高毒肺炎克雷伯K1血清型菌株噬菌体解聚酶的表达纯化及功能验证  

Expression,purification and functional validation of phage depolymerase from hypervirulent Klebsiella pneumoniae serotype K1

作　　者：范政 陈雨晨 刘洪伯 崔晓虎 李周霏 付彤彤 袁静[1] Fan Zheng;Chen Yuchen;Liu Hongbo;Cui Xiaohu;Li Zhoufei;Fu Tongtong;Yuan Jing(Department of Bacteriology,Capital Institute of Pediatrics,Beijing 100020,China;Graduate School of Peking Union Medical College,Beijing 100730,China)

机构地区：[1]首都儿科研究所细菌学研究室,北京100020 [2]北京协和医学院研究生院,北京100730

出　　处：《中华预防医学杂志》2024年第9期1348-1353,共6页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金(32200159)。

摘　　要：目的表达纯化高毒肺炎克雷伯K1血清型菌株噬菌体的解聚酶并对其进行功能验证。方法从医院废水中分离高毒肺炎克雷伯K1血清型菌株的噬菌体。通过观察噬菌斑、透射电镜等技术明确噬菌体的生物学与形态学特征。借助HiSeq 2500高通量测序平台对噬菌体进行全基因组测序。通过观察噬菌斑的晕圈初步判断解聚酶的存在。进一步利用生物信息学分析工具和原核蛋白表达系统预测并鉴定噬菌体的解聚酶。通过PCR获取解聚酶基因片段并克隆到pET28a表达载体,在菌株BL21中完成解聚酶的表达纯化。通过滴板及低速离心法检测解聚酶对K1血清型高毒肺炎克雷伯菌荚膜多糖的裂解活性。结果从医院废水中分离到一株能够特异性杀伤K1血清型高毒肺炎克雷伯菌株的裂解性噬菌体phiA2。噬菌体phiA2属于有尾噬菌体目,短尾噬菌体科,全基因组长度为43526 bp并包含51个编码域序列。噬菌体phiA2所包含的解聚酶phiA2-dep被预测并表达纯化。滴板实验与低速离心实验均表明解聚酶phiA2-dep能够特异性降解多株K1血清型高毒肺炎克雷伯菌的荚膜多糖。结论解聚酶phiA2-dep能够特异性降解K1血清型高毒肺炎克雷伯菌的荚膜多糖,在治疗细菌感染方面具有潜在应用价值。Objective To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae(hv Kp)serotype K1 and validate its function.Methods Phage that infected serotype K1-type hv Kp was isolated from hospital sewage.The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy.The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform.The presence of depolymerase was determined by observing the plaque halo.Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase.The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector,and the expression and purification of the depolymerase were completed in strain BL21.The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay.Results A lytic phage(phiA2)that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage.It was typical of the Caudovirales order and Autographiviridae family,and its whole genome was 43526 bp in length and contained 51 coding domain sequences.The phage phiA2-derived depolymerase phiA2-dep was predicted,expressed and purified.The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates.Conclusion Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp,which has potential application value in treating bacterial infection.

关 键 词：高毒力肺炎克雷伯菌 K1血清型 解聚酶 荚膜 噬菌体 

分 类 号：R378[医药卫生—病原生物学]