阅读说明：本技术 一种携带肿瘤-睾丸抗原10基因的重组腺相关病毒载体及其应用价值 (Recombinant adeno-associated virus vector carrying tumor-testis antigen 10 gene and application value thereof ) 是由 刘勇 刘传新 江孝青 常爱全 李婵 黄家良 王洪亮 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种携带肿瘤-睾丸抗原10(Cancer-testis antigen 10,CT10)基因的重组腺相关病毒(rAAV)载体,并证明了其应用价值。该rAAV载体是将CT10抗原基因插入本发明人构建的AAV出发载体从而构建成功的。本发明的研究证明rAAV的病毒载体可有效地将其携带的CT10抗原基因导入人单核细胞-树突状细胞系中,用于刺激CT10抗原特异性细胞毒性T淋巴细胞(CTL)的产生。实验证明,被本发明的rAAV感染的DC所诱导的CTL能够有效地裂解CT10抗原阳性的恶性肿瘤细胞。因此,本发明的携带CT10抗原基因的重组腺相关病毒载体或其相关产品具有实用价值,可被用于制备CT10抗原特异性CTL,裂解CT10抗原阳性的恶性肿瘤细胞,发挥抗肿瘤细胞免疫治疗的作用。(The invention discloses a recombinant adeno-associated virus (rAAV) vector carrying tumor-testis antigen 10 (CT 10) gene, and proves the application value thereof. The rAAV vector is successfully constructed by inserting the CT10 antigen gene into the AAV starting vector constructed by the inventor. The research of the invention proves that the rAAV viral vector can effectively transfer the carried CT10 antigen gene into a human monocyte-dendritic cell line for stimulating the generation of CT10 antigen specific Cytotoxic T Lymphocyte (CTL). Experiments prove that CTL induced by DC infected by the rAAV can effectively crack CT10 antigen-positive malignant tumor cells. Therefore, the recombinant adeno-associated virus vector carrying the CT10 antigen gene or the related products thereof have practical value, can be used for preparing CT10 antigen-specific CTL, and can crack CT10 antigen-positive malignant tumor cells to play the role of anti-tumor cell immunotherapy.)

1. A recombinant adeno-associated virus vector carrying CT10 antigen gene, which is characterized in that: the recombinant adeno-associated virus vector does not contain structural genes of adeno-associated virus, and is only a recombinant adeno-associated virus vector of CT10 antigen genes.

2. The method for constructing the recombinant adeno-associated virus vector carrying the CT10 antigen gene according to claim 1 comprises:

total mRNA was obtained from tumor tissue positive for CT10 antigen, and total cDNA was obtained by reverse transcription from the total mRNA. PCR amplification was performed under the guide of the upstream primer (ATACGCGTTCCTGAAGAAGTCGTCATGC) and the downstream primer (AT TCTAGACTCTGAAGCTAACTACCTGC) to obtain CT10 cDNA with Mlu I and Xba I restriction enzyme recognition sequences. And then the CT10 cDNA is inserted into an initial vector which is an adeno-associated virus vector and is successfully constructed by the inventor, and a CT10 antigen gene is inserted into the position of the structural gene of the knocked-out adeno-associated virus to obtain the recombinant adeno-associated virus vector.

3. A product related to a recombinant adeno-associated virus vector carrying a CT10 antigen gene, comprising: comprises recombinant adeno-associated virus vector DNA carrying CT10 antigen gene and recombinant adeno-associated virus vector carrying CT10 antigen gene with infectivity. The recombinant adeno-associated virus vector DNA carrying the CT10 antigen gene is prepared by the construction method of claims 1-2; the recombinant adeno-associated virus vector with infectivity and carrying the CT10 antigen gene is prepared by transfecting the recombinant adeno-associated virus vector DNA carrying the CT10 antigen gene and performing cell culture.

4. A cell line expressing CT10 antigen, characterized by: the cell line is a human monocyte-dendritic cell line and is infected or transfected by the recombinant adeno-associated virus vector carrying the CT10 antigen gene according to claims 1-3.

5. A T lymphocyte cell line having the specificity of the CT10 antigen, comprising: the cell line is human lymphocyte, and is produced by the dendritic cell infected or transfected by the recombinant adeno-associated virus vector carrying CT10 antigen gene of claim 4 and human lymphocyte mixed culture.

[ technical field ] A method for producing a semiconductor device

The invention relates to a vector in the biological field and application thereof, in particular to a recombinant adeno-associated virus vector carrying tumor-testis antigen 10 (CT 10) gene, a construction method thereof and application value thereof in preparation of targeted cell immunotherapy for resisting CT10 antigen positive tumor.

[ background of the invention ]

The genetic structure of adeno-associated virus (AAV) has been identified. In 1983, Samulski et al described terminal repeats (upstream 5 'end fragment, downstream 3' end fragment) of AAV (Samulski RJ, Srivastava A, Berns KI, Muzyczka N.Rescus of adono-assisted virus from recombinant plasmids: gene correction with the terminal repeat of AAV. cell.33: 135. 143.). In 1984, Hermonat et al described the low-infective particle (lip) and envelope (cap) genes of AAV (Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N.genetics of infected-infected viruses: isolation and prediction characteristics of infected-infected viruses type 2 tissues.J virol.51:329-339.Hermonat, P.L., and Muzyczka, N.Use of infected-infected viruses as a monoclonal DNA cloning vector: transform of toxin expression in tissue culture.Proc.Natal.Acad.S. 81.66.A.640). In 1986, Labow et al identified the p5 promoter located between the upstream 5' fragment and the rep gene (Labow MA, Hermonat PL, Berns KI. Positive and negative automation of the adono-associated virus type 2 gene. J Virol.160: 251-258.).

In 1984, the AAV vector was demonstrated to be useful for gene therapy of human diseases by Paul l. Currently, clinical trials for AAV-based gene therapy of human diseases are mainly conducted in the european and american countries. According to the statistics of the U.S. food and drug administration, ten AAV-based gene therapy clinical trials are ongoing, and AAV viruses carrying therapeutic genes are mainly injected into patients to express the therapeutic genes in vivo, thereby achieving the purpose of treating diseases. The diseases mainly treated by the method are non-tumor diseases such as Parkinson's syndrome, rheumatoid arthritis, hemophilia, heart failure, progressive muscular atrophy, Alzheimer's syndrome and the like. The 11.11.2.2012 european union approved the Glybera product of UniQure company to be used in 27 member countries in the european union, which is the first approved gene therapy drug in western countries, and is a gene drug for treating lipoprotein lipase deficiency genetic disease (LPLD) by using adeno-associated virus type I (AAV-I) to carry foreign genes. Luxturona, developed by Spark Therapeutics, approved by FDA in the United states, is used to treat inherited retinal diseases caused by mutations in RPE65 by carrying foreign genes with adeno-associated virus type II (AAV-II).

AAV is a non-pathogenic defective virus that requires the assistance of gene products from other viruses (e.g., adenovirus) in order to assemble into infectious viral particles. AAV can be currently classified into 12 serotypes (AAV-1 to AAV-12). Among them, the adeno-associated virus type 2 (AAV-2) has the advantages of no pathogenicity, wide host range, long-term expression of target gene, etc., and thus is one of the most potential viral vectors in current gene therapy. AAV-2 genome is about 4700 base pairs (bp) in length, and has repeated terminal segment (TR) at two ends and virus structural gene in the middle, including Rep and Cap genes.

Tumor-testis antigen 10(Cancer-testis antigen 10, CT10), belongs to the tumor-testis antigen (CT antigen) family, and the gene is localized to X chromosome q 27.2. Under physiological conditions, CT10 is restricted to be expressed in testis, but not in other normal tissues. The research finds that the CT10 can be expressed in various tumor tissues, including melanoma, lung cancer, liver cancer, prostate cancer, ovarian cancer and the like. CT10 was first discovered in Melanoma cells and may therefore be referred to as the melanin antigen C2(Melanoma antigen C2, MAGE-C2). The existing research shows that the CT10 antigen has important significance for the diagnosis, treatment effect and prognosis evaluation of malignant tumor. Furthermore, because testis tissue has no Human Leukocyte Antigen (HLA) or human Major Histocompatibility Complex (MHC) molecules, the expressed CT10 antigen does not cause immune response of the body. The CT10 antigen has the characteristics, so that the CT10 antigen becomes a very important target point for anti-tumor immunotherapy.

Dendritic Cells (DC) have a powerful function of initiating an immune response and are the most prominent antigen-presenting cells. The cellular immune response, i.e., the generation of antigen-specific cytotoxic T lymphocytes (cytotoxic T lymphocytes), plays a very important role in the resistance to malignant tumors and viral infections. However, a large number of research data have demonstrated that the stimulation of DC by either antigenic proteins or peptides is inefficient and results in insufficient ability to induce antigen-specific CTL responses by DC, leading to poor efficacy against malignant tumors and viral infections.

[ summary of the invention ]

The technical problem to be solved by the invention is as follows: the defects of the prior art are overcome, the recombinant adeno-associated virus (rAAV) vector carrying the CT10 antigen gene and capable of efficiently and continuously stimulating the DC is invented, and the construction method and the application value of the rAAV vector are elucidated.

The technical problem of the invention is solved by the following technical scheme:

a rAAV vector carrying a CT10 antigen gene is obtained by inserting a CT10 antigen gene into the position of a structural gene which is already knocked out in an adeno-associated virus (AAV) vector, which is a starting vector constructed by the inventor.

The known AAV vector has a p5 promoter, and in order to improve the transcription level of a target gene, a CT10 antigen gene can be further inserted into the AAV vector of which the successfully constructed promoter is one of a macrophage virus (CMV) promoter, a beta-actin promoter and an SV40 early promoter.

The construction method of the recombinant adeno-associated virus vector carrying the CT10 antigen gene comprises the step of inserting the CT10 antigen gene between sites of restriction endonucleases Mlu I and Xba I at the position of a structural gene which is already removed from an AAV vector to obtain the recombinant adeno-associated virus vector carrying the CT10 antigen gene. Specifically, by using a gene recombination method, the AAV vector DNA and the CT10 cDNA are cut off by MluI and Xba I respectively, and then the specific antigen gene CT10 is connected with the AAV vector DNA by using a DNA connection technology to obtain the recombinant adeno-associated virus vector DNA carrying the CT10 antigen gene. The recombinant adeno-associated virus vector has AAV p5 promoter, macrophage virus (CMV) promoter, beta-actin promoter or SV40 early promoter as promoter.

A product related to a recombinant adeno-associated virus (AAV/CT10) vector carrying a CT10 antigen gene comprises an AAV/CT10 plasmid (DNA) and an AAV/CT10 viral vector (infectious viral particles). The AAV/CT10 plasmid is constructed and prepared by the gene recombination technology; the AAV/CT10 virus vector with infectivity is obtained by cell culture after the AAV/CT10 plasmid is transfected.

The preparation method of the product related to the AAV/CT10 viral vector comprises the following steps: the AAV/CT10 plasmid constructed as above is introduced into competent cell of genetic engineering colibacillus, resistance screening is carried out by using culture medium containing ampicillin, white single colony is selected, plasmid is extracted and purified, and a large amount of AAV/CT10 plasmid is obtained. Preparation of infectious AAV/CT10 viral vector: after AAVp cells are co-transfected by the AAV/CT10 plasmid and the pHelper plasmid, infectious AAV/CT10 virus, namely a virus vector, can be obtained.

The recombinant adeno-associated virus vector carrying the CT10 antigen gene has practical value.

The recombinant adeno-associated virus vector can transfect or infect human monocyte-dendritic cell line, and dendritic cells expressed and stimulated by CT10 antigen can effectively activate and generate Cytotoxic T Lymphocyte (CTL) specific to CT10 antigen. The CTL can target, namely specifically crack CT10 positive malignant tumor cells.

AAV/CT10 virus vector and its related products may be in solvent, powder, etc. The solvent can be selected from various solvents, such as cell culture solution (medium), physiological saline or phosphate buffer solution. If necessary, one or more pharmaceutically acceptable carriers can also be added. The carrier comprises a diluent, an absorption enhancer, a surfactant and the like which are conventional in the pharmaceutical field.

The AAV/CT10 virus vector may be used in separating mononuclear cell and lymphocyte from human peripheral blood and infecting or transfecting mononuclear cell with AAV/CT10 virus vector. During cell culture, monocytes are transformed into Dendritic Cells (DCs). After the mixed culture of DC cells and lymphocytes, the DC stimulates T lymphocytes, induces and generates CT10 antigen-specific CTL, and effectively lyses CT10 positive malignant tumor cells.

Compared with the prior art, the invention has the advantages that:

the invention provides a recombinant adeno-associated virus (AAV/CT10) vector carrying CT10 antigen gene. In the AAV/CT10 viral vector of the present invention, the CT10 antigen gene carried by the AAV/CT10 viral vector can be introduced into a monocyte-dendritic cell line, and Dendritic Cells (DCs) in which the CT10 antigen gene is present are used to stimulate effector cells of the immune system. Experiments prove that Cytotoxic T Lymphocytes (CTL) generated by DC infected by the AAV/CT virus can effectively lyse CT10 antigen-positive malignant tumor cells, and have no lysis effect on CT10 antigen-negative cells, so that the AAV/CT10 viral vector or a product related to the recombinant adeno-associated viral vector can be used for preparing CT10 antigen-specific CTL and plays a role in cellular immunotherapy. The invention has important theoretical and practical significance in the application of the cell immunotherapy of malignant tumors and has wide application prospect.

[ description of the drawings ]

FIG. 1 is the structural schematic diagram of recombinant adeno-associated virus vector (AAV/CT10) carrying CT10 antigen gene.

FIG. 2 is a flow chart of construction of recombinant adeno-associated virus vector (AAV/CT10) carrying CT10 antigen gene and preparation of infectious recombinant adeno-associated virus.

FIG. 3 is a graph showing the results of PCR amplification to obtain CT10 cDNA.

FIG. 4 is a diagram showing the results of double digestion of recombinant adeno-associated virus (AAV/CT10) vector.

FIG. 5 is a comparison of the CT10 antigen Gene sequence obtained with the Gene sequence published by the U.S. NCI Gene Bank (NM-016249).

FIG. 6 is a flow chart of the experiment for lysis of CT10 antigen positive malignant tumor cells by Cytotoxic T Lymphocytes (CTL) based on infection of peripheral blood mononuclear cells by a recombinant adeno-associated virus vector (AAV/CT10) carrying the CT10 antigen gene.

FIG. 7 is a diagram showing the results of the detection of the efficiency of infecting peripheral blood mononuclear cells with a recombinant adeno-associated virus vector (AAV/CT10) carrying the CT10 antigen gene.

FIG. 8 is a graph showing the results of detecting the levels of CD80 and CD86 expressed in DCs infected with recombinant adeno-associated virus vector (AAV/CT10) carrying SV40 promoter.

FIG. 9 is a graph showing the results of measuring IFN-. gamma.levels of CTLs induced by DCs infected with a recombinant adeno-associated virus vector (AAV/CT10) carrying a CMV promoter.

FIG. 10 CTLs induced by DCs infected with recombinant adeno-associated virus vector (AAV/CT10) carrying the CT10 antigen gene lyse CT10 antigen positive and negative cells⁵¹Graph of Cr (chromium-51) experimental results.

FIG. 11 shows the result of MHC Class I restriction test of CTL killing induced by DC infected with recombinant adeno-associated virus vector (AAV/CT10) carrying the CT10 antigen gene.

[ detailed description ] embodiments

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

The inventors have succeeded in deleting all the structural genes of AAV-2 including rep and cap genes in a pBR322 plasmid (pBR-AAV2) containing AAV type 2 whole genome DNA, via retention of terminal repeats and/or p5 promoter, and inserting an oligonucleotide fragment to improve the efficiency of replication of recombinant adeno-associated virus (rAAV) DNA and the stability of recombinant adeno-associated virus, thereby obtaining the basic backbone of AAV-2 vector. In addition, the p5 promoter in the AAV-2 vector was also successfully replaced with the Cytomegalovirus (CMV) promoter, the simian vacuolating virus 40(SV40) early promoter, and the beta actin promoter. And on the basis, rAAV virus particles with infectivity are successfully prepared (see Chinese patent ZL201110125683.X), and a foundation is laid for developing new rAAV products.

The AAV/CT10 is recombinant adeno-associated virus vector obtained by inserting CT10 antigen gene into the skeleton of adeno-associated virus vector successfully constructed by the inventor, and the products related to the vector include plasmid, virus and cell line.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold spring harbor).

The percentage concentration is a mass/volume (W/V) percentage concentration or a volume/volume (V/V) percentage concentration unless otherwise specified.

The primers used, DNA sequence synthesis and DNA sequence determination were all done by Life Technologies, USA.