猪β-防御素-2基因在大肠杆菌中的融合表达及抑菌活性检测

摘要

将猪β-防御素-2基因(pBD-2)片段正确连接到pET32a载体上,从而得到稳定表达pBD-2的大肠杆菌表达株.根据已报道的pBD-2基因序列,按大肠杆菌密码子优化原则合成了5条pBD-2基因片段,通过重叠延伸PCR(SOE PCR)方法将这5段延伸成完整的pBD-2基因序列,将该基因定向插入原核表达质粒pET32a的多克隆位点上,成功地在大肠杆菌BL21(DE3)中表达出TrxA-pBD2融合蛋白.结果显示,表达的融合蛋白用肠激酶切掉TrxA后做琼脂孔穴扩散法检测,pBD-2对金黄色葡萄球菌有较好的抑菌活性,表明试验获得表达pBD-2的大肠杆菌菌株.%To construct the recombinant E. coli BL21 in which porcine β-defensin-2(pBD-2) could be stably expressed and the antimicrobial effect was evaluated. Based on cDNA sequence of porcine defensin pBD-2 gene reported in this study, five gene fragments were synthesized according to the codon preference of E. coli in this experiment. The pBD-2 gene was produced by gene splicing by overlap extension PCR(SOE PCR) , and pBD-2 was inserted into expression vector (pET32a) and recombinant vector transformed into E. coli. where recombinant vector produced fusion protein successfully. The fusion and soluble induced protein were cut by enterokinase to get the recombinant pBD-2. Agarose diffusion assay showed that the pBD-2 had an antibacterial activity against Staphylococcus aureus. The pBD-2 was expressed successfully and casted a light for massive production of the pig defensin, a new antimicrobial agent.