弓形虫sod3基因缺失株的构建及其基本生物学功能研究  

Generation and functional characterization of sod3 knock-out strain ofToxoplasma gondii

作　　者：赵丹钰 李婷婷[1] 王萌[1] 祁淑芸[1] 梁勤立 张芝玮 朱兴全[1,2] 王金磊 ZHAO Dan-yu;LI Ting-ting;WANG Meng;QI Shu-yun;LIANG Qin-li;ZHANG Zhi-wei;ZHU Xing-quan;WANG Jin-lei(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]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,甘肃兰州730046 [2]山西农业大学动物医学学院,山西太谷030801

出　　处：《中国兽医科学》2022年第7期882-891,共10页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFC2300800,2021YFC2300802);中国农业科学院科技创新工程项目(CAAS-ASTIP-2016-LVRI-03);山西省“1331工程”项目(20211331-13)。

摘　　要：超氧化物歧化酶(SOD)是一种重要的抗氧化酶,参与一系列重要的细胞过程,可保护机体免受氧化损伤,在寄生虫氧化还原平衡调节中发挥重要作用。本研究利用CRISPR-Cas9介导的同源重组技术,分别构建了弓形虫Ⅰ型RH虫株和Ⅱ型Pru虫株的sod3基因缺失株(RHΔsod3;PruΔsod3);通过体内和体外试验,探究了sod3基因的基本生物学功能及其在小鼠体内的毒力作用。噬斑试验分析表明,RHΔsod3和PruΔsod3所形成噬斑的大小和数量与野生株相比无显著差异(P>0.05)。研究发现缺失sod3基因不影响弓形虫RH速殖子在体外的入侵效率、细胞内复制能力、逸出能力、活性氧(ROS)水平、总抗氧化能力(T-AOC)。缓殖子转化试验发现,缺失sod3基因不影响弓形虫Pru虫株由速殖子向缓殖子转化。小鼠毒力试验表明,分别单独感染100个RHΔsod3和RH野生株速殖子的昆明鼠均在感染后第8~10天发生死亡;分别单独感染5×105个PruΔsod3和Pru野生株速殖子的昆明鼠均在感染后10 d内发生死亡;分别单独感染5×103个PruΔsod3和Pru野生株速殖子的昆明鼠所形成的脑包囊数量无显著差异(P>0.05)。对弓形虫转录组数据库现有数据分析表明,SOD3在弓形虫孢子化卵囊阶段的表达量显著高于其他时期,说明SOD3可能参与卵囊孢子化过程。本研究成功构建了弓形虫sod3基因缺失株(RHΔsod3;PruΔsod3),为进一步研究sod3基因在Ⅰ型和Ⅱ型虫株的其他生物学功能奠定了基础。Toxoplasma gondii superoxide dismutase(SOD)is involved in a series of important cellular processes,and it is an important antioxidant enzyme that protects the organism from oxidative damage and plays a key role in regulating the redox homeostasis of the parasite.In this study,we successfully constructed sod3 knock-out strains(RHΔsod3 and PruΔsod3)of T.gondii typeⅠRH strain and typeⅡPru strain using the CRISPR-Cas9-mediated homologous recombination technology,and explored the basic biological function in vitro and virulence in vivo of T.gondii sod3.Analysis of plaque assay showed that the size and number of plaques formed by RHΔsod3 and PruΔsod3 were not significantly different from those of wild strains(P>0.05).In addition,it was found that the deletion of sod3 did not affect the invasion efficiency,intracellular replication,egress process,reactive oxygen species(ROS)level,and total antioxidant capacity(T-AOC)of T.gondii in vitro.The bradyzoite differentiation assay showed that the deletion of sod3 did not affect the differentiation of T.gondii Pru strain from tachyzoites to bradyzoites.Mice virulence experiments showed that all Kunming mice infected with 100 RHΔsod3 and RH strain tachyzoites separately died within 8—10 days;that all Kunming mice infected with 5×105 PruΔsod3 and Pru strain tachyzoites separately died within 10 days;there was no significant difference in the number of brain cysts formed in mice infected with 5×103 PruΔsod3 and Pru strain(P>0.05)tachyzoites separately.Analysis of the existing T.gondii transcriptome database showed that the expression of sod3 in sporulated oocysts of T.gondii is significantly higher than that in other stages,indicating that SOD3 may be involved in the sporulation of oocysts.In summary,RHΔsod3 and PruΔsod3 were successfully constructed,which laid a foundation for further research on other biological functions of the sod3 gene in typeⅠand typeⅡstrains.

关 键 词：弓形虫 氧化应激 超氧化物歧化酶(SOD) CRISPR-Cas9 生物学功能 

分 类 号：S852.729[农业科学—基础兽医学]