Construction of eukaryotic expression vector of fusion protein pEGFP/hVEGF165 and its expression in vascular endothelial cells.
- Author:
Miao OUYANG
1
;
Guo-nian LI
;
Yi-you ZOU
Author Information
1. Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha 410008, China.
- Publication Type:Journal Article
- MeSH:
Cells, Cultured;
Endothelial Cells;
metabolism;
Gene Expression;
Genetic Vectors;
Green Fluorescent Proteins;
genetics;
Humans;
Plasmids;
RNA, Messenger;
Recombinant Fusion Proteins;
genetics;
metabolism;
Transfection;
Vascular Endothelial Growth Factor A;
genetics;
metabolism
- From:
Journal of Central South University(Medical Sciences)
2007;32(6):1047-1050
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct the plasmid of human vascular endothelial cell growth factor165 and green fluorescence protein report gene eukaryotic expression vector of fusion protein pEGFP /hVEGF165, and to detect its expression in vascular endothelial cells.
METHODS:We amplified full-length of gene VEGF165 by PCR, cloned in direction in multiple clone sites of pEGFP-N1, constructed recombinant plasmid of pEGFP/hVEGF165. Through enzyme digestion, PCR, and sequencing analysis, we also performed liposome-mediated transfection of vascular endothelial cells of in vitro cultivation, and detected the expression of fusion protein pEGFP/hVEGF165 using fluorescence microscope, RT-PCR, and Western blot.
RESULTS:Both gene VEGF165 and multiple clone site of pEGFP-N1 confirmed by PCR, enzyme digestion, and sequence analysis. EGFP/VEGF protein was expressed in vascular endothelial cells after pEGFP/VEGF165 recombinant plasmid transfected vascular endothelial cells.
CONCLUSION:Fusion protein eukaryotic plasmid of report gene EGFP and VEGF165 is successfully constructed, and EGFP/VEGF can be expressed in vascular endothelial cells, which lays a foundation for the application of VEGF gene in treating ischemia vascular diseases.
