丙型肝炎病毒构象性表位与乙型肝炎病毒S基因嵌合表达质粒的构建及其表达

Construction and expression analysis of a chimeric plasmid containing hepatitis C virus conformational epitope and hepatitis B virus Sgene

目的构建丙型肝炎病毒(HCV)构象性表位AR3与乙型肝炎病毒S抗原(HBs)嵌合的真核表达质粒,并在293T细胞中表达鉴定。方法采用重叠拼接PCR法,将HCV AR3表位的基因插入pSVB-24H质粒中,构建pSVB-24H-AR3重组质粒;再用PCR法扩增出全长的AR3-S嵌合基因,将其克隆到pCMV-HA载体中,构建pCMV-HA-AR3-S真核表达质粒。脂质体和PEI转染法导入293T细胞,Western blot和ELISA法分别检测重组蛋白的表达及分泌情况。结果 PCR法分别扩增出pSVB-24H部分载体、HCV AR3及HBs部分基因共3条基因片段;利用两步重叠拼接PCR,扩增得到1 118bp的三片段拼接产物,双酶切鉴定重组质粒pSVB-24H-AR3位置正确,测序分析表明插入序列及开放读码框正确,但细胞表达产物难以被抗-HBs识别;将完整的1 128bp的AR3-S基因插入带HA标签的表达载体中,酶切鉴定和测序分析表明重组质粒pCMV-HA-AR3-S构建正确,Western blot结果显示重组AR3-S蛋白可与HA抗体特异性反应;ELISA结果也显示细胞内能检测到较高水平的AR3-S重组蛋白。结论成功构建pSVB-24H-AR3和pCMV-HA-AR3-S嵌合表达质粒并在细胞中表达,为后续开展基于HBs的病毒样HCV颗粒疫苗研究提供了实验依据。

Objective To construct a chimeric eukaryotic expression plasmid containing the hepatitis C virus （HCV） conformational epitope AR3 and the hepatitis B virus （HBV） S antigen and detect its expression in 293T cells. Methods Using overqap splicing PCR, HCV AR3 gene was inserted into pSVB-24H to construct a recombinant plasmid pSVB-24H AR3. Then the full-length chimeric AR3-S gene was amplified by PCR, and cloned into pCMV-HA vector to construct the eukaryotic expression vector pCMV-HA-AR3-S. After being transfected into 293T cells by lipofectamine and PEI, the expression and secretion of the recombinant proteins were assayed by Western blot and ELISA. Results Partial fragment of pSVB-24H vector, HCV AR3 and partial HBs gene were amplified by PCR, respectively. The 1 118 bp long splicing product of these 3 fragments was amplified by over-lapping PCR, and its position in plasmid pSVB-24H-AR3 was identified by double digestions with restriction enzymes and DNA sequence was confirmed by sequencing analysis. However, the proteins expressed in cells were hardly recognized by anti-HBs antibody. Therefore, the full-length AR3-S gene （1 128 bp） was inserted into the vector with HA-tag. The resulting recombinant plasmid, named pCMV-HA- AR3-S, was verified by double digestions with restriction enzymes and DNA sequencing. Western blot indicated that the recombinant AR3 S protein could be recognized by HA antibody specifically, and relatively high level of AR3-S protein was detected in transfected ceils by ELISA. Conclusions Both chimeric expression plasmids, pSVB-24H-AR3 and pCMV-HA AR3-S, were constructed and expressed in mammalian cells successfully, these constructs provide experimental tools for the study of HBs-based HCV vaccine.