铜绿假单胞菌噬菌体vB_PaS_IME307的生物学特性研究及全基因组分析  

Biological characteristics and whole genome analysis of Pseudomonas aeruginosa phage vB_PaS_IME307

作　　者：王灿 刘慧莹 高明明 米丽媛 喻鑫婷 米志强 柏长青 WANG Can;LIU Huiying;GAO Mingming;MI Liyuan;YU Xinting;MI Zhiqiang;BAI Changqing(Department of Respiratory Medicine,Fuyang Hospital Affiliated to Anhui Medical University,Fuyang,Anhui Province,236000;Department of Respiratory and Critical Care Diseases,Ffth Medical Centre,Chinese PLA General Hospital,Bejjing,100071;State Key Laboratory of Pathogen and Biosecurity,Institute of Micrbiology and Epidemiology,Academy of Military Medical Sciences,Beijing,100071;Depatment of Respiratory and Crtical Care Diseases,General Hospital of Shenzhen University,Shenzhen,Guangdong Province,518000,China)

机构地区：[1]安徽医科大学附属阜阳医院呼吸内科,安徽阜阳236000 [2]解放军总医院第五医学中心呼吸与危重症医学科,北京100071 [3]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [4]深圳大学总医院呼吸与危重症医学科,广东深圳518000

出　　处：《陆军军医大学学报》2023年第19期2045-2056,共12页Journal of Army Medical University

基　　金：安徽省自然科学基金青年项目(2108085QH310);安徽省卫生健康委科研项目(AHWJ2021b057);安徽医科大学校科研基金项目(2020xkj060)。

摘　　要：目的以实验室保存菌株Pa175为宿主菌,从医院污水中分离得到1株铜绿假单胞菌噬菌体,研究其生物学特性及全基因组学特征。方法利用双层平板法、噬斑法分离并鉴定噬菌体;PEG8000浓缩NaCl沉淀法纯化噬菌体;蔗糖梯度离心进一步纯化噬菌体,经磷钨酸负染染色后,使用透射电子显微镜观察其形态;蛋白酶K/SDS法提取噬菌体DNA,对提取的核酸进行全基因组测序和生物信息学分析。结果得到1株裂解性铜绿假单胞菌噬菌体vB_PaS_IME307,该噬菌体可以形成透明且有晕环的噬菌斑,全基因组大小为51545 bp。透射电镜结果显示vB_PaS_IME307的头部呈正二十面体形状,直径(66±2)nm,尾长(116±2)nm;进化树分析发现该噬菌体为Mccleskeyvirinae病毒属,长尾噬菌体科(Siphoviridae)。生物学特性研究发现:vB_PaS_IME307在pH=4.0~11.0、60℃以下能够保持较高活性,感染周期约为60 min,其中包括10 min潜伏期和50 min暴发期,其暴发量为150 PFU/cell。结论噬菌体vB_PaS_IME307具有宿主特异性,对温度和pH具有较好的耐受能力,具有防治铜绿假单胞菌细菌感染的潜力。Objective To determine the biological properties and whole genome characteristics of a phage against P.aeruginosa isolated from sewage samples as the host strain Pa175.Methods Double-layer agar plate and plaque method were used for phage isolation and identification.The obtained phage was purified through PEG 8000 concentration and NaCl precipitation.The phage was further purified with sucrose gradient centrifugation,stained by phosphotungstic acid negative staining.Transmission electron microscopy was employed to observe its morphology.After its DNA was extracted by using proteinase K/SDS,whole genome sequencing was performed and the results were analyzed by bioinformatics.Results A lytic P.aeruginosa phage was obtained and then named as vB_PaS_IME307,which can form transparent plaque with halo,with a whole genome size of 51545 bp.Transmission electron microscopy showed that the head of vB_PaS_IME307 was icosahedral shape,with a diameter of 66±2 nm and a tail length of 116±2 nm.Phylogenetic tree analysis revealed that IME307 was belonged to Mccleskeyvirinae virus genus,Siphoviridae family.The phage was maintained of high activity at pH value from 4.0 to 11.0 and temperature below 60℃.The infection cycle was about 60 min,including 10 min incubation period and 50 min outbreak period.The outbreak was 150 PFU/cell.Conclusion vB_PaS_IME307 is of host specificity,good tolerance to temperature and pH valule,and has the potential to prevent and treat P.aeruginosa infection.

关 键 词：铜绿假单胞菌 噬菌体 生物学特性 基因组学分析 

分 类 号：Q393.48[生物学—遗传学] R378.991[医药卫生—病原生物学] R394.2[医药卫生—基础医学]