大肠杆菌P88噬菌体gp52和gp53基因对其裂解活性和宿主谱形成影响的研究  

Effect of genes gp52 and gp53 on lytic activity and host range of Escherichia coli bacteriophage P88

作　　者：费定润 吴柔霖 程丽丽 鲁珂蓥 庄楠茜 孙静 程昌勇 陈绵绵 宋厚辉 FEI Ding-run;WU Rou-lin;CHENG Li-li;LU Ke-ying;ZHUANG Nan-xi;SUN Jing;CHENG Chang-yong;CHEN Mian-mian;SONG Hou-hui(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,Hangzhou 311300,China;Taizhou Jiaojiang District Agriculture,Rural and Water Resources Bureau,Taizhou 318000,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]台州市椒江区农业农村和水利局,浙江台州318000

出　　处：《中国预防兽医学报》2023年第10期994-1000,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省自然科学基金(LQ20C180001、LY23C180002);国家自然科学基金(32002253、31972648、31872620);浙江农林大学创新训练项目(S202210341103);浙江省大学生科技创新活动计划暨新苗人才计划(2023R412020)。

摘　　要：为探究大肠杆菌溶原性噬菌体P88中非结构与功能基因gp52和gp53对其裂解活性和宿主谱形成的影响,本研究在P88宿主细菌K88基因组中对其进行基因工程操作,然后诱导出相应噬菌体进行功能研究。首先在大肠杆菌Red同源重组质粒p KD46基础上,融合p ST98-AS质粒的四环素抗性基因和I-Sce I核酸内切酶基因,构建缺失质粒p WRG99。利用质粒p WRG99分别构建大肠杆菌K88的gp52和gp53基因缺失突变株(Δgp52和Δgp53)并经PCR和测序鉴定。利用丝裂霉素C诱导Δgp52和Δgp53,采用双层平板法检测噬菌体的裂解活性,结果显示,Δgp52和Δgp53的诱导液可以在大肠杆菌DE048为宿主菌的平皿上形成清晰的噬菌斑,表明Δgp52和Δgp53能诱导出可以感染DE048的噬菌体(将该噬菌体命名为P88-52和P88-53),并且gp52和gp53基因缺失均不影响噬菌体P88的形成和裂解活性。将P88-52和P88-53纯化并经电镜观察,结果显示,P88-52和P88-53与野生株P88形态相似,具有P2-like噬菌体典型的肌尾和短尾丝特征,表明gp52和gp53基因缺失均不影响P88噬菌体颗粒的形态。利用P88的宿主菌对P88-52和P88-53进行宿主谱分析,结果显示,P88-52和P88-53在DE048为宿主菌的平皿上均可以形成清晰的噬菌斑,在MC1061、DH5α和BL21为宿主菌的平皿上均不能形成噬菌斑,与噬菌体P88宿主谱相同,表明gp52和gp53基因缺失均不影响P88噬菌体的宿主谱。综上所述,在宿主菌K88基因组的基础上缺失gp52和gp53基因后,诱导出的P88突变噬菌体的裂解活性和宿主谱均不受影响。本研究通过在宿主菌中对前噬菌体基因编辑来研究溶原性噬菌体的基因功能,为其他溶原性噬菌体基因功能的研究提供了参考依据。To investigate the effect of non-structural and functional genes gp52 and gp53 on lytic activity and host range of Escherichia coli bacteriophage P88,the genome of P88 will be genetically manipulated in its host bacteria K88,and then the corresponding phages can be induced for functional research.In this study,the tetracycline resistance gene and I-SceI endonuclease gene of plasmid pST98-AS were fused on the basis of Escherichia coli(E.coli)Red homologous recombinant plasmid pKD46,and the plasmid pWRG99 for deletion was successfully constructed.The gp52 and gp53 gene deletion strains(Δgp52 andΔgp53)of E.coli K88 were successfully generated using pWRG99.The deletion mutant phages were induced fromΔgp52 andΔgp53 by mitomycin C,and the lytic activity of mutants was detected by double-layer plate method.The results showed that the induction solution of mutantsΔgp52 andΔgp53 could form clear plaques on the plate of E.coli DE048 as the host bacteria,the induced phage named P88-52 and P88-53,indicating thatΔgp52 andΔgp53 could be induced to produce phages that could infect DE048,and neither gene gp52 nor gp53 affected the formation and lytic activity of P88.Purification of P88-52 and P88-53 and electron microscopy observation showed that P88-52 and P88-53 were similar in morphology to wild strains P88,with typical muscle tail and short tail fibers characteristic of P2-like phage,indicating that genes gp52 and gp53 did not affect the morphology of P88 phage particles.The results of host range analysis showed that P88-52 and P88-53 could form clear plaque on the plates with DE048 as host bacteria,but could not form plaque on the plates with MC1061,DH5αand BL21 as host bacteria,which was the same as the host range of P88,indicating that neither gp52 nor gp53 affected the host range of P88.In summary,deletion of genes gp52 and gp53 from phage P88 did not affected the lytic activity and host range of the induced phages.Genetic manipulating of prophages in host bacteria to study the gene function of lysogenic phages

关 键 词：大肠杆菌 前噬菌体 裂解活性 宿主谱 

分 类 号：S852.61[农业科学—基础兽医学]