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

摘要： 目的　构建刚地弓形虫ME49株致密颗粒蛋白3（dense granule protein 3，GRA3）基因缺陷虫株并鉴定其毒力。 方法　利用规律成簇间隔短回文重复序列及其相关蛋白9 ［clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR⁃associated protein 9，CRISPR/Cas9］技术构建基因缺陷虫株。使用E⁃CRISPR软件设计向导RNA（guide RNA，gRNA），利用Q5定点突变试剂盒突变pSAG1::Cas9⁃U6::sgUPRT质粒上的gRNA，构建pSAG1::Cas9⁃U6::sgGRA3质粒。构建一个含有GRA3基因上游序列、乙胺嘧啶抗性基因二氢叶酸还原酶胸苷酸合酶（dihydrofolate reductase⁃thymidylate synthase，DHFR⁃TS）和GRA3基因下游序列的GRA3基因供体质粒，并通过PCR扩增出GRA3基因供体DNA。将pSAG1::Cas9⁃U6::sgGRA3质粒和GRA3基因供体DNA电穿孔转染野生型刚地弓形虫ME49速殖子，电转后的虫株悬液接种于人包皮成纤维（human foreskin fibroblast，HFF）细胞中，并用乙胺嘧啶进行筛选，获得乙胺嘧啶抗性虫株后进行单克隆筛选，采用PCR和Western blotting鉴定弓形虫GRA3基因缺陷单克隆株ME49Δgra3，采用Western blotting检测弓形虫ME49Δgra3株中GRA3蛋白表达水平。将新鲜裂解的弓形虫ME49株和ME49Δgra3株速殖子各1 000个加入接种有HFF细胞的12孔板中，置于37 ℃含5% CO2的细胞培养箱中培养7 d，以结晶紫染液染色法计数空斑数量。采用吉姆萨染色法对弓形虫ME49株和ME49Δgra3株速殖子感染的HFF细胞进行染色，计数细胞中含有1、2、4个和> 4个弓形虫纳虫泡数量的细胞。比较弓形虫ME49株和ME49Δgra3株感染的C57BL/6小鼠35 d存活率。结果　PCR扩增发现，DHFR⁃TS基因上、下游同源臂条带在弓形虫ME49Δgra3株中均成功扩增，ME49株未扩增出相应条带；GRA3基因条带在弓形虫ME49株中成功扩增，ME49Δgra3株未扩增出GRA3基因条带但扩增出含DHFR⁃TS基因的条带。Western blotting检测发现，弓形虫ME49Δgra3株中未见GRA3蛋白表达。弓形虫ME49株平均形成（352.67 ± 26.39）个空斑、ME49Δgra3株平均形成（235.00 ± 26.29）个空斑，差异有统计学意义（t = 5.472，P < 0.01）。吉姆萨染色结果显示，弓形虫ME49株感染的HFF细胞中，纳虫泡内至少含有4个弓形虫速殖子的比例为（75.67 ± 2.52）%，高于ME49Δgra3株感染组［（59.67 ± 2.31）%］（t = 8.113，P < 0.01）；纳虫泡内含有1个或2个弓形虫速殖子的比例为（24.33 ± 2.52）%，低于ME49Δgra3株感染组［（40.33 ± 2.31）%］（t = -8.113，P < 0.01）。感染弓形虫ME49株和ME49Δgra3株的小鼠分别自感染后第8、9天开始死亡，感染后第35天存活率分别为10.00%（1/10）和70.00%（7/10），差异具有统计学意义（[χ2] = 6.762，P < 0.01）。结论　成功构建了刚地弓形虫ME49Δgra3株，GRA3蛋白可能增强弓形虫ME49株毒力。

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