致猪水肿病大肠杆菌鄂E株SLT-IIeB基因的克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Expression of B Subunit of Shiga-like Toxin IIe Gene in Escherichia coli

作　　者：林艺远[1] 刘国平[1] 吴斌[1] 赵战勤[1] 陈焕春[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2006年第5期474-478,共5页Journal of Huazhong Agricultural University

基　　金：湖北省科技攻关计划项目(2001AA201;2006AA202)资助

摘　　要：以湖北本地分离的致猪水肿病大肠杆菌鄂E株为模板,PCR扩增去掉信号肽和跨膜区的SLT-IIeB基因,将其克隆到原核表达载体pGEX-KG。同时对筛选出的阳性质粒pGEX-SLT-IIeB进行测序,测序结果与Genbank上发表的12个SLT-IIeB基因序列的同源性达100%,表明该基因保守性很好。重组质粒pGEX-SLT-IIeB经IPTG诱导后在大肠杆菌中实现了表达,SDS-PAGE分析表明表达产物GST-SLT-IIeB有特异性表达带,Western-blot检测证实表达产物具有免疫反应性。The SLT-IIeB gene without its signal and transmembrane sequence was amplified by PCR from a Escherichia coli strain called Ee responsible for the edema disease in piglets in Hubei Province. The amplicon was cloned into the prokaryotic expression vector pGEX-KG, the recombinant plasmid pGEX-SI.T-IIeB was then sequenced and compared with other SLTEC isolates. The result showed that the cloned SLT-IIeB presented 100% identity of nucleotide sequence with the 12 published SLT-IIeB in Genbank. So the SLT-IIeB was highly conservative in SLTEC genome. The recombinant plasmid pGEX-SLT-IIeB was expressed in the E. coli BL21 （DE3） induced by IPTG. The expression product, GST-SLT-IIeB, was present in a form of inclusion bodies confirmed by SDS-PAGE analysis. The GSTSI.T-IIeB showed the biological activity of immunity in Western-blot analysis.

关 键 词：产类志贺毒素大肠杆菌 猪水肿病 SLT-IIeB 克隆 序列分析 原核表达 

分 类 号：S852.612[农业科学—基础兽医学] S858.28[农业科学—兽医学]