泛耐药铜绿假单胞菌菌株广谱噬菌体裂解酶的获取及抗菌活性观察  

Obtaining of pandrug-resistant pseudomonas aeruginosa′s broad-spectrum phage lyase and observation of its antimicrobial activity

作　　者：王猛[1] 蔡大敏 王景雨 王策 邵煜涵 穆红 WANG Meng;CAI Damin;WANG Jingyu;WANG Ce;SHAO Yuhan;MU Hong(Department of Clinical Laboratory,Tianjin First Central Hospital Affiliated to Nankai University,Tianjin 300192,China;不详)

机构地区：[1]南开大学附属天津市第一中心医院检验科,天津300192 [2]杭州医学院检验学院 [3]天津医科大学检验学院

出　　处：《山东医药》2023年第6期33-37,共5页Shandong Medical Journal

基　　金：天津市医学重点学科(专科)建设项目资助(TJYXZDXK-015A);浙江省教育厅一般科研项目(00004C4Y201738494);天津市第一中心医院春风基金项目(院CF201817)。

摘　　要：目的获取泛耐药铜绿假单胞菌(PA)广谱噬菌体裂解酶,并观察其抗菌活性。方法(1)取237株PA菌株,采用药敏实验筛选得到84株泛耐药PA菌株。采用噬菌斑测定法,将污水与10株泛耐药PA菌株混合培养,筛选得到9株泛耐药PA菌株噬菌体,并用84株泛耐药PA菌株,筛选出90%以上泛耐药PA菌株的广谱噬菌体。(2)采用Proteinase K裂解法及测序获取泛耐药PA菌株广谱噬菌体裂解酶基因、运用生物信息学工具(ORF finder和BLAST)进行序列确认,获得泛耐药PA菌株广谱噬菌体裂解酶的基因序列(目标序列),将目标序列克隆、转化、诱导纯化后得到泛耐药PA广谱噬菌体裂解酶。取泛耐药PA菌株,将其均匀涂抹至2个培养基,分别滴加5、10μL泛耐药PA菌株广谱噬菌体裂解酶,培养24 h时观察培养基噬菌斑生长情况。结果得到泛耐药PA菌株广谱噬菌体EPA19及EPA19裂解酶lysEPA19。5、10μL的lysEPA19与泛耐药PA菌株混合培养后,培养基上均可见噬菌斑(5μL LysEPA19噬菌斑为11~13mm,10μL LysEPA19噬菌斑为23~27 mm)。结论成功筛选得到泛耐药PA广谱噬菌体裂解酶lysEPA19,可有效抑制泛耐药PA菌株生长。Objective To obtain a broad-spectrum phage lyase of pandrug-resistant pseudomonas aeruginosa(PA)and to observe its antibacterial activity.Methods①237 PA strains were selected and 84 pandrug-resistant PA strains were screened by drug sensitivity test.By using the method of plaque determination,10 strains of pandrug-resistant PA were mixed with sewage,9 strains of pandrug-resistant PA were obtained,and 84 strains of pandrug-resistant PA were used to screen out more than 90%of broad-spectrum phages of pandrug-resistant PA strains.②The broad-spectrum phage lyase gene of pandrug-resistant PA strain was obtained by Proteinase K cleavage method and sequenced,and the sequence was confirmed by bioinformatics tools(ORF finder and BLAST)to obtain the gene sequence(target sequence)of the broad-spectrum phage lyase gene of pandrug-resistant PA strain.The target sequence was cloned,transformed,induced and purified to obtain a wide spectrum phage lyase of pandrug-resistant PA.Pandrug-resistant PA strains were evenly plat⁃ed into two medium,5 and 10μL of phage lyase solution were added into the agar medium,respectively.The growth of phage plaque in the medium was observed after 24 h.Results The broad-spectrum phage EPA19 and its lysEPA19 lyase were obtained.After 5 and 10μL lysEPA19 were mixed with pandrug-resistant PA strains,plaque was visible on the medi⁃um(5μL LysEPA19 plaque was 11-13 mm,10μL LysEPA19 plaque was 23-27mm).Conclusion The phage lysE⁃PA19 is successfully screened out,which can effectively inhibit the growth of pandrug-resistant PA strains.

关 键 词：噬菌体裂解酶 噬菌体 铜绿假单胞菌 泛耐药铜绿假单胞菌 

分 类 号：R372[医药卫生—病原生物学]