产肠毒素性大肠杆菌K99菌毛蛋白抗原基因的克隆与表达  

Cloning and Expression of the K99 Fimbrial Antigen Gene of Enterotoxingenic Escherichia coli

作　　者：黄亚红[1] 梁婉琪[2] 潘爱虎[2] 王阿凤[2] 黄诚[1] 赵主江[1] 陈建秀[1] 周智爱[3] 张大兵[2] 

机构地区：[1]南京大学生命科学院生物科学与技术系,江苏南京210093 [2]上海市农业科学院生物技术中心上海市遗传育种重点实验室,上海201106 [3]上海市农业科学院畜牧兽医研究所上海市遗传育种重点实验室,上海201106

出　　处：《中国兽医学报》2001年第5期471-474,共4页Chinese Journal of Veterinary Science

基　　金：上海市农业科学院青年基金资助项目 ( 2 0 0 0 -0 9-0 1-1)

摘　　要：应用 PCR从产肠毒素性大肠杆菌 ( ETEC)中扩增出不含信号肽序列的 K99菌毛蛋白基因片段 ,克隆测序后 ,将该片段连接到 E.coli表达载体 p ET2 8a( + )中 ,转化 E.coli表达菌株 BL2 1 ( DE3) ,筛选得到可诱导表达 K99抗原的工程菌株。经 IPTG诱导 ,分离纯化 K99重组蛋白 ,以其免疫新西兰大白兔 ,获得重组蛋白的兔抗血清 ;免疫印迹分析表明 ,此重组蛋白制备的抗血清能与标准的A K99fimbrial subunit gene without signal peptide sequence was amplified by PCR from enterotoxigenic E.coli K99strain.After cloning and sequencing,the fragment was inserted into an E.coli expression vector p ET2 8a( +) ,then the recombinant plasmid was transferred into E.coli BL2 1 ( DE3 ) ,and E.coli BL2 1 ( DE3 ) ( +K99) obtained.Cultured and induced with IPTG,the over-ex- pressed recombinantprotein in the solubleprotein of E.coli BL 2 1 ( DE3 ) ( +K99) wasdetected and pu- rified using preparation electrophoresis.The antiserum againstthe recombinantprotein was raised us- ing a rabbit.With theimmunological analysisthe anti-serum showed a specific immunological response to standard antigen of enterotoxingenic E.coli strain.This work provides a new method for preparing genetic engineering vaccine to prevent diarrheal disease in piglets.

关 键 词：产肠毒素性大肠杆菌 K99 基因克隆 K99菌毛蛋白抗血清 表达 

分 类 号：S852.61[农业科学—基础兽医学]