摘要
为了确定大肠埃希氏菌噬菌体swi2裂解系统的穿孔素(holin)与裂解酶(lysin1、lysin2)在裂解宿主菌过程中的协同作用,根据噬菌体swi2裂解系统的基因序列设计引物,搭桥PCR法分别扩增holin-lysin1、holin-lysin2、lysin1-lysin2基因,以pColdTF为载体,构建重组质粒pCold-holin-lysin1、pCold-holin-lysin2、pCold-lysin1-lysin2,在大肠埃希氏菌BL21中诱导表达,并测定各串联蛋白对大肠埃希氏菌BL21的菌内外裂解活性。结果表明,成功构建了噬菌体swi2裂解系统相关基因两两串联的重组质粒,并在原核系统中实现了holin-lysin1、holin-lysin2、lysin1-lysin2蛋白的可溶性表达。lysin1、lysin2具有天然的菌内和菌外裂解活性,而单独的holin未表现出任何抑菌活性;lysin1与lysin2、holin与lysin1之间无协同作用,而holin与lysin2之间存在协同作用,可以显著增强lysin2的菌内外裂解活性(P<0.05)。研究结果证明了lysin1和lysin2在噬菌体swi2增殖后期替代holin完成裂解宿主菌的过程,两者之间不存在协同作用,这将为进一步阐明噬菌体swi2的裂解机制提供参考。
To determine the synergistic effect of holin,lysin1 and lysin2 of the lysis system in the process of E.coli phage swi2 against host bacteria,primers were designed based on the sequences of phage swi2,holin-lysin1,holin-lysin2 and lysin1-lysin2 were amplified by overlap PCR,the recombinant plasmids pCold-holin-lysin1,pCold-holin-lysin2 and pCold-lysin1-lysin2 were constructed and expressed in E.coli BL21,the extracellular and intracellular lytic activities of the recombinant proteins against E.coli BL21 were determined.The results showed that the recombinant plasmids of lysis system of phage swi2 were successfully constructed,and the recombined proteins holin-lysin1,holin-lysin2 and lysin1-lysin2 were successfully and solubly expressed in prokaryotic system.Lysin1 and lysin2 showed natural lytic activities in vitro and in vivo,but holin itself showed no intracellular lytic activity;Lysin1 and lysin2,holin and lysin1 showed no synergistic effect,but there was a synergistic effect between holin and lysin2,which could significantly enhance the antibacterial activity of lysin2 in vitro and in vivo(P<0.05).The results indicated that lysin1 and lysin2 completed the lysis process instead of holin in the late stage of proliferation,and there was no synergistic effect between lysin1 and lysin2,which would provide help to further clarify the lysis mechanism of phage swi2.
作者
邹玲
赵天祎
王馨锐
甄建瑜
刘文华
张灿
ZOU Ling;ZHAO Tian-yi;WANG Xin-rui;ZHEN Jian-yu;LIU Wen-hua;ZHANG Can(College of Veterinary Science,Qingdao Agricultural University,Qingdao,Shandong 266109,China)
出处
《动物医学进展》
北大核心
2024年第4期75-81,共7页
Progress In Veterinary Medicine
基金
山东省自然科学基金项目(ZR2021MC053)
青岛市关键技术攻关及产业化示范类项目(23-1-4-xxgg-21-nsh)
青岛农业大学研究生创新计划项目(QNYCX22005)。
