乙型肝炎病毒PreS基因的克隆表达及亲和层析纯化  

Cloning and fusion expression of HBV PreS gene and its purification using affinity chromatography

作　　者：杨敏[1] 刘新平[1] 韩炯[1] 张晓光[1] 药立波[1] 苏成芝[1] 陈常庆[2] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033 [2]中国科学院上海生物工程中心,上海200032

出　　处：《第四军医大学学报》2001年第16期1493-1496,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金项目 (3 982 5 113 ) ;杰出青年科学基金资助项目 (3 9870 6 99)

摘　　要：目的　获取乙型肝炎病毒前 S区基因 (HBVpre S) ,序列测定正确后进行融合表达 ,为今后研究其机制及应用创造条件 .方法　利用乙型肝炎病毒基因组为模板体外扩增HBVPre S基因后进行基因克隆 .目的基因经测序正确后克隆入融合表达载体 p RSET- C中 ,转化大肠杆菌 JM10 9(DE3) .目的基因经 IPTG诱导 ,由 T7启动子调控表达了氨基端带 6个连续组氨酸残基的 HBV- Pre S蛋白 ,在变性条件下对目的蛋白进行纯化 .结果　成功地扩增到 pre S区全长基因 ,得到融合 6个 His的 HBV- Pre S蛋白纯度大于 90 % .结论　构建了乙型肝炎病毒前 S区基因的重组表达载体 ,获得了稳定表达的工程菌 .AIM To search the novel protein interacted with HBV PreS so as to further study its function and mechanism. METHODS Firstly we cloned the gene of PreS. After sequencing cloned it into the expressing vector pRSET C, itwas transfected into the host strain E.coli JM109 (DE3). After 3 hours induction with IPTG, the strain controlled by T7 promoter expressed the fused PreS protein with a hexahistidine tail in its N terminal. We purified the target protein under denaturing condition. RESULTS SDS PAGE analysis showed that the fusion protein mainly existed in inclusion bodies. We isolated the inclusion bodies from the culture, then purified the fusion protein under denaturing condition using metal chelate affinity chromatography. The purity of HBV PreS was up to 90%. CONCLUSION Construction of the recombinant expressing plasmid lays a basis for further study of the PreS protein.

关 键 词：乙型肝炎病毒 PreS基因 基因克隆 基因表达 亲和层析纯化 

分 类 号：R373.2[医药卫生—病原生物学] Q71[医药卫生—基础医学]