猪大肠杆菌不耐热肠毒素LTB基因的克隆及原核表达

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。

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

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(+) v...