【摘要】 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。更多还原

【Abstract】 Objective To construct a recombinant BCG exressing the fusion protein CFP10 -ESAT6. Methods Mycobacterium tuberculosis fusional gene CFP10 -ESAT6 was amplified by Gene SOEing and cloned to an E.coli - Mycobacterium shuttle expressing plasmid pBCG3000, and then the recombinant shuttle expressing plasmid pBCG3000 - CFP10-ESAT6 was successfully constructed. The recombinant plasmid pBCG3000- CFP10-ESAT6 was transduced into BCG by electroporation. Then the recombinant BCG was induced by heat. The expression and activity of the fusional CFP10- ESAT6 protein were verified by SDS -PAGE and Western blot. Results A positive E.coli -Mycobacterium shuttle expressing plasmid pBCG3000- CFP10-ESAT6 was confirmed by PCR,enzyme digestion and sequencing. The recombinant BCG cell expressed a 22kD protein induced by heat, which could react with antibodies in sera of anti-ESAT6 and CFP10. Conclusions The recombinant BCG vaccine was successfully constructed.更多还原