HIV-1 CN54 Pol P51抗原高效表达、纯化、复性及在抗体检测中的应用  

Expression, purification and renaturation of Pol P51 antigen of HIV-1 strain CN54 and its application in antibody detection

作　　者：侯爵[1,2] 孙静[2] 徐智勇[2] 范文玲[2] 张怡轩[1] 刘勇[2] 郝彦玲[2] 

机构地区：[1]沈阳药科大学生命科学与生物制药学院,沈阳110016 [2]中国疾病预防控制中心性病艾滋病预防控制中心传染病防治国家重点实验室,北京100050

出　　处：《生物工程学报》2010年第2期201-206,共6页Chinese Journal of Biotechnology

基　　金：国家十一五科技重大专项(Nos.2008ZX1001-1010;2009ZX10004-713)资助~~

摘　　要：为获得可溶性高纯度HIV-1中国株CN54PolP51抗原,将携带CN54polp51基因的重组质粒pTHioHisA51转化大肠杆菌BL21codonplus-RIL,用IPTG进行诱导表达。用Chelating SepharoseFF-Ni亲和层析柱及DEAE Sepharose Fast Flow阴离子交换层析柱纯化目的蛋白,采用透析复性法得到可溶性抗原,Western blotting检测目的蛋白。用纯化的P51抗原蛋白标记辣根过氧化物酶及包被酶标板进行双抗原夹心法ELISA检测。结果显示P51以包涵体的形式表达,表达量占菌体总蛋白的50%,经两步层析和透析复性,目的蛋白纯度大于95%。Western blotting和双抗原夹心法ELISA检测均显示了良好的灵敏度和特异性。本研究可以为HIV-1疫苗研究和开发检测试剂提供支持。To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia. coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase （Pol） P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG. We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation. Purified protein was identified by Western blotting. We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals. The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein. After purification and renaturation, the purity of P51 was up to 95%. Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity. The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.

关 键 词：HIV-1CN54 逆转录酶P51 纯化 复性 抗体检测 

分 类 号：Q78[生物学—分子生物学] S943[农业科学—水产养殖]