抗肾细胞癌异种化G250抗原基因疫苗的构建及真核表达

Construction and eukaryotic expression of genetic vaccine encoding G250 complex antigen for renal cell carcinoma

目的:构建含有人肾细胞癌特异性抗原G250主要T细胞表位区域、猴和鼠G250部分片段区域融合基因tG250的真核表达质粒,并在猴肾COS7细胞中表达。方法:通过基因合成和PCR技术构建人、猴和鼠G250区域融合基因tG250,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;将重组质粒pCI-Fc-tG250-GPI转染COS7细胞,流式细胞仪和免疫荧光检测其表达情况。结果:tG250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pCI-Fc-tG250-GPI中;流式细胞仪和免疫荧光检测显示,重组质粒pCI-Fc-tG250-GPI在COS7细胞中得到很好的表达。结论:成功构建了重组质粒pCI-Fc-tG250-GPI,且在COS7细胞中可以有效表达,为以G250抗原为靶点基因疫苗的后续功能研究打下良好基础。

Objective： To construct a eukaryotic expression plasmid of tG250 fusion genes encoding the most Cytotoxic T Lymphocyte Epitopes of human G250 and part of G250 genes of mice and monkeys, and detect the expression in the eukaryotic cell COS7. Methods： tG250 fusion genes were constructed by gene synthesis and PCR. tG250 genes were inserted into a eukary- otic expression vector pCI-Fc-GPI that included the genes of the targeting signal peptide of human Igk, human IgG-Fc and GPI. The recombinant plasmid PCI-Fc-tG250 GPI was transfected to the COS7 cells, and the expression was detected by FACS （Fluo- rescence Activated Cell Sorting） and IMF （immunofluorescence）. Results： The sequence of tG250 fusion genes was consistent with the design. PCR and enzyme digestion analysis showed that the recombinant plasmid PC1-Fc-tG250- GPI was successfully constructed. The expression of this plasmid was demonstrated by FACS and IMF. Conclusion： The recombinant plasmid pCI-Fc- tG250-GPI has been successfully constructed and expressed in the COS7 ceils. These results have provided necessary bases for the study of the anti-tumor effects of this cancer vaccine targeting G250 in the future.