一株大肠埃希菌烈性噬菌体ΦTRI-1的分离及特性分析  

Isolation and Characteristic Analysis of E. coli Lytic Phage ΦTRI-1

作　　者：高苗[1] 杨金广[1] 孙航军 刘旭[1] 刘伟[1] 王凤龙[1] 

机构地区：[1]中国农业科学院烟草研究所/烟草行业烟草病虫害监测与综合治理重点实验室,青岛266101

出　　处：《吉林农业大学学报》2015年第5期535-542,共8页Journal of Jilin Agricultural University

基　　金：国家烟草专卖局科技项目(110200902065);云南省烟草公司科技项目(2013YN37);湖北省烟草公司科技项目(027Y2013-006)

摘　　要：通过在E.coli菌液中加入土壤悬浮液共培养获得噬菌体,利用双层平板法反复纯化4次后得到单一清晰的噬菌斑。将单个噬菌斑加入对数早期的E.coli菌液中增殖培养后利用核酸抽提试剂盒提取总核酸,并分别用DNaseⅠ和RNase A对总核酸进行消化,利用限制性内切酶HindⅢ和EcoRⅠ对其进行酶切;增殖后的噬菌体液进行噬菌体颗粒浓缩,取浓缩液负染后置于电子显微镜下确定噬菌体的形态特征,并进行SDSPAGE电泳分析,确定其蛋白条带;同时测定其他生物学特性,包括滴度、最佳感染复数、一步生长曲线以及其对温度、pH、紫外线、氯仿的敏感性等。结果表明：分离并纯化出1株E.coli烈性噬菌体,命名为ΦTRI-1,形态特征：头部为二十面体的立体对称,直径约为60 nm,有一长尾,长约200 nm;其核酸属于双链DNA,按照国际病毒分类委员会分类标准,其属于有尾噬菌体目（Caudovirales）,长尾噬菌体科（Siphoviridae）的烈性噬菌体。生物学特性测定结果显示ΦTRI-1对E.coli的最佳感染复数为0.1,潜伏期约为20 min,爆发期约为60 min,裂解量约为38;ΦTRI-1在40℃时活性最高,在20-70℃活性均较强,80℃时活性丧失;其酸碱耐受力较强,在偏碱环境下活性相对更高;对紫外线有一定的耐受能力;对氯仿敏感,浓度5%的氯仿即可致其失活。分离到的噬菌体属于有尾噬菌体目,长尾噬菌体科,综合其各项生物学特性可知其潜伏期短,裂解能力强,环境适应能力强,具有很好的杀菌效果,具有开发为抗E.coli菌剂的潜力。Phages were screened by co-cultivating of E. coli bacteria liquid and suspension of soil,and a single clear plaque was obtained after 4-time purification using two layer plating method. The single plaque was added into logarithmic phase E. coli bacteria liquid to cultivate phage culture.The total nucleic acid of the phage was extracted using the Phage DNA extraction kit,and digested with DNase I,RNase A,Hind Ⅲ and EcoR Ⅰ. At the same time,morphological properties of the phage were determined with electron microscope after phage particles concentrated. Coat proteins of phage particles were analyzed by SDS- PAGE. And other biological properties including titer,MOI,one-step growth curve,and sensitivity to temperature,pH,ultraviolet ray and chloroform were determined. A strain of lytic E. coli phages named ΦTRI-1 was isolated and purified. The electron microscope results showed that phage particles had icosahedra head with 60 nm in diameter,and a long tail about 200 nm and nucleic acid digestion showed it was a double-stranded DNA. According to classification standards of international commission on virus classification,it belonged to the order of Cuadovirales,the family of Siphoviridae. Determination of biological properties showed that multiplicity of infection（ MOI） of the Escherichia coli phage was 0. 1 and its growth curve showed that incubation period was 20 min,time of burst phase was 60 min,and burst size was 38. Activity of E. coli phage was higher between 20 ℃ and 70 ℃ and reached the maximum when temperature was40 ℃,but when temperature was 80 ℃ the phage was inactivated. And it had strong receivable ability of pH changes and relatively higher activity in the alkali environment. The E. coli phage was not particularly sensitive to ultraviolet ray,but sensitive to chloroform,and 5% concentration of chloroform could cause inactivation. The E. coli phage isolated in this experiment belonged to the order of Cuadovirales,the family of Siphoviridae,and it had a shorter incubation period,a stronge

关 键 词：ESCHERICHIA COLI 噬菌体 分离 烈性 生物学特性 

分 类 号：R378.21[医药卫生—病原生物学]