乙型肝炎病毒前-X蛋白在大肠埃希菌中的表达及多克隆抗体的制备  

Expression and polyclonal antibody preparation of HBV pre-X protein

作　　者：张延峰[1] 成军[2] 洪源[2] 王琦[2] 

机构地区：[1]军事医学科学院,北京100850 [2]北京地坛医院传染病研究所

出　　处：《解放军医学杂志》2008年第7期801-802,共2页Medical Journal of Chinese People's Liberation Army

基　　金：“973”国家重点基础研究发展计划资助项目(2004CB518908)

摘　　要：目的在大肠埃希菌中表达乙型肝炎病毒(HBV)前-X蛋白,纯化、鉴定并制备多克隆抗体。方法通过PCR技术获得HBV前-X编码基因片段,克隆至载体pET32a(+),构建原核表达重组质粒,转化至大肠埃希菌中诱导表达,表达产物进行Ni+亲和层析纯化,经Western blotting证实该蛋白的免疫原性,然后免疫新西兰兔获得多克隆抗体并经ELISA实验测定效价,经Western blotting证实其免疫反应特异性。结果成功构建原核表达载体并在大肠埃希菌中高效表达,表达产物主要以包涵体形式存在,融合蛋白的相对分子量约为25kD,Western blotting结果表明目的蛋白具有特异性的免疫反应识别。成功获得前-X蛋白的多克隆抗体,ELISA检测抗体效价为>1:128000,Western blotting实验证实获得的抗体能发生特异性免疫反应。结论成功完成了HBV前-X蛋白的体外表达及多克隆抗体制备,为检测前-X蛋白在乙型肝炎患者体内的表达及进一步研究其生物学特性奠定了实验基础。Objective To express and purify HBV pre-X fusion protein in E. coli, and prepare polyclonal antibody for further study on the expression of pre-X protein in vivo in HBV patients. Methods HBV pre-X coding gene fragment obtained by PCR was cloned into vector pET32a（＋） to construct recombinant vector pET32a（＋）-pre-X. The latter was then transformed into competent E. coli BL21 to express fusion protein inducing by IPTG. The expressive products were then purified and re-natured by Ni＋ affinity column chromatogra- phy. The purified pET32a（ ＋）-pre-X fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were detected by Western blotting and ELISA. Results The expression vector pET32a（＋）-preX was successfully constructed and fusion protein was highly expressed. The fusion protein of about 25 kD was mainly produced in the form of inclusion body. The polyclonal antibody was obtained successfully. It was showed by ELISA detection that the titer of polyclonal antibody was over 1 ： 128 000 and its high specificity was testified by Western blotting. Conclusion The expression of pre-X fusion protein in vitro and the polyclonal antibody preparation will be helpful to detect the expression of pre-X protein in patients with chronic ttBV infection. These re suits lay a foundation for further study on biological function and the mechanism of interaction between pre-X protein and liver cells.

关 键 词：肝炎 乙型 慢性 原核表达 前-X蛋白 

分 类 号：R512.62[医药卫生—内科学]