摘要
为研究趋化性基因cheZ对大肠杆菌益生菌Nissle1917的影响,研究以敲除大肠杆菌Nissle1917的cheZ基因为目的.首先,利用诺唯赞ClonExpress®MultiS一步克隆试剂盒构建pTargetTΔcheZ重组质粒,随后将pTargetTΔcheZ重组质粒电转入含pCas质粒的Nissle1917感受态中,以实现对大肠杆菌益生菌Nissle1917染色体中cheZ基因进行靶向敲除,经PCR鉴定和基因测序双重证实,Nissle1917基因组中的cheZ基因已敲除.最后,经IPTG诱导和高温培养,消除缺失株中的pTargetTΔcheZ和pCas2个质粒,最终获得趋化性基因cheZ缺失株Nissle1917ΔcheZ.本试验为验证cheZ基因在益生菌Nissle1917中的生物活性及其扮演的基因功能的后续相关研究打下了前期基础.
In order to study the effect of chemotaxis gene cheZ on Escherichia coli probiotic Nissle1917,the purpose of this study is to knock out the cheZ gene of Escherichia coli Nissle1917.First,the pTargetTAcheZ re-combinant plasmid was constructed by Novozan ClonExpress® MultiS one-step cloning kit,and then the pTar-getTAcheZ recombinant plasmid was electroporated into Nissle1917 competent cells containing pCas plasmid to achieve targeted knockout of the cheZ gene in the chromosome of E.coli probiotic Nissle1917.PCR identifica-tion and gene sequencing confirmed that the cheZ gene in the Nissle1917 genome had been knocked out.Finally,the two plasmids pTargetT△cheZ and pCas in the deletion strain were eliminated by IPTG induction and high temperature culture,and as a result the chemotaxis gene cheZ deletion strain Nissle1917AcheZ was obtained.This experiment is to verify the biological activity of the cheZ gene in the probiotic Nissle1917 and a preliminary foundation has been laid for the follow-up related research on the role of gene function.
作者
区炳明
林鑫
吕海慧
葛桦
朱惠敏
黄丽华
刘文华
李赛男
张敏瑜
OU Bingmingh;LIN Xin;LV Haihuil;GE Hua;ZHU Huimin;HUANG Lihua;LIU Wenhua;LI Sainan;ZHANG Minyu(School of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526061,China;School of Physical Education&Sports Science,South China Normal University,Guangzhou,Guangdong 510006,China)
出处
《肇庆学院学报》
2023年第2期16-22,共7页
Journal of Zhaoqing University
基金
国家自然科学基金项目(32102703)
广东省基础与应用基础联合基金项目(2019A1515111186)
全国大学生平台创新和创业培训计划项目(202110580008)
广东省大学生创新创业训练计划项目(S202010580051).
