Expression Of HGM-CSF In The Pichia Pastoris

Objective:To get Pichia pastoris which express the activity hGM-CSF,hGM-CSF gene was integrated into yeast according to bias codon optimization.Methods:HGM-CSF gene was obtained by being cloned from humam gene,code gene being cloned from the human genome by the primer-5 '-extension,as the same time that code had been optumized partly to gene in the cloned procession.It was integrated into the gene secreted Pichia pastoris expression vector pPIC9K and the recombinant expression vector pPIC9K-CSF was obtained.Pichia pastoris stain GS 115/pPIC9k-CSF was constructed by transfering the expression vector pPIC9k-CSF into Pichia pastoris GS 115 competent cells via electroporation method.Under the induction by methanol,the expressed monellin was secreted into medium at the level of 3.89g/L.Result:PCR,SDS-PAGE and western blotting confirmed hGM-CSF gene was integrated into the yeast genome and expression was successful.Experiments in vivo showed that mice expression of hGM-CSF in mice was activity normal.SDS-PAGE electrophoresis revealed that the hGM-CSF expression had occurred in glycosylation,N-glycosidase F to glycosylation also confirmed this point.Conclusion:the study show that Pichia pastoris of hgm-csf in high expression had been stuctured,providing the new way to produce,to distill and to lower the cost.It makes the foundation for the industrialization.