F18大肠杆菌黏附素在染色体-质粒平衡致死系统中的表达  

FedF adhesin from F18 fimbriaed E.coli expression in chromosome-plasmid balanced-lethal system

作　　者：原志伟[1,2] 吴娟[1] 朱春红[1] 朱军[1] 郁磊[1] 羊扬[1] 王建业[1] 朱国强[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]中华人民共和国荣成出入境检验检疫局,山东荣成264300

出　　处：《中国兽医学报》2010年第11期1450-1455,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571374;30771603);江苏省属高校自然科学重大基础研究项目(08KJA230002)

摘　　要：将含有启动子及核糖体结合位点RBS的E.coliK12菌株源asd基因克隆入表达质粒载体pnirBMisL-fedF中,构建重组表达质粒pMisL-fedF-asd,使该质粒中氨苄抗性基因失活的同时能表达fedF和asd基因。该重组质粒pMisL-fedF-asd转化E.coliDH5α宿主菌asd基因缺失株即DH5α△asd菌株后,表达F18E.coli黏附素的DH5α△asd重组菌能在不含DAP的LB平板生长,构成非抗性平衡致死系统。厌氧条件下诱导培养后,经玻板凝集反应、间接免疫荧光试验及易感仔猪小肠上皮细胞黏附试验表明,F18E.coli黏附素在DH5α△asd菌体表面得到了功能性展呈。经20次传代培养没有发现质粒丢失,F18E.coli黏附素在该系统中持续稳定表达。该染色体-质粒平衡致死系统的成功构建为研究F18E.coli黏附素亚单位疫苗在动物体内的免疫效果提供了一个安全有效的操作平台。The recombinant plasmid pMisL-fedF-asd was constructed by the based expressive vector pnirBMisL-fedF with the insert of E.coli K12 originated（Aspartate-semialdehyde dehydrogenase） asd gene with its own promoter and RBS,which could express both FedF and asd,no bla with inactivation of insertion site.Like prototype E.coli DH5α,the E.coli DH5α△asd mutant transformed with the recombinant plasmid could grow on the LB plate or in LB broth without any supplementation of DAP.After induction in anoxia condition,the results of combination of assays,including slide agglutination,indirect immunofluorescence and the porcine small intestine epithelial cells adhesion,demonstrated that the FedF was expressed in the recombinant bacteria,and the chromosome-plasmid balanced-lethal system was set up successfully,which was stable for 20 generations of passage in vitro culture and expressed the FedF adhesion without antibiotic resistance gene.It could provide the platform with safety and function in the near future study for F18 FedF subunit vaccine in vivo.

关 键 词：asd基因 F18大肠杆菌 菌体表面展示 平衡致死系统 

分 类 号：S852.61[农业科学—基础兽医学]