噬菌体裂解酶Lysin1902原核表达及其与ε-聚赖氨酸联用效果评价  

Phage endolysin Lysin1902:prokaryotic expression and evaluation of its bactericidal activity combined withε-poly-lysine

作　　者：王瑜欣 李静[1] 付开来 吴珮榕 陈鑫茹 薛峰[1] 汤芳[1] 戴建君[1,2] WANG Yuxin;LI Jing;FU Kailai;WU Peirong;CHEN Xinru;XUE Feng;TANG Fang;DAI Jianjun(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China;School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,Jiangsu,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《微生物学报》2023年第2期834-844,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32172858)。

摘　　要：【目的】研究裂解酶Lysin1902与ε-聚赖氨酸(ε-PL)对大肠杆菌O157:H7的协同抗菌作用。【方法】通过Mega构建进化树、使用在线工具等分析大肠杆菌噬菌体裂解酶Lysin1902氨基酸序列组成和结构等;原核表达并纯化Lysin1902;通过平板裂解实验检测Lysin1902对大肠杆菌O157:H7灭活菌株的裂解活性;用96孔板法检测Lysin1902或ε-PL的活菌裂解能力;棋盘法验证Lysin1902和ε-PL联用效果。【结果】体外成功表达并纯化了Lysin1902。Lysin1902对大肠杆菌O157:H7灭活菌株具有裂解活性,但不能有效裂解活的大肠杆菌。噬菌体裂解酶与ε-PL联用结果表明,加入Lysin1902后,ε-PL能够完全控制大肠杆菌O157:H7增殖的使用浓度由0.7mg/mL降低到0.1 mg/mL。【结论】体外原核表达并纯化Lysin1902,其单独使用对活的大肠杆菌O157:H7无裂解活性,但与ε-PL联用可显著提高ε-PL对大肠杆菌O157:H7的裂解能力。[Objective]To investigate the antibacterial effect of the phage endolysin Lysin1902combined withε-poly-lysine(ε-PL)on Escherichia coli O157:H7.[Methods]We employed Mega to construct the phylogenetic tree and used online tools to analyze the amino acid sequence of Lysin1902.After prokaryotic expression,Lysin1902 was purified.We then detected the lytic activity of Lysin1902 against autoclaved E.coli O157:H7 by plate lysis assay and detected the lysis activity of Lysin1902 orε-PL on live E.coli O157:H7 in 96-well plates.Further,we adopted checkerboard assay to measure the bactericidal activity of Lysin1902combined withε-PL.[Results]We successfully expressed and purified Lysin1902.Lysin1902showed lytic activity against autoclaved E.coli O157:H7 but cannot effectively lyse live E.coli strains.After the addition of Lysin1902,the concentration ofε-PL that could completely control the proliferation of E.coli O157:H7 decreased from 0.7 mg/mL to 0.1 mg/mL.[Conclusion]Lysin1902 was successfully expressed and purified.Lysin1902 alone has no lytic activity against E.coli O157:H7,while the combined use of Lysin1902 withε-PL significantly increased the lytic activity ofε-PL against E.coli O157:H7.

关 键 词：裂解酶Lysin1902 Ε-聚赖氨酸 联用 大肠杆菌O157:H7 

分 类 号：Q78[生物学—分子生物学]