HIV-1膜蛋白GP120V1/V2区域的真核表达、纯化和鉴定

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。

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

Objective:To express the gp120 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 CNE1 isolates were fused with the C terminus of N-terminal domain of the murine leukemia virus surface protein,GP70.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 transfection.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 electrophoresis(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 American and the Chinese strains.Conclusion:Two bioreactive V1V2 proteins were successfully expressed and purified.Serological analysis showed that antibodies to V1V2 broadly existed in the sera of HIV-1 positive Chinese patients.