人血小板源性生长因子-B链肽基因的分子克隆、表达及其对猫角膜内皮细胞体外增殖的影响
[Molecular cloning and expression of human PDGF-B chain peptide gene and its effect on cat corneal endothelial cell proliferation in vitro].
作者信息
Luo Wen-juan, Liu Mei-guang, Wang Chuan-fu, Wang Li-mei
机构信息
Department of Ophthalmology, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266001, China.
出版信息
Zhonghua Yan Ke Za Zhi. 2006 May;42(5):415-9.
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 tertrozalium salt (MTT), 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 BL21 (DE3) using western blot; MTT 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.
目的
构建血小板源性生长因子B（PDGF-B）过表达的pET-PDGF-B质粒，并研究其对猫角膜内皮细胞体外增殖的影响。
方法
采用基因重排技术构建插入pET-PDGF-B的原核载体。从大肠杆菌BL21（DE3）中获得重组PDGF-B。通过改良的四氮唑盐（MTT）、倒置相差显微镜和透射电子显微镜检测重组PDGF-B对培养的猫角膜内皮细胞增殖和形态变化的影响。
结果
将PDGF-B肽基因插入原核表达载体pET-28a（+），经序列分析证实；用蛋白质印迹法从大肠杆菌BL21（DE3）提取物中证实pET-PDGF-B有一条27000的蛋白条带；MTT法显示PDGF-BB促进猫角膜内皮细胞的增殖。
结论
构建了PDGF过表达的pET-PDGF-B质粒载体。重组PDGF-BB促进培养的猫角膜内皮细胞增殖。
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