猪传染性胃肠炎检测试剂的研制及快速诊断方法的研究
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摘要
本实验用IPTG诱导含有猪传染性胃肠炎病毒(TGEV)重组N基因质粒(PHN)的大肠杆菌DH5α，表达重组TGEV N蛋白，经Ni~(2+)-NTA金属螯合层析柱纯化后获得纯化的N蛋白。对纯化条件进行优化，得到最佳纯化重组TGEV N蛋白的方法：将大肠杆菌DH5α菌体沉淀物经裂解液(含有1mmol／L PMSF)作用后，依次经过2倍体积的Buffer A(pH8.0)、Buffer B(pH6.3)和Buffer C(pH5.9)洗柱后，收集Buffer C(80mmol／L咪唑)洗脱产物。经SDS-PAGE分析分子量为47ku，Western-Blot检测呈阳性，纯化的N蛋白溶液经Bradford法测定蛋白质含量为154.7μg／ml-479.7μg／ml，推算每克大肠杆菌菌体沉淀物可获得纯化重组TGEV N蛋白1.52mg。
以重组TGEV N蛋白作为诊断抗原建立了两种检测抗体的诊断方法。在Dot-ELISA中其抗原最适包被量为1.25μg／点，抗原预点膜4℃可保存5周以上，特异性实验表明其与猪轮状病毒、猪流行性腹泻病毒等肠道腹泻性病毒均无交叉反应。在间接ELISA中，最佳抗原包被量为0.51μg／孔，封闭液为含5％脱脂奶粉的PBST，待检血清最适稀释倍数为200倍，作用时间为30min，底物液最佳作用时间为10min。
以纯化的重组TGEV N蛋白免疫BALB／c小鼠制备诊断用单克隆抗体。按常规方法进行细胞融合，用间接ELISA方法进行筛选，采用有限稀释法，经过3次克隆筛选，最终获得一株分泌抗重组TGEV N蛋白单克隆抗体的杂交瘤细胞(命名为E10F10D5)，其染色体平均计数为87±10对，间接ELISA检测制备的腹水抗体效价达1:12800。以全病毒为抗原对杂交瘤细胞(E10F10D5)产生的腹水进行Western-Blot及间接ELISA检测分析，结果均为阳性。证明制备的单克隆抗体可以识别天然TGEV N蛋白的某一抗原表位。优化了裂解肠内容物中TGEV的方法。为建立诊断TGEV抗原的检测方法奠定了物质基础。
本研究首次在国内建立以重组TGEV N蛋白为抗原检测TGEV抗体的两种检测方法：DOT-ELISA和间接ELISA；制备了一株抗TGEV N蛋白的杂交瘤细胞系。为TGEV的疫情监测、及时而准确的诊断奠定物质基础。
E.coli DH5a harbouring recombinant plasmid pProExHTb-N was induced with IPTG to express the recombinant nucleoprotein (rNP) of transmissible gastroenteritis virus (TGEV).The rNP collected after cell extraction was applied onto the nickel nitrilo-tri-acetic acid (Ni-NTA) resin. The optimized procedure of purification was lysing the E.coli by Buffer A (containing Immol/LPMSF) .washing the resin with 2+Buffer A (pH8.0),2+BufferB (pH6.3), 2X BufferC (pH5.9) and collecting the elution of Buffer C (80mmol/L imidazole) .The molecular weight of purified rNP was 47ku analyzed by SDS-PAGE.The result of Western-Blot showed that the rNP can act to sera of anti-TGEV. The concentration of the elution including rNP was identified 154.Vug per ml-4V9.Vug per ml by Bradford's assay, at the same ratio 1.52mg rNP can be abtained per gram E.coli sediment
Dot-ELISA and indirect-ELISA were established by the rNP. The optimal amount of the rNP coated in Dot-ELISA was 1.25g per dot. The pre-dot membrane can be kept 5 weeks long at 4 C. The specific assay showed that rNP have no cross-reaction with porcine epidemic diarrhea coronavirus (PEDV) porcine rotavirus (PRV) .In indirect-ELISA the optimal amount of coated antigen was 0.5lg per well,the optimal blocking solution was 5% dry milk,the ideal dilution of serum was 200 and the fit reaction time was 30mins the adequatable reacrion time of substrate was 10min.
The monoclonal antibody (McAb) detecting TGEV was performed by immunizing the BALB/c mice with purified rNP. Cells fusion was carried out by standard method and hybridmas with rNP-antibody-positive supernatants were subcloned third by the limiting dilution. One hybridoma cell line secreting monoclonal antibody specific against rPN was picked out by indirect-ELISA designated E10F10D5. The even number of chromosomes per hybridoma was 8V +10 paires .The antibody liter of ascites v/as 1:12800 determined by indirect-ELISA. TGEV was applicated as antigen in Western-Blot and indirect-ELISA to analyze the McAb. The result showed that the McAb could indetified certain antigenic epitope of TGEV N protein. Method to dispose TGEV in intestinal sample was optimized.
Two methods to detect antibodies of TGEV were established firstly by using rNp as antigen coated in Dot-ELISA and indirect-ELISA in domestic. One hybridoma cell line secreting monoclonal antibody recognizing TGEV N protein was produced. All the work founded material base for TGE diagnosis accurately and rapidly.
Postgraduate: QianJiang Major: Preventive Veterinary Science
Supervisor: Professor Li YiJing
以重组TGEV N蛋白作为诊断抗原建立了两种检测抗体的诊断方法。在Dot-ELISA中其抗原最适包被量为1.25μg／点，抗原预点膜4℃可保存5周以上，特异性实验表明其与猪轮状病毒、猪流行性腹泻病毒等肠道腹泻性病毒均无交叉反应。在间接ELISA中，最佳抗原包被量为0.51μg／孔，封闭液为含5％脱脂奶粉的PBST，待检血清最适稀释倍数为200倍，作用时间为30min，底物液最佳作用时间为10min。
以纯化的重组TGEV N蛋白免疫BALB／c小鼠制备诊断用单克隆抗体。按常规方法进行细胞融合，用间接ELISA方法进行筛选，采用有限稀释法，经过3次克隆筛选，最终获得一株分泌抗重组TGEV N蛋白单克隆抗体的杂交瘤细胞(命名为E10F10D5)，其染色体平均计数为87±10对，间接ELISA检测制备的腹水抗体效价达1:12800。以全病毒为抗原对杂交瘤细胞(E10F10D5)产生的腹水进行Western-Blot及间接ELISA检测分析，结果均为阳性。证明制备的单克隆抗体可以识别天然TGEV N蛋白的某一抗原表位。优化了裂解肠内容物中TGEV的方法。为建立诊断TGEV抗原的检测方法奠定了物质基础。
本研究首次在国内建立以重组TGEV N蛋白为抗原检测TGEV抗体的两种检测方法：DOT-ELISA和间接ELISA；制备了一株抗TGEV N蛋白的杂交瘤细胞系。为TGEV的疫情监测、及时而准确的诊断奠定物质基础。
E.coli DH5a harbouring recombinant plasmid pProExHTb-N was induced with IPTG to express the recombinant nucleoprotein (rNP) of transmissible gastroenteritis virus (TGEV).The rNP collected after cell extraction was applied onto the nickel nitrilo-tri-acetic acid (Ni-NTA) resin. The optimized procedure of purification was lysing the E.coli by Buffer A (containing Immol/LPMSF) .washing the resin with 2+Buffer A (pH8.0),2+BufferB (pH6.3), 2X BufferC (pH5.9) and collecting the elution of Buffer C (80mmol/L imidazole) .The molecular weight of purified rNP was 47ku analyzed by SDS-PAGE.The result of Western-Blot showed that the rNP can act to sera of anti-TGEV. The concentration of the elution including rNP was identified 154.Vug per ml-4V9.Vug per ml by Bradford's assay, at the same ratio 1.52mg rNP can be abtained per gram E.coli sediment
Dot-ELISA and indirect-ELISA were established by the rNP. The optimal amount of the rNP coated in Dot-ELISA was 1.25g per dot. The pre-dot membrane can be kept 5 weeks long at 4 C. The specific assay showed that rNP have no cross-reaction with porcine epidemic diarrhea coronavirus (PEDV) porcine rotavirus (PRV) .In indirect-ELISA the optimal amount of coated antigen was 0.5lg per well,the optimal blocking solution was 5% dry milk,the ideal dilution of serum was 200 and the fit reaction time was 30mins the adequatable reacrion time of substrate was 10min.
The monoclonal antibody (McAb) detecting TGEV was performed by immunizing the BALB/c mice with purified rNP. Cells fusion was carried out by standard method and hybridmas with rNP-antibody-positive supernatants were subcloned third by the limiting dilution. One hybridoma cell line secreting monoclonal antibody specific against rPN was picked out by indirect-ELISA designated E10F10D5. The even number of chromosomes per hybridoma was 8V +10 paires .The antibody liter of ascites v/as 1:12800 determined by indirect-ELISA. TGEV was applicated as antigen in Western-Blot and indirect-ELISA to analyze the McAb. The result showed that the McAb could indetified certain antigenic epitope of TGEV N protein. Method to dispose TGEV in intestinal sample was optimized.
Two methods to detect antibodies of TGEV were established firstly by using rNp as antigen coated in Dot-ELISA and indirect-ELISA in domestic. One hybridoma cell line secreting monoclonal antibody recognizing TGEV N protein was produced. All the work founded material base for TGE diagnosis accurately and rapidly.
Postgraduate: QianJiang Major: Preventive Veterinary Science
Supervisor: Professor Li YiJing
引文
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83 Riffault S, Baric R, Levy L. In vivo induction of inferonalpha in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of inferonalpha producing cells. [J]. J Gen Virol.1997,78 (10): 2483-2487
84 Risco C., Antón I M., Enjuanes L. et al. The transmissible gastroenteritis coronavirus
contains a spherical core shell consisting of M and N proteinm. [J] J Virol.1996,70: 4773-4777
85 Rukhadze GG, Aliper TI,Sergeer VA.Isolation of peplomer glycoprotein E2 of transmissible gastroenteritis virus and application in enzyme-linked immunosoebent assay.[J] J Clin Microbiol.1989,27 (8): 1754-1758
86 Saif J and Wesley R D. pp: 295-325. In. Diseases of Swine,8th ed. (Straw B E and Allarire, S.D., Mengeling W L and Taylor, D J eds) [M]. Lowa state university Press, Ames, Lowa USA. 1999
87 Saif J and Wesley R D. Transmissible gastroenteritis, pp: 362-386. In: Diseases of Swine (Leman A D, Straw B E, Mengeling W L, et al.) [M] 1992.Lowa state university Press, Ames, IA
88 Saif L J., Theill K W. Viral Diarrheas of Man and Animals. Boca Raton, Fla: CRC Press. 1990
89 Schultze B, Krempl C., et al. Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) Binding activity. [J] J Virol.1996,70:5634-5637
90 Shoup D.I, Swayne D.E., Jackword D.J. Saif L.J.Immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues.[J]Vet. Diagn Invest. 1996,8: 161-176
91 Siddle S G, Anderson R, Cavangnagh D, et al. Coronaviridae. [J]Intervirology. 1983,20: 181-189
92 Simikins R A., Weilnau P A., Bias J. et al. Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus stains detected with monoclonal antibodies to the S protein of TGEV. [J] Am J Vet Res. 1992,53: 1253-1258
93 Sirinarumitr T, Paul PS, Halbur PG. et al. An overview of immunological and genetic methods for coronaviruses, transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues. [J] Adv Exp Med Biol.1997,412:37-46
94 Sirinarumitr T, Siddell S, Grahare F, et al. Porcine transmissible gastroenteritis virus induced apoptosis in swine testes cell culture [J]. Archives of Virology. 1998,143 (12): 2471～2485
95 Sirinarumitr T., Paul P.S., Kluge J.P. & Halbur P.G.In sim hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues. [J]J Virol Methods. 1996,56:149-160
96 Span W., Cavanagh D and Horzinek M C. Coronavirus: Structure and genomic expression. [J] J Gen Virol.1988, 69:2939-2952
97 Stepanek J., Mensik J., Franz J. et al. Epizootiology, diagnosis and prevention of viral diarrhea in piglets ander intensive husbandry conditions. Proc 21st World Vet Congr.
Moscow. 1979,6:43-44
98 Van Nieuwstadt A.P., Cornelissen J.B., Zetstra T. Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection. [J] Am J Vet Res. 1988,49:1836-1843
99 Van Nieuwstadt A.P., Cornelissen J.B.Vreeswijk J.Solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs. [J]Res. Vet. Sci.1988,44: 286-294
100 Wainright P. O. et al. Characterization of infectious bronchitis virus using monoclonal antibodies. [J] Avian Dis. 1989,33:482-490
101 Wesley R D, Woods R D., Cheung A K. Genetic basis for pathogenesis of transmissible gastroenteritis virus. [J] J Virol.1990,64:4761-4766
102 Wesley R, Woods R, Cheuny A. Nucleotide sequence of the E2-peplomer protein gene and partial nucleotide sequence of upstream polymerase gene of transmissible gastroenteritis virus (Miller strain). [J]Adv Exp Med Biol. 1990,276:301-306
103 Witte K.H. Micro-colour test for assay of transmissible gastroenteritis virus neutralizing antibodies. [J] Arch. Gesamte Virusforsch. 1971.33: 171-176
104 Woods R D., Cheville N F., Gallagher J E. Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses. [J] Am J Vet Res. 1981,42: 1163-1169
105 Woods R D., Wesley R D. Seroconversion of pigs in contact with dogs exposed to canine coronavirus. [J] Can J Vet Res. 1992,56:78-80
106 Woods R.D.Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates. [J]Can J Vet Res. 1997,61: 167-172
107 Yaling Z., Ederveen J., Egberink H., et al. Porcine epidemic diarrhea virus (CV777) and feline infectious peritonitis virus (FIPV) are antigenically related. [J] Arch Virol 1988,102: 63-71
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86 Saif J and Wesley R D. pp: 295-325. In. Diseases of Swine,8th ed. (Straw B E and Allarire, S.D., Mengeling W L and Taylor, D J eds) [M]. Lowa state university Press, Ames, Lowa USA. 1999
87 Saif J and Wesley R D. Transmissible gastroenteritis, pp: 362-386. In: Diseases of Swine (Leman A D, Straw B E, Mengeling W L, et al.) [M] 1992.Lowa state university Press, Ames, IA
88 Saif L J., Theill K W. Viral Diarrheas of Man and Animals. Boca Raton, Fla: CRC Press. 1990
89 Schultze B, Krempl C., et al. Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) Binding activity. [J] J Virol.1996,70:5634-5637
90 Shoup D.I, Swayne D.E., Jackword D.J. Saif L.J.Immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues.[J]Vet. Diagn Invest. 1996,8: 161-176
91 Siddle S G, Anderson R, Cavangnagh D, et al. Coronaviridae. [J]Intervirology. 1983,20: 181-189
92 Simikins R A., Weilnau P A., Bias J. et al. Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus stains detected with monoclonal antibodies to the S protein of TGEV. [J] Am J Vet Res. 1992,53: 1253-1258
93 Sirinarumitr T, Paul PS, Halbur PG. et al. An overview of immunological and genetic methods for coronaviruses, transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues. [J] Adv Exp Med Biol.1997,412:37-46
94 Sirinarumitr T, Siddell S, Grahare F, et al. Porcine transmissible gastroenteritis virus induced apoptosis in swine testes cell culture [J]. Archives of Virology. 1998,143 (12): 2471～2485
95 Sirinarumitr T., Paul P.S., Kluge J.P. & Halbur P.G.In sim hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues. [J]J Virol Methods. 1996,56:149-160
96 Span W., Cavanagh D and Horzinek M C. Coronavirus: Structure and genomic expression. [J] J Gen Virol.1988, 69:2939-2952
97 Stepanek J., Mensik J., Franz J. et al. Epizootiology, diagnosis and prevention of viral diarrhea in piglets ander intensive husbandry conditions. Proc 21st World Vet Congr.
Moscow. 1979,6:43-44
98 Van Nieuwstadt A.P., Cornelissen J.B., Zetstra T. Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection. [J] Am J Vet Res. 1988,49:1836-1843
99 Van Nieuwstadt A.P., Cornelissen J.B.Vreeswijk J.Solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs. [J]Res. Vet. Sci.1988,44: 286-294
100 Wainright P. O. et al. Characterization of infectious bronchitis virus using monoclonal antibodies. [J] Avian Dis. 1989,33:482-490
101 Wesley R D, Woods R D., Cheung A K. Genetic basis for pathogenesis of transmissible gastroenteritis virus. [J] J Virol.1990,64:4761-4766
102 Wesley R, Woods R, Cheuny A. Nucleotide sequence of the E2-peplomer protein gene and partial nucleotide sequence of upstream polymerase gene of transmissible gastroenteritis virus (Miller strain). [J]Adv Exp Med Biol. 1990,276:301-306
103 Witte K.H. Micro-colour test for assay of transmissible gastroenteritis virus neutralizing antibodies. [J] Arch. Gesamte Virusforsch. 1971.33: 171-176
104 Woods R D., Cheville N F., Gallagher J E. Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses. [J] Am J Vet Res. 1981,42: 1163-1169
105 Woods R D., Wesley R D. Seroconversion of pigs in contact with dogs exposed to canine coronavirus. [J] Can J Vet Res. 1992,56:78-80
106 Woods R.D.Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates. [J]Can J Vet Res. 1997,61: 167-172
107 Yaling Z., Ederveen J., Egberink H., et al. Porcine epidemic diarrhea virus (CV777) and feline infectious peritonitis virus (FIPV) are antigenically related. [J] Arch Virol 1988,102: 63-71