刚地弓形虫巨噬细胞迁移抑制因子基因敲除虫株的构建与鉴定  

Construction and identification of macrophage migration inhibitory factor gene knockout strain of Toxoplasma gondii

作　　者：王杰 温红阳 陈滢 安然[1,2,3,4] 罗庆礼 沈继龙 都建 WANG Jie;WEN Hong-yang;CHEN Ying;AN Ran;LUO Qing-li;SHEN Ji-long;DU Jian(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;The Provincial Key Laboratory of Zoonoses of High Institutions in Anhui,Hefei 230032,China;Research Center for Infectious Diseases,School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;The Key Laboratory of Microbiology and Parasitology of Anhui Province,Hefei 230032,China)

机构地区：[1]安徽医科大学基础医学院生化与分子生物学教研室,合肥230032 [2]人畜共患病安徽高校省级重点实验室,合肥230032 [3]安徽医科大学基础医学院感染性疾病研究中心,合肥230032 [4]病原生物学安徽省重点实验室,合肥230032

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第3期349-354,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82072300,81871674,81902084)。

摘　　要：目的构建并鉴定刚地弓形虫RH株巨噬细胞迁移抑制因子(Tgmif)基因敲除虫株。方法设计刚地弓形虫RH株Tgmif的单向导RNA(sgRNA)和点突变引物,将pSAG1::Cas9-U6::sgUPRT质粒定点突变为针对Tgmif基因的pSAG1::Cas9-U6::sg Tgmif质粒。构建含有Tgmif上游1001 bp(Tgmif-up)、二氢叶酸还原酶-胸苷酸合成酶基因(dhfr-ts)和Tgmif下游1011 bp(Tgmif-down)等3个片段的Tgmif供体质粒,从Tgmif供体质粒PCR扩增获得Tgmif供体DNA。将pSAG1::Cas9-U6::sg Tgmif质粒和Tgmif供体DNA混合后电击转染野生型刚地弓形虫RH株(RH_(WT))速殖子,用乙胺嘧啶培养7 d,筛选获得稳定表达乙胺嘧啶抗性的虫株并进行单克隆筛选,PCR扩增dhfr-ts上、下游同源臂和Tgmif鉴定Tgmif基因敲除单克隆株(RH_(ΔTgmif))。提取RH_(ΔTgmif)和RH_(WT)株虫体蛋白,蛋白质免疫印迹(Western blotting)分析TgMIF蛋白的表达情况。RH_(ΔTgmif)在人包皮成纤维(HFF)细胞中传代10代,吉氏染色,镜检计数100个HFF中速殖子数,评估RH_(ΔTgmif)在体外培养HFF细胞中的增殖能力,以RH_(WT)为对照。将20只BABL/c小鼠随机分为RH_(WT)组和RH_(ΔTgmif)组(10只/组),各组分别腹腔注射RH_(WT)或RH_(ΔTgmif)株速殖子(1000个/鼠),记录小鼠生存情况。弓形虫增殖能力的比较采用独立样本t检验,小鼠生存率的比较采用Log Rank检验。结果PCR检测结果显示,含Tgmif-up、dhfr-ts和Tgmif-down等3个片段的Tgmif供体DNA片段长5049 bp,与预期相符;RH_(ΔTgmif)株分别扩增出1387、1524 bp的dhfr-ts上、下游同源臂条带,RH_(WT)株无相应条带;RH_(WT)株扩增出1837 bp的Tgmif条带,RH_(ΔTgmif)株无相应条带。Western blotting分析结果显示,RH_(WT)株在相对分子质量(M_(r))12500处出现1条蛋白条带,RH_(ΔTgmif)株无相应的蛋白条带。吉氏染色镜检结果显示,100个HFF中RH_(ΔTgmif)株速殖子为(2986±69.20)个,高于RH_(WT)株的(2067±51.08)个(t=18.50,P<0.01)。体内毒力试验结果显示,RH_(WT)组小鼠在第7天出Objective To construct and validate a macrophage migration inhibitory factor (mif) gene knockout strain of Toxoplasma gondii RH strain.Methods Single guide RNA (sgRNA) and site-directed mutation primers the RH strain of T.gondii Tgmif gene were designed.The pSAG1::Cas9-U6::sgUPRT plasmid was mutated into the pSAG1::Cas9-U6::sg Tgmif plasmid targeting the Tgmif gene.A Tgmif donor plasmid containing 3fragments of 1 001 bp upstream of Tgmif (Tgmif-up),dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) and1 011 bp downstream of Tgmif (Tgmif-down) was constructed.The Tgmif donor DNA was amplified from the Tgmif donor plasmid by PCR.The pSAG1::Cas9-U6::sg Tgmif plasmid and Tgmif donor DNA were mixed and electroporated into the tachyzoites of wild-type T.gondii RH strain (RH_(WT)) and cultured with pyrimethamine for 7 days prior to screening to obtain the stable expression of pyrimethamine-resistant tachyzoites.The upper and lower homology arms of dhfr-ts and Tgmif were amplified by PCR to identify Tgmif knockout monoclonal strains (RH_(ΔTgmif)).The protein of RH_(ΔTgmif)and RH_(WT)tachyzoites was extracted and the expression of TgMIF protein was analyzed by Western blotting.RH_(ΔTgmif)was cultured in human foreskin fibroblasts (HFF) cells for 10 passages,and the number of tachyzoites per100 HFF cells was counted by Giemsa staining to evaluate the proliferation of RH_(ΔTgmif)in HFF cells in vitro.RH_(WT )was used as the control.Twenty BABL/c mice were randomly divided into RH_(WT)group and RH_(ΔTgmif)group (10 mice/group).The mice were injected intraperitoneally with tachyzoites of RH_(WT)or RH_(ΔTgmif)strains (1 000/mouse) for each group respectively.The survival of mice was recorded.Independent sample t-test was used to compare the proliferation of T.gondii,and Log Rank test was used to compare the survival rate of mice.Results PCR results showed that the Tgmif donor DNA fragment containing 3 fragments,including Tgmif-up,dhfr-ts and Tgmif-down was5 049 bp long,which was as expected;RH_(ΔTgmif)strain am

关 键 词：刚地弓形虫 巨噬细胞迁移抑制因子 基因敲除 

分 类 号：R382.5[医药卫生—医学寄生虫学]