人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原，制备特异抗血清，为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板，用PCR法获得Nef蛋白编码基因，将其克隆到pET30a载体中，在大肠杆菌中表达Nef融合蛋白；用纯化的融合蛋白免疫BALB／c小鼠获得抗血清，用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达，相对分子质量约为36x103，免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清，ELISA抗体滴度为1：6400；免疫荧光和Westemblot检测表明，该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白，制备了Nef融合蛋白的高效价小鼠免疫血清，该血清能特异性识别HIVNef抗原，为HlVNef抗原检测提供了技术方法。

Expression,Purification and Immunogenicity Analysis of HIV Early Protein Nef

Objective： To obtain specific serum of HIV early protein Nef and provide a detection method of Nef antigen. Methods： Amplify Nef gene by PCR and the PCR fragment was sequenced and subeloned into the pET30a vector, The purified Nef protein expressed in E, coli cell was used to vaccinate BALB/c mice and detect specificity and antibody titer of antiserum, Result： The fusion Nef protein could be expressed effectively in E, coli cell and the relative molecular weight was about 36x103. The ELISA assay titer of antiserum from vaccinated mice reached 1：6 400. The Western blot and IFA assays demonstrated that antiserum could recognize the Nef protein produced by recombinant vaccinia virus. Conclusion： The fusion HIV Nef protein was expressed in E.coli cell and could obtain high titer antiserum from BALB/c mice immunized with purified fusion Nef protein. Then the antiserum had be demonstrated could recognize the Nef antigen by Western blot and IFA assays. The study provided a method to detect Nef antigen.