Expressing Of VEGFR-Fc Fusion Protein In DG44Cell And Determination Of Biological Activity

Objective According to gene BANK,the sequences of the second immunoglobulin domain of VEGFR1gene (VEGFR1D2), the third human immunoglobulin domain of VEGFR2gene(VEGFR2D3) and human IgG1Fc gene are cloned to get a recombinant fusion gene VEGFR-Fc, and recombinant eukaryotic expression plasmid pD2-VEGFR-Fc was constructed,which was integrated into Chinese hamster ovary cells (DG44cells) by lipofectamine,and VEGFR-Fc fusion protein was expressed in DG44cell,a stable cell line expressing high VEGFR-Fc protein was screened out by MTX.VEGFR-Fc activity was detected using microscopy and endothelial cells ECV304model. Methods The sequence of second immunoglobulin domain of VEGFR1(VEGFR1D2) and the third human immunoglobulin domain of VEGFR2(VEGFR2D3) was synthesized, and fused with human IgG1Fc by Overlap PCR to generate the VEGFR-Fc fusion gene,which was then subcloned into eukaryotic expression plasmid pD2by XbaI、EcoRI double digestion,and transformed into competent cell of E.coli DH5a. and identified by blue-white screen, double digestion and DNA sequencing dentification.The recombinant expression plasmid pD2-VEGFR-Fc was transfected to DG44cell by lipofectamine FreeStyleTM MAX Reagent after being linearized.A stable cell line expressing high VEGFR-Fc protein was screened out by MTX.VEGFR-Fc was detectable in cell supernatant by SDS-PAGE and Westernblot, and its expression level in cell supernatant was detected by ELISA, and was purified through HiTrapTM ProteinA FF column. VEGFR-Fc activity was detected using microscopy and endothelial cells ECV304model. Results The PCR product of VEGFR1D2-VEGFR2D3gene is693bp and VEGFR-Fc is1377bp.Restriction analysis and sequencing proved that recombinant plasmid pD2-VEGFR-Fc was constructed correctly, and Western-Blotting showed that VEGFR-Fc protein was detectable in cell supernatant. A cell line expressing0.5g/L of VEGFR-Fc protein was screened out by MTX, A single brand of VEGFR-Fc protein was obtained through HiTrapTM ProteinA FF column, which is about110kDa.Microscopy and Cell model revealed that VEGFR-Fc specifically binds to VEGF to inhibit vascular endothelial cell growth. Conclusion The recombinant pD2-VEGFR-Fc vector was successfμlly constructed and was expressed in DG44cells.The expression of VEGFR-Fc in eukaryotic cells and identification of its functional activity provide experimental basis for further study on its role in angiogenesis inhibition and anticancer research.