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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 a recombinant Mycobacterium Smegmatis (rMs) capable of secreting the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) and human IL-2. Methods: pPRO-hsp65-IL-2 vector containing Hsp65-IL-2 fusion gene was digested with EcoRV and HindIII, and the target gene fragment Hsp65-IL-2 was recovered and subcloned into the same double-digested Escherichia coli Mycobacterium - mycobacterium shuttle secretion expression vector pDE22. The recombinant plasmid pDE22-hsp65-IL-2 identified correctly after electroporation transformed into MS competent, hygromycin resistance pressure screening positive rMs. The expression of target protein in rMs culture supernatant was identified by Western-blot. Results: The recombinant plasmid pDE22-hsp65-IL-2 was digested with about 2000 bp fragment, which was consistent with the expected size. Western-blot results showed that there was a specific reaction band in rMs culture supernatant with a size of 78 kD, which was consistent with the size of Hsp65-IL-2 fusion protein. CONCLUSION: The E. coli-mycobacterial shuttle secretion expression vector pDE22-hsp65-IL-2 was successfully constructed, which lays the foundation for the study of the immunological characteristics of the rMs and the protective effect against Mycobacterium tuberculosis infection.