大肠杆菌-分枝杆菌穿梭表达质粒pBCG-2100的构建及应用

• 作 者: 程继忠 郑波 海涛 皇甫永穆
• 机 构: 同济医科大学实验医学研究中心医学分子生物学研究室,同济医科大学职业医学研究所
• 出 处: 生物工程学报
• 发表日期: 1999年02期
• 关键词: 穿梭表达质粒 pBCG-2100 抗原基因 日本血吸虫 启动子 分子生物学方法 穿梭质粒 定向克隆 全长序列 表达蛋白

利用分子生物学方法，构建了大肠杆菌分枝杆菌（Ｅ．ｃｏｌｉＭｙｃｏｂａｃｔｅｒｉｕｍ）穿梭表达质粒ｐＢＣＧ２１００，研究了编码日本血吸虫中国大陆株谷胱甘肽Ｓ转移酶（ＧｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅ，ＧＳＴ）抗原基因在卡介苗（ＢａｃｉｌｕｓＣａｌｍｅｔｅＧｕｅｒｉｎ，ＢＣＧ）中的表达。以含人结核杆菌热休克蛋白（Ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ，ｈｓｐ）７０基因全长序列的质粒ｐＭＴ７０为模板，扩增出ｈｓｐ７０启动子，测序选出无错配的启动子，将其定向克隆入Ｅ．ｃｏｌｉＭｙｃｏｂａｃｔｅｒｉｕｍ穿梭质粒ｐＢＣＧ２０００中，构建成Ｅ．ｃｏｌｉＭｙｃｏｂａｃｔｅｒｉｕｍ穿梭表达质粒ｐＢＣＧ２１００。再将编码ＧＳＴ的ｃＤＮＡ按正确的阅读框顺序，克隆到ｐＢＣＧ２１００中ｈｓｐ７０启动子的下游，得到分枝杆菌表达质粒ｐＢＣＧＧＳＴ。将ｐＢＣＧＧＳＴ电转化入ＢＣＧ中，筛选出重组ＢＣＧ疫苗，经热诱导后所表达的重组ＧＳＴ（ｒＧＳＴ）抗原，为可溶性蛋白，经纯化后，在ＳＤＳＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带，其表达量占ＢＣＧ菌体总蛋白的１３％。Ｗｅｓｔｅｒｎｂｌｏｔ提示ｒＧＳＴ能与?

Molecular biology method was used to construct the shuttle expression plasmid pBCG2100 of E.coliMycobacterium. The gene encoding Glutathione Stransferase (GST ) Antigen gene in BCG (BacilusCalmete  Guerin, BCG) expression. The hsp70 promoter was amplified from the plasmid pMT70 containing the full-length heat shock protein 70 (hsST) gene of Mycobacterium tuberculosis, and the promoter with no mismatch was selected by sequencing and cloned into E.coli. Mycobacterium shuttle plasmid pBCG 2000, constructed into E. coli Mycobacterium shuttle expression plasmid pBCG  2100. The cDNA encoding GST was then cloned into the downstream of the hsp70 promoter in pBCG-2100 by the correct reading frame to obtain the mycobacterial expression plasmid pBCG-GST. Recombinant BCG vaccine was screened by electroporation of pBCGGST into BCG, and the recombinant GST (rGST) antigen expressed after heat induction was a soluble protein. After purification, the recombinant BCG vaccine showed obvious molecular weight of 26 kD on SDSPAGE Expression of protein bands, the expression of total protein accounted for BCG 13%. Westernblot tips rGST with?