臧 闻 ,董 剑, 高 翔 ,陈其皎,王延鹏 ,李志业 ,史红飞.小麦品种“陕253”PDI基因的克隆、原核表达及蛋白纯化[J].麦类作物学报,2011,31(5):799 |
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” |
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DOI:10.7606/j.issn.1009-1041.2011.05.002 |
中文关键词: 小麦 蛋白质二硫键异构酶(PDI) 基因克隆 原核表达 蛋白纯化 |
英文关键词:Wheat Protein disulfide isomerase (PDI) Gene isolation Prokaryotic expression Purification |
基金项目:国家自然科学基金项目(30900896);陕西省“13115”科技创新工程重大项目(2007ZDKG 01);国家现代农业产业技术体系专项(CARS 3);中央高校基本科研业务费专项资金项目(QN2009007)。 |
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中文摘要: |
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.coli BL21(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。 |
英文摘要: |
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. |
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