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目的构建分泌表达融合蛋白CFP10-ESAT6的重组卡介苗。方法采用基因拼接(Gene SOEing)法,体外扩增结核杆菌CFP10-ESAT6融合基因,插入大肠杆菌-分枝杆菌穿梭表达质粒pBCG3000,构建重组穿梭表达质粒pBCG3000-CFP10-ESAT6,用电穿孔法将pBCG3000-CFP10-ESAT6质粒转化BCG细胞,得到重组卡介苗,热诱导表达CFP10-ESAT6融合蛋白,SDS-PAGE电泳观察CFP10-ESAT6融合蛋白的表达,Westernblot鉴定其生物活性。结果经PCR、酶切及测序鉴定,证实成功构建重组质粒pBCG3000-CFP10-ESAT6,经热诱导表达出约22kDa的CFP10-ESAT6蛋白,可分别被鼠抗ESAT6血清、鼠抗CFP10血清识别。结论分泌性表达融合蛋白CFP10-ESAT6的重组卡介苗构建成功。
Objective To construct a recombinant BCG expressing secreted fusion protein CFP10-ESAT6. Methods The fusion gene of Mycobacterium tuberculosis CFP10-ESAT6 was amplified by gene splicing (Gene SOEing) method. The E. coli-Mycobacterium shuttle plasmid pBCG3000 was inserted into the shuttle plasmid pBCG3000-CFP10-ESAT6. The pBCG3000 -CFP10-ESAT6 plasmid was transformed into BCG cells to obtain recombinant BCG. CFP10-ESAT6 fusion protein was induced by heat induction. The expression of CFP10-ESAT6 fusion protein was observed by SDS-PAGE electrophoresis. The biological activity of CFP10-ESAT6 fusion protein was identified by Western blot. Results The recombinant plasmid pBCG3000-CFP10-ESAT6 was successfully constructed by PCR, restriction enzyme digestion and sequencing. The CFP10-ESAT6 protein of about 22 kDa was induced by heat, and was identified by the serum of mouse anti-ESAT6 and anti-CFP10. Conclusion The recombinant BCG secreting the fusion protein CFP10-ESAT6 was successfully constructed.