HIV-1膜蛋白gp140三聚体的真核表达、纯化、鉴定

摘要：目的构建表达中国流行株HIvl-1cRF_07B／C亚型（CN54）膜蛋白gpl40及其突变体，为HIV-1疫苗设计提供优秀的免疫原。方法构建gpl40、gpl40ST质粒，将质粒瞬时转染293F细胞，120h后收获上清液，经纯化、浓缩、PBS置换后，利用SDS-PAGE电泳、Western-blot检测蛋白的表达，ELISA检测其与HIV-1阳性病人血清的反应性。结果SDS-PAGE电泳、Western-blot试验证明成功表达了gpl40、gpl40ST蛋白，并且gpl40ST是-个稳定的三聚体蛋白质。ELISA试验检测，发现表达的gpl40ST蛋白与HIV-1阳性病人血清的亲和力高于gpl40蛋白。结论经过突变改造的gpl40ST具有稳定的三聚体构象，可作为HIV-1免疫原进行疫苗研究。

Eukaryocyte expression and characterization of HIV-1 envelope trimer gp140

Abstract：Objective To construct the recombinant plasmid that for express gpl40 of HIV-1 CRF_07 subtypes B/C （CN54） and the mutations,two amino acids on gp140 protein was mutated through Q567K and K588R,named gp140 ST in order to make the gp140 become stable trimer conformation produced to be used as a candidate of HIV-1 vaccine. Methods HIV-1 gp140 and gp140 ST plasmids were concstructed by PCR technology. Recombinant plasmids of gp140 and gpl40 ST were transfected into 293F cells,respectively,and the supematants were collected 120 hours after transfection. After purified and concentrated,the proteins of gpl40 and gpl40 ST were identified by SDS-PAGE,Westem-blot,and ELISA were performed to analyze and determine the bioreactivity of the gpl40 and gpl40 ST proteins. Results SDS-PAGE,westem-blot suggested that gpl40 ST was a stable trimer compared with gpl40. ELISA indicated that the recombinant proteins of gp140 ST could strongy bind with the sera of Chinese HIV-1 positive patients than that with gpl40. Conclusions Gpl40 ST trimer was more stable with more immunogenic and can be an excellent immunogen for HIV-1 vaccine design.