一种骨桥蛋白小分子肽的原核表达与生物学活性鉴定  

Prokaryotic expression and identification of a small peptide of osteopontin

作　　者：刘颖慧[1] 田文嘉[1] 郑斌[1] 韩梅[1] 温进坤[1] 

机构地区：[1]河北医科大学生物化学与分子生物学研究室,河北石家庄050017

出　　处：《中国病理生理杂志》2009年第5期848-852,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30770787);国家"973"计划资助项目(No.2008CB517402);河北省创新药物新品种专项资助项目(No.07276403D-2)

摘　　要：目的:用大肠杆菌(E.coli)表达骨桥蛋白13肽(OPN13),经亲和层析纯化后检测其生物学活性。方法:运用基因重组技术,将骨桥蛋白分子中含黏附序列的13肽cDNA片段与携带His编码序列的原核表达载体连接构建融合蛋白表达质粒pET-32c-OPN13。将重组质粒转化E.coliDH5α宿主菌后,对诱导融合蛋白表达的条件进行优化。表达产物His-OPN13经Ni-NT AHis Bind Resin金属离子螯合层析纯化后,分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响。结果:所表达的His-OPN13融合蛋白在宿主菌中以胞浆可溶性的形式存在。经亲和层析可得到高纯度的His-OPN13融合蛋白。产物活性分析表明,His-OPN13融合蛋白能特异性地抑制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移。结论:OPN13肽可在大肠杆菌中高效表达,并可剂量依赖性地抑制血管平滑肌细胞黏附和迁移活性。AIM： To express osteopontin 13 peptide （OPN 13 ） in E. coli, and to test the biological activity of the purified products. METHODS： cDNA fragments containing RGD sequences were cloned into prokaryotic expression vector pET- 32c（ ＋ ） including His coding sequence to construct pET- 32c -OPN 13 plasmid. E. coli Dl-15α transformed by pET- 32c -OPN 13 plasmid was induced by IPTG at different concentrations for different times to identify the optimal induction condition. Expressed His -OPN 13 fusion protein was purified via Ni -NTA His Bind Resin metal chelation chromatography, and detected by VSMCs adhesion and migration analysis. RESULTS： His -OPN 13 fusion protein was expressed in soluble manner. The fusion proteins were purified via Ni -NTA His Bind Resin affinity chromatography. His - OPN 13 fusion protein specifically inhibited adhesion and migration of VSMCs stimulated by osteopontin in dose - dependent manner. CONCLUSION： The OPN 13 peptide is successfully expressed in E. coli DH5α. The purified His - OPN 13 fusion protein could inhibit the adhesion and migration of VSMCs stimulated by osteopontin.

关 键 词：骨桥蛋白 OPN13肽 原核表达 生物学活性 

分 类 号：Q78[生物学—分子生物学]