PC12细胞谷胱甘肽二硫键氧化还原酶cDNA的克隆表达与序列分析

Cloning expression and sequence analysis of glutaredoxin from PC12 cell line

目的获得PC12细胞谷胱甘肽二硫键氧化还原酶全长cDNA及表达的蛋白质,分析比较该cDNA及外推的蛋白质序列。方法用逆转录PCR(RT鄄PCR)获得该蛋白cDNA并将其分别克隆和亚克隆于PUC18和PQE31载体,受体细胞为BL21。亲和层析分离重组蛋白,SDS电泳分析表达产物。结果PC12细胞谷胱甘肽二硫键氧化还原酶全长cDNA含324个碱基,编码含107个氨基酸的蛋白质,并与小鼠和人的该序列有高度同源性,获得了具有活性的谷胱甘肽二硫键氧化还原酶。结论构建了PC12细胞谷胱甘肽二硫键氧化还原酶cDNA在大肠杆菌细胞的克隆表达体系;测定了该蛋白cDNA序列并推导出它的氨基酸排列顺序;证明了该蛋白与人和小鼠具有高度同源性。

Objective To clone glutaredoxin full length cDNA to obtain its expressing protein from PC12 cell, and to compare the hypothetical amino acids sequence of this protein with the cDNA sequence. Methods Firstly, glutaredoxin cDNA was obtained by means of RT-PCR and then it was cloned and subcloned into PUC18 and PQE31 respectively. Secondly, the protein was expressed in E.coli BL21 and purified by affinity chromatography. Finally, the expressed protein was analyzed by SDS-PAGE. Results Our experiment elucidate that, on the one hand, the full cDNA sequence of glutaredoxin from PC12 cell included 324 bases and the protein was encoded by 107 amino acids, on the other hand, our cDNA sequence shared high homology with the cDNA of both human and mouse. As a result, the recombinant glutaredoxin protein from PC12 cell was acquired and exhibited its enzyme activity. Conclusions A cloning and expressing system of glutaredoxin cDNA from PC12 cell in E.coli is constructed. The glutaredoxin cDNA sequence is determined and the amino acid sequence can be inferred later. The cDNA sequence from PC12 cell shares high homology with that of the human and mouse.