融合蛋白MBP-enterocin A的表达、纯化及抗菌活性检测

Fusion expression and purification of enterocin A in Escherichia coli and antibacterial activity determination of MBP-enterocin A

以重组质粒pGAIF为模板,PCR扩增编码细菌素enterocin A的基因片段OAH,克隆到表达载体pMAL-p2x中正确构建重组表达质粒pMA。将pMA转化大肠杆菌DH5α,构建enterocin A融合表达系统。加入IPTG诱导目的基因的表达,然后通过亲和层析方法纯化表达产物,并采用琼脂打孔扩散试验或者琼脂点种试验检测抗菌活性。SDS-PAGE分析结果显示,大肠杆菌表达系统能够高效表达可溶性的融合蛋白MBP-enterocin A,其相对分子质量与推测值(约47 000)相符。抗菌活性结果表明,MBP-enterocin A具有抗李斯特菌活性,而对金黄色葡萄球菌ATCC25923、大肠杆菌O157∶H7均不表现活性;以无害李斯特氏菌LIN3为指示菌,MBP-enterocin A纯化样品的抗菌活性可表示为800 BU/mL。

The gene fragment OAH encoding enterocin A was amplified by PCR,and then cloned into vector pMAL-p2x to properly construct recombinant plasmid pMA.The fusion expression system was successfully developed by transforming pMA into Escherichia coli DH5α.The fusion protein was expressed by IPTG induction and was purified by affinity chromatography of the amylose resin column.Antibacterial activity of samples was determined by the agar well diffusion assay or the agar spot assay.The SDS-PAGE analysis indicated Escherichia coli DH5α/pMA could overexpress soluble fusion protein MBP-enterocin A with approximate molecular weight of 47 000.The results of antibacterial activity showed that MBP-enterocin A was active against 3 Lister strains used,but not active against Staphylococcus aureus ATCC25923 and Escherichia coli O157∶H7,The antibacterial activity of purified MBP-enterocin A was expressed as bacteriocin units per milliliter against Lister innocua LIN3,that is,800 BU/mL.This study provided a basis for further study of enterocin A.