The Expression And Function Identification Of Human Neuritin In Baclovirus Expression System

Neurotrophic factors are polypeptides which can promote neuron survival and growth , and play an important role in development, differentiation, damage and repair of nervous systems. Neuritin was a novel neurotrophic factor that was discovered from the adult rat hippocampus cDNA library by Israel scientist Nedivi in 1993. The function research about neuritin displayed that the expression product of neuritin gene can enhance the development of neuron axon arbors by promoting synaptogenesis and by increasing the addition of new axon branches. Expression of neuritin was induced by neural activity or neurotrophins and limited in nervous , especially the regions related to nerve plasticity. These results indicated that Neuritin was related to the neural activities or neurotrophins function. Therefore the reseach about the function and mechanism of neuritin is important to neural disease. At present the research about neuritin was focused on the product of prokaryotic,there is no report about eukaryotic product of neuritin.Objective:To obtain eukarya product of neuritin using bac-to-bac baclovirus expression system.Methods: The ORF of neuritin was amplified by PCR from full-length neuritin cDNA, and the fragment was inserted into donor vector pfastbac-HT, The plasmid pfastbac-HT -neuritin was transformed into DH10Bac E.coli cells, and the recombinant shuttle vector Bacmid-neuritin was constructed by the site-specific transposition. Bacmid-neuritin was transfected into Sf9 cells via lipofectamine to generate the recombinant baculovirous . And then the recombinant baculovirous was amplified.When the recombined baclovirus infect sf9 cells again,the protein Neuritin was expressed. The expression product was analyzed by SDS-PAGE and verified by western-blot. Finally,Time–analysis of recombinant fusion protein...