特异性识别猪瘟病毒野毒株的单克隆抗体的制备
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摘要
猪瘟(classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种高度接触性传染病,该病被世界动物卫生组织(OIE)列入OIE疫病名录(OIE-listed diseases),为须申报的(notifiable)动物传染病。近年来,我国猪瘟的流行和发病特点发生了很大的变化,在临床表现上趋于非典型化,主要表现为隐性带毒和慢性感染,成为影响养猪业发展的重大隐患。由于猪瘟兔化弱毒疫苗(HCLV)在我国的大规模使用,使得猪群中猪瘟抗体多数呈阳性,这种疫苗抗体很容易与猪瘟野毒感染产生的抗体相混淆,从而不易区分疫苗接种猪与野毒感染猪,给猪瘟诊断带来困难。
E2囊膜糖蛋白是猪瘟病毒的主要结构蛋白,是猪瘟病毒中和抗体的主要靶标,是抗猪瘟病毒感染的主要保护性抗原,因此,猪瘟病毒E2蛋白是开发猪瘟新型疫苗、诊断试剂及研究猪瘟病毒致病机理的重要靶蛋白。
本研究根据昆虫细胞密码子偏爱性,对猪瘟病毒E2基因进行密码子优化,利用昆虫杆状病毒表达系统进行表达,表达水平约为野生型猪瘟病毒E2基因的3倍。此外,用E2蛋白免疫家兔可以使其抵抗猪瘟兔化弱毒的攻击。
采用环磷酰胺免疫抑制法,用纯化的杆状病毒表达的HCLV株E2蛋白(HCLV-E2)作为耐受原,石门株E2蛋白(Shimen-E2)作为免疫原,免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0骨髓瘤细胞融合,经筛选获得一株稳定分泌抗E2蛋白单克隆抗体的杂交瘤细胞株,命名为4A4。经检测,该单克隆抗体能够与石门株发生反应,同时能与1.1、2.1、2.2和2.3基因亚群猪瘟野毒发生特异性反应,并具有中和活性,而与HCLV株不发生反应。该单克隆抗体可以用于鉴别猪瘟野毒株和猪瘟兔化弱毒疫苗株,为建立一种区分猪瘟野毒感染与疫苗接种的诊断方法与研究猪瘟强毒和弱毒之间的分子差异奠定了基础。
Classical swine fever (CSF), also known as hog cholera, one of OIE-listed diseases, is caused by Classical swine fever virus (CSFV). CSF is a highly contagious disease of pigs. In recent years, CSF has changed a lot in clinical manifestations under the pressure of mass vaccination in China, resulting in atypical or chronic forms quite different from acute CSF, and causing great economic losses in pork industry. Extensive vaccination with C-strain (lapinized hog cholera vaccine, HCLV) makes it difficult to differentiate the animals infected with wild-type viruses from those vaccinated with C-strain vaccine using conventional assays.
The envelope glycoprotein E2 of CSFV is responsible for the elicitation of neutralizing antibodies. It is often used for developing new type vaccines, clinical diagnostic reagents and studying immunopathological mechanisms of the virus.
CSFV E2 gene was codon-optimized according to the Sf9 cell codon usage. The codon-optimized E2 gene was expressed in insect baculovirus expression system, with expression level of 3 times higher than that of the wild-type E2 gene. On the other hand, rabbits immunized with E2 protein can be protected from challenge with HCLV.
A monoclonal antibody (mAb) specifically directed against the E2 protein of wild-type CSFV was generated using the recombinant baculovirus-expressed E2 protein of C-strain (HCLV-E2) as tolergen and that of Shimen strain (Shimen-E2) as immunogen using chemical-induced immunosuppression. Cyclophosphamide was used to tolerize mice to HCLV-E2 followed by immunization with Shimen-E2. The mAb was shown to recognize CSFV Shimen strain, but not C-strain. The mAb showed neutralizing activity with several subgroups of wild-type CSFV in neutralizing immunofluorescence assay. The mAb was shown to recognize a conformational epitope on the E2 protein. The mAb was identified to be IgG1 subtype. The mAb can be used to differentiate wild-type CSFV strains from C-strain.
E2囊膜糖蛋白是猪瘟病毒的主要结构蛋白,是猪瘟病毒中和抗体的主要靶标,是抗猪瘟病毒感染的主要保护性抗原,因此,猪瘟病毒E2蛋白是开发猪瘟新型疫苗、诊断试剂及研究猪瘟病毒致病机理的重要靶蛋白。
本研究根据昆虫细胞密码子偏爱性,对猪瘟病毒E2基因进行密码子优化,利用昆虫杆状病毒表达系统进行表达,表达水平约为野生型猪瘟病毒E2基因的3倍。此外,用E2蛋白免疫家兔可以使其抵抗猪瘟兔化弱毒的攻击。
采用环磷酰胺免疫抑制法,用纯化的杆状病毒表达的HCLV株E2蛋白(HCLV-E2)作为耐受原,石门株E2蛋白(Shimen-E2)作为免疫原,免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0骨髓瘤细胞融合,经筛选获得一株稳定分泌抗E2蛋白单克隆抗体的杂交瘤细胞株,命名为4A4。经检测,该单克隆抗体能够与石门株发生反应,同时能与1.1、2.1、2.2和2.3基因亚群猪瘟野毒发生特异性反应,并具有中和活性,而与HCLV株不发生反应。该单克隆抗体可以用于鉴别猪瘟野毒株和猪瘟兔化弱毒疫苗株,为建立一种区分猪瘟野毒感染与疫苗接种的诊断方法与研究猪瘟强毒和弱毒之间的分子差异奠定了基础。
Classical swine fever (CSF), also known as hog cholera, one of OIE-listed diseases, is caused by Classical swine fever virus (CSFV). CSF is a highly contagious disease of pigs. In recent years, CSF has changed a lot in clinical manifestations under the pressure of mass vaccination in China, resulting in atypical or chronic forms quite different from acute CSF, and causing great economic losses in pork industry. Extensive vaccination with C-strain (lapinized hog cholera vaccine, HCLV) makes it difficult to differentiate the animals infected with wild-type viruses from those vaccinated with C-strain vaccine using conventional assays.
The envelope glycoprotein E2 of CSFV is responsible for the elicitation of neutralizing antibodies. It is often used for developing new type vaccines, clinical diagnostic reagents and studying immunopathological mechanisms of the virus.
CSFV E2 gene was codon-optimized according to the Sf9 cell codon usage. The codon-optimized E2 gene was expressed in insect baculovirus expression system, with expression level of 3 times higher than that of the wild-type E2 gene. On the other hand, rabbits immunized with E2 protein can be protected from challenge with HCLV.
A monoclonal antibody (mAb) specifically directed against the E2 protein of wild-type CSFV was generated using the recombinant baculovirus-expressed E2 protein of C-strain (HCLV-E2) as tolergen and that of Shimen strain (Shimen-E2) as immunogen using chemical-induced immunosuppression. Cyclophosphamide was used to tolerize mice to HCLV-E2 followed by immunization with Shimen-E2. The mAb was shown to recognize CSFV Shimen strain, but not C-strain. The mAb showed neutralizing activity with several subgroups of wild-type CSFV in neutralizing immunofluorescence assay. The mAb was shown to recognize a conformational epitope on the E2 protein. The mAb was identified to be IgG1 subtype. The mAb can be used to differentiate wild-type CSFV strains from C-strain.
引文
1. 杜念兴. 猪瘟的回顾与展望. 中国畜禽传染病, 1998, 20 (5):317-319.
2. 郭丽, 周红莉, 屈建国, 等. 人诺如病毒衣壳蛋白的密码子优化及在昆虫细胞中的表达. 中国病毒学, 2006, 21(2):121-125.
3. 侯强, 彭伍平, 孙元, 等. 猪瘟病毒E2蛋白主要抗原区编码基因的原核表达及其单克隆抗体的制备. 中国兽医科学. 2008, 38(1):1-5..
4. 陆芹章. 免疫抑制法制备单克隆抗体的研究. 中国免疫学杂志, 1994, 10(6):350-353.
5. 吕宗吉, 涂长春, 余兴龙, 等. 我国猪瘟的流行病学现状分析. 中国预防兽医学报, 2001, 23(4):300-303.
6. 马刚, 李作生, 余兴龙, 等. 猪瘟病毒保护性抗原E2蛋白单抗的制备及其抗原表位的初步分析. 中国兽医学报, 2002, 22(2):121-124.
7. 彭伍平, 夏照和, 侯强, 等. 猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析. 病毒学报, 2007, 23(5):389-393.
8. 钱建飞, 毛洪先, 邵国青, 等. 环磷酰胺免疫抑制法制备单克隆抗体的研究. 中国农业科学, 1996, 29(3):86-92.
9. 丘惠深, 王在时, 方国安. 猪瘟单克隆抗体诊断试剂的研究. 中国畜禽传染病, 1991, 6:20-26.
10. 孙元, 夏照和, 梁冰冰, 等. 一种基于重组E2蛋白的检测猪瘟病毒抗体的间接ELISA方法. 中国兽医科学, 2008, 38(4):273-277.
11. 王晓波, 宁波, 曾文涛, 等. 抗前列腺特异膜抗原单克隆抗体的制备及鉴定. 中华泌尿外科杂志, 2000, 21(2):72-74.
12. 王在时, 邱惠深, 猪瘟单抗诊断试剂的推广及应用.中国兽医杂志, 1995, 129(4):22-26.
13. 王镇, 阂光伟, 李明义, 等. 猪瘟病毒的形态结构与形态发生. 微生物学报, 2000, 40(3):237-242.
14. 肖昌, 余兴龙, 张茂林, 等. 利用随机肽库鉴定猪瘟病毒E2蛋白抗原表位. 中国兽医学报, 2005, 25(5):449-453.
15. 谢庆阁, 翟中和. 畜禽重大疫病免疫研究进展. 北京:中国农业科技出版社, 1996, 321-338.
16. 杨艳艳, 张改平, 王选年. 猪瘟病毒单克隆抗体的制备及其免疫组织化学研究. 中国兽医科学, 2006, 36(10):782-786.
17. 周璟, 马宝骊, 严俊. 应用环磷酰胺研制细胞表面抗原的单克隆抗体. 微生物学免疫学进展, 2000, 28(3):26-28.
18. Akutagawa E, Konishi M. A monoclonal antibody specific to a song system nuclear antigen in estrildine finches. Neuron, 2001, 31(4):545-556.
19. Alt FW, Blackwell TK, Yancopoulos GD. Development of the primary antibody repertoire. Science, 1987, 238(4830):1079-1087.
20. Bernardi G. Codon usage and genome composition. J Mol Evol, 1985, 22(4): 363-365.
21. Bjorklund H, Lowings P, Stadejek T, et al. Phylogenetic comparison and molecular epidemiology of classical swine fever virus. Virus Genes, 1999, 19(3): 189-195.
22. Bowes T, Wagner ER, Boffey J, et al. Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome. Infect Immun, 2002, 70(9):5008-5018.
23. Briner TJ, Kuo MC, Keating KM, et al. Peripheral T-cell tolerance induced in naive and primed mice by subcutaneous injection of peptides from the major cat allergen Fel d I. Proc Natl Acad Sci USA, 1993, 90(16):7608-7612.
24. Brooks PC, Lin JM, French DL, et al. Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis. J Cell Biol, 1993, 122(6):1351-1359.
25. Bruschke CJM, Hulst MM, Moormann RJM, et al. Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. J Virol, 1997, 71(9):6692-6696.
26. Codon Usage Database. http://www.kazusa.or.jp/codon/.
27. Edwards S, Sands JJ. Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies. Dtsch Tierartl Wochenschr, 1990, 97(2):79-81.
28. Fauquet CM, Mayo MA, Maniloff J, et al (eds.). Virus Taxonomy: The Eighth Report of the International Committee on Taxonomy of Viruses. San Diego, Calif.: Elsevier Academic Press, 2005.
29. Freitas TR, Caldas LA, Rebello MA. Protaglandin A1 inhibits replication of classical swin fever virus. Mem Inst Oswaldo Cruz, 1998, 93(6):815-818.
30. Fuchs EJ, Matzinger P. B cells turn off virgin but not memory T cells. Science, 1992, 258(5085):1156-1159.
31. Golumbeski GS Jr, Dimond RL. The use of tolerization in the production of monoclonal antibodies against minor antigenic determinants. Anal Biochem, 1986, 154(2):373-381.
32. Greiser-Wilke I, Moennig V, Coulibaly CO, et al. Identification of conserved epitopes on a hog cholera virus protein. Arch Virol, 1990, 111(3-4):213-225.
33. Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol, 1996, 6(3):315-324.
34. Handschuh G, Caselmann WH. Bacterial expression and purification of hepatitis C virus capsid proteins of different size. J Hepatol, 1995, 22(2):143-150.
35. Harkness JW. Classical swine fever and its diagnosis: A current view. Vet Rec, 1985, 116(11):288-293.
36. Hasek M, Chutná J. Complexity of the state of immunological tolerance. Immunol Rev, 1979, 46:3-26.
37. Hennecken M, Stegeman JA, Elbers AR, et al. Transmission of classical swine fever virus byartificial insemination during the 1997-1998 epidemic in The Netherlands: a descriptive epidemiological study. Vet Q, 2000, 22(4):228-233.
38. Hockfield S. A Mab to a unique cerebellar neuron generated by immunosuppression and rapid immunization. Science, 1987, 237(4810):67-70.
39. Hulst MM, Himes G, Newbigin E, et al. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology, 1994, 200 (2):558-65.
40. Hulst MM, Moormann RJ. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical fever virus: Erns and E2 interact with different receptors. J Gen Virol, 1997, 78(11):2779-2787.
41. Hulst MM, Panoto FE, Hoekman A, et al. Inactivation of the RNase activity of glycoprotein Erns of classical swine fever virus results in a cytopathogenic virus. J Virol, 1998, 72(1):151-157.
42. Kipps TJ, Benacerraf B, Dorf ME. Regulation of antibody heterogeneity by suppressor T cells: diminishing suppressor T cell activity increases the number of dinitrophenyl clones in mice immunized with dinitrophenyl-poly(Glu,Lys,Phe) or dinitrophenyl-poly(Glu,Lys,Ala). Proc Natl Acad Sci USA, 1978, 75(6):2914-2917.
43. Kocks C, Rajewsky K. Stable expression and somatic hypermutation of antibody V regions in B-cell developmental pathways. Annu Rev Immunol, 1989, 7:537-559.
44. Kohler G, Milstein C. Continuous cultures of fused cell secreting antibody of predefined specificity. Nature, 1975, 256(5517):495-497.
45. Kosmidou A, Ahl R, Thiel HJ, et al. Differentiation of classical swine fever virus strains using monoclonal antibodies against structural glycoproteins. Vet Microbiol, 1995, 47(1-2):111-118.
46. Lebrón JA, Shen H, Bjorkman PJ, et al. Tolerization of adult mice to immunodominant proteins before monoclonal antibody production. J Immunol Methods, 1999, 222(1-2):59-63
47. Lin M, Lin F, Mallory M, et al. Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitope of monnclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol, 2000, 74(24):11619-11625.
48. Matthew WD, Patterson PH. The production of a monoclonal antibody that blocks the action of a neurite outgrowth-promoting factor. Cold Spring Harb Symp Quant Biol, 1983,48(2):625-631
49. Matthew WD, Sandrock AW Jr. Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies. J Immunol Methods, 1987, 100(1-2):73-82.
50. McLoon SC. A monoclonal antibody that distinguishes between temporal and nasal retinal axons. J Neurosci, 1991, 11(5):1470-1477.
51. Meyers G, Thiel HJ. Molecular characterization of pestivirus. Adv Virus Res, 1996, 47:53-118.
52. Mitchison NA. Immunological paralysis induced by brief exposure of cells to protein antigens. Immunology, 1968, 15(4):531-547.
53. Moennig V. Introduction to classical swine fever virus, disease and control policy. Vet Microbiol, 2000, 73(2-3):93-102.
54. Moennig V. The hog cholera virus. Comp Immunol Microbiol Infect Dis, 1992, 15(3):189-201.
55. Monroe JG, Lowy A, Granstein RD, et al. Studies of immune responsiveness and unresponsiveness to the p-azobenzenearsonate (ABA) hapten Immunol Rev, 1984, 80:103-131.
56. Moore M J, Dorfman T, Li W, et al. Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2. J Virol, 2004, 78 (19): 10628-10635.
57. Moormann RJ, van Gennip HG, Miedema GK, et al. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J Virol, 1996, 70:763-770.
58. OIE. Disease Information dated 15 July, 2005, 18(28):201.
59. Ou SK, McDonald C, Patterson PH. Comparison of two techniques for targeting the production of monoclonal antibodies against particular antigens. J Immunol Methods, 1991, 145(1-2):111-118.
60. Quintáns J, Quan ZS. Idiotype shifts caused by neonatal tolerance to phosphorylcholine. J Immunol, 1983, 130(2):590-595.
61. Ramakrishna L, Anand K K, Mohankumar K M, et al. Codonoptimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol, 2004, 78(17):9174-9189.
62. Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Fields BN, and Howley P (eds.), Fundamental Virology, Philadelphia: Lippincott-Rave Publishers, 1996: 931-959.
63. Riggott MJ, Matthew WD. Generating monoclonal antibodies to neuronal antigens. J Neurosci Methods, 1996, 68(2):235-245.
64. Risatti GR, Borca MV, Kutish GF, et al. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J Virol, 2005, 79(6): 3787-96.
65. Risatti GR, Holinka LG, Fernandez Sainz I, et al. N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine. J Virol, 2007, 81(2):924-933.
66. Ruggli N, Tratschin JD, Mittelholzer C, et al. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol, 1996, 70(6): 3478-3487.
67. Rumenapf T, Meyers G, Stark R, et al. Hog cholera virus?Characterization of specific antiserum and identification of cDNA clones. Virology, 1989, 171(1):18-27.
68. Rumenapf T, Stark R, Meyers G, et al. Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. J Virol, 1991, 65(2): 589-597.
69. Rumenapf T, Unger G, Strauss JH, et al. Processing of the envelope glycoproteins of pestiviruses. J Virol, 1993, 67(6):3288-3294.
70. Salata RA, Malhotra IJ, Hampson RK, et al. Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone. Anal Biochem, 1992, 207(1):142-149.
71. Schatz DG, Oettinger MA, Schlissel MS. V(D)J recombination: molecular biology and regulation. Annu Rev Immunol, 1992, 10:359-383.
72. Sleister HM, Rao AG. Subtractive immunization: a tool for the generation of discriminatory antibodies to proteins of similar sequence. J Immunol Methods, 2002, 261(1-2):213-220.
73. Stadejek T. Genetic heterogeneity of classical swine fever virus in central Europe. Virus Genes, 1997, 52(2): 195-204.
74. Stark R, Meyers G, Rümenapf T, et al. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J Virol, 1993, 67(12):7088-7095.
75. Streilein JW. Neonatal tolerance: towards an immunogenetic definition of self. Immunol Rev, 1979, 46:123-146.
76. Thiel HJ, Stark R, Weiland E, et al. Hog cholera virus: molecular composition of virions from a pestivirus. J Viro1, 1991, 65(9):4705-4712.
77. van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol, 2003, 96(4):367-384.
78. van Rijn PA, Miedema GK, Wensvoort G, et al. Antigenic structure of envelope enveloprotein E1 of hog cholera virus. J Virol, 1994, 68(6): 3934-3942.
79. van Rijn PA, van Gennip HG, de Meijer EJ, et al. Epitope mapping of envelope glycoprotein E1 of Hog Cholera Virus strain Brescia. J Gen Virol, 1993, 74(10): 2053-2060.
80. van Rijn PA, van Gennip HG, Moormann RJ. An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV). Vaccine, 1999, 17(5):433-440.
81. van Rijn PA, van Gennip RG, de Meijer EJ, et al. A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Vet Microbiol, 1992, 33(1-4):221-230.
82. van Zijl M, Wensvoort G, de Kluyver E, et al. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J Virol, 1991, 65(5):2761-2765.
83. Vandeputte J, Chappuis G. Classical swine fever: the European experience and a guide for infected areas. Rev Sci Tech, 1999, 18(131): 638-647.
84. Weiland E, Ahl R, Stark R, et al. A second envelope glycoprotein mediates neutralization of a pestivirus Hog Cholera Virus. J Virol, 1992, 66(6): 3677-3682.
85. Weiland E, Stark R, Haas B, et al. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol, 1990, 64(8): 3563-3569.
86. Weiland E, Thiel HJ, Hess G, et al. Development of monoclonal neutralizing antibodies against bovine viral diarrhea virus after pretreatment of mice with normal bovine cells and cyclophosphamide. J Virol Methods, 1989, 24(1-2):237-243.
87. Wensvoort G, Terpstra C, Boonstra J, et al. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis.Vet Microbiol, 1986, 12(2):101-108.
88. Wensvoort G, Terpstra C, de Kluijver EP, et al. Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet Microbiol, 1989, 21(1):9-20.
89. Wensvoort G, Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies. J Gen Virol, 1989, 70(11): 2865-2876.
90. Williams CV, Stechmann CL, McLoon SC. Subtractive immunization techniques for the production of monoclonal antibodies to rare antigens. Biotechniques, 1992, 12(6):842-847.
91. Windisch JM, Schneider R, Stark R, et al. RNase of classical swine fever virus: Biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J Virol, 1996, 70(1):352-358.
92. Yamaguchi Y, O'Doherty U, Peng M, et al. Difficulties in obtaining monoclonal antibodies to subsets of human leukocytes, using neonatal tolerance induction in mice. J Immunol Methods, 1995, 181(1):115-124.
93. Yu M, Wang LF, Shiell BJ, et al. Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses. Virology, 1996, 222(1):289-292.
94. Zhang F, Yu M, Weiland E, et al. Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library. Arch Virol, 2006, 151(1):37-54.
2. 郭丽, 周红莉, 屈建国, 等. 人诺如病毒衣壳蛋白的密码子优化及在昆虫细胞中的表达. 中国病毒学, 2006, 21(2):121-125.
3. 侯强, 彭伍平, 孙元, 等. 猪瘟病毒E2蛋白主要抗原区编码基因的原核表达及其单克隆抗体的制备. 中国兽医科学. 2008, 38(1):1-5..
4. 陆芹章. 免疫抑制法制备单克隆抗体的研究. 中国免疫学杂志, 1994, 10(6):350-353.
5. 吕宗吉, 涂长春, 余兴龙, 等. 我国猪瘟的流行病学现状分析. 中国预防兽医学报, 2001, 23(4):300-303.
6. 马刚, 李作生, 余兴龙, 等. 猪瘟病毒保护性抗原E2蛋白单抗的制备及其抗原表位的初步分析. 中国兽医学报, 2002, 22(2):121-124.
7. 彭伍平, 夏照和, 侯强, 等. 猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析. 病毒学报, 2007, 23(5):389-393.
8. 钱建飞, 毛洪先, 邵国青, 等. 环磷酰胺免疫抑制法制备单克隆抗体的研究. 中国农业科学, 1996, 29(3):86-92.
9. 丘惠深, 王在时, 方国安. 猪瘟单克隆抗体诊断试剂的研究. 中国畜禽传染病, 1991, 6:20-26.
10. 孙元, 夏照和, 梁冰冰, 等. 一种基于重组E2蛋白的检测猪瘟病毒抗体的间接ELISA方法. 中国兽医科学, 2008, 38(4):273-277.
11. 王晓波, 宁波, 曾文涛, 等. 抗前列腺特异膜抗原单克隆抗体的制备及鉴定. 中华泌尿外科杂志, 2000, 21(2):72-74.
12. 王在时, 邱惠深, 猪瘟单抗诊断试剂的推广及应用.中国兽医杂志, 1995, 129(4):22-26.
13. 王镇, 阂光伟, 李明义, 等. 猪瘟病毒的形态结构与形态发生. 微生物学报, 2000, 40(3):237-242.
14. 肖昌, 余兴龙, 张茂林, 等. 利用随机肽库鉴定猪瘟病毒E2蛋白抗原表位. 中国兽医学报, 2005, 25(5):449-453.
15. 谢庆阁, 翟中和. 畜禽重大疫病免疫研究进展. 北京:中国农业科技出版社, 1996, 321-338.
16. 杨艳艳, 张改平, 王选年. 猪瘟病毒单克隆抗体的制备及其免疫组织化学研究. 中国兽医科学, 2006, 36(10):782-786.
17. 周璟, 马宝骊, 严俊. 应用环磷酰胺研制细胞表面抗原的单克隆抗体. 微生物学免疫学进展, 2000, 28(3):26-28.
18. Akutagawa E, Konishi M. A monoclonal antibody specific to a song system nuclear antigen in estrildine finches. Neuron, 2001, 31(4):545-556.
19. Alt FW, Blackwell TK, Yancopoulos GD. Development of the primary antibody repertoire. Science, 1987, 238(4830):1079-1087.
20. Bernardi G. Codon usage and genome composition. J Mol Evol, 1985, 22(4): 363-365.
21. Bjorklund H, Lowings P, Stadejek T, et al. Phylogenetic comparison and molecular epidemiology of classical swine fever virus. Virus Genes, 1999, 19(3): 189-195.
22. Bowes T, Wagner ER, Boffey J, et al. Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome. Infect Immun, 2002, 70(9):5008-5018.
23. Briner TJ, Kuo MC, Keating KM, et al. Peripheral T-cell tolerance induced in naive and primed mice by subcutaneous injection of peptides from the major cat allergen Fel d I. Proc Natl Acad Sci USA, 1993, 90(16):7608-7612.
24. Brooks PC, Lin JM, French DL, et al. Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis. J Cell Biol, 1993, 122(6):1351-1359.
25. Bruschke CJM, Hulst MM, Moormann RJM, et al. Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. J Virol, 1997, 71(9):6692-6696.
26. Codon Usage Database. http://www.kazusa.or.jp/codon/.
27. Edwards S, Sands JJ. Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies. Dtsch Tierartl Wochenschr, 1990, 97(2):79-81.
28. Fauquet CM, Mayo MA, Maniloff J, et al (eds.). Virus Taxonomy: The Eighth Report of the International Committee on Taxonomy of Viruses. San Diego, Calif.: Elsevier Academic Press, 2005.
29. Freitas TR, Caldas LA, Rebello MA. Protaglandin A1 inhibits replication of classical swin fever virus. Mem Inst Oswaldo Cruz, 1998, 93(6):815-818.
30. Fuchs EJ, Matzinger P. B cells turn off virgin but not memory T cells. Science, 1992, 258(5085):1156-1159.
31. Golumbeski GS Jr, Dimond RL. The use of tolerization in the production of monoclonal antibodies against minor antigenic determinants. Anal Biochem, 1986, 154(2):373-381.
32. Greiser-Wilke I, Moennig V, Coulibaly CO, et al. Identification of conserved epitopes on a hog cholera virus protein. Arch Virol, 1990, 111(3-4):213-225.
33. Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol, 1996, 6(3):315-324.
34. Handschuh G, Caselmann WH. Bacterial expression and purification of hepatitis C virus capsid proteins of different size. J Hepatol, 1995, 22(2):143-150.
35. Harkness JW. Classical swine fever and its diagnosis: A current view. Vet Rec, 1985, 116(11):288-293.
36. Hasek M, Chutná J. Complexity of the state of immunological tolerance. Immunol Rev, 1979, 46:3-26.
37. Hennecken M, Stegeman JA, Elbers AR, et al. Transmission of classical swine fever virus byartificial insemination during the 1997-1998 epidemic in The Netherlands: a descriptive epidemiological study. Vet Q, 2000, 22(4):228-233.
38. Hockfield S. A Mab to a unique cerebellar neuron generated by immunosuppression and rapid immunization. Science, 1987, 237(4810):67-70.
39. Hulst MM, Himes G, Newbigin E, et al. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology, 1994, 200 (2):558-65.
40. Hulst MM, Moormann RJ. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical fever virus: Erns and E2 interact with different receptors. J Gen Virol, 1997, 78(11):2779-2787.
41. Hulst MM, Panoto FE, Hoekman A, et al. Inactivation of the RNase activity of glycoprotein Erns of classical swine fever virus results in a cytopathogenic virus. J Virol, 1998, 72(1):151-157.
42. Kipps TJ, Benacerraf B, Dorf ME. Regulation of antibody heterogeneity by suppressor T cells: diminishing suppressor T cell activity increases the number of dinitrophenyl clones in mice immunized with dinitrophenyl-poly(Glu,Lys,Phe) or dinitrophenyl-poly(Glu,Lys,Ala). Proc Natl Acad Sci USA, 1978, 75(6):2914-2917.
43. Kocks C, Rajewsky K. Stable expression and somatic hypermutation of antibody V regions in B-cell developmental pathways. Annu Rev Immunol, 1989, 7:537-559.
44. Kohler G, Milstein C. Continuous cultures of fused cell secreting antibody of predefined specificity. Nature, 1975, 256(5517):495-497.
45. Kosmidou A, Ahl R, Thiel HJ, et al. Differentiation of classical swine fever virus strains using monoclonal antibodies against structural glycoproteins. Vet Microbiol, 1995, 47(1-2):111-118.
46. Lebrón JA, Shen H, Bjorkman PJ, et al. Tolerization of adult mice to immunodominant proteins before monoclonal antibody production. J Immunol Methods, 1999, 222(1-2):59-63
47. Lin M, Lin F, Mallory M, et al. Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitope of monnclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol, 2000, 74(24):11619-11625.
48. Matthew WD, Patterson PH. The production of a monoclonal antibody that blocks the action of a neurite outgrowth-promoting factor. Cold Spring Harb Symp Quant Biol, 1983,48(2):625-631
49. Matthew WD, Sandrock AW Jr. Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies. J Immunol Methods, 1987, 100(1-2):73-82.
50. McLoon SC. A monoclonal antibody that distinguishes between temporal and nasal retinal axons. J Neurosci, 1991, 11(5):1470-1477.
51. Meyers G, Thiel HJ. Molecular characterization of pestivirus. Adv Virus Res, 1996, 47:53-118.
52. Mitchison NA. Immunological paralysis induced by brief exposure of cells to protein antigens. Immunology, 1968, 15(4):531-547.
53. Moennig V. Introduction to classical swine fever virus, disease and control policy. Vet Microbiol, 2000, 73(2-3):93-102.
54. Moennig V. The hog cholera virus. Comp Immunol Microbiol Infect Dis, 1992, 15(3):189-201.
55. Monroe JG, Lowy A, Granstein RD, et al. Studies of immune responsiveness and unresponsiveness to the p-azobenzenearsonate (ABA) hapten Immunol Rev, 1984, 80:103-131.
56. Moore M J, Dorfman T, Li W, et al. Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2. J Virol, 2004, 78 (19): 10628-10635.
57. Moormann RJ, van Gennip HG, Miedema GK, et al. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. J Virol, 1996, 70:763-770.
58. OIE. Disease Information dated 15 July, 2005, 18(28):201.
59. Ou SK, McDonald C, Patterson PH. Comparison of two techniques for targeting the production of monoclonal antibodies against particular antigens. J Immunol Methods, 1991, 145(1-2):111-118.
60. Quintáns J, Quan ZS. Idiotype shifts caused by neonatal tolerance to phosphorylcholine. J Immunol, 1983, 130(2):590-595.
61. Ramakrishna L, Anand K K, Mohankumar K M, et al. Codonoptimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol, 2004, 78(17):9174-9189.
62. Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Fields BN, and Howley P (eds.), Fundamental Virology, Philadelphia: Lippincott-Rave Publishers, 1996: 931-959.
63. Riggott MJ, Matthew WD. Generating monoclonal antibodies to neuronal antigens. J Neurosci Methods, 1996, 68(2):235-245.
64. Risatti GR, Borca MV, Kutish GF, et al. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J Virol, 2005, 79(6): 3787-96.
65. Risatti GR, Holinka LG, Fernandez Sainz I, et al. N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine. J Virol, 2007, 81(2):924-933.
66. Ruggli N, Tratschin JD, Mittelholzer C, et al. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol, 1996, 70(6): 3478-3487.
67. Rumenapf T, Meyers G, Stark R, et al. Hog cholera virus?Characterization of specific antiserum and identification of cDNA clones. Virology, 1989, 171(1):18-27.
68. Rumenapf T, Stark R, Meyers G, et al. Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. J Virol, 1991, 65(2): 589-597.
69. Rumenapf T, Unger G, Strauss JH, et al. Processing of the envelope glycoproteins of pestiviruses. J Virol, 1993, 67(6):3288-3294.
70. Salata RA, Malhotra IJ, Hampson RK, et al. Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone. Anal Biochem, 1992, 207(1):142-149.
71. Schatz DG, Oettinger MA, Schlissel MS. V(D)J recombination: molecular biology and regulation. Annu Rev Immunol, 1992, 10:359-383.
72. Sleister HM, Rao AG. Subtractive immunization: a tool for the generation of discriminatory antibodies to proteins of similar sequence. J Immunol Methods, 2002, 261(1-2):213-220.
73. Stadejek T. Genetic heterogeneity of classical swine fever virus in central Europe. Virus Genes, 1997, 52(2): 195-204.
74. Stark R, Meyers G, Rümenapf T, et al. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J Virol, 1993, 67(12):7088-7095.
75. Streilein JW. Neonatal tolerance: towards an immunogenetic definition of self. Immunol Rev, 1979, 46:123-146.
76. Thiel HJ, Stark R, Weiland E, et al. Hog cholera virus: molecular composition of virions from a pestivirus. J Viro1, 1991, 65(9):4705-4712.
77. van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol, 2003, 96(4):367-384.
78. van Rijn PA, Miedema GK, Wensvoort G, et al. Antigenic structure of envelope enveloprotein E1 of hog cholera virus. J Virol, 1994, 68(6): 3934-3942.
79. van Rijn PA, van Gennip HG, de Meijer EJ, et al. Epitope mapping of envelope glycoprotein E1 of Hog Cholera Virus strain Brescia. J Gen Virol, 1993, 74(10): 2053-2060.
80. van Rijn PA, van Gennip HG, Moormann RJ. An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV). Vaccine, 1999, 17(5):433-440.
81. van Rijn PA, van Gennip RG, de Meijer EJ, et al. A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Vet Microbiol, 1992, 33(1-4):221-230.
82. van Zijl M, Wensvoort G, de Kluyver E, et al. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J Virol, 1991, 65(5):2761-2765.
83. Vandeputte J, Chappuis G. Classical swine fever: the European experience and a guide for infected areas. Rev Sci Tech, 1999, 18(131): 638-647.
84. Weiland E, Ahl R, Stark R, et al. A second envelope glycoprotein mediates neutralization of a pestivirus Hog Cholera Virus. J Virol, 1992, 66(6): 3677-3682.
85. Weiland E, Stark R, Haas B, et al. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol, 1990, 64(8): 3563-3569.
86. Weiland E, Thiel HJ, Hess G, et al. Development of monoclonal neutralizing antibodies against bovine viral diarrhea virus after pretreatment of mice with normal bovine cells and cyclophosphamide. J Virol Methods, 1989, 24(1-2):237-243.
87. Wensvoort G, Terpstra C, Boonstra J, et al. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis.Vet Microbiol, 1986, 12(2):101-108.
88. Wensvoort G, Terpstra C, de Kluijver EP, et al. Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet Microbiol, 1989, 21(1):9-20.
89. Wensvoort G, Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies. J Gen Virol, 1989, 70(11): 2865-2876.
90. Williams CV, Stechmann CL, McLoon SC. Subtractive immunization techniques for the production of monoclonal antibodies to rare antigens. Biotechniques, 1992, 12(6):842-847.
91. Windisch JM, Schneider R, Stark R, et al. RNase of classical swine fever virus: Biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J Virol, 1996, 70(1):352-358.
92. Yamaguchi Y, O'Doherty U, Peng M, et al. Difficulties in obtaining monoclonal antibodies to subsets of human leukocytes, using neonatal tolerance induction in mice. J Immunol Methods, 1995, 181(1):115-124.
93. Yu M, Wang LF, Shiell BJ, et al. Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses. Virology, 1996, 222(1):289-292.
94. Zhang F, Yu M, Weiland E, et al. Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library. Arch Virol, 2006, 151(1):37-54.