酵母双杂交筛选与弓形虫毒力因子ROP18相互作用的宿主蛋白

发布时间：2018-07-27 10:58

【摘要】：目的利用酵母双杂交方法,筛选与弓形虫毒力因子ROP18相互作用的宿主蛋白。方法 RT-PCR扩增弓形虫RH株速殖子ROP18基因片段,扩增产物经双酶切后插入酵母双杂交诱饵载体pGBKT7中,将重组质粒导入酵母AH109菌株中,检测诱饵载体有无毒性和自激活作用,利用酵母双杂交系统从人胚胎脑cDNA文库中筛选与ROP18相互作用的宿主蛋白。结果构建了诱饵载体pGBKT7-ROP18,ROP18具有自激活功能。用ROP18~(25-25lan)为诱饵,筛选获得一系列与ROP18相互作用的宿主蛋白:DNA损伤特异性结合蛋白1(DDB1)、torsin A相互作用蛋白1(TOR1AIP1)、整联蛋白β1(integrinβ1)、溶质运载蛋白3(SLC3A2)、酪氨酸硫化转移酶2(TPST2)、Derl样域家族成员2(DERL2)和OCIA结构域蛋白1(OCIAD1)。结论通过酵母双杂交技术筛选出与弓形虫毒力因子ROP18相互作用的多种宿主蛋白。
[Abstract]:Objective to screen host proteins interacting with toxoplasma virulence factor ROP18 by yeast two-hybrid method. Methods Toxoplasma gondii RH strain Toxoplasma gondii Toxoplasma gondii RH strain tachyzoite ROP18 gene fragment was amplified by RT-PCR. The amplified product was digested by double enzyme and inserted into yeast two-hybrid bait vector pGBKT7. The recombinant plasmid was introduced into yeast AH109 strain to detect the nontoxicity and self-activation of bait vector. Yeast two-hybrid system was used to screen host proteins interacting with ROP18 from human embryonic brain cDNA library. Results the decoy vector pGBKT 7-ROP 18 has self-activation function. Using ROP18 ~ (25-25lan) as bait, A series of host proteins that interact with ROP18, such as TOR1AIP1 (TOR1AIP1), integrin 尾 1 (integrin 尾 1), solute transport protein 3 (SLC3A2), tyrosine sulphide transferase 2 (TPST2), DERL2 and OCIA domain protein 1 (OCIAD1), were obtained. Conclusion several host proteins interacting with toxoplasma virulence factor ROP18 were screened by yeast two-hybrid technique.
【作者单位】：安徽医科大学基础医学院生化与分子生物学教研室;安徽医科大学药学院;安徽医科大学省部级基因研究重点实验室病原生物学教研室;
【基金】：国家自然科学基金(No.81271864,30801329) 教育部霍英东教育基金会高等院校青年教师基金(No.131033) 安徽省杰出青年基金(No.10040606Y19)~~
【分类号】：R382.5

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