[Construction of eukaryotic expression vector of fusion protein pEGFP/hVEGF165 and its expression in vascular endothelial cells].
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.


CONCLUSIONS
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.
Ouyang
Miao
M
Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha 410008, China.
Li
Guo-nian
GN
Zou
Yi-you
YY
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins