherapy and prevention of cancer, and in particular to metastatic cancer vaccines targeting VEGFR 2.

Background

Tumor metastasis is the leading cause of cancer mortality, with over 90% of cancer mortality due to metastasis, and tumor vascularization essential for tumor growth and metastasis (Valastyan and Weinberg). Tumor blood vessels provide sufficient oxygen and nutrients for tumor growth and metastasis. The formation of new blood vessels is a necessary step for the formation of tumor blood vessels. The theory of neovascularization holds that tumors induce the formation of new blood vessels from existing blood vessels. Based on this theory, researchers developed a series of drugs targeting tumor neovascularization, and the current FDA-approved anti-angiogenesis drugs are mainly classified into three classes, monoclonal antibodies, receptor tyrosine kinase inhibitors, and fused polypeptides. For example, bevacizumab, sorafenib and VEGFR1/2 protein extracellular domain fusion protein can be used for treating metastatic cancer, and can effectively improve the progression-free survival period (Cascone and Heymach) of patients. However, many problems still exist in the current anti-angiogenesis drugs, and in the case of bevacizumab, frequent parenteral administration is required, side effects are serious, obvious drug resistance is achieved, and the rebound progress of tumor after metastasis is caused, and the like, and the problems need to be solved urgently.

Vascular Endothelial Growth Factor (VEGF) is the most important regulator of neovascularization and functions primarily by binding to the three receptor tyrosine kinases VEGFR 1/2/3. VEGFR1 has been shown to exert a negative feedback inhibition effect on angiogenesis due to its high affinity for VEGF but low intracellular enzyme activity, VEGFR3 is mainly expressed in adult lymphatic vessels, and VEGFR2 is a promoter that mainly mediates VEGF-induced angiogenesis and increased endothelial cell permeability (Dong et al.). VEGFR2 knockout mice develop to 8.5-9.5 days and die because angiogenesis is impaired (Shalaby et al.). VEGFR2 is highly expressed in tumor-associated endothelial cells in tumor tissues in much higher amounts than normal vascular cells, and VEGFR2 is also highly expressed in certain types of tumor cells, such as B-cell lymphoma, breast cancer, lung cancer, bladder cancer, melanoma, and the like (Decaussin et al, 1999; El-Obeid et al, 2004; Gakiopoulou-Givalou et al, 2003; Kranz et al, 1999) in addition to tumor-associated endothelial cells, thus VEGFR2 is an ideal target for tumor therapy. The bevacizumab and the sorafenib are antitumor drugs developed by taking VEGFR2 as targets.

Although highly expressed in certain tumors and tumor-associated endothelial cells, the vascular endothelial growth factor receptor 2 (VEGFR 2) is an embryonic tumor-associated antigen, and therefore, T cells that recognize and bind with high affinity to VEGFR2 have been eliminated by negative selection in the thymus during immune development of somatic cells (Houghton et al, 2007). Only T cells with lower affinity for VEGFR2 survived selection of the thymus, however these T cells were not activated by the body's own VEGFR 2. Therefore, although tumor cells highly express VEGFR2, the body itself cannot produce effector T cells that effectively recognize tumor cells highly expressing VEGFR2, thereby inhibiting angiogenesis and tumor growth. This phenomenon is called immune tolerance. Previous studies show that the generation of T cells which do not originally recognize tumor-associated antigens such as VEGFR2 can be promoted by mutation optimization of certain epitopes in the tumor-associated antigens such as VEGFR2 with enhanced affinity with the T cells, so that the immune tolerance of VEGFR2 is broken (Guevara-Patino et al, 2006).

Previous studies showed that clinical phase I/II experiments were performed using predicted and validated HLA-a2 and HLA-a24 restricted VEGFR2 immunogenic polypeptides (VEGFR 2169-177: RFVPDGNRI and VEGFR 2773-781: VLAMFFWLL, respectively) to immunize patients with advanced metastatic colorectal cancer as well as patients with neurofibromas, and the results showed that, following stimulation with polypeptide vaccines, an increased proportion of VEGFR2 specific cytotoxic T Cells (CTLs) inhibited tumor neovascularization, increased the number of intratumoral CD8+ T cells, eliminated VEGFR2 positive tumor cells, reduced tumor burden, and increased overall survival of patients (Hazama et al, 2014) and (Tamura et al, 2019). However, the research contents of the human beings are only suitable for people carrying two subtypes of HLA-A2 and A24, and the immune polypeptides of HLA-A2 and HLA-A24 restricted VEGFR may not have any effect on other HLA subtype people because T cell immune response has HLA restriction and polypeptide immunity generally cannot effectively cause antibody-mediated humoral immune response. And the limited extent of immune response that can be elicited by the immunizing polypeptide without epitope optimization may limit the effectiveness of such vaccines. In view of the above, there is a need to design a prophylactic and therapeutic vaccine targeting VEGFR2 that can effectively break immune tolerance, antagonize neovascularization via cell-specific immune responses, and inhibit metastatic cancer foci formation.

Antigen-presenting cells refer to a class of cells that can process antigens and present antigenic peptides to T cells in the form of antigenic peptide-MHC molecule complexes, which play an important role in immune recognition, immune response, and immune regulation in the body. Dendritic Cell (DC) is one of the antigen presenting cells of the body, and is the only antigen presenting cell in the body that can present antigen peptide to the original T cell and induce T cell activation and proliferation, and has the strongest function, and is the initiator of the body's adaptive immune response. DC cells can be classified into two major categories, classical DC and plasma cell-like DC, according to morphology and function. Plasma cell-like DCs mainly mediate adaptive immune responses after viral infection, and classical DCs have the strongest ability to process presented antigens, and mediate processes such as the elimination of dead cells and the identification of tumor cells in the body (Carreno et al, 2017; Minetto et al, 2019). Thus, more efficient presentation of tumor antigens to classical DCs is key to vaccine success, and studies have found that classical DCs express a class of receptors for the chemokine XCL1, and thus XCL1 molecules can efficiently bind to classical DC cells (Dorner et al, 2009).

Disclosure of Invention

In view of this, the present invention provides metastatic cancer vaccines that target VEGFR 2. According to the invention, through fusion expression of the VEGFR2 protein for promoting tumor angiogenesis and the DC cell ligand XCL1, the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein expressed by the nucleic acid vaccine can be effectively combined with DC cells, induces VEGFR2 specific T cell response and obviously inhibits the growth of melanoma with high VEGFR2 expression in various models.

In order to achieve the above object, the present invention provides the following technical solutions:

the fusion protein provided by the invention comprises an extracellular region of VEFGR2 capable of inducing specific CD8+ T response or an extracellular region of MHCI-type molecule binding epitope optimized form thereof for promoting tumor angiogenesis; a joint; and human or murine XCL1 protein that specifically binds DC cells with cross-antigen presentation capability;

wherein:

the extracellular region of VEFGR2 capable of inducing specific CD8+ T reaction or the extracellular region of MHCI-type molecule binding epitope optimized form of the extracellular region has an amino acid sequence shown as amino acids 20 to 764 in SEQ ID No.1 or 2; the human or mouse XCL1 specifically binding the DC cells with antigen cross-presentation capacity is shown as a sequence shown by amino acids 1-114 of SEQ ID NO.3 or 4;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I);

or

(III) an amino acid sequence which has at least 90% sequence identity with the sequence of (I) or (II) and which is functionally identical or similar to the amino acid sequence of (I).

In some embodiments of the invention, the fusion protein comprises the sequence of SEQ ID Nos. 7 and 8 from amino acids 1 to 889.

On the basis of the above research, the present invention also provides a nucleotide encoding the fusion protein, having

(I) a nucleotide sequence as shown in any of SEQ ID Nos. 11 to 16;

or

(II) a complementary nucleotide sequence of the nucleotide sequence shown in any of SEQ ID Nos. 11 to 16; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;

or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III);

or

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

On the basis of the research, the invention also provides a recombinant expression vector, which comprises a vector and the fusion protein or the nucleotide.

In some embodiments of the invention, the vector comprises a mammalian cell expression vector or an insect baculovirus expression vector; the vector includes pcDNA3.1(+), pcDNA3.1(-), pFastbac 1-dual-MBP.

On the basis of the above research, the present invention also provides a recombinant strain or cell comprising the fusion protein or the nucleotide of the present invention.

On the basis of the research, the invention also provides application of the fusion protein, the nucleotide, the recombinant expression vector or the recombinant strain or cell in preparation of a vaccine for metastatic cancer or high-expression VEGFR2 melanoma or preparation of a medicament for preventing and/or treating metastatic cancer or high-expression VEGFR2 melanoma.

In some embodiments of the invention, the metastatic cancer comprises liver cancer, colorectal cancer or lung cancer.

On the basis of the research, the invention also provides a metastatic cancer or a vaccine of the melanoma with high expression of VEGFR2, which comprises the fusion protein, the nucleotide, the recombinant expression vector and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

On the basis of the research, the invention also provides a medicament for preventing and/or treating metastatic cancer or high-expression melanoma of VEGFR2, which comprises the fusion protein, the nucleotide, the recombinant expression vector and pharmaceutically acceptable auxiliary materials.

The vaccine provided by the invention is a protein consisting of an extracellular region of a vascular endothelial cell growth factor receptor VEGFR2 capable of causing specific CD8+ T cell immune response and an extracellular region of an MHCI-type molecule binding epitope optimized form, and an XCL1 molecule of DC which is fused and expressed at the N end and specifically binds to antigen cross-presentation capacity, nucleic acid containing the fusion protein and a carrier containing the nucleic acid, and application of the fusion protein, the nucleic acid and the carrier in preventing and treating melanoma and metastatic cancer. According to the invention, through fusion expression of the VEGFR2 protein for promoting tumor angiogenesis and the DC cell ligand XCL1, the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein expressed by the nucleic acid vaccine can be effectively combined with DC cells, induces VEGFR2 specific T cell response and obviously inhibits the growth of melanoma with high VEGFR2 expression in various models.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a map of a vector encoding a fusion protein nucleotide; wherein FIG. 1A shows the CMV promoter followed by the XCL1 sequence and then by the VEGFR2 wild-type extracellular region for nucleic acid vaccine coding sequences; FIG. 1B shows the CMV promoter followed by the XCL1 sequence and then by the VEGFR2 MHC class I molecule binding optimized mutant extracellular region nucleic acid vaccine coding sequence; FIG. 1C shows the CMV promoter followed by the XCL1 secretory signal peptide sequence and then the extracellular domain of VEGFR2 for nucleic acid vaccine coding sequences; FIG. 1D shows the pFASTbac-dual-MBP-XCL1-VEGFR2 wild-type fusion protein vaccine purification vector; FIG. 1E shows pFASTbac-dual-MBP-XCL1-VEGFR2 mutant fusion protein vaccine purification vectors; FIG. 1F shows the pFASTbac-dual-MBP-VEGFR2 wild-type protein vaccine purification vector;

FIG. 2 is a graph showing the results of prediction of the three-dimensional structure of a fusion protein; inputting a nucleotide sequence for coding the fusion protein into http:// raptorx.uchicago.edu/website to predict the three-dimensional spatial structure of the fusion protein, and illustrating a spatial structure diagram of XCL1-VEGFR2 fusion protein;

FIG. 3 shows the detection of expression of a nucleic acid encoding a fusion protein in HEK293T cells; transfecting HEK292T cells by a plasmid vector carrying nucleic acid for encoding the fusion protein for 48 hours, and detecting the expression condition of the fusion protein encoding nucleotide with Flag tag at the C end by using a western blot technology (Westernblot);

FIG. 4 shows the detection of fusion proteins purified from insect rod cells SF 9; detecting the purification condition of XCL1-VEGFR2 secreted and expressed from insect rod-shaped cells by using Coomassie brilliant blue staining;

FIG. 5 shows that the fusion protein can efficiently bind to MHCII + CD11c + CD8a + antigen cross-presenting DC cells; incubating protein purified by SF9 cells with Flag tags with splenocytes enriched by CD11C magnetic beads, and detecting the binding condition of the fusion protein and MHCII + CD11c + CD8a + antigen cross-presenting DC cells by using Flag fluorescent antibody; wherein, FIG. 5(A) shows a diagram of fusion protein binding DC flow analysis; FIG. 5(B) is a graph showing the statistical analysis of the ratio of fusion protein-bound DC cells in FIG. 5 (A);

FIG. 6 shows the detection of the chemotactic effect of fusion proteins on MHCII + CD11c + CD8a + antigen cross-presenting DC cells; splenocytes containing MHCII + CD11c + CD8a + antigen cross-presenting DC cells were plated in the upper layer, and fusion protein purified from SF9 insect cells was plated in the lower layer using the Transwell assay, and the number of MHCII + CD11c + CD8a + antigen cross-presenting DC cells in the upper layer that entered the lower layer after 2 hours was observed; the results show that the fusion protein has chemotactic effect on MHCII + CD11c + CD8a + antigen cross-presenting DC cells; wherein FIG. 6(A) is a diagram showing a flow analysis of the fusion protein chemotactic DC; FIG. 6(B) is a diagram showing a statistical analysis of the ratio of the fusion protein chemotactic DC cells of FIG. 6 (A);

FIG. 7 shows the detection of VEGFR2 (KDR) expression after melanoma cell line B16 cell nodulation; inoculating tumor cells under the skin, after tumor formation, taking tumor tissues and peripheral skin tissues to extract total RNA, detecting the expression conditions of VEGFR2 in the tumor and the peripheral skin by using a real-time fluorescent quantitative PCR amplification technology, and displaying that the expression of VEGFR2 in the tumor tissues is obviously higher than that of the peripheral normal skin;

FIG. 8 shows the growth of melanoma in mice immunized with the fusion gene encoding the XCL1-VEGFR2/XCL1-VEGFR2 mutant fusion proteins; FIG. 8A shows a diagram of the immunization method and the immunization process of the fusion gene; FIG. 8B shows a graph of tumor growth curves made by measuring tumor volume after melanoma inoculation; FIG. 8C shows tumor growth to 2000mm in control group³Mice were sacrificed at the ethical endpoint and a comparative plot of tumor size was dissected;

FIG. 9 is a graph showing the analysis of the ratio of lymphocyte granzyme B positive CD8aT cells infiltrating into melanoma of a mouse immunized with a fusion gene encoding XCL1-VEGFR2 fusion protein;

FIG. 10 shows the detection of metastatic nodule formation in the lung by spleen cells adoptively implanted into mice injected intravenously with melanoma cells after immunization of the mice with the fusion protein; FIG. 10A is a graph showing the effect of the mode of immunization with the fusion protein by intravenous injection; FIG. 10B shows a nodule map of the mouse lung after adoptive therapy of fusion protein immunization.

Detailed Description

The invention discloses a metastatic cancer vaccine targeting VEGFR2, which can be realized by appropriately modifying process parameters by one skilled in the art with the reference to the content in the specification. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention adopts the amino acid sequence of the extracellular region of VEGFR2 as immunogen to facilitate the purification of soluble protein vaccine, the vaccine of the invention can effectively induce specific T cell immunoreaction aiming at VEGFR2 in human body or mouse body, breaks the immune tolerance of the original tumor-related antigen VEGFR2, can effectively play a role in directly eliminating tumor cells expressing VEGFR2, and indirectly inhibits the growth of tumor and the formation of metastasis by inhibiting the generation of tumor neovascularization.

The invention relates to a fusion protein vaccine for metastatic cancer, which consists of 1) an extracellular region of vascular endothelial growth factor receptor 2 (VEFGR 2) for promoting tumor angiogenesis, which can induce specific CD8+ T response, an extracellular region of MHCI class molecule binding epitope optimized form of the extracellular region, and 2) human or mouse XCL1 protein of which the N end is fused and expressed by a linker and specifically binds DC cells with antigen cross presentation capacity. For example, mouse VEGFR2, amino acids 20 to 764 in SEQ ID No.1 and SEQ ID No.2, and the N-terminus of human or mouse XCL1, which expresses DC cells specifically binding to cells having the ability to cross-present antigen by linker fusion, is composed of the sequence shown by amino acids 1 to 114 of SEQ ID No.3 or SEQ ID No. 4.

In some embodiments, the vaccine for metastatic cancer comprises any of the sequences of amino acids 1-889 of SEQ ID No.7 and SEQ ID No. 8.

Another aspect of the invention relates to a nucleic acid encoding the fusion protein vaccine, the nucleic acid vaccine is composed of 1) a nucleotide sequence encoding the extracellular domain of vascular endothelial growth factor receptor 2 (VEFGR 2) capable of inducing specific CD8+ T response and the extracellular domain of MHC class I molecule binding epitope optimized form thereof, which promotes tumor angiogenesis, and 2) a nucleotide sequence encoding human or mouse XCL1 protein, the N-terminal of which is expressed by fusion through a linker and specifically binds DC cells with antigen cross-presentation capability. I.e. encoding the amino acid sequence from position 20 to 764 of SEQ ID No.1 and SEQ ID No.2 and the human or murine XCL1 sequence specifically binding to DC cells with antigen cross-presenting ability, i.e. the amino acid sequence from position 1 to 114 of SEQ ID No.3 or SEQ ID No. 4.

In some embodiments, the sequences encoding VEFGR2 that promotes tumor neovascularization and human or murine XCL1 that specifically binds DC cells with antigen cross-presenting capability comprise the nucleotide sequences in SEQ ID No.11-SEQ ID No. 16.

The invention also provides a recombinant expression vector loaded with the nucleic acid vaccine, which comprises the fusion protein vaccine, a nucleotide sequence corresponding to a check vaccine and a vector. The vector can be a mammalian cell expression vector or an insect rod cell expression vector, and concretely can be pcDNA3.1(+), pcDNA3.1(-), pFastbac 1-dual-MBP.

The invention finally provides a vaccine for metastatic cancer, which comprises the fusion protein vaccine, the nucleic acid vaccine and the recombinant expression vector.

The vaccine of the invention provides a method for preventing relapse or treating metastatic cancers such as liver cancer, colorectal cancer, lung cancer and the like after operation and high-expression VEGFR2 melanoma. Including administering the vaccines of the invention for other indications.

The invention also provides application of the vaccine, the nucleic acid vaccine and the vector in preparation of a melanoma vaccine with high VEGFR2 expression after metastatic cancer operations such as liver cancer, colorectal cancer, lung cancer and the like.

The invention also provides the vaccine, the nucleic acid vaccine and the carrier for treating melanoma.

The vaccine or medicament of the invention comprises the fusion protein, the nucleic acid, the recombinant expression vector and optionally pharmaceutically acceptable carriers, excipients and/or adjuvants.

In conclusion, the invention provides a vaccine for metastatic cancer, which is a protein consisting of an extracellular region of a vascular endothelial cell growth factor receptor VEGFR2 capable of causing specific CD8+ T cell immune response and an extracellular region of an MHCI-type molecule binding epitope optimized form, wherein the N end of the protein is fused with an XCL1 molecule expressing DC specifically binding to antigen cross-presentation capacity, nucleic acid containing the fusion protein and a vector containing the nucleic acid, and application of the fusion protein, the nucleic acid and the vector in preventing and treating melanoma and metastatic cancer. According to the invention, through fusion expression of the VEGFR2 protein for promoting tumor angiogenesis and the DC cell ligand XCL1, the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein expressed by the nucleic acid vaccine can be effectively combined with DC cells, induces VEGFR2 specific T cell response and obviously inhibits the growth of melanoma with high VEGFR2 expression in various models.

The raw materials and reagents involved in the present invention are all commercially available.

The invention is further illustrated by the following examples:

example 1 design of fusion protein vaccine antigens and construction and preparation of expression plasmids for mammalian cells and insect baculovirus cells

(1) Construction of mammalian cell expression vector of fusion gene:

fusion proteins XCL1-VEGFR2 were constructed based on the amino acid sequences of mouse VEGFR2 (SEQ ID No.1 and SEQ ID No.2) and mouse XCL1 (SEQ ID No.4) proteins, and we retained the secretory signal peptide (SEQ ID No.5) of XCL1 protein while removing the VEGFR 2-mediated transmembrane localization signal peptide (SEQ ID No.6) in order to promote efficient secretion of the fusion protein by translation of the fusion protein-expressing nucleic acid in vivo out of the cell and chemotaxis of MHC-II + CD11c + CD8a + antigen cross-presenting DC cells. The amino acid sequences of the resulting fusion proteins (SEQ ID No.7 and SEQ ID No. 8). For the VEGFR2 protein alone, the secretion signal peptide of XCL1 protein was added to the N-terminus of the protein (SEQ ID nos. 9 and 10) from which the self signal peptide was removed to ensure that the VEGFR2 protein expressed from the VEGFR2 expression vector alone was also secreted extracellularly. Nucleotide sequences corresponding to the amino acid sequences are subjected to codon optimization of mammalian cell expression preference, synthesized by Oncorks and connected to pcDNA3.1/zeo (-) and pFastbac1-dual-MBP expression vectors (SEQ ID No.11-SEQ ID No. 16).

(2) Amplification of fusion gene mammalian cell expression vectors

And (3) transforming bacteria, namely putting the competent bacteria frozen at the temperature of-80 ℃ on ice for thawing, adding 100 ng of plasmid when the competent bacteria are nearly completely thawed, gently and uniformly mixing, and putting the competent bacteria on ice for 30 mins. The competence was placed in a 42 ℃ water bath for 60 s with heat shock and immediately after removal placed on ice for 2 mins. And adding 500 mu L of LB culture medium without antibiotics into the tube, and carrying out shaking culture in a shaking table at 37 ℃ for 1 h. The bacteria were centrifuged at 4000 rpm for 2 mins at room temperature, a portion of the supernatant (about 450 μ L) was discarded and the bacteria were resuspended, and an appropriate amount of the bacterial suspension was spread onto a petri dish containing the corresponding antibiotic. The petri dish was placed face down and incubated overnight in an incubator at 37 ℃. After the clone size was appropriate (about 16 h), the clone was picked up with a gun tip into LB medium supplemented with antibiotics and shake-cultured in a shaker at 37 ℃ until turbid. Taking 15mL of bacterial liquid cultured to a proper concentration, centrifuging at 4000 rpm for 5mins at room temperature, and discarding the supernatant. Bacterial DNA was extracted according to the instructions of the plasmid Mini kit (DP 103) of Beijing Tiangen. First, 250 μ L of RNase-containing cell resuspension P1 was added to thoroughly resuspend the bacterial pellet and the pellet was transferred to a 1.5 mL EP tube. Adding 250 mu L of alkaline cell lysate P2, and gently inverting and mixing until the liquid is clear. 350 mu L of neutralizing liquid P3 is added, and the mixture is inverted and mixed evenly until flocculent precipitates appear. The suspension was centrifuged at 12000 rpm for 10 mins at room temperature. And putting the DNA adsorption column into a recovery tube, adding 500 muL of equilibrium liquid to activate the adsorption membrane, centrifuging at 12000 rpm for 1min, and then discarding the liquid. The supernatant obtained by centrifugation in step 4 was transferred to a DNA adsorption column, centrifuged at 12000 rpm for 1min, and the liquid was discarded. And adding 600 mu L of cleaning solution into the adsorption column, centrifuging at 12000 rpm for 1min, then discarding the solution, and repeatedly washing once. The tube was evacuated at 12000 rpm for 2 mins. The collection tube was replaced with a new 1.5 mL EP tube and the column was allowed to air dry at room temperature for 5 mins. Adding 70 mu L of elution buffer preheated at 65 ℃ or purified water subjected to high pressure, standing at room temperature for 5mins to fully dissolve DNA, centrifuging at 12000 rpm at room temperature for 3 mins, and collecting liquid. And (5) after the adsorption column is discarded, carrying out concentration measurement on the plasmids in the tube and marking the name, the concentration and the extraction date of the plasmids.

(3) Construction of expression vector of fused insect baculovirus

1) Basic principle of blue-white screening: the first component of this expression system is the pFastBac strain used to clone the gene of interest. Based on the selection of pFastBac strain, the expression gene is controlled by PH or p10 promoter, and the high-level expression frame is surrounded by the left and right arms of Tn7 in insect cells, contains a gentamicin resistance point and forms a miniature Tn 7. The second component of this expression system is the DH10Bac strain of e. DH10Bac cells contain a viral plasmid with mini-attTn 7 target sites and a helper plasmid. Once the pFastBac expression plasmid was transformed into DH10Bac cells, transformation occurred between the mini Tn7 unit of pFastBac strain and the target site of mini-attTn 7, resulting in the generation of recombinant plasmid. The viral strain (bacmid) was amplified in the kanamycin-resistant plasmid DH10Bac and was able to complement the deletion of the chromosomal LacZ to form blue spots in the presence of the chromogenic substance X-gal and the inducer IPTG. Insertion of mini Tn7 into the mini attTn7 attachment site interfered with LacZa peptide expression, so clones containing the recombinant plasmid were white on a blue background and white spot analysis was selected. The true white spot clones are larger, so in order to avoid selection as false positives, the largest, most independent white spot is selected.

2) Preparation of blue and white spots: construction of recombinant plasmid pFastBac HTB: experiments such as primer design (SEQ ID No. 17-20), agarose gel recovery, enzyme digestion reaction, ligation reaction, transformation, plasmid extraction and the like are carried out by using the experimental method introduced above, and finally, the recombinant plasmid which clones the target gene into pFastBac HTB is obtained; transformation of the recombinant plasmid pFastBac HTB into DH10Bac competent cells: picking the target plaque (white spot): after 48 hours, until the blue white spots can be clearly distinguished, 2-3 largest and most independent white spots are selected, and 5ml of LB culture medium (added with 50 mug/ml kanamycin, 10 mug/ml tetracycline and 7 mug/ml gentamicin) is shaken at 37 ℃ and 220 rpm for 8-12 hours until bacterial liquid is turbid, and then Bacmid is extracted by the bacterial liquid.

TABLE 1

Example 2 prediction of three-dimensional Structure of fusion protein

Based on the nucleotide sequence of the fusion protein, we used http:// raptorx. uchicago. edu/website to predict the three-dimensional structure of XCL1 and VEGFR2 extracellular domain fusion proteins. XCL1 belongs to the chemokine, and its chemotactic function needs to maintain an intact spatial structure. In order to ensure that XCL1 still has a spatial structure after the XCL1 is fused with the VEGFR2 extracellular region, the three-dimensional structure of the fused amino acid sequence is predicted on a http:// raptorx. uchicago. edu/website, and the result is shown in FIG. 2, after XCL1 is fused with VEGFR2, the XCL1 and the VEGFR2 still respectively have the original spatial structure and do not influence the chemotactic function of XCL 1.

Example 3 detection of the Effect of expression of mammalian cell expression vectors encoding XCL1-VEGFR2 fusion proteins

24 hours prior to transfection, 6-well cell culture plates were inoculated with 1 x 10⁶HEK293T cells, the transfection assay was started when the cell density reached 70% -80%. Preheating in 37 deg.C water bathCell culture medium, Opti-MEM medium without serum. 5 micrograms of empty Vector (Vector), VEGFR2 expression Vector alone, XCL1-VEGFR2 wild type and mutant fusion gene plasmid, and 20. mu.L PEI transfection reagent were added to 200. mu.L of serum-free Opti-MEM in sequence for transfection, mixed well, and then left to stand at room temperature for 10 minutes. The cells to be transfected are replaced by fresh culture medium, and the cells are added into the transfection system gently and shaken up. The cells were returned to the cell incubator and incubated for 6 hours before changing the medium. Cells are harvested after 48 hours of transfer, and expression effect of XCL1-VEGFR2 fusion gene plasmid HEK293T cells is detected by Western Blot.

To facilitate detection of expression effect of fusion gene, a Flag tag consisting of 5 amino acids of DDDDK is connected to C end of fusion protein, so as to detect expression of fusion protein by using Flag tag antibody. Cells were harvested and 60 μ L of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor was added. Resuspend the cells well and spin lyse the cells at 4 ℃ for 30 min. The lysate was centrifuged at 12000 rpm for 10min at 4 ℃ and the supernatant was collected in a fresh 1.5 mL EP tube and the pellet discarded. Adding a 5 xSDS-PAGE protein loading buffer according to the actual volume of the sample, uniformly mixing, placing the sample in an air bath at 100 ℃ for heating for 10 minutes, immediately carrying out Western blot, and detecting by using a Flag tag antibody (Sigma, F3165), wherein the result is shown in figure 3 that an empty Vector (Vector) has no protein expression, a VEGFR2 expression Vector alone, and XCL1-VEGFR2 wild-type and mutant fusion gene plasmids can effectively express and have equivalent expression quantity.

Example 4 insect rod cell expression vector expression and protein purification encoding XCL1-VEGFR2 fusion protein

Selection of cells and media: the cell expression used in the experiment is insect cell SF9 (Spodoptera frugiperda cell), the cell can be attached to the wall or cultured in suspension, and the SF9 cell is generally cultured in suspension to carry out mass expression of target protein. The culture medium is serum-free, and Gibico culture medium is required for adherent culture in the virus coating stage of the experiment. The suspension culture of the culture medium of the SIM SF of the state of Yinqiao is selected in the large-scale preparation stage of the recombinant protein.

(1) Transfection of insect cells

Preparing cells: total cell count was 0.9X 10 using a 6-well culture plate of Polystyrene⁶Moving the cells/holes up and down and left and right repeatedly to make the cells uniformly spread on the culture plate, and standing for 30 min;

(2) preparation of DNA/lipid mixture:

1) adding 100 mul of Gibco culture medium into a 1.5 ml EP tube, and then adding 1 mul of Genejuise, which is a solution 1;

2) adding 100 mu l of Gibco culture medium into another 1.5 ml EP tube, and then adding 1 mu g of Bacmid, which is a solution 2;

3) fully mixing the solution 1 and the solution 2, and incubating for 30 min at room temperature;

(3) transfection: adding 800. mu.l of Gibco medium to the DNA/lipid mixture, blotting the cell culture medium in the culture plate, rapidly transferring 1ml of DNA/lipid mixture to the cell monolayer, shaking gently to homogenize, and standing at 27 ℃ for 5 hours;

(4) culturing: after 5 hours, removing the transfection medium, adding 2ml of Gibco medium, and standing and culturing at 27 ℃;

(5) after 72 hours of incubation, a phenomenon of virus infection was observed, and the P0 virus strain was extracted on days 5 to 6.

(6) Virus strain preparation, amplification and identification

1) Preparation of the virus strain: once transfected cells are obtained and late transfection occurs, virus-containing cells can be collected from wells in a 6-well plate and transferred to sterile 2.0 ml centrifuge tubes. Cells and debris were removed by centrifugation for 5 min. The supernatant was then transferred to a new 2.0 ml centrifuge tube, i.e., P0 strain, and 10% FBS was added and stored at 4 ℃ in the dark.

2) Amplification of the viral strain: adding 100-200 mu l P0 virus into the total cell number of 18-20 multiplied by 10⁶And in a culture medium with a total volume of 15ml, performing virus amplification by using T75; after 4-5 days of culture, the virus-containing medium was collected and transferred to a sterile 15ml centrifuge tube. Centrifuging at 500 Xg for 5min, and collecting supernatant and precipitate respectively

(7) Mass production of recombinant proteins

1) Viral infection: insect cells SF9 are used to express the protein of interest, the cells are expanded to the cell density at which they are suitably infected, and the corresponding amount of virus is added. The cell suspension was collected after about 65 h.

2) Collecting cell supernatant: the protein of interest is a secreted protein and is therefore in the supernatant. And (3) centrifuging the cell suspension expressing the target protein for 30 min at 8000 rpm/4 ℃ by using a high-speed centrifuge to separate the cells from the supernatant, taking the supernatant, and then directly carrying out protein purification of in-vitro affinity chromatography.

3) Protein purification by affinity chromatography using polyhistidine tag (His-tag)

The N-terminal of the constructed target protein is provided with His-tag consisting of six histidine chains in sequence, and the imidazole group of the histidine side chain can be combined with metal ions (nickel or cobalt). If nickel or cobalt ions are fixed on the chromatographic medium, when the mixed solution containing the His-tag fusion protein flows through the chromatographic column, the His-tag fusion protein is adsorbed on the column, and all the foreign protein flows out; thereafter, the His-tag fusion protein is eluted by using a higher concentration of imidazole solution (e.g., 200 mM) to compete the imidazole for the His-tag imidazole ring. The histidine tag is very small, hardly influences the function, activity or structure of the target protein, and has wide application. The main experimental steps are as follows:

1) combining: adding the cell supernatant obtained after centrifugation into a nickel column, combining at 4 ℃, and enabling the supernatant to slowly flow through Ni Beads for one time so as to enable the target protein with the His label to be fully combined with the Ni Beads;

2) washing the hybrid protein: washing with a PBS solution added with 20mM imidazole, and detecting whether the hybrid protein is completely washed or not in a Coomassie brilliant blue dye solution in the washing process;

3) eluting the target protein: eluting the target protein by using PBS (phosphate buffer solution) eluent added with 200mM imidazole, and detecting whether the target protein is completely washed or not in Coomassie brilliant blue dye solution in the elution process;

4) concentrating the target protein solution eluted in the step 3 by using a 4 ℃ centrifuge to a final volume of 500 mu l;

5) separation and purification of a gel filtration chromatographic column: loading the concentrated solution obtained in the step 4 onto a gel filtration chromatography column Superdex 200 pre-equilibrated with PBS, collecting a target protein peak, identifying whether the target protein is the target protein by using Western Blot, and identifying the protein purity by using 12% SDS-PAGE electrophoresis, as shown in FIG. 4;

6) concentrating and freezing: according to the result of 12% SDS-PAGE electrophoretic identification, the protein with the purity meeting the requirement is concentrated, the protein concentration is measured by a micro-spectrophotometer and then split charging is carried out, and the split charging is carried out by quick freezing with liquid nitrogen and the storage is carried out at-80 ℃.

Example 5 detection of the ability of XCL1-VEGFR2 fusion protein to cross-present DC cells in combination with MHC-II + CD11c + CD8a + antigen

After obtaining the purified fusion protein, the present invention first verified whether the protein obtained from insect cells could efficiently bind to MHCII + CD11c + CD8a + antigen cross-presenting DC cells.

(1) Separation and purification of CD11c + DC cells

Taking the whole spleen of a mouse as a CD11c + DC cell source, firstly taking the spleen of the mouse, grinding the spleen of the mouse into single cells in a 1640 culture medium by using a cell screen with 70um aperture, centrifuging the single cells for 10 minutes at 200g, removing supernatant, and using R&The spleen cells were treated with red blood cell lysate (cat # WL 2000) from company D. Diluting lysis solution A by 10 times with distilled water to prepare working solution, adding 2mL of working solution into each spleen for resuspension, standing at room temperature for 10min, and diluting neutralization solution B by 10 times with distilled water to prepare working solution. Ten minutes later, 10mL of the neutralization solution B was added to the lysate for neutralization, followed by centrifugation at 200g for 10 min. The cell pellet was washed once with PBS buffer containing 1% inactivated FBS and counted. Take the total number 1 x 10⁸The next power of cells were resuspended in 400. mu.L of PBS buffer containing 1% inactivated FBS, 100. mu.L of magnetic beads LCD11c (Miltenyi Biotec: 130-. During this time the column adsorbing the magnetic beads was equilibrated with 1% inactivated FBS in PBS buffer. After 20min, the mixed solution of the cells and the magnetic beads is transferred to a magnetic bead adsorption column, the column is placed on a magnetic frame, and when the cells completely enter the column, the cells are washed three times by 3mL of 1% inactivated FBS PBS buffer solution every time. The column was then removed from the magnetic rack and placed over a 15mL centrifuge tube, 5mL1% inactivated FBS in PBS buffer was added and pushed quicklyCD11c positive DC cells were eluted, counted and centrifuged at 200g for 10 min. The finally obtained CD11c positive DC cells were resuspended to 1 x 10 with serum-free 1640⁶Per mL. Spread in 24-well plates, 1mL per well. The cells were divided into a BSA protein control group, a VEGFR2 protein control group and an XCL1-VEGFR2 wild-type protein experimental group, 50 μ g of total protein was added to each group, mixed uniformly, placed in a 37 ℃ carbon dioxide incubator for 40 minutes, and then 500g of the cells were collected, centrifuged, and washed twice with 1% FBS-inactivated PBS buffer. And carrying out flow dyeing: MHC-II APC, Flag-dye light 488, CD8 α -Percp/Cy5.5. Cells were collected at medium speed using a BD LSRII instrument and analyzed for the proportion of MHC-II + Flag-dye light 488+ CD8 α + cells between different groups. Each group was repeated 3 times.

(2) The results showed that the VEGFR2 protein not fused with XCL1 bound MHC-II + CD11c + CD8a + in similar numbers and ratios to the BSA control protein, while VEGFR2 protein fused with XCL1 bound more strongly to MHC-II + CD11c + CD8a + antigen cross-presenting DC cells with more than 20-fold higher binding capacity than the control group, as shown in fig. 5A, 5B.

Example 6 detection of chemotactic Capacity of XCL1-VEGFR2 fusion protein for MHCII + CD11c + CD8a + antigen Cross-presenting DC cells

(1) Since the XCL1-VEGFR2 fusion protein can effectively bind MHCII + CD11c + CD8a + antigen cross-presenting DC cells, the XCL1 chemokine can effectively recruit MHCII + CD11c + CD8a + antigen cross-presenting DC cells according to the expression in previous researches, and a Tanswell test is designed to verify that the fusion protein can effectively chemotact and recruit DC cells. The Transwell assay uses two layers of cell culture plates, one on top of the other, with chemokines added to the bottom layer and target cells, in this case DC cells enriched with CD11c magnetic beads, plated on the top layer. The bottom of the upper culture chamber is a membrane with a specific pore diameter, and according to the previous research report, the membrane with the pore diameter of 5 mu m and the diameter of 6.5mm on the upper layer of a 24-pore Transwell plate is selected in the experiment, target cells are paved on the membrane, and can penetrate through the membrane to enter the lower layer due to the attraction of the chemotactic factors of the lower layer. The amount of cells that enter the lower layer is proportional to the chemotactic capacity of the chemokine. We first resuspended CD11c magnetic bead enriched DC cells to 1 x 10⁷Per mL, 100. mu.L of cells, 1X 10, were added to the upper chamber⁶And (4) respectively. And adding 700 mu L of culture medium of a control group containing 70ng of BSA (bovine serum albumin), 70ng of VEGFR2 protein and 70ng of wild type and mutant protein XCL1-VEGFR2 of the experimental group into the lower layer so as to ensure the liquid level heights of the culture medium of the upper layer and the lower layer to be consistent and prevent the cells of the upper layer from entering the lower layer due to pressure caused by different liquid level heights. The plate was placed in a carbon dioxide incubator for 5 hours. The upper chamber was removed to collect cells in the lower medium, centrifuged at 500g, and washed twice with 1% inactivated FBS in PBS. And carrying out flow dyeing: MHC-II APC, CD11c-PE, CD8 α -Percp/Cy5.5. Cells were harvested at medium speed using a BD Canto II instrument and analyzed for the proportion of MHC-II + CD11c-PE + CD8 α + cells between different groups. Each group was repeated 3 times.

(2) The results show that the VEGFR2 protein not fused with XCL1 has similar numbers and proportions of MHC-II + CD11c + CD8a + under the chemotaxis with BSA control protein, while the VEGFR2 protein fused with XCL1 can chemotaxis more MHC-II + CD11c + CD8a + antigen cross-presenting DC cells no matter the wild type or the mutant, and the statistics shows that the difference is significant through statistical tests, and the statistical method is Unaccessing t test, as shown in FIGS. 6A and 6B.

Example 7 Effect of fusion Gene on intervention in development of B16 melanoma cell Homoplastic tumor

Given that the fusion gene can be normally expressed in mammalian cells, the fusion protein can also effectively chemotaxis and bind to MHC-II + CD11c + CD8a + antigen cross-presenting DC cells. We extracted the individual VEGFR2 and XCL1-VEGFR2 wild-type and mutant plasmids and used the Wealtec company's gene gun (GDS-80) for immunization of mice with plasmid injection. And after the B16 melanoma cells are transplanted in the same species, the inhibition of the fusion gene immunity on the growth of the B16 melanoma cells is observed.

(1) B16 melanoma cell culture and detection of VEGFR2 gene expression after tumor formation

Resuscitation of B16 cells: the cell culture medium (cell culture medium was prepared by adding 10% concentration FBS to DMEM medium from Corning). Taking out the frozen cells to be recovered from the liquid nitrogen or a refrigerator at minus 80 ℃, and quickly putting the frozen cells into a water bath kettle at 37 ℃ for thawing. After the cells were completely lysed, the cells were centrifuged at 1500 rpm for 3min, while 8 mL of the pre-warmed cell culture medium was added to the dish. After centrifugation, the supernatant was discarded, 1mL of pre-warmed cell culture medium was added, the cells were resuspended and transferred to a prepared petri dish, and the cell name, date and passage number were marked. The cells were cultured at 37 ℃ in a 5% CO2 incubator. The next day of passage, cell culture medium, trypsin, PBS buffer were pre-warmed. The old medium in the dish was aspirated by a vacuum pump. Cells were rinsed once with 7 mL PBS buffer and the PBS aspirated. 1mL of trypsin was added and left to digest for the appropriate time at 37 ℃ in an incubator. Digestion was terminated by adding 3mL of medium, cells were pipetted into the single cell suspension and transferred to a 15mL centrifuge tube and centrifuged at 1500 rpm for 3 min while preparing new dishes, each with 8 mL of medium. And (3) after centrifugation, removing the supernatant, adding an appropriate amount of culture medium according to the required passage proportion to resuspend cells, blowing to obtain single cell suspension, adding 1mL of single cell suspension into a new culture dish, and putting back into a 37 ℃ incubator containing 5% CO2 to continue culture. After the cells grow to 80% -90% density, the cell count is digested as above and resuspended in serum-free DMEM medium, and the cell concentration is adjusted to 1 x 10 according to our previous nodulation experience⁶Each mouse was inoculated with 100 μ L of cells subcutaneously in the axilla per mL. After two weeks, the formed melanoma and surrounding normal skin were taken and total RNA was extracted. Appropriate mouse tissues were harvested and 1mL Trizol (Invitrogen) was added. The tissue homogenate instrument is used for cracking for 2 mins, and the tissue homogenate instrument is rotated for 30 mins at 4 ℃ for complete cracking. And adding 200 mu L of chloroform into each 1mL of Trizol, shaking vigorously for 15 s, and standing at room temperature for 15 mins. Precooling the centrifuge at 4 ℃, carrying out 12000 rpm and centrifuging for 10 mins. After centrifugation, the upper aqueous phase was transferred to a new 1.5 mL EP tube, 500 μ L of isopropanol was added, the mixture was inverted and mixed, and centrifuged at 12000 rpm at 4 ℃ for 10 mins. White precipitate was observed after centrifugation, the supernatant was discarded, and the RNA precipitate was washed with 75% ethanol in DEPC water at 7500 rpm and centrifuged at room temperature for 5 mins. The supernatant was discarded, and the dried RNA was left at room temperature until the precipitate became translucent. Appropriate volumes of DEPC water were taken to dissolve RNA. Mixing dissolved RNA, detecting RNA concentration and purity with Nanodrop, labeling type, concentration, and extracting RNA species on EP tube wall and coverTime. The primers (SEQ ID Nos. 21-24) shown in the following table were used for real time PCR detection. The results showed that VEGFR2 expression levels were significantly elevated in the resulting melanoma compared to peripheral normal skin, as shown in fig. 7, suggesting that it is reasonable to evaluate the fusion gene vaccine using B16 cells.

TABLE 2

(2) Prevention effect of B16 melanoma cell allograft tumor generation after fusion gene immunization

After determining that a B16 cell oncogenic model is available, we performed gene gun plasmid injections into mice following the immunization strategy outlined by the time axis of fig. 8A. C57B6 (purchased from Torilow) week-old male mice were divided into three groups of five mice each injected with the respective wild-type and mutant plasmids VEGFR2 and XCL1-VEGFR2, and depilatory treatment was performed on the right side of the mice near the inguinal lymph node using a depilatory cream. Then, 50 μ g of plasmid was injected into the hair removal site by using a gene gun, once per week, four times in total, B16 melanoma cells with tumor formation conditions were searched before inoculation one week after the last injection, the tumor formation time was observed, the major diameter a and the minor diameter B of the tumor were measured every two days, and the tumor volume calculation was performed according to a B/2 to plot the tumor growth curve. The results are shown in FIGS. 8A and 8B, and the results of the immunization with XCL1-VEGFR2 wild-type and mutant plasmids were both effective in delaying the time of tumor development and the growth rate. The therapeutic effect of the fusion gene immunity is proved to be obvious and effective.

(3) Exploring whether fusion gene immunity induces stronger cell-specific T cell response

Tumor growth in control group to ethical end point 2000mm³In time, sacrificed mice each group of mice tumor removal and photograph, after the use of 100 u m aperture screen cells into single cell suspension, 800g centrifugal cell heavy suspension and 80% percoll solution per 1000mm³Adding 4mL of heavy suspension, carefully adding 40% of pecoll solution into the upper layer, centrifuging for 20 minutes at 800g, removing the uppermost layer of tumor cells, and reserving the middle layer of the suspensionThe cells were counted by washing and finally resuspended in 1640 medium containing 10% heat-inactivated FBS at a concentration of 1 x 10⁶Cells were plated in 24-well plates, 1mL per well and stimulated with VEGFR2 epitope polypeptide, while Golgi stop (Biolegend) was added to inhibit intracellular factor secretion. Cells were harvested 12 hours later and washed twice with 1% inactivated FBS in PBS buffer and flow stained: gran B-PE, CD8 alpha-Percp/Cy5.5. The results are shown in fig. 9, the mice immunized with XCL1-VEGFR2 wild-type and mutant plasmids showed significantly more specific T cells against VEGFR2 in tumors than the VEGFR2 alone immunized group, indicating that XCL1-VEGFR2 wild-type and mutant plasmid immunization did induce specifically reacting CD8a cytotoxic T lymphocytes.

Example 8: therapeutic effect of adoptive therapy of T cells on melanoma after immunization with fusion protein

Since the fusion gene successfully induces specific cytotoxic T lymphocytes against VEGFR2 in mice after immunization, the invention utilizes the fusion protein purified from insect rod cells to immunize T cell donor mice, induce specific cytotoxic T lymphocytes against VEGFR2 in the donor mice, and adoptively link the T cell donor mice with B16 tumor cells to see whether the development of tumors in the recipient mice can be effectively inhibited.

(1) Fusion protein immunization of adoptive T cell donor mice

In order to effectively induce specific cytotoxic T lymphocytes aiming at VEGFR2 by the purified fusion protein of insect rod cells, the invention searches and optimizes the administration route by combining the characteristics of XCL1 targeted DC cells in the prior study, firstly, the intravenous administration result is adopted as shown in figure 10A, and the intravenous injection of KDR protein and LPS adjuvant cannot generate the anti-tumor effect. The final fusion protein was determined at a 40 μ g dose plus Poly: after being emulsified by IC 30 micrograms and incomplete Freund's adjuvant, the mixture is immunized subcutaneously once a week and twice in a total way, so that the generation of specific cytotoxic T lymphocytes aiming at VEGFR2 can be effectively induced, and the subsequent treatment can generate an inhibiting effect on B16 melanoma cell allograft tumors.

(2) Therapeutic effect of adoptive treatment of T cells on melanoma

One week after the second subcutaneous protein immunization, mice were sacrificed, spleens were removed and ground as in example 5, red lysed and enriched for cytotoxic T lymphocytes with CD8a magnetic beads, and the obtained cells were finally resuspended in serum-free 1640 medium at a concentration of 2 x 10⁷Every mL, two days ahead, 3 x 10 vaccinations via the tail vein⁵T cells obtained by postero-caudal vein adoptively re-inoculation of mice with B16 melanoma cells amounted to 2 x 10 per 200. mu.L⁶And (4) cells. Two weeks later, the lung tissue of the mice was fixed in 10% paraformaldehyde and photographed the next day, and the results are shown in fig. 10B, and the pulmonary nodules of the recipient mice were significantly reduced after the cytotoxic T lymphocytes of the mice immunized with the XCL1-VEGFR2 wild-type and mutant proteins. It is shown that mice immunized with the wild-type and mutant proteins XCL1-VEGFR2 produced cytotoxic T lymphocytes against VEGFR2 and were effective in killing melanoma cells expressing VEGFR2 in vivo.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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

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Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr

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Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn Val Ser Leu

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Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile

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Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser

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Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr

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Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp

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Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu Lys

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Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp

225 230 235 240

Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val

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

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

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

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Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys

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

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

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Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu Leu Thr Ile

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Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val Ile Leu Thr

370 375 380

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

385 390 395 400

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

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Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr Ala

420 425 430

Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala

435 440 445

Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp

450 455 460

Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val Thr Lys

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

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Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile

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

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Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu

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Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu

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Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly Glu

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

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

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

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Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile

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Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr

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Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn

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Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn

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Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala

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

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

35 40 45

Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly Gly Gly Asp

50 55 60

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

65 70 75 80

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

85 90 95

Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val

100 105 110

Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr

115 120 125

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

130 135 140

Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile

145 150 155 160

Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser

165 170 175

Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr

180 185 190

Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp

195 200 205

Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Asn Lys

210 215 220

Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp

225 230 235 240

Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys

305 310 315 320

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

325 330 335

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

340 345 350

Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu Leu Thr Ile

355 360 365

Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val Met Leu Thr

370 375 380

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

385 390 395 400

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

405 410 415

Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr Ala

420 425 430

Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala

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Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp

450 455 460

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

465 470 475 480

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

485 490 495

Val Ile Met Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile

500 505 510

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

515 520 525

Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu

530 535 540

Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu

545 550 555 560

Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Asn Gly Glu

565 570 575

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

580 585 590

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

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

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Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile

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Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr

645 650 655

Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn

660 665 670

Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu

675 680 685

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

690 695 700

Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn

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Val Leu Gly Leu Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala

725 730 735

Gln Glu Lys Thr Asn Leu Glu

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Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

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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

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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

115 120 125

<210> 5

<211> 20

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<213> secretion signal peptide of Murine XCL1

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Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

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Trp Val Val Glu

20

<210> 6

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Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu

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Thr Arg Ala

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<213> Artificial Sequence

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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 Ala Ser Val

115 120 125

Gly Leu Pro Gly Asp Phe Leu His Pro Pro Lys Leu Ser Thr Gln Lys

130 135 140

Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln

225 230 235 240

His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile

245 250 255

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

260 265 270

Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp

275 280 285

Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser Tyr Ala Gly

290 295 300

Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr Gln Ser Ile

305 310 315 320

Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Ile Leu

325 330 335

Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu Lys Leu Val Leu

340 345 350

Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp Phe Thr Trp

355 360 365

His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val Asn Arg Asp

370 375 380

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

385 390 395 400

Thr Ile Glu Ser Val Thr Lys Ser Asp Gln Gly Glu Tyr Thr Cys Val

405 410 415

Ala Ser Ser Gly Arg Met Ile Lys Arg Asn Arg Thr Phe Val Arg Val

420 425 430

His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys Ser Leu Val

435 440 445

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

450 455 460

Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg Pro Ile Glu

465 470 475 480

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

485 490 495

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

500 505 510

Ser Met Glu Lys Gln Ser His Met Val Ser Leu Val Val Asn Val Pro

515 520 525

Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser Pro Met Asp Ser Tyr Gln

530 535 540

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

545 550 555 560

Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala Cys Ser Tyr

565 570 575

Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp Arg His Val

580 585 590

Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val Thr Lys Asn Gln Tyr

595 600 605

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

610 615 620

Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile Asn Lys Ala

625 630 635 640

Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile Arg Gly Pro Glu

645 650 655

Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu Ser Val Ser

660 665 670

Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu Thr Trp Tyr

675 680 685

Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly Glu Ser Leu Thr

690 695 700

Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn Gly Thr Met

705 710 715 720

Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Val Ala Phe Gln Asn Ala

725 730 735

Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Ser Ala Gln Asp Lys Lys

740 745 750

Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile Leu Glu Arg

755 760 765

Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr Thr Thr Ile

770 775 780

Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn Pro Thr Pro

785 790 795 800

His Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser Gly

805 810 815

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

820 825 830

Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn Val Leu Gly

835 840 845

Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala Gln Glu Lys

850 855 860

Thr Asn Leu Glu

865

<210> 8

<211> 868

<212> PRT

<213> Artificial Sequence

<400> 8

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 Ala Ser Val

115 120 125

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

130 135 140

Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln

225 230 235 240

His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile

245 250 255

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

260 265 270

Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp

275 280 285

Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser Tyr Ala Gly

290 295 300

Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr Gln Ser Ile

305 310 315 320

Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Ile Leu

325 330 335

Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Asn Lys Leu Val Leu

340 345 350

Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp Phe Thr Trp

355 360 365

His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val Asn Arg Asp

370 375 380

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

385 390 395 400

Thr Ile Glu Ser Val Thr Lys Ser Asp Gln Gly Glu Tyr Thr Cys Val

405 410 415

Ala Ser Ser Gly Arg Met Ile Lys Arg Asn Arg Thr Phe Val Arg Val

420 425 430

His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys Ser Leu Val

435 440 445

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

450 455 460

Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg Pro Ile Glu

465 470 475 480

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

485 490 495

Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val Met Leu Thr Asn Pro Ile

500 505 510

Ser Met Glu Lys Gln Ser His Met Val Ser Leu Val Val Asn Val Pro

515 520 525

Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser Pro Met Asp Ser Tyr Gln

530 535 540

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

545 550 555 560

Leu His His Ile Gln Trp Tyr Trp Gln Leu Glu Glu Ala Cys Ser Tyr

565 570 575

Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp Arg His Val

580 585 590

Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Leu Thr Lys Asn Gln Tyr

595 600 605

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

610 615 620

Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile Asn Lys Ala

625 630 635 640

Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile Arg Gly Pro Glu

645 650 655

Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu Ser Val Ser

660 665 670

Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu Thr Trp Tyr

675 680 685

Lys Leu Gly Ser Gln Ala Thr Ser Val His Asn Gly Glu Ser Leu Thr

690 695 700

Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn Gly Thr Met

705 710 715 720

Phe Ser Met Ser Asn Asn Leu Ile Leu Ile Val Ala Phe Gln Asn Ala

725 730 735

Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Ser Ala Gln Asp Lys Lys

740 745 750

Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile Leu Glu Arg

755 760 765

Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr Thr Thr Ile

770 775 780

Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn Pro Thr Pro

785 790 795 800

His Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser Gly

805 810 815

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

820 825 830

Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln Ala Cys Asn Val Leu Gly

835 840 845

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

850 855 860

Thr Asn Leu Glu

865

<210> 9

<211> 763

<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 Ala Ser Val Gly Leu Pro Gly Asp Phe Leu His Pro

20 25 30

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

35 40 45

Thr Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp

50 55 60

Pro Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys

65 70 75 80

Gly Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val

85 90 95

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

100 105 110

Ile Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe

115 120 125

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

130 135 140

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

145 150 155 160

Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp

165 170 175

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

180 185 190

Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn

195 200 205

Asp Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Val Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His

260 265 270

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

275 280 285

Lys Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp

290 295 300

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

305 310 315 320

Asn Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly

325 330 335

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

340 345 350

Ile Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr

355 360 365

Arg Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp

370 375 380

Glu Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr

385 390 395 400

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

405 410 415

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

420 425 430

Ser Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys

435 440 445

Thr Val Tyr Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln

450 455 460

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

465 470 475 480

Cys Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Thr Glu Gln Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr

565 570 575

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

580 585 590

His Met Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Gln Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu

660 665 670

Glu Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro

675 680 685

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

690 695 700

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

705 710 715 720

Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys

725 730 735

Gln Ala Cys Asn Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile

740 745 750

Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu

755 760

<210> 10

<211> 763

<212> PRT

<213> Artificial Sequence

<400> 10

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Ala Ser Val Gly Leu Asn Gly Asp Phe Leu His Pro

20 25 30

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

35 40 45

Thr Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp

50 55 60

Pro Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys

65 70 75 80

Gly Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val

85 90 95

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

100 105 110

Ile Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe

115 120 125

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

130 135 140

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

145 150 155 160

Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp

165 170 175

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

180 185 190

Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn

195 200 205

Asp Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr

210 215 220

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

225 230 235 240

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

245 250 255

Val Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His

260 265 270

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

275 280 285

Lys Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp

290 295 300

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

305 310 315 320

Asn Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly

325 330 335

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

340 345 350

Ile Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr

355 360 365

Arg Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp

370 375 380

Glu Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr

385 390 395 400

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

405 410 415

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

420 425 430

Ser Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys

435 440 445

Thr Val Tyr Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln

450 455 460

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

465 470 475 480

Cys Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Thr Glu Gln Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr

565 570 575

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

580 585 590

His Asn Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Gln Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu

660 665 670

Glu Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro

675 680 685

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

690 695 700

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

705 710 715 720

Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys

725 730 735

Gln Ala Cys Asn Val Leu Gly Leu Ala Arg Ala Glu Thr Leu Phe Ile

740 745 750

Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu

755 760

<210> 11

<211> 2628

<212> DNA

<213> Artificial Sequence

<400> 11

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 ggggcggagg cggaggatca 360

gggggagggg gaggagcctc tgtgggtttg cctggcgatt ttctccatcc ccccaagctc 420

agcacacaga aagacatact gacaattttg gcaaatacaa cccttcagat tacttgcagg 480

ggacagcggg acctggactg gctttggccc aatgctcagc gtgattctga ggaaagggta 540

ttggtgactg aatgcggcgg tggtgacagt atcttctgca aaacactcac cattcccagg 600

gtggttggaa atgatactgg agcctacaag tgctcgtacc gggacgtcga catagcctcc 660

actgtttatg tctatgttcg agattacaga tcaccattca tcgcctctgt cagtgaccag 720

catggcatcg tgtacatcac cgagaacaag aacaaaactg tggtgatccc ctgccgaggg 780

tcgatttcaa acctcaatgt gtctctttgc gctaggtatc cagaaaagag atttgttccg 840

gatggaaaca gaatttcctg ggacagcgag ataggcttta ctctccccag ttacatgatc 900

agctatgccg gcatggtctt ctgtgaggca aagatcaatg atgaaaccta tcagtctatc 960

atgtacatag ttgtggttgt aggatatagg atttatgatg tgattctgag ccccccgcat 1020

gaaattgagc tatctgccgg agaaaaactt gtcttaaatt gtacagcgag aacagagctc 1080

aatgtggggc ttgatttcac ctggcactct ccaccttcaa agtctcatca taagaagatt 1140

gtaaaccggg atgtgaaacc ctttcctggg actgtggcga agatgttttt gagcaccttg 1200

acaatagaaa gtgtgaccaa gagtgaccaa ggggaataca cctgtgtagc gtccagtgga 1260

cggatgatca agagaaatag aacatttgtc cgagttcaca caaagccttt tattgctttc 1320

ggtagtggga tgaaatcttt ggtggaagcc acagtgggca gtcaagtccg aatccctgtg 1380

aagtatctca gttacccagc tcctgatatc aaatggtaca gaaatggaag gcccattgag 1440

tccaactaca caatgattgt tggcgatgaa ctcaccatca tggaagtgac tgaaagagat 1500

gcaggaaact acacggtcat cctcaccaac cccatttcaa tggagaaaca gagccacatg 1560

gtctctctgg ttgtgaatgt cccaccccag atcggtgaga aagccttgat ctcgcctatg 1620

gattcctacc agtatgggac catgcagaca ttgacatgca cagtctacgc caaccctccc 1680

ctgcaccaca tccagtggta ctggcagcta gaagaagcct gctcctacag acccggccaa 1740

acaagcccgt atgcttgtaa agaatggaga cacgtggagg atttccaggg gggaaacaag 1800

atcgaagtca ccaaaaacca atatgccctg attgaaggaa aaaacaaaac tgtaagtacg 1860

ctggtcatcc aagctgccaa cgtgtcagcg ttgtacaaat gtgaagccat caacaaagcg 1920

ggacgaggag agagggtcat ctccttccat gtgatcaggg gtcctgaaat tactgtgcaa 1980

cctgctgccc agccaactga gcaggagagt gtgtccctgt tgtgcactgc agacagaaat 2040

acgtttgaga acctcacgtg gtacaagctt ggctcacagg caacatcggt ccacatgggc 2100

gaatcactca caccagtttg caagaacttg gatgctcttt ggaaactgaa tggcaccatg 2160

ttttctaaca gcacaaatga catcttgatt gtggcatttc agaatgcctc tctgcaggac 2220

caaggcgact atgtttgctc tgctcaagat aagaagacca agaaaagaca ttgcctggtc 2280

aaacagctca tcatcctaga gcgcatggca cccatgatca ccggaaatct ggagaatcag 2340

acaacaacca ttggcgagac cattgaagtg acttgcccag catctggaaa tcctacccca 2400

cacattacat ggttcaaaga caacgagacc ctggtagaag attcaggcat tgtactgaga 2460

gatgggaacc ggaacctgac tatccgcagg gtgaggaagg aggatggagg cctctacacc 2520

tgccaggcct gcaatgtcct tggctgtgca agagcggaga cgctcttcat aatagaaggt 2580

gcccaggaaa agaccaactt ggaagattac aaggatgacg acgataag 2628

<210> 12

<211> 2606

<212> DNA

<213> Artificial Sequence

<400> 12

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 ggggcggagg cggaggatca 360

gggggagggg gaggagcctc tgtgggtttg cctggcgaga ttttctgcat ccccccaagc 420

tgtccaccca gaaagatatt ctgaccatcc tggccaatac caccctgcag atcacctgta 480

ggggccagag ggacctggac tggctgtggc ccaacgccca gcgcgactcc gaagagaggg 540

tgctggtgac cgagtgcggc ggcggagact ccatcttctg caaaaccctg accatcccca 600

gggtggtggg caacgacact ggcgcctaca agtgttccta cagggacgtg gatatcgcat 660

ctaccgtgta cgtgtacgtg agggactaca ggtccccctt catcgccagc gtgtcagacc 720

agcatggaat cgtgtatatt accgagaaca agaacaaaac agtggtgatc cccaaccgcg 780

gctccattag caacctgaac gtgtccctgt gcgccaggta cccagaaaag cgctttgtgc 840

ccgacgggaa caggatctcc tgggacagcg aaatcggctt caccctgccc tcctacatga 900

tctcctacgc cggtatggtg ttctgcgagg ccaaaatcaa cgacgagacc taccagtcca 960

tcatgtacat cgtggtggtg gtgggctaca ggatctacga cgtgatcctg tccccccccc 1020

acgagattga gctgtccgcc gggaacaagc tggtgctgaa ctgcaccgcc agaaccgagc 1080

tgaacgtggg actggatttt acttggcact cccccccttc taagtcccac cacaagaaaa 1140

tcgtgaacag ggacgtgaaa cccttccctg gcaccgtggc caagatgttc ctgtccaccc 1200

tgaccattga gagcgtgacc aagtccgacc agggcgagta cacctgcgtg gcatcctccg 1260

gcaggatgat taagagaaac cggaccttcg tgagggtgca caccaagccc ttcatcgctt 1320

tcggcagtgg catgaaatcc ctggtggaag ccaccgtggg cagccaggtg aggatccccg 1380

tgaagtacct gtcctacccc gcccccgata tcaagtggta caggaacggc cgccccatcg 1440

agtccaacta caccatgatc gtgggagacg aactgacaat catggaagtg accgaaagag 1500

acgccggcaa ctacaccgtg atgctgacca accccatcag catggaaaaa cagagccaca 1560

tggtgtccct ggtggtgaac gtgcccccac agatcggcga gaaggccctg atcagcccaa 1620

tggactccta ccagtatggc accatgcaga ccctgacctg caccgtgtac gccaaccccc 1680

ccctgcacca tattcagtgg tactggcagc tggaagaggc atgctcctac agacccggcc 1740

agacttcccc ctacgcatgc aaggagtgga ggcacgtgga agatttccag ggcggcaaca 1800

aaatcgagct gaccaagaac cagtacgccc tgattgaggg gaagaacaag acagtgtcca 1860

ccctggtgat catggccgcc aacgtgagcg ccctgtacaa gtgcgaggct atcaacaagg 1920

caggccgggg cgagagggtg atcagtttcc acgtgattag gggccccgag attaccgtgc 1980

agcccgccgc tcagcccacc gaacaggaat ctgtgtccct gctgtgcacc gccgacagga 2040

atacctttga gaacctgacc tggtacaaac tgggctcaca ggccacatcc gtgcacaacg 2100

gagaatccct gacccccgtg tgcaaaaacc tggacgccct gtggaaactg aacggcacca 2160

tgttcagcat gtccaacaac ctgatcctga tcgtggcatt ccagaacgcc tccctgcagg 2220

accagggcga ctatgtgtgt agcgcccagg acaagaaaac caaaaagagg cattgcctgg 2280

tgaagcagct gatcatcctg gagcgcatgg cccccatgat caccggaaac ctggagaacc 2340

agacaaccac catcggcgag accatcgaag tgacctgccc cgcatcaggc aaccccaccc 2400

cccacatcac ctggttcaag gataacgaaa ccctggtgga agactccggc attgtgctga 2460

gagacggcaa cagaaacctg accatcagaa gggtgagaaa agaggatggc ggcctgtaca 2520

cctgccaggc ctgcaacgtg ctgggactgg ccagggcaga aaccctgttc attatcgagg 2580

gagctcagga gaaaacaaac ctggag 2606

<210> 13

<211> 2316

<212> DNA

<213> Artificial Sequence

<400> 13

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgcctctg tgggtttgcc tggcgatttt ctccatcccc ccaagctcag cacacagaaa 120

gacatactga caattttggc aaatacaacc cttcagatta cttgcagggg acagcgggac 180

ctggactggc tttggcccaa tgctcagcgt gattctgagg aaagggtatt ggtgactgaa 240

tgcggcggtg gtgacagtat cttctgcaaa acactcacca ttcccagggt ggttggaaat 300

gatactggag cctacaagtg ctcgtaccgg gacgtcgaca tagcctccac tgtttatgtc 360

tatgttcgag attacagatc accattcatc gcctctgtca gtgaccagca tggcatcgtg 420

tacatcaccg agaacaagaa caaaactgtg gtgatcccct gccgagggtc gatttcaaac 480

ctcaatgtgt ctctttgcgc taggtatcca gaaaagagat ttgttccgga tggaaacaga 540

atttcctggg acagcgagat aggctttact ctccccagtt acatgatcag ctatgccggc 600

atggtcttct gtgaggcaaa gatcaatgat gaaacctatc agtctatcat gtacatagtt 660

gtggttgtag gatataggat ttatgatgtg attctgagcc ccccgcatga aattgagcta 720

tctgccggag aaaaacttgt cttaaattgt acagcgagaa cagagctcaa tgtggggctt 780

gatttcacct ggcactctcc accttcaaag tctcatcata agaagattgt aaaccgggat 840

gtgaaaccct ttcctgggac tgtggcgaag atgtttttga gcaccttgac aatagaaagt 900

gtgaccaaga gtgaccaagg ggaatacacc tgtgtagcgt ccagtggacg gatgatcaag 960

agaaatagaa catttgtccg agttcacaca aagcctttta ttgctttcgg tagtgggatg 1020

aaatctttgg tggaagccac agtgggcagt caagtccgaa tccctgtgaa gtatctcagt 1080

tacccagctc ctgatatcaa atggtacaga aatggaaggc ccattgagtc caactacaca 1140

atgattgttg gcgatgaact caccatcatg gaagtgactg aaagagatgc aggaaactac 1200

acggtcatcc tcaccaaccc catttcaatg gagaaacaga gccacatggt ctctctggtt 1260

gtgaatgtcc caccccagat cggtgagaaa gccttgatct cgcctatgga ttcctaccag 1320

tatgggacca tgcagacatt gacatgcaca gtctacgcca accctcccct gcaccacatc 1380

cagtggtact ggcagctaga agaagcctgc tcctacagac ccggccaaac aagcccgtat 1440

gcttgtaaag aatggagaca cgtggaggat ttccaggggg gaaacaagat cgaagtcacc 1500

aaaaaccaat atgccctgat tgaaggaaaa aacaaaactg taagtacgct ggtcatccaa 1560

gctgccaacg tgtcagcgtt gtacaaatgt gaagccatca acaaagcggg acgaggagag 1620

agggtcatct ccttccatgt gatcaggggt cctgaaatta ctgtgcaacc tgctgcccag 1680

ccaactgagc aggagagtgt gtccctgttg tgcactgcag acagaaatac gtttgagaac 1740

ctcacgtggt acaagcttgg ctcacaggca acatcggtcc acatgggcga atcactcaca 1800

ccagtttgca agaacttgga tgctctttgg aaactgaatg gcaccatgtt ttctaacagc 1860

acaaatgaca tcttgattgt ggcatttcag aatgcctctc tgcaggacca aggcgactat 1920

gtttgctctg ctcaagataa gaagaccaag aaaagacatt gcctggtcaa acagctcatc 1980

atcctagagc gcatggcacc catgatcacc ggaaatctgg agaatcagac aacaaccatt 2040

ggcgagacca ttgaagtgac ttgcccagca tctggaaatc ctaccccaca cattacatgg 2100

ttcaaagaca acgagaccct ggtagaagat tcaggcattg tactgagaga tgggaaccgg 2160

aacctgacta tccgcagggt gaggaaggag gatggaggcc tctacacctg ccaggcctgc 2220

aatgtccttg gctgtgcaag agcggagacg ctcttcataa tagaaggtgc ccaggaaaag 2280

accaacttgg aagattacaa ggatgacgac gataag 2316

<210> 14

<211> 2586

<212> DNA

<213> Artificial Sequence

<400> 14

catcaccatc accatcacgt ggggactgaa gtcctagaag agagtagctg tgtgaactta 60

caaacccagc ggctgccagt tcaaaaaatc aagacctata tcatctggga gggggccatg 120

agagctgtaa tttttgtcac caaacgagga ctaaaaattt gtgctgatcc agaagccaaa 180

tgggtgaaag cagcgatcaa gactgtggat ggcagggcca gtaccagaaa gaacatggct 240

gaaactgttc ccacaggagc ccagaggtcc accagcacag cgataaccct gactgggggc 300

ggaggcggag gatcaggggg agggggagga gcctctgtgg gtttgcctgg cgattttctc 360

catcccccca agctcagcac acagaaagac atactgacaa ttttggcaaa tacaaccctt 420

cagattactt gcaggggaca gcgggacctg gactggcttt ggcccaatgc tcagcgtgat 480

tctgaggaaa gggtattggt gactgaatgc ggcggtggtg acagtatctt ctgcaaaaca 540

ctcaccattc ccagggtggt tggaaatgat actggagcct acaagtgctc gtaccgggac 600

gtcgacatag cctccactgt ttatgtctat gttcgagatt acagatcacc attcatcgcc 660

tctgtcagtg accagcatgg catcgtgtac atcaccgaga acaagaacaa aactgtggtg 720

atcccctgcc gagggtcgat ttcaaacctc aatgtgtctc tttgcgctag gtatccagaa 780

aagagatttg ttccggatgg aaacagaatt tcctgggaca gcgagatagg ctttactctc 840

cccagttaca tgatcagcta tgccggcatg gtcttctgtg aggcaaagat caatgatgaa 900

acctatcagt ctatcatgta catagttgtg gttgtaggat ataggattta tgatgtgatt 960

ctgagccccc cgcatgaaat tgagctatct gccggagaaa aacttgtctt aaattgtaca 1020

gcgagaacag agctcaatgt ggggcttgat ttcacctggc actctccacc ttcaaagtct 1080

catcataaga agattgtaaa ccgggatgtg aaaccctttc ctgggactgt ggcgaagatg 1140

tttttgagca ccttgacaat agaaagtgtg accaagagtg accaagggga atacacctgt 1200

gtagcgtcca gtggacggat gatcaagaga aatagaacat ttgtccgagt tcacacaaag 1260

ccttttattg ctttcggtag tgggatgaaa tctttggtgg aagccacagt gggcagtcaa 1320

gtccgaatcc ctgtgaagta tctcagttac ccagctcctg atatcaaatg gtacagaaat 1380

ggaaggccca ttgagtccaa ctacacaatg attgttggcg atgaactcac catcatggaa 1440

gtgactgaaa gagatgcagg aaactacacg gtcatcctca ccaaccccat ttcaatggag 1500

aaacagagcc acatggtctc tctggttgtg aatgtcccac cccagatcgg tgagaaagcc 1560

ttgatctcgc ctatggattc ctaccagtat gggaccatgc agacattgac atgcacagtc 1620

tacgccaacc ctcccctgca ccacatccag tggtactggc agctagaaga agcctgctcc 1680

tacagacccg gccaaacaag cccgtatgct tgtaaagaat ggagacacgt ggaggatttc 1740

caggggggaa acaagatcga agtcaccaaa aaccaatatg ccctgattga aggaaaaaac 1800

aaaactgtaa gtacgctggt catccaagct gccaacgtgt cagcgttgta caaatgtgaa 1860

gccatcaaca aagcgggacg aggagagagg gtcatctcct tccatgtgat caggggtcct 1920

gaaattactg tgcaacctgc tgcccagcca actgagcagg agagtgtgtc cctgttgtgc 1980

actgcagaca gaaatacgtt tgagaacctc acgtggtaca agcttggctc acaggcaaca 2040

tcggtccaca tgggcgaatc actcacacca gtttgcaaga acttggatgc tctttggaaa 2100

ctgaatggca ccatgttttc taacagcaca aatgacatct tgattgtggc atttcagaat 2160

gcctctctgc aggaccaagg cgactatgtt tgctctgctc aagataagaa gaccaagaaa 2220

agacattgcc tggtcaaaca gctcatcatc ctagagcgca tggcacccat gatcaccgga 2280

aatctggaga atcagacaac aaccattggc gagaccattg aagtgacttg cccagcatct 2340

ggaaatccta ccccacacat tacatggttc aaagacaacg agaccctggt agaagattca 2400

ggcattgtac tgagagatgg gaaccggaac ctgactatcc gcagggtgag gaaggaggat 2460

ggaggcctct acacctgcca ggcctgcaat gtccttggct gtgcaagagc ggagacgctc 2520

ttcataatag aaggtgccca ggaaaagacc aacttggaag attacaagga tgacgacgat 2580

aagtaa 2586

<210> 15

<211> 2586

<212> DNA

<213> Artificial Sequence

<400> 15

catcaccatc accatcacgt ggggactgaa gtcctagaag agagtagctg tgtgaactta 60

caaacccagc ggctgccagt tcaaaaaatc aagacctata tcatctggga gggggccatg 120

agagctgtaa tttttgtcac caaacgagga ctaaaaattt gtgctgatcc agaagccaaa 180

tgggtgaaag cagcgatcaa gactgtggat ggcagggcca gtaccagaaa gaacatggct 240

gaaactgttc ccacaggagc ccagaggtcc accagcacag cgataaccct gactgggggc 300

ggaggcggag gatcaggggg agggggagga gcttctgtgg gactgaatgg agattttctg 360

catcccccca agctgtccac ccagaaagat attctgacca tcctggccaa taccaccctg 420

cagatcacct gtaggggcca gagggacctg gactggctgt ggcccaacgc ccagcgcgac 480

tccgaagaga gggtgctggt gaccgagtgc ggcggcggag actccatctt ctgcaaaacc 540

ctgaccatcc ccagggtggt gggcaacgac actggcgcct acaagtgttc ctacagggac 600

gtggatatcg catctaccgt gtacgtgtac gtgagggact acaggtcccc cttcatcgcc 660

agcgtgtcag accagcatgg aatcgtgtat attaccgaga acaagaacaa aacagtggtg 720

atccccaacc gcggctccat tagcaacctg aacgtgtccc tgtgcgccag gtacccagaa 780

aagcgctttg tgcccgacgg gaacaggatc tcctgggaca gcgaaatcgg cttcaccctg 840

ccctcctaca tgatctccta cgccggtatg gtgttctgcg aggccaaaat caacgacgag 900

acctaccagt ccatcatgta catcgtggtg gtggtgggct acaggatcta cgacgtgatc 960

ctgtcccccc cccacgagat tgagctgtcc gccgggaaca agctggtgct gaactgcacc 1020

gccagaaccg agctgaacgt gggactggat tttacttggc actccccccc ttctaagtcc 1080

caccacaaga aaatcgtgaa cagggacgtg aaacccttcc ctggcaccgt ggccaagatg 1140

ttcctgtcca ccctgaccat tgagagcgtg accaagtccg accagggcga gtacacctgc 1200

gtggcatcct ccggcaggat gattaagaga aaccggacct tcgtgagggt gcacaccaag 1260

cccttcatcg ctttcggcag tggcatgaaa tccctggtgg aagccaccgt gggcagccag 1320

gtgaggatcc ccgtgaagta cctgtcctac cccgcccccg atatcaagtg gtacaggaac 1380

ggccgcccca tcgagtccaa ctacaccatg atcgtgggag acgaactgac aatcatggaa 1440

gtgaccgaaa gagacgccgg caactacacc gtgatgctga ccaaccccat cagcatggaa 1500

aaacagagcc acatggtgtc cctggtggtg aacgtgcccc cacagatcgg cgagaaggcc 1560

ctgatcagcc caatggactc ctaccagtat ggcaccatgc agaccctgac ctgcaccgtg 1620

tacgccaacc cccccctgca ccatattcag tggtactggc agctggaaga ggcatgctcc 1680

tacagacccg gccagacttc cccctacgca tgcaaggagt ggaggcacgt ggaagatttc 1740

cagggcggca acaaaatcga gctgaccaag aaccagtacg ccctgattga ggggaagaac 1800

aagacagtgt ccaccctggt gatcatggcc gccaacgtga gcgccctgta caagtgcgag 1860

gctatcaaca aggcaggccg gggcgagagg gtgatcagtt tccacgtgat taggggcccc 1920

gagattaccg tgcagcccgc cgctcagccc accgaacagg aatctgtgtc cctgctgtgc 1980

accgccgaca ggaatacctt tgagaacctg acctggtaca aactgggctc acaggccaca 2040

tccgtgcaca acggagaatc cctgaccccc gtgtgcaaaa acctggacgc cctgtggaaa 2100

ctgaacggca ccatgttcag catgtccaac aacctgatcc tgatcgtggc attccagaac 2160

gcctccctgc aggaccaggg cgactatgtg tgtagcgccc aggacaagaa aaccaaaaag 2220

aggcattgcc tggtgaagca gctgatcatc ctggagcgca tggcccccat gatcaccgga 2280

aacctggaga accagacaac caccatcggc gagaccatcg aagtgacctg ccccgcatca 2340

ggcaacccca ccccccacat cacctggttc aaggataacg aaaccctggt ggaagactcc 2400

ggcattgtgc tgagagacgg caacagaaac ctgaccatca gaagggtgag aaaagaggat 2460

ggcggcctgt acacctgcca ggcctgcaac gtgctgggac tggccagggc agaaaccctg 2520

ttcattatcg agggagctca ggagaaaaca aacctggagg attacaagga tgacgacgat 2580

aagtaa 2586

<210> 16

<211> 2274

<212> DNA

<213> Artificial Sequence

<400> 16

catcaccatc accatcacgc ctctgtgggt ttgcctggcg attttctcca tccccccaag 60

ctcagcacac agaaagacat actgacaatt ttggcaaata caacccttca gattacttgc 120

aggggacagc gggacctgga ctggctttgg cccaatgctc agcgtgattc tgaggaaagg 180

gtattggtga ctgaatgcgg cggtggtgac agtatcttct gcaaaacact caccattccc 240

agggtggttg gaaatgatac tggagcctac aagtgctcgt accgggacgt cgacatagcc 300

tccactgttt atgtctatgt tcgagattac agatcaccat tcatcgcctc tgtcagtgac 360

cagcatggca tcgtgtacat caccgagaac aagaacaaaa ctgtggtgat cccctgccga 420

gggtcgattt caaacctcaa tgtgtctctt tgcgctaggt atccagaaaa gagatttgtt 480

ccggatggaa acagaatttc ctgggacagc gagataggct ttactctccc cagttacatg 540

atcagctatg ccggcatggt cttctgtgag gcaaagatca atgatgaaac ctatcagtct 600

atcatgtaca tagttgtggt tgtaggatat aggatttatg atgtgattct gagccccccg 660

catgaaattg agctatctgc cggagaaaaa cttgtcttaa attgtacagc gagaacagag 720

ctcaatgtgg ggcttgattt cacctggcac tctccacctt caaagtctca tcataagaag 780

attgtaaacc gggatgtgaa accctttcct gggactgtgg cgaagatgtt tttgagcacc 840

ttgacaatag aaagtgtgac caagagtgac caaggggaat acacctgtgt agcgtccagt 900

ggacggatga tcaagagaaa tagaacattt gtccgagttc acacaaagcc ttttattgct 960

ttcggtagtg ggatgaaatc tttggtggaa gccacagtgg gcagtcaagt ccgaatccct 1020

gtgaagtatc tcagttaccc agctcctgat atcaaatggt acagaaatgg aaggcccatt 1080

gagtccaact acacaatgat tgttggcgat gaactcacca tcatggaagt gactgaaaga 1140

gatgcaggaa actacacggt catcctcacc aaccccattt caatggagaa acagagccac 1200

atggtctctc tggttgtgaa tgtcccaccc cagatcggtg agaaagcctt gatctcgcct 1260

atggattcct accagtatgg gaccatgcag acattgacat gcacagtcta cgccaaccct 1320

cccctgcacc acatccagtg gtactggcag ctagaagaag cctgctccta cagacccggc 1380

caaacaagcc cgtatgcttg taaagaatgg agacacgtgg aggatttcca ggggggaaac 1440

aagatcgaag tcaccaaaaa ccaatatgcc ctgattgaag gaaaaaacaa aactgtaagt 1500

acgctggtca tccaagctgc caacgtgtca gcgttgtaca aatgtgaagc catcaacaaa 1560

gcgggacgag gagagagggt catctccttc catgtgatca ggggtcctga aattactgtg 1620

caacctgctg cccagccaac tgagcaggag agtgtgtccc tgttgtgcac tgcagacaga 1680

aatacgtttg agaacctcac gtggtacaag cttggctcac aggcaacatc ggtccacatg 1740

ggcgaatcac tcacaccagt ttgcaagaac ttggatgctc tttggaaact gaatggcacc 1800

atgttttcta acagcacaaa tgacatcttg attgtggcat ttcagaatgc ctctctgcag 1860

gaccaaggcg actatgtttg ctctgctcaa gataagaaga ccaagaaaag acattgcctg 1920

gtcaaacagc tcatcatcct agagcgcatg gcacccatga tcaccggaaa tctggagaat 1980

cagacaacaa ccattggcga gaccattgaa gtgacttgcc cagcatctgg aaatcctacc 2040

ccacacatta catggttcaa agacaacgag accctggtag aagattcagg cattgtactg 2100

agagatggga accggaacct gactatccgc agggtgagga aggaggatgg aggcctctac 2160

acctgccagg cctgcaatgt ccttggctgt gcaagagcgg agacgctctt cataatagaa 2220

ggtgcccagg aaaagaccaa cttggaagat tacaaggatg acgacgataa gtaa 2274

<210> 17

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 17

gcgcatcacc atcaccatca cgcctctgtg ggtttgcctg gcgattt 47

<210> 18

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 18

tcgacgtagg cctttgaatt cttacttatc gtcgtcatcc ttgtaat 47

<210> 19

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 19

ccatgggttg tggaaggtgc ctctgtgggt t 31

<210> 20

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 20

aacccacaga ggcaccttcc acaacccatg g 31

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 21

gccctgctgt ggtctcacta c 21

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 22

caaagcattg cccattcgat 20

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 23

tttggcaaat acaacccttc aga 23

<210> 24

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 24

gcagaagata ctgtcaccac c 21

Claims (9)

1. A fusion protein comprising the extracellular domain of VEFGR2 (KDR) capable of eliciting a specific CD8+ T response or its mhc class i molecule binding epitope-optimized form extracellular domain that promotes tumor angiogenesis; a joint; and human or murine XCL1 protein that specifically binds DC cells with cross-antigen presentation capability;

the amino acid sequences of the fusion protein are shown as SEQ ID No.7 and 8.

2. A nucleotide encoding the fusion protein of claim 1.

3. A recombinant expression vector comprising the fusion protein of claim 1 or the nucleotide of claim 2.

4. The recombinant expression vector of claim 3, wherein the recombinant expression vector comprises a mammalian cell expression vector or an insect rod cell expression vector.

5. A recombinant strain or cell comprising the fusion protein of claim 1 or the nucleotide of claim 2.

6. Use of the fusion protein according to claim 1, the nucleotide according to claim 2, the recombinant expression vector according to claim 3 or 4, or the recombinant strain or cell according to claim 5 for the preparation of a vaccine for metastatic cancer or a melanoma with high expression of VEGFR2, or for the preparation of a medicament for the prevention and/or treatment of metastatic cancer or a melanoma with high expression of VEGFR 2.

7. The use of claim 6, wherein the metastatic cancer comprises liver cancer, colorectal cancer or lung cancer.

8. A vaccine for metastatic cancer or melanoma with high expression of VEGFR2, comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

9. A medicament for preventing and/or treating metastatic cancer or melanoma with high expression of VEGFR2, comprising the fusion protein of claim 1 and a pharmaceutically acceptable excipient.

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