Study On Secretory Expression Of Bovine IL2 Gene In Pichia Pastoris

Interleukin 2(IL2) is a cytokine with extens ve biological activity, which is the key factor for body's immune function. It has been proved that bovine IL2 can defend and cure some bovine diseases. Therefore it has great economic value and theoretical significance to express bovine IL2 gene in eukaryotic system. The methylotrophic yeast Pichia pastoris is now on 3 of the important tools used in molecular biology for the generation of recombinant protein. As yeast, P.pastoris is a single-celled microorganism that is easy to manipulate and culture. It is also a eukaryote and capable of many of the posttranslEtional modifications performed by higher eukaryotic cells, such as proteolytic processing, folding, disulfide bond formation, and glycosylation.The bovine Interleukin2 gene was inserted into the Pichia pastoris expression vector pPICZctA which contains AOX1 promoter and a-factor signal sequence .The recombinant plasmid of pPICZaA- BoIL2 was linearnized by Sac I and transformed into X-33 and KM71H by electroporation .The re:ombinant Pichia strains, identified by PCR and Zeocin resistance, were cultured and induced with methanol. SDS-PAGE analysis and Western blot showed that BoIL2 was expressed in a fused protein form with the a-factor signal peptide but unable to secrete into medium. The fused protein can be purified by nickel column for the C-terminal 6xHis-tag. The fused protein hasn't biological activity in the CTLL2 cells assay.Different signal peptide and its procession may place effection on foreign protein secretory expression. We constructed the expression vector pPICZB-NS with native signal peptide using successive extension PCR In order to improve the cleavage efficiency of Kex2, we constructed the expression vectors pPICZ a A-MG contained Glu-Ala repeats next to Lys-Arg. But the interest protein was still expressedintracellularly.In this study, we have constructed expression vectors. Bovine IL2 has been expressed in Pichia pastoris, but in a fused protein form existing intracellularly. Theeffection of signal peptide and its procession on foreign protein secretion have beenstudied.