工程菌的构建和表达-第三军医大学学报.DOC

鲍曼不动杆菌噬菌体AB3裂解酶基因的表达及其抗菌活性分析 张劼1，2，周丽蓉1，罗永艾1（400016 重庆重庆医科大学附属第一医院呼吸科）鲍曼不动杆菌以ET28a(＋为载体构建重组质粒，在大肠杆菌表达，金属离子合亲和层析法纯化重组蛋白Expression and antibacterial activity assay of bacteriophage AB3 endolysin against Acinetobacter baumannii Zhang Jie , Zhou lirong , Luo Yongai（Department of Respiratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China） [Abstract] Objective To construct the prokaryotic expression vector of the bacteriophage AB3 endolysin (LysAB3), and investigate the antibacterial activity of LysAB3 against Acinetobacter baumannii. Methods The LysAB3 gene was synthesized and inserted into the vector pET28a(+) to construct the recombinant expression plasmid (pET28a-LysAB3). After expression in E. coli BL21(DE3) and purification with Ni2+-NTA Sepharose, we determined the antibacterial activity, lysis spectrum, optimal pH and temperature of LysAB3. The degradation against peptidoglycans in the cell wall was analysed. Scanning elctron microscopy revealed the cells shape of Acinetobacter baumannii under their exposure to LysAB3. Results The gel diffusion assay showed that LysAB3 had bactericidal effects against 40 strains of Acinetobacter baumannii, and it was also able to degrade Acinetobacter baumannii peptidoglycan. Of LysAB3 effects, the optimal pH is 7, and the optimal temperature is 25℃. Under scanning elctron microscopy, LysAB3-treated Acinetobacter baumannii exhibited significant abnormalities, including deep roughening of the cell surface, the collapse of the cell structure and resulting cell debris. Conclusions Bacteriophage AB3 endolysin (LysAB3) has significant antibacterial activity to Acinetobacter baumannii . [Key Words] Bacteriophage ; Endolysin ; Acinetobacter baumannii ; Antibacterial activity Supported by the Scientific Project of Chongqing Municipal Health Bureau(2009-2-081,2011-2-334); Corresponding author: Luo Yongai, E-mail; luoyongai1942@163.com 随着感染耐鲍曼不动杆菌GenBank登录号KC311669），运用ORF finder软件进行开放阅读框（