Nucleic acid fragments and their application in the preparation of tumor vaccines

Abstract

The present invention relates to the field of immunotherapy technology, and in particular to nucleic acid fragments and their use in the preparation of tumor vaccines. The nucleic acid fragments provided by the present invention can be used to construct tumor vaccines, which utilize tumor cells to overexpress XCL1 / CCL21 / IL-7 / IL-12, recruit and activate CD8α+DC and killer effector cells, thereby enhancing tumor immune response, releasing a large amount of TAA, activating multiple TAA-specific killer cells, and then inhibiting tumor growth and development.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy technology, in particular to nucleic acid fragments and their application in preparing tumor vaccines. Background Art

[0002] Cancer is one of the most incurable diseases worldwide. Traditional cancer treatments include surgical resection, radiotherapy, and chemotherapy, but these are not suitable for all patients and have limited efficacy. Since the early 20th century, immunotherapy has gradually emerged as a new cancer treatment approach, including allogeneic hematopoietic cell transplantation, adoptive cell transplantation (including T, NK, and DC cells), targeted monoclonal antibodies, immune checkpoint inhibition, and other cell therapies. Despite achieving some efficacy, targeted therapies and immune checkpoint inhibitors often lead to tumor tolerance; adoptive cell transplantation is currently almost ineffective in the treatment of solid tumors. The failure of cancer treatment is mainly due to tumor heterogeneity and the tumor's immunosuppressive microenvironment. Drug therapy often creates selective pressure, giving drug-resistant tumor cells a survival advantage, leading to tumor recurrence and metastasis. Cells used in adoptive cell therapy have difficulty entering tumor tissue, and the tumor's immunosuppressive microenvironment often desensitizes cytotoxic cells, resulting in a loss of anti-tumor effect. Therefore, new strategies are still needed for cancer treatment. To overcome the heterogeneity of tumor cells, tumor cells derived from tumor tissue can be used as vaccines to provide a rich potential for multiple tumor-associated antigens (TAAs) for tumor treatment, activate multiple TAA-specific T cells, and thus achieve the purpose of tumor treatment.

[0003] Overcoming the difficulty of immune cell infiltration in tumor tissue and the immunosuppressive microenvironment can be achieved by targeting chemokines and immune-activating cytokines. Anti-tumor immune responses rely on the activation of antigen-presenting cells (APCs) and antigen-specific T cells. Only when exogenous antigens are taken up and processed by APCs can they be effectively presented to the cell surface and activate naive T cells. In recent years, numerous studies have shown that in mice, CD8α+ DCs are the most effective APCs for inducing CD8+ T cell activation, capable of transferring antigens from tumors to draining lymph nodes (the corresponding cells in humans are CD141+ DCs, also known as BDCA3+ DCs).

[0004] Among cytokines, interleukin-7 (IL-7) and interleukin-12 (IL-12) play important roles in anti-tumor activities. IL-7, a member of the IL-2 superfamily, exerts its effects by binding to cell surface receptors that share a common γ-chain subunit. IL-7 promotes anti-tumor immune responses, inducing monocytes to produce IL-1α, IL-1β, and TNF-α, and is the most potent cytokine for increasing cytotoxic CD8+ T lymphocytes. IL-7 treatment enhances long-term tumor antigen-specific CD8+ T cell responses. IL-12 consists of two covalently linked subunits, p35 and p40. As one of the most effective anti-tumor immunotherapies, IL-12's immunomodulatory and anti-angiogenic properties underlie its anti-cancer effects. IL-12 bridges innate and adaptive immunity, inducing cellular immunity by promoting IFN-γ production, proliferation, and cytolytic activity of NK and T cells. Furthermore, IL-12 induces anti-angiogenic programs through IFN-γ and lymphocyte-endothelial cell interactions. Therefore, the preparation of vaccines with anti-tumor function using interleukin-7 (IL-7) and interleukin-12 (IL-12) has good application prospects. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide nucleic acid fragments and their use in preparing tumor vaccines.

[0006] The nucleic acid fragment provided by the present invention includes an IL-12 expression module and an IL-7 expression module;

[0007] The IL-12 expression module includes: a nucleic acid encoding CCL21 and a nucleic acid encoding IL-12;

[0008] The IL-7 expression module includes: a nucleic acid encoding XCL1 and a nucleic acid encoding IL-7.

[0009] In the present invention, the IL-12 expression module sequentially comprises promoter A, nucleic acid encoding CCL21, nucleic acid encoding self-cleaving 2A ribosomal skipping peptide, and nucleic acid encoding IL-12;

[0010] In the present invention, the IL-7 expression module sequentially comprises: promoter B, nucleic acid encoding XCL1, nucleic acid encoding self-cleaving 2A ribosomal skipping peptide and nucleic acid encoding IL-7.

[0011] In an embodiment of the present invention, the nucleic acid fragment includes the following sequentially connected: promoter A, nucleic acid encoding CCL21, nucleic acid encoding self-cleavage 2A ribosomal skipping peptide, nucleic acid encoding IL-12B, linker, nucleic acid encoding IL-12A, promoter B, nucleic acid encoding XCL1, nucleic acid encoding self-cleavage 2A ribosomal skipping peptide, nucleic acid encoding IL-7, IRES fragment and reporter gene.

[0012] In the present invention, the nucleic acid encoding IL-12 includes: a nucleic acid encoding IL-12B, a linker and a nucleic acid encoding IL-12A.

[0013] In the present invention, the promoter A and promoter B are independently selected from:

[0014] SV40 promoter,

[0015] EF1a mammalian promoter containing enhancer sequence and intron,

[0016] or the CMV mammalian promoter comprising an enhancer sequence and an intron.

[0017] In some embodiments, promoter A is EF-1α promoter, and promoter B is SV40 promoter.

[0018] In the present invention, the amino acid sequence of the short peptide encoded by the linker is VPGVGVPAVG.

[0019] In the present invention, the reporter gene is mRFP.

[0020] In some specific embodiments, the nucleic acid sequence of the nucleic acid fragment is shown in SEQ ID NO: 1.

[0021] The present invention also provides a recombinant vector comprising the nucleic acid fragment of the present invention and a backbone vector.

[0022] In some embodiments, the backbone vector of the recombinant vector of the present invention further comprises EF1a promoter, IRES, WPRE, 3'LTR, 3'LTR (ΔU), HIV-1Ψ, RRE and cPPT / CTS elements.

[0023] In some specific embodiments, the nucleic acid sequence of the recombinant vector of the present invention is shown in SEQ ID NO: 2.

[0024] The present invention also provides a lentivirus, which is obtained by transfecting cells with the recombinant vector.

[0025] The lentivirus of the present invention is constructed by transfecting cells with the recombinant vector and the pPAX2 and pMD2G plasmids. In some embodiments, the cells are 293T cells. The transfection method is calcium phosphate co-precipitation.

[0026] The host is obtained by transfecting cells with the lentivirus.

[0027] The host cell of the present invention is constructed by transfecting cells with the lentiviral particles, and polybrene is added during the transfection process. The concentration of the polybrene is 8 μg / ml. In some embodiments, the host cell of the present invention is TC-1 cell.

[0028] Use of at least one of the following items I) to IV) of the present invention in the preparation of a tumor vaccine;

[0029] 1), the nucleic acid fragment;

[0030] II), the recombinant vector;

[0031] III), the lentivirus;

[0032] IV), the host.

[0033] The present invention also provides a tumor vaccine, which is prepared from the host of the present invention.

[0034] The tumor vaccine preparation method of the present invention comprises incubating the host with mitomycin and then digesting with trypsin to obtain the tumor vaccine. In some embodiments, the concentration of mitomycin is 1 mg / ml. The incubation conditions are 37°C for 4 hours. After incubation, the host is washed 3-5 times with PBS buffer.

[0035] The invention relates to the use of the tumor vaccine in the preparation of medicines for treating tumors.

[0036] The tumors described in the present invention include cervical cancer, melanoma, non-small cell lung cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer, liver cancer metastasis or Ewing's sarcoma.

[0037] The present invention also provides a method for preventing, treating and / or alleviating cancer symptoms in patients, which comprises administering the tumor vaccine of the present invention.

[0038] The nucleic acid fragments provided by the present invention can be used to construct tumor vaccines, which utilize tumor cells to overexpress XCL1 / CCL21 / IL-7 / IL-12 to recruit and activate CD8α+ DCs and killer effector cells, thereby enhancing tumor immune response, releasing a large amount of TAAs, activating multiple TAA-specific killer cells, and thereby inhibiting tumor growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1-a .Backbone vector map;

[0040] Figure 1-b Schematic diagram of CCL21, IL-12, XCL1, and IL-7 expression vector plasmids;

[0041] Figure 2 .Flow cytometry results of mRFP reporter gene expression in TC-1 cells transfected with CCL21, IL-12, XCL1, and IL-7 expression vector plasmids;

[0042] Figure 3 Gene expression detection results of TC-1 cells transfected with CCL21, IL-12, XCL1, and IL-7 expression vector plasmids;

[0043] Figure 4 .Tumor growth curve of TC-1 cell cancer vaccine treated with mitomycin and transfected with CCL21, IL-12, XCL1, and IL-7 expression vector plasmids in TC-1 cell line tumor-bearing mice (preventive model). The vaccine was injected on days -21, -14, and -7, and tumor cells were inoculated on day 0. DETAILED DESCRIPTION

[0044] The present invention provides nucleic acid fragments and their use in the preparation of tumor vaccines. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0045] The nucleic acid fragments provided by the present invention include four fragments encoding CCL21, IL12, XCL-1, and IL-7; the present invention does not limit the connection order of these four fragments, and these four fragments can all be connected to a promoter, a linker, a self-cleaving 2A ribosomal skipping peptide or a reporter gene.

[0046] In the present invention, IL-12 and CCL21 in the nucleic acid fragment are fused to form an expression module, and IL-7 and XCL1 are fused to form an expression module.

[0047] The IL-12 expression module includes: a nucleic acid encoding CCL21 and a nucleic acid encoding IL-12. Specifically, it includes promoter A, a nucleic acid encoding CCL21, a nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, and a nucleic acid encoding IL-12. More specifically, from the 5' end to the 3' end, the IL-12 expression module includes promoter A, a nucleic acid encoding CCL21, a nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, and a nucleic acid encoding IL-12.

[0048] The IL-7 expression module includes: a nucleic acid encoding XCL1 and a nucleic acid encoding IL-7. Specifically, it includes promoter B, a nucleic acid encoding XCL1, a nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, and a nucleic acid encoding IL-7. More specifically, from the 5' end to the 3' end, the IL-7 expression module includes, in order: promoter B, a nucleic acid encoding XCL1, a nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, and a nucleic acid encoding IL-7.

[0049] In the present invention, the IL-12 expression module and the IL-7 expression module may be present in the same plasmid vector or in two different vectors. When present in the same vector, the IL-12 expression module and the IL-7 expression module may be directly connected or separated by other segments. In an embodiment of the present invention, the IL-12 expression module and the IL-7 expression module are directly connected. The present invention does not limit the order in which the IL-12 expression module and the IL-7 expression module are connected. In the embodiments of the present invention, a nucleic acid fragment with an IL-12 expression module at the 5' end is used as an example for verification.

[0050] Therefore, in a specific embodiment of the present invention, the nucleic acid fragment includes the following sequentially linked sequences: promoter A, nucleic acid encoding CCL21, nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, nucleic acid encoding IL-12B, linker, nucleic acid encoding IL-12A, promoter B, nucleic acid encoding XCL1, nucleic acid encoding a self-cleaving 2A ribosomal skipping peptide, nucleic acid encoding IL-7, an IRES fragment, and a reporter gene.

[0051] The nucleic acid sequences of the fragments involved in the present invention are as follows:

[0052]

[0053]

[0054]

[0055]

[0056] The nucleic acid fragments provided herein are derived from mice. In the examples herein, the efficacy of the tumor vaccine was verified using mouse transplanted tumor cells, TC-1. In clinical applications, for the prevention and treatment of human tumors, the nucleic acid fragments should be human.

[0057] In some embodiments, the sequence of the nucleic acid fragment is:

[0058]

[0059] In some embodiments, the cells used to prepare vaccines by inserting the nucleic acid fragments of the present invention may be autologous tumor cells, but xenotransplanted autologous tumor cells, allogeneic tumor cells, xenotransplanted allogeneic tumor cells, or a combination thereof may also be used.

[0060] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally including an operator sequence, a ribosome binding site, and possibly other sequences. Prokaryotes are known to utilize promoters, enhancers, as well as termination and polyadenylation signals. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, integrate into the genome itself. In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably, as plasmids are currently the most commonly used vector form. However, the present invention is intended to encompass other forms of expression vectors that perform equivalent functions and are known or become known in the art, including but not limited to plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0061] The host cells described herein are prokaryotic or eukaryotic hosts containing nucleic acid vectors and / or target genes. The host cells are transformed or transfected with vectors constructed using recombinant DNA technology. Such transformed host cells are capable of replicating protein-encoding vectors or expressing the desired protein.

[0062] The method of preventing, treating and / or alleviating cancer symptoms in patients of the present invention specifically comprises the steps of: identifying patients who need to prevent, treat and / or alleviate cancer symptoms, and administering a tumor cell vaccine.

[0063] The expression vector plasmid comprising CCL21, IL-12, XCL1, and IL-7 described herein is constructed by inserting the nucleic acid fragment described herein into a backbone vector. During construction, the nucleic acid fragment described herein can be ligated simultaneously as a whole, or portions thereof can be ligated in steps to ultimately form a vector containing the nucleic acid fragment, although this is not a limitation of the present invention.

[0064] The vaccine of the present invention can be used to prevent, treat and / or alleviate the symptoms of cancer, wherein the cancer is selected from cervical cancer, melanoma, non-small cell lung cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer, liver cancer metastasis and Ewing's sarcoma and other cancers from patients.

[0065] In one embodiment, the present invention provides a method for treating, preventing and / or alleviating tumor development in HPV-positive tumor model mice by administering a whole tumor cell cancer vaccine expressing CCL21, IL-12, XCL1, and IL-7, comprising the steps of: identifying an HPV-positive tumor model mouse in need of prevention, treatment, and / or alleviation of cancer symptoms, and administering a whole tumor cell cancer vaccine comprising a CCL21, IL-12, XCL1, and IL-7 expression vector plasmid.

[0066] In one aspect of this method, one or more cancer cells are collected from a patient suffering from a cancer selected from the group consisting of cervical cancer, melanoma, non-small cell lung cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer, liver cancer metastasis, and Ewing's sarcoma, as well as other cancers from patients. In another aspect, the genetically modified cells are rendered incapable of proliferation by treatment with mitomycin or irradiation. In yet another aspect, the genetically modified cells are autologous, allogeneic, or xenograft-expanded cells.

[0067] In some embodiments, for example, the construction of an autologous tumor vaccine comprises the following steps: (i) aseptically collecting one or more cancer cells from a patient, (ii) placing the collected cells in an antibiotic solution in a sterile container, (iii) forming a cell suspension from the collected solution, wherein the cells are suspended by enzymatic digestion, mechanical disaggregation, or both, (iv) genetically modifying the cells by transfection with replication-defective lentiviral particles to produce an autologous cell vaccine containing a CCL21, IL-12, XCL1, or IL-7 expression vector plasmid, (vi) harvesting the vaccine, (vii) treating the vaccine with mitomycin, and (vii) freezing the vaccine.

[0068] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0069] Example 1

[0070] Construction of CCL21, IL-12, XCL1, and IL-7 expression vector plasmids:

[0071] First, the nucleic acid fragment of CCL21-HA-P2A-IL12B-linker-IL12A-SV40promotor-XCL1-FLAG-P2A-IL7 was synthesized in full fragment and replaced with the above fragment by homologous recombination. Figure 1-a The sequence between the vector backbone EF1a promotor and IRES was obtained to obtain plasmid 1 ( Figure 1-bThen, the nucleic acid fragment encoding mRFP was synthesized in full fragment, and the ZsGreen1 sequence in plasmid 1 was replaced with the above fragment by homologous recombination to obtain the final expression vector plasmid.

[0072] Example 2:

[0073] Preparation of TC-1 whole tumor cell cancer vaccine includes the following steps:

[0074] (1) TC-1 cell line was measured at 1×10 6 10 cells / mouse, subcutaneously transplanted into C57 / BL6n mice. After 20 days of large tumor formation, the tumor tissue was removed and minced with surgical scissors under sterile conditions. The tissue was digested with collagenase and trypsin at 37°C for 5-10 minutes. The suspension after enzymatic hydrolysis was filtered through a 40μM mesh and collected into a PE centrifuge tube to obtain allogeneic mouse-derived TC-1 tumor cells.

[0075] (2) Centrifuge at 200-300g for 3 minutes to obtain TC-1 tumor cell pellet; add an appropriate amount of RPMI1640 medium containing 10% FBS to resuspend the cells and inoculate them into 10 cm culture dishes, ensuring that there are approximately 5×10 cells per dish. 6 Cells; confluent cells are passaged by trypsin digestion and can be passaged 3-5 times.

[0076] (3) The CCL21, IL-12, XCL1, and IL-7 expression vector plasmids and the pPAX2 and pMD2G plasmids prepared in Example 1 were transiently transferred into 293T cells by calcium phosphate coprecipitation. After 48 hours, the culture supernatant was collected to obtain a lentiviral particle suspension containing the expression vector.

[0077] (4) The above lentiviral particle suspension was added to TC-1 cells at an MOI value of approximately 10-100, and polybrene (final concentration 8 μg / ml) was added to enhance the viral transfection efficiency. 72 hours after transfection, the expression ratio and intensity of the reporter gene mRFP were detected by flow cytometry ( Figure 2 ), and the expression intensity of CCL21, IL-12a, IL-12b, XCL1 and IL-7 was detected by qPCR ( Figure 3 ).

[0078] The results showed that the mRFP-positive ratio in the prepared cells exceeded 80%, and the expression of CCL21, IL-12a, IL-12b, XCL1 and IL-7 were significantly upregulated, indicating that the constructed cells are qualified and can be further used as cell cancer vaccines.

[0079] (5) The qualified transfected TC-1 cells were incubated with mitomycin (final concentration 1 mg / ml) at 37°C for 4 hours, and then washed 3-5 times with PBS. The obtained cells were trypsinized to obtain the whole tumor cell cancer vaccine, which was frozen at -80°C or in liquid nitrogen for later use.

[0080] Example 3:

[0081] The whole tumor cell cancer vaccine obtained in Example 2 was used to immunize the mouse model. A vaccine containing only CCL21-IL-12 was used as control 1, and a vaccine containing only XCL1 and IL-7 was used as control 2. The immunization experiment included the following steps:

[0082] (1) TC-1 whole tumor cell cancer vaccine suspension was injected into C57 / BL6n mice via the tail vein in a volume of 200 μL. The inoculated cell volume per mouse was 1×10 7 The control group mice were injected with an equal volume of PBS. The vaccinations were repeated three times, with an interval of 7 days.

[0083] (2) On the 21st day after the first vaccination, each mouse was inoculated with TC-1 tumor cells. The inoculation amount of tumor cells per mouse was 1×10 6 The inoculation method was subcutaneous injection. The tumor growth was then observed ( Figure 4 The results showed that the tumor volume of animals given the vaccine of Example 2 was significantly reduced, with a significant difference compared to the control (p < 0.05). In addition, the level of reduction in tumor volume of animals given the vaccine of Example 2 was also significantly different from that of controls 1 and 2 (p < 0.05).

[0084] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Beijing Qitang Technology Co., Ltd. <120> Nucleic acid fragments and their application in the preparation of tumor vaccines <130> MP21026511 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5918 <212> DNA <213> Artificial Sequence <400> 1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300 acttccacgc ccctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg 360 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc 420 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc 480 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt 540 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg 600 ggccgcgggc ggcgacgggg cccgtgcgtc 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agacagagtt ccaggccatc aacgcagcac ttcagaatca caaccatcag cagatcattc tagacaaggg catgctggtg gccatcgatg agctgatgca gtctctgaat cataatggcg agactctgcg 3300. ccagaaacct cctgtgggag aagcagaccc ttacagagtg aaaatgaagc tctgcatcct 3360. gcttcacgcc ttcagcaccc gcgtcgtgac catcaacagg gtgatgggct atctgagctc cgcctagtaa agtttaaaca cgcgtggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 3660 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag 3720 aagtagtgag gaggcttttt tggaggccgc caccatgaga cttctcctcc tgactttcct 3780 gggagtctgc tgcctcaccc catgggttgt ggaaggtgtg gggactgaag tcctagaaga 3840 gagtagctgt gtgaacttac aaacccagcg gctgccagtt caaaaaatca agacctatat 3900 catctgggag ggggccatga gagctgtaat ttttgtcacc aaacgaggac taaaaatttg 3960 tgctgatcca gaagccaaat gggtgaaagc agcgatcaag actgtggatg gcagggccag 4020 taccagaaag aacatggctg aaactgttcc cacaggagcc cagaggtcca ccagcacagc 4080 gataaccctg actggggatt acaaggatga cgacgataag gctactaact tcagcctgct 4140 gaagcaggct ggagacgtgg aggagaaccc tggacctatg ttccatgttt cttttagata 4200 tatctttgga attcctccac tgatccttgt tctgctgcct gtcacatcat ctgagtgcca 4260 cattaaagac aaagaaggta aagcatatga gagtgtactg atgatcagca tcgatgaatt 4320 ggacaaatg acaggaactg atagtattg cccgaataat gaaccaact ttttagaaa 4380 acatgtatgt gatgatacaa aggaagctgc ttttctaaat cgtgctgctc gcaagttgaa 4440 gcaatttctt aaatgaata tcagtgaaga attcaatgtc cacttactaa cagtatcaca 4500 aggcacacaa acactggtga actgcacaag taaggaaaaaaacgtaa aggaacagaa 4560 aaagaatgat gcatgttttcc taagagact actgagagaa ataaaactt gttggataa 4620 aattttgaag ggcagtatat agtaagctag ctcgagtcta gaggatccct cccccccccc 4680 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgttttgtct atatgttatt 4740 ttccaccata ttgccgtctt ttggcaatgt gaggccccgg aaacctggcc ctgtctctt 4800 gacgagcatt cctaggggtc ttcccctct cgccaaagga atgcaaggtc tgttgaatgt 4860 cgtgaaggaa gcagttccctc tggaagctc tgagacaa acacgtctg tagcgaccct 4920 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 4980 atagataca cctgcaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 5040 ggaaaggtc aaatggctct cctcaagcgt attchacaag gggctgaagg atgcccagaa 5100 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 5160 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa 5220 aacacgataa taccatgacc atggccctcct ccgaggacgt catcaggag ttcatgcgct 5280 tcaggtgcg catggaggc tccgtgaacg gccacgagtt cgagatcgag ggcgaggggcg 5340 agggccgcccc ctacgaggggc acccagaccg ccaagctga ggtgaccaag ggcggccccc 5400 tgcccttcgc ctgggacatc ctgtcccctc agttccagta cggctccaag gcctacgtga 5460 agcaccccgc cgacatcccc gactacttga agctgtcctt cccgagggc ttcaagtggg 5520 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc 5580 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caactcccc tccgacggcc 5640 ccgtaatgca gagagacc atgggctggg aggcctccac cgagcggatg taccccgagg 5700 acggcgccct gaagggcgag atcagatga ggctgaagct gaggacggc ggccactacg 5760 acgccgaggt caagaccacc tacatggcca agaagcccgt gcagctgccc ggcgcctaca 5820 agaccgacat caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt 5880 acgagcgcgc cgagggccgc cactccaccg gcgcctaa 5918 <210> 2 <211> 11764 <212> DNA <213> Artificial Sequence <400> 2 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 3,60 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200 tgaattatat aaatataaag tagtaaaat tgaaccatta ggagtagcac ccaccaaggc 1260 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800 tataaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920 cccaaccccg aggggacccg acaggcccga agaatagaa gaagaaggtg gagagagaga 1980 2040 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2100 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2160 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt 2220 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 2280 ggggaggggt cggcaattga accggtgcct agaaggtg gcgcggggta aactgggaaa 2340 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2400 2460 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 2520 ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 2580 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 2640 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 2700 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 2760 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 2820 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 2880 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 2940 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 3000 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 3060 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 3120 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 3180 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 3240 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg 3300 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 3360 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaagcgg ccgcgccacc 3420 atggctcaga tgatgactct gagcctcctt agcctggtcc tggctctctg catcccctgg 3480 acccaaggca gtgatggagg gggtcaggac tgctgcctta agtacagcca gaaaaatt 3540 ccctacagta ttgtccgagg ctataggaag aagaaccaa gtttaggctg tcccatcccg 3600 gcaatcctgt tctcaccccg gaagcactct aagcctgagc tatgtgcaaa ccctgaggaa 3660 ggctgggtgc agaacctgat gcgccgcctg gaccagcctc cagccccagg gaaaaaagc 3720 cccggctgca ggagaaccg gggaacctct aagtctggaa agaaaggaaa gggctccaag 3780 ggctccaaga gaccagaca gacacagccc tcaagaggat acccctatga cgtcccagac 3840 3900 cctatgtgtc ctcagaagct aaccatctcc tggtttgcca tcgttttgct ggtgtctcca 3960 ctcatggcca tgtgggagct ggagaaagac gtttatgttg tagaggtgga ctggactccc 4020 gatgcccctg gagaacagt gaacctcacc tgtgacacgc ctgaagaaga tgacatcacc 4080 tggacctcag accagagaca tggactcata ggctctggaa agaccctgac catcactgtc 4140 aaagagtttc tagatgctgg ccagtacacc tgccacaag gaggcgagac tctgagccac 4200 tcacatctgc tgctccaca gaaggaaat ggaatttggt tcacatctgt ttaaaaat 4260 ttcaaaaaca agactttcct gaagtgtgaa gcaccaatt actccggacg gttcacgtgc 4320 tcatggctgg tgcaagaaa catggacttg aagttcaca tcagagcag tagcagttcc 4380 cctgactctc gggcagtgac atgtggaatg gcgtctctgt ctgcagagaa gtcacactg 4440 gaccaaaggg actatgagaa gtattcagtg tcctgccagg aggatgtcac ctgcccaact 4500 gccgaggaga ccctgcccat tgaactggcg tggaagcac ggcagcagaa taataatgag 4560 aactacagca ccagctctt catcagggac atcatcaac cagacccgcc cagaacttg 4620 cagatgaagc ctttgaagaa ctcacaggtg gaggtcagct gggagtaccc tgactcctgg 4680 agcactcccc attcctactt ctccctcaag ttctttgttc gatccagcg caaaagaa 4740 aagatgaagg agacagaga ggggtgtaac cagaaaggtg cgttccctcgt agagagaca 4800 tctaccgaag tccaatgcaa aggcgggaat gtctgcgtgc aagctcagga tcgctattac 4860 aattcctcgt gcagcaagtg ggcatgtgtt ccctgcaggg tccgatccgt accaggagta 4920 ggagtaccag cagtaggaat ggtcagcgtt ccaacagcct caccctcggc atccagcagc 4980 tcctctcagt gccggtccag catgtgtcaa tcacgctacc tcctcttttt ggccaccctt 5040 gccctcctaa accacctcag tttggccagg gtcattccag tctctggacc tgccaggtgt 5100 cttagccagt cccgaaacct gctgaagacc acagatgaca tggtgaagac ggccagagaa 5160 aaactgaaac attattcctg cactgctgaa gacatcgatc atgaagacat cacacgggac 5220 caaaccagca cattgaagac ctgtttacca ctggaactac acaagaacga gagttgcctg 5280 gctactagag agacttcttc cacaacaaga gggagctgcc tgcccccaca gaagacgtct 5340 ttgatgatga ccctgtgcct tggtagcatc tatgaggact tgaagatgta ccagacagag 5400 ttccaggcca tcaacgcagc acttcagaat cacaaccatc agcagatcat tctagacaag 5460 ggcatgctgg tggccatcga tgagctgatg cagtctctga atcataatgg cgagactctg 5520 5580. cgccagaaac ctcctgtggg agaagcagac ccttacagag tgaaaatgaa gctctgcatc 5640. ctgcttcacg ccttcagcac ccgcgtcgtg accatcaaca gggtgatggg ctatctgagc tccgcctagt aaagtttaaa cacgcgtggt gtggaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 5880 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agagtagtg aggaggcttt tttggaggcc gccaccatga gacttctcct cctgactttc ctgggagtct gctgcctcac cccatgggtt gtggaaggtg tggggactga agtcctagaa 6060 gagagtagct gtgtgaactt acaaacccag cggctgccag ttcaaaaaaat caagacctat atcatctggg agggggccat gagagctgta atttttgtca ccaaacgagg actaaaatt tgtgctgatc cagaagccaa atgggtgaaa gcagcgatca agactgtgga tggcagggcc agtaccagaa agaacatggc tgaactgtt cccacaggag cccagaggtc caccagcaca 6300 gcgataaccc tgactgggga ttacaaggat gacgacgata aggctacta cttcagccctg 6360 ctgaagcagg ctggagacgt ggaggagaac cctgaccta tgtccatgt ttctttaga 6420 tatactttg gattcctcc actgatcctt gttctgctgc ctgtcacatc atctgagtgc 6480 cacattaaag aaagaagg windowcat gagagtgtac tgatgatcag catcgatgaa 6540 ttggacaaaa tgacaggaac tgatagtaat tgcccgaata atgaccaaa cttttttaga 6600 aaacatgtat gtgatgatac aaaggaagct gcttttctaa atcgtgctgc tcgcaagttg 6660 aagcaatttc ttaaaatgaa tatcagtga gattcaatg tccacttact aacagtatca 6720 aaggcacac aaacactggt gaacgcaca agtaggaag aaaaaacgt aaggaacag 6780 aaaaagaatg atgcatgttt cctaagaga ctactgagag aaaaaaac ttgttggaat 6840 aaaattttga agggcagtat aagctagct agctcgagtc taggattcc ctcccccccc 6900 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgttttgt ctatatgtta 6960 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc 7020 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat 7080 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc 7140 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt 7200 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt 7260 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag 7320 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt 7380 tagtcgaggt taaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga 7440 aaaacacgat aataccatga ccatggcctc ctccgaggac gtcatcaagg agttcatgcg 7500 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg 7560 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc 7620 cctgcccttc gcctgggaca tcctgtcccc tcagttccag tacggctcca aggcctacgt 7680 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg 7740 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct 7800 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg 7860 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgga tgtaccccga 7920 ggacggcgcc ctgaagggcg agatcaagat gaggctgaag ctgaaggacg gcggccacta 7980 cgacgccgag gtcaagacca cctacatggc caagaagccc gtgcagctgc ccggcgccta 8040 caagaccgac atcaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca 8100 gtacgagcgc gccgagggcc gccactccac cggcgcctaa atcgatagat cctaatcaac 8160 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 8220 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 8280 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 8340 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 8400 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 8460 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 8520 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 8580 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 8640 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 8700 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc 8760 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga 8820 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg 8880 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 8940 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 9000 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc 9060 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag 9120 gcccgggtta attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac 9180 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9240 ttcgggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9300 9360 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 9420 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 9480 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 9540 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 9600 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 9660 ttgagtactc accagtcaca gaaagcatc ttacggatgg catgacagta agagaattat 9720 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 9780 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 9840 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 9900 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 9960 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 10020 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 10080 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 10140 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 10200 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 10260 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 10320 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 10380 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 10440 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 10500 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 10560 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 10620 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 10680 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 10740 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 10800 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 10860 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 10920 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 10980 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 11040 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 11100 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 11160 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc 11220 tcatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 11280 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt 11340 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 11400 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 11460 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca 11520 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 11580 cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca 11640 ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt 11700 cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag 11760 tagt 11764

Claims

1. A nucleic acid fragment, the nucleic acid sequence of which is shown in SEQ ID NO:

1.

2. A recombinant vector, characterized in that: It comprises the nucleic acid fragment and a backbone vector according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The backbone vector also includes EF1a promoter, IRES, WPRE, 3'LTR, 3'LTR (ΔU), HIV-1 Ψ, RRE and cPPT / CTS elements.

4. The recombinant vector according to claim 2 or 3, characterized in that Its nucleic acid sequence is shown in SEQ ID NO:

2.

5. A lentivirus, characterized in that Obtained by transfecting cells with the recombinant vector according to any one of claims 2 to 4.

6. A host cell obtained by transfecting the cell with the lentivirus according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell is TC-1 cell.

8. A tumor vaccine, characterized in that Produced by the host cell according to claim 6 or 7.

9. The method for preparing the tumor vaccine according to claim 8, characterized in that: The host cells are incubated with mitomycin and then digested with trypsin to obtain the tumor vaccine.

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