弓形虫MIC11基因敲除株的构建及鉴定  

Construction and identification of the knock-out strains of the MIC11 gene of Toxoplasma gondii

作　　者：千慧燕 王浩[1,2] 杨冰祎 闵鹏飞 张爽 贾立军[1,2] QIAN Huiyan;WANG Hao;YANG Bingyi;MIN Pengfei;ZHANG Shuang;JIA Lijun(Engineering Research Center of North-East Cold Region Beef Cattle Science&Technology Innovation,Ministry of Education/Yanbian University,Yanji 133002,China;College of Agriculture of Yanbian University,Yanji 133002,China)

机构地区：[1]东北寒区肉牛科技创新教育部工程研究中心/延边大学,吉林延吉133002 [2]延边大学农学院,吉林延吉133002

出　　处：《畜牧与兽医》2021年第11期93-96,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31760729);吉林省中青年科技创新领军人才及团队项目(20200301034RQ);高等学校学科创新引智111计划资助(D20034);延边大学科研创新团队项目(延大校发[2018]203号)。

摘　　要：本试验利用CRISPR/Cas9技术构建弓形虫顶端复合体微线体蛋白11(MIC11)基因敲除虫株,通过乙胺嘧啶药物筛选、绿色荧光蛋白(GFP)观察及PCR技术进行体外和小鼠体内MIC11基因鉴定,确定单克隆敲除株KO-MIC11。结果表明,经乙胺嘧啶药物筛选和GFP确认,KO-MIC11株可在Vero细胞和小鼠体内稳定增殖,经PCR鉴定Vero细胞和接种后小鼠的心、肝、脾、肺、肾、脑及生殖器官中均能扩增出KO-MIC11株B1基因和GFP基因片段,但扩增不出MIC11基因片段。说明本试验成功构建并筛选出MIC11基因敲除的单克隆虫株,为弓形虫MIC11基因敲除株的致病性研究奠定了基础。In this study, the MIC11 gene knock-out strains of Toxoplasma gondii was constructed by the CRISPR/Cas9 technology. The MIC11 gene was identified by pyrimethamine screening, green fluorescent protein(GFP) observation and PCR in vitro and in vivo on mice to determine the stability of the MIC11 gene knock-out strains. The results showed that Ko-MIC11 strains, screened by pyrimethamine and confirmed by GFP, could proliferate normally in Vero cells and mice. PCR analysis showed that the B1 gene and GFP gene fragments of the Ko-MIC11 strains could be amplified from Vero cells and the heart, liver, spleen, lung, kidney, brain and reproductive organs of inoculated mice, while the MIC11 gene fragment could not. It suggested that the MIC11 gene knock-out monoclonal strains were successfully constructed and screened out here, laying a foundation for pathogenicity research on the Toxoplasma gondii MIC11 gene knock-out strains.

关 键 词：弓形虫 顶端复合体微线体蛋白11 绿色荧光蛋白 基因敲除 鉴定 

分 类 号：S852.7[农业科学—基础兽医学]