弓形虫泛素特异性蛋白酶TgUSP10的定位及其功能  

Localization and function of TgUSP10 in Toxoplasma gondii

作　　者：陈璐璐 孙洪超 阳毅敏[1] 盛楷茵 姚晨倩 陈学秋[1] 杨怡[1] 杜爱芳[1] CHEN Lulu;SUN Hongchao;YANG Yimin;SHENG Kaiyin;YAO Chenqian;CHEN Xueqiu;YANG Yi;DU Aifang(Zhejiang Province Key Laboratory of Preventive Veterinary Medicine/Institute of Animal Preventive Medicine,College of Animal Science,Zhejiang University,Hangzhou 310012,China;Institute of Animal Husbandry and Veterinary Medicine,Zhejiang Academy of Agricultural Science,Hangzhou 31002l,China)

机构地区：[1]浙江大学动物科学学院、浙江省动物预防医学重点实验室/浙江大学动物预防医学研究所,浙江杭州310012 [2]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《中国兽医学报》2022年第12期2444-2451,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31802183,31672543);浙江省自然科学基金资助项目(LQ21C180002)。

摘　　要：利用CRISPR/Cas9技术构建弓形虫泛素特异性蛋白酶TgUSP10的内源性标记虫株,经单克隆后通过PCR和Western blot对内源性标记虫株进行鉴定,并通过间接免疫荧光试验分析了TgUSP10蛋白的亚细胞定位与表达水平;同时,构建TgUSP10敲除虫株,经单克隆后进行PCR鉴定,并通过噬斑、黏附/入侵、复制和逸出试验评估TgUSP10缺失对弓形虫体外增殖的影响。IFA结果显示,TgUSP10定位于细胞质,并在G1期、S期和分裂后期表达量较高;成功构建了TgUSP10敲除虫株,与RH△ku80虫株相比,TgUSP10敲除虫株形成噬斑的能力显著减弱;进一步分析体外增殖的具体过程后发现,TgUSP10敲除虫株的复制能力显著减弱,而黏附/入侵和逸出能力没有显著变化。结果表明,成功构建了TgUSP10的内源性标记虫株,TgUSP10定位于虫体细胞质,并对弓形虫体外增殖具有重要作用,为后续进一步阐明其功能和作用机制奠定基础。To understand the localization and function of the ubiquitin-specific protease TgUSP10 in T.gondii,we generated TgUSP10 endogenously tagged strain using the CRISPR/Cas9 system.After screening,the endogenously tagged strain was confirmed by PCRand Western blot.Immunofluorescence analysis(IFA)was conducted lately to examine the subcellular localization and expression level of TgUSP10.Simultaneously,TgUSP10 knockout strain was generated and confirmed by PCR after screening.Plaque,attachment/invasion,replication and egress assay were carried out to evaluate the effect of TgUSP10 deletion on the in vitro growth of T.gondii.The results showed that TgUSP10 endogenously tagged strain was generated successfully.IFA results revealed that TgUSP10 localized in the cytoplasm and had a high expression level in the G1,S and late M phases.TgUSP10 knockout strain was generated successfully.Compared to RHΔku80,the TgUSP10 knockout strain exhibited a significant reduction of the ability to form plaques.After further analysis of the steps influenced,the results showed that deletion of TgUSP10 inhibited replication but had no significant effect on the attachment/invasion and egress.Together,TgUSP10 localizes in the cytoplasm and plays a vital role in the in vitro growth of T.gondii,this study laid the foundation for further investigation of its function and mechanism.

关 键 词：弓形虫 去泛素化酶 TgUSP10 CRISPR/Cas9 

分 类 号：S852.2[农业科学—基础兽医学] S852.7[农业科学—兽医学]