牛传染性鼻气管炎病毒gC结构蛋白的真核表达及其反应原性分析
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摘要
目的真核表达牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gC结构蛋白,并对其反应原性进行鉴定。方法以GenBank中登录的IBRV基因序列合成gC基因全长,构建重组真核表达质粒p CDNA4-gCHis,并进行双酶切鉴定。将鉴定正确的质粒转染MDBK细胞,收集上清,镍柱纯化带有His标签的重组gC蛋白,经Dot blot和间接免疫荧光法鉴定其反应原性。结果质粒p CDNA4-gC-His经双酶切鉴定证明构建正确,表达的重组gC蛋白相对分子质量约54 000,纯化后的浓度为0. 03 mg/mL,可与IBRV免疫的兔多抗发生反应。结论成功构建了真核表达质粒pCDNA4-gC-His,并能在MDBK细胞系中正确表达,表达的蛋白与天然IBRV gC蛋白具有相同的反应原性,为后续免疫学诊断方法的建立奠定了基础。
Objective To express the infectious bovine rhinotracheitis virus(IBRV) gC structural protein in eukaryotic cells and identify its immunogenicity. Methods Full-length gC gene was synthesized according to the IBRV gC gene sequence in GenBank,based on which recombinant plasmid p CDNA4-gC-His was constructed,identified by restriction analysis and transfected to MDBK cells. The supernatant was collected,from which the recombinant gC protein with His tag was purified by nickel ion affinity chromatography and identified for reactogenicity by dot blot and indirect immunofluorescent assay(IFA). Results Restriction analysis proved that recombinant plasmid pCDNA4-gC-His was constructed correctly. The expressed recombinant gC protein,with a relative molecular mass of about 54 000,reached a concentration of 0. 03 mg/mL and showed specific reaction with rabbit polyclonal antibody against IBRV. Conclusion Eukaryotic expression vector p CDNA4-gC-His was successfully constructed and expressed in MDBK cells. The expressed protein showed the same reactogenicity as that of natural IBRV gC protein,which laid a foundation of further development of immunological diagnostic method.
Objective To express the infectious bovine rhinotracheitis virus(IBRV) gC structural protein in eukaryotic cells and identify its immunogenicity. Methods Full-length gC gene was synthesized according to the IBRV gC gene sequence in GenBank,based on which recombinant plasmid p CDNA4-gC-His was constructed,identified by restriction analysis and transfected to MDBK cells. The supernatant was collected,from which the recombinant gC protein with His tag was purified by nickel ion affinity chromatography and identified for reactogenicity by dot blot and indirect immunofluorescent assay(IFA). Results Restriction analysis proved that recombinant plasmid pCDNA4-gC-His was constructed correctly. The expressed recombinant gC protein,with a relative molecular mass of about 54 000,reached a concentration of 0. 03 mg/mL and showed specific reaction with rabbit polyclonal antibody against IBRV. Conclusion Eukaryotic expression vector p CDNA4-gC-His was successfully constructed and expressed in MDBK cells. The expressed protein showed the same reactogenicity as that of natural IBRV gC protein,which laid a foundation of further development of immunological diagnostic method.
引文
[1] VINGS R L,STOLL I R,WARG J V,et al. Generation by self re-fusion of bovin 3x murine2 heterohybridomas secreting virusneutralizing bovine monoclonal antibodies to bovine herpesvirus1 glycoproteins gB,gC,and gD[J]. Vet Immunol Immunopathol,2014,159(1-2):58-73.
[2] MA H,BIAN C Z,WANG Y F,et al. Development and preliminary application of monoclonal antibody against IBRV gC and identification of its biological characteristic[J]. Acta Vet Zootech Sin,2013,44(10):1637-1644.(in Chinese)马辉,边传周,王永芬,等.牛传染性鼻气管炎病毒gC蛋白单克隆抗体的制备与初步应用[J].畜牧兽医学报,2013,44(10):1637-1644.
[3] ZHANG F,LIU R N,CHEN Y Y,et al. Progress on diagnosis method of infectious bovine rhinotracheitis[J]. Prog in Vet Med,2017,38(4):93-97.(in Chinese)张芳,刘瑞宁,陈颖钰,等.牛传染性鼻气管炎诊断方法研究进展[J].动物医学进展,2017,38(4):93-97.
[4] RAAPERI K,ORRO T,VILTROP A. Epidemiology and control of bovine herpesvirus 1 infection in Europe[J]. Vet J.2014,201(3):249-256.
[5] NEWCOMER B W,GIVENS D. Diagnosis and control of viral diseases of reproductive importance:infectious bovine rhinotracheitis and bovine viral diarrhea[J]. Vet Clin North Am Food Anim Pract,2016,32(2):425-441.
[6] JONES C,CHOWDHURY S. Bovine herpesvirus type 1(BHV-1)is an important cofactor in the bovine respiratory disease complex[J]. Vet Clin North Am Food Anim Pract,2010,26(2):303-321.
[7] LEVINGS R L,COLLINS J K,PATTERSON P A,et al. Virus,strain,and epitope specificities of neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB,gC,and gD,with sequence and molecular model analysis[J].Vet Immunol Immunopathol,2015,164(3-4):179-193.
[8] BI Y,WEN X B,NI H B. Preparation and identification of monoclonal antibody against gD protein of bovine infectious rhinotracheitis virus[J]. Chin J Biologicals,2017,30(10):1050-1054.(in Chinese)毕莹,闻晓波,倪宏波.牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定[J].中国生物制品学杂志,2017,30(10):1050-1054.
[9] TIKOO S K,FITZPATRICK D R,BABIUK L A,et al. Molecular cloning,sequening,and expression of functional bovine herpesvirus 1 glycoprotein gⅣin transfected bovine cells[J]. J Virol,1990,64(10):5132-5142.
[10] MARSHALL R L,RODRIGUEZ L L,LETCHWORTH G J3rd. Characterization of envelope proteins of infectious bovine rhinotracheitis virus(bovine herpesvirus 1)by biochemical and immunoloical methods[J]. J Virol,1986,57(3):745-753.
[11] LEROY B,GILLET L,VANDERPLASSCHEN A,et al. Structural proteomics of herpesviruses[J]. Viruses,2016,8(2):50. DOI:10.3390/v8020050.
[12] CHEN R,ZENG C Y,LUO Q,et al. Cloning and expression of antigenic region of glycoprotein gC gene from infectious bovine rhinotrachetis virus[J]. Anim Husb Vet Med,2008,40(2):16-19.(in Chinese)陈茹,曾彩云,罗琼,等.牛传染性鼻气管炎病毒糖蛋白gC基因抗原活性区的克隆与表达[J].畜牧与兽医,2008,40(2):16-19.
[13] XU J,WU J,JIANG B,et al. Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D[J]. Appl Microbiol Biotechnol,2017,101(23-24):8331-8344.
[14] CHOWDHURY S I. Identification of an epitope within the bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals[J]. J Virol Methods,2016,233(1):97-104.
[15] RIJSWEIJK F A,KAASHOEK M J,LANGEVELD J P,et al.Epitopes on glycoprotein C of bovine herpesvirus-1(BHV-1)that allow differentiation between BHV-1.1 and BHV-1.2 strains[J]. J Gen Virol,1999,80(Pt 6):1477-1483.
[2] MA H,BIAN C Z,WANG Y F,et al. Development and preliminary application of monoclonal antibody against IBRV gC and identification of its biological characteristic[J]. Acta Vet Zootech Sin,2013,44(10):1637-1644.(in Chinese)马辉,边传周,王永芬,等.牛传染性鼻气管炎病毒gC蛋白单克隆抗体的制备与初步应用[J].畜牧兽医学报,2013,44(10):1637-1644.
[3] ZHANG F,LIU R N,CHEN Y Y,et al. Progress on diagnosis method of infectious bovine rhinotracheitis[J]. Prog in Vet Med,2017,38(4):93-97.(in Chinese)张芳,刘瑞宁,陈颖钰,等.牛传染性鼻气管炎诊断方法研究进展[J].动物医学进展,2017,38(4):93-97.
[4] RAAPERI K,ORRO T,VILTROP A. Epidemiology and control of bovine herpesvirus 1 infection in Europe[J]. Vet J.2014,201(3):249-256.
[5] NEWCOMER B W,GIVENS D. Diagnosis and control of viral diseases of reproductive importance:infectious bovine rhinotracheitis and bovine viral diarrhea[J]. Vet Clin North Am Food Anim Pract,2016,32(2):425-441.
[6] JONES C,CHOWDHURY S. Bovine herpesvirus type 1(BHV-1)is an important cofactor in the bovine respiratory disease complex[J]. Vet Clin North Am Food Anim Pract,2010,26(2):303-321.
[7] LEVINGS R L,COLLINS J K,PATTERSON P A,et al. Virus,strain,and epitope specificities of neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB,gC,and gD,with sequence and molecular model analysis[J].Vet Immunol Immunopathol,2015,164(3-4):179-193.
[8] BI Y,WEN X B,NI H B. Preparation and identification of monoclonal antibody against gD protein of bovine infectious rhinotracheitis virus[J]. Chin J Biologicals,2017,30(10):1050-1054.(in Chinese)毕莹,闻晓波,倪宏波.牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备及鉴定[J].中国生物制品学杂志,2017,30(10):1050-1054.
[9] TIKOO S K,FITZPATRICK D R,BABIUK L A,et al. Molecular cloning,sequening,and expression of functional bovine herpesvirus 1 glycoprotein gⅣin transfected bovine cells[J]. J Virol,1990,64(10):5132-5142.
[10] MARSHALL R L,RODRIGUEZ L L,LETCHWORTH G J3rd. Characterization of envelope proteins of infectious bovine rhinotracheitis virus(bovine herpesvirus 1)by biochemical and immunoloical methods[J]. J Virol,1986,57(3):745-753.
[11] LEROY B,GILLET L,VANDERPLASSCHEN A,et al. Structural proteomics of herpesviruses[J]. Viruses,2016,8(2):50. DOI:10.3390/v8020050.
[12] CHEN R,ZENG C Y,LUO Q,et al. Cloning and expression of antigenic region of glycoprotein gC gene from infectious bovine rhinotrachetis virus[J]. Anim Husb Vet Med,2008,40(2):16-19.(in Chinese)陈茹,曾彩云,罗琼,等.牛传染性鼻气管炎病毒糖蛋白gC基因抗原活性区的克隆与表达[J].畜牧与兽医,2008,40(2):16-19.
[13] XU J,WU J,JIANG B,et al. Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D[J]. Appl Microbiol Biotechnol,2017,101(23-24):8331-8344.
[14] CHOWDHURY S I. Identification of an epitope within the bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals[J]. J Virol Methods,2016,233(1):97-104.
[15] RIJSWEIJK F A,KAASHOEK M J,LANGEVELD J P,et al.Epitopes on glycoprotein C of bovine herpesvirus-1(BHV-1)that allow differentiation between BHV-1.1 and BHV-1.2 strains[J]. J Gen Virol,1999,80(Pt 6):1477-1483.