贵州医科大学学报

2019, v.44;No.226(07) 751-756

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艰难梭菌sigB基因簇CRISPR-Cas9敲除载体的构建及验证
Construction of CRISPR-Cas9 Knockout Vectors against sig B Gene Cluster of Clostridium Difficile

陈相好;蔡梦迪;陈峥宏;谷俊莹;文学琴;洪伟;崔古贞;
CHEN Xianghao;CAI Mengdi;CHEN Zhenghong;GU Junying;WEN Xueqin;HONG Wei;CUI Guzhen;Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou;School of Basic Medical Science,Guizhou Medical University;School of Clinical Laboratory Science,Guizhou Medical University;Key Laboratory of Molecular Biology of Guizhou Province;

摘要(Abstract):

目的:构建艰难梭菌rsbV(编码anti-anti-sigma因子)、rsbW(编码anti-sigma因子)及sigB基因(编码sigma-B因子)的CRISPR-Cas9敲除载体。方法:利用重叠延伸PCR技术分别扩增针对艰难梭菌rsbV、rsbW及sigB基因的gRNA序列及同源臂序列,并分别将其克隆到pJZ23载体上,采用菌落PCR结合DNA测序验证所构建的基因敲除载体。结果:扩增获得艰难梭菌rsbV、rsbW、sigB基因的gRNA序列及同源臂序列,菌落PCR及测序均表明成功构建了艰难梭菌rsbV、rsbW、sigB基因的敲除载体。结论:成功构建了艰难梭菌rsbV、rsbW、sigB基因CRISPR-Cas9敲除载体。
Objective:To construct CRISPR-Cas9 vectors to knockout sigB gene cluster including rsbV(encoding anti-anti-sigma factor),rsbW(encoding anti-sigma factor) and sigB gene(encoding sigma-B factor) of Clostridium difficile(C.difficile).Methods:The gRNA sequences against rsbV,rsbW and sigB genes and homologous arm sequences of C.difficile were amplified by overlap extension PCR,and cloned into pJZ23 vector of CRISPR-Cas9 system,respectively.The recombinant pJZ23 vectors were verified by colony PCR and DNA sequencing.Results:The gRNA sequences against rsbV,rsbW and sigB genes and homologous arm sequences of C.difficile were amplified.Colony PCR and DNA sequencing showed that rsbV,rsbW and sigB genes were correctly amplified and successfully cloned into pJZ23 vector.Conclusion:The CRISPR-Cas9 knockout vectors against rsbV,rsbW and sigB genes of C.difficile was successfully constructed.

关键词(KeyWords): 艰难梭菌;CRISPR-Cas9;sigma-B因子;rsbW;rsbV
Clostridium difficile;CRISPR-Cas9;sigma-B factor;rsbW;rsbV

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31760318,31500078,31601012);; 贵州省科技计划项目[黔科合基础(2018)1132,2019(1441)];; 贵州医科大学培育项目[黔科合平台人才(2018)5779-17];; 贵州省大学生创新创业训练计划项目(201710660022)

作者(Author): 陈相好;蔡梦迪;陈峥宏;谷俊莹;文学琴;洪伟;崔古贞;
CHEN Xianghao;CAI Mengdi;CHEN Zhenghong;GU Junying;WEN Xueqin;HONG Wei;CUI Guzhen;Key Laboratory of Medical Microbiology and Parasitology of Education Department of Guizhou;School of Basic Medical Science,Guizhou Medical University;School of Clinical Laboratory Science,Guizhou Medical University;Key Laboratory of Molecular Biology of Guizhou Province;

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DOI: 10.19367/j.cnki.1000-2707.2019.07.002

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