人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究

目的：制备人MCM7 N-端的GST融合蛋白（GST-MCM7N），研究其是否和雄激素受体（AR）蛋白存在直接的相互作用。方法：利用RT-PCR获得长度为744 bp的人mcm7 基因N-端cDNA碱基片段，把该片段构建到原核表达载体pGEX-5x-3中，转化大肠杆菌BL-21菌株。经IPTG诱导菌株基因表达产生了GST-MCM7N融合蛋白；使用Glutathione Sepharose 4B球珠分离纯化。利用GST pull-down 技术把纯化的融合蛋白分别和前列腺癌细胞LNCaP细胞裂解液及基因工程获得的AR蛋白孵育，检测MCM7蛋白的 N-端是否和AR蛋白直接相互作用。结果：酶切鉴定及基因测序表明，mcm7 基因cDNA的 N-端片段被构建到表达载体pGEX-5x-3中；SDS-PAGE及Western blotting结果分别显示，本研究获得了GST和GST-MCM7N融合蛋白；GST pull-down 结果证实GST-MCM7N和AR蛋白存在相互作用。结论：MCM7蛋白的N-端和AR蛋白至少在体外可以发生直接的相互作用。

Expression, Purification of N-terminus Fusion Human MCM7 and a Study on Its Interaction with AR

744 b Pof N-terminal of mcm7 gene was amplified by PCR and was cloned into prokaryotic expressing vector pGEX-5x-3 to construct pGEX-5x-3/MCM7N.A batch of E.coli BL-21 were transformed with pGEX-5x-3 and pGEX-5x-3/MCM7N,respectively.GST protein and GST-MCM7N fusion protein were obtained from the recombinant E.coli BL-21 after IPTG induction and were purified with Glutathione Sepharose 4B.The fusion proteins with 54 kDa molecular weight were specifically recognized by both anti-GST antibody and anti-MCM7 ant...