摘要
目的构建血小板源性生长因子B(PDGF B)原核表达载体,表达足量的PDGF BB蛋白,作用于体外培养的猫角膜内皮细胞,观察角膜内皮细胞增殖情况。方法从健康剖宫产妇胎盘组织中提取总RNA,通过逆转录聚合酶链反应(RT PCR),扩增出PDGF BcDNA,将其克隆至含T7启动子的质粒pET28a(+)中,构建表达质粒pET PDGF B,转化大肠杆菌BL21(DE3),获得表达菌株BL PDGF B,进行PDGF BB蛋白的表达,以Ni2+NTA树脂对表达蛋白纯化、复性;将得到的PDGF BB蛋白作用于体外培养的猫角膜内皮细胞,用MTT法分析其对细胞增殖的影响;通过倒置相差显微镜、透射电镜观察细胞形态变化以及细胞内部超微结构。结果经基因测序证明,成功构建出pET PDGF B质粒;凝胶自动扫描分析表明,PDGF BB在BL21(DE3)大肠杆菌中得到了高效表达,表达的PDGF B单体蛋白经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS PAGE),显示了1条特异蛋白带,相对分子质量约为27000;MTT法证明所表达的PDGF BB蛋白可促进体外培养的猫角膜内皮细胞增殖。结论pET PDGF B原核表达载体的成功构建和PDGF BB蛋白制备纯化为生产活性PDGF BB蛋白及其进一步功能研究奠定了基础。PDGF BB蛋白具有促进猫角膜内皮细胞增殖的作用。
Objective To construct pET-PDGF-B plasmid with over expression of PDGF and investigate its effects on cat corneal endothelial cells proliferation in vitro. Methods Gene rearrangement technique was used to construct the prokaryotic vector with insertion of pET-PDGF-B. The recombinant PDGF-B was obtained from E. coli BL21 （DE3）. The effects of recombinant PDGF-B on proliferation and morphologic changes of cultured cat corneal endothelial cells were assayed by modified tertrosalium salt （MTY）, inverted phase contrast microscope and transmission electron microscope. Results PDGF-B peptide gene was inserted into prokaryotic expression vector pET-28a（ ＋ ）, which was confirmed by sequence analysis; a 27 000 protein band was demonstrated as pET-PDGF-B from extraction of E. coli BI21 （DE3） using western blot; MTY assay showed PDGF-BB promoted the proliferation of cat corneal endothelial cells. Conclusions A constructed pET-PDGF-B plasmid vector over expression of PDGF is produced. Recombinant PDGF-BB promotes cultured cat corneal endothelial cells proliferation. （Chin J Ophtalmol,2006, 42 ： 415-419）
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2006年第5期415-419,共5页
Chinese Journal of Ophthalmology
基金
国家自然科学基金资助项目(30572011)
关键词
血小板源性生长因子
基因表达
内皮
角膜
内皮细胞
细胞分裂
Platelet-derived growth factor
Gene expression
Endothelium, corneal
Endothelial cells
Cell division
