ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Animals , Female , Humans , Mice , Cell Proliferation , Escherichia coli , Genetics , Metabolism , Gene Expression , Genetic Vectors , Human Umbilical Vein Endothelial Cells , Mice, Inbred BALB C , Plasmids , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor Receptor-2 , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.</p><p><b>METHODS</b>The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.</p><p><b>RESULTS</b>The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.</p><p><b>CONCLUSION</b>Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.</p>