短期重复口服鸡α干扰素体内抑制H9N2亚型流感病毒复制
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摘要
为研究口服鸡α干扰素最佳的给药频率及与灭活病毒联合使用对鸡群的影响,文中将构建的原核表达质粒pET-22b-ChIFN-α转入大肠杆菌Escherichia coli BL21 (DE3),宿主菌经诱导表达收获重组包涵体蛋白,经变性、纯化、复性后获得重组鸡α干扰素。SDS-PAGE结果分析显示,目的蛋白可在原核表达载体中高效表达,发酵液上清中目的蛋白浓度高达0.2 mg/mL,分子量约为20 kDa。将鸡α干扰素稀释至活性为2.5×10~4 U/羽份,与灭活H9N2亚型流感病毒联用以口服的方式免疫无特定病原(SPF)鸡群,试验结果表明,短期(96 h)重复免疫3次,鸡α干扰素具有较好的安全性,可诱导鸡群的外周血、脾脏及胸腺产生较高水平的抗病毒相关的诱导基因,攻毒结果显示鸡α干扰素使用次数为连续使用3–5 d鸡群排毒率最低,体现出较好的抗流感病毒能力。本研究结果获得了鸡α干扰素的最佳免疫频率及免疫时间,为干扰素的最佳临床应用方法提供理论支撑。
To evaluate the optimal administration frequency for interferon-α(IFN-α) and the effect of its combined use with inactive virus on chicken flocks, the prokaryotic expression plasmid pET-22 b-ChIFN-α was constructed and transferred into Escherichia coli BL21(DE3) host bacteria to induce the expression of chicken IFN-α and to harvest recombinant proteins inclusion bodies. The expression of recombinant chicken IFN-α was confirmed by SDS-PAGE, and the results demonstrated that the chicken IFN-α(20 kDa) was highly expressed using the prokaryotic expression vector with a concentration of 0.2 mg/mL in the medium. Chicken IFN-α was diluted to 2.5×10~4 U/fowls and administered to immunized specific-pathogen-free chickens orally in combination with inactivated H9N2 subtype influenza virus. Chicken that received chicken IFN-α were safe after three repeated immunizations(96 h). In addition, chicken IFN-α could induce higher levels of antiviral-related inducible genes in peripheral blood, spleen, and thymus of chicken flocks. The results of a challenge assay revealed that the lowest detoxification rates of chicken IFN-α ranged from three to five days, suggesting a higher capacity to resist H9N2 subtype avian influenza virus. The present study obtained the optimal immune frequency and immunization period for chicken IFN-α to provide theoretical support for the optimal clinical application of IFN-α.
To evaluate the optimal administration frequency for interferon-α(IFN-α) and the effect of its combined use with inactive virus on chicken flocks, the prokaryotic expression plasmid pET-22 b-ChIFN-α was constructed and transferred into Escherichia coli BL21(DE3) host bacteria to induce the expression of chicken IFN-α and to harvest recombinant proteins inclusion bodies. The expression of recombinant chicken IFN-α was confirmed by SDS-PAGE, and the results demonstrated that the chicken IFN-α(20 kDa) was highly expressed using the prokaryotic expression vector with a concentration of 0.2 mg/mL in the medium. Chicken IFN-α was diluted to 2.5×10~4 U/fowls and administered to immunized specific-pathogen-free chickens orally in combination with inactivated H9N2 subtype influenza virus. Chicken that received chicken IFN-α were safe after three repeated immunizations(96 h). In addition, chicken IFN-α could induce higher levels of antiviral-related inducible genes in peripheral blood, spleen, and thymus of chicken flocks. The results of a challenge assay revealed that the lowest detoxification rates of chicken IFN-α ranged from three to five days, suggesting a higher capacity to resist H9N2 subtype avian influenza virus. The present study obtained the optimal immune frequency and immunization period for chicken IFN-α to provide theoretical support for the optimal clinical application of IFN-α.
引文
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[22]Tseng LP,Chiou CJ,Chen CC,et al.Effect of lipopolysaccharide on intranasal administration of liposomal Newcastle disease virus vaccine to SPFchickens.Vet Immunol Immunopathol,2009,131(3/4):285-289.
[23]Zhang AG,Lai HZ,Xu JH,et al.Evaluation of the protective efficacy of poly I:C as an adjuvant for H9N2 subtype avian influenza inactivated vaccine and its mechanism of action in ducks.PLoS ONE,2017,12(1):e0170681.
[24]Swayne DE.Avian influenza vaccines and therapies for poultry.Comp Immunol Microbiol Infect Dis,2009,32(4):351-363.
[25]Tang YH,Lu JH,Wu PP,et al.Inactivated vaccine with adjuvants consisting of pattern recognition receptor agonists confers protection against avian influenza viruses in chickens.Vet Microbiol,2014,172(1/2):120-128.
[26]Vignali DAA,Kuchroo VK.IL-12 family cytokines:immunological playmakers.Nat Immunol,2012,13(8):722-728.
[27]Gainey MD,Rivenbark JG,Cho H,et al.Viral MHCclass I inhibition evades CD8+T-cell effector responses in vivo but not CD8+T-cell priming.Proc Natl Acad Sci USA,2012,109(47):E3260-3267.
[28]Schroder K,Hertzog PJ,Ravasi T,et al.Interferon-γ:an overview of signals,mechanisms and functions.J Leukoc Biol,2004,75(2):163-189.
[29]Chen Z,Bozec A,Ramming A,et al.Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis.Nat Rev Rheumatol,2019,15(1):9-17.
[30]Mebius RE,Kraal G.Structure and function of the spleen.Nat Rev Immunol,2005,5(8):606-616.
[2]Savan R,Ravichandran S,Collins JR,et al.Structural conservation of interferon gamma among vertebrates.Cytokine Growth Factor Rev,2009,20(2):115-124.
[3]Schindler C,Levy DE,Decker T.JAK-STATsignaling:from interferons to cytokines.J Biol Chem,2007,282(28):20059-20063.
[4]Goossens KE,Ward AC,Lowenthal JW,et al.Chicken interferons,their receptors and interferon-stimulated genes.Dev Comp Immunol,2013,41(3):370-376.
[5]Karpala AJ,Morris KR,Broadway MM,et al.Molecular cloning,expression,and characterization of chicken IFN-λ.J Interferon Cytokine Res,2008,28(6):341-350.
[6]Feller L,Altini M,Khammissa RAG,et al.Oral mucosal immunity.Oral Surg Oral Med Oral Pathol Oral Radiol,2013,116(5):576-583.
[7]Srivastava A,Gowda DV,Madhunapantula SV,et al.Mucosal vaccines:a paradigm shift in the development of mucosal adjuvants and delivery vehicles.APMIS,2015,123(4):275-288.
[8]Zdrojewicz Z,Pachura E,Pachura P.The thymus:a forgotten,but very important organ.Adv Clin Exp Med,2016,25(2):369-375.
[9]Zhang Z,Zhang HF,Chen DW,et al.Response to interferon-αtreatment correlates with recovery of blood plasmacytoid dendritic cells in children with chronic hepatitis B.J Hepatol,2007,47(6):751-759.
[10]Crouse J,Kalinke U,Oxenius A.Regulation of antiviral T cell responses by type I interferons.Nat Rev Immunol,2015,15(4):231-242.
[11]Rouzaut A,Garasa S,Teijeiraá,et al.Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN-α.Eur J Immunol,2010,40(11):3054-3063.
[12]Parlato S,Santini SM,Lapenta C,et al.Expression of CCR-7,MIP-3β,and Th-1 chemokines in type IIFN-induced monocyte-derived dendritic cells:importance for the rapid acquisition of potent migratory and functional activities.Blood,2001,98(10):3022-3029.
[13]AricòE,Belardelli F.Interferon-αas antiviral and antitumor vaccine adjuvants:mechanisms of action and response signature.J Interferon Cytokine Res,2012,32(6):235-247.
[14]Santini SM,Lapenta C,Logozzi M,et al.Type Iinterferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-Scid mice.J Exp Med,2000,191(10):1777-1788.
[15]Frydecka D,Paw?owski T,Pawlak D,et al.Functional polymorphism in the interleukin 6(IL6)gene with respect to depression induced in the course of interferon-αand ribavirin treatment in chronic hepatitis patients.Arch Immunol Ther Exp(Warsz),2016,64(S1):169-175.
[16]Dai MM,Wu SY,Feng M,et al.Recombinant chicken interferon-alpha inhibits the replication of exogenous avian Leukosis virus(ALV)in DF-1cells.Mol Immunol,2016,76:62-69.
[17]Qu HR,Yang LM,Meng SS,et al.The differential antiviral activities of chicken interferonα(ChIFN-α)and ChIFN-βare related to distinct interferon-stimulated gene expression.PLoS ONE,2013,8(3):e59307.
[18]Woodrow KA,Bennett KM,Lo DD.Mucosal vaccine design and delivery.Annu Rev Biomed Eng,2012,14:17-46.
[19]Kang SM,Guo LZ,Yao QZ,et al.Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens.J Virol,2004,78(18):9624-9632.
[20]Akbari O,DeKruyff RH,Umetsu DT.Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen.Nat Immunol,2001,2(8):725-731.
[21]Hagenaars N,Mastrobattista E,Glansbeek H,et al.Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines:effect of composition,spatial organization and immunization route on the immunogenicity in a murine challenge model.Vaccine,2008,26(51):6555-6563.
[22]Tseng LP,Chiou CJ,Chen CC,et al.Effect of lipopolysaccharide on intranasal administration of liposomal Newcastle disease virus vaccine to SPFchickens.Vet Immunol Immunopathol,2009,131(3/4):285-289.
[23]Zhang AG,Lai HZ,Xu JH,et al.Evaluation of the protective efficacy of poly I:C as an adjuvant for H9N2 subtype avian influenza inactivated vaccine and its mechanism of action in ducks.PLoS ONE,2017,12(1):e0170681.
[24]Swayne DE.Avian influenza vaccines and therapies for poultry.Comp Immunol Microbiol Infect Dis,2009,32(4):351-363.
[25]Tang YH,Lu JH,Wu PP,et al.Inactivated vaccine with adjuvants consisting of pattern recognition receptor agonists confers protection against avian influenza viruses in chickens.Vet Microbiol,2014,172(1/2):120-128.
[26]Vignali DAA,Kuchroo VK.IL-12 family cytokines:immunological playmakers.Nat Immunol,2012,13(8):722-728.
[27]Gainey MD,Rivenbark JG,Cho H,et al.Viral MHCclass I inhibition evades CD8+T-cell effector responses in vivo but not CD8+T-cell priming.Proc Natl Acad Sci USA,2012,109(47):E3260-3267.
[28]Schroder K,Hertzog PJ,Ravasi T,et al.Interferon-γ:an overview of signals,mechanisms and functions.J Leukoc Biol,2004,75(2):163-189.
[29]Chen Z,Bozec A,Ramming A,et al.Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis.Nat Rev Rheumatol,2019,15(1):9-17.
[30]Mebius RE,Kraal G.Structure and function of the spleen.Nat Rev Immunol,2005,5(8):606-616.