摘要
为比较3种噬菌体裂解基因M-lysis、mE和E对大肠杆菌的致死效果,本研究将噬菌体裂解基因M-lysis、mE和E克隆于严谨调控的高效表达载体pN15E6中,构建重组质粒pN15E6-M-lysis、pN15E6-mE和pN15E6-E,分别转化入JM109和DH31soplacI二种大肠杆菌中,观察重组大肠杆菌在含IPTG诱导剂平板培养基上的生长情况,判定M-lysis、mE、E基因过表达对大肠杆菌宿主的影响。结果显示,在大肠杆菌JM109菌株中,mE基因过表达仅表现出抑制细菌生长的效果;M-lysis基因过表达部分致死细菌,存活菌生长受到抑制;E基因过表达能够致死绝大部分细菌,但有少量耐受菌生长。在大肠杆菌DH31soplacI菌株中,M-lysis与mE基因过表达也能致死绝大部分细菌,有少量耐受菌生长;而E基因过表达则完全致死涂布于平板上经106稀释的菌株,未见任何菌落生长。将重组菌株稀释后经平板菌落计数,测得M-lysis、mE、E基因过表达后对DH31soplacI菌株致死率分别为99.03%、99.68%和99.9998%,表明E基因过表达后对宿主菌的致死作用最强;同时定期测定培养的3种重组菌OD600nm值,并绘制生长曲线,结果显示过表达E基因能够迅速致死和裂解细菌。本研究为进一步开发噬菌体裂解肽奠定基础,也为探究细菌致死机理提供了一种新模型。
In order to investigate the lethality of three phage lysis genes to host Escherichia coli strains,the bacteriophage lysis genes M-lysis,mE and E were cloned into the highly regulated expression vector pN15E6,respectively,in this study.The lethality of over-expression of M-lysis,mE and E genes to E.coli strains JM109 and DH31soplacI was evaluated through plating.The results indicated that over-expression of mE and M-lysis genes only inhibited bacterial growth in JM109 strain,while over-expression of E gene was lethal to its host bacteria,with a small proportion of resistant colonies grown.In DH31soplacI strain,over-expression of M-lysis and mE genes could also kill bacteria,with a small proportion of resistant colonies grown,while over-expression of E gene killed almost all bacteria plated.Statistically,the lethality of M-lysis,mE and E genes over-expressed in DH31soplacI strain were 99.03%,99.68% and 99.9998%,further confirming that the E gene over-expression exhibited the highest lethality.Growth curves showed that over-expression of E gene rapidly killed and lysed bacteria after IPTG induction.This study laid a foundation for further development of phage lysis peptides,and also provided a new model for exploring the mechanism of bacteria death.
作者
张远星
李统战
余子豪
查帆
李倩文
余旭平
ZHANG Yuan-xing;LI Tong-zhan;YU Zi-hao;ZHA Fan;LI Qian-wen;YU Xu-ping(College of Animal Science,Zhejiang University,Hangzhou 310058,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第12期1268-1274,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31272582)。
关键词
噬菌体裂解基因
过表达
致死性
bacteriophage lysis gene
over-expression
lethality
