鸡干扰素γ的原核表达及其抗传染性法氏囊病毒的活性分析
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摘要
目的原核表达鸡干扰素γ(chicken interferonγ,ChIFNγ),并对其抗传染性法氏囊病毒(infectious bursal disease virus,IBDV)活性进行检测。方法 PCR法扩增Ch IFNγ基因,克隆至载体p ET-28a中,转化感受态E. coli BL21,经IPTG诱导表达,His-binding-resin柱进行纯化。取200 ng纯化蛋白,加至鸡胚成纤维细胞,37℃孵育24 h,接种IBDV,攻毒48 h观察细胞病变情况。将纯化蛋白经腹腔注射14日龄雏鸡,200μg/只,24 h后再注射1次,48 h后用IBDV进行感染,103 TCID50/只,感染48 h后无菌取鸡法氏囊组织,进行病理学及qRT-PCR检测。结果纯化蛋白相对分子质量约18 000,可与抗His标签单克隆抗体发生特异性结合,浓度约0. 8 mg/mL。加入纯化蛋白的鸡成纤维细胞病变明显减轻;注射纯化蛋白雏鸡的法氏囊组织病变减轻,病毒载量明显降低(P <0. 001)。结论本实验成功表达了Ch IFNγ重组蛋白,且于体内、外均有抑制IBDV的作用,为该蛋白的应用提供了实验依据。
Objective To express chicken interferon γ(Ch IFNγ) in prokaryotic cells and determine its activity against infectious bursal disease virus(IBDV). Methods Ch IFNγ gene was amplified by PCR and cloned into vector pET-28 a.The constructed recombinant plasmid was transformed to competent E. coli BL21 and induced with IPTG. The expressed ChIFNγ was purified by His-binding-resin column chromatography. Chicken embryo fibroblasts were treated with 200 ng of purified ChIFNγ and incubated at 37 ℃ for 24 h,then inoculated with IBDV and observed for CPE 48 h later. Chicks at age of 14 d were injected i.p. with 200 μg of purified ChIFNγ and boosted 24 h later,then challenged with IBDV 48 h after the first injection,103 TCID50 for each. Bursal tissue was collected aseptically for pathological examination and qRTPCR. Results Purified Ch IFNγ,with a relative molecular mass of about 18 000,showed specific binding to anti-His tag monoclonal antibody,of which the concentration was about 0. 8 mg/mL. The CPE of chicken embryo fibroblasts treated with purified chicken embryo fibroblasts was relieved significantly. However,the pathological change of bursal tissue of chicks injected with ChIFNγ was relieved,while the virus load decreased significantly(P < 0. 001). Conclusion Recombinant Ch IFNγ was expressed in prokaryotic cells,which inhibited IBDV in vitro and in vivo. It provided an experimental basis for application of ChIFNγ.
Objective To express chicken interferon γ(Ch IFNγ) in prokaryotic cells and determine its activity against infectious bursal disease virus(IBDV). Methods Ch IFNγ gene was amplified by PCR and cloned into vector pET-28 a.The constructed recombinant plasmid was transformed to competent E. coli BL21 and induced with IPTG. The expressed ChIFNγ was purified by His-binding-resin column chromatography. Chicken embryo fibroblasts were treated with 200 ng of purified ChIFNγ and incubated at 37 ℃ for 24 h,then inoculated with IBDV and observed for CPE 48 h later. Chicks at age of 14 d were injected i.p. with 200 μg of purified ChIFNγ and boosted 24 h later,then challenged with IBDV 48 h after the first injection,103 TCID50 for each. Bursal tissue was collected aseptically for pathological examination and qRTPCR. Results Purified Ch IFNγ,with a relative molecular mass of about 18 000,showed specific binding to anti-His tag monoclonal antibody,of which the concentration was about 0. 8 mg/mL. The CPE of chicken embryo fibroblasts treated with purified chicken embryo fibroblasts was relieved significantly. However,the pathological change of bursal tissue of chicks injected with ChIFNγ was relieved,while the virus load decreased significantly(P < 0. 001). Conclusion Recombinant Ch IFNγ was expressed in prokaryotic cells,which inhibited IBDV in vitro and in vivo. It provided an experimental basis for application of ChIFNγ.
引文
[1] ISAACS A,LINDENMANN J. Pillars article:virus interference.I. The interferon[J]. J Immunol,2015,195(5):1911-1920.
[2] SONG B F,LI Y C,ZOU S,et al. Construction of Eukaryotic expression vector of cow IFN-γgene[J]. J Heilongjiang Bayi Agri Univ,2010,22(1):53-57.(in Chinese)宋佰芬,李迎春,邹珊,等.牛干扰素-γ基因的克隆与真核表达载体的构建[J].黑龙江八一农垦大学学报,2010,22(1):53-57.
[3] AL-YAHYA S,MAHMOUD L,AL-ZOGHAIBI F,et al. Human cytokinome analysis for interferon response[J]. J Virol,2015,89(14):7108-7019.
[4] MAC MICKING J D. Recognizing macrophage activation and host defense[J]. Cell Host Microbe,2009,5(5):405-407.
[5] LOWENTHAL J W,LAMBRECHT B,VAN DEN BERG T P,et al. Avian cytokines-the natural approach to therapeutics[J].Dev Comp Immunol,2000,24(2-3):355-365.
[6] ESPINOSA V,DUTTA O,MCELRATH C. Type III interferon is a critical regulator of innate antifungal immunity[J]. Sci Immunol,2017,2(16):pii:eaan5357. doi:10.1126/sciimmunol.aan5357.
[7] LIN F C,YOUNG H A. Interferons:Success in anti-viral immunotherapy[J]. Cytokine Growth Factor Rev,2014,25(4):369-376.
[8] PANE J A,FLEMING F E,GRAHAM K L,et al. Rotavirus acceleration of type 1 diabetes in non-obese diabetic mice depends on type I interferon signaling[J]. Sci Rep,2016,6:29697. doi:10.1038/srep29697.
[9] DIGBY M R,LOWENTHAL J W. Cloning and expression of the chicken interferon-gamma gene[J]. J Interferon Cytokine Res,1995,15(11):939-945.
[10] LI X T. Prokaryotic expression and anti-IBDV activity of chicken IL-18 and IFN-γ[D]. Daqing:Heilongjiang Bayi Agricultural University,2017.(in Chinese)李晓婷.鸡IL-18和IFN-γ原核表达及抗IBDV活性分析[D].黑龙江大庆:黑龙江八一农垦大学,2017.
[11] FENIMORE J,A YOUNG H. Regulation of IFN-γexpression[J]. Adv Exp Med Biol,2016,941:1-19.
[12] CHEN J,FENG X,DESIERTO M J,et al. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure[J]. Blood,2015,126(24):2621-2631.
[13] ANDERSEN S H,VERVELDE L,SUTTON K,et al. Quantification and phenotypic characterisation of peripheral IFN-γproducing leucocytes in chickens vaccinated against Newcastle disease[J]. Vet Immunol Immunopathol,2017(193-194):18-28.
[14] QIN Y,ZHANG C. The regulatory role of IFN-γon the proliferation and differentiation of hematopoietic stem and progenitor cells[J]. Stem Cell Rev,2017,13(6):705-712.
[15] BIERING S B,CHOI J,HALSTROM R A,et al. Viral replication complexes are targeted by LC3-guided interferon-inducible GTPases[J]. Cell Host Microbe,2017,22(1):74-85.e7. doi:10.1016/j.chom.2017.06.005.
[2] SONG B F,LI Y C,ZOU S,et al. Construction of Eukaryotic expression vector of cow IFN-γgene[J]. J Heilongjiang Bayi Agri Univ,2010,22(1):53-57.(in Chinese)宋佰芬,李迎春,邹珊,等.牛干扰素-γ基因的克隆与真核表达载体的构建[J].黑龙江八一农垦大学学报,2010,22(1):53-57.
[3] AL-YAHYA S,MAHMOUD L,AL-ZOGHAIBI F,et al. Human cytokinome analysis for interferon response[J]. J Virol,2015,89(14):7108-7019.
[4] MAC MICKING J D. Recognizing macrophage activation and host defense[J]. Cell Host Microbe,2009,5(5):405-407.
[5] LOWENTHAL J W,LAMBRECHT B,VAN DEN BERG T P,et al. Avian cytokines-the natural approach to therapeutics[J].Dev Comp Immunol,2000,24(2-3):355-365.
[6] ESPINOSA V,DUTTA O,MCELRATH C. Type III interferon is a critical regulator of innate antifungal immunity[J]. Sci Immunol,2017,2(16):pii:eaan5357. doi:10.1126/sciimmunol.aan5357.
[7] LIN F C,YOUNG H A. Interferons:Success in anti-viral immunotherapy[J]. Cytokine Growth Factor Rev,2014,25(4):369-376.
[8] PANE J A,FLEMING F E,GRAHAM K L,et al. Rotavirus acceleration of type 1 diabetes in non-obese diabetic mice depends on type I interferon signaling[J]. Sci Rep,2016,6:29697. doi:10.1038/srep29697.
[9] DIGBY M R,LOWENTHAL J W. Cloning and expression of the chicken interferon-gamma gene[J]. J Interferon Cytokine Res,1995,15(11):939-945.
[10] LI X T. Prokaryotic expression and anti-IBDV activity of chicken IL-18 and IFN-γ[D]. Daqing:Heilongjiang Bayi Agricultural University,2017.(in Chinese)李晓婷.鸡IL-18和IFN-γ原核表达及抗IBDV活性分析[D].黑龙江大庆:黑龙江八一农垦大学,2017.
[11] FENIMORE J,A YOUNG H. Regulation of IFN-γexpression[J]. Adv Exp Med Biol,2016,941:1-19.
[12] CHEN J,FENG X,DESIERTO M J,et al. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure[J]. Blood,2015,126(24):2621-2631.
[13] ANDERSEN S H,VERVELDE L,SUTTON K,et al. Quantification and phenotypic characterisation of peripheral IFN-γproducing leucocytes in chickens vaccinated against Newcastle disease[J]. Vet Immunol Immunopathol,2017(193-194):18-28.
[14] QIN Y,ZHANG C. The regulatory role of IFN-γon the proliferation and differentiation of hematopoietic stem and progenitor cells[J]. Stem Cell Rev,2017,13(6):705-712.
[15] BIERING S B,CHOI J,HALSTROM R A,et al. Viral replication complexes are targeted by LC3-guided interferon-inducible GTPases[J]. Cell Host Microbe,2017,22(1):74-85.e7. doi:10.1016/j.chom.2017.06.005.