鸡源大肠杆菌噬菌体LHE71的生物学特性和全基因组序列分析  

Biological Characteristics and Whole Genome Sequence Analysis of Chicken-Origin Escherichia coli Phage LHE71

作　　者：荣家庆 任慧英[1] 刘文华[1] 张灿[1] RONG Jia-qing;REN Hui-ying;LIU Wen-hua;ZHANG Can(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医杂志》2023年第12期11-19,共9页Chinese Journal of Veterinary Medicine

基　　金：山东省自然基金面上项目(ZR2021MC053);山东省现代农业产业技术体系家禽创新团队建设项目(SDAIT-11-03);青岛农业大学研究生创新计划(QNYCX22005)。

摘　　要：为了获得具有高效广谱裂解特征的噬菌体,本试验以96株鸡源大肠杆菌(E.coli)临床分离株为宿主菌,用双层平板法从鸡场采集的5份粪便样品中分离纯化噬菌体,测定噬菌体的裂解谱,并对筛选到的噬菌体进行形态观察、生物学特性测定和全基因组序列分析。结果显示,从鸡场采集的粪样中分离到1株烈性大肠杆菌噬菌体,命名为LHE71,在菌苔上形成透亮带晕环的噬斑。透射电镜观察该噬菌体形态为长尾噬菌体,正二十面体的头部直径约为70 nm,弯曲的尾部长约150 nm。以E.coli K12为宿主菌增殖噬菌体,当感染复数为0.1时效价最高,可达到2.6×10^(15)PFU/mL;该噬菌体增殖的潜伏期约为40 min,爆发量约为10^(4)PFU/cell。噬菌体LHE71在60℃以下、pH 7~9的范围内活性稳定,对紫外线有一定的抵抗力。噬菌体LHE71基因组全长50158 bp,预测到81个开放阅读框,其中24个编码已知功能蛋白,无tRNA,不含毒力基因和耐药基因。GenBank数据库中共有32株噬菌体与LHE71有同源性,基于噬菌体衣壳蛋白和末端酶大亚基的进化分析结果显示,LHE71为Tunavirus属噬菌体。结果表明,本试验从鸡场粪样中成功分离并鉴定1株高效宽裂解谱噬菌体LHE71,具有良好的生物学特性,为进一步开发为噬菌体制剂防控鸡源大肠杆菌病提供了基础。In order to obtain a phage with highly efficient and broad-spectrum lytic features,this study used 96 clinical isolates of chicken-origin Escherichia coli(E.coli)as host bacteria.Phages were isolated and purified from 5 fecal samples collected from chicken farms using the double-layer agar plate method.The phage's lytic spectrum was determined,and the isolated phages were subjected to morphological observation,biological characteristic determination,and whole genome sequence analysis.The results showed that one virulent E.coli phage was isolated from the chicken fecal sample,named LHE71,which formed clear plaques with halos on bacterial lawns.Transmission electron microscopy revealed that the phage had a long-tailed morphology,with a head diameter of about 70 nm and a curved tail of about 150 nm in length.The phage proliferated in the presence of E.coli K12 as the host bacterium,with the highest efficacy observed when the infection multiplicity was 0.1,reaching a titer of 2.6×10^(15)PFU/mL.The latent period of phage LHE71 proliferation was approximately 40 min,and the burst size was about 104 PFU/cell.Phage LHE71 exhibited stability in activity at temperatures below 60°C and within a pH range of 7 to 9,and it also demonstrated some resistance to ultraviolet light.The whole genome length of phage LHE71 was 50158 bp,with 81 predicted open reading frames(ORFs),of which 24 encoded known functional proteins.The phage lacked tRNA and did not contain virulence or antibiotic resistance genes.In the GenBank database,there were 32 phages with homology to LHE71.Evolutionary analysis based on major capsid proteins and terminase large subunits revealed that LHE71 belonged to the Tunavirus genus.These results demonstrate the successful isolation and identification of a highly efficient and broad-spectrum lytic phage,LHE71,from chicken fecal samples,which exhibited promising biological charac-teristics.This study provides a foundation for further development of phage formulations for controlling chicken-origin E.coli-associat

关 键 词：大肠杆菌 噬菌体 生物学特性 基因组分析 

分 类 号：S855.1[农业科学—临床兽医学]