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目的 从感染HCV的中国患者血清中扩增编码HCV丝氨酸蛋白酶的cDNA ,在大肠杆菌中进行表达。方法 从血清中提取HCVRNA ,应用RT PCR方法扩增HCV丝氨酸蛋白酶cDNA ,Sanger双脱氧链终止法测定其序列 ,与pBV2 2 0载体构建重组质粒 ,在大肠杆菌中表达 ,SDS PAGE和Westernblot分析重组蛋白。结果 用RT PCR方法扩增得到了HCV丝氨酸蛋白酶基因 ,序列分析表明其读码框架正确 ,丝氨酸蛋白酶活性中心高度保守 ,同源性比较表明该区段来源于HCVⅡ / 1b中国株 ,构建重组载体后 ,在大肠杆菌中获得了丝氨酸蛋白酶的表达。结论 HCVⅡ / 1b型中国株丝氨酸蛋白酶基因的克隆及其在大肠杆菌中的成功表达 ,为进一步研究该蛋白酶活性及影响因素打下了基础。
Objective To amplify the cDNA encoding HCV serine protease from the serum of Chinese patients infected with HCV and express in E. coli. Methods HCV RNA was extracted from serum, cDNA of HCV serine protease was amplified by RT PCR and sequenced by Sanger dideoxy chain termination method. The recombinant plasmid was constructed with pBV220 vector and expressed in E. coli. The recombinant protein was analyzed by SDS PAGE and Western blot . Results The HCV serine protease gene was amplified by RT PCR. The sequence analysis showed that the reading frame was correct and the serine protease activity center was highly conserved. The homology comparison indicated that this region originated from HCV Ⅱ / 1b Chinese strain. After construction of the recombinant vector , Serine protease expression was obtained in E. coli. Conclusion The cloning of serine protease gene of HCV Ⅱ / 1b strain and its successful expression in E. coli provide the basis for further study on the protease activity and influencing factors.