HIV-1 p24和gp41融合基因表达载体的构建及融合蛋白的表达纯化  

Construction,expression and purification of HIV-1 p24-gp41 fusion protein in E.coli

作　　者：蒋卫民[1] 卢洪洲[1] 潘孝彰[1] 康来仪[1] 尹春煜[1] 胡越凯[1] 翁心华[1] 

机构地区：[1]复旦大学上海医学院附属华山医院传染科,上海200040

出　　处：《中华传染病杂志》2005年第3期170-173,共4页Chinese Journal of Infectious Diseases

基　　金：教育部归国人员基金资助(教育部26)

摘　　要：目的用基因重组技术将HIV-1 p24基因以及gp41基因具有抗原线性中和表位的部分重组连接,构建重组质粒,并在大肠埃希菌中高效表达融合蛋白。方法设计带有酶切位点的引物,分别PCR扩增p24和gp41两个基因,将它们分别连接到pMD18-T载体中,测序验证后,挑选出含有目的基因的正确克隆。将p24片段酶切后连接到gp41基因所在的pMD18-T载体中,再将连接后的两个基因酶切,重新连接到pET21a表达载体中。将表达载体转染大肠埃希菌诱导表达,经Western-blot验证表达正确。结果融合蛋白p24-gp41在大肠埃希菌中高效表达。结论融合蛋白p24-gp41可以在pET21a表达载体中高效表达。Objective To construct recombinant plasmid with p24 and gp41 gene, express fusion protein in E. coli. Methods To design primer with restriction endonuclease position,and amplify p24 and gp41 by RT-PCR, link both into pMD18-T vector. To choose correct clone with target gene. Then p24 fragment will be cleaved and linked into pMD18 T vector within gp41 gene. Both post-linked gene will be cleaved and linked into pET21a vector. The vector will be transformed into E. coll. And protein is highly effective expressed in E coli. Western blotting proved that the expressed product could react with 6 × his antibody. Result Fusion protein p24-gp41 is highly effective expressed in E. eoli. Conclusion Fusion protein p24-gp41 is highly effective expressed in E. coli in pET21a vector.

关 键 词：HIV核心蛋白质p24 HIV包膜蛋白质gp41 基因表达 重组融合蛋白质类 基因表达载体 gp41基因 p24基因 HIV-1 融合蛋白 WESTERN-BLOT 

分 类 号：R346[医药卫生—基础医学]