分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定  

Construction and identification of Mycobacterium shuttle expression plasmid pJHSP70

作　　者：吕琳[1] 曹红丹[1] 王丕龙[1] 刘少宁[1] 王丽娟[1] 向廷秀[2] 

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆 400016 [2]重庆医科大学附属第一医院实验研究中心,重庆 400016

出　　处：《第三军医大学学报》2008年第10期890-893,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30371318);重庆市卫生局重点项目(07-1-007)~~

摘　　要：目的用结核分枝杆菌热休克蛋白70(heat shock protein70,HSP70)启动子改建分枝杆菌穿梭质粒pJEM11为分枝杆菌穿梭表达质粒pJHSP70,并对其功能进行鉴定。方法以卡介苗(BCG)基因组DNA为模板,利用聚合酶链反应(polymerase chain reaction,PCR)扩增HSP70启动子,克隆至pMD18-T载体,经鉴定后,再定向克隆入pJEM11的ApaⅠ位点与SnaBⅠ位点之间,构建pJHSP70载体,并对载体进行PCR及酶切鉴定,然后再将幽门螺杆菌(Helicobacter pylori,HP)尿素酶B亚单位(urease B subunit,UreB)基因克隆入pJHSP70载体,评价其在耻垢分枝杆菌(Mycobacteria smegmatis,M.smegmatis)mc2155中的表达情况。结果从BCG基因组中扩增出160bp的基因片段,所构建的穿梭质粒经酶切和PCR鉴定与预期结果一致。测序结果证实插入片段正确,UreB基因在M.smegmatis中成功表达。结论成功改建穿梭质粒pJEM11为穿梭表达质粒pJHSP70。Objective To reconstruct shuttle plasmid pJEM11 into Mycobacterium shuttle expression plasmid （pJHSP70） with Mycobacterium tuberculosis HSP70 promoter and identify the function of pJHSP70. Methods The HSP70 promoter was amplified from the BCG DNA by PCR and cloned into pMD18-T vector. After identified by enzyme digestion analysis and sequencing, HSP70 promoter was subcloned into pJEMll that was digested with Apa I and SnaB I , to form the plasmid pJHSP70. After pJHSP70 was amplified by PCR and identified with enzyme digestion analysis, Helicobacter pylori UreB （Hp UreB） gene was cloned into pJH-SP70. The expression of Hp UreB protein in the Mycobacteria smegmatis mc2 155 was detected by SDS-PAGE. Results The fragment of 160 bp was amplified from the BCG DNA by PCR. Sequencing and homologous analysis showed that the homology between the cloned HSP70 gene and related genes in GenBank was 100%. SDS-PAGE analysis showed that Hp UreB protein expressed in Mycobacteria smegmatis mc2 155. Conclusion The Mycobacterium shuttle expression plasmid pJHSP70, which was based on shuttle plasmid pJEM11, has been constructed successfully.

关 键 词：结核分枝杆菌 热休克蛋白70 启动子 基因表达 

分 类 号：Q75[生物学—分子生物学] Q781