人结核杆菌热休克蛋白70启动子对外源基因在分枝杆菌中表达的影响  

Effect of human Mycobacterium tuberculosis heat shock protein70 promoter on the expression of foreign gene in Mycobacterium

作　　者：程继忠[1] 皇甫永穆[1] 冯作化[1] 梁驹卿[1] 肖红[1] 

机构地区：[1]同济医科大学实验医学研究中心医学分子生物学研究室

出　　处：《中华微生物学和免疫学杂志》1997年第6期410-415,共6页Chinese Journal of Microbiology and Immunology

基　　金：总理基金;国家自然科学基金

摘　　要：利用聚合酶链反应技术（Ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）扩增得到１５０ｂｐ的人结核杆菌热休克蛋白（Ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ，ＨＳＰ）７０启动子和约６５０ｂｐ的外源基因日本血吸虫谷胱甘肽硫转移酶（ＧｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅ，ＧＳＴ）的ｃＤＮＡ片段。并采用Ｓａｎｇｅｒ双脱氧链终止法测定了人结核杆菌ＨＳＰ７０起始密码ＡＴＧ上游１５０ｂｐ的启动子序列。在这一区域可见与大肠杆菌（Ｅ．ｃｏｌｉ）－３５和－１０区同源的碱基顺序ＴＴＧＡＧ和ＡＴＣＡＴＡ，以及ＡＴＧ上游－１２～－８处的核糖体结合位点ＧＧＡＧＧ。然后将人结核杆菌ＨＳＰ７０启动子和日本血吸虫ＧＳＴ编码基因克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中，构成能表达日本血吸虫ＧＳＴ基因的Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达载体ｐＢＣＧ－Ｓｊ２６。电转化耻垢分枝杆菌后，用卡那霉素筛选出阳性重组子。含有ｐＢＣＧ－Ｓｊ２６的耻垢分枝杆菌在热和过氧化氢的作用下均诱导出ＧＳＴ的表达。其表达产物分子量为２６×１０３，经聚丙烯酰胺凝胶（ＳＤＳ－ＰＡＧＥ）后，扫描分析，表明重组ＧＳＴ（ｒＧＳＴ）占菌体总蛋白的１０％。本研究为Ｈ?The cDNA fragments of 150bp length promoter of human Mycobacterium(M) tuberculosis heat shock protein(HSP) 70 and 650bp length foreign gene,the Schistosoma japonicum glutathione S transferase(GST) gene,were obtained by amplification with polymerase chain reaction.And the upstream 150bp DNA sequence of initiation codon ATG of the human M.tuberculosis HSP70 promoter was determined by Sanger Dideoxy mediated Chain termination method.This region contained the sequences TTGAG and ATCATA which were consistent with those of E.coli promoters 35 and 10 region,respectively,as well as the ribosome binding site GGAGG at upstream position 12￣ 8 of ATG.Then,the human M.tuberculosis HSP70 promoter and Schitosoma japonicum GST cDNA were cloned into the E.coli mycobacteria shuttle plasmid pBCG 2000 to construct E.coli mycobacterium expression shuttle plasmid pBCG Sj26 that could express Schistosoma japonicum GST gene.The mycobacteria smegmatis were electroporated and the positive colonies were selected with kanamycin.The mycobacteria smegmatis containing the vector pBCG Sj26 could be induced by heating and hydrogen peroxide(H 2O 2) to express GST.The molecular weigth of the recombinant GST(rGST) was 26000.The rGST contents that were about 10 percent of the total bacterial protein were analysed by density scanning after running SDS PAGE.This study would supply scientific evidences for application of HSP70 promoter in expressing foreign gene in mycobacterium and the development of mycobacterium as multiple valent vectoral vaccine.

关 键 词：结核分枝杆菌 热休克蛋白 启动子 基因表达 

分 类 号：R378.911[医药卫生—病原生物学]