多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达

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Survivin2B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IgK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sig2PAG-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。

Construction and Eukaryotic Expression of Cancer Vaccine Encoding Multitarget Complex Antigen

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.