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当前位置: 首页 > 文章 > 禽致病性大肠杆菌噬菌体vB_EcoM_JS09裂解酶的表达及其活性分析 中国兽医科学 2014 (11) 1109-1113
Position: Home > Articles > Expression and antibacterial activity of lysin of avian pathogenic Escherichia coli phage vB_EcoM_JS09 Chinese Veterinary Science 2014 (11) 1109-1113

禽致病性大肠杆菌噬菌体vB_EcoM_JS09裂解酶的表达及其活性分析

作  者：
周艳;包红朵;张辉;张莉莉;王冉
单  位：
江苏省农业科学院食品质量安全与检测研究所江苏省畜禽产品安全性研究重点实验室
关键词：
革兰阴性菌;禽致病性大肠杆菌噬菌体;噬菌体裂解酶
摘  要：
为研究禽致病性大肠杆菌噬菌体vB_EcoM_JS09编码的裂解酶对宿主大肠杆菌的裂解作用,克隆表达了禽致病性大肠杆菌噬菌体vB_EcoM_JS09的裂解酶,并检测了裂解酶的菌体内外裂解活性。利用PCR扩增了禽致病性大肠杆菌噬菌体vB_EcoM_JS09裂解酶基因(Lysin基因),构建了重组表达质粒pET-32a(+)-LysJS09,测序验证后转化至大肠杆菌BL21(DE3)进行IPTG诱导表达。在37℃IPTG浓度为1.0mmol/L诱导4h表达效果最好。重组融合蛋白LysJS09在BL21(DE3)中获得了高表达,在N端具有His标签,其分子质量约为38.9ku。重组融合蛋白以分泌型方式表达,经His-trap HP(GE)亲和层析柱纯化后获得的重组蛋白LysJS09的纯度为97%。裂解活性试验表明,该裂解酶单独使用和加入EDTA(0.1mol/L)均无体外酶活性;但菌体内裂解试验表明,表达的裂解酶对宿主菌具有一定的裂解作用。
译  名：
Expression and antibacterial activity of lysin of avian pathogenic Escherichia coli phage vB_EcoM_JS09
作  者：
ZHOU Yan;BAO Hong-duo;ZHANG Hui;ZHANG Li-li;WANG Ran;Key Laboratory of Animal-derived Food Safety of Jiangsu Province/Jiangsu Academy of Agricultural Sciences;
关键词：
Gram-negative bacteria;;avian pathogenic Escherichia coli phage;;phage lysin
摘  要：
In order to investigate the lytic activity of avian pathogenic Escherichia coli phage vB_EcoM_JS09lysin on its host avian pathogenic E.coli,LysJS09,lysin of JS09 phage was successfully cloned and expressed,and the antibacterial activity of LysJS09 was analyzed in vitro and in vivo.The complete lysin gene(Lysin gene)of JS09 phage was amplified by PCR,and the targeted fragment was cloned into expression E.coli vector pET-32a(+).Recombinant vector pET-32a(+)-LysJS09 was transformed into BL21(DE3),then expressed and induced with IPTG.The results showed that 1.0mmol/L IPTG and 4hof induction time were optimal conditions for expression.And exogenous protein with His tags in the N terminal was highly expressed in BL21(DE3),with a molecular weight of about 38.9ku.The recombinant fusion protein expressed in the secretory form,after purification with His-trap HP(GE)affinity chromatography column,the recombinant LysJS09 with more than 97%of purity was obtained.Lytic activity test indicated that the recombinant LysJS09 used alone or with EDTA(0.1mol/L)had no lytic enzyme activity in vitro,but in vivo bacteria lysis test showed that the recombinant LysJS09 had a lytic effect on the host bacteria.

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