Expression, Purification And Activity Determination Of Recombinant Human PDGF-Fc Fusion Protein

Background and objective: human platelet-derived growth factor-BB(PDGF-BB)can promote the proliferation of a variety of cells and participate in neovascularization and anti-apoptosis.It is widely used in clinic to treat skin trauma in patients with skin burn and severe diabetes mellitus.The expression vector of human platelet-derived gorwth factor-BB(PDGF-BB)was constructed,and the stable genetic and high expression PDGF-BB strain X33 Pichia pastoris was screened,and the purification conditions of PDGF-BB recombinant protein were explored.The protein quantity and activity were determined.Methods: by querying the genomic library and designing the witness sequence,the pPICZ?A-PDGF-BB-Fc expression vector was constructed by Zhongding Biological Company,and the PDGF-BB protein was transformed into Pichia pastoris engineering strain X33 by electroporation with BTX square wave electroporator.Stable PDGF-BB expression strains were screened by YPD solid medium containing bleomycin.And through the optimization of different induction expression time and induced expression of methanol concentration conditions.Then the expressed protein was purified by ammonium sulfate crude purification,DEAE anion exchange chromatography and Superdex75 gel filtration chromatography,which provided a reference scheme and process for further large-scale fermentation.Results: the expression vector of PDGF-BB recombinant protein was successfully constructed,and the recombinant protein strain with stable inheritance and relatively high expression was obtained.A complete separation and purification system of PDGF-BB recombinant protein was established.the purity of the purified recombinant protein was 90% by SDS-PAGE electrophoresis.the results of activity determination showed that the purity of the purified recombinant protein was 90%.The recombinant PDGF-BB protein has biological activity and can stimulate the proliferation of fibroblasts.Conclusion: by constructing the expression vector of PDGF-BB recombinant protein and optimizing the expression and purification conditions of the recombinant protein,the recombinant protein of PDGF-BB was obtained.the purity of the recombinant protein was 90%,and the recombinant protein had good biological characteristics.