刚地弓形虫巨噬细胞迁移抑制因子基因敲除虫株的构建与鉴定

doi:10.12140/j.issn.1000-7423.2022.03.011

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (3): 349-354.doi: 10.12140/j.issn.1000-7423.2022.03.011

刚地弓形虫巨噬细胞迁移抑制因子基因敲除虫株的构建与鉴定

王杰¹^,²^,³^,⁴(), 温红阳¹^,²^,⁴, 陈滢²^,³^,⁴, 安然¹^,²^,³^,⁴, 罗庆礼²^,⁴, 沈继龙²^,⁴, 都建¹^,²^,³^,⁴^,^*()

1.安徽医科大学基础医学院生化与分子生物学教研室,合肥 230032
2.人畜共患病安徽高校省级重点实验室,合肥 230032
3.安徽医科大学基础医学院感染性疾病研究中心,合肥 230032
4.病原生物学安徽省重点实验室,合肥 230032

收稿日期:2021-10-15 修回日期:2021-12-01 出版日期:2022-06-30 发布日期:2022-07-06
通讯作者: 都建
作者简介:王杰（1996-）,男,硕士研究生,从事弓形虫致病机制研究。E-mail： 15256969837@163.com
基金资助:
国家自然科学基金(82072300);国家自然科学基金(81871674);国家自然科学基金(81902084)

Construction and identification of macrophage migration inhibitory factor gene knockout strain of Toxoplasma gondii

WANG Jie¹^,²^,³^,⁴(), WEN Hong-yang¹^,²^,⁴, CHEN Ying²^,³^,⁴, AN Ran¹^,²^,³^,⁴, LUO Qing-li²^,⁴, SHEN Ji-long²^,⁴, DU Jian¹^,²^,³^,⁴^,^*()

1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
2. The Provincial Key Laboratory of Zoonoses of High Institutions in Anhui, Hefei 230032, China
3. Research Center for Infectious Diseases, School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
4. The Key Laboratory of Microbiology and Parasitology of Anhui Province, Hefei 230032, China

Received:2021-10-15 Revised:2021-12-01 Online:2022-06-30 Published:2022-07-06
Contact: DU Jian
Supported by:
National Natural Science Foundation of China(82072300);National Natural Science Foundation of China(81871674);National Natural Science Foundation of China(81902084)

摘要/Abstract

摘要：

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_Δ_Tgmif）。提取RH_Δ_Tgmif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_Δ_Tgmif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_Δ_Tgmif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_Δ_Tgmif组（10只/组）,各组分别腹腔注射RH_WT或RH_Δ_Tgmif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_Δ_Tgmif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_Δ_Tgmif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_Δ_Tgmif株无相应的蛋白条带。吉氏染色镜检结果显示,100个HFF中RH_Δ_Tgmif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_Δ_Tgmif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_Δ_Tgmif,RH_Δ_Tgmif株的毒力比RH_WT株强。

关键词: 刚地弓形虫, 巨噬细胞迁移抑制因子, 基因敲除

Abstract:

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_Δ_Tgmif). The protein of RH_Δ_Tgmif and RH_WT tachyzoites was extracted and the expression of TgMIF protein was analyzed by Western blotting. RH_Δ_Tgmif 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_Δ_Tgmif 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_Δ_Tgmif group (10 mice/group). The mice were injected intraperitoneally with tachyzoites of RH_WT or RH_Δ_Tgmif 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_Δ_Tgmif 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_Δ_Tgmif 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_Δ_Tgmif strain had no corresponding protein band. The results of Giemsa staining showed that the tachyzoites of RH_Δ_Tgmif 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_Δ_Tgmif 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_Δ_Tgmif was successfully constructed, and the RH_Δ_Tgmif strain of Toxoplasma was more virulent than the RH_WT strain.

Key words: Toxoplasma gondii, Macrophage migration inhibitory factor, Gene knockout

中图分类号:

R382.5

王杰, 温红阳, 陈滢, 安然, 罗庆礼, 沈继龙, 都建. 刚地弓形虫巨噬细胞迁移抑制因子基因敲除虫株的构建与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 349-354.

WANG Jie, WEN Hong-yang, CHEN Ying, AN Ran, LUO Qing-li, SHEN Ji-long, DU Jian. Construction and identification of macrophage migration inhibitory factor gene knockout strain of Toxoplasma gondii[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(3): 349-354.

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参考文献 21

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引物名称 Primer name	引物序列（5′→3′） Primer sequence （5′→3′）
sgTgmif	F: GCCCTCTTGAGTTTTAGAGCTAGAAATAGCAAG
	R: GTCCTGCTGCAACTTGACATCCCCATTTAC
Tgmif-up	F: AAAACGACGGCCAGTGAATTCACACAAGTCCTTCG CCTTCTG
	R: GGGGGTGAAAATCGAATGACATTTGTGGGGGAACT TGAAAGG
dhfr-ts	F: TGTCATTCGATTTTCACCCCC
	R: GTCCAGAGCGCTCTCAGCGGATCGATCCCCCCGGGC TGC
Tgmif-down	F: GCAGCCCGGGGGGATCGATCCGCTGAGAGCGCTCTG GAC
	R: GACCATGATTACGCCAAGCTTGGCCTTCGTAGATCG TTGTG
Ⅰ	F: CTGCCTTCCACTCCGAAAGAAGAAA
	R: TTACCAGTCATGGACGAGATCGTTC
Ⅱ	F: AGACCCCTGTGTCCTTTATCGAAGA
	R: GTTCTTCTCTGTATTGGATCCAGGC
Ⅲ	F: ATGCCCAAGTGCATGATCTTCT
	R: TCAGCCGAAAGTTCGGTCGC

刚地弓形虫巨噬细胞迁移抑制因子基因敲除虫株的构建与鉴定

Construction and identification of macrophage migration inhibitory factor gene knockout strain of Toxoplasma gondii

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