重组IL-18BP拮抗重组鸡IL-18刺激外周血单核细胞产生IFN-γ的作用  

Recombinant cIL-18-binding protein as an antagonist to cIL-18 enhanced PBMCs secreting IFN-γ

作　　者：刘文强[1] 刘桂芹[1] 秦绪岭[1] 王桂英[1] 

机构地区：[1]聊城大学农学院,聊城大学畜禽疫病防制技术研究所,聊城252059

出　　处：《微生物学报》2010年第4期506-511,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30800130);山东省中青年科学家奖励基金(2006BS02012);聊城大学博士基金(31805)~~

摘　　要：【目的】白细胞介素-18通过激活Th1细胞和NK细胞产生IFN-γ而发挥关键的免疫调节作用。人和小鼠分泌的白细胞介素-18结合蛋白(IL-18BP)可以拮抗其活性。推测在鸡痘病毒基因组中也含有IL-18BP基因的同源物,对其表达的蛋白质进行了活性鉴定,为拮抗IL-18主导的疾病提供理论依据。【方法】根据鸡痘疫苗病毒的基因组序列设计特异性引物,使用PCR方法从中分离cIL-18BP基因,将该基因克隆到酵母表达载体pPICZαA中,甲醇诱导后在酵母GS115中进行表达。对表达的重组蛋白进行了活性鉴定。【结果】从鸡痘病毒中克隆到cIL-18BP基因,SDS-PAGE鉴定了该基因在酵母系统中的高效表达。ELISA检测表明纯化后的cIL-18BP与重组鸡(c)IL-18发生特异性结合;通过测定IFN-γ的浓度,表明该蛋白具有拮抗IL-18刺激外周血单核细胞(PBMCs)和MSB1细胞分泌IFN-γ的活性。【结论】实验表明,cIL-18BP通过抑制cIL-18刺激相关免疫细胞分泌IFN-γ而发挥对IL-18的拮抗作用,敲除该基因可能有助于研制更安全和高效的鸡痘疫苗。Objective Interleukin-18（IL-18） is a proinflammatory cytokines that plays a key role of immune regulation through activating the Th 1 cell and NK cell secreting interferon-γ（IFN-γ）.IL-18-binding proteins（IL-18BP） secreted in human and mouse can antagonise the activity of IL-18.The homologue gene of IL-18BP from fowlpox virus genome was predicted and identified of its expressed protein,to use as an antagonist to IL-18 guiding disease.MethodsThe cIL-18BP gene was isolated from the genome of fowlpox vaccine virus by PCR through a pair of special primers and cloned into vectors pPICZαA,then expressed in yeast GS115.The biologic activity of recombinant proteins binding with cIL-18 was measured,inhibiting the activity of cIL-18 enhancing relative cells secreting IFN-γ was confirmed through ELISA.ResultsThe cIL-18BP gene was cloned from fowlpox virus and acquired high performance expression in yeast systems induced with methanol identified by SDS-PAGE.Recombinant cIL-18BP purified can bind specifically with recombinant cIL-18 determined by ELISA test.cIL-18BP can antagonise the activity of cIL-18 with determining the IFN-γ concentration secreting in PBMCs and MSB1 cells stimulated by cIL-18 respectively.Conclusion The experiment indicates that recombinant cIL-18BP can inhibit the activity of cIL-18 stimulating related immune cells secreting IFN-γ.Deletion of the cIL-18BP from virus may improve the safety and immunogenicity of fowlpox virus vaccine.

关 键 词：鸡痘病毒 IL-18结合蛋白 活性 

分 类 号：S852.65[农业科学—基础兽医学]