人类免疫缺陷病毒-1包膜糖蛋白gp41的表达及优化

目的 优化HIV-1本地流行株06044 gp41蛋白表达设计,为gp41免疫原的制备提供实验基础.方法 以含HIV-1 06044 gp41基因的质粒为模板,采用聚合酶链反应(PCR)扩增gp41目的基因,分别构建原核表达载体pET26b-gp41T(含546～683 aa片段)及去掉N-端(NHR)和C-端(CHR)七价重复序列中间部分loop区(582～627aa)的pET26b-gp41 T(△L),经测序确认后,转化至大肠杆菌感受态细胞BL21 (DE3)中,IPTG诱导表达.用聚丙烯酰氨凝胶电泳(SDS-PAGE)及Western blot检测并鉴定重组表达蛋白.优化了重组蛋白的诱导表达条件.结果 pET26b-gp41T和pET26b-gp4 1T (△L)重组表达质粒均能表达gp41蛋白,gp41T (△L)蛋白表达量高于gp41T;不同IPTG浓度诱导的蛋白表达量没有区别;用1 mmol/L IPTG诱导后,37℃条件下表达量最高.Western blot结果显示,gp41T (△L)与His抗体结合性能好.结论 实验获得了稳定表达HIV-1 06044 gp41的原核表达载体以及表达条件,为大量制备gp41蛋白免疫原奠定了基础.

Optimization of expression of human immunodeficiency virus type 1 envelope gp41 in E coli

Objective To optimize the expression of HIV-1 envelope gp41 in E coli.Methods The HIV-1 06044 gp41 gene encoding 546 ～ 683 aa was amplified by PCR and the prokaryotic expression plasmid pET26b-gp41T was constructed.Meanwhile,plasmid pET26b-gp41T (△L) lacking the loop (582 ～ 627aa) between NHR and CHR of gp41 was also constructed.After identification by sequencing,the plasmids were transformed into E.coli BL21 (DE3) and the protein expression was induced by Isopropyl β-D-1-thiogalactopyrano-side (IPTG) and analyzed by polypropylene acyl ammonia gel electrophoresis (SDS-PAGE) and Western Blot.The protein expression condition was optimized.Results The pET26b-gp41T and pET26b-gp41T (△L) can express specific gp41 protein.Under 1 mmol/L IPTG induction,the gp41T(△L) protein expression quantity was higher than gp41T and the best expression was at 37 ℃.Comparing with gp41T,gp41T (△L) protein showed better affinity to his antibody.Conclusion We successfully constructed a recombinant plasmid that can express gp41 protein successfully in E coli with well exposure of His-Tag.This will facilitate the production and purification of gp41 immunogen.