小麦品种“陕253”PDI基因的克隆、原核表达及蛋白纯化

Isolation,Prokaryotic Expression and Protein Purification of PDI Gene from Wheat Cultivar "Shaan 253"

为进一步研究控制小麦蛋白二硫键形成的关键蛋白即蛋白质二硫键异构酶(PDI),运用RT-PCR方法克隆出小麦品种"陕253"PDI基因的cDNA序列,基因登陆号为HQ911363。序列分析表明,HQ911363全长为1 539 bp,编码512个氨基酸,含有PDI典型的异构酶活性的催化位点-CGHC-和内质网驻留信号肽-KDEL-。构建该基因的原核表达载体,在宿主菌E.coliBL21(DE3)中经IPTG诱导表达融合蛋白,并对表达蛋白进行了纯化。SDS-PAGE及Western-blot检测证实融合蛋白诱导表达并纯化成功。

To further explore the function of PDI gene,a key protein regulating the disulfide formation of proteins in wheat,a cDNA fragment was isolated from wheat cultivar ＂Shaan 253＂ via RT-PCR（GenBank accession No.HQ911363）.The full-length is 1 539 bp,encoding 512 amino acid and containing typical isomerase catalytic site-CGHC-and endoplasmic reticulum retention signal peptide-KDEL-.The expression vector was constructed and transformed into the host bacteria Escherichia coli BL21（DE3）,then the expressed protein induced by IPTG was purified.The fusion protein was successfully expressed and purified by the dual tests of SDS-PAGE and Western-blot.