饶凤琴;程玉梅;吴昌学;王义;崔古贞;齐晓岚;洪伟;
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;Department of Critical Care Medicine, the Affiliated Hospital of Guizhou Medical University;Department of Biosystems Engineering, Auburn University;School of Basic Medical Sciences, Guizhou Medical University;

• 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
• 2:贵州医科大学附院综合ICU
• 3:美国奥本大学系统生物学系
• 4:贵州医科大学基础医学院

摘要(Abstract)：

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 the ΔPaLoc mutant had an allelic double exchange at the design site. Conclusion: The C. difficile ΔPaLoc mutant was successfully constructed.

关键词(KeyWords)： 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
Clostridioides difficile;CRISPR-Cpf1 system;PaLoc loci;gene knockout;live vaccine;toxic gene;Clostridium difficile infection;vector;plasmid

Abstract:

Keywords:

基金项目(Foundation): 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]

作者(Authors): 饶凤琴;程玉梅;吴昌学;王义;崔古贞;齐晓岚;洪伟;
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;Department of Critical Care Medicine, the Affiliated Hospital of Guizhou Medical University;Department of Biosystems Engineering, Auburn University;School of Basic Medical Sciences, Guizhou Medical University;

DOI: 10.19367/j.cnki.1000-2707.2019.10.003

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