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

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。

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

The SLT-IIeB gene without its signal and transmembrane sequence was amplified by PCR from a Escherichia colistrain called Ee responsible for the edema disease in piglets in Hubei Province.The amplicon was cloned intothe prokaryotic expressionvector pGEX-KG, the recombinant plasmid pGEX-SLT-IIeB was then sequenced and compared with other SLTECisolates.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-IIeBwas highly conservative in SLTECgenome.The recombinant plasmid pGEX-SLT-IIeBwas expressed in the E.coli BL21 (DE3) induced byIPTG.The expression product,GST-SLT-IIeB,was present in a form of inclusion bodies confirmed by SDS-PAGE analysis.The GST-SLTIIeB showed the biological activity of immunityinWestern-blotanalysis.