乙型肝炎病毒PreS基因的克隆表达及亲和层析纯化

目的：获取乙型肝炎病毒前S区基因（HBVpreS)，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HBVPreS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pRSET-C中，转化大肠杆菌JM109（）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－PreS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preS区全长基因，得到融合6个His的HBV－PreS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，为以后的深入研究奠定了基础。

Cloning and fusion expression of HBV-PreS gene and its purification us ing affinity chromatography

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.