胞壁表达HSP65-IL-2融合蛋白的重组耻垢分枝杆菌的构建和鉴定

Construction and Identification of Recombinant Mycobacterium smegmatis Expressing Heat Shock Protein 65 and Interleukin 2 Fusion Protein on its Surface

目的:获得胞壁表达结核分枝杆菌热激蛋白65(HSP65)和人白细胞介素2(IL-2)融合蛋白的重组耻垢分枝杆菌。方法:将HSP65和IL-2融合基因克隆入大肠杆菌-卡介苗(E.coli-BCG)穿梭质粒pCW,构建成重组质粒HSP65-IL-2-pCW,电穿入耻垢分枝杆菌,经潮霉素抗性筛选和PCR方法选取阳性克隆;用间接免疫荧光法对重组耻垢分枝杆菌进行表型鉴定;用重组耻垢分枝杆菌免疫BALB/c小鼠,检测其诱导的抗体水平。结果:筛选获得的重组耻垢分枝杆菌增殖特性与普通耻垢分枝杆菌无明显区别;与抗人的IL-2抗体和HSP65抗体均可形成免疫印迹条带;间接荧光染色后,可见细菌表面有极强的绿色荧光;重组耻垢分枝杆菌免疫BALB/c小鼠2周后,小鼠血清中HSP65抗体平均滴度为1∶4000,而生理盐水组的抗体几乎为阴性。结论:结核分枝杆菌HSP65和人IL-2融合基因在耻垢分枝杆菌胞壁获得表达,有望为结核病的预防提供有效的疫苗。

Objective：To construct recombinant Mycobacterium smegmatis（M.S） expressing M.tuberculosis heat shock protein 65（HSP65） and human interleukin-2（IL-2） fusion protein on its surface. Methods： HSP65 and IL-2 fusion gene was cloned into E.coli-BCG shuttle plasmid pCW, named as HSP65-IL-2-pCW. The recombinant plasmid HSP65-IL-2-pCW was transfeeted into M.S by electroporation, and the positive bacterium strain was selected by hygromycin resistance screening and PCR method. The expression of fusion protein was determined by indirect immunofluorescence. The specific antibody titers in the sera of BALB/c mice immunized with recombinant M.S were detected by ELISA. Results： Compared with the conventional M.S strain, the positive recombinant M.S strain showed no significant difference in proliferation characteristics. Western blot analysis showed that there were specific bindings at 76 kD with the specific monoclonal antibody against human IL-2 and HSP65 respectively, equalivalent to the sum of IL-2 and HSP65. The strong positive immunofluorescence was observed on the surface of the recombinant M.S. The titer of anti-HSP65 antibody in BALB/c mice sera immunized with recombinant M.S strain was 1：4 000, and that of saline group was nearly negative. Conclusion： The fusion protein of HSP65 and IL-2 was expressed on the surface of recombinant M.S successfully. It may be one of the useful candidate for the development of tuberculosis vaccine.