重组IFN-α-IL-18体外对鸡外周血淋巴细胞转化及核因子κB的影响

摘要

目的：：探讨鸡IFN-α-IL-18融合蛋白对鸡外周血淋巴细胞转化及核因子κB( NF-κB）的影响。方法：构建重组酵母表达质粒pPICZ-IFN-α、pPICZ-IL-18和pPICZ-IFN-α-IL-18，电转化酵母菌X-33后以1％甲醇诱导，SDS-PAGE和Western blot鉴定表达IFN-α、IL-18及IFN-α-IL-18蛋白，淋巴细胞转化试验初步检测表达产物生物学活性，双抗体夹心法测定不同时段淋巴细胞中总NF-κB和细胞核中NF-κB的含量。结果：酵母培养上清液中存在分子量分别为22、23和43 kD的目的蛋白；表达的IFN-α-IL-18和IL-18均能明显促进鸡外周血淋巴细胞转化(P＜0.05），IFN-α对淋巴细胞转化有促进作用，但总体差异不显著(P＞0.05）。与对照组相比，经IFN-α-IL-18和IL-18作用的鸡淋巴细胞中总NF-κB含量明显增加(P＜0.05），细胞核中NF-κB含量差异极显著(P＜0.01），IFN-α作用的淋巴细胞中总NF-κB含量与对照组相比差异不显著(P＞0.05），细胞核中NF-κB含量显著增加(P＜0.05）。结论：IFN-α-IL-18及IL-18能够明显促进淋巴细胞转化，IFN-α促进淋巴细胞转化活性较弱，这种促淋转机制与NF-κB的表达、活化及核质转运有关，为进一步研究IFN-α-IL-18融合蛋白的功能及其在疫病防控中的作用奠定了基础。%Objective:To discuss the effects of recombinant IFN-α-IL-18 fusion protein on chicken lymphocyte transformation and the chicken nuclear factor kappa B ( NF-κB ) activity.Methods: The recombinant plasmids pPICZ-IFN-α, pPICZ-IL-18 and pPICZ-IFN-α-IL-18 were constructed,and transformed into P.Pastoris X-33 strain by electroporation.The recombinant proteins were ex-pressed under the induction of 1% methanol,and detected by SDS-PAGE and Western blot.The biological activities of the expressed products were detected by the lymphocyte transformation test,the NF-κB concentration in chicken lymphocyte and lymphocytic nucleus were detected at different times with ELISA.Results:SDS-PAGE and Western blot showed that the expressed products existed in super-natant,and the molecular weights were about 22 kD,23 kD and 43 kD,respectively.The expressed products IL-18 and IFN-α-IL-18 could stimulate chicken lymphocyte transformation (P<0.05),the biological activity of IFN-αstimulating lymphocyte transform was feeble (P>0.05).Compared with the control group,the total NF-κB concentration in chicken lymphocyte induced by IL-18 and IFN-α-IL-18 were increased (P<0.05),and the NF-κB in lymphocytic nucleus was increased significantly (P<0.01).Otherwise,the total NF-κB in lymphocyte induced by IFN-αincrease was limited (P>0.05),but the NF-κB in lymphocytic nucleus showed the remarkable increase ( P<0.05 ) .Conclusion: IFN-α-IL-18 and IL-18 can promote lymphocyte transformation significantly, the activity of IFN-αinduced lymphocyte transformation is imperfect.The biological activity of stimulating lymphocyte transformation is associated with the NF-κB expression,activation and nucleo-cytoplasmic transport.The study built foundation for the function of IFN-α-IL-18 fusion protein and the exploration of the role of controlling epidemic diseases.