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

Abstract

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

CLC Number:

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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Generation and Basic Functional Characterization of Four Hypothetical Protein Genes Deletion Strains of Toxoplasma gondii

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