Expression And Biological Activity Of Fibroblast Growth Factor 14 Protein

Fibroblast growth factor 14 is a member of the FGF family and is mainly expressed in the central nervous system.Its gene mutation is closely related to abnormal nervous system diseases.However,the research on FGF14 and related diseases and its mechanism of action is still in its infancy.Therefore,this study cloned FGF14 gene fragment by genetic engineering technology,constructed engineering strain and expression and purification process in E.coli,and studied the proliferation of FGF14 protein on NIH3T3 and PC12 cells and the establishment of A?_25-35 induced PC12 cells.Neuroprotective effects of the Alzheimer's disease model.The research results of this topic are as follows:The FGF14 gene fragment was ligated with the pET15b expression vector to form a recombinant plasmid,which was transformed into competent BL21?DE3?,and the recombinant FGF14 protein was expressed at a high density in the E.coli expression system.The bacterial cells were disrupted by ultrasonication to release the target protein.It was verified by SDS-PAGE that most of the FGF14 protein was expressed in the form of inclusion bodies.The inclusion body was washed,the guanidine hydrochloride was denatured,and the protein was restored to its normal spatial structure by dilution and renaturation method.The higher purity FGF14 protein was obtained by purifying the expression vector with histidine tag and nickel column specific affinity.The establishment of expression purification engineering technology provides a guarantee for the application and mechanism of FGF14 protein.NIH3T3 and PC12 cells were selected to detect the proliferation-promoting biological activity of FGF14 protein by CCK8.It was found that FGF14 protein had no or proliferative activity against NIH3T3 and PC12 cells in the absence of heparin,and FGF14 had obvious proliferative activity in PC12 cells under the condition of binding to a certain concentration of heparin.The establishment of activity detection methods provides an important basis for the qualitative and quantitative determination of FGF14 protein.The neuroprotective effect of FGF14 protein was studied by using A?_25-35 to damage PC12 cells.The nerve injury model was successfully constructed by using A?_25-35 with a concentration of 40?mol/L and a treatment time of 48 h.The dose of FGF14 protein was found to be in the range of 4-16 ng/mL,which alleviated the neurological damage caused by A?_25-35.Dose-dependent,and can significantly inhibit the release of LDH and MDA in this concentration range,which proves that FGF14 protein has neuroprotective effect on PC12 cells.Fluorescence and flow-through techniques have also demonstrated that FGF14 protein has a good neuroprotective effect.Further,Western Blot technology was used to demonstrate that FGF14 protein play a neuroprotective role through the MAPK signaling pathway.