NALP3富含亮氨酸重复序列结构域识别人细胞周期蛋白H和禽冠状病毒蛋白

摘要

AIM: To investigate the proteins that interact with the domain of leucine-rich repeats in NALP3 (NALP3-LRR). METHODS: The DNA sequence of NALP3-LRR domain was cloned and verified by DNA sequencing. Using yeast two-hybrid system, the plasmid of NALP3-LRR-pSos as bait was constructed to screen the cDNA library of human embryonic lung. The positive clones were verified and selected by the method of yeast backcross verification. Co-im-munoprecipitation assay was also performed to further confirm the reliability of the interactions of NALP3-LRR with the positive proteins. RESULTS: DNA sequence of NALP3-LRR domain was successfully cloned. Four positive clones interacted with NALP3-LRR domain were verified by the yeast two-hybrid screening from the cDNA library of human embryonic lung, in which Homo sapiens cyclin H and avian infectious bronchitis virus strain Cal99 ORFla were further confirmed by the assay of co-immunoprecipitation. CONCLUSION: NALP3-LRR domain interacts with the proteins of Homo sapiens cyclin H and avian infectious bronchitis virus.%目的:寻找与NALP3富含亮氨酸重复序列(leucine-rich repeat,LRR)结构域相互作用的蛋白质分子.方法:克隆NALP3富含亮氨酸重复序列(NALP3-LRR)结构域的DNA序列并经测序检验.应用酵母双杂交技术,构建以NALP3-LRR结构域为诱饵基因的酵母双杂交载体,对人胚肺cDNA文库进行杂交筛选,经酵母回交实验验证蛋白质在酵母细胞内的相互作用并对阳性克隆的DNA进行序列测定和生物信息学分析.将筛选到的阳性克隆进一步用免疫共沉淀实验验证NALP3-LRR结构域与阳性蛋白之间相互作用的可靠性.结果:成功克隆NALP3-LRR结构域的DNA序列并经测序检验正确.用酵母双杂交技术对人胚肺cDNA文库进行酵母杂交筛选共获得4个阳性克隆.免疫共沉淀实验证实能与NALP3-LRR结构域发生相互作用的阳性蛋白是人细胞周期蛋白H和禽传染性支气管炎病毒株Cal99的ORF1a.结论:NALP3的富含亮氨酸重复序列结构域能与人细胞周期蛋白H和禽冠状病毒蛋白发生相互作用.