HCV E1/E2-Fc融合基因真核载体的构建及表达产物的鉴定

Construction of eukaryotic expression vector of HCV gene E1 + E2 or E2 fused with human IgG Fc and identification of the expression productions

目的 构建表达HCV包膜糖蛋白 E1+E2661及E2661与人IgG Fc融合蛋白的真核表达载体并在COS-7中表达E1+E2661及E2661与人IgG Fc的融合蛋白,为研究HCV包膜糖蛋白的功能及其与细胞表面受体相互作用奠定基础。方法 利用PCR 方法从HCV基因组序列中扩增出编码HCV包膜糖蛋白El+E2661及E2661的基因,再将两段基因片段亚克隆到含有人IgG Fc基因的Pblue-scriptⅡSK-hc γ1载体中,获取El+E2661及E2661与hc γ1的融合基因并插入真核表达载体pEF BOSneo,构建表达HCV E1+E2661及E2661与 hc γ1的融合蛋白的真核表达载体;用脂质体介导转染COS-7细胞;RT-PCR检测COS-7细胞内融合基因,直接免疫荧光、ELISA、Western blot检测COS-7细胞培养上清及细胞内融合蛋白的表达。结果DNA测序结果证实插入片段即是HCV E1+E2661及E2661与HC γ1的融合基因;直接免疫荧光、ELISA、Western blot均检测到融合蛋白的表达。结论 pEF BOSHCV E1+E2661/Fc pEF—BOSHCV E2661/Fc真核表达载体构建成功,并能够在COS-7细胞中表达分泌性有活性的融合蛋白。

Objective To construct the eukaryotic expression vector of HCV gene E1 + E2661 or E2661 fused with human IgG Fc and express them in COS-7 cells for investigating the function of HCV envelope proteins and the coreaction of HCV envelope proteins with their receptors on the suface of cells. Methods The sequences encoding HCV envelope proteins E1 + E2661 and E2661 were amplified by polymlerase chain reaction (PCR) method from HCV genome and were cloned into the vector Pbluescript Ⅱ SK-hc γ1 which contained the gene of human IgG Fc to obtain the genes that fused HCV envelope protein genes with human IgG-Fc.Cloned fuse genes into pEF BOSneo, a eukaryotic expression vector, to construct recombinant plasmids pEFBOS-HCV E1 + E2661/Fc and pEFBOS-HCV E2661/Fc. These recombinant plasmids were transfected into COS-7 cells by Lipofactamine 2000 regent and fuse genes of HCV E1 + E2661/Fc and HCV E2661/Fc were detected by reverse transcripted polymerase chain reaction (RT-PCR) .The fuse proteins expressed in COS-7 cells were detected by direct immunofluorescence, ELISA and Western-blot methods. Results The analysis of DNA sequence approved that the hc γ1 gene fuse with HCV E1 + E2661 or E2661 were cloned into the recombinant plasmids successfully. The positive results had obtained when the fuse proteins of hc γ1 fused with HCV E1+ E2661 or E2661 were detected by direct immunofluorescence, ELISA and Western-blot methods. Conclusion The eukaryotic expression vector of pEFBOSHCV E1 + E2661/Fc and pEFBOSHCV E2661/FC were constructed successfully and the active proteins were expressed in COS-7 cells.