HIV-1 gag基因p17+24重组抗原表达、纯化及抗原性分析

Expression,purification and identification of HIV-1 gag p17+24 recombinant antigen

目的获得HIV-1型gag基因p17＋2，4重组抗原，分析其免疫原性和探讨开发诊断试剂的可能性。方法采用PCR技术扩增HIV-1gag基因p17＋p24核酸片段，并克隆入pTreHis2A表达质粒，在大肠杆菌BL21（DE3）株中表达。用Ni—NTA金属螯合层析法纯化目的蛋白，并用免疫印迹法分析纯化蛋白的抗原特异性。结果重组质粒在BL21（DF3）菌株中经IPm诱导表达一个相对分子质量（肘，）约为51×10^3的融合重组蛋白，重组蛋白经HIV-1p17、p24抗原单克隆抗体鉴定，具有抗原特异性。25份HIV阳性血清、180份HIV阴性血清标本初步经l临床验证，具有较高的检出敏感性和特异性。结论成功构建HIV-1型gag基因p17＋24抗原表达载体，表达纯化的p17＋24重组抗原具有较好的抗原性，可用于诊断试剂的开发。

Objective To express and purify p17 ＋ 24 recombinant protein of HIV-1 gag gene for research and clinical assay. Methods Coding sequences of gag p17 ＋ 24 of HIV-1 by PCR were cloned in prokaryotic expression vector pTreHis2A. Recombinant plasmids were transformed into E. coli BL21（DE3） and induced for protein expression by IPTG. Recombinant proteins were purified by Ni-NTA column chromatography and identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed a band with molecular relative mass （Mr） of 51 ×10^3. The purified protein was recognized by HIV-1 p17 and p24 McAb. The specificity and sensitivity of p17 ＋ 24 protein were tested by Western blot using 25 HIV-1 Ab positive and 180 negative sera. Conclusion Recombinant p17 ＋ 24 antigen showes high specificity and sensitivity and is a potential diagnostic reagent for HIV-Ab.