猪大肠杆菌不耐热肠毒素LTB基因的克隆及原核表达  

Cloning and prokaryotic expression of heat-labile enterotoxin B subunit gene of Escherichia coli from swine

作　　者：刘媛[1] 左玉柱[1] 范京惠[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001

出　　处：《中国兽医科学》2011年第2期163-166,共4页Chinese Veterinary Science

基　　金：河北农业大学校长基金项目

摘　　要：从河北省部分地区病猪中分离大肠杆菌,根据GenBank中登录的大肠杆菌不耐热肠毒素B亚基(LTB)基因序列,设计了1对引物,采用PCR技术扩增LTB的编码基因,得到一条375bp的片段。将其连入Simple-T载体中,经酶切、PCR及序列测定法进行鉴定。测序正确后,将该基因插入到pET-28a(+)载体中构建原核表达载体,将此重组质粒转化到Rosetta(DE3)感受态细胞中,并用IPTG诱导,将诱导产物用SDS-PAGE和Western-blot进行分析。结果显示,LTB基因可以在大肠杆菌中获得表达,表达产物分子质量约为14ku,与预计的蛋白分子质量大小一致。Western-blot分析表明,该蛋白可以与大肠杆菌免疫血清产生特异性结合反应,具有良好的抗原性,为大肠杆菌亚单位疫苗的研究奠定了基础。Escherichia coli were derived from the diseased swine in some areas of Hebei Province.A pair of primers was designed according to B subunit of heat-labile enterotoxin（LTB） gene sequence in GenBank.Subsequently,the LTB gene of 375 bp was amplified by PCR.Then,the gene was cloned into the Simple-T vector,and identified by digestion with restriction endonucleases,PCR and sequencing.After the sequences were confirmed correct,a prokaryotic expression vector was constructed by inserting the gene into pET-28a（＋） vector.The recombinant plasmid was transformed into Rosetta（DE3） competent cells,and was induced with IPTG.The expressed product was analysed by SDS-PAGE and Western-blot.In result,the LTB gene was highly expressed in E.coli and the expressed protein was 14 ku in size,as was expected.Western-blot test demonstrated that the expressed protein could had specifically react with E.coli antiserum.The results showed that the expressed protein with satisfactory antigenicity,laid the foundation for the development of E.coli subunit vaccines.

关 键 词：不耐热肠毒素 LTB基因 原核表达 

分 类 号：S852.612[农业科学—基础兽医学]