人类免疫缺陷病毒-1 Vif蛋白的表达及生物学功能

Expression of viral infectivity factor protein of human immunodificiency viruses and its biological function

目的分析上海HIV-1分离株病毒感染因子（Vif）蛋白编码基因突变特点，构建HIV-1 vif基因原核表达质粒，并了解其免疫原性。方法采用RTPCR法扩增23例上海HIV-1分离株vif基因并测序，与HIV-1国际标准毒株比较；将vif编码基因克隆人原核表达载体pET32b（＋），构建pET32b（＋）-HIv-1／Vif重组质粒；将该质粒转化人细胞BL21D3（Star）表达，并纯化HIV-1 Vif蛋白，制备Vif蛋白鼠多克隆抗体，并以ELISA检测Vif蛋白及其鼠多克隆抗体的免疫原性。统计学处理采用t检验。结果上海HIV-1分离株vif基因与国际标准毒株相比较，核苷酸突变率为（0.179±0.006）％，并具有多处相似的变异位点，上海HIV-1分离株Vif蛋白第151-240位氨基酸相对保守；成功构建HIV-1 vif基因原核表达质粒pET32b（＋）-HIV-1／Vif，表达并纯化Vif蛋白，制备了Vif多克隆抗体；重组HIV-1 Vif蛋白与HIV感染者及健康人血清反应比较，差异无统计学意义（P〉0.05）；鼠多克隆抗体与重组Vif蛋白反应与健康对照组血清比较差异有统计学意义（t=178．61，P〈0．01）。结论上海HIV-1分离株vif基因与HIV-1国际标准毒株比较存在较高突变，成功地构建了HIV-1 vif基因原核表达载体pET32b（＋）-HIV-1／Vif，制备了Vif多克隆抗体。

Objectives To analyze the characteristic of HIV-1 viral infectivity factor （Vif） gene variants isolated from Shanghai. To construct the prokaryotic expression vector of HIV-1 vif gene and understand its immunogenicity. Methods HIV-1 vif genes were amplified and sequenced from 23 serum samples of HIV-1 infected patients in Shanghai and then compared with the international standard HIV-1 strain. Subsequently, these amplified Vif fragments were sub cloned into pET32b（＋） expression vector. The recombinant prokaryotic plasmids pET32b （＋）-HIV-1/Vif were then transferred into BL21D3 （Star）cells for expressing and purifying HIV-1 Vif protein. HIV-1 Vif rat polyclonal antibody was then preparing by injecting the purified Vif proteins into the mice. ELISA was used to determine the purity of Vif proteins and the immunogenicity of its polyclonal antibodies. Results The nucleotide acid mutation rate of HIV-1 vif gene in Shanghai AIDS patient was （0.179±0. 006）% compared with the international standard HIV-1 strain. Some similar mutations in vif gene were found in HIV-1 strains isolated from Shanghai while the amino acid sequence between 151 and 240 in Vif protein was conserved. The construction of HIV-1 Vif prokaryotic expression plamids and the preparation of Vif polyclonal antibodies were successfully done in this study. The reaction between recombinant HIV-1 Vif protein and the serum from HIV-1 infected patient was not significantly different from that between the recombinant protein and healthy control serum（P〉0.05）. HIV-1 Vif polyclonal antibody reacted differently with recombined HIV-1 Vif protein compared with healthy control serum samples（t=178.61, P〈0.01）. Conelmiora The Vif gene mutation rate is high in HIV-1 strains isolated from Shanghai compared with international standard HIV-1 strain. The prokaryotic expression plasmids of HIV-1 vif antigen are successfully constructed and Vif polyclonal antibodies are prepared well.