目的 鉴定刚地弓形虫假定蛋白TGGT1_310420在速殖子阶段的功能及其蛋白N端序列的亚细胞定位作用。 方法 在线设计针对TGGT1_310420的sgRNA,通过定点突变弓形虫CRISPR/Cas9载体pSAG1::CAS9-GFP-U6::sgUPRT上的sgRNA序列获得针对TGGT1_310420的CRISPR/Cas9载体。利用CRISPR/Cas9编辑技术将含有荧光蛋白mCherry和弓形虫次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶（HXGPRT）的PCR片段mCherry-Ty_HXGPRT插入至TGGT1_310420内源基因编码N端前20个氨基酸残基之后。通过PCR鉴定片段是否正确插入;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹分析检测融合蛋白的表达情况。间接免疫荧光分析和共聚焦显微镜观察融合蛋白亚细胞定位;空斑实验检测基因缺失株虫体表型的变化。 结果 PCR和测序结果显示,成功构建了针对TGGT1_310420的CRISPR/Cas9载体,mCherry-Ty_HXGPRT序列定点插入至靶点位置。蛋白质印迹分析结果显示,mCherry融合蛋白的相对分子质量（M_r）约为36 000;间接免疫荧光实验表明mCherry融合蛋白与弓形虫滑行相关蛋白45（TgGAP45）共定位于虫体的表膜。空斑实验检测结果显示,TGGT1_310420的缺失并未引起虫体出现可测的表型变化。 结论 TGGT1_310420编码蛋白前20个氨基酸残基序列具有定位到弓形虫表膜的功能,TGGT1_310420在弓形虫速殖子阶段为非必需基因。

Objective To characterize the function and localization of a putative protein TGGT1_310420 expressed on tachyzoite of Toxoplasma gondii. Methods The single guide RNA (sgRNA) targeting TGGT1_310420 was designed online. CRISPR/Cas9 construct targeting GGT1_310420 was obtained by mutating sgRNA of TgUPRT on pSAG1::CAS9-GFP-U6::sgUPRT. The knockout of TGGT1_310420 gene was performed through tagging the mCherry-Ty_HXGPRT sequence after the first 60 nt coding sequences of the endogenous copy using CRISPR/Cas9 genome-editing strategy. The correct insert was confirmed by PCR and DNA sequencing. The expressed fusion protein was detected by SDS-PAGE and Western blotting analysis, and its subcellular localization was determined by immunofluorescence microscopy. The phenotype of the knockout parasites was examined by a plaque assay. Results The CRISPR/Cas9 construct targeting TGGT1_310420 was successfully constructed and confirmed by sequencing. PCR analysis confirmed the integration of the mCherry-Ty_HXGPRT sequence into the correct locus and Western blotting assay detected a single protein band with an apparent relative molecular weight of 36 000. Immunofluorescent assay demonstrated that the first 20 amino acids of TGGT1_310420 fused to mCherry-Ty_HXGPRT was co-localized with T. gondii gliding associated protein 45(TgGAP45)to the parasite’s pellicle. Knockout of TGGT1_310420 gene revealed no measurable alteration in tachyzoites. Conclusion The first 20 amino acids of TGGT1_310420 are sufficient for pellicle targeting and the knockout of TGGT1_310420 did not change the phenotype of T. gomdii tachyzoite.