无抗性表达致PWD和ED大肠杆菌保护性抗原基因的重组菌构建  

Construction of Non-antibiotic Recombinant Strain Expressing the Major Protective Antigens of Shiga-like Toxigenic Escherichia coli Caused PWD and ED

作　　者：杨健美[1] 张小荣[1] 高崧[1] 韦栋平[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,江苏扬州225009

出　　处：《动物医学进展》2005年第7期45-48,68,共5页Progress In Veterinary Medicine

基　　金：江苏省高技术研究计划资助(No.BG2002316)

摘　　要：利用平衡致死系统成功地将构建表达致PWD和ED大肠杆菌保护性抗原基因的重组菌BL21(pFSFaeG)中所含氨苄抗性基因敲除,并转化沙门氏菌宿主菌X4550。经过质粒酶切分析和表达产物的SDSPAGE证实,氨苄抗性基因已经成功敲除,并且目的蛋白仍与谷胱甘肽转移酶相融合,得到有效表达,重组融合蛋白的分子质量约为80ku。经体外连续传代试验,重组菌X4550(pFSFaeG)均能在无抗生素压力下大量扩增并稳定传代,质粒稳定性和蛋白表达稳定性良好。无抗性重组菌的成功构建解决了基因工程疫苗抗性基因潜在危险问题,并有望通过沙门氏菌载体的递送作用,为研制口服疫苗奠定了基础。By introducing asd gene from sallmonella typhimurium into a prokaryotic expression plasmid vector of E.coli and destroying ampicillin resistance gene at the same time, a new prokaryotic expression recombinant strain which contained no antibiotic resistance gene was constructed .It was based on the recombinant strain BL21(pFSFaeG) which contained ampicillin resistance gene.Restriction endonuclease analysis was used to indentify the new recombinant plasmid.The results indicated that the plasmid was correctly constructed and the ampicillin resistance gene was koncked out.Partly soluble GST-FedA-Stx2eB-FaeG fusion protein was expressed in recombinant strain X4550 (pFSFaeG) after induced by IPTG at 37 ℃, which was confirmed by SDS-PAGE analysis.The new recombinant strain can be amplified in large amount in vitro without the need of antibiotic pressure.The stability of plasmid and expression of protein were confirmed very well, which constituted a solid foundation for the further study of oral immunization.

关 键 词：平衡致死系统 基因敲除 蛋白表达 

分 类 号：S852.612[农业科学—基础兽医学] Q789[农业科学—兽医学]