老年神经变性疾病蛋白基因研究:3种α-突触核蛋白提取纯化方法的比较  

Protein and gene of neural degeneration disease in the elderly:comparison of three methods to extract and purify alpha-synuclein

作　　者：李淑婷[1] 陈彪[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神经生物学室,北京市100053

出　　处：《中国临床康复》2004年第25期5294-5296,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家科技部973项目资助(G2000057005)~~

摘　　要：目的:用经济、简便、快捷的方法得到大量、高纯度的α-突触核蛋白。方法:将重组的proEXNACP,pGEXNACPH和pTYBNACP质粒分别转入大肠杆菌DH5α,BL21和ER2566细胞进行培养增菌,IPTG诱导产生α-突触核蛋白;超声破碎、离心粗提取得到融合的α-突触核蛋白;分别经Ni-NTAresin,GlutathioneSepharose4B和ChitinBeads纯化,得到α-突触核蛋白。用SDS-PAGE电泳观察纯度、Western杂交检测正确性、用BCA蛋白定量法测定蛋白含量。结果:3种方法均能得到较高纯度的α-突触核蛋白,其中用Ni-NTAresin提取纯化的α-突触核蛋白的纯度最高;蛋白收获量分别是10mg/L,5mg/L和5mg/L;经济耗费和实验的繁简程度没有较大差异。结论:3种方法均可作为α-突触核蛋白的提取方法。AIM:To obtain a large quantitative and highly purified alpha-synuclein(α-synuclein) with economical,convenient and quick methods.METHODS:The recombined proEXNACP,pGEXNACP and pTYBNACP were transferred into escherichia coli DH5α,BL21 and ER2566 competence cells,respectively.The α-synuclein were induced by IPTG and confluencing α-synuclein were extracted by ultrasonic breaking,centrifugation.α-synuclein were obtained by purification of Ni-NTA resin,Glutathione Sepharose 4B and Chitin Beads.Purity was observed by SDS-PAGE electrophoresis,correctness was detected by using Western Blot and the protein content was determined by BCA protein quantitative method. RESULTS:α-synuclein of high purity could be obtained by all the three methods,with Ni-NTA resin ranking first in purity.The protein productions were 10 mg/L,5 mg/L and 5 mg/L,respectively.There were no obvious differences in the economical consumption and degree of complex and simple. CONCLUSION:All the three methods can be used to purify α-synuclein.

关 键 词：老年 神经变性疾病 蛋白基因 Α-突触核蛋白 提取 纯化 

分 类 号：R741[医药卫生—神经病学与精神病学]