禽流感病毒单克隆抗体的研制及初步应用
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摘要
禽流感(Avian Influenza，AI)是由A型流感病毒感染引起的一种禽类传染病，有重要的公共卫生意义。本研究的目的是以高致病性禽流感病毒A／Chicken／Shanghai／1／2000(H5N1)作为免疫原研制单克隆抗体(McAbs)，并将研制的McAbs初步应用于AIV检测。尿囊液用甲醛灭活，差速离心法提纯，三次免疫BALB／c小鼠，取免疫小鼠的脾细胞与骨髓瘤细胞SP2／0-Ag-14融合。用间接ELISA和血凝抑制(HI)试验筛选阳性细胞株。将获得的阳性细胞株用有限稀释法进行多次亚克隆，筛选稳定分泌McAbs细胞株。
本研究一共获得14株能够稳定分泌特异性McAbs的杂交瘤细胞株，分别命名为2B6、3A5、4B3、5A5、5C2、F2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4，其中2B6和3A5的免疫球蛋白亚类为IgM，4B3、F2、A6、1D3、4A6和6C4的免疫球蛋白亚类为IgG_(2a)，其余6株McAbs均为IgG_1。McAb A6具有HI特性，并可以与重组鸡痘病毒表达的H5亚型AIV HA蛋白反应，能与64株H5亚型AIV中的2株病毒发生HI反应。用间接ELISA试验测定McAbs与125株AIV分离株的反应性，结果显示有11株McAbs即2B6、3A5、5C2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4能与H5亚型AIV反应，反应的比例均低于56％(36／64)；3株McAbs(5A5、4B3与F2)能与H5亚型和H9亚型AIV发生反应，其中5A5与H5亚型AIV反应的比例为48％(31／64)、与H9亚型AIV反应的比例为49％(30／61)；4B3与H5亚型AIV反应的比例为89％(57／64)、与H9亚型AIV反应的比例为83％(51／61)；F2与H5亚型AIV反应的比例为89％(57／64)、与H9亚型AIV反应的比例为90％(55／61)。用McAbs(4B3和F2)进行交叉反应实验，确定了McAbs可用于双抗体夹心ELISA的最佳包被和酶标方式，以McAb 4B3为基础建立的双抗体夹心ELISA对64株H5亚型AIV、61株H9亚型AIV、36株H3亚型AIV、6株H1亚型AIV、2株H4亚型AIV、2株H6亚型AIV和2株H11亚型AIV进行检测，阳性检出率分别为98％(63／64)、90％(55／61)、97％(35／36)、
扬州大学硕士学位论文
100%(2/2)、100%(2/2)、100%(2/2)和100%(2/2);与30株新城疫病毒和l株传染性
支气管炎病毒、1株传染性法氏囊病毒和1株产蛋下降综合征病毒进行交叉反应，
结果均呈阴性。以McAb 4B3为基础建立的双抗体夹心EUSA对AIV的最小检测
限为0.4卜g/ml。
Avian influenza has raised great concerns for its economic importance to poultry industry and potential threat to the public health. In this representation, a highly pathogenic avian influenza virus, A/Chicken/Shanghai/2000(H5Nl), was chosen to immunize BALB/c mice in order to product the monoclonal antibodies (McAbs). The allantoic fluid was inactivated by 0.1% formaldehyde, purified and concentrated by differential centrifugation. The spleen cells of immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) and hemagglutinatin inhibition (HI) test were used to screen hybridoma cells and limited dilution method was performed to subclone positive clones.
Fourteen of McAbs specifically against avian influenza virus (AIV) were obtained, designated as 2B6, 3A5, 4B3, 5A5, 5C2, F2, A6, 1A2, 1A4, ID3, 4A6, 5A2, 6B5 and 6C4 respectively. Two of these 14 McAbs, 2B6 and 5A5, were of IgM subtype. Four McAbs, 4B3, F2, A6 and 6C4, were of IgG2a and the others were of IgG1 subtype. One of these McAbs, A6, had the ability of HI, and could be detected in two samples of 64 H5 subtype AIV tested. Furthermore, A6 could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell (CEF). It was verified that 11 McAbs could react with H5 subtype AIV and all the rates of reaction were lower than 56% (36/64) in indirect ELISA. Three McAbs, 5A5, 4B3 and F2, could react with H5 and H9 subtype AIV and the rates reacting with H5 subtype AIV were 48% (31/64), 89% (57/64) and 89% (57/64) respectively. And that of H9 subtype AIV were 49% (30/61), 83% (51/61) and 90% (55/61) respectively. Two McAbs, 4B3 and F2, were coated and conjugated with horseradish peroxides (HRP). The s
pecificity of the coated/HRP-conjugated McAbs was detected respectively by sandwich ELISA and direct ELISA respectively.
Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated. By using the selected 4B3 for both coating and conjugating of HRP, the allantoic fluids of 64 strains of H5 subtype AIV, 61 strains of H9 subtype AIV, 36 strains of H3 subtype AIV, 6 strains of H1 subtype AIV, 2 strains of H4 subtype AIV, 2 strains of H6 subtype AIV, 2 strains of H1 1 subtype AIV were tested, the positive testing rates were 98(63/64), 90% (55/61), 97% (35/36), 100% (2/2), 100% (2/2), 100% (2/2) and 100% (2/2) respectively. No cross-reaction was observed in the samples of NDV, IBV, IBDV and EDSV, additionally, the lowest detection concentration of AIV was 0.4g/ml.
本研究一共获得14株能够稳定分泌特异性McAbs的杂交瘤细胞株，分别命名为2B6、3A5、4B3、5A5、5C2、F2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4，其中2B6和3A5的免疫球蛋白亚类为IgM，4B3、F2、A6、1D3、4A6和6C4的免疫球蛋白亚类为IgG_(2a)，其余6株McAbs均为IgG_1。McAb A6具有HI特性，并可以与重组鸡痘病毒表达的H5亚型AIV HA蛋白反应，能与64株H5亚型AIV中的2株病毒发生HI反应。用间接ELISA试验测定McAbs与125株AIV分离株的反应性，结果显示有11株McAbs即2B6、3A5、5C2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4能与H5亚型AIV反应，反应的比例均低于56％(36／64)；3株McAbs(5A5、4B3与F2)能与H5亚型和H9亚型AIV发生反应，其中5A5与H5亚型AIV反应的比例为48％(31／64)、与H9亚型AIV反应的比例为49％(30／61)；4B3与H5亚型AIV反应的比例为89％(57／64)、与H9亚型AIV反应的比例为83％(51／61)；F2与H5亚型AIV反应的比例为89％(57／64)、与H9亚型AIV反应的比例为90％(55／61)。用McAbs(4B3和F2)进行交叉反应实验，确定了McAbs可用于双抗体夹心ELISA的最佳包被和酶标方式，以McAb 4B3为基础建立的双抗体夹心ELISA对64株H5亚型AIV、61株H9亚型AIV、36株H3亚型AIV、6株H1亚型AIV、2株H4亚型AIV、2株H6亚型AIV和2株H11亚型AIV进行检测，阳性检出率分别为98％(63／64)、90％(55／61)、97％(35／36)、
扬州大学硕士学位论文
100%(2/2)、100%(2/2)、100%(2/2)和100%(2/2);与30株新城疫病毒和l株传染性
支气管炎病毒、1株传染性法氏囊病毒和1株产蛋下降综合征病毒进行交叉反应，
结果均呈阴性。以McAb 4B3为基础建立的双抗体夹心EUSA对AIV的最小检测
限为0.4卜g/ml。
Avian influenza has raised great concerns for its economic importance to poultry industry and potential threat to the public health. In this representation, a highly pathogenic avian influenza virus, A/Chicken/Shanghai/2000(H5Nl), was chosen to immunize BALB/c mice in order to product the monoclonal antibodies (McAbs). The allantoic fluid was inactivated by 0.1% formaldehyde, purified and concentrated by differential centrifugation. The spleen cells of immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) and hemagglutinatin inhibition (HI) test were used to screen hybridoma cells and limited dilution method was performed to subclone positive clones.
Fourteen of McAbs specifically against avian influenza virus (AIV) were obtained, designated as 2B6, 3A5, 4B3, 5A5, 5C2, F2, A6, 1A2, 1A4, ID3, 4A6, 5A2, 6B5 and 6C4 respectively. Two of these 14 McAbs, 2B6 and 5A5, were of IgM subtype. Four McAbs, 4B3, F2, A6 and 6C4, were of IgG2a and the others were of IgG1 subtype. One of these McAbs, A6, had the ability of HI, and could be detected in two samples of 64 H5 subtype AIV tested. Furthermore, A6 could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell (CEF). It was verified that 11 McAbs could react with H5 subtype AIV and all the rates of reaction were lower than 56% (36/64) in indirect ELISA. Three McAbs, 5A5, 4B3 and F2, could react with H5 and H9 subtype AIV and the rates reacting with H5 subtype AIV were 48% (31/64), 89% (57/64) and 89% (57/64) respectively. And that of H9 subtype AIV were 49% (30/61), 83% (51/61) and 90% (55/61) respectively. Two McAbs, 4B3 and F2, were coated and conjugated with horseradish peroxides (HRP). The s
pecificity of the coated/HRP-conjugated McAbs was detected respectively by sandwich ELISA and direct ELISA respectively.
Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated. By using the selected 4B3 for both coating and conjugating of HRP, the allantoic fluids of 64 strains of H5 subtype AIV, 61 strains of H9 subtype AIV, 36 strains of H3 subtype AIV, 6 strains of H1 subtype AIV, 2 strains of H4 subtype AIV, 2 strains of H6 subtype AIV, 2 strains of H1 1 subtype AIV were tested, the positive testing rates were 98(63/64), 90% (55/61), 97% (35/36), 100% (2/2), 100% (2/2), 100% (2/2) and 100% (2/2) respectively. No cross-reaction was observed in the samples of NDV, IBV, IBDV and EDSV, additionally, the lowest detection concentration of AIV was 0.4g/ml.
引文
[1] 甘盂候．2002．禽流感 第二版．北京：中国农业出版社
[2] Nishidawa, F., T. Sugiyama, K. Yamamoto. 1997. Isolation of influenza type A virues from imported pet birds. Jpn J Med Sci Boil. 30:31-36
[3] Kaplan, M. M., R. G. Webster. 1997. The epidemiology of influenza. Sci. Am. 237(6):88-106
[4] Taisuke, H., K. Yoshihiro. 2001. Pandemic threat posed by avian influenza A virus. Clinical Microbiology Review. 14(1): 129-149
[5] 田国彬 冯菊艳 李艳冰等．2002．我国禽流感的流行与防制．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集．四川成都．125-128
[6] Pensaert, M., K. Ottis, J. Vandeputte. 1981. Evidence for the natural transmission of influenza A virus from wild ducks to swine and its potential importance for man. Bull. WHO. 59:75-78
[7] 于康震 崔尚金 付朝阳等．2000．禽流感与养禽业发展和人类健康．中国预防兽医学报．4：312-315
[8] Yuen, K. Y., P. K. S. Chan, M. Peiris, et al. 1998. Members of the H5N1 study group, Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancci. 351:467-471
[9] Wood, G. W., G. Parsons, D. J. Alexander. 1995. Replication of influenza A viruses of high and low pathogenicity for chickens at different sites in chickens and ducks following intranasal inoculation. Avian Pathol. 24:545-551
[10] Castrucci, M., I. Donatelli, R. G. Webster, et al. 1992. Ecology of avian influenza virus. Vet. Micro. 1:17-27
[11] Marine, W. M., J. E. McGowan, J. E. Thomas. 1976. Influenza detection: a prospective comparison of surveillance methods and analysis of isolates. Am J Epidemiol. 104:248-255
[12] Rott, R., H. D. Klenk, Y. Nagai, et al. 1995. Influenza viruses, cell enzymes and pathogenicity. Am. J. Respir. Crit. Care Med. 152:16-19
[13] Suarez, D. L. 2000. Immunology of avian influenza virus: a review. Developmental and Compatative Immunology. 24:269-283
[14] 何雷堂 薛云德 郑志学．2000．禽流感HA和HI试验中的问题及对策．疾病诊治．21(8)：4-25
[15] 郭元吉 程小雯．1997．流行性感冒病毒及其实验技术(第一版)．北京：中国三峡出版社
[16] 赵增连．1997．一种敏感的禽流感病毒快速定型双扩散法．中国兽医学报．17(3)：
[17] Van, W. K., J. W. Yewdell, L. J. Reck, et al. 1984. Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies. J Virol. 49:248-252
[18] Sarkkinen, H. K., P. E. Halonen, A. A. Sclmi. 1981. Detection of influenza A virus by radiommunoassay and enzyme-immunoassay from nasopharyngeal specimens. J Med Virol. 7:213-220
[19] Rohm, C., T. Horimoio, Y. Kawaoka, et al. 1995, Do haemagglutinin genes of highly pathogenic avian influenza viruses constitute unique phylogenetic lineages? virology. 209:664-670
[20] Senne, D. A., B. Panigrahy, Y. Kawaoka, et al. 1998. Survey of the haemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the cleavage site as a marker of pathogenicity potential. Avian Dis. 40:425-437
[21] Bosch, F. X., W. Gaten, H. D. Klenk, et al. 1981. Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses. Virology. 113:725-735
[22] Slemons, R. D., M. Brugh. 1998. Rapid antigen detection as an aid in early diagnosis and control of avian influenza. Virginia USA. 313-317
[23] 陈忠斌．1998．分子信标核酸检测技术研究进展．生物化学与生物物理研究进展．25(6)：488-492
[24] Rahman, A., J. D. Fox, D. Westmoreland. 2000. Development and evaluation of NASBA and real-time RT-PCR for diagnosis of viral respiratory tract infections. J. Clin. Virol. 18:174
[25] Collins, R. A., T. Ellis. 2002. Detection of highly pathogenic and low pathogenic avian influenza subtype H5 using NASBA. J. Virol. Methods. 102:213-225
[26] Dciman, B., P. Sillckens. 2002. Characteristics and application of nucleic acid sequence-based amplification (NASBA). Mol. Biotechnol. 20:163-179
[27] Compton. J. 1991. Nucleic acid sequence-based amplification. Nature. 350:91-92
[28] 万秀峰．1998．禽流感病毒的分离及其特性的研究．华南农业大学硕士学位论文．
[29] 殷震 刘景华．1997．动物病毒学 第二版．北京：科学出版社．
[30] Espy, M. J., T. F. Smith, M. W. Harmon, et al. 1986. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J clin Microbiol. 24:677-679
[31] 王敏文．2002．流感病毒对福乐马林敏感性的研究．微生物与免疫学进展．1(30)：28-31
[32] 焦新安 刘秀梵．1993．实验室手册
[33] 刘秀梵．1994．单克隆抗体在农业上的应用．安徽科学技术出版社
[34] 徐宜为．1997．免疫检测技术(第二版)．科学出版社．167-182
[35] Mohamed, K., S. Shawky. 1996. Comparison between antigen-capture ELISA and Conventional Methods used for titration of IBDV. Avian Dis. 40:562-566
[36] 张海艇 袁彦平．2000．抗人早期T细胞单克隆抗体杂交瘤细胞株的建立及在不同BALB／c小鼠亚系中的应用．黑龙江畜牧兽医．7：26
[37] Brodeur, B. R., E Tsang, Y. Larose. 1984. Parameters affecting ascites tumor formation in mice and monoclonal antibody production, J Immunol Meth. 71:265-272
[38] 刘玉斌．1989．动物免疫实验技术．吉林科学技术出版社．
[39] 曹澍泽．1992．兽医微生物学及免疫学技术．中国农业大学出版社．
[40] 严隽端 王秀荣等．1997．抗鸡传染性喉气管炎病毒单克隆抗体的研制及在诊断上的应用．中国畜禽传染病．4：47-52
[41] Snyder, D. B., D. P. Lana, B. R. Cho, et al. 1988. Group and strain-specific neutralization sites of infectious bursal disease virus defined with monoclonal antibodies. Avian Dis. 32:527-534
[42] 王京杭．1993．ELISA方法中几个环节的探讨．上海医学检验杂志．8(1)：48
[2] Nishidawa, F., T. Sugiyama, K. Yamamoto. 1997. Isolation of influenza type A virues from imported pet birds. Jpn J Med Sci Boil. 30:31-36
[3] Kaplan, M. M., R. G. Webster. 1997. The epidemiology of influenza. Sci. Am. 237(6):88-106
[4] Taisuke, H., K. Yoshihiro. 2001. Pandemic threat posed by avian influenza A virus. Clinical Microbiology Review. 14(1): 129-149
[5] 田国彬 冯菊艳 李艳冰等．2002．我国禽流感的流行与防制．中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集．四川成都．125-128
[6] Pensaert, M., K. Ottis, J. Vandeputte. 1981. Evidence for the natural transmission of influenza A virus from wild ducks to swine and its potential importance for man. Bull. WHO. 59:75-78
[7] 于康震 崔尚金 付朝阳等．2000．禽流感与养禽业发展和人类健康．中国预防兽医学报．4：312-315
[8] Yuen, K. Y., P. K. S. Chan, M. Peiris, et al. 1998. Members of the H5N1 study group, Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancci. 351:467-471
[9] Wood, G. W., G. Parsons, D. J. Alexander. 1995. Replication of influenza A viruses of high and low pathogenicity for chickens at different sites in chickens and ducks following intranasal inoculation. Avian Pathol. 24:545-551
[10] Castrucci, M., I. Donatelli, R. G. Webster, et al. 1992. Ecology of avian influenza virus. Vet. Micro. 1:17-27
[11] Marine, W. M., J. E. McGowan, J. E. Thomas. 1976. Influenza detection: a prospective comparison of surveillance methods and analysis of isolates. Am J Epidemiol. 104:248-255
[12] Rott, R., H. D. Klenk, Y. Nagai, et al. 1995. Influenza viruses, cell enzymes and pathogenicity. Am. J. Respir. Crit. Care Med. 152:16-19
[13] Suarez, D. L. 2000. Immunology of avian influenza virus: a review. Developmental and Compatative Immunology. 24:269-283
[14] 何雷堂 薛云德 郑志学．2000．禽流感HA和HI试验中的问题及对策．疾病诊治．21(8)：4-25
[15] 郭元吉 程小雯．1997．流行性感冒病毒及其实验技术(第一版)．北京：中国三峡出版社
[16] 赵增连．1997．一种敏感的禽流感病毒快速定型双扩散法．中国兽医学报．17(3)：
[17] Van, W. K., J. W. Yewdell, L. J. Reck, et al. 1984. Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies. J Virol. 49:248-252
[18] Sarkkinen, H. K., P. E. Halonen, A. A. Sclmi. 1981. Detection of influenza A virus by radiommunoassay and enzyme-immunoassay from nasopharyngeal specimens. J Med Virol. 7:213-220
[19] Rohm, C., T. Horimoio, Y. Kawaoka, et al. 1995, Do haemagglutinin genes of highly pathogenic avian influenza viruses constitute unique phylogenetic lineages? virology. 209:664-670
[20] Senne, D. A., B. Panigrahy, Y. Kawaoka, et al. 1998. Survey of the haemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the cleavage site as a marker of pathogenicity potential. Avian Dis. 40:425-437
[21] Bosch, F. X., W. Gaten, H. D. Klenk, et al. 1981. Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses. Virology. 113:725-735
[22] Slemons, R. D., M. Brugh. 1998. Rapid antigen detection as an aid in early diagnosis and control of avian influenza. Virginia USA. 313-317
[23] 陈忠斌．1998．分子信标核酸检测技术研究进展．生物化学与生物物理研究进展．25(6)：488-492
[24] Rahman, A., J. D. Fox, D. Westmoreland. 2000. Development and evaluation of NASBA and real-time RT-PCR for diagnosis of viral respiratory tract infections. J. Clin. Virol. 18:174
[25] Collins, R. A., T. Ellis. 2002. Detection of highly pathogenic and low pathogenic avian influenza subtype H5 using NASBA. J. Virol. Methods. 102:213-225
[26] Dciman, B., P. Sillckens. 2002. Characteristics and application of nucleic acid sequence-based amplification (NASBA). Mol. Biotechnol. 20:163-179
[27] Compton. J. 1991. Nucleic acid sequence-based amplification. Nature. 350:91-92
[28] 万秀峰．1998．禽流感病毒的分离及其特性的研究．华南农业大学硕士学位论文．
[29] 殷震 刘景华．1997．动物病毒学 第二版．北京：科学出版社．
[30] Espy, M. J., T. F. Smith, M. W. Harmon, et al. 1986. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J clin Microbiol. 24:677-679
[31] 王敏文．2002．流感病毒对福乐马林敏感性的研究．微生物与免疫学进展．1(30)：28-31
[32] 焦新安 刘秀梵．1993．实验室手册
[33] 刘秀梵．1994．单克隆抗体在农业上的应用．安徽科学技术出版社
[34] 徐宜为．1997．免疫检测技术(第二版)．科学出版社．167-182
[35] Mohamed, K., S. Shawky. 1996. Comparison between antigen-capture ELISA and Conventional Methods used for titration of IBDV. Avian Dis. 40:562-566
[36] 张海艇 袁彦平．2000．抗人早期T细胞单克隆抗体杂交瘤细胞株的建立及在不同BALB／c小鼠亚系中的应用．黑龙江畜牧兽医．7：26
[37] Brodeur, B. R., E Tsang, Y. Larose. 1984. Parameters affecting ascites tumor formation in mice and monoclonal antibody production, J Immunol Meth. 71:265-272
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