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当前位置: 首页 > 文章 > 基于生长素诱导降解系统条件性敲除弓形虫ATG6蛋白的研究 中国预防兽医学报 2019,41 (1) 1015-1020
Position: Home > Articles > Conditional knockout of Toxoplasma gondii ATG6 protein based on auxin-induced degradation system Chinese Journal of Preventive Veterinary Medicine 2019,41 (1) 1015-1020

基于生长素诱导降解系统条件性敲除弓形虫ATG6蛋白的研究

作  者：
陈运通;陈赫明;付佳雯;庞宇;郑君;贾洪林
单  位：
中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室
关键词：
弓形虫;植物生长激素;条件性敲除;TgATG6
摘  要：
为构建生长素诱导降解系统的弓形虫条件性基因缺失虫株,本研究首先利用CRISPR/Cas9方法和流式细胞术筛选获得TgKu80基因缺失虫株,然后采用该缺失虫株,利用CRISPR/Cas9方法和流式细胞术筛选,将生长素诱导降解因子(AID)插入该弓形虫TgATG6基因序列的5'端得到TgATG6条件性敲除虫株TgATG6-iKD.PCR扩增及测序结果显示,以TgATG6-iKD虫株为模板可扩增出AID标签,而在野生型虫株中,不能有效扩增该靶序列,经测序验证也得到相符结果,表明AID标签序列能够高效定点插入到弓形虫TgKu80基因缺失虫株中;通过蛋白免疫印迹试验能够在TgATG6条件性敲除虫株检测到AID标签和TgATG6蛋白的融合表达;蛋白免疫印迹试验及间接免疫荧光实验结果显示,利用生长素(NAA)能够快速诱导融合表达蛋白的降解.本研究通过蛋白N端融合AID共表达,建立了高效条件性敲除弓形虫基因的方法,为进一步研究弓形虫相关基因的功能奠定基础.
译  名：
Conditional knockout of Toxoplasma gondii ATG6 protein based on auxin-induced degradation system
作  者：
CHEN Yun-tong;CHEN He-ming;FU Jia-wen;PANG Yu;ZHENG Jun;JIA Hong-lin;State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
Toxoplasma gondii;;auxin;;conditional knockout;;TgATG6
摘  要：
To construct conditional gene-deficient strains in Toxoplasma gondii based on auxin-inducible degradation system,the TgKu80 gene-deficient strain was used in this study. The gene sequence of auxin-induced degradation factor(AID) was inserted into the 5' end of TgATG6 gene. A TgATG6 conditional knockout strain was obtained using CRISPR/Cas9 method and flow cytometry screening. The results of PCR amplification and sequencing results showed that the AID tag could be successfully amplified using the TgATG6 conditional knockout strain as a template,but the target sequence could not be amplified effectively in the wild type strain, indicating that the AID sequence have been efficiently inserted into the Toxoplasma gondii Ku80-deficient strain. The fusion expression of AID tag and Tg ATG6 protein could be detected by western blot. The results of western blot and indirect immunofluorescence showed that auxin(NAA) can rspidly induce the degradation of the fusion protein. In this study, a method for conditional gene-deficient strain with high efficiency was establlished by N-terminally fusion AID co-expression, which laid a foundation for further investigation of the function of Toxoplasma gondii gene.

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