重组耻垢分枝杆菌Msmc^2-CFP10-ESAT6的构建  

Preparation of recombinant Mycobacterium smegmatis pBCG3000-CFP10-ESAT6

作　　者：吴雯[1] 张红宇[2] 黄汉菊[1] 范兴[2] 罗道泉[3] 吴少庭[2] 

机构地区：[1]华中科技大学同济医学院,湖北武汉430000 [2]深圳市疾病预防控制中心,广东深圳518020 [3]深圳市慢性病防治院,广东深圳518020

出　　处：《中国病原生物学杂志》2009年第3期187-190,共4页Journal of Pathogen Biology

摘　　要：目的构建能表达结核分枝杆菌早期分泌蛋白CFP10-ESAT6融合蛋白的重组耻垢分枝杆菌。方法采用基因拼接(Gene SOEing)法体外扩增结核杆菌1hp-ESAT6融合基因,插入大肠埃希菌-分枝杆菌穿梭表达质粒pB-CG3000,构建重组穿梭表达质粒pBCG3000-CFP10-ESAT6,电转化耻垢分枝杆菌mc2155,构建耻垢疫苗株Msmc2155-CFP10-ESAT6,热诱导表达CFP10-ESAT6融合蛋白,Western blot分析其免疫原性。结果经PCR、酶切及测序鉴定,证实成功构建重组质粒pBCG3000-CFP10-ESAT6,并在耻垢分枝杆菌中经热诱导表达出分子质量单位约为22 ku的CFP10-ESAT6蛋白,该蛋白可被结核病患者血清、鼠抗ESAT6血清和鼠抗CFP10血清识别。结论结核分枝杆菌lhp-ESAT6融合基因在耻垢分枝杆菌中成功表达。Objective To construct a recombinant Mycobacterium smegmatis mc^2155（Msmc^2155） expressing the fusional CFP10-ESAT6 protein secreted early by Mycobacterium tuberculosis. Methods The M.tuberculosis fusion gene was amplified by Gene SOEing and cloned to an Escherichia 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 Msmc^2155 by electroporation.The expression and activity of the fusional CFP10-ESAT6 protein induced by heat was evaluated using Western blot.Results A positive E.coli-Mycobacterium shuttle expressing plasmid pBCG3000-CFP10-ESAT6 was confirmed by PCR,enzyme digestion,and sequencing.The recombinant Msmc^2155-CFP10-ESAT6 expressed a 22 ku protein induced by heat that may react with antibodies in sera of tuberculosis patients,anti-ESAT6,and CFP10.Conclusion The M.tuberculosis fusional lhp-ESAT6 gene is expressed in Msmc^2155.

关 键 词：分枝杆菌 结核 CFP10 ESAT6 穿梭表达质粒 分枝杆菌 耻垢 

分 类 号：R378.911[医药卫生—病原生物学]