弓形虫4个假定蛋白基因缺失株的构建及其基本生物功能学研究

doi:10.11843/j.issn.0366-6964.2022.10.031

摘要/Abstract

摘要： 旨在探究4个与MIC相关的假定蛋白在弓形虫速殖子阶段的作用及基因缺失对毒力的影响。在线设计TGME49_222975、TGME49_275798、TGME49_238220和TGME49_238210的sgRNA序列，用PCR将pSAG1：：CAS9-U6：：sgUPRT模板上的sgUPRT突变为相应的sgRNA，获得目的基因的CRISPR质粒。将获得的目的基因质粒和DHFR片段混合后电转入弓形虫速殖子，经乙胺嘧啶单克隆筛选后，PCR鉴定成功即得目的基因缺失株。再用目的基因缺失株和RH野生株进行复制、逸出、噬斑和小鼠毒力试验，比较二者各项试验结果，以评定目的基因对于弓形虫速殖子功能和毒力的影响。结果显示：目的基因缺失株和野生株RH在相同时间内形成的含有1、2、4、8、16个速殖子的纳虫空泡数占比差异不显著（P>0.05），纳虫空泡内速殖子在钙离子刺激下2 min内均可全部逸出，相同条件和时间培养形成的噬斑大小和面积差异不显著（P>0.05），接种相同数目速殖子的小鼠自开始死亡到全部死亡时间差别不显著（P>0.05）。本研究成功构建了4个弓形虫MIC相关基因的缺失株（RHΔTGME49_222975、RHΔTGME49_275798、RHΔTGME49_238200和RHΔTGME49_238210），但毒力与野生株毒力均差异不显著，说明这4个基因均不是弓形虫的重要“独立”毒力因子，但可能在其他方面发挥着重要的生物学功能。本研究初步解析了4个弓形虫MIC相关蛋白的基本生物学功能，为后期全面解析弓形虫蛋白功能提供数据。

关键词: 弓形虫, 微线体蛋白, CRISPR/Cas9, 基因敲除

Abstract: The aim of this study was to investigate the role of four hypothetical MIC-related proteins in the tachyzoite stage of Toxoplasma gondii and the effect of gene deletion on virulence. The sgRNA sequences of TGME49_222975, TGME49_275798, TGME49_238220 and TGME49_238210 were designed online at the website, respectively, and the sgUPRT on the pSAG1::CAS9-U6::sgUPRT template was mutated to sgRNA by PCR to obtain the target gene of the CRISPR plasmid. The obtained target gene plasmids and DHFR fragments were subsequently mixed and electrotransferred to T. gondii tachyzoites, and the target gene deletion strains were obtained by successful PCR identification after ethidium monoclonal screening. Subsequently, the target gene deletion strains and the RH wild strain were used for replication, egress, plaques formed and virulence in mouse assays, and the results were compared to evaluate the effect of the target genes on the function and virulence of T. gondii tachyzoites. There was no significant difference in the number of vacuoles containing 1, 2, 4, 8 and 16 tachyzoites formed by of the deletion and wild strains within the same period of time (P>0.05), and all the tachyzoites in the vacuoles could egress within 2 min under the stimulation of calcium ions, plaques in the size and area of plaques in 12-well plates infected with the same number of tachyzoites for the same period of time (P>0.05). The differences in the time from the beginning of the death to the total death were not significant (P>0.05). In this study, four deletion strains of T. gondii MIC-related genes (RHΔTGME49_222975, RHΔTGME49_275798, RHΔTGME49_238200 and RHΔTGME49_238210) were successfully constructed, but the virulence of the deletion strains was not significantly different from that of the wild strain, indicating that the four genes are not important independent virulence factors of T. gondii. But the four genes may play other important biological functions. This study preliminarily analyzed the basic biological functions of four T. gondii MIC-related proteins, and provided data for the later comprehensive analysis of T. gondii protein functions.

Key words: Toxoplasma gondii, micronemes, CRISPR/Cas9, gene knockout

中图分类号:

王沛, 王萌, 李婷婷, 郑晓楠, 梁勤立, 陈小庆. 弓形虫4个假定蛋白基因缺失株的构建及其基本生物功能学研究[J]. 畜牧兽医学报, 2022, 53(10): 3598-3608.

WANG Pei, WANG Meng, LI Tingting, ZHENG Xiaonan, LIANG Qinli, CHEN Xiaoqing. Generation and Basic Functional Characterization of Four Hypothetical Protein Genes Deletion Strains of Toxoplasma gondii[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3598-3608.

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