羊传染性脓疱病毒VEGF基因的克隆及PCR诊断方法的研究
摘要
本试验对羊传染性脓疱病毒吉林省分离株Orf-ys株进行传代培养,待出现典型的细胞病变后收毒并进行DNA的提取,根据已发表的羊传染性脓疱病毒B2L基因序列,设计引物,进行VEGF基因的特异性扩增,并将其克隆到pMD18-T载体后进行测序。结果表明：Orf-ys株的VEGF基因长为414bp。经核苷酸序列比较、分析结果表明：Orf-ys株与Matsumoto同源性最高,达98%,与IT01最低,为84%,说明Orf-ys株VEGF基因与参考毒株之间差异不大。
本试验根据已发表的羊传染性脓疱病毒NZ2株的序列,设计引物,对Orf-ys株、羊痘病毒和新生犊牛睾丸原代细胞的核酸进行PCR扩增,结果表明：只有Orf-ys株扩增出414bp左右的特异性片段,其它均为阴性,说明该PCR扩增体系具有良好的特异性。对Orf-ys株进行敏感性试验,结果表明：该PCR扩增体系可检测出4.5ng模板,说明该PCR扩增体系具有较高的敏感性。
The Orf-ys virus was propagated and the total DNA of virus was extracted.According to the published VEGF gene sequence of ORF V strain in GenBank, one pair of primer was designed. The VEGF gene of virus was amplificated with the primer by PCRand then cloned into pMD18-T plasmids and then sequenced and analysed in homology.The acquired sequences contained 414bp, The homology analysis revealed that thehomology of the strain Matsumoto and the strain Orf-ys was 98%, the IT01 and the Orf-ys was 84%. There were a little variation between Orf-ys strain and the referenced virus strains.
According to the published sequence of strain NZ2 in GenBank, another pair of primer was designed. The virus DNA was amplificated with the primer by PCR, then PCR diagnostic method was established, and this method's specificity and sensitivity was detected. The results indicated that the PCR method could detect the nucleic acid of Orf-ys virus only and a 414bp DNA fragment was amplificated. The minimum ORF V DNA's detection level was 4.5ng. This illustrated that this method was specific and sensitive for the detection of ORF V.
本试验根据已发表的羊传染性脓疱病毒NZ2株的序列,设计引物,对Orf-ys株、羊痘病毒和新生犊牛睾丸原代细胞的核酸进行PCR扩增,结果表明：只有Orf-ys株扩增出414bp左右的特异性片段,其它均为阴性,说明该PCR扩增体系具有良好的特异性。对Orf-ys株进行敏感性试验,结果表明：该PCR扩增体系可检测出4.5ng模板,说明该PCR扩增体系具有较高的敏感性。
The Orf-ys virus was propagated and the total DNA of virus was extracted.According to the published VEGF gene sequence of ORF V strain in GenBank, one pair of primer was designed. The VEGF gene of virus was amplificated with the primer by PCRand then cloned into pMD18-T plasmids and then sequenced and analysed in homology.The acquired sequences contained 414bp, The homology analysis revealed that thehomology of the strain Matsumoto and the strain Orf-ys was 98%, the IT01 and the Orf-ys was 84%. There were a little variation between Orf-ys strain and the referenced virus strains.
According to the published sequence of strain NZ2 in GenBank, another pair of primer was designed. The virus DNA was amplificated with the primer by PCR, then PCR diagnostic method was established, and this method's specificity and sensitivity was detected. The results indicated that the PCR method could detect the nucleic acid of Orf-ys virus only and a 414bp DNA fragment was amplificated. The minimum ORF V DNA's detection level was 4.5ng. This illustrated that this method was specific and sensitive for the detection of ORF V.
引文
[1]蔡宝祥,肖传发.实用传染病学[M].上海：上海科学技术出版社,1989,42-44
[2]潘淑惠,周永连.羊传染性脓疱油乳剂灭活苗中间试验[J].贵州畜牧兽医.2000,24(6)：6
[3]殷震,刘景华.动物病毒学[M].第二版.北京：科学出版社,1997,977-980
[4]蔡宝祥,沈正达,王锡祯,等.家畜传染病学[M].第二版.北京：农业出版社,1989,283～286
[5]陈燕军,沈正达,王锡贞.羊口疮研究—病原学及实验病理学研究研究[J].兽医科技杂志.1982,(6)：2-8
[6]沈正达,靳诚,沈斌元,等.羊口疮的研究[J].甘肃农大学报,1989,(1)：1-16
[7]詹述宣,薛双虎,薛掌林等.犊牛睾丸细胞培养羊传染性脓疱皮炎病毒的研究[J].1983,兽医科技杂志.(7)：5-7
[8]何水林等,羊口疮病毒的分离与鉴定,中国兽医科技。2002,(9)：22-23。
[9]白积荣,沈正达,于锡贞等.羊口疮病毒形态发生的电镜观察[J].中国兽医科技.1985,(11)：34-36
[10]Buddle B M,Pulford H D.Effect of passively acquired andvaccination of the immune response to contagious ecthymavirus Vet.Microbiol,1984,9:515-522
[11]Robinson A J,BarnsG,FraserKetal.Conservation andvariation in orf virus genomes. Virology,1987,156:13-23
[12]左玉婷,靳成等,羊口疮病毒的提纯及其囊膜亚单位的分离鉴定[J],中国兽医杂志。1998,14(7)：2-4
[13]Robinson A J,Ellis G,Balassu T C.The genome of orfvirus:Restrictionendonucle seanalys is of viral DNA isolatedfrom lesion so for finsheep[J].ArchivesofVirology,1982,71:43-55
[14]郭素珍,张惠安,黄守明等.骆驼、绵羊、山羊口疮病原的血清学关系比较[J].中国兽医科技,1988,5：35-37
[15]Robinson A J,Balassu T C.Contagious pustular dermatit is(orf)[J].VetBull,1981,51:771-781
[16]Gassmann U, Wyler R, Wittek R. Analysis of parapoxvirus genomes[J]. Archives of Virology,1985, 83:17-31
[17]Cottone R, Buttner M, Bauer B, et al. Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J]. Virus Res,1998,56:53～67
[18]Delhon G, Tulman E R, Afonso C L. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus[J]. J Virol,2004,78(1):168～177。
[19]Sullivan J T, Mercer A A, Flening S B, et al. Identification and characterization ofan orf virus homologue of the vaccinia virusgene encoding the major envelop antigen p37k[J]. Virol,1994,202:968-973
[20]王廷璞,薛双虎,吕武夫,等.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
[21]Haig DM. Orf virus infection and host immunity[J]. Curr Opin Infect Dis.2006,19(2):127～31
[22]Wise LM, Savory LJ, Dryden NH, et al. Major amino acid sequence variants of viral vascular endothelial growth factor are functionally equivalent during Orf virus infection of sheep skin[J]. Virus Res, 2007,128(2):115-25
[23]Marsland B J,D.J.Tisdall1,D.D.Heath,and A. A.Mercer Construction of a recombinant orf virus that expresses an Echinococcus granulosus vaccine antigenfrom a novel genomic insertion site[J].Arch Virol,2003,148:555-562
[24]Andrew A, Mercer A A, Fraser K, et al. The structure and cloning of orf virus DNA[J]. Virology,1987, 157(1):1-12
[25]左玉婷,靳诚,王锡祯,等.羊口疮病毒的提纯及其囊膜亚单位的分离鉴定[J].中国兽医杂志,1988,14(7)：1-4
[26]黄引贤,李乃津.羊传染性脓疱病毒某些特性的初步研究[J].中国兽医杂志,1986,(12)：2-5
[27]Boughton and Hardy. Texas Agr. Exp. Sta. Bull.1935,504
[28]左玉婷,王锡祯,靳诚.羊传染性脓疱皮炎[J].中国兽医科技.1987,(3)：56-58
[29]薛双虎,吕武夫,薛掌林,等.羊传染性脓疱病毒弱毒株的培育研究[J].甘肃畜牧兽医,1988,(3)：1-2
[30]薛掌林,薛双虎,吕武大,等.中和试验对羊传染性脓疱皮炎抗体的检测[J].甘肃畜牧兽医,1990,2：13～14
[31]McKeever D J, Reid H W, Highs N F, et al. A qualititative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection[J]. Vet, Microbial,1987,15:229～241
[32]Buddle B M, Dellers R W, Schurig G G. Contagious ecthyma virusvaccination failures[J]. American Journal of Veterinary Research,1984,45,262～266
[33]王廷璞,薛双虎,吕武夫,等.利用醋酶染色对羊传染性脓疱皮炎病羊和免疫羊T琳巴细胞值的测定[J].甘肃畜牧兽医,1989,(06)：7-8
[34]Seppo A M, Saari Eeva Rudback, Mirjami Niskanen, et al. Contagious mucocutaneous dermatitis of the mountain hare (Lepus timidus):pathology and cause[J]. J Wildl Dis,2005,41(4):775～82
[35]Turid Vikoren, Atle Lillehaug, Johan Akerstedt, et al. A severe outbreak of contagious ecthyma (orf) in a free～ranging musk ox (Ovibos moschatus) population in Norway[J]. Veterinary Microbiology,2008, 127:10～20
[36]靳诚,沈正达.羊口疮的流行病学调查[J].甘肃农大学报,1988,(1)：85～88
[37]范春玲,孙斌,政伟.羊传染性脓疱病的病理组织学观察[J].畜牧兽医科技信息,2007,(8)：32～33
[38]Gardiner, Craig, Nairn. Austral. Vet Jour.1967,43,163
[39]Nettleton P F, Munro R, Pow I, et al. Isolation of a parapoxvirus from a grey seal (Halichoerus grypus)[J]. The Veterinary Record,137(22):562～564
[40]Uzel M, Sasmaz S, Bakaris S, et al. A viral infection of the hand commonly seen after the feast of sacrifice:human orf (orf of the hand) [J]. Epidemiol Infect.2005,133(4):653～7
[41]Key S J, Catania J, Mustafa S F, et al. Unusual presentation of human giant orf (ecthyma contagiosum) [J]. Craniofac Surg,2007,18(5):1076～8
[42]Al-Salam S, Nowotny N, Sohail MR, et al. Ecthyma contagiosum (orf)-report of a human case from the United Arab Emirates and review of the literature[J]. Cutan Pathol,2008,35(6):603～7
[43]W.T.休伯特,W.F.麦卡洛克,P.R.施努伦贝格尔.人兽共患病.上海科学技术出版社,1985,518-521。
[44]Savory L J, Stacker S A, Fleming S B, et al. Viral vascular endo thelial growth factor plays a critical role in orf virus infection[J]. Virol,2000,74:10699～10706
[45]Delhon G, Tulman E R, Afonso C L. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus[J]. J Virol,2004,78(1):168～177
[46]Bruce T, Catherine S, McCaughan A, et al. Analysis of an orf virus chemokine-binding protein:Shifting ligand specificities among a family of poxvirus viroceptors[J]. PNAS,2003,100(25):15137～15142
[47]王良娟,马维卿,张再清.应用对流免疫电泳检测细胞培养物中羊口疮病毒抗原[J].甘肃畜牧兽医,1992,(3)：78
[48]Robinson A J, Barns G, Fraser K, et al. Conservation and variation in orf virus genomes[J]. Virology, 1987,156:13～23
[49]王廷璞,赵菲佚,孙春香,等.光敏生物素和DIG标记Orf病毒DNA探针的研究[J].生物技术,2002,12(6)：21-22
[50]Nitsche A, Buttner M, Wilhelm S, et al. Real-time PCR detection of parapoxvirus DNA[J]. Clinical Chemistry,2006,52(2):316～319
[51]王辉,赵晓军,易建刚.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检.2007,(6)24
[52]吕玉杰,王波.小议山羊传染性脓疱的病理及其防治[J].科技咨询导报,2007,23：242
[53]庞理化,谢文华,吴维忠等.青海羊口疮病的研究—弱毒细胞冻干苗的试制.青海畜牧兽医杂志,1986,6：5-10
[54]王辉,赵晓军,易建刚.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检.2007,(6)24
[55]改树清,王志明,刘慧纯.用湿苗防治羊传染性脓疱口疮病[J].现代畜牧兽医,1988,(04)：16～17
[56]潘淑惠,周永连.羊传染性脓疱油乳剂灭活苗中间试验[J].贵州畜牧兽医,2000,24(6)：6
[57]王志军.羊传染性脓疱病的诊断和防治[J].河南畜牧兽医,1999,20(1)：23
[2]潘淑惠,周永连.羊传染性脓疱油乳剂灭活苗中间试验[J].贵州畜牧兽医.2000,24(6)：6
[3]殷震,刘景华.动物病毒学[M].第二版.北京：科学出版社,1997,977-980
[4]蔡宝祥,沈正达,王锡祯,等.家畜传染病学[M].第二版.北京：农业出版社,1989,283～286
[5]陈燕军,沈正达,王锡贞.羊口疮研究—病原学及实验病理学研究研究[J].兽医科技杂志.1982,(6)：2-8
[6]沈正达,靳诚,沈斌元,等.羊口疮的研究[J].甘肃农大学报,1989,(1)：1-16
[7]詹述宣,薛双虎,薛掌林等.犊牛睾丸细胞培养羊传染性脓疱皮炎病毒的研究[J].1983,兽医科技杂志.(7)：5-7
[8]何水林等,羊口疮病毒的分离与鉴定,中国兽医科技。2002,(9)：22-23。
[9]白积荣,沈正达,于锡贞等.羊口疮病毒形态发生的电镜观察[J].中国兽医科技.1985,(11)：34-36
[10]Buddle B M,Pulford H D.Effect of passively acquired andvaccination of the immune response to contagious ecthymavirus Vet.Microbiol,1984,9:515-522
[11]Robinson A J,BarnsG,FraserKetal.Conservation andvariation in orf virus genomes. Virology,1987,156:13-23
[12]左玉婷,靳成等,羊口疮病毒的提纯及其囊膜亚单位的分离鉴定[J],中国兽医杂志。1998,14(7)：2-4
[13]Robinson A J,Ellis G,Balassu T C.The genome of orfvirus:Restrictionendonucle seanalys is of viral DNA isolatedfrom lesion so for finsheep[J].ArchivesofVirology,1982,71:43-55
[14]郭素珍,张惠安,黄守明等.骆驼、绵羊、山羊口疮病原的血清学关系比较[J].中国兽医科技,1988,5：35-37
[15]Robinson A J,Balassu T C.Contagious pustular dermatit is(orf)[J].VetBull,1981,51:771-781
[16]Gassmann U, Wyler R, Wittek R. Analysis of parapoxvirus genomes[J]. Archives of Virology,1985, 83:17-31
[17]Cottone R, Buttner M, Bauer B, et al. Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701[J]. Virus Res,1998,56:53～67
[18]Delhon G, Tulman E R, Afonso C L. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus[J]. J Virol,2004,78(1):168～177。
[19]Sullivan J T, Mercer A A, Flening S B, et al. Identification and characterization ofan orf virus homologue of the vaccinia virusgene encoding the major envelop antigen p37k[J]. Virol,1994,202:968-973
[20]王廷璞,薛双虎,吕武夫,等.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
[21]Haig DM. Orf virus infection and host immunity[J]. Curr Opin Infect Dis.2006,19(2):127～31
[22]Wise LM, Savory LJ, Dryden NH, et al. Major amino acid sequence variants of viral vascular endothelial growth factor are functionally equivalent during Orf virus infection of sheep skin[J]. Virus Res, 2007,128(2):115-25
[23]Marsland B J,D.J.Tisdall1,D.D.Heath,and A. A.Mercer Construction of a recombinant orf virus that expresses an Echinococcus granulosus vaccine antigenfrom a novel genomic insertion site[J].Arch Virol,2003,148:555-562
[24]Andrew A, Mercer A A, Fraser K, et al. The structure and cloning of orf virus DNA[J]. Virology,1987, 157(1):1-12
[25]左玉婷,靳诚,王锡祯,等.羊口疮病毒的提纯及其囊膜亚单位的分离鉴定[J].中国兽医杂志,1988,14(7)：1-4
[26]黄引贤,李乃津.羊传染性脓疱病毒某些特性的初步研究[J].中国兽医杂志,1986,(12)：2-5
[27]Boughton and Hardy. Texas Agr. Exp. Sta. Bull.1935,504
[28]左玉婷,王锡祯,靳诚.羊传染性脓疱皮炎[J].中国兽医科技.1987,(3)：56-58
[29]薛双虎,吕武夫,薛掌林,等.羊传染性脓疱病毒弱毒株的培育研究[J].甘肃畜牧兽医,1988,(3)：1-2
[30]薛掌林,薛双虎,吕武大,等.中和试验对羊传染性脓疱皮炎抗体的检测[J].甘肃畜牧兽医,1990,2：13～14
[31]McKeever D J, Reid H W, Highs N F, et al. A qualititative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection[J]. Vet, Microbial,1987,15:229～241
[32]Buddle B M, Dellers R W, Schurig G G. Contagious ecthyma virusvaccination failures[J]. American Journal of Veterinary Research,1984,45,262～266
[33]王廷璞,薛双虎,吕武夫,等.利用醋酶染色对羊传染性脓疱皮炎病羊和免疫羊T琳巴细胞值的测定[J].甘肃畜牧兽医,1989,(06)：7-8
[34]Seppo A M, Saari Eeva Rudback, Mirjami Niskanen, et al. Contagious mucocutaneous dermatitis of the mountain hare (Lepus timidus):pathology and cause[J]. J Wildl Dis,2005,41(4):775～82
[35]Turid Vikoren, Atle Lillehaug, Johan Akerstedt, et al. A severe outbreak of contagious ecthyma (orf) in a free～ranging musk ox (Ovibos moschatus) population in Norway[J]. Veterinary Microbiology,2008, 127:10～20
[36]靳诚,沈正达.羊口疮的流行病学调查[J].甘肃农大学报,1988,(1)：85～88
[37]范春玲,孙斌,政伟.羊传染性脓疱病的病理组织学观察[J].畜牧兽医科技信息,2007,(8)：32～33
[38]Gardiner, Craig, Nairn. Austral. Vet Jour.1967,43,163
[39]Nettleton P F, Munro R, Pow I, et al. Isolation of a parapoxvirus from a grey seal (Halichoerus grypus)[J]. The Veterinary Record,137(22):562～564
[40]Uzel M, Sasmaz S, Bakaris S, et al. A viral infection of the hand commonly seen after the feast of sacrifice:human orf (orf of the hand) [J]. Epidemiol Infect.2005,133(4):653～7
[41]Key S J, Catania J, Mustafa S F, et al. Unusual presentation of human giant orf (ecthyma contagiosum) [J]. Craniofac Surg,2007,18(5):1076～8
[42]Al-Salam S, Nowotny N, Sohail MR, et al. Ecthyma contagiosum (orf)-report of a human case from the United Arab Emirates and review of the literature[J]. Cutan Pathol,2008,35(6):603～7
[43]W.T.休伯特,W.F.麦卡洛克,P.R.施努伦贝格尔.人兽共患病.上海科学技术出版社,1985,518-521。
[44]Savory L J, Stacker S A, Fleming S B, et al. Viral vascular endo thelial growth factor plays a critical role in orf virus infection[J]. Virol,2000,74:10699～10706
[45]Delhon G, Tulman E R, Afonso C L. Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus[J]. J Virol,2004,78(1):168～177
[46]Bruce T, Catherine S, McCaughan A, et al. Analysis of an orf virus chemokine-binding protein:Shifting ligand specificities among a family of poxvirus viroceptors[J]. PNAS,2003,100(25):15137～15142
[47]王良娟,马维卿,张再清.应用对流免疫电泳检测细胞培养物中羊口疮病毒抗原[J].甘肃畜牧兽医,1992,(3)：78
[48]Robinson A J, Barns G, Fraser K, et al. Conservation and variation in orf virus genomes[J]. Virology, 1987,156:13～23
[49]王廷璞,赵菲佚,孙春香,等.光敏生物素和DIG标记Orf病毒DNA探针的研究[J].生物技术,2002,12(6)：21-22
[50]Nitsche A, Buttner M, Wilhelm S, et al. Real-time PCR detection of parapoxvirus DNA[J]. Clinical Chemistry,2006,52(2):316～319
[51]王辉,赵晓军,易建刚.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检.2007,(6)24
[52]吕玉杰,王波.小议山羊传染性脓疱的病理及其防治[J].科技咨询导报,2007,23：242
[53]庞理化,谢文华,吴维忠等.青海羊口疮病的研究—弱毒细胞冻干苗的试制.青海畜牧兽医杂志,1986,6：5-10
[54]王辉,赵晓军,易建刚.羊传染性脓疱病毒内蒙分离株F1L基因的克隆与序列分析[J].中国动物检.2007,(6)24
[55]改树清,王志明,刘慧纯.用湿苗防治羊传染性脓疱口疮病[J].现代畜牧兽医,1988,(04)：16～17
[56]潘淑惠,周永连.羊传染性脓疱油乳剂灭活苗中间试验[J].贵州畜牧兽医,2000,24(6)：6
[57]王志军.羊传染性脓疱病的诊断和防治[J].河南畜牧兽医,1999,20(1)：23