HIV-1膜蛋白GP120V1/V2区域的真核表达、纯化和鉴定

Eukaryocyte expression and characterization of HIV-1 envelope V1/V2 subdomain

目的:真核表达北美HIV-1分离株BAL和中国分离株CNE1两种毒株的V1V2,并纯化、鉴定其生物活性,为血清学分析鉴别HIV-1感染病人抗体反应及性质提供实验基础。方法:本研究将HIV-1北美分离株BAL和中国分离株CNE1两种毒株的V1V2基因序列定向克隆到多系统表达载体pTriEx-3 Hygro vector(Merck&Co.,Inc.)中,构建了表达质粒,并将表达质粒瞬时转染293T细胞,144小时后收获上清液,经浓缩、纯化后,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:经SDS-PAGE电泳、Western blot、ELISA方法证实确实得到了有免疫反应活性的V1V2蛋白,从表观分子量判断,表达的V1V2蛋白被糖基化修饰。使用V1V2蛋白作为抗原,分析发现中国HIV-1阳性病人体内比较广泛的存在着V1V2的抗体,为血清学的分析奠定了实验基础。

Objective：To express the gpl20 V1/V2 domain of BAL （isolate from North America） and CNE1 （isolate from China） in eukaryocyte and characterize the expressed proteins. Methods：Full length of the V1V2 sequences from BAL and CNEI iso- lates were fused with the C terminus of N-terminal domain of the murine leukemia virus surface protein, GPT0. TPA signal peptide and a 6 his-tag were also fused to facilitate protein secretion and purification, respectively. Recombinant plasmids were transfected into 293T cells and the supernatants were collected 144 hours after transfeetion. The proteins were concentrated through a TFF System and purified by nickel chelate discs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） , Western blot and ELISA were performed to analyze and determine the bioreactivity of the V1V2 proteins. Results：Sodium dodecyl sulfate polyacrylamide gel e- lectrophoresis （SDS-PAGE）, Western blot and ELISA indicated that the expressed V1V2 proteins were glycosylated and could reactive with the serum of Chinese HIV-1 positive patients and the serum reacted differentially with the V1V2 proteins derived from the North A- merican and the Chinese strains. Conclusion：Two bioreactive V1 V2 proteins were successfully expressed and purified. Serological analysis showed that antibodies to V1V2 broadly existed in the sera of HIV-1 positive Chinese patients.