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多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达 被引量:4

Construction and Eukaryotic Expression of Cancer Vaccine Encoding Multitarget Complex Antigen
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摘要 目的:构建含有人存活蛋白(survivin)-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域融合基因的真核表达质粒,并在人胚肾293T细胞中进行表达。方法:通过基因合成和搭桥PCR技术构建含有Survivin-2B主要T细胞表位区域、人和猴CTP37区域基因的融合基因2PAG,将其插入含有人Igκ链前导信号肽(sig)、人IgG-Fc和糖基磷脂酰肌醇(GPI)锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI-Fc-GPI中,继而又将酶切后的sig-2PAG-FC-GPI融合基因导入含有细小病毒内部核糖体结合位点(IRES)基因且可以共表达人GM-CSF和B7.1融合基因的真核表达载体pVAX1-IRES-GM/B7中;将构建的重组质粒pVAX1-sig-2PAG-FC-GPI-GM/B7(简称pVAX1-2PFcGB)转染293T细胞,利用流式细胞仪和免疫荧光检测其表达情况。结果:2PAG融合基因经测序正确,PCR和酶切鉴定证明已成功连入真核表达载体pVAX-IRES-GM/B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-2PFcGB在293T细胞中得到很好的表达。结论:成功构建了重组质粒pVAX1-2PFcGB,且在293T细胞中可以有效表达,为对该基因疫苗的后续功能研究奠定了基础。 Objective: To construct an eukaryotic expression plasmid of 2PAG fusion gene which encoding the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain CTP37 region of human and monkey, and detect the expression in eukaryotic cell 293T. Methods: 2PAG fusion gene was constructed by gene synthesis and PCR, and was inserted into an eukaryotic expression vector pCI-Fc-GPI that included the gene of the targeting signal peptide of human Igκ, human IgG-Fc and GPI, and then inserted into another eukaryotic expression vector pVAX-IRES- GM-B7 which included IRES and human GM-CSF and B7.1 gene. The recombinant plasmid pVAX-sig-2PAG-FC-GPI- GM/B7 was transfected to the 293T cells, and the expression was detected by FACS and IMF. Results: The sequence of 2PAG fusion gene was consistent with that of design. PCR and enzyme digestion analysis showed that the recombinant plasmid pVAX-sig-2PAG-FC-GPI-GM/B7 was constructed successfully. The expression of this plasmid had been demonst.rated by FACS and IMF. Conclusion: The recombinant plasmid pVAX-sig-2PAG-FC-GPI-GM/B7 has been constructed and expressed successfully in the 293T cells. These results have provided necessary basises to research the anti-tumor effects of this cancer vaccine in the future.
出处 《生物技术通讯》 CAS 2008年第1期1-4,共4页 Letters in Biotechnology
基金 国家高技术研究发展计划项目(2003AA216093)
关键词 肿瘤抗原 基因疫苗 真核表达 cancer antigen genetic vaccine eukaryotic expression
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