论文部分内容阅读
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 the protein interacting with the new gene AngRem104 and to verify the interaction of AngRem104 and BardetBiedl syndrome 2 (BBS2) protein in mammalian cells and lay the foundation for further study on the physiological function of AngRem104. Methods AngRem104 yeast eukaryotic expression vector AngRem104-pGBKT7 / c-myc was constructed and transformed into yeast AH109. The fusion protein was then ligated with yeast Y187 transformed with an adult kidney cDNA library to screen the protein interacting with AngRem104. The Bardet-Biedl syndrome 2 protein was selected for co-immunoprecipitation to construct AngRem104-pcDNA3.1 / V5-His and BBS2-pCMV / c-myc fusion expression vector and co-transfected into HEK293T cells. Immunoprecipitation and immunoblotting were performed with mouse anti-human c-myc monoclonal antibody and mouse anti-human V5-HRP antibody respectively to verify the interaction between AngRem104 and BBS2 proteins. Results A total of 7 proteins interacting with BBS2, including BBS2, were screened in human kidney cDNA library using AngRem104 as bait protein. Coimmunoprecipitation results showed that AngRem104-V5 fusion protein could be detected in immunoprecipitates of cmyc antibody: BBS2-c-myc fusion protein was also detected in the immunoprecipitate of V5 antibody. Conclusion There is some interaction between AngRem104 and BBS2 eggs in mammalian cells, which provides valuable clues for exploring the function of AngRem104 and the pathogenesis of Bardet-Biedl syndrome.