【摘要】 目的应用基因芯片技术筛选获得p7蛋白反式调节的新基因,即丙型肝炎病毒(HCV)p7蛋白反式激活基因p7TP2,研究其生物学功能。方法应用聚合酶链反应(PCR)技术扩增其全长cDNA序列,并构建原核表达载体pET-32a(+)-p7TP2,转入大肠埃希菌Rosetta-gami B中,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达重组融合蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析在相对分子量约35kD处有一条特异的蛋白质条带,以包涵体形式表达,并通过蛋白质变性、重折叠及纯化后得到了高纯度融合蛋白,将重组蛋白免疫新西兰大耳白兔,制备抗-p7TP2多克隆抗体。结果经酶联免疫吸附法(ELISA)测定、蛋白印迹(Western blot)和免疫组织化学鉴定,制备的多克隆抗体具有较高效价和特异性。结论 p7TP2蛋白的表达及多克隆抗体的制备为进一步研究HCV p7TP2蛋白的功能及丙型肝炎的发病机理创造了条件。更多还原

【Abstract】 Objective:To screen the new gene p7TP2 transregulated by HCV p7 protein through gene chip technique and investigate its biological functions. Methods The p7TP2 cDNA was amplified by PCR and prokaryotic expression vector pET-32a(+) -p7TP2 was constructed by gene recombination techniques,which were transformed into E. coli Rosetta-gami B,for IPTG induced expression. The p7TP2 fusion protein was expressed in the form of inclusion body. A band with 35 kD appeared on SDS-PAGE gel. The protein expressed by carboxyl terminal coding sequence had a high purity after denaturation,refolding and purification. New Zealand rabbits were immunized with p7TP2 fusion protein to prepare polyclonal antibody against p7TP2. The p7TP2 antiserun was obtained and characterized by ELISA,Western blot,immunohistochemistry. Results Results showed that the polyclonal antibody had high titer,affinity and specificity. Conclusions The study provide a favorable tool for further functional study of HCV p7TP2 and the pathogenesis of hepatitis C.更多还原