结核杆菌Hsp65与人IL-2融合蛋白在耻垢杆菌表达  

Expression of Heat Shock Protein 65 of Mycobacterium Tuberculosis and Human IL-2 Fused Gene in Mycobacterium Smegmatis

作　　者：王丽梅[1] 师长宏[2] 柏银兰[1] 张海[2] 康健[1] 张薇[1] 徐志凯[1] 

机构地区：[1]第四军医大学基础部微生物学教研室,陕西西安710032 [2]第四军医大学实验动物研究中心,陕西西安710032

出　　处：《现代生物医学进展》2011年第18期3405-3407,3430,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(No.30801055);"十一五"传染病重大专项项目(No.2008ZX10003-013)

摘　　要：目的:构建能够分泌表达结核分枝杆菌热休克蛋白65(Hsp65)与人IL-2融合蛋白的重组耻垢分枝杆菌(recombinant Mycobacterium Smegmatis,rMs)。方法:用EcoRⅤ和HindⅢ双酶切含Hsp65-IL-2融合基因的pPRO-hsp65-IL-2载体,回收目的基因片断Hsp65-IL-2,并将其亚克隆入同样双酶切的大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22中。重组质粒pDE22-hsp65-IL-2酶切鉴定正确后,电穿孔转化MS感受态,潮霉素抗性压力筛选阳性rMs。Western-blot鉴定rMs培养上清蛋白中目的蛋白的表达。结果:重组pDE22-hsp65-IL-2质粒酶切后可获得约2000 bp片段,与预期大小一致。Western-blot结果表明,rMs培养上清蛋白中有特异性反应条带,大小为78kD,与Hsp65-IL-2融合蛋白大小相一致。结论:成功构建了大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22-hsp65-IL-2,为该rMs的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。Objective： To construct the recombinant Mycobacterium Smegmatis which can express the fused protein of the heat shock protein 65 of Mycobacterium tuberculosis with human IL-2. Methods： The EcoRV/Hind III fragment of the fused gene Hsp65-IL-2 digested from the plasmid pPRO-hsp65-IL-2 was subcloned into the E.coli-Mycobacterium shuttle vector pDE22 predigested by the same restrictive endoenzymes. The positive pDE22-hsp65-IL-2 recombinant plasmid was identified by restriction enzymatic digestion and then transformed into Ms cells by electroporation. The expression of the fusion protein Hsp65-IL-2 in the culture of recombinant Ms（rMs） was detected by Western-blot. Results： A 2000bp fragment was obtained from the shuttle vector pDE22-hsp65-IL-2 by restriction enzyme digestion, which was identical with the predicted. The Western-blot showed that there was a specific reactive band at the size of 78kD in the culture filtrate proteins of rMs, which was identical with the size of Hsp65-IL-2 fusion protein. Conclusion： The shuttle expression vector pDE22-hsp65-IL-2 was constructed successfully, which provided the foundation for the study of the immunogenicity and protective efficacy of rMs against MTB infection.

关 键 词：结核分枝杆菌 HSP65 IL.2 耻垢分枝杆菌 

分 类 号：R378.911[医药卫生—病原生物学]