异种化人肾细胞癌特异性抗原G250真核表达载体的构建及表达

目的：构建含有人肾细胞癌特异性抗原G250（CAⅨ）主要T细胞表位区域、猴和鼠CAⅨ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,继而又将酶切后的sig-tG250-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-tG250-Fc-GPI-GM/B7转染COS7细胞,利用流式细胞仪和免疫荧光检测其表达。结果：测序结果表明tG250融合基因序列正确,PCR和酶切鉴定证明已将其连入真核表达载体pVAX1-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-sig-tG250-Fc-GPI-GM/B7在COS7细胞中得到很好的表达。结论：构建了重组质粒pVAX1-sig-t G250-Fc-GPI-GM/B7,且在COS7细胞中有效表达,为以G250为靶点的抗肾细胞癌基因疫苗的构建与功能研究奠定了基础。

Construction and Expression of Eukaryotic Expression Vector Encoding Xenogeneic G250 Complex Antigen for Renal Cell Carcinoma

Objective： To construct a eukaryotic expression plasmid of tG250 fusion gene which encoding the most cytotoxic T lymphocyte epitopes of human G250 and part （3250 of mouse and monkey, and detect its expression in eukaryotic cell COS7. Methods： tG250 fusion gene was constructed by gene synthesis and PCR, and was inserted into a eukaryotic expression vector pCI-Fc-GPI that included the gene of the signal peptide of human Igκ, human IgG-Fc and GPI, and then inserted into another eukaryotic expression vector pVAX1-IRES-GM/B7 which included IRES and human GM-CSF as well as B7.1 gene. The recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM/B7 was transfected to the COS7 cells, and the expression was detected by fluorescence activated cell sorting（FACS） and immunofluorescence（IMF）. Results： The sequence of tG250 fusion gene was consistent with that of design. PCR and enzyme digestion analysis showed that the recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM/B7 was constructed successfully. The expression of this plasmid had been confirmed by FACS and IMF. Conclusion： The recombinant plasmid pVAX1-sig-tG250-FC-GPI-GM/B7 has been constructed and expressed successfully in the COS7 cells. These results are necessary and basic for construction of DNA vaccine targeting G250 and research on the anti-tumor effects in the future.