Expression Of Recombinant Human Fibroblast Growth Factor 17 In The Insect Cells And Its Effect On Osteoblast Differentiation

Fibroblast growth factor 17 was isolated from murine embryos by Hoshikawa,a Japanese scientist in 1998.It is a member of the protein type fibroblast growth factor family.The same subfamily as FGF8 and FGF18 is called the FGF8 subfamily.During embryonic development,FGF17 is preferentially expressed in the brain,particularly the brain and cerebellar cortex.In the process of embryonic development,it plays important roles in the development of different many organs.While on the development of nervous system,bone,arteries,tumors and other aspects also play critical roles.As a key protein,FGF17 has clinical value and research significance,but how to obtain enough amount of FGF17 with high purity and good activity for studying its effect on MC3T3-E1 pre-osteoblast is the main content in the theses.The research contents are as follows:(1)The human FGF17 gene with insect preference codon was optimized by codon optimization tool OPTIMWIZ,the recombinant baculovirus vector Bacmid-rh FGF17 was successfully constructed by restriction endonuclease digestion,ligation,competent cell transfection and blue-white screening.(2)Using liposome transfection method,the recombination plasmid was transfected into Sf9 insect cells to get the first baculovirus.In order to obtain high-yield and high-concentration protein,constantly optimize expression conditions include the Multiplicity of infection(MOI)and Time of infection(TOI).Heparin affinity chromatography column was used to purify the target protein.The results showed that when Bacmid-rh FGF17 infected Sf9 cells,the cell volume became larger,ruptured and granules.The optimal expression condition: MOI was 8 pfu/m L,TOI was 72 h.The eluent Na Cl concentration was 1.0 mol/L.The molecular weight of rh FGF17 was 22.6 KDa,the yield was 2.60 mg/L.(3)MTT assay was used to detect the effect of rh FGF17 protein on the proliferation activity of NIH3T3 and MC3T3-E1 cells,the protein was expressed in different expression systems.The effect of rh FGF17 on the differentiation of MC3T3-E1 cells was investigated by Alkaline phosphatase assay,Alkaline phosphatase staining,Quantitative Real-time PCR(RT-PCR),Alizarin red staining and quantitative.Finally,the signal pathway of rh FGF17 protein regulating the differentiation of MC3T3-E1 cell was studied by Western Blot.The results showed that the activity of rh FGF17 protein expressed in insect cell baculovirus expression system was stronger than that expressed in Escherichia coli expression system,which both can promote the proliferation of NIH3T3 and MC3T3-E1 cells.But rh FGF17 protein inhibitted the differentiation of MC3T3-E1 cells,mainly through activating the ERK and p38 on the mitogen-activated protein kinase(MAPK)signaling pathway.