草鱼干扰素3蛋白多克隆抗体制备及其应用

为探讨干扰素3(Interferon 3,IFN3)在抗病毒免疫应答中的作用,以草鱼(Ctenopharyngodon idella)巨噬细胞cDNA为模板,PCR扩增IFN3成熟肽基因序列,制备草鱼IFN3蛋白的多克隆抗体,同时研究了IFN3在草鱼不同免疫组织中的蛋白表达,以及草鱼呼肠孤病毒(grass carp reovirus,GCRV)感染草鱼肾细胞系(Ctenopharyngodon idella kidney,CIK)后不同时间点的表达。结果显示,细菌表达的重组IFN3大小约为45 kD,主要以包涵体形式存在;抗体效价约为1∶3 200,制备的多克隆抗体既能识别原核表达的重组蛋白,也能识别个体水平上和细胞水平上的内源蛋白。草鱼主要免疫组织中,肝胰腺和皮肤检测到相应条带。CIK细胞感染病毒后12 h开始检测到IFN3蛋白,随感染时间的延长,IFN3蛋白表达量有所增加。蛋白水平上检测IFN3的表达,为深入研究草鱼的抗病毒免疫机制奠定了基础。

Preparations and applications for polyclonal antibody of Ctenopharyngodon idella interferon 3

Interferon 3 ( IFN3)is an important member of the type I interferon family and plays an important role in the antiviral immune response.In this study,the polyclonal antibody of IFN3 protein was prepared and applied to individuals and cell levels. Ctenopharyngodon idella macrophage cDNA was used as template,IFN3 mature peptide gene was amplified by PCR,ligated to pGEX-4T-1 vector,transformed E.coli BL21 ( DE3)pLysS and induced expression with IPTG.After purification,the recombinant IFN3 protein was used to immunize BALB/c mice to prepare polyclonal antibody,antibody titer was determined by indirect ELISA method,and polyclonal antibody specificity was identified by western blot.The protein levels of IFN3 in different immune tissues of C.idella and in its kidney ( CIK)cells post grass carp reovirus ( GCRV)infection at different time points were studied.The results indicated that the recombinant IFN3 was about 45 kD,mainly existed in the form of inclusion body.The antibody titer was about 1∶ 3200.The polyclonal antibody could bind recombinant protein from prokaryotic expression,and also bind the corresponding proteins in individuals and cells.Among the main immune tissues in C.idella ,corresponding bands were detectable in hepatopancreas and skin samples.The IFN3 protein was detectable at 12 h post GCRV infection in CIK cells.The expression levels increased over time.The expression of IFN3 was detected at the protein level,which laid a foundation for in-depth studies on the antiviral mechanism in grass carp.