Expression And Production Optimization Of Porcine Beta Interferon In Pichia Pastoris

There were many functions of interferons including antivirus,adjusting auxesis and activating immunity.PoIFN-? which is mainly secreted by fibrocyte possesses the abilities of induce the production of multiple antivirus protein,raise the molecular expression level of MHC I and enhance the liveness of NK cell.Though PoIFN-? is obtainable by stimulating some cells with virus infection or ployI:C,but the PoIFN-? gained by this method is low at quantity and hard to purify.Therefore,obtaining PoIFN-? through vitro expression has become the key to the problem above.There were two kinds of vitro expression:prokaryotic expression and eukaryotic expression,but because there were three glycosylation site in PoIFN-?,and prokaryotic expression is not compatible with galactosylated modification,so the Pichia pastoris of eukaryotic expression system was chosen as carrier to carry out the vitro expression of PoIFN-?.DNA sequence of PoIFN-? mature peptide has been redesigned in this study,119 bases were replaced,99 optimized trinucleotides account for 60% of all trinucleotides.The content of GC base was lowered to 41.1%,and the genic trinucleotide adaptation index was raised from 0.69 to 0.85 due to this.The optimized gene was successfully built into inducive expression vector pPICZ?A and constitutive expression vector pGAPZ?A,both of them were expressed in Pichia pastoris X33.Constitutively secretory expression of PoIFN-? in Pichia pastoris was gained for the first time,the basis for its expression in pGAP expression system was laid.But both of them were low in output,and difference between them is not considerable.Because methanol which is toxic,flammable and explosive was used as revulsant in the inducive Pichia pastoris expression system,the procedure of turning PoIFN-? into industrialized product has been affected.Therefore,on the basis of constitutively secretory expression of PoIFN-? in Pichia pastoris,the optimum condition was determined through investigating the effection on expression of time,pH,immunity,carbon source,substrate and inoculum concentration.The optimum condition was: inoculate 1% of 2mg immune bacterial strain on BMGY substrate,supplement 1% propanetriol as carbon source after 24 h.The highest expression output was reached in 48 h as 11.25mg/L approximately.The expression time was shortened by 48 h comparing to traditional YPD substrate and the output was raised by 4.3 times approximately.The highly efficient expression of exogenetic gene in Pichia pastoris was affected by many factors especially technics,carbon source and so on.In this study,the optimal expression conditions of PoIFN-? in pGAP expression system were explored,thus simplifying the downstream expression of PoIFN-?(without adding methanol,replacing carbon source).The basis of continuously production inside fermentation cylinder of PoIFN-? has been laid.