酵母双杂交筛选与Fbxl16相互作用的蛋白质

发布时间：2018-04-25 13:42

本文选题：脊髓损伤 + Fbxl16　；参考：《第四军医大学》2008年硕士论文

【摘要】： 目的:前期工作中以改良的锚定消减杂交技术构建了大鼠脊髓全横断模型的差异表达cDNA文库,经点杂交排除假阳性和半定量PCR验证,从中筛选到一批差异表达EST序列。其中的新基因Scirr1编码蛋白具有“F-box+LRR”结构域,称为Fbxl16(又称SCIRR1)。Fbxl16是急性脊髓损伤后高调表达的新蛋白之一,本文目的在于寻找体内能够与Fbxl16相互作用的蛋白质,用以揭示该蛋白的功能及其在脊髓损伤修复过程中的作用,以期通过该蛋白的研究寻找促进损伤脊髓修复的药物靶标。方法:制作大鼠脊髓全横断损伤模型,提取损伤段脊髓总RNA;通过RT-PCR技术克隆新Scirr1的编码区序列;测序正确后,将该基因的EST序列构建入载体pSos的多克隆位点构建质粒pSos-scirr1;酵母双杂交预实验明确系统效率、排除pSos空载体对RAS系统的自激活活性;把该重组质粒表达的融合蛋白hSos-Fbxl16作为诱饵蛋白,以改良的酵母双杂交技术筛选大鼠脑cDNA文库,筛选出能够与Fbxl16假定阳性相互作用的蛋白质;最后用酵母双杂交回转实验对获得的假定相互作用的可靠性加以验证。结果:成功构建了融合蛋白表达质粒pSos-scirr1之后,筛选出了11个与Fbxl16假定阳性相互作用的蛋白质;通过回转实验验证,确定其中两个蛋白能与Fbxl16相互作用,两者分别是cAMP依赖蛋白激酶α型催化亚基(protein kinase, cAMP-dependent, catalytic,alpha;Prkaca)和衣被蛋白复合物ζ亚基(coatomer protein complex, subunit zeta 1;Copz1).结论: Prkaca作为cAMP依赖蛋白激酶α型催化亚基,是体内该酶催化亚基的主要存在形式。Prkaca与Fbxl16相互作用的发现是关注的要点,它和脊髓损伤有关,其传导通路cAMP依赖蛋白激酶传导通路(PKA)在脊髓损伤修复中表现出重要意义。脊髓损伤后中枢受损时,相应外周感觉神经元损伤后不能再生。在腰髓背根神经节微量注射膜通透cAMP类似物能够促进受损的中枢神经再生;刺激cAMP信号通路能够提升受损的感觉神经元的内在的生长能力。神经营养因子能增加神经元的cAMP水平,激活PKA通路,继而阻断鞘磷脂相关糖蛋白对脊髓损伤后神经元再生的抑制。在脊髓损伤局部和大脑运动皮层给入cAMP能诱导大鼠脊髓损伤后神经再生。细胞移植和cAMP联合治疗脊髓损伤再生已经受到许多学者的关注。cAMP亦能介导调控BDNF水平。所以,脊髓损伤修复相关蛋白Fbxl16和蛋白激酶A催化亚基PKA C-alpha相互作用的发现无疑为解释cAMP通路与脊髓损伤的关系,进而为阐明脊髓损伤的分子机理带来新的思路,也为脊髓损伤治疗的药物干预提供新的方向。细胞内膜系统各个部分之间的物质传递常常通过膜泡运输方式进行。衣被蛋白(coatomer protein complex)是一种构成运输膜泡表面衣被的蛋白复合体。它们具有两个主要作用:①选择性的将特定蛋白聚集在一起,形成运输小泡;②如同模具一样决定运输小泡的外部特征,相同性质的运输小泡之所以具有相同的形状和体积,与衣被蛋白的组成有关。已知三类具有代表性的衣被蛋白,即:笼形蛋白(clathrin)、COPI和COPII,各自介导不同的运输途径。Copz1是衣被蛋白I(coatomer protein complex I ,COPI)的组成蛋白,COPI是一种构成运输膜泡表面衣被的蛋白复合体,负责从高尔基体向内质网的膜泡运输。Copz1与Fbxl16相互作用的发现提示Fbxl16可能与细胞内物质转运有关。
[Abstract]:Objective : To construct the differentially expressed cDNA library of rat spinal cord full transection model with modified anchoring subtractive hybridization technique . The new gene Scirr1 encoded protein has an " F - box + LRR " domain , called Fbx16 ( also known as SCIRR1 ) . The aim of this paper is to find out the function of the protein and its role in the repair of spinal cord injury , in order to find a drug target to promote the repair of injured spinal cord through the research of the protein .

Methods : The rat spinal cord injury model was made and the total RNA of spinal cord was extracted . The coding region sequence of new Scirr1 was cloned by RT - PCR . After sequencing , the EST sequence of the gene was constructed into the multi - cloning site of vector pSos - scirr1 . The fusion protein hSos - FbX16 expressed by the recombinant plasmid was used as the bait protein to screen the cDNA library of rat brain with modified yeast two - hybrid technique . Finally , the reliability of the obtained putative interaction was verified by yeast two - hybrid rotary experiment .

Results : After the fusion protein expression plasmid pSos - scirr1 was successfully constructed , eleven proteins were screened for the positive interaction between the two proteins . Conclusion : Prkaca as a type of cAMP - dependent protein kinase 伪 - type catalytic subunit is the main form of the enzyme catalytic subunit in vivo . The discovery of interaction between Prkaca and Fbx16 is the main point of attention . It is related to spinal cord injury , and its pathway of conduction cAMP - dependent protein kinase is very important in the repair of spinal cord injury .

When the spinal cord injury is damaged , the peripheral sensory neurons can not be regenerated after injury . cAMP analog can promote the damaged central nerve regeneration in the dorsal root ganglion of lumbar spinal dorsal root ganglion . The stimulation of cAMP signal pathway can improve the intrinsic growth ability of the damaged sensory neurons .

Neurotrophic factors can increase the cAMP level of neurons , activate the pathway , and then block the inhibition of neuronal regeneration after spinal cord injury by sphingomylein - associated glycoprotein . In the local and cerebral cortex of the spinal cord , cAMP can induce nerve regeneration after spinal cord injury in rats .

Cell transplantation and cAMP combined treatment of spinal cord injury have been paid attention to by many scholars . cAMP can also mediate the level of BDNF .

Therefore , the discovery of the interaction between the protein Fbx16 and the protein kinase A catalytic subunit of protein kinase A in spinal cord injury is no doubt the relation between cAMP pathway and spinal cord injury , which provides a new way to elucidate the molecular mechanism of spinal cord injury , and also provides a new direction for drug intervention in the treatment of spinal cord injury .

The transfer of substance between the various parts of the cellular membrane system is often carried out by means of membrane vesicle transport . It has two main functions : ( 1 ) selective binding of specific proteins together to form transport vesicles ; ( 2 ) the transport of small bubbles of the same shape and volume as in the mould ; and ( 2 ) the transport of proteins from the golgi body to the endoplasmic reticulum . Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R651.2;R341

【参考文献】