类志贺邻单胞菌噬菌体ФP4-7裂解酶Gp2的表达及活性测定  

Characterization and Activity Determination of Gp2 Endolysin from Plesiomonas shigelloides Phage ФP4-7

作　　者：何洋[1] 荆兆元 杨洪江[1] 

机构地区：[1]工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津科技大学生物工程学院,天津300457

出　　处：《天津科技大学学报》2017年第3期16-22,共7页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金资助项目(31370205,30970114)

摘　　要：多重耐药菌株的出现给临床治疗带来了困难,噬菌体编码的裂解酶能够杀死病原菌,是潜在的重要杀菌因子.本实验采用PCR技术,扩增类志贺邻单胞菌(Plesiomonas shigelloides)噬菌体ФP4-7裂解酶gp2基因并克隆至质粒p QE30上进行表达,重组蛋白采用Ni-NTA亲和层析法进行纯化.重组裂解酶Gp2最适反应p H为9;裂解酶40,℃保温15,min,酶活剩余56.4%,.底物专一性实验结果显示,该裂解酶能够高效裂解5种革兰氏阴性菌,即铜绿假单胞菌(Pseudomonas aeruginosa)、大肠杆菌(Escherichia coli)、粘质沙雷氏菌(Serratia marcescens)、摩氏摩根菌(Morganella morganii)、弗氏柠檬酸杆菌(Citrobacter freundii),以及3种革兰氏阳性菌,即金黄色葡萄球菌(Staphylococcus aureus)、枯草芽胞杆菌(Bacillus subtilis)、地衣芽胞杆菌(Bacillus licheniformis).杀菌实验结果表明,裂解酶Gp2可与EDTA、LAB-35以及SDS配合使用,进一步提高该酶对铜绿假单胞菌和枯草芽胞杆菌的杀菌活性.裂解酶Gp2具有高效广谱裂菌活性,具有潜在的应用价值.The emergence of multi-drug resistant pathogens has brought difficulties to clinical treatments. The endolysin encoded phage could lyse pathogenic microorganisms, and as an important bactericidal agent, it is a potential alternative to antibiotics. In this study,gp2 gene of Plesiomonas shigelloides phage ΦP4-7 was amplified and cloned into pQE30 plas- mid. The recombined protein was purified by Ni-NTA agarose resin. The optimal acidity of recombined endolysin was pH 9. After treatment at 40 ℃ for 15 min, it retained approximately 56.4% of its lyric activity. The GP2 endolysin could cleave 5 G-strains（Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Morganella morganii, Citrobacter freundii） and 3 G＋strains （Staphylococcus aureus, Bacillus subtilis,Bacillus licheniformis）. In combination with EDTA, LAB35, or SDS, respectively, Gp2 displayed synergetic antimicrobial activities to both Pseudomonas aeruginosa and Bacil- lus subtilis. Our results indicate that Gp2 endolysin is effective in killing some pathogens and might be used as an antibacte- rial agent.

关 键 词：噬菌体裂解酶 类志贺邻单胞菌噬菌体ΦP4—7 蛋白表达 杀菌活性 

分 类 号：Q786[生物学—分子生物学]