猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达

The Fusion and Expression of Genes Encoding Heat-stable and Heat-labile Enterotoxins of Escherichia coli from Porcine Origin

用聚合酶链反应扩增出猪源大肠杆菌编码ST前体 (pro ST)和LT的B亚单位 (LTB)成熟多肽的序列 ,再通过套式PCR将pro ST编码序列 3′端和LTB编码序列 5′端融合 ,并置于同一阅读框内 ,得到ST和LTB的融合基因 ,将此序列克隆到pGEM T质粒中 ,序列分析后 ,亚克隆到表达载体pQE30中 ,在大肠杆菌细胞中得到表达 ,表达的融合蛋白同时具有ST和LTB的抗原性 ,且无ST和LT的生物毒性。

The genes encoding precursor heat-stabile (pro-ST) and mature peptide of B unit of heat-labile (LT) enterotoxins of Escherichia coli from piglet were amplified by polymerase chain reaction (PCR). The 3′ terminus of gene encoding pro-ST was genetically fused to the 5′ terminus of the LTB subunit gene in nest-PCR. The fusion genes encoding pro-ST and LTB were cloned into pGEM-T vector and subcloned into the pQE30. The recombinant plasmid was expressed in E.coli by IPTG induction. The fusion protein possessed both ST and LTB antigenicity, as well as it had lost the biological toxicity of ST or LT toxin.