金黄色葡萄球菌噬菌体裂解酶Lys239表达纯化及活性分析  

Expression,Purification and Activity Analysis of Lys239 Phage Lyase from Staphylococcus aureus

作　　者：李君 孙虎芝 田延军 潘强 任慧英 刘文华[1] LI Jun;SUN Hu-zhi;TIAN Yan-jun;PAN Qiang;REN Hui-ying;LIU Wen-hua(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong,266109,China;Qingdao NABT Phagepharm Co.,Ltd,Qingdao,Shandong,266000,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109 [2]青岛诺安百特生物技术有限公司,山东青岛266000

出　　处：《动物医学进展》2024年第9期27-31,共5页Progress In Veterinary Medicine

基　　金：山东省产业技术体系驴产业创新团队项目(SDAIT-27)。

摘　　要：为获得具有高效裂解活性的金黄色葡萄球菌噬菌体裂解酶,将测序后的噬菌体全基因序列在GenBank数据库中进行比对,找出裂解酶基因序列,对其克隆后在BL21(DE3)中表达蛋白并纯化,选择大蜡螟幼虫作为模型模拟体内细菌感染以探究其抑菌效果。结果显示,裂解酶合成基因构建到载体pET22b中,得到重组质粒pET22b-lys239。并将重组质粒转入BL21(DE3)感受态细胞后诱导其表达,经纯化验证后,获得浓度为2μg/μL的具有裂解活性的裂解酶Lys239。大蜡螟幼虫体内细菌感染试验结果显示,Lys239处理组存活率明显高于对照组,血淋巴负荷测定显示各处理组与对照组差异极显著(P<0.01)且Lys239浓度越高,对于金黄色葡萄球菌抑制效果越显著。说明重组表达的裂解酶Lys239具有良好的抑菌效果,可作为一种潜在的新型抑菌剂。In order to obtain phage lyase of Staphylococcus aureus with high lytic activityy,the entire gene sequence of the sequenced phage was compared in the GenBank database,and the lytic enzyme gene sequence was excavated.After cloning,the protein was expressed in BL21(DE3)and purified.The Galleria mellonella was selected as a model to simulate bacterial infection in vivo to explore its antibacterial effect.The results showed that the lyase synthesis gene was successfully constructed into the vector pET22b,resulting in a recombinant plasmid pET22b-lys239.And the recombinant plasmid was transfected into BL21(DE3)receptive cells to induce its expression.After purification and validation,Lys239 with lysis activity at a concentration of 2μg/μL was obtained.The results of the bacterial infection test in the body of the Galleria mellonella showed that the survival rate of the Lys239 treatment group was significantly higher than that of the control group.The hemolymph load test showed a significant difference between the control group and each treatment group(P<0.01),and the higher the concentration of Lys239,the more significance the inhibitory effect on Staphylococcus aureus.The recombinant lysase Lys239 expressed in this study has good antibacterial effects and can be used as a potential new antibacterial agent.

关 键 词：金黄色葡萄球菌 噬菌体 裂解酶 大蜡螟幼虫 抑菌作用 

分 类 号：S852.611[农业科学—基础兽医学]