伪狂犬病病毒gE基因在大肠杆菌中的表达
摘要
伪狂犬病病毒是疱疹病毒科、α-疱疹病毒亚科的成员，可以引起猪、牛、羊及野生动物的伪狂犬病(又称Aujeszky氏病)，猪是该病毒的天然宿主、也是主要感染源。伪狂犬病给养猪业造成了巨大经济损失，其危害仅次于猪瘟和口蹄疫。作为主要的诊断抗原，伪狂犬病病毒糖蛋白E(glycoprotein,gE)在伪狂犬根除计划中发挥重要的作用。
为获得大量的、成本较低的诊断抗原，本研究利用大肠杆菌表达系统对伪狂犬病病毒gE主要抗原区域进行了表达，为gE-ELISA试剂盒的研制提供一定的理论依据和借鉴。本研究主要内容：
根据GenBank中伪狂犬病毒gE基因序列，设计了一对引物，PCR扩增长度为558bp的gE基因去信号肽后的主要抗原区域(157-714bp)，将其克隆进测序载体PGEM-Teasy中，成功构建质粒PGEMTeasy-gE。用BamhI和HindⅢ将gE基因主要抗原区域从PGEM-Teasy中切出，然后与同样酶处理的原核表达载体pet32a连接，命名为pet-gE。
在IPTG的诱导后，经SDS-PAGE电泳发现43ku处出现特异性蛋白条带。
研究了目的蛋白表达量与各个培养条件的关系，优化后的最佳表达条件为IPTG浓度为1mM，诱导时间为5小时时的蛋白表达量最大。
经过western-blot分析，表达产物具有很好的抗原性。
该研究成功表达出具有抗原性的目的蛋白，为血清学鉴别诊断方法gE-ELISA的建立打下很好的基础，同时gE-ELISA也能对野毒和疫苗毒进行鉴别。
Pseudorabies virus(prv),is a member of Alphaherpesvirinae,herpesviridae,which can causeAujeszky's disease in swine,bovine,sheep and wild animals.PRV infection could cause its naturaland main storing host-swine(also its main infectious origination)a huge loses,the most hugeeconomical lose in all swine infectious diseases except swine plague and aftosa. Glyprotein E ofPseudorabies Virus is known to be an important diagnostic antigen in pseudorabies eradicationcampaign.
In order to obtain the gE antigen in large quantity and at a low cost, a gene fragment encodingthe main antigtie domain (157-714bp) of PRV gE without signal peptide was expressed in E coliexpression system. This study may supply theoretical evidence for establishment of gE-ELISA.
Using a pair of primers designed according to the revelant nucleotide sequence fromGenBank, the specific genes encoding the main antigetic domains of PRV gE were amplified. ThePCR products of 557bp were cloned into the sequencing vectors PGEM-Teasy.The plasmidPGEMTeasy-gE was constructed successfully. Cut out the main antigetic of PRV gE fromPGEMTeasy-gE with BamhⅠand HindⅢ, and linked with prokaryotic expression vector pet32awhich has been cut by the two enzyme beforehand. The plasmid construced was named PET-gE.
After induced by IPTG,a specific strip of 43 Ku appeard in SDS-PAGE.
According to the study on the relationship bwteen expression quantity of protein and revelantcultivating conditions, the optimal expression condition were: 5 hours inducing range, the 1mMconcentration of IPTG.
On analysis by Western-blot, the expression product has good antigenicity.
The target protein with antigenicity was expressed successfully in the study. It laid thefoundation for the establishment of the diagnostic method of serology gE-ELISA,which can tellvaccine virus from virulence virus.
为获得大量的、成本较低的诊断抗原，本研究利用大肠杆菌表达系统对伪狂犬病病毒gE主要抗原区域进行了表达，为gE-ELISA试剂盒的研制提供一定的理论依据和借鉴。本研究主要内容：
根据GenBank中伪狂犬病毒gE基因序列，设计了一对引物，PCR扩增长度为558bp的gE基因去信号肽后的主要抗原区域(157-714bp)，将其克隆进测序载体PGEM-Teasy中，成功构建质粒PGEMTeasy-gE。用BamhI和HindⅢ将gE基因主要抗原区域从PGEM-Teasy中切出，然后与同样酶处理的原核表达载体pet32a连接，命名为pet-gE。
在IPTG的诱导后，经SDS-PAGE电泳发现43ku处出现特异性蛋白条带。
研究了目的蛋白表达量与各个培养条件的关系，优化后的最佳表达条件为IPTG浓度为1mM，诱导时间为5小时时的蛋白表达量最大。
经过western-blot分析，表达产物具有很好的抗原性。
该研究成功表达出具有抗原性的目的蛋白，为血清学鉴别诊断方法gE-ELISA的建立打下很好的基础，同时gE-ELISA也能对野毒和疫苗毒进行鉴别。
Pseudorabies virus(prv),is a member of Alphaherpesvirinae,herpesviridae,which can causeAujeszky's disease in swine,bovine,sheep and wild animals.PRV infection could cause its naturaland main storing host-swine(also its main infectious origination)a huge loses,the most hugeeconomical lose in all swine infectious diseases except swine plague and aftosa. Glyprotein E ofPseudorabies Virus is known to be an important diagnostic antigen in pseudorabies eradicationcampaign.
In order to obtain the gE antigen in large quantity and at a low cost, a gene fragment encodingthe main antigtie domain (157-714bp) of PRV gE without signal peptide was expressed in E coliexpression system. This study may supply theoretical evidence for establishment of gE-ELISA.
Using a pair of primers designed according to the revelant nucleotide sequence fromGenBank, the specific genes encoding the main antigetic domains of PRV gE were amplified. ThePCR products of 557bp were cloned into the sequencing vectors PGEM-Teasy.The plasmidPGEMTeasy-gE was constructed successfully. Cut out the main antigetic of PRV gE fromPGEMTeasy-gE with BamhⅠand HindⅢ, and linked with prokaryotic expression vector pet32awhich has been cut by the two enzyme beforehand. The plasmid construced was named PET-gE.
After induced by IPTG,a specific strip of 43 Ku appeard in SDS-PAGE.
According to the study on the relationship bwteen expression quantity of protein and revelantcultivating conditions, the optimal expression condition were: 5 hours inducing range, the 1mMconcentration of IPTG.
On analysis by Western-blot, the expression product has good antigenicity.
The target protein with antigenicity was expressed successfully in the study. It laid thefoundation for the establishment of the diagnostic method of serology gE-ELISA,which can tellvaccine virus from virulence virus.
引文
程由铨，吴平，林天龙等．4种检测伪狂犬病病毒抗原方法的比较[J]．中国兽医杂志，1992，18(1)：628．
程由铨，吴平，林天龙等．4种检测伪狂犬病病毒抗原方法的比较[J]．中国兽医杂志，1992，18(1)：6～9．
初秀．国外猪伪狂犬病研究的概况[J]．中国兽医科技，1985，12：21～25．
黄骏明，李亚香，魏良治等．应用斑点ELISA检测猪伪狂犬病[J]．中国畜禽传染病，1989，49(6)：25229．
黄骏明，李亚香，朱庆虎等．应用免疫荧光抗体技术快速检测猪伪狂犬病病毒[J]．中国畜禽传染病，1995，85(6)：38～42．
李健强，张国祥，王晶钰等．Dot-ELISA检测猪血清中伪狂犬病病毒抗体方法的建立与评价[J]．中国畜禽传染病，1990，50(1)：27230．
李文刚，甘孟侯．猪伪狂犬病研究进展(下)[J]．国外畜牧科技，1995，22(1)：42-45．
陆承平，兽医微生物学[M]，第三版，北京，中国农业出版社，2001，411-483．
罗廷荣，余克伦，粱家权等．SPA-ELISA检测猪伪狂犬病病毒血清抗体[J]．广西畜牧兽医，2000，16(6)：324．
苗得园，杨兵，李富强等．单抗夹心LAB-ELISA检测猪伪狂犬病病毒的研究[J]．华北农学报，2001，16(1)：1272131．
彭志领，时庆华潴伪狂犬病的诊断及防治研究进展[J]．中国动物检疫，1997，14(5)：34-35．
邱德新，陈焕春，何启盖等．间接ELISA检测猪伪狂犬病血清抗体[J]．中国兽医学报，2002，22(2)：1492152．
冉智光，童光志，张绍杰等．伪狂犬病毒Min-A株gE基因序列分析及其真核表达质粒的构建[J]．西南农业学报，1999，12(2)：1-6．
石建平，宣华，卢强等．伪狂犬病PCR检测方法的建立及初步应用[J]．中国畜禽传染病，1996，5：16-20．
王勤．猪伪狂犬病病毒gE基因的研究进展[J]．畜牧与兽医，2002，10(34)：42-44．
A fshar A, Wright PF, Dulac GC. Evaluation of an enzyme immunoassay for detection of antibodies to Pseudorabies virus in porcine field sera[J].Can J V et Res, 1987, 51: 5392541,
Bush CE, Pitchett RF. Immuno logic comparison of the proteins of Pseudorabies(Aujeszky's disease) virus and bovine berpesvirusl [J]. Am J Vet Res, 1986, 47: 170821712.
Caims TM,Milne M,Ponce-De-leon DK, et al.2003.Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL:Clues from gDgH Chimeras.J, virol.77:6731-6742.
Cheung AK,J Fang,Wesley RD. 1994.Characterization of a pseudorabies virus that is defective in the early proteinO and latency genes.Am.J.Vet.Res.55:1710-1716.
Crabb BS, Nagesha HS , Studdert MJ.1992.Idendification of equine herpesvirus glycoprotein G:a type specific,secreted glycoprotein. Virology 190:143-154.
David Kinker.Sabrina L,Swenson,Lie-Ling Wu,et al.Evaluation of serological tests for the detection of pseudorabies gE antibodies during Earlyinfection[J]. Veterinary Microbiology 1997,55:159-166.
Demmin GL, Clase AC. 2001 .Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection.J Virol.75:10856-10869.
Dijkstra JM,Visser, Mettenleiter TC.Identification and characterization of pseudorabies virus glycoprotein gM as a nonessential virion component J.Virol. 1996,70(8):5684-5688.
Ducatelle R,Coursement W,Hoorens J.Immunoperoxidase study of Aujeszky's disease in pigs [J].Res Vet Sci ,1982,32:294-302.
Elioit M,Forgeaud D,Haridon R L,et al.Identification of the pseudorabies virus glycoprotein gp50 as a major target of neutralizing antibodies.Arch,virol, 1988,99:45-46.
Favoreel HW,VanMinnebruggen HJ,Nauwynck LW, et al.2002.A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced intemalization of viral glycoproteins and efficient cell-to-cell spread. J spread.J.virol.76:6845-6851.
Firkins LD ,Weigel RM,.Biehl LG , et al.l997.Field trial to evaluate the immunogenicity of pseudorabies virus vaccines with deletions for glycoproteins G and E Am J Vet Res 58:976-9 84.
Geraghty RJ,Krummenacher C,Cohen GH, et al.Entry of alphaherpesvirus mediated by poliovirus receptor-related Protein 1 and poliovirus receptor [J].Science,1998,280:1618-1620.
Granzow HF, Weiland A , Jons B, et al. 1997 Ultrasturctural analysis of the replication cycle of pseudoradies virus in cell culture,a reassessment J Virol 71:2072-2082.
Hanebe H,Wheeler JG.Osorio FA.Gene specific assey to differentiate stain of pseudorabies virus [J].VetMicrobiol,1993,34,221-231.
Harding MJ,Prud'homme I, Rola.Specificity and nucleotyping studies of a gp50 based polyme -rase chain reaction assey for detection of pseudorabies virus[J].Can J Vet Res,1997,61:157-160.
Jacobs L.Kimman TG, Bianchi A ,et al. Lack of serum antibodies against gE in maternally immune pigs exposed to a mildly virulent strain of PRV [J ]. Am J Vet Res,1996,(57) :152521528.
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程由铨，吴平，林天龙等．4种检测伪狂犬病病毒抗原方法的比较[J]．中国兽医杂志，1992，18(1)：6～9．
初秀．国外猪伪狂犬病研究的概况[J]．中国兽医科技，1985，12：21～25．
黄骏明，李亚香，魏良治等．应用斑点ELISA检测猪伪狂犬病[J]．中国畜禽传染病，1989，49(6)：25229．
黄骏明，李亚香，朱庆虎等．应用免疫荧光抗体技术快速检测猪伪狂犬病病毒[J]．中国畜禽传染病，1995，85(6)：38～42．
李健强，张国祥，王晶钰等．Dot-ELISA检测猪血清中伪狂犬病病毒抗体方法的建立与评价[J]．中国畜禽传染病，1990，50(1)：27230．
李文刚，甘孟侯．猪伪狂犬病研究进展(下)[J]．国外畜牧科技，1995，22(1)：42-45．
陆承平，兽医微生物学[M]，第三版，北京，中国农业出版社，2001，411-483．
罗廷荣，余克伦，粱家权等．SPA-ELISA检测猪伪狂犬病病毒血清抗体[J]．广西畜牧兽医，2000，16(6)：324．
苗得园，杨兵，李富强等．单抗夹心LAB-ELISA检测猪伪狂犬病病毒的研究[J]．华北农学报，2001，16(1)：1272131．
彭志领，时庆华潴伪狂犬病的诊断及防治研究进展[J]．中国动物检疫，1997，14(5)：34-35．
邱德新，陈焕春，何启盖等．间接ELISA检测猪伪狂犬病血清抗体[J]．中国兽医学报，2002，22(2)：1492152．
冉智光，童光志，张绍杰等．伪狂犬病毒Min-A株gE基因序列分析及其真核表达质粒的构建[J]．西南农业学报，1999，12(2)：1-6．
石建平，宣华，卢强等．伪狂犬病PCR检测方法的建立及初步应用[J]．中国畜禽传染病，1996，5：16-20．
王勤．猪伪狂犬病病毒gE基因的研究进展[J]．畜牧与兽医，2002，10(34)：42-44．
A fshar A, Wright PF, Dulac GC. Evaluation of an enzyme immunoassay for detection of antibodies to Pseudorabies virus in porcine field sera[J].Can J V et Res, 1987, 51: 5392541,
Bush CE, Pitchett RF. Immuno logic comparison of the proteins of Pseudorabies(Aujeszky's disease) virus and bovine berpesvirusl [J]. Am J Vet Res, 1986, 47: 170821712.
Caims TM,Milne M,Ponce-De-leon DK, et al.2003.Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL:Clues from gDgH Chimeras.J, virol.77:6731-6742.
Cheung AK,J Fang,Wesley RD. 1994.Characterization of a pseudorabies virus that is defective in the early proteinO and latency genes.Am.J.Vet.Res.55:1710-1716.
Crabb BS, Nagesha HS , Studdert MJ.1992.Idendification of equine herpesvirus glycoprotein G:a type specific,secreted glycoprotein. Virology 190:143-154.
David Kinker.Sabrina L,Swenson,Lie-Ling Wu,et al.Evaluation of serological tests for the detection of pseudorabies gE antibodies during Earlyinfection[J]. Veterinary Microbiology 1997,55:159-166.
Demmin GL, Clase AC. 2001 .Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection.J Virol.75:10856-10869.
Dijkstra JM,Visser, Mettenleiter TC.Identification and characterization of pseudorabies virus glycoprotein gM as a nonessential virion component J.Virol. 1996,70(8):5684-5688.
Ducatelle R,Coursement W,Hoorens J.Immunoperoxidase study of Aujeszky's disease in pigs [J].Res Vet Sci ,1982,32:294-302.
Elioit M,Forgeaud D,Haridon R L,et al.Identification of the pseudorabies virus glycoprotein gp50 as a major target of neutralizing antibodies.Arch,virol, 1988,99:45-46.
Favoreel HW,VanMinnebruggen HJ,Nauwynck LW, et al.2002.A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced intemalization of viral glycoproteins and efficient cell-to-cell spread. J spread.J.virol.76:6845-6851.
Firkins LD ,Weigel RM,.Biehl LG , et al.l997.Field trial to evaluate the immunogenicity of pseudorabies virus vaccines with deletions for glycoproteins G and E Am J Vet Res 58:976-9 84.
Geraghty RJ,Krummenacher C,Cohen GH, et al.Entry of alphaherpesvirus mediated by poliovirus receptor-related Protein 1 and poliovirus receptor [J].Science,1998,280:1618-1620.
Granzow HF, Weiland A , Jons B, et al. 1997 Ultrasturctural analysis of the replication cycle of pseudoradies virus in cell culture,a reassessment J Virol 71:2072-2082.
Hanebe H,Wheeler JG.Osorio FA.Gene specific assey to differentiate stain of pseudorabies virus [J].VetMicrobiol,1993,34,221-231.
Harding MJ,Prud'homme I, Rola.Specificity and nucleotyping studies of a gp50 based polyme -rase chain reaction assey for detection of pseudorabies virus[J].Can J Vet Res,1997,61:157-160.
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