Heterologous expression of biologically active chicken granulocyte-macrophage colony stimulating factor in Pichia pastoris
Granulocyte-macrophage colony stimulating factor (GM-CSF) is an effective nucleic acid adjuvant, which can enhance the body immunity, especially in the immune response to the DNA vaccine. In this study, we investigated the function of recombinant chicken GM-CSF (rchGM-CSF). Chicken GM-CSF (chGM-CSF) gene sequence was cloned, the signal peptide in the N terminal sequence was cut, and 3’ end of the stop codon was changed. The rchGM-CSF was labeled with a C-terminal c-myc and His antigen. The recombinant Pichia pastoris expression vector pPICZαA-rchGM-CSF was constructed by inserting the reformed cytokines genes in pPICZαA. The pPICZαA-rchGM-CSF was expressed in GS115 cells. After being screened by yeast peptone dextrose (YPD) containing high concentrations of Zeocin and direct PCR, the positive clone was cultured in flask with buffered minimal methanol (BMMY) and expression induced by methanol. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that rchGM-CSF was expressed and secreted into BMMY and the secreted peak was generally at 72 h in BMMY medium. After large-scale induced expression, the supernatant was collected by centrifuge, concentrated by centrifugal ultrafiltration and purified by His Bind Ni-NTA chromatography. The MTT (3-(4, 5-dimethylthiazolyl-2) -2, 5-diphenyltetrazoliumbromide) assay results showed that rchGM-CSF could stimulate lymphocyte proliferation. The experiment laid the foundation for further study of immune adjuvant.
Key words: Granulocyte-macrophage colony stimulating factor (GM-CSF), Pichia pastoris, secretory expression, biological activity.