Wu Zhong-Jun, Yang Xiao-Hong, Zheng Shu-Sen, Yang Su-Fen, Shi De
Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Hepatobiliary Pancreat Dis Int. 2004 Aug;3(3):355-9.
The highly specific vascular endothelial growth factor (VEGF) induces the growth of vascular endothelial cell. This study was to construct the eukaryotic expression plasmid of vascular endothelial growth factor165 (VEGF165) and observe its expression in vascular smooth muscles (VSMCs).
The primers were designed and synthesized according to the gene sequences of human VEGF165. The VEGF165 gene was obtained from umbilic artery tissue by the method of RT-PCR, then it was cloned to eukaryotic expression plasmid pBudCE4.1 by recombination strategy. The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion, and was sequenced. The pBudCE4.1/VEGF165 was transfected into VSMCs by using lipofection. The VEGF165 expression of mRNA and protein was detected by RT-PCR and Western blot respectively.
VEGF165 was shown about 576bp by RT-PCR. Sequencing revealed the amplified VEGF165 gene was identical with that in the GeneBank. Restrictive enzyme (Hind III, Bam HI) digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by RT-PCR and Western blot.
The recombinant eukaryotic expression plasmid pBudCE4.1/VEGF165 has been successfully constructed and expressed in transformed VSMCs. The present study has laid a foundation for VEGF165 gene therapy of vascular stenosis in the transplant organ.
高度特异性的血管内皮生长因子（VEGF）可诱导血管内皮细胞生长。本研究旨在构建血管内皮生长因子165（VEGF165）的真核表达质粒，并观察其在血管平滑肌（VSMC）中的表达。
根据人VEGF165的基因序列设计并合成引物。采用RT-PCR方法从脐动脉组织中获取VEGF165基因，然后通过重组策略将其克隆至真核表达质粒pBudCE4.1。通过限制性酶切鉴定并测序命名为pBudCE4.1/VEGF165的真核表达质粒。利用脂质体转染法将pBudCE4.1/VEGF165转染至VSMC。分别通过RT-PCR和Western blot检测VEGF165的mRNA和蛋白表达。
RT-PCR显示VEGF165约为576bp。测序表明扩增的VEGF165基因与基因库中的序列一致。限制性酶（Hind III、Bam HI）切分析显示重组表达质粒pBudCE4.1/tVEGF165构建成功。RT-PCR和Western blot证实了转染的VSMC中VEGF165在mRNA和蛋白水平的表达。
重组真核表达质粒pBudCE4.1/VEGF165已成功构建并在转染的VSMC中表达。本研究为移植器官血管狭窄的VEGF165基因治疗奠定了基础。
