构建α-synuclein原型基因能在大肠杆菌中高效表达的质粒 医学论文.docVIP

构建α-synuclein原型基因能在大肠杆菌中高效表达的质粒 摘要 α-synuclein蛋白在体内异常折叠是帕金森病发生发展的重要原因，从α-synuclein来源的小分子多肽和分子伴侣DJ-1可以抑制α-synuclein的聚集。利用定向PCR技术体外扩增出GFP和α-synuclein原型的全长基因，将GFP重组到由PBR323改构的V2载体中，得到重组体LG；然后以LG 作为载体，以合成的9肽RGGAVVTGR为插入片断进行亚克隆，得到LG9重组体；最后以LG9为载体，α-synuclein原型为目的基因，进行克隆。偶联蛋白相互作用在细菌中的可视化技术，可以准确快速地用于鉴定α-synuclein蛋白在体内的折叠变化，和作为快速有效地筛选大量突变体的经济简便的方法。促进α-synuclein正确折叠的多肽的应用，将有希望能直接阻断α-synuclein蛋白错误折叠的继续产生，和阻断甚至逆转由异常聚集蛋白所造成的神经损伤。( Visualization of the Coupled Protein Folding and Binding in Bacteria ) 关键词：α-synuclein PD 蛋白质折叠 GFP 克隆 Abstract Abstract : The abnormal folding of α-synuclein is the important reason for Parkinson disease in the body. The peptide and chaperones can inhibit α-synuclein aggregation. Using direction PCR increase the full-length gene of GFP and α-synuclein, insert GFP to the V2 plasmid which is changed from PBR323 and get the combination LG, then utilize the peptide( RGGAVVTGR) as insert to recombine LG to get LG9.Coning the full-length gene ofα-synuclein to LG9 in the end. Visualization of the Coupled Protein Folding and Binding in Bacteria can nicety and rapidly to identify the abnormal folding changes of α-synuclein in the body, and as a sppendiness and availably economy method to pick up the abundance of mutation. The peptide which can improving the α-synuclein aggregation maybe interdict abnormal folding of α-synuclein directly，even reverse the neuropathic damnification which lead by protein aggregation. keywords：α-synuclein PD protein folding GFP clone 1．前言 1 1.1 帕金森疾病（PD）简介 1 1.1.1 PD简介 1 1.1.2 PD研究史 1 1.1.3 PD发病机理 2 1.1.4 PD的治疗方法 3 1.2 α-synuclein与PD的研究 4 1.2.1 α-synuclein的发现 4 1.2.2. α-synuclein的突变与家族性PD 5 1.2.3. α-synuclein的聚集 7 1.2.4．α-synuclein的聚集和解聚集的研究 8 1.2.5 α-synuclein基因的功能，表达和调节 9 1.3. 绿色荧光蛋白（GFP）与α-synuclein的研究 10 1.3.1绿色荧光蛋白（GFP） 10 1.3.2 GFP与α-synuclein 的关系 12 2. 材料与方法 13 2.1．材料 13 2.1.1． 实验用基因 13 2.1.2． 菌种 13 2.1.3． 试剂 13 2.1.4 酶及其缓冲液 13 2.1.5． 主要仪器设备 13 2.2 方法 14 2.2.1．缓冲液的配制 14 2.2.2 实验方案和路线： 15 2.2.3 感受态细胞的制备 16 2