丙型肝炎病毒F蛋白反式激活基因2的表达纯化及多克隆抗体制备  

Expression and polyclonal antibody preparation of F protein transactivating gene 2 of HCV

作　　者：王丹琼[1] 郭江[1] 成军[1] 张健康[2] 赵龙凤[2] 洪源[1] 张黎颖[1] 

机构地区：[1]北京地坛医院传染病研究所,北京100011 [2]山西医科大学第一医院传染科

出　　处：《解放军医学杂志》2008年第7期798-800,共3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：目的构建丙型肝炎病毒(HCV)F蛋白反式激活基因2(FTP2)的原核表达载体并诱导其表达,制备多克隆抗体并探讨其在临床病理组织中表达的意义。方法通过PCR获得HCVFTP2基因,将其克隆至原核表达载体pET32a(+)上,构建重组表达载体,在大肠埃希菌BL21中诱导表达。表达产物经SDS-PAGE及Western blotting验证后进行大量表达并纯化。将纯化的重组蛋白免疫家兔获得多克隆抗体,并将此抗体作为诊断试剂观察其在正常肝组织和临床病理组织中的表达情况。结果以pET32a(+)-FTP2表达载体转化BL21菌后,经IPTG诱导,成功获得大量重组蛋白,分子量约为25kD。将此重组蛋白免疫新西兰兔获得多克隆抗体,经抗体间接ELISA检测证明,多克隆抗体效价>1:128000,免疫组化显示多克隆抗体能够与丙型肝炎、肝硬化、肝细胞癌等临床病理组织产生FTP2抗原反应。结论成功表达了HCV的FTP2蛋白并制备了其多克隆抗体,证实FTP2与丙型肝炎、肝硬化的发病机制密切相关。Objective To construct prokaryotic expression vector of F protein transactivating gene 2 （FTP2） of HCV and induce the expression of FTP2 in E, coli, and prepare the polyclonal antibody, and study the significance of its expression in clinical pathological tissue. Methods HCV FTP2 gene was amplified by PCR, and then cloned into prokaryotic expression vector pET32a（＋） to construct the recombinant vector pET32a（＋） FTP2. The latter was then transformed into E, cdi BL21 and induced with IPTG. After analyzed by SDS-PAGE and Western blotting, the expression products were abundantly expressed and purified. The purified protein was used to im munize normal rabbits and prepare polyclonal antibody. The polyclonal antibody was then used as a diagnostic reagent to detect the FTP2 expression in normal liver tissue and clinical pathological tissue. Results After transferring pET32a（＋）-FTP2 into BL21 and induced with IPTG, the FTP2 fusion protein of about 25kD was highly expressed. The purified protein and polyclonal antibody were obtained successfully. It was manifested by ELISA that the titer of polyclonal antibody was over 1 ： 128 000. lmmunohistochemistry showed that the polydonal antibody underwent a FTP2 antigen reaction with the clinical pathological tissue including pathological tissue of HCV, liver cirrhosis tissue and hepatic carcinoma tissue. Conclusions HCV FTP2 is successfully expressed in prokaryotic system and its polyclonal antibodies were successfully prepared. These results brought some new clues for studying the immunogenicity and biological function of HCV FTP2 protein.

关 键 词：肝炎病毒 F蛋白反式激活基因2 原核表达 

分 类 号：R512.63[医药卫生—内科学] R34-33[医药卫生—临床医学]