免疫共沉淀结合质谱筛选绵羊BMPR-1B互作蛋白的研究

Screening of Sheep BMPR-1B Interacting Proteins by CoIP and MS

为研究与绵羊骨形态发生蛋白受体1B(BMPR-1B)相互作用的蛋白,试验通过构建的pcDNA3.1a-BMPR-1B真核表达载体并在Sf9昆虫细胞中特异性表达,采用免疫共沉淀的方法富集绵羊卵巢中与BMPR-1B蛋白的结合蛋白,SDS-PAGE凝胶电泳分离免疫共沉淀复合物,MALDI-TOF/TOF结合数据库检索方法筛选鉴定与BMPR-1B相互作用蛋白。结果表明,试验获得的GDF5、BMP2、BMP4、RhoD和HSP 10蛋白与BMPR-1B互作,GDF5和BMP4作为BMPR-1B蛋白的配体来发挥其生物学功能,BMP2、RhoD和HSP 10在卵泡发育上起重要作用。RhoD和HSP 10与棉羊繁殖主效基因相关联。研究结果为进一步研究BMPR-1B的功能和研究BMPR-1B作为绵羊高繁主效基因的机理和分子调控机制提供了新的思路与方法。

In order to screen the interacting proteins of BMPR-1B, the eukaryotic expression vector of pcDNA3. la-BMPR-lB was constructed and specifically expressed in Sf9 cells. The binding proteins of BM- PR-1B were enriched in sheep＇s ovary using CoIP assay. The CoIP assay were separated on SDS-polyacrylamide gel, then analyzed by MALDI-TOF/TOF and the database. In this study, there were 5 interacting proteins, GDF5, BMP2 , BMP4,RhoD and HSP 10. GDF5 and BMP4 served as ligand to play its biological fundion, BMP2, RhoD and HSP10 played a key role in ovary development. RhoD and HSP10 correlated with the major genes of sheep production. The study provids a new idea for the further study on mechanism of BMPR-1B with ewes reproductive performance.