百日咳杆菌毒素S_1亚单位与谷胱甘肽S-转移酶融合基因在大肠杆菌中表达及其一步纯化  

Expression of Fused Gene for PTX S 1 with GST in Eschrichia Coli and Single step Purification of Its Products

作　　者：郑波[1,2] 程继忠[1,2] 皇甫永穆[1,2] 海涛 戴五星[1,2] 王建平[1,2] 

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 [2]同济医科大学公共卫生学院劳动卫生与职业病教研室

出　　处：《同济医科大学学报》1999年第1期17-20,共4页Acta Universitatis Medicinae Tongji

基　　金：国家自然科学基金;卫生部基金

摘　　要：研究了百日咳杆菌毒素Ｓ１亚单位与谷胱甘肽Ｓ－转移酶融合蛋白（ＧＳＴ－Ｓ１）在大肠杆菌（Ｅ．ｃｏｌｉ）中的表达，并一步纯化了重组ＧＳＴ－Ｓ１。首先用聚合酶链反应（ＰＣＲ）扩增出长度为５６０ｂｐ的编码百日咳毒素Ｓ１亚单位的ＤＮＡ片段。该片段比原编码Ｓ１蛋白的ＤＮＡ片段在其３′端缺失了１３８个碱基，将其按正确的阅读框克隆入质粒ｐＧＥＸ中ＧＳＴ基因的３′端，构成ＧＳＴ－Ｓ１融合基因表达载体ｐＧＥＸ－Ｓ１。经异丙基硫代－β－Ｄ－半乳糖苷（ＩＰＴＧ）诱导表达和十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ），可见所表达的ＧＳＴ－Ｓ１融合蛋白存在于细菌上清，为可溶性蛋白。分子量为４９ｋｕ处有明显的蛋白表达带。用谷胱甘肽（ＧＳＨ）亲和层析系统，一步纯化出ＧＳＴ－Ｓ１融合蛋白。最后经凝血酶的消化，得到２３ｋｕ的重组百日咳杆菌毒素Ｓ１亚单位蛋白。The expression of fused gene for Bordetella pertussis toxin (PTX) S 1 subunit with glutathion S transferase (GST S 1) was studied in Eschrichia coli , and the recombinant GST S 1 was purified by single step procedure A 560bp length DNA fragment encoding PTX S 1 subunit, the hydrophobic region of which, consisting of 46 amino acids, was deleted at the C terminal, was firstly amplified by PCR The GST S 1 fusion gene expression plasmid pGEX S 1 was constructed by cloning the S 1 gene into 3′ terminal of GST gene in plasmid pGEX in according to correct reading frame The existing proteins from fusion gene in bacterial supernatant were soluable and could be observed on SDS PAGE at molecular weigth of about 49 ku after induced with IPTG The fusion products were purified in single step by GSH affinity chromatography and the recombinant PTX S 1 subunit which was 23 ku were finally obtained by digestion with thrombin

关 键 词：百日咳杆菌毒素 S1亚单位 基因表达 疫苗 

分 类 号：R378.42[医药卫生—病原生物学] R516.603[医药卫生—基础医学]