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重组人CD40胞外结构域在大肠杆菌中的表达和纯化鉴定 被引量：2

Purification and identification of human CD40 extracellular domain expressed in Escherichia coli
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摘要 目的构建人CD40胞外结构域(exCD40)的原核表达载体,获得可溶性产物。方法以RT-PCR方法从人白细胞总RNA中扩增编码CD40胞外区的cDNA,构建羧基端融合His6标签的exCD40的原核表达载体,在大肠杆菌中表达,并对表达产物进行复性、纯化和鉴定。结果构建了exCD40的原核表达载体,并在大肠杆菌中获得高表达,Mr为23 000,与理论大小相符,表达产物主要存在于包涵体中,经Ni2+-NTA柱上复性和纯化获得纯度达95%的可溶性exCD40蛋白,该蛋白能与细胞上的CD40L结合。结论从大肠杆菌中成功获得具有配基结合活性的可溶性exCD40蛋白。 Objective To construct a prokaryotic expression vector for human CD40 extracellular domain （exCD40） and to obtain a soluble product. Methods cDNA encoding human CD40 extracellular domain was amplified by RT-PCR from the total RNA of human leukocytes, and then a prokaryotic expression vector for exCD40 fused at its carboxyl terminus with a polyhistidine tag was constructed. The exCD40 protein was expressed in Escherichia coli （E.coli） and its refolded and purified products were identified. Results The prokaryotic expression vector for exCD40 was constructed. The recombinant protein was expressed in high yield in E.coli. The relative molecular weight of the protein was 23 000, agreed with theoretical calculation. The expressed exCD40 could bind to CD40L on cells and was mainly presented in inclusion bodies. After refolding on Ni^2＋-NTA column, purity of the soluble exCD40 could reach 95%. Conclusion Soluble exCD40 protein possessing ligand-binding activity is successfully obtained from E.coli.
作者 何贤辉 徐丽慧 刘毅 杜丽蕊
机构地区 暨南大学组织移植与免疫教育部重点实验室 暨南大学生物工程研究所
出处 《免疫学杂志》 CAS CSCD 北大核心 2006年第2期195-198,共4页 Immunological Journal
基金 国家自然科学基金(30371651) 国家自然科学基金重点(30230350) 国家重点基础研究发展规划("973")(G200057006)资助项目
关键词 CD40 原核表达 包涵体 复性 CD40 Prokaryotic expression Inclusion body Refolding
分类号 Q78 [生物学—分子生物学]
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参考文献12

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同被引文献18

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免疫学杂志
2006年 第2期

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