The Expression Of Porcine Granulocyte-macrophage Colony Stimulating Factor Gene In Pichia Pastoris

Granulocyte-macrophage colony-stimulating factor(GM-CSF) is a cytokine with multifunctional biological activity.GM-CSF has a better therapeutic effect of inhibiting or adjuvant therapy on systemic inflammatory, cancer and other diseases. It can enhance the immune response of vaccines significantly, which can play a great role in clinical applications. So to express GM-CSF protein with biological activity in vitro has high value to study.In the study, porcine GM-CSF(pGM-CSF) gene was expressed in Pichia pastoris.Construction of Pichia pastoris expression vector pPIC9K-pGMFor 19T-GM which had the whole pGM-CSF coding region gene, primers were designed to amplify pGM-CSF mature sequence without signal peptide,and the gene was 384bp.By T-A cloning, pMD19-T-pGM plasmid was constructed successfully; The plasmid of pMD19-T-pGM was digested by EcoR I and Not I, pGM-CSF gene was obtained and inserted into the pPIC9K plasmid which was also digested by EcoR I and Not I. The Pichia pastoris expression vector pPIC9K-pGM was successfully constructed.Screening of recombinant GS115/pPIC9K-pGMThe expression vector pPIC9K-pGM was electrotransporation into host yeast Pichia pastoris GS115 after the liberalization by restriction endonuclease Sac I and recovering by ethanol precipitation.Milky white colonies grew on the YPD medium 3 days later,which indicated that electroporation was successful;High-copy recombinants was picked out successfully on 4mg/mL G418/YPD medium through screening by G418/YPD concentration gradient; Mut+high-copy recombinant was screened successfully by identified.When Mut+High-copy recombinants were amplified by PCR, a DNA fragments(384bp) had been gained. The results show that recombinant screening was right, which was named GS115/pPIC9K-pGM.Expression of recombinant GS 115/pPIC9K-pGM in Pichia pastoris GS115/pPIC9K-pGM were activated in YPD medium,and collected it when OD600 reached 2.0-2.5.Then diluted the collection with BMMY medium until OD600=1.0.GS115/pPIC9K-pGM was induced with 1% methanol at 30℃for target protein expression.Expressed protein was analysised by indirect ELISA and SDS-PAGE,the results showed that the target protein was up to the highest yield after4 days induced, and the product was about 18kD.Western blotting assay showed that the expressed protein could act with the positive serum of pGM-CSF.The pGM-CSF protein was used for TF-1 cell proliferation assay after filter sterilization, the results proved the protein has the ability to promote TF-1 cells proliferation, and the activity was up to (0.88～1.05)×104 IU/μg. It Indicated that the protein has powerful biologic activity.