【2017年整理】(完整)HIV-P24蛋白的原核表达及单克隆抗体的制备-8.24修改.doc

HIV-1 P24蛋白的原核表达及单克隆抗体的制备 关键字：HIV-1 P24，原核表达，单克隆抗体，ELISA Expression of HIV-1 P24 protein and Preparation of its monoclonal antibodies WANG GONG ZE , LIU CHAOQI ,YANGJIANLIN,QINGXIAOLIN,LVBAIRUI Institute of Molecular Biology; Three Gorges University; Yichang HUBEI 443002; China ABSTRACT: Objective　To construct Prokaryotic expression plasmid of HIV-1 P24, purify recombinant HIV-1 P24 protein and prepare its monoclonal antibodies. Methods HIV-1 P24 cDNA was cloned into prokaryotic expression vector pET28a (+) and was further transformed into E.coli BL21(DE3). The recombinant HIV-1 P24 protein was used to immunize Balb/c female mouse. The splenocytes were fused with SP2/0 hybridoma to produce monoclonal antibody against HIV-1 p24 protein. Western blotting and ELISA were carried out to identify the monoclonal antibody produced by these hybridomas. Results　 HIV-1 P24 plasmid was successfully constructed and identified by enzyme digestion and sequencing. HIV-1 P24 protein was expressed in the E.coli BL21(DE3) and purified by affinity chromatography. After the immunized murine splenocytes were fused with SP2/0 hybridoma, we selected hybridoma cell lines which were able to produce monoclonal antibodies against HIV-1 P24 stablely. Competition ELISA assay was established and the p24 protein of HIV-1 positive plasma was significant correlated with the HIV-1 viral load. Conclusions We successfully expressed HIV-1 p24 and prepare hybridomas which can stablely produce monoclonal antibodies against HIV-1 P24. KEY WORDS: HIV-1 P24; prokaryotic expression; monoclonal antibody;ELISA HIV是艾滋病的病原体，主要通过性接触、血液和母婴传播。近年来，HIV感染患者数量一直呈上升趋势。根据卫生部统计，中国自1985年发现第一例艾滋病病人以来，截至2009年10月底，累计报告艾滋病病毒感染者和病人319 877例。2009年当年新发艾滋病病毒感染者4.8万人，我国艾滋病疫情形势依然严峻，性传播正在成为主要传播途径[1]。而目前测定血清HIV抗体是诊断HIV感染的常规实验方法,但是测定HIV抗体有局限性:有超过70%的HIV感染者在感染6个月后才能检测出抗体,在同性恋群体,这个数字超过80%,检测抗体方法增加了HIV “窗口期” 传播的危险[2]; 另外新生儿产生抗体需要出生1年后,来自母亲的HIV抗体会致使假阳性产生;由于HIV抗体在疾病过程中持续存在,只是到艾滋病晚期时消失,无法作为治疗监测的稳定指标[3]。 P24是HIV病毒颗粒的主要结构蛋白, 是结构基因gag