羊口疮病毒榆林株B2L和F1L串联基因在昆虫杆状病毒系统的表达
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摘要
【目的】克隆分析羊口疮病毒(ORFV)榆林株(ORFV-Yulin)的F1L基因序列,通过昆虫杆状病毒表达B2L和F1L串联基因。【方法】采用PCR方法扩增ORFV-Yulin株F1L全长基因,测序后对其进行序列分析和遗传进化分析。扩增ORFV-Yulin株B2L融合基因(r B2L)和F1L融合基因(c F1L),利用碱基linker连接,通过重叠PCR方法扩增获得ORFV r B2L-linker-c F1L重组基因(r B2Lc F1L),将其与昆虫杆状病毒表达载体pBac5连接构建表达载体pBac-rB2LcF1L。将pBac-rB2LcF1L包装出杆状病毒,侵染sf9细胞,通过SDS-PAGE、Western-blotting以及质谱鉴定等方法验证目的基因r B2Lc F1L的表达。【结果】克隆获得了1 029bp的F1L全长基因,该基因高度保守,与FJ-MH2015株的序列相似性最高,核苷酸和氨基酸的相似性分别为99.0%和99.4%。系统进化分析表明,ORFV-Yulin株与XP(KU199840.1)、FJ-MH2015(KM675409.1)以及Shanxi(HQ221964.1)株同处于一个分支。PCR扩增获得了855bp的c F1L基因、1 180bp的r B2L基因及1 983bp的r B2Lc F1L基因,成功构建了pBacrB2LcF1L载体,并包装出相应的杆状病毒。B2L和F1L串联基因通过昆虫杆状病毒表达系统获得成功表达,表达产物的分子质量为80ku,且有部分重组蛋白能分泌到细胞培养基中。【结论】ORFV-Yulin株的B2L和F1L串联基因在昆虫杆状病毒系统表达成功。
【Objective】This study cloned and analyzed the sequence of F1 Lgene of Orf virus(ORFV)strain Yulin(ORFV-Yulin)and its co-expressed B2 Land F1 Lgenes in baculovirus system.【Method】The full-length gene of F1 Lof ORFV-Yulin was amplified by PCR,and the sequenced gene was analyzed by genetic evolution.The fused gene of B2 L(r B2 L)and F1 L(cF1 L)of ORFV-Yulin was amplified by PCR and the ORFV B2 L-linker-cF1 Lrecombinant gene(r B2 LcF1 L)was amplified by overlapping PCR method with linker.The baculovirus expression vector pBac-rB2 LcF1 Lwas constructed with pBac5 and r B2 LcF1 Lto get baculovirus expressing r B2 LcF1 Lgene after the infection of sf9 cells.The expressed product was identified by SDS-PAGE,mass spectrometry and western blot.【Result】The length of F1 Lgene obtained by cloning was 1 029 bp.The gene was highly conserved and had the highest similarity with FL-MH2015 strain with the nucleotide and amino acid similarity of 99.0%and 99.4%,respectively.The phylogenetic analysis showed that the ORFV-Yulin strain belonged to a branch with XP(KU199840.1),FJ-MH2015(KM675409.1)and Shanxi(HQ221964.1).The lengths of cF1 L,r B2 L,and r B2 LcF1 Lobtained by PCR were 855 bp,1 180 bp and 1 983 bp,respectively.The pBac-rB2 LcF1 Lvector was successfully constructed,followed by packaging the corresponding baculovirus.The tandem gene of r B2 Land cF1 L was successfully expressed as 80 ku protein by baculovirus expression system and some recombinant proteins secreted into cell culture medium.【Conclusion】The recombinant B2 Land F1 Lgene of ORFV-Yulin was successfully expressed in baculovirus expression system.
【Objective】This study cloned and analyzed the sequence of F1 Lgene of Orf virus(ORFV)strain Yulin(ORFV-Yulin)and its co-expressed B2 Land F1 Lgenes in baculovirus system.【Method】The full-length gene of F1 Lof ORFV-Yulin was amplified by PCR,and the sequenced gene was analyzed by genetic evolution.The fused gene of B2 L(r B2 L)and F1 L(cF1 L)of ORFV-Yulin was amplified by PCR and the ORFV B2 L-linker-cF1 Lrecombinant gene(r B2 LcF1 L)was amplified by overlapping PCR method with linker.The baculovirus expression vector pBac-rB2 LcF1 Lwas constructed with pBac5 and r B2 LcF1 Lto get baculovirus expressing r B2 LcF1 Lgene after the infection of sf9 cells.The expressed product was identified by SDS-PAGE,mass spectrometry and western blot.【Result】The length of F1 Lgene obtained by cloning was 1 029 bp.The gene was highly conserved and had the highest similarity with FL-MH2015 strain with the nucleotide and amino acid similarity of 99.0%and 99.4%,respectively.The phylogenetic analysis showed that the ORFV-Yulin strain belonged to a branch with XP(KU199840.1),FJ-MH2015(KM675409.1)and Shanxi(HQ221964.1).The lengths of cF1 L,r B2 L,and r B2 LcF1 Lobtained by PCR were 855 bp,1 180 bp and 1 983 bp,respectively.The pBac-rB2 LcF1 Lvector was successfully constructed,followed by packaging the corresponding baculovirus.The tandem gene of r B2 Land cF1 L was successfully expressed as 80 ku protein by baculovirus expression system and some recombinant proteins secreted into cell culture medium.【Conclusion】The recombinant B2 Land F1 Lgene of ORFV-Yulin was successfully expressed in baculovirus expression system.
引文
[1]McKeever D J,Jenkinson D M,Hutchison G,et al.Studies on the pathogenesis of Orf virus infection in sheep[J].J Comp Pathol,1988,99:317-328.
[2]Mercer A,Haig D.Parapoxviruses[J].Encyclopedia of Virology,1999,18(2):1140-1146.
[3]Delhon G,Tulman E R,Afonso C L,et al.Genomes of the Parapoxviruses Orf virus and bovine papular stomatitis virus[J].J Virol,2004,78(1):168-177.
[4]Cottone R,Büttner M,Bauer B,et al.Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J].Virus Research,1998,56:53-67.
[5]Balassu T C,Robinson A J.Orf virus replication in bovine testis cells:kinetics of viral DNA,polypeptide,and infectious virus production and analysis of virion polypeptides[J].Archives of Virology,1987,97(3):267-281.
[6]于永忠,吴志军,朱战波,等.羊口疮病毒分子特征与免疫逃逸策略[J].病毒学报,2012(3):278-284.Yu Y Z,Wu Z J,Zhu Z B,et al.Molecular characteristics and immune evasion strategies of ORFV[J].Chinese Journal of Virlogy,2012(3):278-284.
[7]赵魁.羊传染性脓疱病毒重组DNA疫苗的构建与实验免疫研究[D].长春:吉林大学,2010.Zhao K.Study on the construction and experimental immunity of recombinant DNA vaccine against Orf virus[D].Changchun:Jilin University,2010.
[8]林裕胜,江锦秀,林甦,等.羊传染性脓疱病毒研究进展[J].动物医学进展,2016,37(2):91-96.Lin Y S,Jiang J X,Lin S,et al.Progress on Contagious ecthyma virus[J].Progress in Veterinary Medicine,2016,37(2):91-96.
[9]邵洪泽.羊传染性脓疱病毒FIL基因克隆表达及分子生物学检测方法的建立与初步应用[D].长春:吉林农业大学,2013.Sao H Z.Cloning and expression of F1Lgene of CEV and establishment and preliminary application of molecular biological detection methods[D].Changchun:Jilin Agriculture University,2013.
[10]Hosamani M,Bhanuprakash V,Scagliarini A,et al.Comparative sequence analysis of major envelope protein gene(B2L)of Indian orf viruses isolated from sheep and goats[J].Veterinary Microbiology,2006,116(4):317-324.
[11]王廷璞,薛双虎.羊接触传染性脓疱性皮炎病毒核酸及蛋白类型分析[J].中国兽医学报,1998(4):328-333.Wang T P,Xue S H.Analysis of nucleic acid and envelop protein of Contagious ecthyma virus[J].Chinese Journal of Veterinary Science,1998(4):328-333.
[12]Gallina L,Scagliarini A,Ciulli S,et al.Cloning and expression of the Orf virus F1Lgene:possible use as a subunit vaccine[J].Veterinary Research Communications,2004,28(S1):291-293.
[13]Housawi F M T,Roberts G M,Gilray J A,et al.The reactivity of monoclonal antibodies against Orf virus with other parapoxviruses and the identification of a 39kD a immunodominant protein[J].Archives of Virology,1998,143(12):2289-2303.
[14]Haig D M,Mc Innes C J,Deane D,et al.The immune and inflammatory response to Orf virus[J].Comp Immunol Microbiol Infect Dis,1997,20:197-204.
[15]Azwai S M,Carter S D,Woldehiwet Z.Immune responses of the camal to Contagious ecthyma(Orf)virus infection[J].Vet Micro,1995,47:119-131.
[16]Guo J,Zhang Z,Edwards J F,et al.Characterization of a North American Orf virus isolated from a goat with persistent,proliferative dermatitis[J].Virus Res,2003,93(2):169-179.
[17]Zheng M,Liu Q,Jin N,et al.A duplex PCR assay for simultaneous detection and differentiation of Capripoxvirus and Orf virus[J].Mol Cell Probes,2007,21(4):276-281.
[18]余波,冉懋韬,谭诗文,等.羊传染性脓疱病毒PCR检测方法的建立及CBP基因的序列分析[J].畜牧与兽医,2013,45(7):30-34.Yu B,Ran M T,Tan S W,et al.Establishment of PCR method for detection of Orf virus and sequencing analysis of CBP gene[J].Animal Husbandry and Veterinary Medicine,2013,45(7):30-34.
[19]闫丰超,邵佳,窦永喜.羊口疮病毒分子生物学的研究进展[J].中国兽医科学,2013(1):103-109.Yan F C,Shao J,Dou Y X.Progress on molecular characteristics of Orf virus[J].Chinese Veterinary Science,2013(1):103-109.
[20]Czerny C P,Waldmann R,Scheubeck T.Identification of three distinct antigenic sites in parapoxviruses[J].Archives of Virology,1997,142(4):807-821.
[21]Scagliarini A,Ciulli S,Battilani M,et al.Characterisation of immunodominant protein encoded by the F1Lgene of Orf virus strains isolated in Italy[J].Archives of Virology,2002,147(10):1989-1995.
[22]李杰,李前瑞,田婷婷,等.羊口疮病毒B2L和VIR基因原核表达及抗原性鉴定[J].动物医学进展,2013,34(3):1-6.Li J,Li Q R,Tian T T,et al.Prokaryotic expression of Orf virus B2L,VIR genes and antigenicity evaluation[J].Progress in Veterinary Medicine,2013,34(3):1-6.
[23]赵文博,李瑞航,贺鹏亮,等.羊口疮病毒B2L基因克隆及表达[J].黑龙江八一农垦大学学报,2015,27(2):42-45.Zhao W B,Li R H,He P L,et al.Cloning and expression of ORFV B2L gene[J].Journal of Heilongjiang Bayi Agricultural University,2015,27(2):42-45.
[24]Zhao K,He W,Wei G,et al.Orf virus DNA vaccines expressing ORFV 011and ORFV 059chimeric protein enhances immunogenicity[J].Virol J,2011,8(1):562.
[25]张克山,刘永杰,孔汉金,等.羊口疮病毒B2L基因DNA疫苗载体的构建及其在BHK-21细胞中的表达[J].中国人兽共患病学报,2013,29(10):951-954.Zhang K S,Liu Y J,Kong H J,et al.Construction of DNA vaccine carrier for ORFV B2L gene and its expression in BHK-21cells[J].Chinese Journal of Zoonoses,2013,29(10):951-954.
[26]王廷璞,赵菲佚,安建平,等.羊传染性脓疱病毒42K囊膜蛋白基因克隆及表达[J].中国预防兽医学报,2006,28(1):29-32.Wang T P,Zhao F Y,An J P,et al.The cloning and expression of Contagious ecthyma virus 42K envelop protein gene[J].Chinese Journal of Prevent Veterinary Medicine,2006,28(1):29-32.
[27]冯平,李云章,孙丰廷,等.ORFV-Yulin株的分离鉴定及其B2L基因在昆虫杆状病毒系统中的表达[J].西北农林科技大学学报(自然科学版),2017,45(9):32-38.Feng P,Li Y Z,Sun F T,et al.Isolation and identification of ORFV-Yulin strain and the expression of B2Lgene in baculovirus system[J].Journal of Northwest A&F University(Nat Sci Ed),2017,45(9):32-38.
[28]李雯丽.陕西白绒山羊和奶山羊羊口疮病原调查及灭活疫苗免疫效果评估[D].陕西杨凌:西北农林科技大学,2016.Li W L.Molecular eiologies of Orf in cashmere and dairy goats in Shaanxi province and the effects of the inactivatied avicine[D].Yangling,Shaanxi:Northwest A&F University,2016.
[2]Mercer A,Haig D.Parapoxviruses[J].Encyclopedia of Virology,1999,18(2):1140-1146.
[3]Delhon G,Tulman E R,Afonso C L,et al.Genomes of the Parapoxviruses Orf virus and bovine papular stomatitis virus[J].J Virol,2004,78(1):168-177.
[4]Cottone R,Büttner M,Bauer B,et al.Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J].Virus Research,1998,56:53-67.
[5]Balassu T C,Robinson A J.Orf virus replication in bovine testis cells:kinetics of viral DNA,polypeptide,and infectious virus production and analysis of virion polypeptides[J].Archives of Virology,1987,97(3):267-281.
[6]于永忠,吴志军,朱战波,等.羊口疮病毒分子特征与免疫逃逸策略[J].病毒学报,2012(3):278-284.Yu Y Z,Wu Z J,Zhu Z B,et al.Molecular characteristics and immune evasion strategies of ORFV[J].Chinese Journal of Virlogy,2012(3):278-284.
[7]赵魁.羊传染性脓疱病毒重组DNA疫苗的构建与实验免疫研究[D].长春:吉林大学,2010.Zhao K.Study on the construction and experimental immunity of recombinant DNA vaccine against Orf virus[D].Changchun:Jilin University,2010.
[8]林裕胜,江锦秀,林甦,等.羊传染性脓疱病毒研究进展[J].动物医学进展,2016,37(2):91-96.Lin Y S,Jiang J X,Lin S,et al.Progress on Contagious ecthyma virus[J].Progress in Veterinary Medicine,2016,37(2):91-96.
[9]邵洪泽.羊传染性脓疱病毒FIL基因克隆表达及分子生物学检测方法的建立与初步应用[D].长春:吉林农业大学,2013.Sao H Z.Cloning and expression of F1Lgene of CEV and establishment and preliminary application of molecular biological detection methods[D].Changchun:Jilin Agriculture University,2013.
[10]Hosamani M,Bhanuprakash V,Scagliarini A,et al.Comparative sequence analysis of major envelope protein gene(B2L)of Indian orf viruses isolated from sheep and goats[J].Veterinary Microbiology,2006,116(4):317-324.
[11]王廷璞,薛双虎.羊接触传染性脓疱性皮炎病毒核酸及蛋白类型分析[J].中国兽医学报,1998(4):328-333.Wang T P,Xue S H.Analysis of nucleic acid and envelop protein of Contagious ecthyma virus[J].Chinese Journal of Veterinary Science,1998(4):328-333.
[12]Gallina L,Scagliarini A,Ciulli S,et al.Cloning and expression of the Orf virus F1Lgene:possible use as a subunit vaccine[J].Veterinary Research Communications,2004,28(S1):291-293.
[13]Housawi F M T,Roberts G M,Gilray J A,et al.The reactivity of monoclonal antibodies against Orf virus with other parapoxviruses and the identification of a 39kD a immunodominant protein[J].Archives of Virology,1998,143(12):2289-2303.
[14]Haig D M,Mc Innes C J,Deane D,et al.The immune and inflammatory response to Orf virus[J].Comp Immunol Microbiol Infect Dis,1997,20:197-204.
[15]Azwai S M,Carter S D,Woldehiwet Z.Immune responses of the camal to Contagious ecthyma(Orf)virus infection[J].Vet Micro,1995,47:119-131.
[16]Guo J,Zhang Z,Edwards J F,et al.Characterization of a North American Orf virus isolated from a goat with persistent,proliferative dermatitis[J].Virus Res,2003,93(2):169-179.
[17]Zheng M,Liu Q,Jin N,et al.A duplex PCR assay for simultaneous detection and differentiation of Capripoxvirus and Orf virus[J].Mol Cell Probes,2007,21(4):276-281.
[18]余波,冉懋韬,谭诗文,等.羊传染性脓疱病毒PCR检测方法的建立及CBP基因的序列分析[J].畜牧与兽医,2013,45(7):30-34.Yu B,Ran M T,Tan S W,et al.Establishment of PCR method for detection of Orf virus and sequencing analysis of CBP gene[J].Animal Husbandry and Veterinary Medicine,2013,45(7):30-34.
[19]闫丰超,邵佳,窦永喜.羊口疮病毒分子生物学的研究进展[J].中国兽医科学,2013(1):103-109.Yan F C,Shao J,Dou Y X.Progress on molecular characteristics of Orf virus[J].Chinese Veterinary Science,2013(1):103-109.
[20]Czerny C P,Waldmann R,Scheubeck T.Identification of three distinct antigenic sites in parapoxviruses[J].Archives of Virology,1997,142(4):807-821.
[21]Scagliarini A,Ciulli S,Battilani M,et al.Characterisation of immunodominant protein encoded by the F1Lgene of Orf virus strains isolated in Italy[J].Archives of Virology,2002,147(10):1989-1995.
[22]李杰,李前瑞,田婷婷,等.羊口疮病毒B2L和VIR基因原核表达及抗原性鉴定[J].动物医学进展,2013,34(3):1-6.Li J,Li Q R,Tian T T,et al.Prokaryotic expression of Orf virus B2L,VIR genes and antigenicity evaluation[J].Progress in Veterinary Medicine,2013,34(3):1-6.
[23]赵文博,李瑞航,贺鹏亮,等.羊口疮病毒B2L基因克隆及表达[J].黑龙江八一农垦大学学报,2015,27(2):42-45.Zhao W B,Li R H,He P L,et al.Cloning and expression of ORFV B2L gene[J].Journal of Heilongjiang Bayi Agricultural University,2015,27(2):42-45.
[24]Zhao K,He W,Wei G,et al.Orf virus DNA vaccines expressing ORFV 011and ORFV 059chimeric protein enhances immunogenicity[J].Virol J,2011,8(1):562.
[25]张克山,刘永杰,孔汉金,等.羊口疮病毒B2L基因DNA疫苗载体的构建及其在BHK-21细胞中的表达[J].中国人兽共患病学报,2013,29(10):951-954.Zhang K S,Liu Y J,Kong H J,et al.Construction of DNA vaccine carrier for ORFV B2L gene and its expression in BHK-21cells[J].Chinese Journal of Zoonoses,2013,29(10):951-954.
[26]王廷璞,赵菲佚,安建平,等.羊传染性脓疱病毒42K囊膜蛋白基因克隆及表达[J].中国预防兽医学报,2006,28(1):29-32.Wang T P,Zhao F Y,An J P,et al.The cloning and expression of Contagious ecthyma virus 42K envelop protein gene[J].Chinese Journal of Prevent Veterinary Medicine,2006,28(1):29-32.
[27]冯平,李云章,孙丰廷,等.ORFV-Yulin株的分离鉴定及其B2L基因在昆虫杆状病毒系统中的表达[J].西北农林科技大学学报(自然科学版),2017,45(9):32-38.Feng P,Li Y Z,Sun F T,et al.Isolation and identification of ORFV-Yulin strain and the expression of B2Lgene in baculovirus system[J].Journal of Northwest A&F University(Nat Sci Ed),2017,45(9):32-38.
[28]李雯丽.陕西白绒山羊和奶山羊羊口疮病原调查及灭活疫苗免疫效果评估[D].陕西杨凌:西北农林科技大学,2016.Li W L.Molecular eiologies of Orf in cashmere and dairy goats in Shaanxi province and the effects of the inactivatied avicine[D].Yangling,Shaanxi:Northwest A&F University,2016.