我国HIV-1流行株gp41抗原表位筛选与串联表达

Selection of HIV-1 gp41 epitope and tandem expression of two epitopes gene

目的 探讨HIV 1gp4 1抗原表位串联表达蛋白用于HIV抗体检测的可行性。方法用HIV 1gp4 1蛋白亲和层析柱制备HIV 1感染者血清中的多克隆抗体 ,用噬菌体展示随机十二肽库进行生物淘洗 ,反向吸附非特异性噬菌体。经ELISA鉴定阳性克隆 ,DNA测序 ,确定优势表位。将优势表位与文献报道的另一个优势表位串联 ,克隆入pQE30载体进行蛋白表达。结果 成功筛选到位于HIV 1gp4 1蛋白上的优势抗原表位 (YGPKDAETTAIW ) ,串联表位 (YGPKDAETTAIW GGGS SC SAKFTCTTQI)在pQE30载体中实现可溶性表达。重组蛋白具有良好的抗原性 ,能与不同的HIV 1抗体阳性血清呈特异反应。结论 HIV 1gp4 1抗原表位串联表达设计是可行的 ,串联表位重组蛋白可用于HIV 1抗体检测 ,但检测灵敏性低于常规方法。

Objective To explore the feasibility of testing anti-HIV antibody using a fusion protein of two tandem HIV-1 gp41 epitopes. Methods The polyclonal antibodies specific to HIV-1 gp41 protein were prepared from HIV-1 B′ IgG positive plasma by affinity chromatography 4B coupled with HIV-1 gp41 protein. The phage display 12 peptides libraries were biopanned first with the antibody, and then non-specific phages were subtracted by purified IgG from non-HIV sera. After three round screening, the positive phages were tested by ELISA for their reactivity with anti-HIV IgG antibodies, and their major display peptides were determined by DNA sequencing. One of major epitopes was ligated tandem with a reference gp41 epitope, and the tandem epitope genes were expressed as fusion proteins in pQE30 vector. Results A major HIV-1 gp41 epitope (YGPKDAETTAIW) was determined and a fusion protein of two-tandem-epitope (YGPKDAETTAIW-GGGS-SCSAKFTCTTQI) gene was expressed in pQE30 vector. Recombinant proteins showed an actual positive reactivity with HIV-1 IgG antibody from different sera. Conclusion The design of expressing two-tandem-HIV-1 IgG gp41-epitope gene is practical. Recombinant protein of two tandem epitopes gene could test the anti-HIV-1 antibody, but the sensitivity is lower than routine methods.