关键词: 刚地弓形虫, 致密颗粒蛋白3, 基因缺陷, 规律成簇间隔短回文重复序列及其相关蛋白9, 毒力

Abstract: Objective　To generate a dense granule protein 3 (GRA3) gene⁃deficient mutant of the Toxoplasma gondii ME49 strain and to test the virulence of the mutant. Methods　Gene⁃deficient parasites were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR⁃associated protein 9 (CRISPR/Cas9) system. Guide RNA (gRNA) was designed using the E⁃CRISPR software, and mutated on the pSAG1::Cas9⁃U6::sgUPRT plasmid using the Q5 site⁃directed mutagenesis kit to generate the pSAG1::Cas9⁃U6::sgGRA3 plasmid. A GRA3 donor plasmid containing GRA3 gene upstream sequences, pyrimethamine resistant gene dihydrofolate reductase⁃thymidylate synthase (DHFR⁃TS) and GRA3 gene downstream sequence was generated, and GRA3 donor DNA was amplified using PCR assay. The pSAG1::Cas9⁃U6::sgGRA3 plasmid and GRA3 donor DNA were electroporated into tachyzoites of the wild⁃type T. gondii ME49 strain. Then, parasite suspensions were inoculated into human foreskin fibroblast (HFF) cells and screened with pyrimethamine to yield pyrimethamine⁃resistant parasites for monoclonal screening. The GRA3 gene deficient monoclonal strain (ME49Δgra3) of T. gondii was identified using PCR and Western blotting assays, and the expression of GRA3 protein was determined in the T. gondii ME49Δgra3 strain using Western blotting. Subsequently, 1 000 freshly lysed tachyzoites of T. gondii ME49 and ME49Δgra3 strains were transferred to 12⁃well plates seeded with HFF cells, and incubated at 37 ℃ containing 5% CO2 for 7 days, and the number of plaques was counted by staining with crystal violet solutions. HFF cells infected with tachyzoites of T. gondii ME49 and ME49Δgra3 strains were stained using Giemsa solutions, and the numbers of cells containing 1, 2, 4, and > 4 T. gondii parasitophorous vacuoles were counted. In addition, the survival rates of C57BL/6 mice infected with T. gondii ME49 and ME49Δgra3 strains were compared 35 days post⁃infection. Results　PCR assay revealed successful amplification of both the upstream and downstream homologous arm bands of the DHFR⁃TS gene in the T. gondii ME49Δgra3 strain, and no corresponding bands were amplified in the ME49 strain. The GRA3 band was amplified in the ME49 strain, and the DHFR⁃TS band, rather than GRA3 band, was amplified in the ME49Δgra3 strain. Western blotting determined absence of GRA3 protein expression in the ME49Δgra3 strain. Crystal violet staining showed that the T. gondii ME49 strain produced more plaques than the ME49Δgra3 strain [(352.67 ± 26.39) plaques vs. (235.00 ± 26.29) plaques; t = 5.472, P < 0.01], and Giemsa staining revealed that the proportion of T. gondii parasitophorous vacuoles containing at least four T. gondii tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(75.67 ± 2.52)% vs. (59.67 ± 2.31)%; t = 8.113, P < 0.01], and the proportion of T. gondii parasitophorous vacuoles containing at least 1 or 2 T. gondii tachyzoites was higher in HFF cells infected with the T. gondii ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(24.33 ± 2.52)% vs. (40.33 ± 2.31)%; t = -8.113, P < 0.01]. In addition, mice infected with the T. gondii ME49 and ME49Δgra3 strains started to die 8 and 9 days post⁃infection, and the 35⁃day mortality rates of mice infected with T. gondii ME49 and ME49Δgra3 strains were 10.00% and 70.00% post⁃infection ([χ2] = 6.762, P < 0.01). Conclusions　The T. gondii ME49Δgra3 strain has been successfully generated, and GRA3 protein may increase the virulence of the T. gondii ME49 strain.

Key words: Toxoplasma gondii, Dense granule protein 3, Gene deficiency, Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR?associated protein 9, Virulence