人类免疫缺陷病毒-1包膜糖蛋白gp41的表达及优化

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

目的优化HIV．1本地流行株06044sp4t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．106044gp41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—683f1．a片段）及去掉N一端（NHR）和C一端（CHR）七价重复序列中间部分loop区（582～627aa）的pET26b—Sr41T（AL），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Westernblot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—gp41T（AL）重组表达质粒均能表达gp41蛋白，glMlT（△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1mmol／LIPTG诱导后，37℃条件下表达量最高。Westernblot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-106044gp41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。

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） be- tween NHR and CHR of gp41 was also constructed. After identification by sequencing,the plasmids were trans- formed into E. coli BL21 （ DE3 ） and the protein expression was induced by Isopropyl β-D-l-thiogalactopyrano- side （IPTG） and analyzed by polypropylene aeyl ammonia gel electrophoresis （SDS-PAGE） and Western Blot. The protein expression condition was optimized. Results The pET26b-gp41T and pET26b-gp41T （AL） can express specific gp41 protein. Under lmmol/L IPTG induction,the gp41T（AL） protein expression quantity was higher than gp41T and the best expression was at 37 ℃. Comparing with gp41T,gp41T （AL） protein showed better affinity to his antibody. Conclusion We successfully constructed a recombinant plasmid that can express gp41 protein successfully in E eoli with well exposure of His-Tag. This will facilitate the production and purifica- tion of gp41 immunogen.