采用卡介苗结核蜡酸合酶启动子构建分枝杆菌穿梭表达质粒  

Construction of Mycobacterium Shuttle Expression Plasmid with Mycocerosic Acid Synthase Promoter of BCG

作　　者：曾瀚庆[1] 吕琳[1] 梅浙川[1] 王丕龙[2] 娄世锋[1] 

机构地区：[1]重庆医科大学附属第二医院,重庆400010 [2]重庆医科大学附属第一医院消化内科,重庆400016

出　　处：《中国生物制品学杂志》2010年第10期1043-1045,1054,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30371318);重庆市卫生局重点项目(渝卫教[2007]1号文07-1-007)

摘　　要：目的采用卡介苗(BCG)结核蜡酸合酶(Mycocerosic acid synthase,Mas)启动子,将分枝杆菌穿梭质粒pJEM11改建为分枝杆菌穿梭表达质粒。方法 PCR扩增BCG基因组中Mas启动子,定向克隆至质粒pJEM11的ApaⅠ与SnaBⅠ位点之间,构建分枝杆菌穿梭表达质粒pJMas。将幽门螺杆菌(Helicobacter pylori,Hp)UreB基因克隆至pJMas质粒,转化耻垢分枝杆菌(Mycobacteria smegmatis,M.smegmatis)mc2155,SDS-PAGE检测重组UreB蛋白在M.smegmatis mc2155中的表达,Western blot分析其反应原性。结果克隆的Mas启动子序列与结核杆菌H37Rv株的Mas启动子序列(gi31619628)同源性达99%;重组穿梭表达质粒pJMas经PCR及双酶切鉴定证明构建正确;UreB基因在M.smegmatis mc2155中成功表达,表达的蛋白可与Hp阳性病人血清发生特异性反应。结论成功构建分枝杆菌穿梭表达质粒pJMas,为构建以耻垢分枝杆菌为载体的多价疫苗奠定了基础。Objective To improve mycobacterium shuttle plasmid pJEM11 into a mycobacterium shuttle expression plasmid by using the mycocerosic acid synthase （Mas）promoter of BCG. Methods The Mas promoter in genome of BCG was amplified by PCR and cloned into plasmid pJEM11 between sites ApaⅠ and SnaBⅠ to construct a mycobacterium shuttle expression plasmid pJ-Mas. The UreB gene of Helicobacter pylori was cloned into plasmid pJMas, and the constructed recombinant plasmid was transformed to Mycobacteria smegmatis mc2 155. The expressed recombinant UreB protein was identified by SDS-PAGE and analyzed for reacto-genicity by Western blot. Results The sequence of cloned Mas promoter showed a homology of 99% to that of Mas promoter of My-cobacterium tuberculosis H37Rv strain （gi31619628）. Both PCR and restriction analysis proved that recombinant shuttle expression plasmid pJMas was constructed correctly. Recombinant UreB protein was successfully expressed in Mycobacteria smegmatis mc2 155, which showed specific reaction with Hp positive human sera. Conclusion Mycobacterium shuttle expression plasmid pJMas was successfully constructed, which laid a foundation of construction of polyvalent vaccine using Mycobacteria smegmatis as a vector.

关 键 词：分枝杆菌 结核 分枝杆菌 耻垢 结核蜡酸合酶 启动子 基因表达 

分 类 号：Q78[生物学—分子生物学] R378.91[医药卫生—病原生物学]