Expression And Activity Identification Of CD25Binding Epitope Modiifed HIL-2in Pichia Pastoris

Interleukin2(IL-2) is a monomeric globular glycoprotein of15kDa. Its molecular formed fourα-helices and further folded into a structure of typical type I cytokines. IL-2is mainly secreted byactivated T cells in vivo and contributes to T cells, B cells and NK cells proliferation. IL-2also couldpromote differentiation of B cells and secretion of antibody. HIL-2is a natural immune enhancer,which has strong anti-infective, anti-tumor and multiple activities. IL-2was able to inducelymphokine-activated killer cells (LAK) to proliferate in vitro, whereby people started to use IL-2combined with LAK cells to treat patients with advanced cancer, and achieved a certain effect. However,the effect of IL-2treatment of tumor was limited by serious toxic side effects. In recent years, lots ofresearch results revealed that the major role of IL-2in vivo is to maintain the survival and proliferationof regulatory T cells (Treg), which give further play to the immunosuppressive effects. For years,scientists dedicated to exploit new modification of hIL-2, expecting to enhance its antitumor activitywhile reducing its toxic side effects.In this study, according to the amino acid sequence and three-dimensional structure of hIL-2,site-directed mutagenesis of gene sequence of binding sites between hIL-2and CD25was conducted tosynthesize the modified hIL-2gene fragment by artificial synthesis method. Modified hIL-2gene wasinserted into pPIC9K to construct an efficient eukaryotic expression vector pPIC9K-mhIL-2, which wasused to be transformed into pichia pastoris genome to express mhIL-2protein. After induced expressionof mhIL-2, SDS-PAGE and Western blot were used to detect the expression level of mhIL-2in thesupernatant. The result revealed that mhIL-2was highly expressed, and had an expected15kDa sizeband. The mhIL-2protein was efficiently purified by desalting column chromatography, cationexchange column chromatography and gel filtration column chromatography. The purified mhIL-2protein had a single band of15kDa in SDS-PAGE. The concentration of the purified mhIL-2proteinwas approximately0.45g/L.In the activity experiment, mhIL-2could stimulate IL-2-dependent CTLL-2cell proliferationobviously. In the activity experiments in vivo, mhIL-2can effectively enhance the immunity of mouses.This study will lay a foundation for further study on a more effective immunopotentiator of mhIL-2with less side effects.