神经元蛋白3.1结合蛋白的筛选与鉴定

Screening and Identification of P311 Binding Proteins

哺乳动物肺组织发育的基本模式总体分为上皮分支的形态发生和分隔膜的形成两个部分.基因p311是克隆到的一个在分隔膜形成阶段特异表达的基因,可能在肺泡发育中起重要作用.为进一步探讨神经元蛋白3.1(P311)对肺发育过程的影响,构建了小鼠肺组织cDNA文库,以融合Gal4 DNA结合结构域的重组P311蛋白为诱饵,利用酵母双杂交技术从文库中筛选P311结合蛋白.通过免疫共沉淀和双分子荧光互补等技术进一步验证,SPARC(secreted protein,acidic and rich incysteine)被确定为P311相互作用蛋白.进一步的研究发现,SPARC在肺组织中具有与P311相似的表达时序特征,双重免疫组织化学染色显示SPARC和P311在小鼠肺组织中共定位于肺泡上皮细胞和肌成纤维细胞中.提示P311可能通过与SPARC的相互作用影响肺泡发育.

Alveoli are the key functional units of the lungs where gas exchange take place.But the regulation of alveolar morphogenesis is not completely understood.The basic strategies of mammalian lung development can be generally divided into two stages： epithelial branching morphogenesis,and septal formation.P311 was identified previously as a gene that specifically expressed during lung septal formation.In order to further explore the potential effects of P311 during lung development,a yeast two-hybrid screen was performed to identify P311 interacting partners.A recombinant P311 fused with the Gal4 DNA binding domain was used as the bait protein to screen a cDNA library constructed from developing mouse lungs.After confirmed by coimmunoprecipitation（CoIP） and bimolecular fluorescence complementation（BiFC） experiments,SPARC（secreted protein,acidic and rich in cysteine） was identified as a P311 binding protein.In further studies,it was found that SPARC showed similar temporal expression pattern with P311 during lung development.Double immunostaining indicated SPARC and P311 colocalized in alveolar epithelium and myofibroblast in P11 mouse lung sections.Taken together,the data suggested that P311 might have a close connection with SPARC on its influences on lung development.