[Construction and expression of soluble vascular endothelial growth factor receptor-1 eukaryotic expression vector and its effect on proliferation of vascular endothelial cells].
This study sought to construct recombinant eukaryotic plasmid pcDNA3. 1-sFlt-1 and observe its effect on proliferation of vascular endothelial cells. Total RNA was extracted from human umbilical vein endothelial cells (HUVECs) firstly. The 1st-3rd Ig-like domains of Flt were amplified by polymerase chain reaction (PCR) from the full-length cDNA. Subsequently, the PCR product was cloned into the eukaryotic plasmid pcDNA3.l(+)/myc-His. The constructed recombinant plasmid pcDNA3. 1-sFlt-1 was sequenced. Then recombinant plasmid was transfected into Lewis lung cancer cells. RT-PCR and SDS-PAGE were used to detect the expression of soluble vascular endothelial growth factor (VEGF) receptor mRNA and protein, respectively. MTT method was used to evaluate the effect of sFlt-1 protein on proliferation of HUVECs induced by VEGF. The results showed: (1) The sequence of inserted fragment was correct. (2) Lewis lung cancer cells with recombinant plasmid transfection could express the soluble VEGF receptor mRNA and protein stably. (3) Culture supernatant of Lewis lung cancer cells with sFlt-1 could significantly inhibit the proliferation of HUVECs induced by VEGF. These data suggested that recombinant eukaryotic plasmids pcDNA3. 1-sFlt-1 was constructed successfully, sFlt-1 mRNA and protein were expressed in eukaryotic system stably and sFlt-1 protein could significantly inhibit the proliferaton of endothelial cells induced by VEGF.
Ji
Lingling
L
Department of Pathophysiology, West China School of Preclinical and Forensic Medicine, Sichuan Unitersity, Chengdu 610041, China.
Mao
Shuhua
S
Liu
Hong
H
Xu
Shiye
S
Yang
Yu
Y
Yi
Cheng
C
Huang
Ying
Y
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*
D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1