含有CRISPR序列的噬菌体耐受菌的筛选  

Screen for the phage-resistant bacteria containing CRISPR sequences

作　　者：滑玉会[1,2] 黄勇[2] 张志毅[2] 安小平[2] 米志强[2] 尹秀云[3] 陈建魁[3] 童贻刚[1,2] 

机构地区：[1]安徽医科大学,合肥 230032 [2]军事医学科学院微生物流行病研究所、病原微生物生物安全国家重点实验室,北京 100071 [3] 军事医学科学院附属医院临床检验科,北京100071

出　　处：《安徽医科大学学报》2014年第6期833-836,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家高技术研究发展计划“863计划”(编号:2012AA022003);“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项“十二五”实施计划(编号:2013ZX10004-605、2013ZX10004-217、2013ZX10004-607、2011ZX10004-001);“重大新药创制”科技重大专项“十二五”实施计划(编号:2011ZX09401-023);国家自然科学基金(编号:81072350);病原微生物生物安全国家重点实验室开放课题(编号:SKLPBS1113)

摘　　要：人工设计合成规律成簇的间隔短回文重复(CRISPR)序列特异性引物,将从临床分离的大肠埃希菌株作为模板,采用PCR扩增方法筛选含有CRISPR系统的大肠埃希菌;利用噬菌斑法从医院未经消毒处理的污水中分离大肠埃希菌噬菌体,经过PEG沉淀的方法浓缩得到高滴度的噬菌体,再用该筛选噬菌体感染上述含有CRISPR的大肠埃希菌以筛选耐受该噬菌体的大肠埃希菌菌株。最后从70株大肠埃希菌中筛选到42株含有CRISPR序列的菌株,然后采用噬菌斑法分离到1株大肠埃希菌E.coli 147-30的裂解性噬菌体IME-EC1,在此基础上利用E.coli 147-30筛选到1株耐受噬菌体IME-EC1的大肠埃希菌菌株,命名为E.coli 147-30R1。By using PCR method with primers specific to CRISPR sequences, clinical isolates of pathogenic Esche-richia coli strains were screened for the presence of CRISPR system. The resultant CRISPR E. coli strain was used for screening phages from hospital sewage by plaque methods. The purified phage through PEG precipitation was then used to infect the original CRISPR E. coli strain for screening of the phage-resistant E. coli strains. As a result, 42 pathogenic E. coli strains containing CRISPR were identified from 70 strains and one lytic phage IME-EC1 was i-solated. By infecting E. coli 147-30 with phage IME-EC1, a phage-resistant E. coli strain designated as 147-30R1 was identified.

关 键 词：CRISPR系统 大肠埃希菌 噬菌体 噬菌体耐受 

分 类 号：R378.21[医药卫生—病原生物学]