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

摘要:

为获得可溶性高纯度HIV-1中国株CN54 Pol P51抗原，将携带CN54 pol p51基因的重组质粒pTHioHisA51转化大肠杆菌BL21 codonplus-RIL，用IPTG进行诱导表达。用Chelating Sepharose FF-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。

关键词:HIV-1 CN54，逆转录酶P51，纯化，复性，抗体检测

Abstract:

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.

Key words:HIV-1 CN54, reverse transcriptase P51, purification, renaturation, antibody detection