(完整)HIV-P24蛋白原核表达和单克隆抗体制备-8.24修改.doc

HIV-1 P24蛋白的原核表达及单克隆抗体的制备 汪龚泽，刘朝奇，杨建林，覃晓琳，吕佰瑞，姚佳红 三峡大学分子生物学研究所，湖北 宜昌 443002 摘要：目的：制备HIV-1 P24蛋白及单克隆抗体，建立ELISA方法用于检测HIV感染。方法：用PCR方法扩增P24基因，将其克隆到原核表达载体PET-28a（+）中, 经大肠杆菌表达并纯化HIV-1 P24蛋白。以纯化的P24蛋白抗原免疫Balb/c小鼠，取其脾淋巴细胞与SP2/0细胞融合,制备能产生P24.单克隆抗体的杂交瘤细胞株,进一步采用Western blotting、ELISA等技术对制备的单克隆抗体进行初步鉴定，建立了P24的ELISA竞争法。结果：原核表达重组质粒在大肠杆菌中能高效表达P24蛋白,并进一步从杂交瘤细胞中选取了能稳定分泌P24单抗的杂交瘤细胞株。应用ELISA竞争法测定的HIV-1阳性病人血浆P24与病毒载量显著相关。结论：成功制备了P24蛋白及特异性单克隆抗体，建立了P24的ELISA竞争法。 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 a