Expression of recombinant human β-nerve growth factor in insect cells and purification and biological activity of expressed product

Objective To express recombinant human β nerve growth factor(rhβ-NGF) in insect cells,purify the expressed product and determine its biological activity.Methods Recombinant plasmid Bacmid +[rhβ-NGF]was constructed and transfected to insect cell line Sf9 in mediation of liposome cellfection Ⅱ.Sf9 cells were repeatedly infected with the supernatant of transfected cells,of which the culture supernatant containing target protein was collected in a large quantity.The rhβ-NGF was purified by ion exchange and molecular sieve chromatography,identified by SDSPAGE and Western blot and determined for biological activity by chick embryo dorsal root nervous ganglion culture method.Results Restriction analysis and sequencing proved that recombinant plasmid Bacmid +[rhβ-NGF]was constructed correctly.The rhβ-NGF was expressed in Sf9 cells infected with recombinant virus,which showed a single band with relative molecular mass of about 14 200 on SDS-PAGE profile and reached a purity of more than 98% after purification.The expressed protein showed specific binding to rabbit anti-human monoclonal antibody against NGF and stimulated the growth of nerve ganglion process,of which the specific activity was about 600 000 AU / mg.Conclusion The rhβ-NGF with biological activity was expressed successfully in insect cells,which laid a foundation of preparation of rhβNGF in a large quantity.