Prokaryotic Expression And Characterization Of Recombinant VEGF-SLC Protein Targeting To Tumor

Objective:In order to combine the targeted therapy and immune enhancement therapy of lung cancer, to amplify the fusion gene of VEGF and SLC, then to construct its prokaryotic expression plasmid, and to express the recombinant protein in E.coli M15 and observe its biological activity, which would provide a basis for further study and development of the new biotherapeutics targeting to tumor.Methods:1. The VEGF gene fragment was amplified by RT-PCR by using the A549 mRNA as template, and the SLC gene fragment was amplified by PCR by using the pET32a(+)/SLC plasmid as template, then the VEGF-SLC fusion gene was amplified by Gene SOEing method and inserted into pQE30 vector to construct the recombinant pQE30-VEGF-SLC plasmid. After identified by restriction digestion and DNA sequencing, the recombinant plasmid was transformed into E.coli M15, then the recombinant E.coli was induced with IPTG, and the product was analyzed by SDS-PAGE and Western bolt. Finally, the expressed protein was purified by 6×His-Tagged Protein Purification Kit.2. The tumor targeting of rVEGF-SLC in vitro was evaluated by cell adhesion assay. The different concentration gradient of rVEGF-SLC fusion protein was coated on 96-well plates and cultured with A549 cells and MRC-5 control cells for 1 hour respectively after blocking and rinsing. After being stained with crystal violet, the tumor adhesion effect of rVEGF-SLC was observed.3. The chemotaxis ability of rVEGF-SLC on human peripheral blood lymphocyte (PBL) was detected by chemotaxis assays. The different concentration gradient of rVEGF-SLC fusion protein and control protein was added in 96-well plates, and each group had 3 multiple wells. The PBL was added in the up room of each wells, and co-cultured for 4 hours, then the cell migration and cell number was observed under Microscope, and the Chemotactic Index was calculated.Results:1. The results of restriction endonuclease analysis and DNA sequencing confirmed that the VEGF-SLC fusion gene was correctly inserted into pQE30 plasmid. The expressed protein induced by IPTG in E.coli M15 as inclusion bodies had relative molecular mass of about 28 kDa. The purity of purified recombinant fusion protein reached 90%. 2. The cell adhesion assay indicated that the A549 cells adhered to the wells coated with rVEGF-SLC significantly, and the number of adherent cells increased with increasing concentration of rVEGF-SLC fusion protein. But, as the control wells and the wells with MRC-5 control cells, no cells adhered to the wells obviously.3. The chemotaxis assays indicated that the rVEGF-SLC fusion protein induced the migration of PBL obviously. At the concentration of 3μg/L, the Chemotactic Index of rVEGF-SLC fusion protein reached a maximum.Conclusions:1. The recominant prokaryotic expression plasmid pQE30-VEGF-SLC was constructed successfully, and the high level recombinant protein was expressed and purified, which provides a basis for explorations on new ways of biological therapy for tumor.2. The experimental results that the A549 cells adhered to the rVEGF-SLC fusion protein and the MRC-5 cells did not adhere it obviously proved that the recombinant protein has the effect of tumor targeting. At the same time, the lymphocyte chemotaxis effect of this protein was confirmed by chemotaxis assays. So the rVEGF-SLC fusion protein have both the biological effects of tumor targeting as VEGF and lymphocyte chemotaxis as SLC, which lay the foundations for the study of integrated approach which combined the targeted therapy and immunotherapy of lung cancer and other tumor.