HIV11C亚型Gp120蛋白表达、纯化及其抗体制备的研究  

Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation

作　　者：杨海儒[1] 冯霞[1] 余双庆[1] 张晓光[1] 张晓梅[1] 陈国敏[1] 李泽琳[2] 曾毅[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,100052 [2]北京工业大学生命科学与生物医学工程学院药理室

出　　处：《中华实验和临床病毒学杂志》2009年第2期94-96,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：基金项目：国家自然科学基金资助（30872397）

摘　　要：目的制备HIV-1C亚型Gp120蛋白及其抗体。方法用PCR技术从表达C亚型HIV-1全长gp160基因的质粒上扩增gp120基因羧基末端部分片段，其长度为612个核苷酸，编码204个氨基酸。将测序鉴定正确的gP120基因片段克隆到原核表达载体pET-30a上，以包涵体的形式在大肠埃希菌B121（DE3）中高效表达，Gp120蛋白c端融合6×His标签便于纯化。将纯化的蛋白免疫新西兰大白兔制备C亚型Gp120特异性兔多克隆抗体，用间接ELISA法测定抗体滴度，并用WesternBlot方法验证该抗体是否可识别在哺乳动物细胞内表达的C亚型Gp160蛋白。结果成功地获得高纯度的C亚型Gp120融合蛋白，用其制备的多克隆抗体滴度可达1：204800，该抗体可特异性识别在COS-1细胞中瞬时表达的C亚型Gp160蛋白。结论获得了高纯度的C亚型Gp120融合蛋白及其高效价的抗体。Objective To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies. Methods A C-terminal fragment of gp120 gene was amplified by PCR from a plasntid expressing fulllength HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector （pET-30a） and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 （DE3） as an insoluble protein. The vector also contained a six-histidine （His6） tag at the C-terntinus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays （ELISA） to determine the level of antibodies. And Westem blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gpl60 protein expressed in mammalian cells. Results HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1 ： 204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 ceils. Conclusion HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.

关 键 词：HIV 病毒包膜蛋白质类 基因表达 抗体 病毒 

分 类 号：R392[医药卫生—免疫学]