hCuZn-SOD基因的克隆、表达及其产物的初步纯化和表征

为获得hCuZn-SOD基因,根据GenBank中人铜锌超氧化物歧化酶(hCuZn-SOD)基因碱基序列,设计扩增引物,用RT-PCR方法从人的肝细胞中克隆出hCuZn-SODcDNA序列,并将它插入pUCm-T载体中,经过DNA序列测定证实,该片段序列的一个碱基发生突变(与报道基因相比),引起其编码第116个氨基酸由G变为D.采用定向克隆的方法将hCu Zn-SOD的cDNA片段克隆到表达载体pGEX-2T上,构建表达质粒pGEX-SOD,并转化大肠杆菌BL21(DE3).SDS-PAGE分析证实,经IPTG诱导,融合蛋白GST-SOD获得高效表达.破碎菌体、上清经谷胱甘肽亲和层析初步纯化后,得到纯度达90%的目的蛋白.活性实验表明,GST-SOD具有生物活性.

Molecular cloing, expression of hCuZn-SOD and its preliminary purification and characterization

To obtain the hCuZn-SOD cDNA, primers were designed according to the full cDNA sequence in the GenBank. Full gene was obtained by RT-PCR from liver cell, subcloned into pUCm-T vector and sequenced. The data indicate that there was one base substitution leading subsequently to the change of one amino acid(116)compared to that previously reported.And then, to construct expression plasmid pGEX-SOD, hCuZn-SOD cDNA was inserted into expression vector pGEX-2T. After IPTG induction, the fusion protein GST-SOD was highly expressed in E.coli BL21(DE3).The product(molecular weight 45?KD)was obtained with 40% yield of total bacterial proteins in soluble form. The results of activity experiment suggested that GST-SOD has biological activity. After preliminary purification through GST-Sepharose 4B affinity chromatograghy, the purity of the product was above 90%.