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弓形虫srs19d基因敲除株的构建及其在宿主细胞中的生物学功能分析OA北大核心

Construction of Toxoplasma gondii srs19d Gene Knockout Strain and Analysis of Its Biological Functions in Host Cells

中文摘要英文摘要

为探究弓形虫表面抗原1相关序列(SRS)蛋白家族新成员SRS19D的功能,本试验以弓形虫Ⅱ型Pru虫株为亲本,运用成簇规律间隔短回文重复-CRISPR相关蛋白9(CRISPR/Cas9)技术构建了PruΔsrs19d敲除虫株,并进行了噬斑试验、细胞内增殖试验、体外缓殖子转化试验、小鼠毒力试验和小鼠脑包囊检测试验.噬斑试验和细胞内增殖试验结果显示,与Pru虫株相比,PruΔsrs19d敲除虫株在人包皮成纤维(HFF)细胞上形成的噬斑显著变小(P<0.05),纳虫泡内形成的速殖子个数显著减少(P<0.05).体外缓殖子转化试验发现,与Pru虫株相比,PruΔsrs19d敲除虫株的体外缓殖子转化率极显著降低(P<0.01).小鼠毒力试验结果显示,与Pru虫株相比,弓形虫PruΔsrs19d敲除虫株不会引起ICR小鼠的死亡(P<0.05).小鼠脑包囊检测试验结果显示,与Pru虫株相比,PruΔsrs19d敲除虫株感染小鼠后形成的脑包囊数量极显著减少(P<0.01).本试验成功构建了弓形虫PruΔsrs19d敲除虫株,发现srs19d基因在弓形虫的体外生长、复制、毒力和慢性感染中发挥重要作用,为进一步探究SRS19D的功能和作用机制提供参考.

To explore the function of a new member of the Toxoplasma gondii surface antigen 1-related sequence(SRS)protein family,SRS19D,this study used the Pru strain of Toxoplasma gondii type Ⅱ as a parent to construct the PruΔsrs19d knockout strain using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)technology.Plaque assays,intracellular proliferation assays,in vitro bradyzoite conversion assays,mouse virulence assays,and mouse brain cyst detection assays were performed.The results from plaque assays and intracellular proliferation assays showed that,compared to the Pru strain,the PruΔsrs19d knockout strain formed significantly smaller plaques on human foreskin fibroblast(HFF)cells(P<0.05)and produced significantly fewer tachyzoites within the parasitophorous vacuole(P<0.05).The in vitro bradyzoite conversion assay revealed that the bradyzoite conversion rate of the PruΔsrs19d knockout strain was significantly lower than that of the Pru strain(P<0.01).The mouse virulence assay showed that the PruΔsrs19d knockout strain did not cause death in ICR mice,in contrast to the Pru strain(P<0.05).The mouse brain cyst detection assay indicated that the number of brain cysts formed after infection with the PruΔsrs19d knockout strain was significantly reduced compared to the Pru strain(P<0.01).This study successfully constructed the Toxoplasma gondii PruΔsrs19d knockout strain and discovered that the srs 19d gene plays a crucial role in the parasite's in vitro growth,replication,virulence,and chronic infection,providing a reference for further investigation into the function and mechanism of SRS19D.

牛水珠;潘明;葛层层;黄思扬

扬州大学兽医学院江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州 225009扬州大学兽医学院江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州 225009||教育部农业与农产品安全国际合作联合实验室,江苏扬州 225009

畜牧业

弓形虫表面抗原1相关序列(SRS)srs19d成簇规律间隔短回文重复-CRISPR相关蛋白9(CRISPR/Cas9)生物学功能

Toxoplasma gondiisurface antigen 1-related sequences(SRS)srs19dCRISPR/Cas9biological function

《中国兽医杂志》 2024 (008)

10-17 / 8

江苏省杰出青年基金项目(BK20190046);江苏高校优势学科建设工程资助项目(PAPD)

10.20157/j.cnki.zgsyzz.2024.08.002

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