结核杆菌Hsp65与人IL-2融合蛋白在耻垢杆菌表达

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。Westem—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2000bp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。

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

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.