鸭肠炎病毒UL18基因的转录、表达特征及原核表达蛋白的初步应用
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摘要
为了探讨鸭肠炎病毒(duck enteritis virus,DEV)UL18基因的特性和功能,本研究运用生物信息学软件对DEV UL18基因及其编码蛋白进行了分析。构建原核表达质粒pET32a-UL18,并将其转化大肠杆菌BL21(DE3),经IPTG诱导后获得了大小约为55 000的pET32a/DEV-UL18重组蛋白。将该重组蛋白纯化后免疫家兔,获得了兔抗pET32a/DEV-UL18重组蛋白高免血清。采用荧光定量RT-PCR和Western blot对DEV感染鸭胚成纤维细胞(DEF)后UL18基因的转录和表达情况进行检测,采用间接免疫荧光对DEV感染鸭的肠道和食道进行检测。生物信息学分析结果表明,DEV UL18基因大小969bp,编码1条由322个氨基酸残基组成的多肽,含15个潜在的磷酸化位点和2个N-糖基化位点,含15个潜在的B细胞表位;系统进化树分析表明,DEV UL18属于ɑ-疱疹病毒的1个成员,与MeHV-1亲缘关系最近;密码子偏爱性分析结果表明,若对DEV UL18基因进行外源表达,真核表达系统可能更容易,若选择原核表达系统,则需对宿主表达菌进行选择和条件优化。荧光定量RT-PCR和Western blot结果表明,DEV感染DEF后2h,UL18基因开始转录,感染后12h开始表达,感染24h后转录和表达产物急剧上升,到36和48h后转录和表达产物分别达最大值,之后逐渐下降。间接免疫荧光结果表明,被DEV感染鸭肠道、食道等组织能检测到明显的阳性信号,表明制备的DEV UL18原核表达蛋白及其兔抗多克隆抗体可用于DEV的诊断试剂材料。
To investigate the characteristics and function of duck enteritis virus(DEV)UL18 gene,the DEV UL18 gene and its encoded protein were analyzed using bioinformatics analysis software.The prokaryotic expression plasmid pET32 a-UL18 was constructed and transformed into E.coli BL21(DE3).After induction by IPTG,recombinant pET32 a/DEV-UL18 protein with a size of approximately 55 000 was obtained.After the recombinant protein was purified,the rabbit was immunized to obtain rabbit anti-pET32 a/DEV-UL18 hyperimmune serum.The transcription and expression of DEV UL18 gene in DEV-infected duck embryo fibroblasts(DEF)was detected by fluorescent quantitative RT-PCR and Western blot.The intestine and esophagus of DEV-infected ducks were detected by indirect immunofluorescence.Bioinformatics analysis showed that the DEV UL18 gene is 969 bp in size and encodes a polypeptide consisting of 322 amino acid residues,containing 15 potential phosphorylation sites,2 N-glycosylation sites,and 15 potential B cell epitopes.Phylogenetic tree analysis showed that DEV UL18 belongs to a member ofα-herpesvirus and has a close relationship with MeHV-1.The results of codon bias analysis showed that if the DEV UL18 gene is expressed exogenously,the eukaryotic expression system may be easier.If the prokaryotic expression system is selected,the host expression bacteria must be selected and the conditions of expression need to be optimized.The results of fluorescent quantitative RT-PCR and Western blot showed that the UL18 gene was transcribed at 2 hafter DEV infection with DEF,and it began to express at 12 hafter infection.The transcription and expression products increased rapidly after infection for 24 h,and the transcription and expression products reached the maximum after 36 hand48 hrespectively,then decreased gradually.The results of indirect immunofluorescence showed that the DEV-infected duck intestinal and esophageal tissues could detect significant positive signals,indicating that the prepared DEV UL18 prokaryotic expression protein and its rabbit antipolyclonal antibody can be applied to DEV diagnostic reagent materials.
To investigate the characteristics and function of duck enteritis virus(DEV)UL18 gene,the DEV UL18 gene and its encoded protein were analyzed using bioinformatics analysis software.The prokaryotic expression plasmid pET32 a-UL18 was constructed and transformed into E.coli BL21(DE3).After induction by IPTG,recombinant pET32 a/DEV-UL18 protein with a size of approximately 55 000 was obtained.After the recombinant protein was purified,the rabbit was immunized to obtain rabbit anti-pET32 a/DEV-UL18 hyperimmune serum.The transcription and expression of DEV UL18 gene in DEV-infected duck embryo fibroblasts(DEF)was detected by fluorescent quantitative RT-PCR and Western blot.The intestine and esophagus of DEV-infected ducks were detected by indirect immunofluorescence.Bioinformatics analysis showed that the DEV UL18 gene is 969 bp in size and encodes a polypeptide consisting of 322 amino acid residues,containing 15 potential phosphorylation sites,2 N-glycosylation sites,and 15 potential B cell epitopes.Phylogenetic tree analysis showed that DEV UL18 belongs to a member ofα-herpesvirus and has a close relationship with MeHV-1.The results of codon bias analysis showed that if the DEV UL18 gene is expressed exogenously,the eukaryotic expression system may be easier.If the prokaryotic expression system is selected,the host expression bacteria must be selected and the conditions of expression need to be optimized.The results of fluorescent quantitative RT-PCR and Western blot showed that the UL18 gene was transcribed at 2 hafter DEV infection with DEF,and it began to express at 12 hafter infection.The transcription and expression products increased rapidly after infection for 24 h,and the transcription and expression products reached the maximum after 36 hand48 hrespectively,then decreased gradually.The results of indirect immunofluorescence showed that the DEV-infected duck intestinal and esophageal tissues could detect significant positive signals,indicating that the prepared DEV UL18 prokaryotic expression protein and its rabbit antipolyclonal antibody can be applied to DEV diagnostic reagent materials.
引文
[1]METTENLEITER T C,KLUPP B G,GRANZOW H.Herpesvirus assembly:A tale of two membranes[J].Curr Opin Microbiol,2006,9(4):423-429.
[2]SANDHU T S,METWALLY S A.Duck virus enteritis(duck plague)[M].12th ed.Singapore:Blackwell Publishing,2008:384-393.
[3]BAUDET A E R F.Mortality in ducks in the Netherlands caused by a filtrable virus:Fowl plague[J].Tijdschr Diergeneeskd,1923,50:455-459.
[4]黄引贤.拟鸭瘟的研究[J].华南农学院学报,1959,1(1):1-13.
[5]甘孟侯.中国禽病学[M].北京:中国农业出版,1999:107-119.
[6]FAUQUET C M,MAYO M A,MANILOFF J,et al.Virus taxonomy:Ⅷth report of the international committee on taxonomy of viruses[M].San Diego:Elsevier Academic Press,2005:1-12.
[7]LI Y,HUANG B,MA X,et al.Molecular characterization of the genome of duck enteritis virus[J].Virology,2009,391(2):151-161.
[8]CHANG H,CHENG A C,WANG M S,et al.Cloning,expression and characterization of gE protein of duck plague virus[J].Virol J,2010,7(1):1-11.
[9]CAI M S,CHENG A C,WANG M S,et al.Characterization of synonymous codon usage bias in the duck plague virus UL35 gene[J].Intervirology,2009,52(5):266-278.
[10]XIANG J,ZHANG S C,CHENG A C,et al.Expression and characterization of recombinant VP19cprotein and N-terminal from duck enteritis virus[J].Virol J,2011,8(1):1-8.
[11]GKLUPP B,GRANZOW H,METTENLEITER T C.Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34gene product[J].J Virol,2000,74(21):10063-10073.
[12]DONNELLY M,ELLIOTT G.Fluorescent tagging of herpes simplex virus tegument protein VP13/14in virus infection[J].J Virol,2001,75(6):2575-2583.
[13]程安春,汪铭书,文明,等.鸭瘟病毒基因文库的构建及其核衣壳蛋白基因的发现、克隆与鉴定[J].高技术通讯,2006,16(9):948-953.
[14]韩俊英,曾瑞萍.荧光定量PCR技术及其应用[J].国外医学(遗传学分册),2000,23(3):117-120.
[15]LUKASIAK S,BREUHAHN K,SCHILLER C,et al.Quantitative analysis of gene expression relative to18SrRNA in carcinoma samples using the light cycler instrument and a SYBR greeni-based assay:determining FAT10mRNA levels in hepatocellular carcinoma[J].Methods Mol Biol,2008,429(1):59-72.
[16]KUBISTA M,ANDRADE J M,BENGTSSON M,et al.The real-time polymerase chain reaction[J].Mol Aspects Med,2006,27(2/3):95-125.
[17]PINZANI P,LIND K,MALENTACCHI F,et al.Prostate-specific antigen mRNA and protein levels in laser microdissected cells of human prostate measured by real-time reverse transcriptase-quantitative polymerase chain reaction and immuno-quantitative polymerase chain reaction[J].Hum Pathol,2008,39(10):1474-1482.
[18]FELDMAN L,RIXON F J,JEAN J H,et al.Transcription of the genome of pseudorabies virus(A herpesvirus)is strictly controlled[J].Virology,1979,97(2):316-327.
[19]寸韡.HSVI立即早期基因研究进展[J].国外医学(病毒学分册),2003,10(5):129-132.
[20]寸韡,张莹,刘龙丁,等.单纯疱疹Ⅰ型病毒即刻早期基因上游调控序列分析[J].生物化学与生物物理进展,2006,33(1):77-82.
[21]SPEAR P,ROIZMAN B,TOOZE J.Molecular biology of tumor viruses,part ii,dna tumor viruses[M].2nd ed.New York:Cold Spring Harbor Laboratory,1980:958-963.
[22]WEIR J P,NARAYANAN P R.The use of B-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1glycoprotein C gene in recombinant herpesviruses[J].Nucleic Acids Res,1988,16(26):10267-10282.
[23]徐耀基,黄引贤.应用琼脂凝胶沉淀试验检测鹅鸭病料中的鸭瘟病毒[J].中国畜禽传染病,1992(4):30-31.
[24]MALMARUGAN S,SULOCHANA S.Comparison of Dot-ELISA passive haemagglutination test for the detection of antibodies to duck plague[J].Indian Vet J,2002,79(7):648-651.
[25]LU L T,CHENG A C,WANG M S,et al.Polyclonal antibody against the DPV UL46Mprotein can be a diagnostic candidate[J].Virol J,2010,83(7):1-10.
[26]TORO H,KALETA E F.Absence of neutralising antibodies to duck plague virus in the commercial duck and goose populations in West Germany(1980-1985)[J].Avian Pathol,1986,15(1):57-62.
[27]宋涌,程安春,汪铭书,等.聚合酶链反应(PCR)检测鸭瘟病毒方法的建立和应用[J].中国兽医杂志,2005,41(2):17-20.
[28]李凯年.用PCR诊断水禽的鸭瘟[J].中国家禽,2002,24(2):50.
[29]CHENG A C,WANG M S,LIU F,et al.The preliminary application of PCR in research of clinical diagnosis and mechanisms of immunity and pathogeny of duck plague virus(DPV)[J].Chin J Virol,2004,20(4):364-370.
[30]HANSEN W R,NASHOLD S W,DOCHERTY D E,et al.Diagnosis of duck plague in waterfowl by polymerase chain reaction[J].Avian Dis,2000,44(2):266-274.
[31]索化夷,汤承,岳华,等.实时荧光定量TaqManPCR检测鸭瘟病毒方法的建立[J].中国预防兽医学报,2006,28(3):332-335,340.
[32]QI X F,YANG X Y,CHENG A C,et al.The pathogenesis of duck virus enteritis in experimentally infected ducks:aquantitative time-course study using TaqMan polymerase chain reaction[J].Avian Pathol,2008,37(3):307-310.
[33]ISLAM M R,NESSA J,HALDER K M.Detection of duck plague virus antigen in tissues by immunoperoxidase staining[J].Avian Pathol,1993,22(2):389-393.
[34]ISLAM M R,KHAN M A.An immunocytochemical study on the sequential tissue distribution of duck plague virus[J].Avian Pathol,1995,24(1):189-194.
[35]SHAWKY S.Target cells for duck enteritis virus in lymphoid organs[J].Avian Pathol,2000,29(6):609-616.
[36]徐超,程安春,汪铭书,等.间接酶免疫组化检测DPV在感染鸭体内细胞定位的研究和应用[J].中国兽医学报,2007,27(5):640-644.
[37]程安春,韩晓英,朱德康,等.间接免疫荧光染色检测石蜡切片中的鸭病毒性肠炎病毒和抗原定位[J].中国兽医学报,2008,28(8):871-875.
[38]HARDY V B,DAGENAIS A,SIMARD R.In situlocalization of the major capsid protein during lytic infection by herpes simplex virus[J].J Gen Virol,1985,66(4):897-901.
[39]MATSUOKA T,TAVASSOLI M.A modified method for application of indirect immunofluorescent staining for factorⅧ/vWF to capillary endothelia[J].Am J Med Sci,1988,296(2):107-110.
[40]SHEN F X,MA G P,CHENG A C,et al.Development and application of an indirect immunohistochemical method for the detection of duck plague virus vaccine antigens in paraffin sections and localization in the vaccinated duckling tissues[J].Poult Sci,2010,89(9):1915-1923.
[41]SALGUERO F J,SANCHEZ-CORDON P J,NUNEZ A,et al.Histopathological and ultrastructural changes associated with herpesvirus infection in waterfowl[J].Avian Pathol,2002,31(2):133-140.
[2]SANDHU T S,METWALLY S A.Duck virus enteritis(duck plague)[M].12th ed.Singapore:Blackwell Publishing,2008:384-393.
[3]BAUDET A E R F.Mortality in ducks in the Netherlands caused by a filtrable virus:Fowl plague[J].Tijdschr Diergeneeskd,1923,50:455-459.
[4]黄引贤.拟鸭瘟的研究[J].华南农学院学报,1959,1(1):1-13.
[5]甘孟侯.中国禽病学[M].北京:中国农业出版,1999:107-119.
[6]FAUQUET C M,MAYO M A,MANILOFF J,et al.Virus taxonomy:Ⅷth report of the international committee on taxonomy of viruses[M].San Diego:Elsevier Academic Press,2005:1-12.
[7]LI Y,HUANG B,MA X,et al.Molecular characterization of the genome of duck enteritis virus[J].Virology,2009,391(2):151-161.
[8]CHANG H,CHENG A C,WANG M S,et al.Cloning,expression and characterization of gE protein of duck plague virus[J].Virol J,2010,7(1):1-11.
[9]CAI M S,CHENG A C,WANG M S,et al.Characterization of synonymous codon usage bias in the duck plague virus UL35 gene[J].Intervirology,2009,52(5):266-278.
[10]XIANG J,ZHANG S C,CHENG A C,et al.Expression and characterization of recombinant VP19cprotein and N-terminal from duck enteritis virus[J].Virol J,2011,8(1):1-8.
[11]GKLUPP B,GRANZOW H,METTENLEITER T C.Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34gene product[J].J Virol,2000,74(21):10063-10073.
[12]DONNELLY M,ELLIOTT G.Fluorescent tagging of herpes simplex virus tegument protein VP13/14in virus infection[J].J Virol,2001,75(6):2575-2583.
[13]程安春,汪铭书,文明,等.鸭瘟病毒基因文库的构建及其核衣壳蛋白基因的发现、克隆与鉴定[J].高技术通讯,2006,16(9):948-953.
[14]韩俊英,曾瑞萍.荧光定量PCR技术及其应用[J].国外医学(遗传学分册),2000,23(3):117-120.
[15]LUKASIAK S,BREUHAHN K,SCHILLER C,et al.Quantitative analysis of gene expression relative to18SrRNA in carcinoma samples using the light cycler instrument and a SYBR greeni-based assay:determining FAT10mRNA levels in hepatocellular carcinoma[J].Methods Mol Biol,2008,429(1):59-72.
[16]KUBISTA M,ANDRADE J M,BENGTSSON M,et al.The real-time polymerase chain reaction[J].Mol Aspects Med,2006,27(2/3):95-125.
[17]PINZANI P,LIND K,MALENTACCHI F,et al.Prostate-specific antigen mRNA and protein levels in laser microdissected cells of human prostate measured by real-time reverse transcriptase-quantitative polymerase chain reaction and immuno-quantitative polymerase chain reaction[J].Hum Pathol,2008,39(10):1474-1482.
[18]FELDMAN L,RIXON F J,JEAN J H,et al.Transcription of the genome of pseudorabies virus(A herpesvirus)is strictly controlled[J].Virology,1979,97(2):316-327.
[19]寸韡.HSVI立即早期基因研究进展[J].国外医学(病毒学分册),2003,10(5):129-132.
[20]寸韡,张莹,刘龙丁,等.单纯疱疹Ⅰ型病毒即刻早期基因上游调控序列分析[J].生物化学与生物物理进展,2006,33(1):77-82.
[21]SPEAR P,ROIZMAN B,TOOZE J.Molecular biology of tumor viruses,part ii,dna tumor viruses[M].2nd ed.New York:Cold Spring Harbor Laboratory,1980:958-963.
[22]WEIR J P,NARAYANAN P R.The use of B-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1glycoprotein C gene in recombinant herpesviruses[J].Nucleic Acids Res,1988,16(26):10267-10282.
[23]徐耀基,黄引贤.应用琼脂凝胶沉淀试验检测鹅鸭病料中的鸭瘟病毒[J].中国畜禽传染病,1992(4):30-31.
[24]MALMARUGAN S,SULOCHANA S.Comparison of Dot-ELISA passive haemagglutination test for the detection of antibodies to duck plague[J].Indian Vet J,2002,79(7):648-651.
[25]LU L T,CHENG A C,WANG M S,et al.Polyclonal antibody against the DPV UL46Mprotein can be a diagnostic candidate[J].Virol J,2010,83(7):1-10.
[26]TORO H,KALETA E F.Absence of neutralising antibodies to duck plague virus in the commercial duck and goose populations in West Germany(1980-1985)[J].Avian Pathol,1986,15(1):57-62.
[27]宋涌,程安春,汪铭书,等.聚合酶链反应(PCR)检测鸭瘟病毒方法的建立和应用[J].中国兽医杂志,2005,41(2):17-20.
[28]李凯年.用PCR诊断水禽的鸭瘟[J].中国家禽,2002,24(2):50.
[29]CHENG A C,WANG M S,LIU F,et al.The preliminary application of PCR in research of clinical diagnosis and mechanisms of immunity and pathogeny of duck plague virus(DPV)[J].Chin J Virol,2004,20(4):364-370.
[30]HANSEN W R,NASHOLD S W,DOCHERTY D E,et al.Diagnosis of duck plague in waterfowl by polymerase chain reaction[J].Avian Dis,2000,44(2):266-274.
[31]索化夷,汤承,岳华,等.实时荧光定量TaqManPCR检测鸭瘟病毒方法的建立[J].中国预防兽医学报,2006,28(3):332-335,340.
[32]QI X F,YANG X Y,CHENG A C,et al.The pathogenesis of duck virus enteritis in experimentally infected ducks:aquantitative time-course study using TaqMan polymerase chain reaction[J].Avian Pathol,2008,37(3):307-310.
[33]ISLAM M R,NESSA J,HALDER K M.Detection of duck plague virus antigen in tissues by immunoperoxidase staining[J].Avian Pathol,1993,22(2):389-393.
[34]ISLAM M R,KHAN M A.An immunocytochemical study on the sequential tissue distribution of duck plague virus[J].Avian Pathol,1995,24(1):189-194.
[35]SHAWKY S.Target cells for duck enteritis virus in lymphoid organs[J].Avian Pathol,2000,29(6):609-616.
[36]徐超,程安春,汪铭书,等.间接酶免疫组化检测DPV在感染鸭体内细胞定位的研究和应用[J].中国兽医学报,2007,27(5):640-644.
[37]程安春,韩晓英,朱德康,等.间接免疫荧光染色检测石蜡切片中的鸭病毒性肠炎病毒和抗原定位[J].中国兽医学报,2008,28(8):871-875.
[38]HARDY V B,DAGENAIS A,SIMARD R.In situlocalization of the major capsid protein during lytic infection by herpes simplex virus[J].J Gen Virol,1985,66(4):897-901.
[39]MATSUOKA T,TAVASSOLI M.A modified method for application of indirect immunofluorescent staining for factorⅧ/vWF to capillary endothelia[J].Am J Med Sci,1988,296(2):107-110.
[40]SHEN F X,MA G P,CHENG A C,et al.Development and application of an indirect immunohistochemical method for the detection of duck plague virus vaccine antigens in paraffin sections and localization in the vaccinated duckling tissues[J].Poult Sci,2010,89(9):1915-1923.
[41]SALGUERO F J,SANCHEZ-CORDON P J,NUNEZ A,et al.Histopathological and ultrastructural changes associated with herpesvirus infection in waterfowl[J].Avian Pathol,2002,31(2):133-140.