丙型肝炎病毒丝氨酸蛋白酶基因的克隆和表达  

Cloning and expression of hepatitis C virus serine protease gene

作　　者：范涛[1] 程计林[1] 梁庆华[1] 陶其敏[1] 

机构地区：[1]北京医科大学人民医院肝病研究所,100044

出　　处：《中华微生物学和免疫学杂志》2000年第6期511-513,共3页Chinese Journal of Microbiology and Immunology

摘　　要：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 and clone the HCV cDNA fragment that encode serine protease from the serum of a Chinese patient infected with HCV and express it in E.coli. Methods The HCV RNA was extracted from serum, the cDNA that encode HCV serine protease was amplified by RT PCR. Its sequence was determined by dideoxy chain termination method. It was subcloned into vector pBV220 and expressed in E.coli. The expressed recombinant protein was analysed by SDS PAGE electrophoresis and Western blot. Results The HCV serine cDNA was cloned. Sequence analysis showed that its reading frame is correct and no insert or deletion mutation happened. Homology comparison suggested it is from a HCV typeⅡ/1b isolate. This cDNA fragment was efficiently expressed in E.coli. Conclusion The serine protease cDNA was amplified successfully from the serum of a Chinese patient with HCV(Ⅱ/1b) infection. The expression of the serine protease in E.coli rollouted a basis for the study of its activity.

关 键 词：丙型肝炎病毒 丝氨酸蛋白酶 基因表达 

分 类 号：R373.2[医药卫生—病原生物学]