犬细小病毒VP2基因的分子生物学特性的研究
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摘要
犬细小病毒(Canine Parvovirus CPV)的VP2基因编码主要衣壳蛋白，VP2基因在犬细小病毒感染过程中起着极为重要的作用，其编码CPV的主要抗原决定簇，可诱导机体产生中和抗体。
根据基因库中犬细小病毒基因序列设计合成了一对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，从犬细小病毒扬州分离株提取基因组DNA，并以此为模板，进行聚合酶链式反应(polymerase chain reaction，PCR)扩增出VP2基因的1740bp的扩增产物，将PCR产物克隆进pUC18载体，并转化大肠杆菌TG_1，通过LacZ基因的插入失活，初步筛选出重组质粒。通过限制性内切酶分析，得到CPV VP2阳性重组质粒。
对该分离株的VP2基因进行序列测定，结果表明：所扩增基因片段长度为1740bp,同美国参考株CPV-d(type 2)、CPV-15(type 2a)、CPV-39(type 2b)比较，核苷酸同源性分别高达99.5％、99.7％、99.7％，氨基酸同源性分别高达99.0％、99.7％、99.7％，表明VP2基因变异相对较少。
为探讨VP2蛋白的生物学特性，本研究还设计合成了另两对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，以扬州分离株病毒DNA为模板，扩增了VP2基因的5’端和3’端大小为794bp、1041bp的两个片段。将PCR产物按预定的阅读框架插入表达性载体pGEX-6P-1中的谷胱甘肽-S-转移酶下游，获得的重组质粒在1.0mM IPTG和37℃条件下诱导，通过对重组大肠杆菌裂解物的SDS-PAGE电泳分析，鉴定出两条分子量分别为54kD和61kD表达条带。将表达产物从SDS-PAGE凝胶中切出回收，并用其免疫6-8周龄ICR小鼠，每周免疫一次，5次免疫后，采血分离血清，所得抗血清与犬细小病毒扬州分离株感染的胎猫肾细胞(FK81)进行间接免疫荧光试验(IFA)，呈现特异性荧光，表明大肠杆菌表达的VP2基因产物保留了天然VP2蛋白的抗原性。
本研究对CPV扬州分离株的VP2基因序列分析，为探索CPV抗原漂变奠定了基础；而借助于大肠杆菌表达系统制备VP2的融合蛋白，为进一步研制VP2蛋白特异单抗及筛选CPV基因工程苗创造了条件。
A pair of primers were designed and synthesized based on VP2 gene of CPV from Genebank. The genomic DNA samples of canine parvovirus strain isolated in Yangzhou were extracted and used as templates for polymerase chain reaction (PCR) to amplify the VP2 gene. The specific 1740 bp band was amplifed and cloned into pUC18 vector at the restriction endonuclease EcoRI and Sail sites. The recombinant palsmids were identified by restriction endonuclease analysis. The exogenous insert of recombinant plasmid was further confirmed by sequence analysis. After sequencing, VP2 gene of Yangzhou isolation was compared with that of American reference strains CPV-d (type 2) ,CPV-15 (type2a) , CPV-39(type 2b). The results showed 99.5%, 99.7%, 99,7% homology among these different strains.
VP2 fragments from CPV Yangzhou strain were amplified with another two pairs of primers. PCR fragments were cloned to the downstream of GST gene according to the right Open Reading Frame in pGEX-6p-l vector. After inducing with 1.0mM IPTG and 37℃, the inserts were expressed as two fusion proteins containing the GST protein. According to SDS-PAGE analysis, two GST-VP2 fusion proteins were found as 54kD and 61kD bands, respectively. The expressed products were extracted from the gel and added as immunogen to immunize mice intraperitoneally, 5 doses with one-week intervals. The specificity of antibody was detected positively against CPV-infected FK81 cell in the indirect immunofluorescent assay (IFA). The results of the study showed that the in vitro expressed products of VP2 gene maintained the naive antigenicity of CPV.
The sequence analysis of VP2 gene will contribute to the study of antigenic drift, and the fusion proteins will be useful for the generation of specific monoclonal antibodies and recombinant vaccines.
根据基因库中犬细小病毒基因序列设计合成了一对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，从犬细小病毒扬州分离株提取基因组DNA，并以此为模板，进行聚合酶链式反应(polymerase chain reaction，PCR)扩增出VP2基因的1740bp的扩增产物，将PCR产物克隆进pUC18载体，并转化大肠杆菌TG_1，通过LacZ基因的插入失活，初步筛选出重组质粒。通过限制性内切酶分析，得到CPV VP2阳性重组质粒。
对该分离株的VP2基因进行序列测定，结果表明：所扩增基因片段长度为1740bp,同美国参考株CPV-d(type 2)、CPV-15(type 2a)、CPV-39(type 2b)比较，核苷酸同源性分别高达99.5％、99.7％、99.7％，氨基酸同源性分别高达99.0％、99.7％、99.7％，表明VP2基因变异相对较少。
为探讨VP2蛋白的生物学特性，本研究还设计合成了另两对引物，其两端分别含EcoRⅠ和SalⅠ酶切位点，以扬州分离株病毒DNA为模板，扩增了VP2基因的5’端和3’端大小为794bp、1041bp的两个片段。将PCR产物按预定的阅读框架插入表达性载体pGEX-6P-1中的谷胱甘肽-S-转移酶下游，获得的重组质粒在1.0mM IPTG和37℃条件下诱导，通过对重组大肠杆菌裂解物的SDS-PAGE电泳分析，鉴定出两条分子量分别为54kD和61kD表达条带。将表达产物从SDS-PAGE凝胶中切出回收，并用其免疫6-8周龄ICR小鼠，每周免疫一次，5次免疫后，采血分离血清，所得抗血清与犬细小病毒扬州分离株感染的胎猫肾细胞(FK81)进行间接免疫荧光试验(IFA)，呈现特异性荧光，表明大肠杆菌表达的VP2基因产物保留了天然VP2蛋白的抗原性。
本研究对CPV扬州分离株的VP2基因序列分析，为探索CPV抗原漂变奠定了基础；而借助于大肠杆菌表达系统制备VP2的融合蛋白，为进一步研制VP2蛋白特异单抗及筛选CPV基因工程苗创造了条件。
A pair of primers were designed and synthesized based on VP2 gene of CPV from Genebank. The genomic DNA samples of canine parvovirus strain isolated in Yangzhou were extracted and used as templates for polymerase chain reaction (PCR) to amplify the VP2 gene. The specific 1740 bp band was amplifed and cloned into pUC18 vector at the restriction endonuclease EcoRI and Sail sites. The recombinant palsmids were identified by restriction endonuclease analysis. The exogenous insert of recombinant plasmid was further confirmed by sequence analysis. After sequencing, VP2 gene of Yangzhou isolation was compared with that of American reference strains CPV-d (type 2) ,CPV-15 (type2a) , CPV-39(type 2b). The results showed 99.5%, 99.7%, 99,7% homology among these different strains.
VP2 fragments from CPV Yangzhou strain were amplified with another two pairs of primers. PCR fragments were cloned to the downstream of GST gene according to the right Open Reading Frame in pGEX-6p-l vector. After inducing with 1.0mM IPTG and 37℃, the inserts were expressed as two fusion proteins containing the GST protein. According to SDS-PAGE analysis, two GST-VP2 fusion proteins were found as 54kD and 61kD bands, respectively. The expressed products were extracted from the gel and added as immunogen to immunize mice intraperitoneally, 5 doses with one-week intervals. The specificity of antibody was detected positively against CPV-infected FK81 cell in the indirect immunofluorescent assay (IFA). The results of the study showed that the in vitro expressed products of VP2 gene maintained the naive antigenicity of CPV.
The sequence analysis of VP2 gene will contribute to the study of antigenic drift, and the fusion proteins will be useful for the generation of specific monoclonal antibodies and recombinant vaccines.
引文
1．殷震，刘景华．动物病毒学．第二版．北京：科学出版社，1997
2．梁士哲等．犬出血性肠炎的研究．上海畜牧兽医通讯，1982，2(4)：172
3．蔡宝祥．家畜传染病学．第三版．北京：中国农业出版社，1996
4. Langeveld JP, Casal JI, Vela C, Dalsgaard K. B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface. J Virol,1993, 67(2): 765-772
5．孔繁德，陆承平．犬细小病毒病原特性的研究概况．中国兽医科技，1995，25(1)：18-19
6. Parrish CR. Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones. Virology, 1991,183(1): 195-205
7. Truyen U, Agbandje M, Parrish CR. Characterization of the feline host range and a specific epitope of feline panleukopenia virus. Virology, 1994, 200(2): 194-503
8. Chang SF, Sgro JY, Parrish CR. Multiple amino acids in the capsid structure of canine parvovims coordinately determine the canine host range and specific antigenic and hemagglutination properties. J Virol, 1992,66(12): 6858-6867
9. Rhode SL, et al. Nucleotide sequence of the coat protein gene of canine parvovirus.J Virol, 1985, 54(2): 630-633
10. Reed AP, Jones EV, Miller TJ. Nucleotide sequence and genome organization of canine parvovirus. J Virol., 1988, 62(1): 266-276
11．张英．细小病毒的分子生物学．中国病毒学，1996，11(3)：193-200
12. Chapman MS, Rossmann MG. Structure, sequence and function correlations among parvoviruses. Virology, 1993, 194(2): 491-508
13. Parrish CR. Host range relationships and the evolution of canine parvovirus. Vet Microbiot, 1999, 69: 29-40
14. Vihinen-Ranta M, Wang D, Weichert WS, Parrish CR. The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection. J Virol, 2002, 76(4): 1884-91
15. Appel MJ, Scott FW, Carmichael LE. Isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis. Vet Rec,1979,105(8): 156-159
16. Parrish CR, Hare P, Foreyt WJ, et al. The global spread and replacement of canine parvovirus strains. J Gen Virol, 1988, 69: 1111-1116
17. Steinel A, Venter EH, Van Vuuren M. Antiginic and genetic analysis of canine parvoviruses in southern Africa. Onderstepoort J Vet Res, 1998, 65:239-242
18. Pereira CA, Monezi TA, Mehnert DH, et al. Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay. Vet Microbiol,
2000,75(2): 127-133
19. Hirasawa T, Yonok K, Mikazuki K, et al. Detection and genomic analysis of canine parvovirus by the polymerase chain reaction. Zentralbl Veterinarmed,1996, 43(9): 545-554
20. Parrish CR, O'Connell PH, Evermann JF, et al. Natural variation of canine parvovirus. Science, 1985, 230(4729): 1046-1048
21. Parrish CR, Aquadro CF, Strassheim ML, et al. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J Virol, 1991, 65: 6544-6552
22．范泉水，齐桂凤，王度林等．血凝和血凝抑制试验诊断犬细小病毒性肠炎．云南畜牧兽医，1998，3：8-10
23. Mathys A, Mueller R, Peersen NC, et al. Comparison of hemagglutination and competitive enzyme-linked immunosorbent assay procedures for detecting canine parvovirus in feces. Am J Vet Res, 1983, 44(1): 152-154
24. Mildbrand MM, Teramoto YA, Collins JK, et al. Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay. Am J Vet Res, 1984,45: 2281-2284
25. Drane DP, Hamilton RC, Cox JC. Evaluation of a novel diagnostic test for canine oarvouirus. Vet Microbiol, 1994, 41(3): 293-302
26．刘宏伟，田克恭，梁川玫等．犬细小病毒酶标试剂盒的研制．中国畜禽传染病，1994，6：10-13
27．董江丽，李武，额日贺等．应用聚合酶链式反应检测犬细小病毒．内蒙古大学学报(自然科学版)，2000，31(3)：289-292
28. Mochizuki M, San Gabriel MC, Nakatani H, et al. Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens. Res Vet Sci, 1993, 55(1): 60-63
29. Esfancdiari J, Klingeborn B. A Comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats and mink. J vet Med B Infect Dis Vet Public Health, 2000, 47(2): 145-153
30. Schunck B, Truyen U. Effect of maternal antibodies on the vaccination against canine parvovirus. Tierarztl Prax.1995, 23(2): 185-186
31. Carmichael LE. Immunization strategies in puppies-Why failures? 32nd gaines symposium on small animal pediatrics. The compendium on continuing education,1983, 5(12): 1043-1051
32．刘晓松，李京玉，宋爱军等．犬细小病毒与犬瘟热病毒特异性免疫核糖核酸制剂的应用．内蒙古畜牧科学，1999，(2)：37-38
33. Pratelli A, Cavalli A, Martella V, et al. Canine parvovirus (CPV) vaccination: comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine. Clin Diagn Lab Immunol, 2001,8(3): 612-615
34. Loopz de Turiso JA, Cortee E, Martinezc C, et al. Recombinant vaccine for canine
parvovirus in dogs. J Virol, 1992,66(5): 2748-2753
35. Langeveld JP, Casal JI, Osterhaus AD, et al. First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs. J Virol, 1994, 68(7): 4506-4513
36. Buonavoglia C, Caialli A, Gravino E, et al. Intranasal vaccination of pups with maternally derived antibodies with a modified live canine parvovirus. Zentralbl Veterinarmed, 1994,41(30): 3-8
37. Jiang W, Baker HJ, Swango LJ, et al. Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus. Vaccine, 1998,16(6): 601-607
38．杨德威，刘福安．不同种类犬细小病毒疫苗临床应用效果的检测．广东畜牧兽医科技，2000，22(2)：18-21
39. Langeveld JP, Brennan FR, Martinez-Torrecuadrada JL. Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus. Vaccine,2001,19(27): 3661-3670
40. Mochizuki M, Horiuchi M, Hiragi H, et al. Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia. J Cli Microbiol,1996,34: 2101-2015
41．王彦红．2001届扬州大学硕士论文．犬细小病毒YZ株的分离鉴定、单抗的制备和单抗的初步应用
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43. Parrish CR, Aquadro CF, Carmichael LE. Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses. Virology, 1988,166: 293-307
44. Pollock RV, Coyne MJ. Canine parvovirus. Vet Clin North Am Small Animal Pract. 1993, 232: 555-568
45. Truyen U. Emergence and recent evolution of canine parvovirus. Vet Microbiol,1999, 69: 47-50
46．卢圣栋．现代分子生物学实验技术．北京：高等教育出版社，1993
47．丁家波．2001届扬州大学硕士论文．马立克氏病病毒囊膜糖蛋白gI基因的分子生物学特性的研究
48．成大荣，徐建生等．类志贺菌毒素Ⅱ型变异体B亚单位基因的表达与鉴定．中国预防兽医学报，1999，5(21)：349-353
49．隋广超，胡美浩．影响大肠杆菌中外源基因表达的因素．生物化学与生物物理进展，1994，21(2)：128-132
50. Ileda Y, Mochizuki M, Naito R, et al. Prominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of New antigenic types of CPVs in cats.Virology, 2000, 278(1): 13-19
2．梁士哲等．犬出血性肠炎的研究．上海畜牧兽医通讯，1982，2(4)：172
3．蔡宝祥．家畜传染病学．第三版．北京：中国农业出版社，1996
4. Langeveld JP, Casal JI, Vela C, Dalsgaard K. B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface. J Virol,1993, 67(2): 765-772
5．孔繁德，陆承平．犬细小病毒病原特性的研究概况．中国兽医科技，1995，25(1)：18-19
6. Parrish CR. Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones. Virology, 1991,183(1): 195-205
7. Truyen U, Agbandje M, Parrish CR. Characterization of the feline host range and a specific epitope of feline panleukopenia virus. Virology, 1994, 200(2): 194-503
8. Chang SF, Sgro JY, Parrish CR. Multiple amino acids in the capsid structure of canine parvovims coordinately determine the canine host range and specific antigenic and hemagglutination properties. J Virol, 1992,66(12): 6858-6867
9. Rhode SL, et al. Nucleotide sequence of the coat protein gene of canine parvovirus.J Virol, 1985, 54(2): 630-633
10. Reed AP, Jones EV, Miller TJ. Nucleotide sequence and genome organization of canine parvovirus. J Virol., 1988, 62(1): 266-276
11．张英．细小病毒的分子生物学．中国病毒学，1996，11(3)：193-200
12. Chapman MS, Rossmann MG. Structure, sequence and function correlations among parvoviruses. Virology, 1993, 194(2): 491-508
13. Parrish CR. Host range relationships and the evolution of canine parvovirus. Vet Microbiot, 1999, 69: 29-40
14. Vihinen-Ranta M, Wang D, Weichert WS, Parrish CR. The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection. J Virol, 2002, 76(4): 1884-91
15. Appel MJ, Scott FW, Carmichael LE. Isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis. Vet Rec,1979,105(8): 156-159
16. Parrish CR, Hare P, Foreyt WJ, et al. The global spread and replacement of canine parvovirus strains. J Gen Virol, 1988, 69: 1111-1116
17. Steinel A, Venter EH, Van Vuuren M. Antiginic and genetic analysis of canine parvoviruses in southern Africa. Onderstepoort J Vet Res, 1998, 65:239-242
18. Pereira CA, Monezi TA, Mehnert DH, et al. Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay. Vet Microbiol,
2000,75(2): 127-133
19. Hirasawa T, Yonok K, Mikazuki K, et al. Detection and genomic analysis of canine parvovirus by the polymerase chain reaction. Zentralbl Veterinarmed,1996, 43(9): 545-554
20. Parrish CR, O'Connell PH, Evermann JF, et al. Natural variation of canine parvovirus. Science, 1985, 230(4729): 1046-1048
21. Parrish CR, Aquadro CF, Strassheim ML, et al. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J Virol, 1991, 65: 6544-6552
22．范泉水，齐桂凤，王度林等．血凝和血凝抑制试验诊断犬细小病毒性肠炎．云南畜牧兽医，1998，3：8-10
23. Mathys A, Mueller R, Peersen NC, et al. Comparison of hemagglutination and competitive enzyme-linked immunosorbent assay procedures for detecting canine parvovirus in feces. Am J Vet Res, 1983, 44(1): 152-154
24. Mildbrand MM, Teramoto YA, Collins JK, et al. Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay. Am J Vet Res, 1984,45: 2281-2284
25. Drane DP, Hamilton RC, Cox JC. Evaluation of a novel diagnostic test for canine oarvouirus. Vet Microbiol, 1994, 41(3): 293-302
26．刘宏伟，田克恭，梁川玫等．犬细小病毒酶标试剂盒的研制．中国畜禽传染病，1994，6：10-13
27．董江丽，李武，额日贺等．应用聚合酶链式反应检测犬细小病毒．内蒙古大学学报(自然科学版)，2000，31(3)：289-292
28. Mochizuki M, San Gabriel MC, Nakatani H, et al. Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens. Res Vet Sci, 1993, 55(1): 60-63
29. Esfancdiari J, Klingeborn B. A Comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats and mink. J vet Med B Infect Dis Vet Public Health, 2000, 47(2): 145-153
30. Schunck B, Truyen U. Effect of maternal antibodies on the vaccination against canine parvovirus. Tierarztl Prax.1995, 23(2): 185-186
31. Carmichael LE. Immunization strategies in puppies-Why failures? 32nd gaines symposium on small animal pediatrics. The compendium on continuing education,1983, 5(12): 1043-1051
32．刘晓松，李京玉，宋爱军等．犬细小病毒与犬瘟热病毒特异性免疫核糖核酸制剂的应用．内蒙古畜牧科学，1999，(2)：37-38
33. Pratelli A, Cavalli A, Martella V, et al. Canine parvovirus (CPV) vaccination: comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine. Clin Diagn Lab Immunol, 2001,8(3): 612-615
34. Loopz de Turiso JA, Cortee E, Martinezc C, et al. Recombinant vaccine for canine
parvovirus in dogs. J Virol, 1992,66(5): 2748-2753
35. Langeveld JP, Casal JI, Osterhaus AD, et al. First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs. J Virol, 1994, 68(7): 4506-4513
36. Buonavoglia C, Caialli A, Gravino E, et al. Intranasal vaccination of pups with maternally derived antibodies with a modified live canine parvovirus. Zentralbl Veterinarmed, 1994,41(30): 3-8
37. Jiang W, Baker HJ, Swango LJ, et al. Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus. Vaccine, 1998,16(6): 601-607
38．杨德威，刘福安．不同种类犬细小病毒疫苗临床应用效果的检测．广东畜牧兽医科技，2000，22(2)：18-21
39. Langeveld JP, Brennan FR, Martinez-Torrecuadrada JL. Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus. Vaccine,2001,19(27): 3661-3670
40. Mochizuki M, Horiuchi M, Hiragi H, et al. Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia. J Cli Microbiol,1996,34: 2101-2015
41．王彦红．2001届扬州大学硕士论文．犬细小病毒YZ株的分离鉴定、单抗的制备和单抗的初步应用
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