HIV-1 B'/C亚型Rev蛋白的表达、纯化与抗体制备  

Expression,Purification and Antibody Preparation of Rev Protein of HIV-1 B'/C Subtype

作　　者：齐香荣[1] 高瑛瑛[1] 陆柔剑[1] 谭文杰[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《生物技术通讯》2011年第3期330-333,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2003AA219080);中国综合性国际艾滋病研究项目(CIPRA)(1U19 A151915 01)

摘　　要：目的:为方便实验室工作中对HIV-1 B'/C亚型Rev蛋白的检测,制备相应的Rev蛋白及其抗体。方法:将我国HIV-1 B'/C亚型流行株的rev基因按大肠杆菌优势密码子进行改造后人工合成,在原核系统中与pET30a(+)载体中的His.Tag、Trx.Tag及S.Tag进行融合表达,目的蛋白经Ni2+金属螯合层析柱纯化后用于免疫家兔,制备多克隆抗体。结果与结论:合成基因在原核系统中融合表达得到相对分子质量约18×103的融合蛋白,目的蛋白的表达量约占菌体总蛋白量的36%;用纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹及间接免疫荧光检测结果显示,获得的多克隆抗体与HIV-1 B'/C亚型的Rev蛋白能产生特异性反应,可用于检测HIV-1 B'/C亚型Rev蛋白的表达。Objective：In order to detect the Rev protein of HIV-1 B＇/C subtype conveniently in lab work,the corresponding Rev protein and its antibody was prepared.Methods：The codons of rev gene of HIV-1 B＇/C subtype were replaced with the preferred codons of E.coli through gene synthesizing.The optimized target gene was fused with the gene of His·Tag,Trx·Tag and S·Tag in pET30a（＋） vector.Then,the Rev fusion protein was expressed in prokaryotic system and purified by nickel-chelating chromatography.The purified Rev fusion protein was used to immune rabbit to prepare polyclonal antibody.Results Conclusion：The approximate 18 kD fusion protein was expressed efficiently in prokaryotic system.The ratio of Rev fusion protein to total bacteria proteins was 36%.The purified protein was immunized to rabbit in order to prepare polyclonal antibody.The polyclonal antibody could react well with Rev protein of HIV-1 B＇/C subtype demonstrated by Western blot and indirect immunofluorescence,and can be used to detect Rev protein of HIV-1 B＇/C subtype.

关 键 词：HIV-1 Rev蛋白 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]