多价噬菌体内溶素的阳离子肽修饰及抗菌效果

Development of cationic peptide chimeric lysins based on polyvalent phage lysin and their antibacterial activities

多价噬菌体vB_SEqdws-315因具备对沙门氏菌和大肠杆菌的跨谱裂解能力而具有良好的应用潜力,为进一步研究其内溶素Lysin315的抗菌效果,该研究尝试构建其大肠杆菌重组表达载体并进行诱导表达。结果表明,诱导表达得到的重组内溶素Lysin315分子质量接近25 kDa,可以在膜通透剂EDTA的参与下裂解沙门氏菌和大肠杆菌,但无法单独发挥抗菌效果。该实验尝试通过将含5~15个氨基酸的阳离子短肽克隆至内溶素末端以使其摆脱膜通透剂依赖性,实现对目标菌株的直接裂解作用。构建修饰内溶素的大肠杆菌重组表达载体,经诱导表达和亲和层析纯化,获得了高纯度的3种阳离子肽修饰内溶素(Lysin315-5aa、Lysin315-10aa、Lysin315-15aa)。3种修饰内溶素均可以在体外独立发挥抗菌作用。同时,修饰内溶素Lysin315-10aa在4℃和25℃条件下对牛奶中的沙门氏菌(10^(6) CFU/mL)也具有良好的抗菌效果。该研究初步为革兰氏阴性菌噬菌体内溶素的独立应用提供了解决策略。

Polyvalent phage vB_SEqdws-315 can lyse both Salmonella and Escherichia coli strains,which has the potential to be used as antibacterial agents.In order to further study the antibacterial effect of its endolysin Lysin315,the gene was cloned and expressed in Escherichia coli BL21(DE3)with the induction of IPTG.High-purity recombinant Lysin315 was obtained by affinity chromatography.The molecular weight of Lysin315 was close to 25 kDa.Lysin315 needed EDTA as membrane permeability agents to inhibit the gram-negative bacteria because of the limitation of the cell wall structure of gram-negative bacteria.In order to enhance the direct antibacterial activity,Lysin315 was modified by cationic peptides to obtain chimeric lysins(Lysin315-5aa,Lysin315-10aa and Lysin315-15aa).All of the recombinant lysins were cloned and expressed in E.coli BL21(DE3)induced by IPTG and purified by affinity chromatography to acquire high purity.The results showed that cationic lysins could independently inhibit the hosts with high concentration without any membrane permeabilities.Lysin315-10aa also showed good antibacterial activities against Salmonella(10^(6) CFU/mL)in milk at 4℃ and 25℃.The development of cationic peptide chimeric lysins provided a solution for the independent application of gram-negative bacteriophages lysins.