重组鸡IFN-α-IL-18融合基因的酵母分泌表达及其抗IBDV活性研究  

Expression and the anti-IBDV activity of recombinant chicken IFN-α-IL-18 fusion protein in Pichia pastoris

作　　者：杨惠[1] 李银聚[1] 刘一尘[1] 程相朝[1] 杨丹芳[1] 韩海峰[1] 金修哲[1] 颜云飞 何雷[1] 

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室/河南省动物疫病与公共安全院士工作站,河南洛阳471003

出　　处：《中国兽医学报》2015年第11期1759-1764,共6页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关项目(132102110117)

摘　　要：为探讨鸡IFN-α和IL-18及其融合蛋白抗IBDV活性,本研究将鸡IFN-α、IL-18及其融合基因分别置于酵母表达载体pPICZ-αA的AOX1启动子下游,构建重组质粒pPICZ-IFN-α、pPICZ-IL-18和pPICZ-IFN-α-IL-18。将重组质粒分别用PmeⅠ酶切线性化,电穿孔方法导入酵母Pichia Pastoris X-33感受态细胞,以1%甲醇诱导表达,SDS-PAGE和Western blot检测表达蛋白,淋巴细胞转化试验初步检测表达产物生物学活性,鸡胚接种试验对表达产物的抗IBDV效果进行评价。结果表明,在酵母培养上清液中存在相对分子质量分别为22 000、23 000和43 000的目的蛋白,表达的IL-18和IFN-α-IL-18对鸡淋巴细胞具有明显的诱导转化作用(P<0.05)。IFN-α-IL-18融合蛋白能够显著抑制IBDV在鸡胚中的增殖(P<0.01),IFN-α和IL-18酵母表达产物对IBDV也有明显的抑制作用(P<0.05)。这为进一步开展IFN-α-IL-18融合蛋白的功能研究及其在鸡病毒性疫病防控中作用的探索奠定了基础。To discuss the antiviral activity of IFN-α-IL-18 fusion protein,IFN-α,IL-18 and the fusion gene IFN-α-IL-18 was cloned into the downstream of the promoter AOX1 of the yeast expression vector pPICZ-αA to construct the recombinant plasmids pPICZ-IFN-α,pPICZ-IL-18 and pPICZ-IFN-α-IL-18.The recombinant plasmids was transformed into P.Pastoris X-33 strain by electroporation after pPICZ-IFN-α-IL-18,pPICZ-IFN-αwas linearized by Pme I enzyme.The expression protein was expressed under the induction of 1% methanol,and detected by SDS-PAGE and Western-blot.The biological activity of the expression products was determined by the lymphocyte transformation test initially,and was the anti-IBDV effect of the expression products evaluated by embryonic vaccinated.The results showed that the expression products existed in supernatant,and its molecular weight was about 22 000,23 000 and 43 000,the expression products IL-18 and IFN-α-IL-18 had a distinct transformation for chicken lymphocyte（P〈0.05）.IFN-α-IL-18 fusion protein can significantly inhibit the amplification of IBDV（P〈0.01）,IFN-αand IL-18 showed the obvious anti-IBDV activity of（P〈0.05）.The study builta foundation for study on the function of IFN-α-IL-18 fusion protein and the exploration of the role in controlling viral diseases in chicken.

关 键 词：IFN-α基因 IL-18基因 融合蛋白 酵母表达 淋巴细胞转化试验 IBDV 

分 类 号：S852.4[农业科学—基础兽医学]