Expression And Identification Of A CD25Binding Related Sites Modified Ovine Interleukin-2

The recombinant human IL-2which being wildly used in clinic has a mutation of Cys125to Ala125,.in order to ensure the rest of two Cys residue form a correct disulfide bond.In the last five years, researchers blocked the IL-2R binding related sites of IL-2byusing an monoclonal antibody of IL-2. It turns out that this new IL-2/IL-2mAb complexshow a remarkable increase in its ability of proliferate effector T lymphocytes that morethan two times to native IL-2, and a prolonged half time in vivo. The following researchshow that in the treatment of metastatic melanoma, IL-2/IL-2mAb complex can reach asimilar therapeutic effect to native IL-2, only but in1/40dose of native IL-2and extremelytrifling side effects.In our study, we mutated the IL-2R binding related sites of ovine IL-2, in order toeliminate the high affinity of IL-2to IL-2R βγ. This study intends to explore a newapproach to a similar effect to IL-2/IL-2mAb complex.We synthesized the full length of roIL-2gene with several IL-2R binding related sitesmutated. In addition, we add an EcoR I restriction enzyme cutting site in5’ end and a SnaBI restriction enzyme cutting site in3’ end. After double digestion by SnaB I and EcoR I,roIL-2was inserted in excretive expression vector pPIC9K of Pichia pastoris. Theeukaryotic expression vector pPIC9K-roIL2was constructed and then identified byrestriction enzyme digestion analyses.We transformed pPIC9K-roIL2into Pichia pastoris GS115strain, and gained thehigh-copy methanol utilization plus (Mut+) phenotype clone, named GS115-IL2.Supernatant during different time length under the induction of0.5~1.0%methanol werecollected and quantitative analysis by ovine IL-2ELISA kit, the result show that roIL-2protein reached a more than1mg/L yield in supernatant undergo72h methanol induction.SDS-PAGE analysis show that the molecular weight of roIL-2is15kDa approximately, ofwhich accordance to the theory.roIL-2was injected in mouse in order to analysis it’s vivo activity. Positive controlgroups were injected with native ovine IL-2in same dose whereas PBS for negative control.In normal state, the rate of CD4/CD8of mouse serum in roIL-2group is2.3945,1.9times topositive control (1.2510) and been significant difference; In morbid state, the rate of CD4/CD8of mouse serum in roIL-2group is2.3712,2.3times to positive control (1.0168)and been significant difference. This result indicates that roIL-2has a remarkable increase inimmunological activity compared to native ovine IL-2and been significant difference.The purification technology, broad scale fermentation condition and immunologicalactivity improved mechanisms needs further study.