猪瘟病毒E2蛋白结合细胞受体的鉴定
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摘要
猪瘟病毒(classical swine fever virus,CSFV)属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus),为单股正链RNA病毒。CSFV含有4种结构蛋白:C、Erns、E1和E2。其中,Erns和E2作为囊膜蛋白存在于成熟病毒粒子表面,能够诱导机体产生中和抗体;而E1与Erns通过二硫键形成异源二聚体,包埋于病毒囊膜内,不能诱导猪产生中和抗体。猪瘟病毒进入宿主细胞是通过病毒囊膜蛋白与细胞受体蛋白之间相互作用介导的内吞作用,Erns作用的受体蛋白为硫酸乙酰肝素,而E2作用的受体蛋白目前尚不清楚。
抗独特型抗体(anti-idiotype antibody,anti-Id)是指机体针对Ab1抗原抗体结合区的独特位产生的抗体,其中Ab2β具有“内影像”特性,能够模拟病毒抗原,产生针对初始抗原的特异性抗体或细胞免疫应答。抗独特型抗体相当于病毒粒子的探针,通过其与宿主细胞之间的相互作用可以鉴定出病毒感染过程中所需的受体蛋白。
我们以针对猪瘟病毒E2蛋白的单克隆抗体6E10为免疫原,经皮下免疫新西兰白兔后制备了抗独特型多克隆抗体。竞争抑制ELISA证明其能够模拟E2抗原,免疫组化试验及Western blot发现anti-Id能够识别猪只外周血淋巴细胞以及不同组织中一个分子量大小约54 kDa的蛋白,命名为p54,质谱分析其与IgG同源性最高。为了进一步揭示p54蛋白的特征及其生物学功能,我们参考大量文献后发现p54蛋白分子量大小与CD46蛋白十分接近,因此我们根据NCBI提供的猪CD46基因设计一对引物,从猪只血液中扩增得到目的基因,经测序分析后分别将其克隆至原核表达载体pET32a和真核表达载体pcDNA3.1,并利用原核表达的蛋白制备了兔抗CD46高免血清,阻断试验证明,病毒感染的抑制作用与抗CD46多克隆抗体表现出剂量依赖性。但我们发现兔anti-Id不与CD46蛋白反应,同样利用抗CD46多抗也无法在PBMC中检测到p54,由此我们认为p54与CD46蛋白是两种截然不同的蛋白质。在此基础之上,我们进行了受体重建工作:将已经构建的pcDNA-CD46转染猪瘟病毒非敏感细胞BHK-21,该细胞经RT-PCR检测是不存在猪CD46基因的。转染后加G418筛选,传至第三代后的BHK-CD46细胞进行病毒感染试验与激光共聚焦试验。病毒感染试验显示,猪瘟病毒能够吸附或进入BHK-CD46细胞但不增殖。激光共聚焦结果显示,猪瘟病毒能够吸附在表达CD46蛋白的BHK-CD46细胞膜表面。免疫共沉淀试验证明猪瘟病毒E2蛋白能够沉淀PK15细胞表面的CD46分子。以上结果表明,CD46蛋白与猪瘟病毒E2蛋白之间存在相互作用,可能是猪瘟病毒吸附过程中所必需的受体蛋白之一。
Classical swine fever virus (CSFV) is classified within the genus Pestivirus in the family Flaviviridae. It is an enveloped, non-segmented, positive-stranded RNA virus encoding four structural proteins: C, Erns (formerly known as E0), E1, and E2. Erns and E2 reside on the outer surface of CSFV virion and transmembrane glycoprotein E1 is present as an E1-E2 heterodimer by disulfide linkage. Glycoprotein E2 is the most immunogenic protein of CSFV and glycoprotein Erns is the second target for neutralizing antibodies. Glycoproteins, which present in the viral envelope, are involved in the attachment to susceptible cells and mediate the CSFV entry. Interaction of Erns with heparin sulfate (HS) immobilizes CSFV at the cell surface but E2 also associates with cellular receptor(s) by an as yet unknown mechanism.
Anti-idiotype antibodies (anti-Ids) elicited by an antibody molecule are directed against antigenic determinants in or close to the antibody’s combing site. Besides using anti-Ids as immunogens for a protective response against a particular infectious agent, Ab2βinternal image antibodies have been used as probes for receptors by mimicking viral epitopes that bind to cell receptors.
Polyclonal anti-Ids were produced in New Zealand White rabbits by immunizing monoclonal antibody (MAb) 6E10 via subcutaneous injection. Characterization of anti-Ids, which can mimic an epitope on the CSFV glycoprotein E2, was performed by competitive ELISA. In immunohistochemistry assay and Western blot analysis, a 54 kDa cell surface protein (p54) was detected by the anti-Ids from PBMC. Microchemical analysis showed that the most similar molecule of p54 was IgG-like. To identify the structure and function of p54, we searched a mass of literatures and found that the molecular masses between p54 and CD46 are similar. To verify our hypothesis, the CD46 gene of pig was cloned and expressed in vectors pET32a and pcDNA3.1. Polyclonal rabbit anti-CD46 sera were produced and they could block CSFV infection in porcine kidney cells. We also found that p54 and CD46 were different proteins because anti-Ids could not recognize CD46 and p54 could not be recognized by anti-CD46 serum. In further study, BHK-21 cells were transfected with the expression plasmid pcDNA-CD46. Stable CD46-expressing cell lines (BHK-CD46) were selected by adding G418 to the culture medium after transfection. CSFV infection on BHK-CD46 cells expressing CD46 was detected by antigen-capture ELISA and laser confocal light microscopy. Co-IP showed that CSFV E2 could precipitate CD46 from PK15 cells. Our data showed that the expression of CD46 did not confer full susceptibility to non-permissive cells but binding. This study indicates that CD46 is a cellular receptors that is involved in attachment to susceptible cells of CSFV.
抗独特型抗体(anti-idiotype antibody,anti-Id)是指机体针对Ab1抗原抗体结合区的独特位产生的抗体,其中Ab2β具有“内影像”特性,能够模拟病毒抗原,产生针对初始抗原的特异性抗体或细胞免疫应答。抗独特型抗体相当于病毒粒子的探针,通过其与宿主细胞之间的相互作用可以鉴定出病毒感染过程中所需的受体蛋白。
我们以针对猪瘟病毒E2蛋白的单克隆抗体6E10为免疫原,经皮下免疫新西兰白兔后制备了抗独特型多克隆抗体。竞争抑制ELISA证明其能够模拟E2抗原,免疫组化试验及Western blot发现anti-Id能够识别猪只外周血淋巴细胞以及不同组织中一个分子量大小约54 kDa的蛋白,命名为p54,质谱分析其与IgG同源性最高。为了进一步揭示p54蛋白的特征及其生物学功能,我们参考大量文献后发现p54蛋白分子量大小与CD46蛋白十分接近,因此我们根据NCBI提供的猪CD46基因设计一对引物,从猪只血液中扩增得到目的基因,经测序分析后分别将其克隆至原核表达载体pET32a和真核表达载体pcDNA3.1,并利用原核表达的蛋白制备了兔抗CD46高免血清,阻断试验证明,病毒感染的抑制作用与抗CD46多克隆抗体表现出剂量依赖性。但我们发现兔anti-Id不与CD46蛋白反应,同样利用抗CD46多抗也无法在PBMC中检测到p54,由此我们认为p54与CD46蛋白是两种截然不同的蛋白质。在此基础之上,我们进行了受体重建工作:将已经构建的pcDNA-CD46转染猪瘟病毒非敏感细胞BHK-21,该细胞经RT-PCR检测是不存在猪CD46基因的。转染后加G418筛选,传至第三代后的BHK-CD46细胞进行病毒感染试验与激光共聚焦试验。病毒感染试验显示,猪瘟病毒能够吸附或进入BHK-CD46细胞但不增殖。激光共聚焦结果显示,猪瘟病毒能够吸附在表达CD46蛋白的BHK-CD46细胞膜表面。免疫共沉淀试验证明猪瘟病毒E2蛋白能够沉淀PK15细胞表面的CD46分子。以上结果表明,CD46蛋白与猪瘟病毒E2蛋白之间存在相互作用,可能是猪瘟病毒吸附过程中所必需的受体蛋白之一。
Classical swine fever virus (CSFV) is classified within the genus Pestivirus in the family Flaviviridae. It is an enveloped, non-segmented, positive-stranded RNA virus encoding four structural proteins: C, Erns (formerly known as E0), E1, and E2. Erns and E2 reside on the outer surface of CSFV virion and transmembrane glycoprotein E1 is present as an E1-E2 heterodimer by disulfide linkage. Glycoprotein E2 is the most immunogenic protein of CSFV and glycoprotein Erns is the second target for neutralizing antibodies. Glycoproteins, which present in the viral envelope, are involved in the attachment to susceptible cells and mediate the CSFV entry. Interaction of Erns with heparin sulfate (HS) immobilizes CSFV at the cell surface but E2 also associates with cellular receptor(s) by an as yet unknown mechanism.
Anti-idiotype antibodies (anti-Ids) elicited by an antibody molecule are directed against antigenic determinants in or close to the antibody’s combing site. Besides using anti-Ids as immunogens for a protective response against a particular infectious agent, Ab2βinternal image antibodies have been used as probes for receptors by mimicking viral epitopes that bind to cell receptors.
Polyclonal anti-Ids were produced in New Zealand White rabbits by immunizing monoclonal antibody (MAb) 6E10 via subcutaneous injection. Characterization of anti-Ids, which can mimic an epitope on the CSFV glycoprotein E2, was performed by competitive ELISA. In immunohistochemistry assay and Western blot analysis, a 54 kDa cell surface protein (p54) was detected by the anti-Ids from PBMC. Microchemical analysis showed that the most similar molecule of p54 was IgG-like. To identify the structure and function of p54, we searched a mass of literatures and found that the molecular masses between p54 and CD46 are similar. To verify our hypothesis, the CD46 gene of pig was cloned and expressed in vectors pET32a and pcDNA3.1. Polyclonal rabbit anti-CD46 sera were produced and they could block CSFV infection in porcine kidney cells. We also found that p54 and CD46 were different proteins because anti-Ids could not recognize CD46 and p54 could not be recognized by anti-CD46 serum. In further study, BHK-21 cells were transfected with the expression plasmid pcDNA-CD46. Stable CD46-expressing cell lines (BHK-CD46) were selected by adding G418 to the culture medium after transfection. CSFV infection on BHK-CD46 cells expressing CD46 was detected by antigen-capture ELISA and laser confocal light microscopy. Co-IP showed that CSFV E2 could precipitate CD46 from PK15 cells. Our data showed that the expression of CD46 did not confer full susceptibility to non-permissive cells but binding. This study indicates that CD46 is a cellular receptors that is involved in attachment to susceptible cells of CSFV.
引文
1.常天明,孙元,李宏宇,等.猪瘟病毒NS3蛋白的重组表达及其单克隆抗体的制备.中国兽医科学, 2010, 2: 144~149.
2.丁天兵,任君萍,马文煜,等.病毒受体的研究方法.中国病毒学, 2006, 21: 189~193.
3.丁天兵,马文煜,肖毅,等.日本脑炎病毒内影响组抗独特型单克隆抗体的制备及初步鉴定.细胞与分子免疫学杂志, 2000, 16 : 481~483.
4.高美华,付凤华,程颖,等.乙酰胆碱酯酶抗独特型抗体的研制.免疫学杂志, 2003, 19: 30~32.
5.郭爱珍,陆承平.病毒的细胞膜受体.中国病毒学, 1997, 12: 295~300.
6.龚非力(主编).医学免疫学,北京,科学出版社, 2001.
7.胡永浩,龚振华,蒋正军.抗独特型抗体对猪繁殖与呼吸综合征病毒感染的免疫作用.畜牧兽医学报, 2005, 36: 272~277.
8.景涛.抗独特型抗体及其在人体寄生虫学中的应用.地方病通讯, 1999, 14(4) :92295.
9.卡恩.分子病毒学原理,北京,科学出版社, 2006.
10.彭伍平.利用噬菌体展示技术鉴定猪瘟病毒E2蛋白抗原表位[硕士学位论文].北京:中国农业科学院研究生院, 2007.
11.王祝鸣,冯振卿,仇镇宁,等.日本血吸虫单克隆anti-anti-id的建株及初步鉴定.中国血吸虫病防治杂志, 2001, 13: 204~206.
12.王万利.应用噬菌体展示技术筛选抗猪瘟病毒E2蛋白单克隆抗体的模拟表位[硕士学位论文].北京:中国农业科学院研究生院, 2009.
13.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.
14.夏照和.特异性识别猪瘟病毒野毒株的单克隆抗体的制备[硕士学位论文],北京:中国农业科学院研究生院, 2008.
15.夏海滨,金伯泉,马文煜,等.基因免疫诱导9.1C3分子内影像类抗独特型抗体的产生.细胞与分子免疫学杂志, 1997, 13: 125~130.
16.杨汉春,孔繁瑶.伊氏锥虫抗独特型抗体疫苗的研究.中国兽医杂志, 1995, 21: 325~330.
17.赵建军.鉴别猪瘟病毒野毒株和兔化弱毒疫苗株的复合实时荧光定量RT-PCR方法的建立与评价[硕士学位论文].北京:中国农业科学院研究生院, 2007.
18.赵青,李冬.病毒受体及其研究现状.动物医学进展, 2009, 30: 95~100.
19.宗益民,史敏言,姚立人,等.免疫学基础与临床.合肥:安徽科技出版社, 1980.
20.周文来,何倩倪,袁燕,等.牛型布氏菌单克隆抗独特型抗体苗生产工艺的研究.中国生物制品学杂志, 1997, 10: 86~88.
21. Agnello V, Abel G, Elfahal M, et al. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci U S A, 1999, 96: 12766~12771.
22. Barilla ML, Liszewski MK, Lambris JD, et al. Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. J Immunol, 2002, 168:6298~6304.
23. Bartosch B, Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the sr-b1 scavenger receptor. J Biol Chem, 2003, 43: 41624~41630.
24. Browning J, Horner JW, Pettoello Mantovani M, et al. Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc Natl Acad Sci U S A, 1997, 94: 14637~14641.
25. Cattaneo R. Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’magnet. J Virol, 2004, 78:4385–4388.
26. Ryu CJ, Cho DY, Gripon P, et al. An 80-kilodalton protein that binds to the pre-s1 domain of hepatitis B virus. J Virol, 2000, 72: 110~116.
27. Dainiak MB, Muronetz VI, Izumrudov VA. Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes. Anal Biochemi, 2000, 277: 58~66.
28. Delputte PL, Costers S, Nauwynck HJ. Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin. J Gen Virol, 2005, 86: 1441~1445.
29. Delputte PL, Vanderheijden N, Nauwynck HJ, et al. Involvement of the matrix protein in the attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. J Virol, 2002, 76: 4312~4320.
30. Dimitrov DS. Cell biology of virus entry. Cell, 2000, 101: 697~702.
31. Dorig R, Marcel A,Chopra A, et al. The human CD46 molecule is a receptor for measles virus. Cell, 1993, 75:295~305.
32. Hiroshi IT, Watanabe S, Takada A, Kawaoka Y. Ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies. J Virol, 2001, 75(3):1576~1580.
33. Hofer F, Gruenberger M, Kowalski H, et al. Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus. Proc Natl Acad Sci U S A, 1994, 91:1839~1842.
34. Hulst MM, Moormann RJ. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors. J Gen Virol, 1997, 78: 2779~2787.
35. Hulst MM, Panoto FE, Hoekman A, et al. Inactivation of the RNase activity of glycoprotein Erns of classical swine fever results in a cytopathogenic virus. J Virol, 1998, 72: 151~157
36. Hulst MM, van Gennip HG, Moormann RJ, et al. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J Virol, 2000, 74: 9553~9561.
37. Hulst MM, van Gennip HG, Vlot AC, et al. Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence. J Virol, 2001, 75: 9585~9595.
38. Iqbal M, Flick-Smith H, McCauley JW. Interactions of bovineviral diarrhoea virus glycoproteinE(rns) with cell surface glycosaminoglycans. J Gen Virol, 2000, 81: 451~459.
39. Iqbal M, McCauley JW. Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis. J Gen Virol, 2002, 83:2153~2159.
40. Kopecky J, Grubhoffer L, Kovár V, et al. A putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblotting. Intervirology, 1999, 42: 9~16.
41. Jerne NK. Towards a network theory of the immune system. Ann Immunol Inst Pasteur (Paris) 1974, 125: 373~389.
42. Kim TY, Choi Y, Cheong HS, et al. Identification of a cell surface 30Ku protein as a candidate receptor for hantaan virus. J Gen Virol, 2002, 83: 767~773.
43. Kreutz LC and Ackerman MR. Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus Res, 1996, 42:137~147.
44. Kreutz LC. Cellular membrane factors are the major determinants of porcine reproductive andrespiratory syndrome virus tropism. Virus Res, 1998, 53:121~128.
45. Krey T, Himmelreich A, Heimann M, et al. Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1. J Virol, 2006. 80: 3912~3922.
46. Krey T, Thiel HJ, Ru¨menapf T. Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry. J Virol, 2005, 79:4191~4200.
47. Kunkel HG, Mannik M, Williams RC. Individual antigenic specificities of isolated antibodies. Science, 1963, 140:12~18.
48. Lambot M, Frétier S, Beeck A, et al. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem, 2002, 277: 20625~2063.
49. Lee C, Hodgins D, Calvert JG, et al. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication. Virology, 2006, 346: 238~250.
50. Lee C, Yoo D. Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity. J Gen Virol, 2005, 86: 3091~3096.
51. Lee C, Yoo D. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties. Virology, 2006, 355: 30~43.
52. Liebert UG and Finke D. Measles virus infections in rodents. Curr Top Microbiol Immunol, 1995, 191: 149～166.
53. 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 monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol, 2000, 74: 11619~11625.
54. Liu JJ, Wong ML, Chang TJ. The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator. Virus Res, 1998, 53: 75~80.
55. Manchester M, Naniche D, Stehlet T. CD46 as a measles receptor: form follows function. Virology, 2000, 274:5～10.
56. Maurer K, Krey T, Moennig V, et al. CD46 is a cellular receptor for bovine viral diarrhea virus. J Virol, 2004, 78: 1792~1799.
57. Mettenleiter C. Brief overview on cellular virus receptors. Virus Res, 2002, 82: 3~8.
58. Meyers G, Rumenapf T, Thiel HJ, Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology, 1989, 171:555~567.
59. Moenning V, 1990. Pestiviruses: a review. Vet Microbiol, 23: 35~54.
60. Moennig V. The hog cholera virus. Comp Immunol Microbiol Infect Dis, 1992, 15: 189~201.
61. Monazahian M, Bohme I, Bonk S, et al. Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol, 1999, 57: 223~229.
62. Mori Y, Okabayashi T, Yamashita T, et al. Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. J Virol, 2005, 79: 3448~3458.
63. Nepom GT, Tardieu M, Epstin RL et al. Virus-binding receptors: similarities to immune receptors as determined by anti-idiotypic antibodies. Surv Immunol Res, 1982, 1: 255~261.
64. Oudin J, Michel M. Une nouvelle forme d'allotypie des immunoglobulines du serum de lapin. CR Hebd Seances Acad Sci,1963, 257:805~808.
65. Peng WP, Hou Q, Xia ZH, et al. Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library. Virus Res, 2008, 135: 267~272.
66. Santoro F, Greenstone HL, Insinga A, et al. Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J Biol Chem, 2003, 278: 25964~25969.
67. Schelp C, Greiser-Wilke I, Wolf G, Beer M, et al. Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhea virus infection. Arch Virol, 1995, 140: 1997~2009.
68. Schneider-Schaulies J. Cellular receptors for viruses: links to tropism and pathogenesis. J Gen Virol, 2000, 81: 1413~1429.
69. Smith GP. Filamentous Fusion phage: novel expression vector that display cloned antigens on the viron surface. Science, 1985, 228: 1315~1317.
70. Suzuki T, Ishii K, Aizaki H, et al. Hepatitis C viral life cycle. Adv Drug Deliv Rev, 2007, 59: 1200~1212.
71. Thaker SR, Stin DL, Zamb TJ, et al. Identification of a putative cellular receptor for bovine herpesvirus 1. J Gen Virol,1994, 75:2303~2309.
72. Krey T, Moussay E, Thiel HJ, et al. Role of the low-density lipoprotein receptor in entry of bovine viral diarrhea virus. J Virol, 2006, 80:10862~10867.
73. Van Rijn PA, Miedema GK, Wensvoort G, et al. Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J Virol, 1994, 68: 3934~3942.
74. Van Rijn PA, Van Gennip HG, De Meijer EJ, et al. Epitope mapping of envelope glycoprotein E1 ofhog cholera virus strain Brescia. J Gen Virol, 1993, 74: 2053~2060.
75. van Rijn PA, Bossers A, Wensvoort G, et al. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J Gen Virol, 1996, 77: 2737~2745.
76. Varthakavi V, Minocha HC. Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies. J Gen Virol, 1996, 77:1875~1882.
77. Wunschmann S, Medh JD, Klinzmann D, et al. Characterization of hepatitis C virus (HCV) and HCV E2 interaction with CD81 and the low-density lipoprotein receptor. J Virol, 2000, 74: 10055~10062.
78. Xue W, Orten DJ, Abdelmagid OY, et al. Anti-idiotypic antibodies mimic viral diarrhea virus antigen. Vet Microbiol, 1991, 29:201~212.
79. Xue W, Zhang S, Minocha. HC. Characterization of a putativereceptor protein for bovine viral diarrhea virus. Vet Microbiol, 1997, 57:105~118.
80. Xue W, Minocha HC. Identification of the cell surface receptor for bovine diarrhoea virus by using anti-idiotypic antibodies. J Gen Virol, 1993, 74:73~79.
81. Zhou EM, Afshar A, Heckert RA, et al. Anti-idiotypic antibodies generated by sequential immunization detect the shared idiotypeon antibodies to pseudorabies virus antigens. J Virol Methods, 1994, 48:301~313.
82. Zhou EM, Huang W. Anti-idiotypic antibody as a potential serodiagnostic reagent for detection of bluetongue virus infection. J Clin Microbiol, 1995, 33: 850~854.
83. Zhou EM, Qin QS. Identification and characterization of a putative cellar receptor for PRRS virus. 2005 International PRRS Symposium. St. Louis Missouri, 2005, Dec 2~3, 24.
84. Zhou EM, Xiao YH, Shi YF, et al. Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus. J Med Virol, 2008, 149:300~308.
2.丁天兵,任君萍,马文煜,等.病毒受体的研究方法.中国病毒学, 2006, 21: 189~193.
3.丁天兵,马文煜,肖毅,等.日本脑炎病毒内影响组抗独特型单克隆抗体的制备及初步鉴定.细胞与分子免疫学杂志, 2000, 16 : 481~483.
4.高美华,付凤华,程颖,等.乙酰胆碱酯酶抗独特型抗体的研制.免疫学杂志, 2003, 19: 30~32.
5.郭爱珍,陆承平.病毒的细胞膜受体.中国病毒学, 1997, 12: 295~300.
6.龚非力(主编).医学免疫学,北京,科学出版社, 2001.
7.胡永浩,龚振华,蒋正军.抗独特型抗体对猪繁殖与呼吸综合征病毒感染的免疫作用.畜牧兽医学报, 2005, 36: 272~277.
8.景涛.抗独特型抗体及其在人体寄生虫学中的应用.地方病通讯, 1999, 14(4) :92295.
9.卡恩.分子病毒学原理,北京,科学出版社, 2006.
10.彭伍平.利用噬菌体展示技术鉴定猪瘟病毒E2蛋白抗原表位[硕士学位论文].北京:中国农业科学院研究生院, 2007.
11.王祝鸣,冯振卿,仇镇宁,等.日本血吸虫单克隆anti-anti-id的建株及初步鉴定.中国血吸虫病防治杂志, 2001, 13: 204~206.
12.王万利.应用噬菌体展示技术筛选抗猪瘟病毒E2蛋白单克隆抗体的模拟表位[硕士学位论文].北京:中国农业科学院研究生院, 2009.
13.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.
14.夏照和.特异性识别猪瘟病毒野毒株的单克隆抗体的制备[硕士学位论文],北京:中国农业科学院研究生院, 2008.
15.夏海滨,金伯泉,马文煜,等.基因免疫诱导9.1C3分子内影像类抗独特型抗体的产生.细胞与分子免疫学杂志, 1997, 13: 125~130.
16.杨汉春,孔繁瑶.伊氏锥虫抗独特型抗体疫苗的研究.中国兽医杂志, 1995, 21: 325~330.
17.赵建军.鉴别猪瘟病毒野毒株和兔化弱毒疫苗株的复合实时荧光定量RT-PCR方法的建立与评价[硕士学位论文].北京:中国农业科学院研究生院, 2007.
18.赵青,李冬.病毒受体及其研究现状.动物医学进展, 2009, 30: 95~100.
19.宗益民,史敏言,姚立人,等.免疫学基础与临床.合肥:安徽科技出版社, 1980.
20.周文来,何倩倪,袁燕,等.牛型布氏菌单克隆抗独特型抗体苗生产工艺的研究.中国生物制品学杂志, 1997, 10: 86~88.
21. Agnello V, Abel G, Elfahal M, et al. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci U S A, 1999, 96: 12766~12771.
22. Barilla ML, Liszewski MK, Lambris JD, et al. Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. J Immunol, 2002, 168:6298~6304.
23. Bartosch B, Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the sr-b1 scavenger receptor. J Biol Chem, 2003, 43: 41624~41630.
24. Browning J, Horner JW, Pettoello Mantovani M, et al. Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection. Proc Natl Acad Sci U S A, 1997, 94: 14637~14641.
25. Cattaneo R. Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’magnet. J Virol, 2004, 78:4385–4388.
26. Ryu CJ, Cho DY, Gripon P, et al. An 80-kilodalton protein that binds to the pre-s1 domain of hepatitis B virus. J Virol, 2000, 72: 110~116.
27. Dainiak MB, Muronetz VI, Izumrudov VA. Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes. Anal Biochemi, 2000, 277: 58~66.
28. Delputte PL, Costers S, Nauwynck HJ. Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin. J Gen Virol, 2005, 86: 1441~1445.
29. Delputte PL, Vanderheijden N, Nauwynck HJ, et al. Involvement of the matrix protein in the attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. J Virol, 2002, 76: 4312~4320.
30. Dimitrov DS. Cell biology of virus entry. Cell, 2000, 101: 697~702.
31. Dorig R, Marcel A,Chopra A, et al. The human CD46 molecule is a receptor for measles virus. Cell, 1993, 75:295~305.
32. Hiroshi IT, Watanabe S, Takada A, Kawaoka Y. Ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies. J Virol, 2001, 75(3):1576~1580.
33. Hofer F, Gruenberger M, Kowalski H, et al. Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus. Proc Natl Acad Sci U S A, 1994, 91:1839~1842.
34. Hulst MM, Moormann RJ. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors. J Gen Virol, 1997, 78: 2779~2787.
35. Hulst MM, Panoto FE, Hoekman A, et al. Inactivation of the RNase activity of glycoprotein Erns of classical swine fever results in a cytopathogenic virus. J Virol, 1998, 72: 151~157
36. Hulst MM, van Gennip HG, Moormann RJ, et al. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. J Virol, 2000, 74: 9553~9561.
37. Hulst MM, van Gennip HG, Vlot AC, et al. Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence. J Virol, 2001, 75: 9585~9595.
38. Iqbal M, Flick-Smith H, McCauley JW. Interactions of bovineviral diarrhoea virus glycoproteinE(rns) with cell surface glycosaminoglycans. J Gen Virol, 2000, 81: 451~459.
39. Iqbal M, McCauley JW. Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis. J Gen Virol, 2002, 83:2153~2159.
40. Kopecky J, Grubhoffer L, Kovár V, et al. A putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblotting. Intervirology, 1999, 42: 9~16.
41. Jerne NK. Towards a network theory of the immune system. Ann Immunol Inst Pasteur (Paris) 1974, 125: 373~389.
42. Kim TY, Choi Y, Cheong HS, et al. Identification of a cell surface 30Ku protein as a candidate receptor for hantaan virus. J Gen Virol, 2002, 83: 767~773.
43. Kreutz LC and Ackerman MR. Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus Res, 1996, 42:137~147.
44. Kreutz LC. Cellular membrane factors are the major determinants of porcine reproductive andrespiratory syndrome virus tropism. Virus Res, 1998, 53:121~128.
45. Krey T, Himmelreich A, Heimann M, et al. Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1. J Virol, 2006. 80: 3912~3922.
46. Krey T, Thiel HJ, Ru¨menapf T. Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry. J Virol, 2005, 79:4191~4200.
47. Kunkel HG, Mannik M, Williams RC. Individual antigenic specificities of isolated antibodies. Science, 1963, 140:12~18.
48. Lambot M, Frétier S, Beeck A, et al. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem, 2002, 277: 20625~2063.
49. Lee C, Hodgins D, Calvert JG, et al. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication. Virology, 2006, 346: 238~250.
50. Lee C, Yoo D. Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity. J Gen Virol, 2005, 86: 3091~3096.
51. Lee C, Yoo D. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties. Virology, 2006, 355: 30~43.
52. Liebert UG and Finke D. Measles virus infections in rodents. Curr Top Microbiol Immunol, 1995, 191: 149～166.
53. 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 monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol, 2000, 74: 11619~11625.
54. Liu JJ, Wong ML, Chang TJ. The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator. Virus Res, 1998, 53: 75~80.
55. Manchester M, Naniche D, Stehlet T. CD46 as a measles receptor: form follows function. Virology, 2000, 274:5～10.
56. Maurer K, Krey T, Moennig V, et al. CD46 is a cellular receptor for bovine viral diarrhea virus. J Virol, 2004, 78: 1792~1799.
57. Mettenleiter C. Brief overview on cellular virus receptors. Virus Res, 2002, 82: 3~8.
58. Meyers G, Rumenapf T, Thiel HJ, Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology, 1989, 171:555~567.
59. Moenning V, 1990. Pestiviruses: a review. Vet Microbiol, 23: 35~54.
60. Moennig V. The hog cholera virus. Comp Immunol Microbiol Infect Dis, 1992, 15: 189~201.
61. Monazahian M, Bohme I, Bonk S, et al. Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol, 1999, 57: 223~229.
62. Mori Y, Okabayashi T, Yamashita T, et al. Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. J Virol, 2005, 79: 3448~3458.
63. Nepom GT, Tardieu M, Epstin RL et al. Virus-binding receptors: similarities to immune receptors as determined by anti-idiotypic antibodies. Surv Immunol Res, 1982, 1: 255~261.
64. Oudin J, Michel M. Une nouvelle forme d'allotypie des immunoglobulines du serum de lapin. CR Hebd Seances Acad Sci,1963, 257:805~808.
65. Peng WP, Hou Q, Xia ZH, et al. Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library. Virus Res, 2008, 135: 267~272.
66. Santoro F, Greenstone HL, Insinga A, et al. Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J Biol Chem, 2003, 278: 25964~25969.
67. Schelp C, Greiser-Wilke I, Wolf G, Beer M, et al. Identification of cell membrane proteins linked to susceptibility to bovine viral diarrhea virus infection. Arch Virol, 1995, 140: 1997~2009.
68. Schneider-Schaulies J. Cellular receptors for viruses: links to tropism and pathogenesis. J Gen Virol, 2000, 81: 1413~1429.
69. Smith GP. Filamentous Fusion phage: novel expression vector that display cloned antigens on the viron surface. Science, 1985, 228: 1315~1317.
70. Suzuki T, Ishii K, Aizaki H, et al. Hepatitis C viral life cycle. Adv Drug Deliv Rev, 2007, 59: 1200~1212.
71. Thaker SR, Stin DL, Zamb TJ, et al. Identification of a putative cellular receptor for bovine herpesvirus 1. J Gen Virol,1994, 75:2303~2309.
72. Krey T, Moussay E, Thiel HJ, et al. Role of the low-density lipoprotein receptor in entry of bovine viral diarrhea virus. J Virol, 2006, 80:10862~10867.
73. Van Rijn PA, Miedema GK, Wensvoort G, et al. Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J Virol, 1994, 68: 3934~3942.
74. Van Rijn PA, Van Gennip HG, De Meijer EJ, et al. Epitope mapping of envelope glycoprotein E1 ofhog cholera virus strain Brescia. J Gen Virol, 1993, 74: 2053~2060.
75. van Rijn PA, Bossers A, Wensvoort G, et al. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J Gen Virol, 1996, 77: 2737~2745.
76. Varthakavi V, Minocha HC. Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies. J Gen Virol, 1996, 77:1875~1882.
77. Wunschmann S, Medh JD, Klinzmann D, et al. Characterization of hepatitis C virus (HCV) and HCV E2 interaction with CD81 and the low-density lipoprotein receptor. J Virol, 2000, 74: 10055~10062.
78. Xue W, Orten DJ, Abdelmagid OY, et al. Anti-idiotypic antibodies mimic viral diarrhea virus antigen. Vet Microbiol, 1991, 29:201~212.
79. Xue W, Zhang S, Minocha. HC. Characterization of a putativereceptor protein for bovine viral diarrhea virus. Vet Microbiol, 1997, 57:105~118.
80. Xue W, Minocha HC. Identification of the cell surface receptor for bovine diarrhoea virus by using anti-idiotypic antibodies. J Gen Virol, 1993, 74:73~79.
81. Zhou EM, Afshar A, Heckert RA, et al. Anti-idiotypic antibodies generated by sequential immunization detect the shared idiotypeon antibodies to pseudorabies virus antigens. J Virol Methods, 1994, 48:301~313.
82. Zhou EM, Huang W. Anti-idiotypic antibody as a potential serodiagnostic reagent for detection of bluetongue virus infection. J Clin Microbiol, 1995, 33: 850~854.
83. Zhou EM, Qin QS. Identification and characterization of a putative cellar receptor for PRRS virus. 2005 International PRRS Symposium. St. Louis Missouri, 2005, Dec 2~3, 24.
84. Zhou EM, Xiao YH, Shi YF, et al. Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus. J Med Virol, 2008, 149:300~308.