【作者】 王晓晶；

【导师】 王志钢；

【作者基本信息】 内蒙古大学 ， 微生物学， 2012， 硕士

【摘要】 mTOR信号通路调控细胞的生长、增殖和分化,Rheb和FKBP38是该通路中两个重要的调节蛋白。Rheb是Ras超家族的一员,它是mTOR信号通路中的上游调节因子,具有整合营养和促细胞分裂信号的功能；FKBP38是免疫亲和素FKBP家族中的一员,可与mTOR结合抑制其活性,从而调节细胞生长。2007年首次报道Rheb通过FKBP38调节mTOR活性,即Rheb通过与FKBP38结合释放mTOR而使其具有活性;之后又有研究者采用不同的方法证明Rheb与FKBP38没有相互作用,各实验室的研究报道出现不同的结论。值得指出的是,以上研究报道都是采用人细胞为材料的,关于绒山羊胎儿成纤维细胞中Rheb和FKBP38的研究尚未见报道。为了探讨Rheb和FKBP38之间的相互关系,我们在已有报道的基础上,采用蛋白质组学技术探讨绒山羊胎儿成纤维细胞中Rheb和FKBP38之间的相互作用关系,以期通过新技术和新材料的应用为二者之间关系的研究提供新的佐证,同时为绒山羊胎儿成纤维细胞中mTOR信号通路的研究提供数据。本研究首先利用RT-PCR克隆绒山羊Rheb和FKBP38基因cDNA的CDS区,分析其表达模式；构建真核表达载体共转染绒山羊胎儿成纤维细胞(GFb),通过免疫共沉淀和Western-Blot鉴定蛋白Rheb和FKBP38的共同存在；最后利用酵母双杂交技术并结合免疫共沉淀结果确定Rheb与FKBP38的相互作用关系。结果表明,内蒙古白绒山羊Rheb基因ORF全长555bp,编码184个氨基酸(HM569224),该基因在脑组织中表达量较高,在心脏组织中表达量较低；得到的FKBP38基因cDNA全长1248bp,包含了1236bp的ORF,编码411个氨基酸(JF714970),该基因在睾丸组织中表达量较高,在肾脏组织中表达量较低。利用得到的基因构建真核表达载体pIRES2-EGFP-Rheb和pIRES2-DsRed2-FKBP38,共转染GFb细胞,G418筛选两周后提取蛋白进行免疫共沉淀和Western-Blot鉴定,结果在免疫共沉淀复合物中鉴定出Rheb与FKBP38,利用所得基因成功构建酵母双杂交诱饵表达载体pGBKT7-Rheb、 pGBKT7-FKBP38和靶载体pGADT7-Rheb、pGADT7-FKBP38,结果显示Rheb与FKBP38具有相互作用关系。综合免疫共沉淀和酵母双杂交结果,可以认为二者可以发生直接相互作用。本文通过蛋白质组学研究证明了在绒山羊胎儿成纤维细胞中Rheb与FKBP38之间存在直接的相互作用关系,为二者之间关系研究提供了新的佐证,结果丰富了Rheb和FKBP38的基因及其产物的遗传及功能信息,为阐明绒山羊胎儿成纤维细胞中mTOR信号通路的功能与作用机制提供科学依据。更多还原

【Abstract】 mTOR signal pathway coordinates the regulation of cell growth, proliferation as well as differentiation and Rheb along with FKBP38are two important regulatory proteins in this pathway. Rheb is a member of the Ras super family and it is a upstream regulator factor in mTOR signal pathway, with the function of integrating nutrition and promoting cell division; As a member of FKBPs family, FKBP38can inhibit the activity of mTOR so as to control cell growth. Early in2007, it was first reported Rheb could regulate the activity of mTOR by FKBP38meaning that Rheb could activate mTOR by antagonizing FKBP38. But later some researchers proved that they did not detect the interaction between Rheb and FKBP38with different methods. What is worth to point out is, those different conclusions were according to human cells, but there was no such report about the research in cashmere goat fetal fibroblasts cells (GFb cells). In order to discuss the interaction between Rheb and FKBP38, we studied the relationships of them in GFb cells using proteomics technology appending previously reports, expecting it may provide new evidence by using of new technology and new material and offer data for research of mTOR signal pathway in GFb cells at the same time.Firstly, we made use of RT-PCR to clone the CDS fragments in cDNA of Rheb and FKBP38genes, analyzing their expressing pattern; Secondly, two recombinant plasmids were transfected into the GFb cells, and then co-immunopricipitation (co-IP) and Western-Blot were used to detect the co-existed of Rheb and FKBP38. Finally, we defined the interaction relationship between Rheb and FKBP38by using yeast two-hybrid and Co-IP.The results showed that, the ORF of Rheb gene in Inner Mongolia Cashmere Goat is555bp long encoding184amino acids (HM569224), this mRNA expresses comparatively higher in brain but lower in heart. The cDNA of FKBP38gene is1248bp long with1236bp ORF, encoding411amino acids (JF714970), this mRNA expressed comparatively higher level in testis but lower in kidney.According to the results of gene cloning, the recombinating eukaryotic expression vector pIRES2-EGFP-Rheb and pIRES2-DsRed2-FKBP38were got, following co-transfected GFB cells, two weeks later, we screened by G418and extracted their proteins to detect by co-IP and Western-Blot. The results showed Rheb and FKBP38were identified in Co-IP complex. Bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38) were got. The screening of yeast mating showed that there was an interaction between Rheb and FKBP38. We concluded that the two of them could interact with each other directly.We proved the direct interactions between Rheb and FKBP38in GFb cells by proteomics technology, offering a new evidence for correlation between Rheb and FKBP38. The results enriched the genetical and functional information of Rheb and FKBP38gene, and providing experimental evidence for clarifying the function and mechanism of mTOR Signal Pathway.更多还原