目的 构建并鉴定刚地弓形虫RH株巨噬细胞迁移抑制因子（Tgmif）基因敲除虫株。 方法 设计刚地弓形虫RH株Tgmif的单向导RNA(sgRNA)和点突变引物,将pSAG1::Cas9-U6::sgUPRT质粒定点突变为针对Tgmif基因的pSAG1::Cas9-U6::sgTgmif质粒。构建含有Tgmif上游1 001 bp（Tgmif-up）、二氢叶酸还原酶-胸苷酸合成酶基因（dhfr-ts）和Tgmif下游1 011 bp（Tgmif-down）等3个片段的Tgmif供体质粒,从Tgmif供体质粒PCR扩增获得Tgmif供体DNA。将pSAG1::Cas9-U6::sgTgmif质粒和Tgmif供体DNA混合后电击转染野生型刚地弓形虫RH株（RH_WT）速殖子,用乙胺嘧啶培养7 d,筛选获得稳定表达乙胺嘧啶抗性的虫株并进行单克隆筛选,PCR扩增dhfr-ts上、下游同源臂和Tgmif鉴定Tgmif基因敲除单克隆株（RH_ΔTgmif）。提取RH_ΔTgmif和RH_WT株虫体蛋白,蛋白质免疫印迹（Western blotting）分析TgMIF蛋白的表达情况。RH_ΔTgmif在人包皮成纤维（HFF)细胞中传代10代,吉氏染色,镜检计数100个HFF中速殖子数,评估RH_ΔTgmif在体外培养HFF细胞中的增殖能力,以RH_WT为对照。将20只BABL/c小鼠随机分为RH_WT组和RH_ΔTgmif组（10只/组）,各组分别腹腔注射RH_WT或RH_ΔTgmif株速殖子（1 000个/鼠）,记录小鼠生存情况。弓形虫增殖能力的比较采用独立样本t检验,小鼠生存率的比较采用Log Rank检验。 结果 PCR检测结果显示,含Tgmif-up、dhfr-ts和Tgmif-down等3个片段的Tgmif供体DNA片段长5 049 bp,与预期相符;RH_ΔTgmif株分别扩增出1 387、1 524 bp的dhfr-ts上、下游同源臂条带,RH_WT株无相应条带;RH_WT株扩增出1 837 bp的Tgmif条带,RH_ΔTgmif株无相应条带。Western blotting分析结果显示,RH_WT株在相对分子质量（M_r）12 500处出现1条蛋白条带,RH_ΔTgmif株无相应的蛋白条带。吉氏染色镜检结果显示,100个HFF中RH_ΔTgmif株速殖子为（2 986 ± 69.20）个,高于RH_WT株的（2 067 ± 51.08）个（t = 18.50,P < 0.01）。体内毒力试验结果显示,RH_WT组小鼠在第7天出现死亡,在第9天全部死亡;RH_ΔTgmif组小鼠在第5天出现死亡,第7天全部死亡,两组之间的差异有统计学意义（χ² = 17.45,P < 0.01）。 结论 成功构建Tgmif基因敲除弓形虫虫株RH_ΔTgmif,RH_ΔTgmif株的毒力比RH_WT株强。

Objective To construct and validate a macrophage migration inhibitory factor (mif) gene knockout strain of Toxoplasma gondii RH strain. Methods Single guide RNA (sgRNA) and site-directed mutation primers the RH strain of T. gondii Tgmif gene were designed. The pSAG1::Cas9-U6::sgUPRT plasmid was mutated into the pSAG1::Cas9-U6::sgTgmif plasmid targeting the Tgmif gene. A Tgmif donor plasmid containing 3 fragments of 1 001 bp upstream of Tgmif (Tgmif-up), dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) and 1 011 bp downstream of Tgmif (Tgmif-down) was constructed. The Tgmif donor DNA was amplified from the Tgmif donor plasmid by PCR. The pSAG1::Cas9-U6::sgTgmif plasmid and Tgmif donor DNA were mixed and electroporated into the tachyzoites of wild-type T. gondii RH strain (RH_WT) and cultured with pyrimethamine for 7 days prior to screening to obtain the stable expression of pyrimethamine-resistant tachyzoites. The upper and lower homology arms of dhfr-ts and Tgmif were amplified by PCR to identify Tgmif knockout monoclonal strains (RH_ΔTgmif). The protein of RH_ΔTgmif and RH_WT tachyzoites was extracted and the expression of TgMIF protein was analyzed by Western blotting. RH_ΔTgmif was cultured in human foreskin fibroblasts (HFF) cells for 10 passages, and the number of tachyzoites per 100 HFF cells was counted by Giemsa staining to evaluate the proliferation of RH_ΔTgmif in HFF cells in vitro. RH_WT was used as the control. Twenty BABL/c mice were randomly divided into RH_WT group and RH_ΔTgmif group (10 mice/group). The mice were injected intraperitoneally with tachyzoites of RH_WT or RH_ΔTgmif strains (1 000/mouse) for each group respectively. The survival of mice was recorded. Independent sample t-test was used to compare the proliferation of T. gondii, and Log Rank test was used to compare the survival rate of mice. Results PCR results showed that the Tgmif donor DNA fragment containing 3 fragments, including Tgmif-up, dhfr-ts and Tgmif-down was 5 049 bp long, which was as expected; RH_ΔTgmif strain amplified dhfr-ts upstream and downstream homology arm bands of 1 387 bp and 1 524 bp, respectively, while RH_WT strain had no corresponding bands; a 1 837 bp Tgmif band was amplified in RH_WT strain, while no corresponding band in RH_ΔTgmif strain. The results of Western blotting showed that the RH_WT strain had a protein band at a relative molecular mass (M_r) of 12 500, while the RH_ΔTgmif strain had no corresponding protein band. The results of Giemsa staining showed that the tachyzoites of RH_ΔTgmif strain were (2 986 ± 69.20) per 100 HFF cells, which was higher than the RH_WT strain (2 067 ± 51.08) (t = 18.50, P < 0.01). The results of the in vivo virulence test showed that the mice in the RH_WT group started to die on the 7th day and all died on the 9th day; the mice in the RH_ΔTgmif group started to die on the 5th day and all died on the 7th day, and the difference between the two groups was statistically significant (χ² = 17.45, P < 0.01). Conclusion The Tgmif gene knockout strain RH_ΔTgmif was successfully constructed, and the RH_ΔTgmif strain of Toxoplasma was more virulent than the RH_WT strain.