Expression Of Fibroblast Growth Factor 18 In Insect Cells And Its Pharmacological Activity

Fibroblast growth factor 18 was first isolated from mouse embryos in 1998 and arranged into the FGF8 subfamily with FGF17. Studies have shown that FGF18 plays an important role in angiogenesis, morphogenesis and cell development. In addition, it is a secreted signaling molecules in the tissue development and it plays an important role in cartilage and bone development. However, the poor stability and low yield have been affected the progress of FGF18 on drugs. While FGF18 also tried expression in plant and prokaryotic cells, its stability and yield have not been resolved. Therefore, this paper presents a new, rapid and effective expression and purification strategy.Using the baculovirus expression system(IC-BEVS), FGF18 is expressed in insect cells. Then the characteristic of insect expression system and the nature of the FGF18 protein were studied. The optimized hFGF18 gene was enzymed and inserted into pFastBac vector. pFastBac-rhFGF18 plasmid was transformed into Escherichia coli(E coli) DH10 Bac to obtain the recombinant Bacmid-hFGF18 by using the principle of bacterial transposon. By blue-white selection, the positive recombinant plasmids were picked out. Using liposome transfection method, the recombinant plasmid was transfected into Sf9 insect cells. Repeating viral infection amplified to obtain high titer virus. The timing of harvest and the conditions of insect cells-baculovirus expression system had a significant effect on protein yield and protein quality. Therefore, the best conditions rhFGF18 protein expression were optimized to increase protein production. First, cells in the logarithmic growth phase were collected at 24, 36, 48, 60, 72, 84, 96 and 120 h post infection(hpi), and the sample at different MOIs was identified by Western Blot method. Under the optimal expression conditions, culture samples were produced. According to the isoelectric point and heparin affinity characteristics of FGF18, the protein samples were purified by heparin affinity column and cation exchange column SP-FF, and the purified protein were confirmed by Western Blot. Finally, the pharmacological activity of the purified rhFGF18 were analyzed using MC3T3-E1 cells in vitro. The effect of promoting proliferation was detected by the MTT method; Alkaline phosphatase(ALP) and osteocalcin(OCN) secretions were examined to validate the osteogenic differentiation effects of rhFGF18.By transposon principle, the optimized hFGF18 by Insect preference codon was successfully constructed a recombinant vector Bacmid-hFGF18, and the molecular weight of rhFGF18 was 22 kDa. The rhFGF18 reached a maximum at 72 h post infection with MOI of 3. In the protein purification process, we directly the purificd rhFGF18 in the heparin-affinity chromatography column with different salt concentration buffer. The rhFGF18 were eluted using 25 mM Tris containing 1.0 M NaCl. After ultrafiltration desalting, rhFGF18 were eluted with 25 mM Tris containing 0.8M NaCl. The purified protein concentration was approximately 80mg/L by BCA assay. In vitro activity assay, rhFGF18 could significantly stimulate the proliferation of mice MC3T3-E1 cells. Compared to the control, the rhFGF18 showed stimulatory effects on ALP and OCN activity after the day 7.