POSTER NO: 323

Construction of eukaryotic expression vector for human vascular endothelial growth factor C gene

Zhiyu Liu, Jie Gao
Department of Human Anatomy, School of Medicine, Shandong University, Jinan, Shandong 250012, P.R. China

Objective: To construct eukaryotic expression vector for human vascular endothelial growth factor C (VEGF-C) gene for further study on the role of VEGF-C gene in lymphangiogenesis. Methods: According to human VEGF-C cDNA sequence, we designed and constructed a pair of specific primers which contained respectively digestion site of EcoR1 and BamH1 on the 5' end. Then reverse transcript polymerase chain reaction (RT-PCR) was employed to clone VEGF-C cDNA from human breast cancer cell MDA-MB-231. After being purified, the product of RT-PCR (1.28Kb) was ligated into a clone vector pUMT-18. The recombinant plasmids pUMT-18, first propagated in Esherichia coli DH5alpha, then extracted, purified and digested with EcoR1 and BamH1, were confirmed to contain full length of VEGF-C cDNA by agarose gel analysis and DNA sequence analysis. The resulting EcoR1-BamH1 fragment (1.27Kb) which contained the full length of human VEGF-C cDNA was ligated into eukaryotic expression vector pcDNA3.1(-) digested with EcoR1 and BamH1. The pcDNA3.1(-)/VEGF-C, digested with EcoR1 and BamH1, was found to contain the VEGF-C cDNA sequence by agarose gel electrophoresis. Results: The product of RT-PCR contained the human VEGF-C cDNA. The recombinant pUMT-18 contained correct nucleotide sequence for full length of human VEGF-C cDNA fragment by DNA sequence analysis. The VEGF-C cDNA fragment had been inserted into the eukaryotic expression vector pcDNA3.1(-). Conclusion: The pcDNA3.1(-)/VEGF-C, a eukaryotic expression vector for human VEGF-C , is constructed successfully.