原核重组表达的鲍曼不动杆菌噬菌体裂解酶LysZHSHW的表征与抗菌活性  

Characterization and antibacterial activity of prokaryotic recombinantly expressed phage lysin LysZHSHW of Acinetobacter baumannii

作　　者：林玉倩 刘俊林 邓嘉丽 黄静琳 石哲铭 冯枫琳 孙永学 LIN Yuqian;LIU Junlin;DENG Jiali;HUANG Jinglin;SHI Zheming;FENG Fenglin;SUN Yongxue(Guangdong Provincial Key Laboratory for Veterinary Pharmaceutics Development and Safety Evaluation/National Laboratory of Veterinary Drug Safety Evaluation(Environment Estimation),College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院国家兽药安全评价(环境评估)实验室/广东省兽药研制与安全评价重点实验室,广东广州510642

出　　处：《中国兽医学报》2024年第9期1965-1975,共11页Chinese Journal of Veterinary Science

基　　金：广东省自然科学基金资助项目(2023A1515011239)。

摘　　要：为评估能对抗鲍曼不动杆菌感染的噬菌体裂解酶LysZHSHW的体内外抗菌潜力和研究其特征。以表达质粒pET28a为骨架,使用PCR和酶切连接方法构建含有裂解酶LysZHSHW编码基因的pET28a-Lys重组质粒并在E.coli BL21(DE3)表达。通过Western blot方法验证裂解酶LysZHSHW的表达后,对其进行表征和体内外应用潜力的评估。pET28a-Lys重组质粒构建成功,LysZHSHW蛋白表达正确。经纯化的酶的质量浓度为4086 mg/L,可用于后续试验。LysZHSHW的酶活性为630 U/μg,其在25℃,pH值为9.0时活性最强。在体外,1000或750 mg/L的酶在EDTA的协助下能使细菌数量下降4.0个数量级,且无细胞毒性;在体内,酶联合EDTA作用,2.5μg的酶能使大蜡螟感染模型24 h后的存活率达92.86%,0.15 mg的酶能使小鼠大腿载菌量下降2.8个数量级,并减轻肌纤维间的炎性反应。由此,鲍曼不动杆菌噬菌体来源的LysZHSHW具有常温、弱碱、安全的特点,其作为新型抗菌剂具有应用前景。The aim of this study is to evaluate the in vitro and in vivo antibacterial potential of LysZHSHW,a phage lysin against Acinetobacter baumannii infections,and to study its characteristics.The pET28a-Lys recombinant plasmid containing LysZHSHW coding gene was constructed by PCR,enzyme digestion and ligation using the expression plasmid pET28a as the backbone and expressed in E.coli BL21(DE3).After confirming the expression of the LysZHSHW through Western blot analysis,its characterization and potential applications were assessed both in vitro and in vivo.The results showed that the pET28a-Lys recombinant plasmid was successfully constructed and the LysZHSHW protein was expressed correctly.The mass concentration of the purified enzyme was 4086 mg/L,which could be used for subsequent experiments.The enzymatic activity of LysZHSHW was determined to be 630 U/μg,with maximum activity observed at 25℃and pH9.0.In vitro experiments demonstrated that 1000 or 750 mg/L of LysZHSHW,in the presence of EDTA,resulted in a four-log reduction in bacterial counts without any cytotoxicity.In vivo,2.5μg of LysZHSHW combined with EDTA could increase the survival rate of Galleria mellonella larvae infection model to 92.86%after 24 hours,and 0.15 mg of LysZHSHW reduced the bacterial load in the thighs of mice by 2.8 logs and alleviated the inflammatory response in muscle fibers.In conclusion,LysZHSHW derived from Acinetobacter baumannii bacteriophage exhibited characteristics such as stability at room temperature,alkaline pH,and safety,making it a promising candidate as a novel antimicrobial agent.

关 键 词：鲍曼不动杆菌 裂解酶 异源表达 酶活性 抗菌活性 

分 类 号：Q786[生物学—分子生物学]