果蝇NOMPC相互作用蛋白的筛选与鉴定

NOMPC是果蝇感受触觉的机械力敏感离子通道,属于瞬时受体电位(TRP)离子通道家族,机械力敏感离子通道通过怎样的机制被机械力激活?与NOMPC相互作用的蛋白有哪些?为了研究这些问题,本研究从果蝇NOMPC全长cDNA中扩增得到其N端的29个锚蛋白重复序列(ARs),ORF长度为3 039bp.然后将这29个ARs构建到表达载体pUAST-EGFP上,转染果蝇S2细胞后,通过免疫沉淀、质谱分析和Western blot筛选和鉴定NOMPC相互作用的蛋白,实验筛选出NOMPC N端的29ARs和NOMPC全长的相互作用蛋白,通过免疫共沉淀(Co-IP)实验,对可能与NOMPC存在相互作用的蛋白质进行验证,鉴定出Annexin B9、CG3195、CG3731、CG5374与NOMPC存在相互作用,为进一步研究NOMPC介导触觉转导的分子机制提供了基础.

Screening and Identification of Proteins Interacting with Drosophila NOMPC

Drosophila NOMPC,a member of the transient receptor potential(TRP) family of ion channels,is a mechanotransduction channel subunit for gentle-touch sensation.However,the transduction mechanism of NOMPC and the protein complexes of touch transduction are largely unclear.In order to study these questions,we obtained NOMPC 29 Ankyrin Repeats(ARs)at N-terminal from NOMPC cDNA,and the open reading frame (ORF) of NOMPC 29 Ankyrin Repeats(ARs)was 3 039 bp in length.Then we constructed pUAST-NOMPC 29 ARs-EGFP expression vector,and transfected it into S2 cells.Using immunoprecipitation(IP),mass spectrometry (MS) and Western blot approaches,we screened many proteins interacting with NOMPC 29 ARs or NOMPC full length.The interactions between NOMPC and some selected proteins were confirmed by Co-Immunoprecipitation (Co-IP).We found that there were interactions among Annexin B9,CG3731,CG3195,CG5374 and NOMPC in S2 cell.Our data provides clues for further understanding the transduction mechanism of NOMPC.