重组犬α干扰素在昆虫细胞中的高效表达与抗病毒活性鉴定
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摘要
目的利用杆状病毒表达系统制备犬α干扰素(Canine interferon-α,CaIFN-α)。方法根据昆虫细胞密码子偏爱性优化CaIFN-α基因,构建含双拷贝CaIFN-α优化基因的载体pFastBacDual-CaIFNα-CaIFNα,转化DH10Bac感受态后获得重组基因组rBacmid-CaIFNα-CaIFNα,转染Sf9细胞获得重组杆状病毒rBac-CaIFNα-CaIFNα,接种Sf9细胞表达rCaIFN-α。采用PCR和透射电子显微镜鉴定重组杆状病毒的外源基因与病毒形态,采用间接免疫荧光染色法检测rCaIFN-α蛋白的表达,采用细胞病变抑制法测定rCaIFN-α抑制水疱性口炎病毒(VSV-GFP)在MDCK细胞的复制能力。结果重组杆状病毒rBac-CaIFNα-CaIFNα构建成功,rCaIFN-α在rBac-CaIFNα-CaIFNα感染的Sf9细胞中表达,其抗病毒活性为8.73×105 IU/ml。结论构建的杆状病毒表达系统能高效表达rCaIFN-α,该干扰素具有高效抗病毒活性,这为犬干扰素制剂的研究奠定了基础。
Objective To efficiently express canine interferon-αusing a baculovirus/insect cell expression system.Methods The canine interferon-αgene(CaIFN-α)was optimized based on preferred codons in insect cells.The vector pFastBacDual-CaIFNα-CaIFNαcontaining the 2 copies of the CaIFN-αgene was transformed into DH10 Bac host bacteria to obtain the recombinant plasmid rBacmid-CaIFNα-CaIFNα.The recombinant baculovirus rBac-CaIFNα-CaIFNαwas obtained by transfecting Sf9 cells,and rCaIFN-αwas expressed by inoculation of Sf9 cells.The recombinant baculovirus was identified using PCR and transmission electron microscopy.CaIFN-αprotein was identified using IFA.Cytopathic inhibition was used to determine the ability of CaIFN-αprotein to inhibit the replication of vesicular stomatitis virus(VSVGFP)in MDCK cells. Results The rBacmid-CaIFNα-CaIFNαplasmid was verified as correct using restriction endonuclease analysis and DNA sequencing.The rBac-CaIFNα-CaIFNαbacmid was verified as correct using PCR and transmission electron microscopy.In IFA,specific green fluorescence was observed in Sf9 cells infected with rBac-CaIFNα-CaIFNα,indicating the expression of interferon.An antiviral activity assay indicated that recombinant CaIFN-αefficiently inhibits the replication of VSV-GFP in MDCK cells.The level of biological activity of recombinant CaIFN-αwas as high as 8.73×105 IU/ml. Conclusion This study successfully developed Sf9 cells expressing CaIFN-αwith a high level of antiviral activity.This finding lays the foundation for the further development and utilization of canine interferon.
Objective To efficiently express canine interferon-αusing a baculovirus/insect cell expression system.Methods The canine interferon-αgene(CaIFN-α)was optimized based on preferred codons in insect cells.The vector pFastBacDual-CaIFNα-CaIFNαcontaining the 2 copies of the CaIFN-αgene was transformed into DH10 Bac host bacteria to obtain the recombinant plasmid rBacmid-CaIFNα-CaIFNα.The recombinant baculovirus rBac-CaIFNα-CaIFNαwas obtained by transfecting Sf9 cells,and rCaIFN-αwas expressed by inoculation of Sf9 cells.The recombinant baculovirus was identified using PCR and transmission electron microscopy.CaIFN-αprotein was identified using IFA.Cytopathic inhibition was used to determine the ability of CaIFN-αprotein to inhibit the replication of vesicular stomatitis virus(VSVGFP)in MDCK cells. Results The rBacmid-CaIFNα-CaIFNαplasmid was verified as correct using restriction endonuclease analysis and DNA sequencing.The rBac-CaIFNα-CaIFNαbacmid was verified as correct using PCR and transmission electron microscopy.In IFA,specific green fluorescence was observed in Sf9 cells infected with rBac-CaIFNα-CaIFNα,indicating the expression of interferon.An antiviral activity assay indicated that recombinant CaIFN-αefficiently inhibits the replication of VSV-GFP in MDCK cells.The level of biological activity of recombinant CaIFN-αwas as high as 8.73×105 IU/ml. Conclusion This study successfully developed Sf9 cells expressing CaIFN-αwith a high level of antiviral activity.This finding lays the foundation for the further development and utilization of canine interferon.
引文
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[13] 王艳,王海震,曹瑞兵,等.犬α干扰素基因的高效表达及其活性测定[J].中国病毒学,2005,20(2):189-92.
[14] 王照,王铁峰,王铁成,等.重组犬α干扰素的制备及生物学活性分析[J].中国病原生物学杂志,2013,(4):307-9.
[15] 王晶宇,欧阳伟,王永山,等.犬干扰素α4在CHO细胞中的稳定表达[J].中国兽医科学,2017,(4):448-54.
[16] Ye Y,Cheng X,Zhang J,et al.Induction of robust immunityresponse in mice by dual-expression-system-based recombinantbaculovirus expressing the capsid protein of porcine circovirus type2[J].Virol J,2013(10):316.
[17] Gao J,Meng C,Chen Z,et al.Codon optimization of the rabbithemorrhagic disease virus(RHDV)capsid gene leads to increasedgene expression in Spodoptera frugiperda 9(Sf9)cells[J].J VetSci,2013,14(4):441-7.
[2] Samuel CE.Antiviral actions of interferons[J].Clin MicrobiolRev,2001,14(4):778-809.
[3] Yang L,Xu L,Li Y,et al.Molecular and functional character-ization of canine interferon-epsilon[J].J Interferon CytokineRes,2013,33(12):760-8.
[4] Taira O,Watanugi I,Hagiwara Y,et al.Cloning and expressionof canine interferon-alpha genes in Escherichia coli[J].J VetMed Sci,2005,67(10):1059-62.
[5] Himmler A,Hauptmann R,Adolf GR,et al.Structure and ex-pression in Escherichia coli of canine interferon-alpha genes[J].JInterferon Res,1987,7(2):173-83.
[6] Na Z,Huipeng Y,Lipan L,et al.Efficient production of canineinterferon-alpha in silkworm Bombyx mori by use of a BmNPV/Bac-to-Bac expression system[J].Appl Microbiol Biotechnol,2008,78(2):221-6.
[7] 宋天琪,金红岩,张通明,等.重组犬α2干扰素的原核表达及其抗病毒活性评价[J].中国畜牧兽医,2018,45(1):14-21.
[8] 王玉,朱艳平,郭霄峰.犬干扰素α1基因的修饰及在毕赤酵母中的表达[J].中国生物制品学杂志,2011,24(9):1064-7.
[9] 王照.毕赤酵母表达犬长效干扰素融合蛋白的研究[D].长春:吉林农业大学,2013.
[10] Chang SF,Sgro JY,Parrish CR.Multiple amino acids in thecapsid structure of canine parvovirus coordinately determine thecanine host range and specific antigenic and hemagglutination prop-erties[J].J Virol,1992,66(12):6858-67.
[11] Nagata T,Ishikawa S,Shimokawa E,et al.High level expres-sion and purification of bioactive bovine interleukin-18using a bac-ulovirus system[J].Vet Immunol Immunopathol,2002,87(1-2):65-72.
[12] Wen Z,Ye L,Gao Y,et al.Immunization by influenza virus-likeparticles protects aged mice against lethal influenza virus challenge[J].Antiviral Res,2009,84(3):215-24.
[13] 王艳,王海震,曹瑞兵,等.犬α干扰素基因的高效表达及其活性测定[J].中国病毒学,2005,20(2):189-92.
[14] 王照,王铁峰,王铁成,等.重组犬α干扰素的制备及生物学活性分析[J].中国病原生物学杂志,2013,(4):307-9.
[15] 王晶宇,欧阳伟,王永山,等.犬干扰素α4在CHO细胞中的稳定表达[J].中国兽医科学,2017,(4):448-54.
[16] Ye Y,Cheng X,Zhang J,et al.Induction of robust immunityresponse in mice by dual-expression-system-based recombinantbaculovirus expressing the capsid protein of porcine circovirus type2[J].Virol J,2013(10):316.
[17] Gao J,Meng C,Chen Z,et al.Codon optimization of the rabbithemorrhagic disease virus(RHDV)capsid gene leads to increasedgene expression in Spodoptera frugiperda 9(Sf9)cells[J].J VetSci,2013,14(4):441-7.
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