敲除PaLoc毒力岛的无毒性艰难梭菌菌株的构建  

Knockout of PaLoc Toxicity Loci to Construct Non-toxic C. difficile Strain

作　　者：饶凤琴 程玉梅[2] 吴昌学[1] 王义 崔古贞 齐晓岚[1] 洪伟[1] RAO Fengqin;CHENG Yumei;WU Changxue;WANG Yi;CUI Guzhen;QI Xiaolan;HONG Wei(Key Laboratory of Endemic and Ethnic Diseases (Ministry of Education),Key Laboratory of Medical Molecular Biology, Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Critical Care Medicine,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Biosystems Engineering,Auburn University,Auburn 36849,Alabama,United States;School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学附院综合ICU,贵州贵阳550004 [3]美国奥本大学系统生物学系,美国奥本阿拉巴马36849 [4]贵州医科大学基础医学院,贵州贵阳550025

出　　处：《贵州医科大学学报》2019年第10期1128-1133,共6页Journal of Guizhou Medical University

基　　金：国家自然科学基金(31560318;31601012;31760318);贵州省科技计划项目[黔科合平台人才(2017)5652]

摘　　要：目的:使用CRISPR-Cfp1系统敲除艰难梭菌毒性(PaLoc)毒力基因岛,构建无毒性艰难梭菌菌株。方法:使用分子克隆方法,构建针对PaLoc毒力岛的基因打靶质粒pWH53;将pWH53质粒转化艰难梭菌630菌株后,诱导Cpf1蛋白表达,PCR筛选发生双交换的PaLoc敲除突变株,测序验证设计位点是否发生等位双交换。结果:PCR结果显示,获得的16株转化子中有8株为敲除PaLoc突变株,阳性率为50%;测序结果显示,获得的8株敲除PaLoc突变株均在设计位点发生了等位双交换。结论:成功构建艰难梭菌毒性毒力岛敲除PaLoc突变株。Objective:To construct a PaLoc loci deleted mutant of C.difficile based on the CRISPR-Cfp1 system and obtain a non-toxic C.difficile strain.Methods:The standard molecular cloning method was used to construct the gene targeting pWH53 against PaLoc loci(virulence island).After conjugation of the pWH53 plasmid into C.difficile 630 strain,Cpf1 protein expression was induced,and a double-exchanged PaLoc knockout mutant strain was screened by colony PCR,allelic double exchange at the design site was verified by sequencing.Results:The results of clony PCR showed that 8 of the 16 transformants were PaLoc knockout mutants with a positive rate of 50%.The sequencing results showed that theΔPaLoc mutant had an allelic double exchange at the design site.Conclusion:The C.difficileΔPaLoc mutant was successfully constructed.

关 键 词：艰难梭菌 CRISPR-Cpf1系统 PaLoc毒力岛 基因敲除 活菌疫苗 毒性基因 梭菌感染 载体 质粒 

分 类 号：R378.8[医药卫生—病原生物学]