草鱼干扰素3蛋白多克隆抗体制备及其应用
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摘要
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。
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-4 T-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∶3 200.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.
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-4 T-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∶3 200.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.
引文
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[19]王红权,章怀云,张学文,等.转hu-IFN-α基因草鱼F1代人α-干扰素表达水平的研究[J].湖南农业大学学报(自然科学版),2006,32(1):60-62.
[20]吴初新,李东明,余新建,等.重组干扰素、趋化因子合剂对草鱼免疫保护作用[J].南昌大学学报(理科版),2014,(1):91-95.
[21]邵健忠,项黎新,李亚南.草鱼干扰素诱生条件的研究[J].水产学报,1999,23(2):122-127.
[2]李贤,曾伟伟,王庆,等.草鱼呼肠病毒研究进展[J].动物医学进展,2016,37(7):94-101.
[3] Rao Y,Su J.Insights into the antiviral immunity against grass carp(Ctenopharyngodon idella)reovirus(GCRV)in grass carp [J].J Immunol Res,2015,2015:670437.
[4] Liao Z,Wan Q,Su J.Bioinformatics analysis of organizational and expressional characterizations of the IFNs,IRFs and CRFBs in grass carp Ctenopharyngodon idella [J].Dev Comp Immunol,2016,61:97-106.
[5]郑青亮.I型干扰素诱导沙门菌感染的巨噬细胞发生坏死性凋亡[J].中国肿瘤生物治疗杂志,2012,(6):667-667.
[6] Wang Y,Lu Y,Zhang Y,et al.The draft genome of the grass carp(Ctenopharyngodon idellus)provides insights into its evolution and vegetarian adaptation [J].Nat Genet,2015,47:625-631.
[7] Wan Q,Yang C,Rao Y,et al.MDA5 induces a stronger interferon response than RIG-I to GCRV infection through a mechanism involving the phosphorylation and dimerization of IRF3 and IRF7 in CIK cells [J].Front Immunol,2017,(8):189-196.
[8] Lindenmann J,Burke D C,Isaacs A.Studies on the production,mode of action and properties of interferon [J].Br J Exp Pathol,1957,38:551-559.
[9] Ippolito G,Di C A,Capobianchi M R.The Chronology of the international response to Ebola in western Africa:lights and shadows in a frame of conflicting position and figures [J].Infect Dis Rep,2015,(7):5957-5966.
[10]尹湘艳,胡国斌,张建业,等.鱼类干扰素系统的研究进展[J].生命科学仪器,2009,7(9):11-15.
[11] Altmann S M,Mellon M T,Distel D L,et al.Molecular and functional analysis of an interferon gene from the zebrafish,Danio rerio [J].J Virol,2003,77:1992-2002.
[12]王晓丽,王永明,朱万光,等.干扰素研究进展[J].动物医学进展,2008,29(12):60-63.
[13] Li Z,Xu X,Huang L,et al.Administration of recombinant IFN1 protects zebrafish(Danio rerio)from ISKNV infection [J].Fish Shellfish Immunol,2010,29:399-406.
[14] Belardelli F,Ferrantini M,Parmiani G,et al.International meeting on cancer vaccines:how can we enhance efficacy of therapeutic vaccines [J].Cancer Res,2004,64(18):6827-683.
[15] Angelico M,Cepparulo M,Barlattani A,et al.Unfavourable effects of colchicine in combination with interferon-α in the treatment of chronic hepatitis C [J].Aliment Pharmacol Ther,2000,14:1459-1467.
[16]邵健忠,项黎新.草鱼干扰素的免疫调节功能[J].动物学报,2001,47(4):404-411.
[17]余新建,李东明,马梅生,等.重组干扰素上调草鱼干扰素系统基因的表达[J].水生生物学报,2013,37(6):1184-1188.
[18]张学文,章怀云,符少辉,等.可在鱼体内表达hu-IFN-α的基因重组构建及转化[J].高技术通讯,2001,11(4):1-5.
[19]王红权,章怀云,张学文,等.转hu-IFN-α基因草鱼F1代人α-干扰素表达水平的研究[J].湖南农业大学学报(自然科学版),2006,32(1):60-62.
[20]吴初新,李东明,余新建,等.重组干扰素、趋化因子合剂对草鱼免疫保护作用[J].南昌大学学报(理科版),2014,(1):91-95.
[21]邵健忠,项黎新,李亚南.草鱼干扰素诱生条件的研究[J].水产学报,1999,23(2):122-127.