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CN115433734B - Nucleic acid fragments and their application in the preparation of tumor vaccines - Google Patents

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

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CN115433734B
CN115433734B CN202210615397.XA CN202210615397A CN115433734B CN 115433734 B CN115433734 B CN 115433734B CN 202210615397 A CN202210615397 A CN 202210615397A CN 115433734 B CN115433734 B CN 115433734B
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nucleic acid
tumor
cells
cell
cancer
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CN115433734A (en
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孙忠杰
刘鹭
齐海龙
谢皇帆
苏红
冯晶
李姝�
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Sun Zhongjie
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Beijing Qitang Technology Co ltd
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Abstract

The invention relates to the technical field of immunotherapy, in particular to a nucleic acid fragment and application thereof in preparing tumor vaccines. The nucleic acid fragment provided by the invention can be used for constructing tumor vaccines, and the effects of enhancing tumor immune response, releasing a large amount of TAA, activating a plurality of TAA specific killer cells and further inhibiting tumor growth and development are achieved by utilizing tumor cells to overexpress XCL1/CCL21/IL-7/IL-12, recruiting and activating CD8α+DC and killer effector cells.

Description

Nucleic acid fragment and application thereof in preparation of tumor vaccine
Technical Field
The invention relates to the technical field of immunotherapy, in particular to a nucleic acid fragment and application thereof in preparing tumor vaccines.
Background
Cancer is one of the most refractory diseases in the world. Traditional cancer treatments include surgical excision, radiation therapy, and chemotherapy, but are not suitable for all patients and have limited efficacy. From the beginning of the 20 th century, immunotherapy (immunotherapy) has become a new approach to tumor treatment, including allogeneic hematopoietic cell transplantation, adoptive cell transplantation (including T, NK, DC, etc. cells), targeted monoclonal antibodies, immune checkpoint inhibition, and other cell therapies, among others. Although achieving a certain curative effect, the targeted therapy and immune checkpoint inhibitor often have tumor tolerance phenomenon, and the adoptive cell transplantation is almost ineffective in treating the solid tumor at present. The reasons for failure of tumor treatment are mainly the heterogeneity of the tumor and the immunosuppressive microenvironment of the tumor. The drug treatment often forms screening pressure, so that drug-resistant tumor cells have survival advantages to cause tumor recurrence and metastasis, cells used in the adoptive cell treatment are difficult to enter tumor tissues, and the immunosuppressive microenvironment of the tumor often desensitizes killer cells to lose anti-tumor effect. Thus, there remains a need for developing new strategies for tumor treatment. In order to overcome the heterogeneity of tumor cells, tumor cells derived from tumor tissues can be used as vaccines, so that abundant potential multiple Tumor Associated Antigens (TAA) are provided for tumor treatment, and T cells with multiple TAA specificities are activated, thereby realizing the purpose of tumor treatment.
Overcoming the difficulty of immune cell infiltration of tumor tissue and the immunosuppressive microenvironment can be initiated by chemokines and immunocompetent cytokines. The anti-tumor immune response relies on activation of Antigen Presenting Cells (APCs) and antigen specific T cells. Exogenous antigen can be effectively presented to the cell surface only by uptake and processing by APC, and the initial T cell is activated. In recent years, a great deal of research has considered that in mice, CD8α+DC is the most potent APC inducing activation of CD8+ T cells, and that antigen can be transferred from a tumor to draining lymph nodes (the corresponding cells in humans are CD141+DC, also known as BDCA 3+DC).
Among cytokines, interleukin-7 (IL-7) and interleukin-12 (IL-12) play an important role in combating tumors. IL-7 is one of the members of the IL-2 superfamily, exerting its effect by binding to receptors with a common gamma chain subunit on the cell surface. IL-7 has anti-tumor immune response, can induce monocytes to produce IL-1 alpha, IL-1 beta and TNF-alpha, and is the most effective cytokine for increasing cytotoxicity CD8+T lymphocytes. IL-7 treatment may enhance long-term tumor antigen-specific CD8+ T cell responses. IL-12 consists of two covalently linked p35 and p40 subunits. As one of the most effective drugs for anti-tumor immunotherapy, the immunomodulatory and anti-angiogenic functions of IL-12 are the basis for their anti-cancer effects. IL-12 can link innate and adaptive immunity, inducing cellular immunity by promoting IFN-gamma production, proliferation and cytolytic activity of NK cells and T cells. In addition, IL-12 induces anti-angiogenic programs through IFN-gamma and lymphocyte-endothelial cell interactions. Therefore, the preparation of the vaccine with the anti-tumor function by using the interleukin-7 (IL-7) and the interleukin-12 (IL-12) has good application prospect.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a nucleic acid fragment and its application in preparing tumor vaccine.
The nucleic acid fragment provided by the invention comprises an IL-12 expression module and an IL-7 expression module;
the IL-12 expression module comprises nucleic acid encoding CCL21 and nucleic acid encoding IL-12;
The IL-7 expression module comprises a nucleic acid encoding XCL1 and a nucleic acid encoding IL-7.
In the invention, the IL-12 expression module sequentially comprises a promoter A, nucleic acid encoding CCL21, nucleic acid encoding self-shearing 2A ribosome jump peptide and nucleic acid encoding IL-12;
in the invention, the IL-7 expression module sequentially comprises a promoter B, nucleic acid encoding XCL1, nucleic acid encoding self-shearing 2A ribosome jump peptide and nucleic acid encoding IL-7.
In an embodiment of the invention, the nucleic acid fragment comprises a promoter A, a nucleic acid encoding CCL21, a nucleic acid encoding a self-cleaving 2A ribosomal jump peptide, a nucleic acid encoding IL-12B, a linker, a nucleic acid encoding IL-12A, a promoter B, a nucleic acid encoding XCL1, a nucleic acid encoding a self-cleaving 2A ribosomal jump peptide, a nucleic acid encoding IL-7, an IRES fragment, and a reporter gene, which are sequentially linked.
In the present invention, the nucleic acid encoding IL-12 includes nucleic acid encoding IL-12B, linker and nucleic acid encoding IL-12A.
In the present invention, the promoter a and the promoter B are independently selected from:
SV40 promoter,
An EF1a mammalian promoter comprising an enhancer sequence and an intron,
Or a CMV mammalian promoter comprising an enhancer sequence and an intron.
In some embodiments, promoter A is an EF-1. Alpha. Promoter and promoter B is an SV40 promoter.
In the invention, the amino acid sequence of the linker-encoded short peptide is VPGVGVPAVG.
In the present invention, the reporter gene is mRFP.
In some embodiments, the nucleic acid sequence of the nucleic acid fragment is shown in SEQ ID NO. 1.
The invention also provides a recombinant vector which comprises the nucleic acid fragment and the backbone vector.
In some embodiments, the recombinant vector of the present invention further comprises EF1apromotor, IRES, WPRE, 3' LTR (ΔU), HIV-1 ψ, RRE and cPPT/CTS elements on the backbone vector.
In some embodiments, the nucleic acid sequence of the recombinant vector is shown in SEQ ID NO. 2.
The invention also provides a lentivirus, which is obtained by transfecting cells with the recombinant vector.
The slow virus of the invention is constructed by transfecting cells with the recombinant vector and pPAX, pMD2G plasmids. In some embodiments, the cell is a 293T cell. The transfection method adopts a calcium phosphate coprecipitation method.
The host is obtained from the lentivirus transfected cells.
The construction of the host of the invention is obtained by transfecting cells with the lentiviral particles, and polybrene is added during transfection. The polybrene concentration was 8 μg/ml. In some embodiments, the cell of the host of the invention is a TC-1 cell.
The invention discloses application of at least one of the following I) to IV) in preparing tumor vaccines;
I) Said nucleic acid fragment;
II) the recombinant vector;
III) said lentivirus;
IV), said host.
The invention also provides tumor vaccines made from the hosts of the invention.
The preparation method of the tumor vaccine comprises the steps of incubating the host with mitomycin and then digesting the host with trypsin to obtain the tumor vaccine. In some embodiments, the concentration of mitomycin is 1mg/ml. The incubation conditions were 37 ℃ for 4 hours. After the incubation, the incubation is performed for 3-5 times by using PBS buffer solution.
The application of the tumor vaccine in preparing a medicine for treating tumors is provided.
The tumor of the present invention includes cervical cancer, melanoma, non-small cell lung cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer metastasis or ewing's sarcoma.
The invention also provides methods for preventing, treating and/or alleviating symptoms of cancer in a patient by administering the tumor vaccine of the invention.
The nucleic acid fragment provided by the invention can be used for constructing tumor vaccines, and the effects of enhancing tumor immune response, releasing a large amount of TAA, activating a plurality of TAA specific killer cells and further inhibiting tumor growth and development are achieved by utilizing tumor cells to overexpress XCL1/CCL21/IL-7/IL-12, recruiting and activating CD8α+DC and killer effector cells.
Drawings
FIG. 1-a. Backbone vector map;
FIG. 1-b schematic representation of CCL21, IL-12, XCL1, IL-7 expression vector plasmids;
FIG. 2 flow assay results of reporter gene mRFP expression of TC-1 cells transfected with CCL21, IL-12, XCL1, IL-7 expression vector plasmids;
FIG. 3 results of gene expression assays of TC-1 cells transfected with CCL21, IL-12, XCL1, IL-7 expression vector plasmids;
FIG. 4 tumor growth curves (prophylactic model) of TC-1 cell line tumor-bearing mice transfected with CCL21, IL-12, XCL1, IL-7 expression vector plasmid mitomycin-treated TC-1 cell cancer vaccine, injected on days-21, -14, -7, and vaccinated on day 0.
Detailed Description
The invention provides a nucleic acid fragment and application thereof in preparing tumor vaccines, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The nucleic acid fragment provided by the invention comprises 4 fragments for encoding CCL21, IL12, XCL-1 and IL-7, the connection sequence of the 4 fragments is not limited, and the 4 fragments can be connected with a promoter, a linker, a self-shearing 2A ribosome jump peptide or a reporter gene.
In the present invention, IL-12 in the nucleic acid fragment is fused to CCL21 as an expression module, and IL-7 is fused to XCL1 as an expression module.
Included among the IL-12 expression modules are nucleic acids encoding CCL21 and nucleic acids encoding IL-12. Specifically included are promoter A, nucleic acid encoding CCL21, nucleic acid encoding self-cleaving 2A ribosomal jump peptide, and nucleic acid encoding IL-12. More specifically, from the 5 'end to the 3' end, the IL-12 expression module includes, in order, a promoter A, a nucleic acid encoding CCL21, a nucleic acid encoding a self-cleaving 2A ribosomal jump peptide, and a nucleic acid encoding IL-12.
Included in the IL-7 expression module are nucleic acids encoding XCL1 and nucleic acids encoding IL-7. Specifically included are promoter B, nucleic acid encoding XCL1, nucleic acid encoding self-cleaving 2A ribosomal jump peptide, and nucleic acid encoding IL-7. More specifically, the IL-7 expression module comprises, in order from the 5 'end to the 3' end, a promoter B, a nucleic acid encoding XCL1, a nucleic acid encoding a self-cleaving 2A ribosomal jump peptide, and a nucleic acid encoding IL-7.
In the present invention, the IL-12 expression module and the IL-7 expression module may exist in the same plasmid vector or may exist in two different vectors, respectively. When present in the same vector, the IL-12 expression module and the IL-7 expression module may be directly linked or may be separated by other fragments. In embodiments of the invention, the IL-12 expression module and IL-7 expression module are directly linked. The invention does not limit the connection sequence of the IL-12 expression module and the IL-7 expression module. In the embodiment of the invention, the 5' end is IL-12 expression module of the nucleic acid fragment as an example for verification.
Thus, in a specific embodiment of the invention, the nucleic acid fragment comprises, in sequential order, promoter A, nucleic acid encoding CCL21, nucleic acid encoding a self-cleaving 2A ribosomal jump 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 jump peptide, nucleic acid encoding IL-7, IRES fragment, and reporter gene.
The nucleic acid sequences of the fragments involved in the present invention are shown in the following table:
In the nucleic acid fragment provided by the invention, each sequence is from a mouse, and in the embodiment of the invention, the effect of the tumor vaccine is verified by taking a mouse transplanted tumor cell TC-1 as an experimental object. In clinical application, the kit is used for preventing and treating human tumors, and each fragment should be selected from human nucleic acid fragments.
In some embodiments, the sequence of the nucleic acid fragment is:
ggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaagcggccgcgccaccatggctcagatgatgactctgagcctccttagcctggtcctggctctctgcatcccctggacccaaggcagtgatggagggggtcaggactgctgccttaagtacagccagaagaaaattccctacagtattgtccgaggctataggaagcaagaaccaagtttaggctgtcccatcccggcaatcctgttctcaccccggaagcactctaagcctgagctatgtgcaaaccctgaggaaggctgggtgcagaacctgatgcgccgcctggaccagcctccagccccagggaaacaaagccccggctgcaggaagaaccggggaacctctaagtctggaaagaaaggaaagggctccaagggctgcaagagaactgaacagacacagccctcaagaggatacccctatgacgtcccagactacgcagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctatgtgtcctcagaagctaaccatctcctggtttgccatcgttttgctggtgtctccactcatggccatgtgggagctggagaaagacgtttatgttgtagaggtggactggactcccgatgcccctggagaaacagtgaacctcacctgtgacacgcctgaagaagatgacatcacctggacctcagaccagagacatggagtcataggctctggaaagaccctgaccatcactgtcaaagagtttctagatgctggccagtacacctgccacaaaggaggcgagactctgagccactcacatctgctgctccacaagaaggaaaatggaatttggtccactgaaattttaaaaaatttcaaaaacaagactttcctgaagtgtgaagcaccaaattactccggacggttcacgtgctcatggctggtgcaaagaaacatggacttgaagttcaacatcaagagcagtagcagttcccctgactctcgggcagtgacatgtggaatggcgtctctgtctgcagagaaggtcacactggaccaaagggactatgagaagtattcagtgtcctgccaggaggatgtcacctgcccaactgccgaggagaccctgcccattgaactggcgttggaagcacggcagcagaataaatatgagaactacagcaccagcttcttcatcagggacatcatcaaaccagacccgcccaagaacttgcagatgaagcctttgaagaactcacaggtggaggtcagctgggagtaccctgactcctggagcactccccattcctacttctccctcaagttctttgttcgaatccagcgcaagaaagaaaagatgaaggagacagaggaggggtgtaaccagaaaggtgcgttcctcgtagagaagacatctaccgaagtccaatgcaaaggcgggaatgtctgcgtgcaagctcaggatcgctattacaattcctcgtgcagcaagtgggcatgtgttccctgcagggtccgatccgtaccaggagtaggagtaccagcagtaggaatggtcagcgttccaacagcctcaccctcggcatccagcagctcctctcagtgccggtccagcatgtgtcaatcacgctacctcctctttttggccacccttgccctcctaaaccacctcagtttggccagggtcattccagtctctggacctgccaggtgtcttagccagtcccgaaacctgctgaagaccacagatgacatggtgaagacggccagagaaaaactgaaacattattcctgcactgctgaagacatcgatcatgaagacatcacacgggaccaaaccagcacattgaagacctgtttaccactggaactacacaagaacgagagttgcctggctactagagagacttcttccacaacaagagggagctgcctgcccccacagaagacgtctttgatgatgaccctgtgccttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaacgcagcacttcagaatcacaaccatcagcagatcattctagacaagggcatgctggtggccatcgatgagctgatgcagtctctgaatcataatggcgagactctgcgccagaaacctcctgtgggagaagcagacccttacagagtgaaaatgaagctctgcatcctgcttcacgccttcagcacccgcgtcgtgaccatcaacagggtgatgggctatctgagctccgcctagtaaagtttaaacacgcgtggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggccgccaccatgagacttctcctcctgactttcctgggagtctgctgcctcaccccatgggttgtggaaggtgtggggactgaagtcctagaagagagtagctgtgtgaacttacaaacccagcggctgccagttcaaaaaatcaagacctatatcatctgggagggggccatgagagctgtaatttttgtcaccaaacgaggactaaaaatttgtgctgatccagaagccaaatgggtgaaagcagcgatcaagactgtggatggcagggccagtaccagaaagaacatggctgaaactgttcccacaggagcccagaggtccaccagcacagcgataaccctgactggggattacaaggatgacgacgataaggctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctatgttccatgtttcttttagatatatctttggaattcctccactgatccttgttctgctgcctgtcacatcatctgagtgccacattaaagacaaagaaggtaaagcatatgagagtgtactgatgatcagcatcgatgaattggacaaaatgacaggaactgatagtaattgcccgaataatgaaccaaacttttttagaaaacatgtatgtgatgatacaaaggaagctgcttttctaaatcgtgctgctcgcaagttgaagcaatttcttaaaatgaatatcagtgaagaattcaatgtccacttactaacagtatcacaaggcacacaaacactggtgaactgcacaagtaaggaagaaaaaaacgtaaaggaacagaaaaagaatgatgcatgtttcctaaagagactactgagagaaataaaaacttgttggaataaaattttgaagggcagtatatagtaagctagctcgagtctagaggatccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgataataccatgaccatggcctcctccgaggacgtcatcaaggagttcatgcgcttcaaggtgcgcatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtcccctcagttccagtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctccaccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagatgaggctgaagctgaaggacggcggccactacgacgccgaggtcaagaccacctacatggccaagaagcccgtgcagctgcccggcgcctacaagaccgacatcaagctggacatcacctcccacaacgaggactacaccatcgtggaacagtacgagcgcgccgagggccgccactccaccggcgcctaa
In some embodiments, the cells into which the nucleic acid fragments of the invention are inserted can be autologous tumor cells, but xenograft expanded autologous tumor cells, xenograft expanded allogeneic tumor cells, or a combination thereof can also be used.
The recombinant vector of the present invention, referred to as a recombinant nucleic acid vector, is a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences necessary for expression of an operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes interchangeable, as the plasmid is the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors that perform equivalent functions, which are or will become known in the art, including but not limited to plasmids, phage particles, viral vectors and/or just potential genomic inserts.
The host cell of the invention is a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a target gene. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells have the ability to replicate a vector encoding the protein or express the desired protein.
The method for preventing, treating and/or alleviating cancer symptoms in a patient according to the present invention specifically comprises the steps of identifying a patient in need of preventing, treating and/or alleviating cancer symptoms and administering a tumor cell vaccine.
The expression vector plasmid containing CCL21, IL-12, XCL1 and IL-7 is obtained by inserting the nucleic acid fragment in a skeleton vector. In the construction process of the nucleic acid fragment, a complete fragment can be connected at the same time, or a part of the complete fragment can be connected in steps respectively, so that a vector connected with the nucleic acid fragment is finally formed, and the invention is not limited to the above.
The vaccine of the present invention can be used to prevent, treat and/or alleviate symptoms of cancer selected from cervical cancer, melanoma, non-small cell lung cancer, gall bladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer metastasis and ewing's sarcoma, as well as other cancers from patients.
In one embodiment, the present invention is a method of treating, preventing and/or alleviating tumor development in an HPV positive tumor model mouse by administering a whole tumor cell cancer vaccine that expresses CCL21, IL-12, XLC 1, IL-7 comprising the steps of determining an HPV positive tumor model mouse in need of prevention, treatment and/or alleviation of symptoms of cancer, and administering a whole tumor cell cancer vaccine comprising a CCL21, IL-12, XLC 1, IL-7 expression vector plasmid.
In one aspect of the method, one or more cancer cells are collected from a patient having a cancer selected from cervical cancer, melanoma, non-small cell lung cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer, liver cancer metastasis and ewing's sarcoma, and other cancers from the patient. In another aspect, the genetically modified cells are rendered unable to proliferate by mitomycin treatment or irradiation. In yet another aspect, the genetically modified cell is an autologous, allogeneic or xenogeneic expanded cell.
In some embodiments, taking the construction of a vaccine for an autologous tumor as an example, the method comprises the steps of (i) aseptically collecting one or more cancer cells from a patient, (ii) placing the collected cells in an antibiotic solution in an aseptic container, (iii) forming a cell suspension from the collected solution, wherein cell formation (iv) is achieved by enzymatic decomposition, mechanical deagglomeration or both, (v) genetically modifying the cells by replication-defective lentiviral particle transfection to produce an autologous cell vaccine comprising CCL21, IL-12, xcl1, IL-7 expression vector plasmids. (vi) Harvesting the vaccine, (vii) mitomycin treating the vaccine and (vii) freezing the vaccine.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
Example 1
Construction of CCL21, IL-12, XCL1, IL-7 expression vector plasmids:
First, a nucleic acid fragment of CCL21-HA-P2A-IL12B-linker-IL12A-SV40promotor-XCL1-FLAG-P2A-IL7 was synthesized in full fragment, and the sequence between the vector backbone EF1a promotor and IRES shown in FIG. 1-a was replaced with the above fragment by homologous recombination to obtain plasmid 1 (FIG. 1-B). Then, the nucleic acid fragment encoding mRFP is synthesized from the whole fragment, and the ZsGreen1 sequence in the plasmid 1 is replaced by the fragment in a homologous recombination mode, so that the final expression vector plasmid is obtained.
Example 2:
The preparation of TC-1 whole tumor cell cancer vaccine comprises the following steps:
(1) The TC-1 cell line is prepared by transplanting C57/BL6n mice subcutaneously according to 1X 10 6 cells/mouse, after large tumors are formed after 20 days, removing tumor tissues by surgical scissors under aseptic conditions, shearing, digesting for 5-10min at 37 ℃ by collagenase and trypsin, filtering the enzymatic suspension by a 40 mu M screen, and collecting the suspension into a PE centrifuge tube to obtain the TC-1 tumor cells from allogeneic mice.
(2) Centrifuging 200-300g for 3min to obtain TC-1 tumor cell precipitate, adding appropriate amount of RPMI 1640 culture medium containing 10% FBS to resuspend cells, inoculating to 10cm culture dish, ensuring about 5×10 6 cells per dish, and subjecting the grown cells to continuous passage by trypsin digestion for 3-5 times.
(3) The CCL21, IL-12, XCL1, IL-7 expression vector plasmid and pPAX, pMD2G plasmids prepared in example 1 were transiently transferred into 293T cells by a calcium phosphate coprecipitation method, and culture medium supernatant was collected after 48 hours, thus obtaining lentiviral particle suspension containing the expression vector.
(4) The above lentiviral particle suspension was added to TC-1 cells at an MOI value of about 10-100, while polybrene (final concentration 8. Mu.g/ml) was added to enhance the viral transfection efficiency, and the expression ratio and intensity of reporter mRFP were examined by flow (FIG. 2) 72 hours after transfection, and the expression intensities of CCL21, IL-12a, IL-12b, XCT 1 and IL-7 were examined by qPCR (FIG. 3).
The results show that the positive proportion of mRFP in the prepared cells exceeds 80%, and the expression of CCL21, IL-12a, IL-12b, XCL1 and IL-7 is obviously up-regulated, which proves that the constructed cells are qualified and can be further used as cell cancer vaccines.
(5) Qualified TC-1 cells after transfection are incubated with mitomycin (final concentration 1 mg/ml) for 4 hours at 37 ℃, and then washed with PBS for 3-5 times, and the cells obtained by trypsin digestion, namely the whole tumor cell cancer vaccine, are frozen in-80 ℃ or liquid nitrogen for later use.
Example 3:
The whole tumor cell cancer vaccine obtained in example 2 was used as a control 1 for the immunization of mice, and a vaccine containing only CCL21-IL-12 was used as a control 2 for the immunization of mice. The immunoassay comprises the following steps:
(1) TC-1 whole tumor cell cancer vaccine suspension is injected into C57/BL6n mice by tail vein according to the volume of 200 mu L, the cell inoculation amount of each mouse is 1X 10 7, and the control group mice are injected with equal volume PBS. The inoculation was continued 3 times, 1 time every 7 days.
(2) Each mouse was vaccinated with TC-1 tumor cells 21 days from the first vaccination. Tumor cells were inoculated in an amount of 1X 10 6 per mouse by subcutaneous injection. Tumor growth was then observed (fig. 4). The results showed that the tumor volume of the animals given the vaccine of example 2 was significantly reduced, with a significant difference (p < 0.05) from the control. Also, the vaccine of example 2 also showed significant differences in the level of tumor volume reduction in animals compared to control 1 and control 2 (p < 0.05).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Beijing Qiyang technologies Co., ltd
<120> Nucleic acid fragment and application thereof in preparing tumor vaccine
<130> MP21026511
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5918
<212> DNA
<213> Artificial sequence (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 ccagcgcaca tgttcggcga ggcggggcct 660
gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt 720
gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc 780
accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg 840
gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt 900
tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct 960
cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc 1020
gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat 1080
gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca 1140
gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaagcggcc gcgccaccat 1200
ggctcagatg atgactctga gcctccttag cctggtcctg gctctctgca tcccctggac 1260
ccaaggcagt gatggagggg gtcaggactg ctgccttaag tacagccaga agaaaattcc 1320
ctacagtatt gtccgaggct ataggaagca agaaccaagt ttaggctgtc ccatcccggc 1380
aatcctgttc tcaccccgga agcactctaa gcctgagcta tgtgcaaacc ctgaggaagg 1440
ctgggtgcag aacctgatgc gccgcctgga ccagcctcca gccccaggga aacaaagccc 1500
cggctgcagg aagaaccggg gaacctctaa gtctggaaag aaaggaaagg gctccaaggg 1560
ctgcaagaga actgaacaga cacagccctc aagaggatac ccctatgacg tcccagacta 1620
cgcagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc 1680
tatgtgtcct cagaagctaa ccatctcctg gtttgccatc gttttgctgg tgtctccact 1740
catggccatg tgggagctgg agaaagacgt ttatgttgta gaggtggact ggactcccga 1800
tgcccctgga gaaacagtga acctcacctg tgacacgcct gaagaagatg acatcacctg 1860
gacctcagac cagagacatg gagtcatagg ctctggaaag accctgacca tcactgtcaa 1920
agagtttcta gatgctggcc agtacacctg ccacaaagga ggcgagactc tgagccactc 1980
acatctgctg ctccacaaga aggaaaatgg aatttggtcc actgaaattt taaaaaattt 2040
caaaaacaag actttcctga agtgtgaagc accaaattac tccggacggt tcacgtgctc 2100
atggctggtg caaagaaaca tggacttgaa gttcaacatc aagagcagta gcagttcccc 2160
tgactctcgg gcagtgacat gtggaatggc gtctctgtct gcagagaagg tcacactgga 2220
ccaaagggac tatgagaagt attcagtgtc ctgccaggag gatgtcacct gcccaactgc 2280
cgaggagacc ctgcccattg aactggcgtt ggaagcacgg cagcagaata aatatgagaa 2340
ctacagcacc agcttcttca tcagggacat catcaaacca gacccgccca agaacttgca 2400
gatgaagcct ttgaagaact cacaggtgga ggtcagctgg gagtaccctg actcctggag 2460
cactccccat tcctacttct ccctcaagtt ctttgttcga atccagcgca agaaagaaaa 2520
gatgaaggag acagaggagg ggtgtaacca gaaaggtgcg ttcctcgtag agaagacatc 2580
taccgaagtc caatgcaaag gcgggaatgt ctgcgtgcaa gctcaggatc gctattacaa 2640
ttcctcgtgc agcaagtggg catgtgttcc ctgcagggtc cgatccgtac caggagtagg 2700
agtaccagca gtaggaatgg tcagcgttcc aacagcctca ccctcggcat ccagcagctc 2760
ctctcagtgc cggtccagca tgtgtcaatc acgctacctc ctctttttgg ccacccttgc 2820
cctcctaaac cacctcagtt tggccagggt cattccagtc tctggacctg ccaggtgtct 2880
tagccagtcc cgaaacctgc tgaagaccac agatgacatg gtgaagacgg ccagagaaaa 2940
actgaaacat tattcctgca ctgctgaaga catcgatcat gaagacatca cacgggacca 3000
aaccagcaca ttgaagacct gtttaccact ggaactacac aagaacgaga gttgcctggc 3060
tactagagag acttcttcca caacaagagg gagctgcctg cccccacaga agacgtcttt 3120
gatgatgacc ctgtgccttg gtagcatcta tgaggacttg aagatgtacc agacagagtt 3180
ccaggccatc aacgcagcac ttcagaatca caaccatcag cagatcattc tagacaaggg 3240
catgctggtg gccatcgatg agctgatgca gtctctgaat cataatggcg agactctgcg 3300
ccagaaacct cctgtgggag aagcagaccc ttacagagtg aaaatgaagc tctgcatcct 3360
gcttcacgcc ttcagcaccc gcgtcgtgac catcaacagg gtgatgggct atctgagctc 3420
cgcctagtaa agtttaaaca cgcgtggtgt ggaaagtccc caggctcccc agcaggcaga 3480
agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 3540
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 3600
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
ggacaaaatg acaggaactg atagtaattg cccgaataat gaaccaaact tttttagaaa 4380
acatgtatgt gatgatacaa aggaagctgc ttttctaaat cgtgctgctc gcaagttgaa 4440
gcaatttctt aaaatgaata tcagtgaaga attcaatgtc cacttactaa cagtatcaca 4500
aggcacacaa acactggtga actgcacaag taaggaagaa aaaaacgtaa aggaacagaa 4560
aaagaatgat gcatgtttcc taaagagact actgagagaa ataaaaactt gttggaataa 4620
aattttgaag ggcagtatat agtaagctag ctcgagtcta gaggatccct cccccccccc 4680
taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt 4740
ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt 4800
gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 4860
cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct 4920
ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 4980
ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 5040
ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa 5100
ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 5160
gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa 5220
aacacgataa taccatgacc atggcctcct ccgaggacgt catcaaggag ttcatgcgct 5280
tcaaggtgcg catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 5340
agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc 5400
tgcccttcgc ctgggacatc ctgtcccctc agttccagta cggctccaag gcctacgtga 5460
agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg 5520
agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc 5580
aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc 5640
ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcggatg taccccgagg 5700
acggcgccct gaagggcgag atcaagatga ggctgaagct gaaggacggc 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 (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 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat 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
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 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 agagaaggtg gcgcggggta aactgggaaa 2340
gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2400
cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 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 gaagaaaatt 3540
ccctacagta ttgtccgagg ctataggaag caagaaccaa gtttaggctg tcccatcccg 3600
gcaatcctgt tctcaccccg gaagcactct aagcctgagc tatgtgcaaa ccctgaggaa 3660
ggctgggtgc agaacctgat gcgccgcctg gaccagcctc cagccccagg gaaacaaagc 3720
cccggctgca ggaagaaccg gggaacctct aagtctggaa agaaaggaaa gggctccaag 3780
ggctgcaaga gaactgaaca gacacagccc tcaagaggat acccctatga cgtcccagac 3840
tacgcagcta ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga 3900
cctatgtgtc ctcagaagct aaccatctcc tggtttgcca tcgttttgct ggtgtctcca 3960
ctcatggcca tgtgggagct ggagaaagac gtttatgttg tagaggtgga ctggactccc 4020
gatgcccctg gagaaacagt gaacctcacc tgtgacacgc ctgaagaaga tgacatcacc 4080
tggacctcag accagagaca tggagtcata ggctctggaa agaccctgac catcactgtc 4140
aaagagtttc tagatgctgg ccagtacacc tgccacaaag gaggcgagac tctgagccac 4200
tcacatctgc tgctccacaa gaaggaaaat ggaatttggt ccactgaaat tttaaaaaat 4260
ttcaaaaaca agactttcct gaagtgtgaa gcaccaaatt actccggacg gttcacgtgc 4320
tcatggctgg tgcaaagaaa catggacttg aagttcaaca tcaagagcag tagcagttcc 4380
cctgactctc gggcagtgac atgtggaatg gcgtctctgt ctgcagagaa ggtcacactg 4440
gaccaaaggg actatgagaa gtattcagtg tcctgccagg aggatgtcac ctgcccaact 4500
gccgaggaga ccctgcccat tgaactggcg ttggaagcac ggcagcagaa taaatatgag 4560
aactacagca ccagcttctt catcagggac atcatcaaac cagacccgcc caagaacttg 4620
cagatgaagc ctttgaagaa ctcacaggtg gaggtcagct gggagtaccc tgactcctgg 4680
agcactcccc attcctactt ctccctcaag ttctttgttc gaatccagcg caagaaagaa 4740
aagatgaagg agacagagga ggggtgtaac cagaaaggtg cgttcctcgt agagaagaca 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
cgccagaaac ctcctgtggg agaagcagac ccttacagag tgaaaatgaa gctctgcatc 5580
ctgcttcacg ccttcagcac ccgcgtcgtg accatcaaca gggtgatggg ctatctgagc 5640
tccgcctagt aaagtttaaa cacgcgtggt gtggaaagtc cccaggctcc ccagcaggca 5700
gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 5760
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 5820
ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 5880
gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 5940
agaagtagtg aggaggcttt tttggaggcc gccaccatga gacttctcct cctgactttc 6000
ctgggagtct gctgcctcac cccatgggtt gtggaaggtg tggggactga agtcctagaa 6060
gagagtagct gtgtgaactt acaaacccag cggctgccag ttcaaaaaat caagacctat 6120
atcatctggg agggggccat gagagctgta atttttgtca ccaaacgagg actaaaaatt 6180
tgtgctgatc cagaagccaa atgggtgaaa gcagcgatca agactgtgga tggcagggcc 6240
agtaccagaa agaacatggc tgaaactgtt cccacaggag cccagaggtc caccagcaca 6300
gcgataaccc tgactgggga ttacaaggat gacgacgata aggctactaa cttcagcctg 6360
ctgaagcagg ctggagacgt ggaggagaac cctggaccta tgttccatgt ttcttttaga 6420
tatatctttg gaattcctcc actgatcctt gttctgctgc ctgtcacatc atctgagtgc 6480
cacattaaag acaaagaagg taaagcatat gagagtgtac tgatgatcag catcgatgaa 6540
ttggacaaaa tgacaggaac tgatagtaat tgcccgaata atgaaccaaa cttttttaga 6600
aaacatgtat gtgatgatac aaaggaagct gcttttctaa atcgtgctgc tcgcaagttg 6660
aagcaatttc ttaaaatgaa tatcagtgaa gaattcaatg tccacttact aacagtatca 6720
caaggcacac aaacactggt gaactgcaca agtaaggaag aaaaaaacgt aaaggaacag 6780
aaaaagaatg atgcatgttt cctaaagaga ctactgagag aaataaaaac ttgttggaat 6840
aaaattttga agggcagtat atagtaagct agctcgagtc tagaggatcc ctcccccccc 6900
cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 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 taaaaaaacg 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
ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9300
atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 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 gaaaagcatc 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 (9)

1. The nucleic acid fragment has a nucleic acid sequence shown in SEQ ID NO. 1.
2. A recombinant vector comprising the nucleic acid fragment of claim 1 and a backbone vector.
3. The recombinant vector of claim 2, wherein the backbone vector further comprises EF1a promotor, IRES, WPRE, 3' ltr (Δu), HIV-1 ψ, RRE, and cPPT/CTS elements thereon.
4. The recombinant vector according to claim 2 or 3, wherein the nucleic acid sequence is shown in SEQ ID NO. 2.
5. A lentivirus obtained by transfecting a cell with the recombinant vector of any one of claims 2 to 4.
6. A host cell obtained from the lentiviral transfected cell of claim 5.
7. The host cell of claim 6, wherein the host cell is a TC-1 cell.
8. Tumor vaccine, characterized in that it is produced from a host cell according to claim 6 or 7.
9. The method for preparing the tumor vaccine according to claim 8, wherein the tumor vaccine is obtained by subjecting the host cells to trypsin digestion after incubation with mitomycin.
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