摘要
目的:构建血管内皮生长因子(VEGF)165原核表达载体,在大肠杆菌中表达,获得重组VEGF165,并通过细胞和鸡胚尿囊膜试验鉴定其生物学活性。方法:本实验室保存的T-VEGF165质粒,经BamHⅠ和EcoRⅠ双酶切,将其克隆至原核表达载体pET28a(+)中,获得含人VEGF165全序列基因的表达载体pET28a(+)-VEGF165,将其转化至大肠杆菌BL21(DE3),用IPTG诱导表达,对表达产物进行纯化,获得VEGF165蛋白。通过体外促进HUVEC细胞增殖及鸡胚尿囊膜血管增生实验鉴定其生物学活性。结果:成功构建了VEGF165蛋白的原核表达载体,表达纯化了VEGF165蛋白,经过细胞增殖试验及鸡胚尿囊膜血管增生实验,证明该蛋白具有良好的促进血管内皮细胞增殖的生物学活性。结论:成功地获得了人VEGF重组蛋白,并证实了该蛋白具有促进血管内皮细胞增殖活性。
Objective： To construct expression vector of vascular endothelial growth factor165, and express in Escherichia coli （ E. coli）. The biological activity of this protein was tested by cell test and effect of chicken embryo allantoic membrane （CAM）. Methods： T- VEGF165 was digested by restriction endonucleases, Then it was cloned into pET-28a（ ＋ ） expression vector, and transformed into Escherichia coli （E. coli） DH5α. The plasmid DNA was extracted, and The right insertion was retransformed into E. coli BL21 （DE3）. The cultures induced by Isopropy-β-D-thiogalactoside（IPTG） was purified and renaturated on the Ni^2＋ -NTA resin. The biological activity of VEGF165 was tested through the effect on proliferation of human umbilical vascular endothelial ceil （HUVEC） and the CAM. Results： Expression recombinants of VEGF165 were obtained. This protein has the biological activity of promote angiogenesis. Conclusion： The VEGF165 expressed in E. coli, and the protein possesses the activity of promote angiogenesis
出处
《江苏大学学报（医学版）》
CAS
2007年第2期121-123,共3页
Journal of Jiangsu University：Medicine Edition
作者简介
陈小波（1982-），男，江苏沭阳人，硕士研究生；康向东（联系人），教授，硕士生导师，E-mail：Xd_kang@msn。
