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作 者:张红宇[1] 李晓恒[1] 范兴[1] 罗道泉[2] 吴少庭[1]
机构地区:[1]深圳市疾病预防控制中心,广东深圳518020 [2]深圳市慢性病防治院,广东深圳518020
出 处:《中国热带医学》2009年第5期787-789,840,共4页China Tropical Medicine
基 金:深圳市科技计划资助项目(200702112)
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。Objective To construct a recombinant BCG exressing the fusion protein CFP10-ESAT6. Methods Mycobacterium tuberculosis fusional gene CFPIO-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.
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