中国人血清中丙型肝炎病毒聚合酶全长基因的克隆表达及鉴定

Cloning, Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum

构建中国人丙型肝炎病毒(HCV)复制的RNA聚合酶原核表达载体pET30aNS5b,并在大肠杆菌中获得NS5B聚合酶蛋白的高效表达,为建立HCV NS5b聚合酶细胞外分子复制模型的方法创造条件。使用高保真Pfu DNA聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性血清中扩增出HCV NS5b RNA多聚酶全基因序列,经BamHI和SalI酶切,将其克隆至同样酶切的pET-30a载体中:转化大肠杆菌BL21,IPTG诱导表达。用抗HCV NS5b单克隆抗体做Western-Blot进行鉴定。结果表明构建了原核表达载体,pET30aNS5bpET30aNS5b明显表达出12-His-NS5b聚合酶蛋白。测序结果表明,与已发表的相关HCV NS5b RNA聚合酶序列比较,其核苷酸和氨基酸的同源性分别在69％～92.7％及88.8％～96.8％之间。在最佳表达条件下,可高效诱导表达融合蛋白(65kDa),最高表达量占菌体蛋白18.9％。Western-Blot结果显示表达蛋白为HCV NS5b酶。HCV聚合酶蛋白全长基因可以成功地克隆在pET-30a载体上并有效表达出目的蛋白,为研究建立HCV NS5b聚合酶细胞外分子复制模型莫定了基础。

To amplify Hepatitis C virus(HCV) polymerase sequence and express HCV NS5b protein in order to, in the next, establish a molecular model of HCV replication. RT-PCR method was used to amplify HCV NS5b polymerase gene from a Chinese HCV(+) patient's serum. The polymerase fragment was constructed in the plasmid pET-30a and expressed in E.coli. HCV NS5b protein was identified by Western blot with a monoclonal antibody against HCV NS5b. Sequence analysis showed that the isolate had 69%-95% and 89%-97% homologies to the reported HCV sequences in respect to nucleotide and amino acid respectively. HCV NS5b gene was expressed at a high level and the molecular weight of the expressed product was 65kDa, which was within the range of expectation. This protein was confirmed to be HCV NS5b by Western blot. HCV NS5b from a Chinese patient can be success- fully cloned and expressed with pET-30a plasmid and E.coli.This protein could be used to establish a molecular model of HCV replication.