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当前位置: 首页 > 文章 > 弓形虫MORN2基因敲除株的构建及其表型鉴定 中国畜牧兽医 2018 (9) 2386-2393
Position: Home > Articles > Construction and Phenotype Identification of Toxoplasma gondii MORN2 Gene Knockout Strain China Animal Husbandry & Veterinary Medicine 2018 (9) 2386-2393

弓形虫MORN2基因敲除株的构建及其表型鉴定

作  者：
陈凯;王金磊;白梦捷;杨文彬;黄思扬
单  位：
西北农林科技大学动物医学院;中国农业科学院兰州兽医研究所;家畜疫病病原生物学国家重点实验室
关键词：
弓形虫;CRISPR/Cas9;MORN2基因;基因敲除
摘  要：
本研究旨在构建弓形虫(Toxoplasma gondii)Ⅰ型RH虫株的膜占位与识别联结蛋白2(membrane occupation and recognition nexus protein,MORN2)基因敲除株,探究MORN2基因的表型功能。利用CRISPR/Cas9技术在sgRNA设计网站筛选出以MORN2基因为靶标的sgRNA并设计引物,以pSAG1∷CAS9-U6∷sgUPRT质粒为模板,在Q5酶作用下构建出MORN2靶向CRISPR的质粒;在MORN2靶点两翼约500bp设计同源臂引物,以pUPRT∷DHFR-D质粒为模板构建含有乙胺嘧啶药物抗性基因(DHFR)的同源片段;将CRISPR质粒与同源片段电转染到弓形虫速殖子中,进行乙胺嘧啶药物筛选、96孔板单克隆筛选及PCR鉴定。结果显示,试验成功获得MORN2基因敲除株,在体外噬斑试验中,该敲除株的生长速度与野生型RH虫株基本一致;在体内感染试验中,分别以100和1 000个敲除株的速殖子腹腔注射小鼠,小鼠的死亡时间与对照组基本一致,说明MORN2基因的缺失几乎不影响RH虫株在体外培养时的生长速度和感染小鼠的毒力。本研究证明了MORN2基因在RH虫株中无表型功能,为进一步探究弓形虫的功能基因奠定了基础。
译  名：
Construction and Phenotype Identification of Toxoplasma gondii MORN2 Gene Knockout Strain
作  者：
CHEN Kai;WANG Jinlei;BAI Mengjie;YANG Wenbin;HUANG Siyang;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;State Key Laboratory of Veterinary Etiological Biology;College of Veterinary Medicine,Northwest A & F University;
关键词：
Toxoplasma gondii;;CRISPR/Cas9;;MORN2 gene;;gene knockout
摘  要：
This study was aimed to construct membrane occupation and recognition nexus protein2(MORN2)gene knockout strain of Toxoplasma gondii type Ⅰ RH strain,and evaluate the functions of MORN2 gene.The sgRNA target of MORN2 gene was screened to design primers of sgRNA using CRISPR/Cas9 technology.MORN2 CRISPR-targeted plasmid(sgMORN2)was constructed using pSAG1∷CAS9-U6∷sgUPRT as a template under the effect of Q5 enzyme.One pair of homologous primers was designed according to the sgMORN2 target sequence to amplify a DHFR fragment with pUPRT∷DHFR-D plasmid as a template.The MORN2-CRISPR plasmid and the homologous DHFR fragment were cotransfected into Toxoplasma gondii tachyzoites to perform pyrimethamine screening,monoclonal selection in 96 well plates and PCR identification.The results showed that the MORN2 knockout strain(ΔMORN2)was successfully created.Theplaque assays showed that the knockout strain had the same growth rate as the wild strainin vitro plaque assay.Mice were injected intraperitoneally with tachyzoites of 100 and 1 000 knockout strains in vivo infection test,and the death time of mice was basically the same as that of control group.These results indicated MORN2 gene knockout had no effect on the growth rate of Toxoplasma gondii RH strain in vitro and the virulence of mouse infection.This study demonstrated that MORN2 gene had no phenotypic function in RH strain,which laid the foundation for further exploration of the functional genes of Toxoplasma gondii.

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