[Prokaryotic expression, purification and antigenicity identification of mouse VEGFR2 extracellular 1-4 IgG-like domains].
OBJECTIVE
To 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.


METHODS
The 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.


RESULTS
The 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.


CONCLUSIONS
The mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.
Department of Urology, General Hospital of PLA, Beijing, China. wayewong261@yahoo.cn
Wei
W
Wang
Xiaotao
X
Yin
Yunqi
Y
Li
Renli
R
Tian
Jinqi
J
Yan
Jiangping
J
Gao
Jiyun
J
Yu
D000818:Animals; D049109:Cell Proliferation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2