新基因AngRem104与Bardet-Biedl综合征2蛋白的相互作用

Interaction of novel gene AngRem104 and Bardet-Biedl syndrome 2 protein in mammalian cells

目的 筛选与新基因AngRem104相互作用的蛋白,并验证AngRem104和BardetBiedl综合征2(BBS2)蛋白在哺乳动物细胞中的相互作用,为进一步研究AngRem104的生理功 能奠定基础。方法 构建AngRem104的酵母真核表达载体AngRem104-pGBKT7／c-myc.用以转 化酵母菌AH109,并与转化了成人肾脏cDNA文库的酵母菌Y187接合,筛选与AngRem104相互 作用的蛋白。从中挑选Bardet-Biedl综合征2蛋白进行免疫共沉淀,构建AngRem104-pcDNA3.1／ V5-His和BBS2-pCMV／c-myc融合表达载体,共转染HEK293T细胞。分别用小鼠抗人c-myc单 克隆抗体和小鼠抗人V5-HRP抗体进行免疫沉淀和免疫印迹,以验证AngRem104和BBS2蛋白 间的相互作用。结果 以AngRem104为诱饵蛋白在人肾脏cDNA文库中共筛选到包括BBS2在 内的7个与之存在相互作用的蛋白。免疫共沉淀结果显示,AngRem104-V5融合蛋白能够在cmyc抗体的免疫沉淀物中检测出来:在V5抗体的免疫沉淀物中也能检测到BBS2-c-myc融合 蛋白。结论AngRem104和BBS2蛋自在哺乳动物细胞中存在某种相互作用,为新基因 AngRem104功能探讨和Bardet-Biedl综合征发病机制的研究提供了有价值的线索。

Objective To screen for proteins interacting with novel gene AngRem104 and to identify the putative interaction of novel gene AngRem104 and Bardet-Bied1 syndrome 2 (BBS2) protein in mammalian cells. Methods The yeast strain AH109 was transformed with AngRem104pGBKT7/c-myc and yeast-mating was utilized to screen for interacting proteins with AngRem104 in pretransformed human kidney cDNA library. The human embryonic kidney (HEK 293T) cells were transformed with two recombined plasmids,AngRem104-pcDNA3.1/V5-His and BBS2-pCMV/c-myc. Mouse anti-human V5 monoclonal antibody and mouse anti-human c-myc monoclonal antibody were used in immunoprecipitation and immunoblot analysis, respectively. Results Seven proteins that interact with AngRem104, including BBS2 were identified. The AngRem104-V5 and the BBS2-c-myc fusion protein were detected respectively in the immunoprecipitation by anti-c-myc and anti-V5 antibody. Conclusion The novel gene AngRem104 may interact with BBS2 protein in mammalian cells,which provides insights as to the function exploration of novel gene AngRem104 and the pathogenesis investigation of Bardet-Biedl syndrome.