乙型肝炎病毒e抗原结合蛋白2原核表达载体的构建及诱导表达  

Construction and proeukaryotic expression of a novel HBeAg binding protein 2 of hepatitis B virus in vitro

作　　者：李小权[1] 王琦[2] 成军[2] 张树林[1] 洪源[2] 向天新[3] 

机构地区：[1]陕西省西安交通大学医学院第一附属医院儿科,710061 [2]北京地坛医院传染病研究所 [3]南昌大学第一附属医院

出　　处：《实用肝脏病杂志》2008年第5期289-291,299,共4页Journal of Practical Hepatology

基　　金：国家自然科学基金资助项目(30371288);国家重点基础研究项目(973项目)(2004CB518908)

摘　　要：目的体外构建乙型肝炎病毒e抗原结合蛋白2原核表达载体并观察其表达情况。方法以含有HBEBP2全长序列的质粒为模板,通过PCR扩增获得HBEBP2基因片段,将HBEBP2连接到pGEM-T载体,在测序证实正确后将其插入至原核表达载体pET-32a(+)中,转化BL21大肠埃希菌,经IPTG诱导,并通过SDS-PAGE和Western blot分析鉴定融合蛋白的表达。结果扩增获得的HBEBP2基因片断被成功构建到大肠埃希菌原核表达载体中。经IPTG诱导,得到了融合蛋白的表达,经Western blot分析证实其分子量为34kD,具有抗原性。结论体外成功表达HBEBP2蛋白,为进一步研究HBEBP2蛋白的免疫原性和生物学特性奠定了基础。Objective To construct proeukaryotie expressive vector of HBEBP2 gene,and to observe the expression of recombinant protein in vitro. Methods The DNA fragment of HBeAg binding protein 2 was amplified by polymerase chainraction reaction,and cloned into pGEM-T vector. The constructed recombinant plasmid was identified by restriction analysis and DNA sequencing. The correct DNA fragment was inserted into inducible proeukaryotic expressive vector pET- 32a （＋） and transformed into E.coli BI21. The protein was induced with IPTG and analyzed with sodium dodecylsulfate- polyacrylamide gel electrophoresis and Western blot hybridization. Results The results of restriction analysis,PCR and DNA sequencing proved that HBEBP2 fragment was correctly inserted into vector pET-32a （＋）. After induction with IlYFG,recombinant target protein with about Mr 34kD was expressed. Western blot analysis showed that the protein had good antigenicity. Conclusions The recombinant HBEBP2 gene was expressed successfully. These results lay the foundation for studying the immunogenieity and bionomies of the HBEBP2 protein.

关 键 词：乙型肝炎病毒 HBeAg结合蛋白2 原核表达 体外 

分 类 号：R392[医药卫生—免疫学]