Expression Of RhBMP-7 By U2-OS Cells

Human bone morphogenetic protein-7(hBMP-7),also known as osteogenic protein-1(OP-1),belongs to the transforming growth factor-beta superfamily.h BMP-7 can induce the differentiation of animal or human mesenchymal stem cells into bone,cartilage,ligament,tendon and nerve tissue,which can be used to promote spinal fusion and treat fractures,bone defects nonunion,avascular necrosis of the femoral head and it is broadly applied in the field of clinical orthopedics,However,some problems was difficult to overcome in the gene engineering expression of the protein encountered in the prokaryotic expression system,It is difficult for the refolding of this protein due to the lack of post-translational processing,modification mechanism,In addition the difference existed in glycosylation between Pichia pastoris and human are not beneficial for the expression of this protein.The recombinant human bone morphogenetic protein-7(rh BMP-7)expressed in CHO cells is active,but the expression level is very low,and it does not yet have the industrial conditions.Therefore,few companies at home and abroad can produce the protein,so it is very few expensive.so it is crucial for the industrialization of rhBMP-7 to overcome the above difficultiesObjectives:Human cancer cells characterized as possessing complete post translational mechanism,infinitely proliferating and easy to be cultivate in vitro.It is challenging to make them used to express protein with clinical value andcomplex structure.In this study,human umbrella tumor U2-OS cells was applied for the expression of rh BMP-7 protein to study the practicability of U2-OS cells used for the expression of rhBMP-7 and may provide a novel solution.for the production of complex structural proteins.Methods:(1)Expression of rhBMP-7 in U2-OS cells:The eukaryotic expression plasmid pcDNA3.1-rhBMP-7 was constructed and the eukaryotic expression plasmid was transfected into U2-OS cells by Lipofectamine2000.Real-time fluorescence quantitative PCR was used to test the expression of rhBMP-7 at gene level and the expression of rhBMP-7 was detected by Western Blot at protein level.(2)Secretory rhBMP-7 expression: Analysing the effect of three signal peptides(hBMP-7 self-signal peptide B7,human matrix metalloproteinase-9 signal peptide M9,human plasminogen peptide SP)on U2-OS cell secretion.Construction of eukaryotic expression plasmid pcDNA3.1-rhB7-hBMP7,pcDNA3.1-rh SP-hBMP7,pcDNA3.1-rhM9-hBMP7.The eukaryotic expression plasmid was transfected into U2-OS cells by Lipofectamine2000.The expression of rhBMP-7 gene was detected by real-time fluorescence quantitative PCR at gene level.Western blot was used to detect the expression of rhBMP-7 in U2-OS cells at protein level.The expression of rhBMP-7 in U2-OS cells was detected by enzyme-linked immunosorbent assay(ELISA).(3)Purification and analysis of rhBMP-7: rhBMP-7 protein was purified by affinity chromatography.The rhBMP-7 protein was digested by restriction endonuclease digestion with rhBMP-7,and purified rhBMP 7 on NIH3T3 cells,and the biological activity of rhBMP-7 was analyzed by detecting the ALP activity of NIH3T3 cells.Results:(1)Western Blot showed that the molecular weight of rhBMP-7 protein was 65-75 KDa,and the expression of rhBMP-7 in the experimental group was higher than that in the negative control group.The results showed that rhBMP-7 could be expressed in U2-OS cells.(2)To study the expression of secretory rhBMP-7 in rhBMP-7,the expression of rhBMP-7 in U2-OS cells was detected by Western Blot and ELISA.The results showed that rhBMP-7 was expressed in U2-OS cells.The results showed that the expression of rhBMP-7 was higher than that of rhBMP-7.(3)rhBMP-7 protein was purified by nickel column.The concentration of rhBMP-7 protein was 28.3 mg / L by BCA method,and it could be completely dissolved in buffer.RhBMP-7 showed a single band by SDS-PAGE and had good purity.(4)Glycosylation of rhBMP-7 by restriction endonuclease(Endo H)was carried out.The molecular weight of rh BMP-7 protein was 34-40 KDa and the molecular weight of rhBMP-7 was 65-75 KDa.The molecular weight of rhBMP-7 protein was 17-20 KDa,and the molecular weight of rhBMP-7 was 34-40 KDa.According to Endo H digestion rhBMP-7 results: U2-OS cells expressed rhBMP-7 protein glycosylation modification.(5)The effect of rh BMP-7 on alkaline phosphatase(ALP)in NIH3T3 cells showed that ALBMP-7 culture medium significantly enhanced the ALP activity of NIH3T3 cells,and showed a significant relationship between U2-OS Cells expressing rhBMP-7 are biologically active.Conclusion:(1)The results of this study show that rh BMP-7 can be expressed by U2-OS cells,and the signal peptide can affect the secretion efficiency of the expressed protein.HBMP-7 itself mediates the expression of rhBMP-7 in U2-OS cells Protein is high.(2)U2-OS cells were glycosylated to express rhBMP-7 protein,and the protein was soluble.The results of bioassay showed that rhBMP-7 expressed in U2-OS cells had biological activity.(3)The results of this experiment show that U2-OS cells can be used as expression system for the preparation of complex structural proteins.This is the basis for large-scale expression and preparation of complex cytokines,human antibodies and vaccines.