口蹄疫病毒vp2基因的克隆表达及抗体检测方法的建立
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摘要
口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouthdisease virus,FMDV)引起的急性热性高度接触性传染病,主要侵害偶蹄动物,偶见于人和其它动物。现今发达国家往往通过捕杀感染动物、同居动物及可疑畜群来控制和消灭本病。而发展中国家多采用注射疫苗的方法以控制该病的发生和流行。
我国主要通过注射疫苗来预防和控制口蹄疫,因此免疫后快速方便的抗体水平检测显得尤为重要。目前,国外学者研究表明VP_2蛋白具有免疫原性,动物体内的FMDV抗体均可与VP_2蛋白发生特异性反应。另外,口蹄疫病毒四种结构蛋白的遗传变异顺序依次是:VP_1＞VP_3＞VP_2＞VP_4,VP_1变异性最大,VP4位于病毒衣壳的内部对免疫贡献相对较少,动物机体很少产生针对VP_4蛋白的抗体。相比之下VP_2基因相对保守,以VP_2蛋白作为抗原检测动物体内口蹄疫抗体有其自身的优点。单克隆抗体因具有高度均质性和特异性的特点,在生物医学领域得到广泛的应用。本研究分别克隆和表达了O型及亚洲I型口蹄疫病毒VP_2基因,建立了针对这两种病毒抗体的ELISA方法,同时制备了亚洲I型口蹄疫VP2蛋白的单克隆抗体,建立了FMDV VP_2结构蛋白竞争ELISA抗体检测方法,经临床试验证明该方法有较高的灵敏度和特异性。具体研究内容如下:
1.克隆表达O型和亚洲I型口蹄疫病毒VP_2基因
根据GenBank公布的序列,分别设计合成了AsiaI型和O型口蹄疫病毒的结构蛋白VP_2引物。PCR扩增得到了AsiaI型口蹄疫病毒VP_2片段大约693bp,O型的VP_2片段大约660bp序列分析表明是VP_2基因,将两种VP_2基因亚克隆至表达载体,实现目的基因的原核表达。
2.O型和AsiaI型口蹄疫病毒抗体间接ELISA方法的建立
以纯化的原核表达O型或AsiaI型口蹄疫病毒VP_2蛋白包被酶标板,分别建立了O型和AsiaI型口蹄疫病毒抗体间接ELISA方法。对影响ELISA反应条件的各种因素进行了优化和选择,如蛋白的包被浓度、温度及时间,各步骤反应的温度及时间,封闭液的选择及浓度确定等,使得该方法有较好的稳定性和重复性。
3.Asial型口蹄疫病毒VP_2蛋白单克隆抗体的制备
以纯化的VP_2蛋白免疫Balb/C小鼠,将脾细胞与骨髓瘤细胞SP2/0融合,用间接ELISA方法筛选,采用有限稀释法对其进行克隆,经过3次融合共获得2株能稳定分泌抗VP_2蛋白单克隆抗体的杂交瘤细胞株,命名为2F_(12),5F_5。两株单抗的腹水效价分别为2~9×100,2~(12)×100且单克隆抗体特异性良好,与载体蛋白无交叉反应。
4.Asial型口蹄疫病毒竞争ELISA方法的建立及初步应用
以VP_2蛋白为抗原,以辣根过氧化物酶标记的单抗5F_5为第二抗体,建立了快速检测FMD抗体的竞争ELISA方法,对于影响ELISA的各种因素进行了优化和选择,包括蛋白的包被浓度、温度及时间,各步反应的温度及时间,封闭液的选择及浓度确定等,使得试剂盒具有较好的稳定性和重复性,具有较好的临床应用价值。
Foot-and-mouth disease(FMD) is a highly contagious and acute disease of cattle, goats,pigs and sheep which is caused by Foot-and-mouth disease virus(FMDV).In developed country,killing all infected and exposed animal could be the first choice to control and eliminate the disease.But in developing country,vaccination is more acceptable.
In China,vaccination is the main measures to prevent and control the disease.So, evaluation of antibody level after vaccination is important.In the past,some researches indicated that structual protein VP_2 of FMDV could induce specific antibodies against FMDV.Otherwise,the four structural protein's mutation probability is VP_1＞VP_3＞VP_2＞VP_4.VP_1 is more variable and VP_2 is relatively conserved.So,VP_2 protein has many advantages as a tool to detect antibodies against FMDV.Monoclonal antibody has high sensitivity and specificity,so it is widely used in biomedicine.In this study,VP_2 gene of O and Asia-1 sreotype FMDV were cloned and expressed in E.coli respectively.monoclonal antibodies against VP_2 protein of Asia-1 type FMDV were prepared.Based on the protein expressed and monoclonal antibodies,competitive ELISA for the detection of VP_2 antibodies was established.clinical tests indicated that the ELISA method had high sensitivity and specificity.Research contents are as follows:
1.cloning and expression of VP_2 genes of o type FMDV and Asia 1 type FMDV
According to the gene sequences pulicated in Genbank,primers were designed and synthesizd.660bp VP_2 genes of O type FMDV and 693bp Asia-1 type FMDV were amplified by PCR.Sequence analysis showed the gene fragments were VP_2 genes of FMDV.Then,the VP_2 genes were subcloned into vector and expressed in E.coli.
2.establishment of indirect ELISA using VP_2 protein of o type and asial type FMDV
Indirect ELISA was established by coating VP_2 protein as detection antigen.All impact factors to reaction were optimizated,including consentration of VP_2 protein,temperature,reaction time and silling buffer,et al.,these work would contribute to the application of the ELISA kit.
3.Generation of monoclonal antibodies against AsiaI type foot-and-mouth disease virus:
Balb/c mice were immunized with the E.coli expressed fusion protein.The splenocytes from immunized mice were fused with myeloma cells SP2/0.The hybridism cells were screened by indirect ELlSA and limited dilution method.Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained.The ELISA titers of the ascites induced by the two hybridism cells were above 100×2~9. Specificity of mAbs was analyzed by Western-blot.All the monoclonal antibodies were proved to be specific.No cross-reactions with nontarget proteins were found.
4.Establishment and prime application of the competitive ELISA for the detection of asial FMDV antibody
The competitive ELISA method used to detect the antibody against FMDV was established by using VP_2 protein as antigen and the HRP-labled monoclonal antibody 5F5 as detection antibody.All the items which may influence the reaction were perfected,including temperature in each steps and final concentration of each solution.At last the best way of this method was found,which provide the foundation for the next step-use of the ELISA kit.
我国主要通过注射疫苗来预防和控制口蹄疫,因此免疫后快速方便的抗体水平检测显得尤为重要。目前,国外学者研究表明VP_2蛋白具有免疫原性,动物体内的FMDV抗体均可与VP_2蛋白发生特异性反应。另外,口蹄疫病毒四种结构蛋白的遗传变异顺序依次是:VP_1＞VP_3＞VP_2＞VP_4,VP_1变异性最大,VP4位于病毒衣壳的内部对免疫贡献相对较少,动物机体很少产生针对VP_4蛋白的抗体。相比之下VP_2基因相对保守,以VP_2蛋白作为抗原检测动物体内口蹄疫抗体有其自身的优点。单克隆抗体因具有高度均质性和特异性的特点,在生物医学领域得到广泛的应用。本研究分别克隆和表达了O型及亚洲I型口蹄疫病毒VP_2基因,建立了针对这两种病毒抗体的ELISA方法,同时制备了亚洲I型口蹄疫VP2蛋白的单克隆抗体,建立了FMDV VP_2结构蛋白竞争ELISA抗体检测方法,经临床试验证明该方法有较高的灵敏度和特异性。具体研究内容如下:
1.克隆表达O型和亚洲I型口蹄疫病毒VP_2基因
根据GenBank公布的序列,分别设计合成了AsiaI型和O型口蹄疫病毒的结构蛋白VP_2引物。PCR扩增得到了AsiaI型口蹄疫病毒VP_2片段大约693bp,O型的VP_2片段大约660bp序列分析表明是VP_2基因,将两种VP_2基因亚克隆至表达载体,实现目的基因的原核表达。
2.O型和AsiaI型口蹄疫病毒抗体间接ELISA方法的建立
以纯化的原核表达O型或AsiaI型口蹄疫病毒VP_2蛋白包被酶标板,分别建立了O型和AsiaI型口蹄疫病毒抗体间接ELISA方法。对影响ELISA反应条件的各种因素进行了优化和选择,如蛋白的包被浓度、温度及时间,各步骤反应的温度及时间,封闭液的选择及浓度确定等,使得该方法有较好的稳定性和重复性。
3.Asial型口蹄疫病毒VP_2蛋白单克隆抗体的制备
以纯化的VP_2蛋白免疫Balb/C小鼠,将脾细胞与骨髓瘤细胞SP2/0融合,用间接ELISA方法筛选,采用有限稀释法对其进行克隆,经过3次融合共获得2株能稳定分泌抗VP_2蛋白单克隆抗体的杂交瘤细胞株,命名为2F_(12),5F_5。两株单抗的腹水效价分别为2~9×100,2~(12)×100且单克隆抗体特异性良好,与载体蛋白无交叉反应。
4.Asial型口蹄疫病毒竞争ELISA方法的建立及初步应用
以VP_2蛋白为抗原,以辣根过氧化物酶标记的单抗5F_5为第二抗体,建立了快速检测FMD抗体的竞争ELISA方法,对于影响ELISA的各种因素进行了优化和选择,包括蛋白的包被浓度、温度及时间,各步反应的温度及时间,封闭液的选择及浓度确定等,使得试剂盒具有较好的稳定性和重复性,具有较好的临床应用价值。
Foot-and-mouth disease(FMD) is a highly contagious and acute disease of cattle, goats,pigs and sheep which is caused by Foot-and-mouth disease virus(FMDV).In developed country,killing all infected and exposed animal could be the first choice to control and eliminate the disease.But in developing country,vaccination is more acceptable.
In China,vaccination is the main measures to prevent and control the disease.So, evaluation of antibody level after vaccination is important.In the past,some researches indicated that structual protein VP_2 of FMDV could induce specific antibodies against FMDV.Otherwise,the four structural protein's mutation probability is VP_1＞VP_3＞VP_2＞VP_4.VP_1 is more variable and VP_2 is relatively conserved.So,VP_2 protein has many advantages as a tool to detect antibodies against FMDV.Monoclonal antibody has high sensitivity and specificity,so it is widely used in biomedicine.In this study,VP_2 gene of O and Asia-1 sreotype FMDV were cloned and expressed in E.coli respectively.monoclonal antibodies against VP_2 protein of Asia-1 type FMDV were prepared.Based on the protein expressed and monoclonal antibodies,competitive ELISA for the detection of VP_2 antibodies was established.clinical tests indicated that the ELISA method had high sensitivity and specificity.Research contents are as follows:
1.cloning and expression of VP_2 genes of o type FMDV and Asia 1 type FMDV
According to the gene sequences pulicated in Genbank,primers were designed and synthesizd.660bp VP_2 genes of O type FMDV and 693bp Asia-1 type FMDV were amplified by PCR.Sequence analysis showed the gene fragments were VP_2 genes of FMDV.Then,the VP_2 genes were subcloned into vector and expressed in E.coli.
2.establishment of indirect ELISA using VP_2 protein of o type and asial type FMDV
Indirect ELISA was established by coating VP_2 protein as detection antigen.All impact factors to reaction were optimizated,including consentration of VP_2 protein,temperature,reaction time and silling buffer,et al.,these work would contribute to the application of the ELISA kit.
3.Generation of monoclonal antibodies against AsiaI type foot-and-mouth disease virus:
Balb/c mice were immunized with the E.coli expressed fusion protein.The splenocytes from immunized mice were fused with myeloma cells SP2/0.The hybridism cells were screened by indirect ELlSA and limited dilution method.Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained.The ELISA titers of the ascites induced by the two hybridism cells were above 100×2~9. Specificity of mAbs was analyzed by Western-blot.All the monoclonal antibodies were proved to be specific.No cross-reactions with nontarget proteins were found.
4.Establishment and prime application of the competitive ELISA for the detection of asial FMDV antibody
The competitive ELISA method used to detect the antibody against FMDV was established by using VP_2 protein as antigen and the HRP-labled monoclonal antibody 5F5 as detection antibody.All the items which may influence the reaction were perfected,including temperature in each steps and final concentration of each solution.At last the best way of this method was found,which provide the foundation for the next step-use of the ELISA kit.
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57. Done,S.H., Morris,D., Paton,P., Kitching. FMD break out in UK.International pig veterinary society 17th congress. 2002, AMES, IOWA, USA
58. Dunn C.S., Donsldson A.L. Natural adaptation to pigs:Taiwanese isolate of foot-and-mouth disease virus. Vet Rec, 1997,141: 174-5
59. Esteban Domingo , Cristina Escarmys, Eric Baranowski, Carmen M. Ruiz-Jarabo, Elisa Carrillo, Juan Ignacio Nunez ,Francisco Sobrino. Evolution of foot-and-mouth disease virus, Virus Research, 2003, 91: 47-63
60. Falk M M, Grigera P R, Bergmann I E, Zibert A, Multhaup G, Beck E. Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3. J Virol, 1990, 64: 748 - 756
61. Ferris N, Reid S, Hutchings G, Kitching P, Danks C, Barker I, Preston S. Pen-side test for investigating FMD. Vet Rec, 2001, 148(26): 823-824
62. Foeter M, Cook A, Cedillo I, et al. Serological and cellular immune response to non-structural proteins in animals infected with foot-and-mouth disease virus [J]. Vet Q, 1998,20:28-30
63. Forss S, Strebel K, Beck E, Schaller H. Nucleotide sequence and genome organization of foot-and-mouth disease virus. Nucleic Acids Res, 1984, 12(16): 6587-6601
64. Francis MJ, Ouldridge EJ, Black L. Antibody response in bovine pharyngeal fluid following foot-and-mouth disease vaccination and, or, exposure to live virus. Res Vet Sci, 1983, 35 (2): 206-210
65. Grubman M J, Chinsangaram J. Foot-and-mouth disease virus: The role of the leader Proteinase in viral pathogenesis. Recent Res Dev Virol, 2000, 2: 123 - 134
66. Jackson J, Sheppard D, Denyer M, Blakemore W, King A M. The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus. J Virol, 2000, 74: 4949 -4956
67. Keeling MJ, Woolhouse ME, May RM, Davies G, Grenfell BT. Modelling vaccination strategies against foot-and-mouth disease. Nature, 2003, 421(6919): 136-142
68. Mackay D, Parida S, Paton D, Anderson J. Making a vaccinate-to-live policy a reality in foot-and-mouth disease. Dev Biol (Basel), 2004, 119: 261-6
69. Mayr G A, Chinsangaram J, Grubman M. Development of replication adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate [J]. Virology, 1999, 263: 496-506
70. Moonen P, Jacobs L, Costa H, Crienen A, Schrijver RS. Diagnosis of persisting infections of foot-and-mouth disease virus in cattle: IgA ELISA virus isolation and RT-PCR[ C ]. Report of the FAO research group of the standing technical committee of the European Commission for the control of foot-and-mouth disease, Borovets, Bulgaria, 5-8 Septenber 2000
71. Moraes MP, Mayr GA, Mason PW, Grubman MJ. Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24.Vaccine. 2002, Feb 22;20(11-12): 1631-9
72. Nikki R. Rhodin, Marloes L. J. A. Van Tilburg, Monika W. Oli, William P. McArthur, and L. Jeannine Brady*. Further Characterization of Immunomodulation by a Monoclonal Antibody against Streptococcus mutans Antigen P1. Infect Immun, 2004, January, 72(1): 13-21
73. OIE (Office International des Epizooties). Manual of Standards for Diagnostic Tests and Vaccines, Chapter 2.1.1. (2000) http://www.oie.int/
74. Pluimers FH. Foot-and-Mouth disease control using vaccination: the Dutch experience in 2001. Dev Biol (Basel). 2004, 119: 41-9
75. Robert W, Maitta, Kausik Datta, Qing Chang, Robin X. Luo,Bradley Witover, Krishanthi Subramaniam,and Liise-anne Pirofskil. Protective and Nonprotective Human Immunoglobulin M Monoclonal Antibodies to Cryptococcus neoformans Glucuronoxylomannan Manifest Different Specificities and Gene Use Profiles. Infect Immun.,2004, August, 72(8): 4810-4818
76. Tomasula PM, Konstance RP. The survival of foot-and-mouth disease virus in raw and pasteurized milk and milk products. J Dairy Sci, 2004, 87(4): 1115-21
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