弓形虫TGME49_287040基因在Na/Ca离子通道中的生物学功能

弓形虫病是一种严重危害人类健康的机会致病性人兽共患寄生虫病，目前没有针对该病的特效药和疫苗。申请人前期研究发现弓形虫TGME49_287040基因参与了钾依赖性钠钙离子通道交换，而该基因在离子通道中的生物学功能尚不清楚。本研究在已经利用CRISPR/Cas9技术构建了该基因缺失株及相关质粒的基础上，采用全细胞记录仪评价该基因缺失株对Na/Ca离子通道交换的影响；使用抗坏血酸过氧化酶2(APEX2)对该基因编码的蛋白进行生物素标记，并结合免疫荧光对其进行亚细胞定位；借用APEX2生物素标记技术对该基因编码蛋白的互作蛋白进行标记、纯化和质谱鉴定；通过免疫共沉淀（CO-IP）对基因TGME49_287040编码蛋白的互作蛋白进行验证，明确其在细胞内的分子组成和具体的生物学功能。预期研究成果将为后期靶向抗弓形虫药物的研发奠定基础。

Toxoplasmosis is an opportunistic zoonotic parasitic disease that poses a serious health risk to humans. Unfortunately, there is currently no specific drug or vaccine for this disease until now. The applicant’s previous study revealed that Toxoplasma gondii (T. gondii) TGME49_287040 gene is involved in a K+-dependent Na/Ca channel exchange, but the biological function of the gene in the ion channel is not yet clear. In this study, the gene deletion strain and the associated plasmid has been constructed by using CRISPR/Cas9, the whole cell recorder will be used to evaluate the effect of the gene-deleted strain on the exchange of Na/Ca ion channel; the protein encoded by the gene will be biotin-labeled with ascorbate peroxidase 2 (APEX2), accompanied by immunofluorescence and biotin labeling technique in order to detect it’s subcellular lolization; as well as its interacting protein will be labeled, purified and identified. Furthermore, the interacting proteins of the TGME49_287040-encoded protein will be validated by CO-IP to clarify their molecular composition and specific biological functions in the cells. The expected research results will lay the foundation for the later development of targeted anti-toxoplasma drugs.