PC12细胞APE/ref-1cDNA的克隆和表达  

Cloning and Expression of APE/ref-1 cDNA Gene from PC12 Cells in E.coli

作　　者：谢振华[1] 王爱民[1] 刘长振[1] 马春 

机构地区：[1]哈尔滨医科大学生物化学和分子生物学教研室 [2]神经生物学教研室,哈尔滨150086

出　　处：《中国生物化学与分子生物学报》2001年第5期585-589,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：黑龙江省自然科学资金资助项目 (No.9717)

摘　　要：APE Ref 1是双功能核蛋白 ,它既能在碱基切除修复过程中切除脱嘌呤 脱嘌啶位点 ,又能促进包括AP 1、Myb和NF κB等受氧化还原调节的转录因子对DNA的结合 .从PC12细胞中抽提总RNA ,经逆转录PCR(RT PCR)扩增出APE ref 1cDNA并克隆到pQE3 1表达质粒上的BamHⅠ和PstⅠ位点间 .经测序表明 ,PC12细胞的APE ref 1cDNA以正确的阅读框架重组进入表达质粒 ,表达重组质粒pQE3 1 APE在宿主菌BL2 1中得到稳定表达 .SDS PAGE鉴定表明 ,带 6个组氨酸的融合蛋白分子量为 3 8kD ,并经Ni NTA琼脂糖亲和纯化得到电泳纯融合蛋白 .Western印迹证明重组蛋白为APE ref 1融合蛋白 .APE/Ref\|1 is a bifunctional nuclear protein that cleaves apurinic/apyrimidinic sites in DNA during base excision repair and is capable of stimulating the DNA binding activity of redox\|regulated transcription factor, including AP\|1, Myb and NF\|κB. Total RNA was extracted from the PC12 cells, and the cDNA including all coding region was obtained by RT\|PCR. The cDNA was cloned into expression vector pQE31 at \%Bam\%HⅠ and \%Pst\%Ⅰ sites with correct open reading frame. After 4 hours induced by IPTG at 37℃, the expression of 38 kD 6 histidine tagged fusion protein in \%E.coli\% BL21 was confirmed by SDS\|PAGE, and(His)6\|APE/Ref\|1 protein was purified from lysate by Ni\|NTA\|Agarose. Western blot proved that the recombinant protein is the fusion protein of APE/Ref\|1.

关 键 词：PC12细胞 APE/REF-1 RT-PCR 克隆 表达 限速酶 

分 类 号：Q78[生物学—分子生物学] Q556.5