人早老素15’端390碱基片段的真核表达载体的构建与鉴定  

Construction and identification of eukaryotic expression vector containing human presenlins-1 cDNA 5'390 bp

作　　者：王鹏[1,2] 温玉新[1] 何欣[1] 于顺[3] 李尧华[3] 

机构地区：[1]北华大学医学院人体解剖教研室 [2]首都医科大学宣武医院北京老年病研究所神经生物研究室北京市100053 [3]首都医科大学宣武医院北京老年病研究所神经生物研究室

出　　处：《中国临床康复》2006年第10期86-88,共3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)~~

摘　　要：目的:构建表达人早老素1N端部分肽段的基因重组质粒。方法:实验于2004-12/2005-01在北京老年医学研究所神经生物研究室完成。参照人早老素1mRNA结构设计合成含适当酶切位点的聚合酶链反应引物,从质粒pBS-Presenilins1扩增其5’端390bp的cDNA片段,插入质粒pGEX-4T-1,转化入大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷诱导表达,使其在大肠杆菌中表达以谷胱甘肽S转移酶为标签的融合蛋白质。用凝血酶定点分解融合蛋白,并应用亲和层析法纯化早老素1N130肽。结果:聚合酶链反应扩增产物约400bp,核酸序列分析结果显示,扩增产物为393bp编码130个氨基酸。重组质粒在原核细胞中表达受异丙基-β-D-硫代半乳糖苷诱导呈时间依赖性递增。纯化的目的蛋白在聚丙烯酰胺凝胶电泳上表现为单一条带,其表观相对分子质量为13000。每升菌液可收获目的蛋白4.5mg。结论:构建人早老素1N端部分肽段的基因重组质粒,制备了基因重组型早老素1N130肽,并确认其亲水特征。AIM： To construct and express the gene recombinant plasmid in human presenlins-1 cDNA 5 ＇390 bp. METHODS： The experiment was carried out in the Research Room of Neurobiology, Beijing Institute of Geriatrics between December 2004 and January 2005. The extracellular domain of presenlinsl eDNA 5＇390 bp was cloned into the recombinant pET28a-chaperonin10 prokaryotic expression vector to construct a recombinant pGEX-Presenilinsl 5＇ 390 prokaryotie expression vector. Then the recombinant plasmid was transducted into E.Coli. expression host BL21 （DE3）, the expression of fusion protein was induced by isopropyl-β-D-thiogalactopyranosid （IPTG） and labeled by glutathione S-transferase. The fusion protein was decomposed with thrombase at fixed points, presenlins-1 N130 peptide was purified affinity chromatography. RESULTS： The amplified product of polymerase chain reaction （PCR） was about 400 bp, the results of nucleic acid sequence analysis showed that the amplified products were 393 bp coded 130 amino acid. The expression of IPTG-induced recombinant plasmid in prokaryocyte was time-dependently increased. The purified target proteins showed a single band on polyacrylamidedel eleetruphoresis, and its relative molecular mass was 13 000. 4.5 mg target protein could be obtained from bacteria liquid per liter. CONCLUSION： Geue recombinant plasmid was constructed at presenilin- 1 5＇390 bp, the gene recombinant presenilin-1 N130 peptide was established, and its hydrophilic character was identified.

关 键 词：阿尔茨海默病 聚合酶链反应 质粒 

分 类 号：R749.053[医药卫生—神经病学与精神病学]