Prokaryotic expression, purification and antigenicity identification of mouse VEGFR2 extracellular 1-4 IgG-like domains.
- Author:
Wei WANG
1
;
Xiaotao YIN
;
Yunqi LI
;
Renli TIAN
;
Jinqi YAN
;
Jiangping GAO
;
Jiyun YU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Proliferation; Escherichia coli; genetics; metabolism; Female; Gene Expression; Genetic Vectors; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Inbred BALB C; Plasmids; Recombinant Fusion Proteins; genetics; immunology; isolation & purification; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor Receptor-2; genetics; immunology; isolation & purification
- From: Journal of Southern Medical University 2013;33(1):13-17
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity.
METHODSThe gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures.
RESULTSThe mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro.
CONCLUSIONThe mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.
