大肠杆菌O157 Stx噬菌体裂解酶的克隆表达及活性分析

为研究大肠杆菌O157Stx噬菌体编码的裂解酶(内溶素)对肠出血性大肠杆菌的抗菌作用,克隆表达了大肠杆菌O157Stx噬菌体裂解酶,并检测其裂解作用和裂菌谱。利用PCR方法扩增大肠杆菌O157Min27株中Stx2噬菌体裂解酶基因(R基因),构建表达质粒pET28a(+)-lysEC1,并转化至大肠杆菌BL21(DE3)。重组质粒在BL21中获得了高表达,目的蛋白表达量占菌体总蛋白的34%,经His-trapTM亲和层析柱纯化后,获得的重组LysEC1的纯度大于97%。体外裂解活性试验证实纯化的噬菌体裂解酶LysEC1对肠出血性大肠杆菌菌株具有很好的裂解作用,这为新型噬菌体抗菌生物制剂的研发提供了新的研发方向。

Expression and Antibacterial Activity of Stx Phage Endolysin of Escherichia coli O157

To investigate the antibacterial activity of Stx phage endolysin of Escherichia coli O157 toward Enterohemorrhagic Escherichia coli(EHEC),LysEC1,endolysin of Stx2 phage from Escherichia coli O157 Min27,was successfully constructed,purified and characterized,and then antibacterial activity and lysis spectrum of LysEC1 were analyzed.The complete endolysin gene(R gene) of Stx2 phage Min27 was amplified by PCR,and targeted fragment was cloned into expression Escherichia coli vector pET28a.Recombinant vector pET-28a(+)-LysEC1 was transformed into BL21.The results showed that exogenous protein was highly expressed in BL21,which accounted for about 34% of total protein.After purification with His-trapTM column,the recombinant LysEC1 was obtained with more than 97% of purity.Lysis test in vitro showed that LysEC1 exerted efficient lysis activity to several E.coli O157 strains.Those results were the foundation for the future application of LysEC1 in the treatment of EHEC infection.