制备与鉴定导致家族性帕金森病突变蛋白质的α-突触核蛋白聚合体  

Preparation and identification of the aggregates of mutant alpha synucleins linked to familial Parkinson disease

作　　者：张晨[1] 李昕[1] 李尧华[1] 陈彪[1] 于顺[1] 

机构地区：[1]首都医科大学宣武医院,北京老年医学研究所,北京市100053

出　　处：《中国临床康复》2005年第13期39-41,i001,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30270482;30271437);北京市自然科学基金资助项目(7022011);市委组织部优秀人才专项经费~~

摘　　要：目的:制备和鉴定与家族性帕金森病发病有关的α-突触核蛋白突变体A53T与A30P的聚合体,为帕金森病患者的治疗乃至康复干预提供重要理论基础及靶点。方法:实验于2004-01/05在首都医科大学宣武医院北京老年医学研究所神经生物研究室完成。设计含适当酶切位点的聚合酶链反应引物,用聚合酶链反应法从质粒pBS-α-突触核蛋白(A53T)和pBS-α-突触核蛋白(A30P)合成α-突触核蛋白突变DNA,并将其亚克隆至谷胱甘肽巯基转移酶融合表达载体pGEX-4T-1。转化大肠杆菌BL21,以异丙基-D-硫代半乳糖苷诱导,使其表达融合蛋白GST-α-突触核蛋白(A53T)和GST-α-突触核蛋白(A30P)。用凝血酶定点分解融合蛋白,并用亲和层析法纯化突变的基因重组型人α-突触核蛋白。将重组蛋白在37℃下孵育2h制备蛋白聚合体。所制备聚合体用Westernblot分析法和透射电镜鉴定。结果:DNA测序结果证明构建载体插入正确α-突触核蛋白突变体A53T与A30P基因。基因表达产物在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳上表现为单一条带,分子量约为18ku,与所报道的该蛋白的分子量一致。Westernblot分析表明,所表达蛋白可被α-突触核蛋白特异性抗体识别,证明表达的重组蛋白为α-突触核蛋白,聚合后的α-突触核蛋白在108ku左右,相当于六聚体。聚合体在电镜下呈短的丝?AIM:To prepare and identify the aggregates of A53T and A30P mutant of α synucleins(α Syns) linking to familial Parkinson disease to provide important theoretical bases and targets for the treatment and rehabilitative intervention of Parkinson disease. METHODS:The experiment was performed in Research Department of Neurobiology,Beijing Institute of Geriatrics,Xuanwu Hospital,Capital University of Medical Sciences from January to May 2004.The PCR primers containing proper enzymatic sites were designed.The encoding sequences for human A53T and A30P α Syns were subcloned into the pGEX 4T 1 expression vector of glutathione S transferase(GST) fusion protein synthesized from pBS α Syn(A53T) and pBS α Syn(A30P).The reconstructed plasmids pGEX α Syn(A53T) and pGEX α Syn(A30P) were transformed into E.coil BL21,and the fusion proteins GST α Syn(A53T) and GST α Syn(A30P) were induced by isopropyl D thiogalactopyranoside(IPTG),purified by Glutatione Sepharose 4B column affinity chromatography and digested with thrombin to obtain recombinant A53T andA30P α Syn proteins.After lyophilized,the proteins were incubated at 37 ℃C for 2 hours to make aggregated mutant α Syns in vitro.The protein aggregates were identified using Western blot analysis and transmission electron microscopy. RESULTS:DNA sequence analysis proved correct reconstruction of A53T and A30P.The purified recombinant A53T and A30P α Syns were separated on SDS PAGE as a single band of 18 ku,which was identical to the reported molecular size of this protein.Western blot analysis showed that the mutant proteins and their aggregates could be recognized by an antibody specifically against α Syn.The size of the aggregated α Syns was about 108 ku,equal to the size of α Syn hexamers.The aggregated α Syns under electron microscope represented short filamentous structures. CONCLUSION:The above results suggest that the aggregate models of the mutant human α Syn A53T and A30P have been successfully produced in vitro.The result

关 键 词：帕金森病 突触蛋白类 突变 

分 类 号：R742.5[医药卫生—神经病学与精神病学]