Abstract：Velvet is a natural cell growth factor library. The top tissue of the antler as the most active growth point of the deer body to store a large number of growth factors, they are the biochemical basis of the clinical role of deer antler. A variety of growth factors have important roles in promoting the proliferation of antler. The rapid growth of velvet and the generation of blood vessels in cartilage tissue are inseparable and vascular endothelial growth factor (VEGF) can promote blood cell formation and promote the formation of blood vessels. Therefore, the study of sika deer (Cervus nippon) VEGF will provide a reference for the mechanism of rapid growth of velvet. To obtain the biologically active protein, the pET-30a prokaryotic expression vector and pMD-18T-VEGF were digested with HindⅢ and BamHⅠ. The digested product was recovered separately. VEGF mature peptide gene from Cervus nippon was inserted to expression vector pET-30a by T4 DNA ligase to obtain recombinant plasmid. The recombinant plasmids were identified by PCR and identified by double digestion. The correct recombinant plasmid was made into glycerol. Proteins were expressed (0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 20 ℃) in the host Rosetta cells in this study. The results were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Target protein was purified by Ni-Agarose affinity column and renatured to obtain the biologically active protein. Target protein on the proliferation of mouse embryonic fibroblast cell line (NIH3T3) cell was analysed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). A specific band of about 519 bp was obtained by double digestion of pET-30a-VEGF with 2 restriction endonucleases BamHⅠ and HindⅢ, which were consistent with the expected results. The PCR results of pET-30a-VEGF were used as templates to show a band corresponding to the size of the target band. The results showed that the VEGF gene of sika deer was successfully inserted into pET-30a vector. Restriction enzyme mapping showed that pET-30a-VEGF expression vector was constructed successfully. The recombinant plasmid pET-30a-VEGF was transformed into Escherichia coli Rosetta competent cells and induced by IPTG. SDS-PAGE results showed that there was a specific band at about 23.4 kD as shown, which was consistent with the expected results. Optimization of the optimal IPTG concentration showed that the protein was highest expressed when the IPTG concentration was 0.6 mmol/L at 37 ℃. The best IPTG induction temperature was optimized, and when the temperature was 20 ℃, the protein was the highest expressed. The induction time was optimized. The results showed that the expression level of recombinant protein was the highest when the induction time was 16 h. Therefore, when the IPTG concentration was 0.6 mmol/L, the induction temperature was 20 ℃ and the induction time was 16 h, the recombinant protein was the highest. The purified VEGF mature peptide appeared at a molecular mass of about 23.4 kD, which was consistent with the molecular weight of VEGF mature peptide. The results of MTT showed that proliferation of NIH3T3 was promoted by different concentration of recombinant protein, and the proliferation rate of the cells increased with the increase of the concentration. Among them, the most significant impact on NIH3T3 cell proliferation was at 48 h, and the final concentration was 200 ng/mL. The results of the study will further explore the function of VEGF in the rapid value-added process of velvet and regulation mechanism to provide experimental basis.
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