蓝舌病病毒VP7蛋白的原核表达及单克隆抗体的制备
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摘要
蓝舌病(Bluetongue disease，BT)是由蓝舌病病毒(Bluetongue virus，BTV)引起，由库蠓传播的反刍动物病毒性传染病。世界动物卫生组织(OIE)将其列为A类动物疫病。该病主要集中在各热带、亚热带和温带国家，给畜牧业造成巨大的损失。在蓝舌病病毒编码的各种蛋白质中，VP7蛋白为群特异性抗原。VP7蛋白是蓝舌病病毒主要的核心抗原，携带有群特异性抗原决定簇，能刺激被感染机体产生强的群特异性免疫反应。因此，VP7基因已成为BTV研究的热点。本研究在克隆并鉴定蓝舌病病毒VP7基因基础上，进行VP7蛋白的原核表达，并制备相应单克隆抗体，为诊断试剂研制奠定了基础。
1．蓝舌病病毒VP7蛋白的原核表达及纯化产物反应原性鉴定
根据已发表BTV VP7基因序列，设计并合成一对引物。利用RT-PCR方法扩增BTV-5型毒株编码群特异性抗原的VP7基因片段。将扩增片段克隆至pGEM-T Easy载体中，构建VP7基因重组载体。经核苷酸序列测定，克隆的基因片段长1050bp，为S7基因开放性读码框的全长序列，编码349个氨基酸。将VP7基因片段连接至pET-DsbA、pET-GST和pET-His表达载体中，经抗性筛选、PCR扩增、限制性内切酶分析，筛选获得BTV VP7基因片段正向插入、有正确读码框的阳性克隆。阳性克隆质粒在E.coli BL21(DE3)pLysS感受态细胞中经IPTG诱导表达，筛选到高表达重组载体pET-DsbA／VP7。镍琼脂糖凝胶亲和层析分离重组载体pET-DsbA／VP7表达的带His标签的重组VP7蛋白。SDS-PAGE、Western blotting和ELISA实验表明，表达蛋白为融合蛋白，分子量约为63.2kDa，具有良好的群特异反应原性。
2．蓝舌病病毒VP7蛋白的单克隆抗体制备及鉴定
本研究通过BHK-21接种，增殖蓝舌病病毒，将收获的病毒液用甲醛灭活。用纯化病毒免疫Balb／c小鼠，取免疫鼠脾细胞与SP2／0骨髓瘤细胞融合。用ELISA试验筛选阳性株，采用有限稀释法对其进行克隆，经过融合共获得2株能稳定分泌抗BTV VP7蛋白单克隆抗体的杂交瘤细胞株，分别命名为3E2和1C11。细胞培养上清ELISA效价分别为2~9和2~(10)，腹水ELISA效价可达4×10~6和16×10~6。所有单抗特异性良好，与鹿流行性出血热病毒、背景源蛋白无交叉反应。腹水中纯化的单抗3E2与不同BTV血清型的绵羊抗血清进行竞争ELISA反应，证实有良好的竞争抑制性。
Bluetongue disease (BT) is an arthropod-borne viral disease affecting ruminants,caused by Bluetongue virus (BTV). At present, bluetongue disease is listed in A infectiousdiseases by World Organization for Animal Health (OIE). The disease broke out primarilyin tropical and temperate regions of the world, and resulted in a serious economic loss.VP7 is the major core antigen of bluetongue virus, which can induce the production ofgroup-specific antibodies against the virus. And there are group-specific antigenic sitesmapped on the VP7. In this study group-specific antigen VP7 of BTV was expressed inE.coli by a recombinant vector, and highly expressed recombinant VP7 for serologicaltests of BTV as a diagnostic reagent antigen. Two McAbs against group-specific antigenVP7 of BTV were prepared. This study gives us a clue to research the diagnosis reagent.
1. Expression and antigentic characterization of the major core protein VP7 ofBluetongue virus
According to published VP7 gene sequence of BTV, primers were designed andsynthesized. The complete cDNA clone of VP7 gene in the cloning procedure wasamplified by RT-PCR. The group-specific antigen VP7 gene of BTV was cloned intopGEM-T Easy vector and sequenced. The length of VP7 gene was 1050 bp, whichencoded 349 amino acids. The recombinant vector containing BTV VP7 cDNA wasconstructed. Then the fragment of VP7 gene was subcloned into prokaryotic expressionvector pET-DsbA、pET-GST and pET-His by PCR and restriction endonucleases. Therecombinant expression vectors were transformed into E.coli BL21 (DE3) pLysS andinduced by IPTG. The higher expression vector pET-DsbA/VP7 was screened. TheHis-tagged recombinant protein was purified using a His-tag affinity chromatographycolumn on Ni~(2+)-nitrilotriacetate (NTA) resin. The results of SDS-polyarcylamide gelelectrophoresis (SDS-PAGE) and Western blotting revealed that the recombinant fusionprotein was expressed with a molecular mass of approximately 63.2 kDa. The indirectenzyme-linked immunosorbent assay (ELISA) indicated that the expressed BTV VP7 washighly immunogenic.
2. Preparation of Monoclonal Antibodies against serogroup-specific antigen VP7 ofBluetongue virus
BTV-5 was cultured in BHK-21 cell. The obtained virus was inactivated with 0.1% formaldehyde. Balb/c mice were immunized with the purified BTV antigens. Splenocytesfrom the immunized mice were fused with SP2/0 myeloma cells, and positive hybridomaclones were screened by ELISA and limited dilution method was performed to subclonethe positive clones. After three cycles of subcloning, two McAbs against the VP7 ofBTV-5 were obtained, designated as 3E2 and 1Cll. The ELISA titer of culturesupernatant were 2~9 and 2~(10), and ascites were 4×10~6 and 16×10~6, respectively. TheseMcAbs had good specificity to BTV-5. No cross-reactions were found when epizootichemorrhagic disease virus (EHDV), BHK-21 cell, and E.coli cell were tested. McAb 3E2purified from wild waterfowl had high activity of binding the recombinant fusion proteinVP7, detected by competitive ELISA with different serotypes of sheep anti-BTVantibodies.
1．蓝舌病病毒VP7蛋白的原核表达及纯化产物反应原性鉴定
根据已发表BTV VP7基因序列，设计并合成一对引物。利用RT-PCR方法扩增BTV-5型毒株编码群特异性抗原的VP7基因片段。将扩增片段克隆至pGEM-T Easy载体中，构建VP7基因重组载体。经核苷酸序列测定，克隆的基因片段长1050bp，为S7基因开放性读码框的全长序列，编码349个氨基酸。将VP7基因片段连接至pET-DsbA、pET-GST和pET-His表达载体中，经抗性筛选、PCR扩增、限制性内切酶分析，筛选获得BTV VP7基因片段正向插入、有正确读码框的阳性克隆。阳性克隆质粒在E.coli BL21(DE3)pLysS感受态细胞中经IPTG诱导表达，筛选到高表达重组载体pET-DsbA／VP7。镍琼脂糖凝胶亲和层析分离重组载体pET-DsbA／VP7表达的带His标签的重组VP7蛋白。SDS-PAGE、Western blotting和ELISA实验表明，表达蛋白为融合蛋白，分子量约为63.2kDa，具有良好的群特异反应原性。
2．蓝舌病病毒VP7蛋白的单克隆抗体制备及鉴定
本研究通过BHK-21接种，增殖蓝舌病病毒，将收获的病毒液用甲醛灭活。用纯化病毒免疫Balb／c小鼠，取免疫鼠脾细胞与SP2／0骨髓瘤细胞融合。用ELISA试验筛选阳性株，采用有限稀释法对其进行克隆，经过融合共获得2株能稳定分泌抗BTV VP7蛋白单克隆抗体的杂交瘤细胞株，分别命名为3E2和1C11。细胞培养上清ELISA效价分别为2~9和2~(10)，腹水ELISA效价可达4×10~6和16×10~6。所有单抗特异性良好，与鹿流行性出血热病毒、背景源蛋白无交叉反应。腹水中纯化的单抗3E2与不同BTV血清型的绵羊抗血清进行竞争ELISA反应，证实有良好的竞争抑制性。
Bluetongue disease (BT) is an arthropod-borne viral disease affecting ruminants,caused by Bluetongue virus (BTV). At present, bluetongue disease is listed in A infectiousdiseases by World Organization for Animal Health (OIE). The disease broke out primarilyin tropical and temperate regions of the world, and resulted in a serious economic loss.VP7 is the major core antigen of bluetongue virus, which can induce the production ofgroup-specific antibodies against the virus. And there are group-specific antigenic sitesmapped on the VP7. In this study group-specific antigen VP7 of BTV was expressed inE.coli by a recombinant vector, and highly expressed recombinant VP7 for serologicaltests of BTV as a diagnostic reagent antigen. Two McAbs against group-specific antigenVP7 of BTV were prepared. This study gives us a clue to research the diagnosis reagent.
1. Expression and antigentic characterization of the major core protein VP7 ofBluetongue virus
According to published VP7 gene sequence of BTV, primers were designed andsynthesized. The complete cDNA clone of VP7 gene in the cloning procedure wasamplified by RT-PCR. The group-specific antigen VP7 gene of BTV was cloned intopGEM-T Easy vector and sequenced. The length of VP7 gene was 1050 bp, whichencoded 349 amino acids. The recombinant vector containing BTV VP7 cDNA wasconstructed. Then the fragment of VP7 gene was subcloned into prokaryotic expressionvector pET-DsbA、pET-GST and pET-His by PCR and restriction endonucleases. Therecombinant expression vectors were transformed into E.coli BL21 (DE3) pLysS andinduced by IPTG. The higher expression vector pET-DsbA/VP7 was screened. TheHis-tagged recombinant protein was purified using a His-tag affinity chromatographycolumn on Ni~(2+)-nitrilotriacetate (NTA) resin. The results of SDS-polyarcylamide gelelectrophoresis (SDS-PAGE) and Western blotting revealed that the recombinant fusionprotein was expressed with a molecular mass of approximately 63.2 kDa. The indirectenzyme-linked immunosorbent assay (ELISA) indicated that the expressed BTV VP7 washighly immunogenic.
2. Preparation of Monoclonal Antibodies against serogroup-specific antigen VP7 ofBluetongue virus
BTV-5 was cultured in BHK-21 cell. The obtained virus was inactivated with 0.1% formaldehyde. Balb/c mice were immunized with the purified BTV antigens. Splenocytesfrom the immunized mice were fused with SP2/0 myeloma cells, and positive hybridomaclones were screened by ELISA and limited dilution method was performed to subclonethe positive clones. After three cycles of subcloning, two McAbs against the VP7 ofBTV-5 were obtained, designated as 3E2 and 1Cll. The ELISA titer of culturesupernatant were 2~9 and 2~(10), and ascites were 4×10~6 and 16×10~6, respectively. TheseMcAbs had good specificity to BTV-5. No cross-reactions were found when epizootichemorrhagic disease virus (EHDV), BHK-21 cell, and E.coli cell were tested. McAb 3E2purified from wild waterfowl had high activity of binding the recombinant fusion proteinVP7, detected by competitive ELISA with different serotypes of sheep anti-BTVantibodies.
引文
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71. Murray PK, Eaton BT. Vaccines for bluetongue. Australian Veterinary Journal, 1996, 73 (6) : 207-210
72. Osburn B I, McGowan B, Heron B, Loomis E, Bushnell R, Stott J, Utterback W. Epizootiologic study of bluetongue: virologic and serologic results. Am J Vet Res, 1981,42(5): 884-887
73. Parker J, Herniman K A J, Gibbs E P J, Sellers R F. An experimental inactivated vaccine against bluetongue. Veterinary Record, 1975, 96 (13): 284-287
74. Parsonson I M. Pathology and pathogenesis of bluetongue infections. Curr Top Microbiol Immunol, 1990,162: 119-41
75. Pearson J E, Carbrey E A, Gustafson G A. Bluetongue and orbivirus diagnosis in the United States. Prog Clin Biol Res, 1985, 178 : 469-475
76. Pearson J E. Diagnosis bluetongue and epizootic hemorrhagic disease. In: walton TE, Osburn B 1, Bluetogue, African Horse Sickness and Related Orbiviruses. 1991. 533-546
77. Pearson J. E. .Protocol for the immunodiffusion test for bluetongue. Proc Amer Assoc Vet lab Diagn, 1979,22:463-471
78. Purdy M, Petre J, Roy P. Cloning of the bluetongue virus L3 gene. J Virol, 1984, 51 (3) : 754-759
79. Ritter D G, Roy P. Genetic relationships of bluetongue virus serotypes isolated from different parts of the world. Virus Research, 1988, 11(1): 33-47
80. Roy P, Marshall J J A, French T J. Structure of the bluetongue virus genome and its encoded proteins. Current Topics in Microbiology and Immunology, 1990, 162 :43-87
81. Roy P, Ritter G D, Akashi H, Collisson E, Inaba Y. A genetic probe for identifying bluetongue virus infections in vivo and in vitro. J Gen Virol, 1985, 66 (7): 1613-1619
82. Roy P. Bluetongue virus genetics and genome structure. Virus Research, 1989, 13 (3): 179-206
83. Sawyer M M, et al.The use of the competitive ELISA to detect neutralizing antibodies resuling from superinfection with ah eterotypic bluetongue virus serotype. In: walton T E, Osburn B I, Bluetongue, African Horse Sickness and Related Orbiviruses. 1991. 617-625
84. Spreull. Malarial catarrhal fever (bluetongue) of sheep in South Africa. Journal of Comp Path Therap, 1905, 18:321-37
85. Squire KRE, Chuang R Y, Chuang L F, Doi R H, Osbum B I. Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe. Journal of Virological Methods 1985, 10 (1): 59-68
86. St George T D, St George T D, Peng Kegao . A short history of the discovery of bluetongue in the world. Bluetongue Disease in Southeast Asia and the Pacific: Proceedings of the First Southeast Asia and Pacific Regional Bluetongue Symposium, 1996, Kunming, China
87. Stott J L, Osburn B I, and Alexander L. Ornithodoros coriaceus (pajaroello tick) as a vector of bluetongue virus. American Journal of Veterinary Research, 1985, 46 (5): 1197-1199
88. Stott J L, Osburn B I, Barber T L. The current status of research on an experimental inactivated bluetongue virus vaccine. Proceedings of the United States Animal Health Association, 1979, USA
89. Verwoerd D, Huismans H. Studies on the in vitro and the in vivo transcription of the bluetongue virus genome. Onderstepoort Journal of Veterinary Research, 1972, 39 (4) : 185-191
90. Wade-Evans A M, Mertens P P, Bostock C J. Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples. J Virol Methods, 1990, 30 (1) : 15-24
91. Walton T E, Walton T E, and Osburn B I. Attenuated and inactivated orbiviral vaccines. Bluetongue, African Horse Sickness, and Related Orbiviruses: Proceedings of the Second International Symposium. 1992, USA
92. Wang L F, Hyatt A D, Whiteley P L, Andrew M, Li J K, Eaton B T. Topography and immunogenicity of bluetongue virus VP7 epitopes. Arch Virol, 1996, 141 (1): 111-123
93. Wang L, Kemp M C, Roy P, Collisson E W. Tissue tropism and target cells of bluetongue virus in the chicken embryo. J Virol, 1988, 62 (3): 887-893
94. Wechsler S J, Austin K J, Wilson W C. Limits of detection of bluetongue virus with different assay systems. J Vet Diagn Invest, 1990, 2 (2): 103-106
95. Wilson W C, Chase C C. Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis. J Virol Methods, 1993, 45 (1): 39-47
96. Wilson W C, Ma H C, Venter E H, van Djik A A, Seal B S, Mecham J O. Phylogenetic relationships of bluetongue viruses based on gene S7. Virus Res, 2000, 67 (2): 141-151
97. Xu G, Wilson W, Mecham J, Murphy K, Zhou E M, Tabachnick W. VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis. Journal of General Virology, 1997, 78,(7): 1617-1623
98. Yu Y, Fukusho A, Ritter DG, Roy P. Complete nucleotide sequence of the group-reactive antigen VP7 of Bluetongue virus. Nucleic Acids Research, 1988,16 (4): 16-20
99. Zhang Nianzu, Li Zihua, Peng Kegao. Bluetongue history ,serology and virus isolation in China, ACIAR proceedings, 1995, 66:43-50
2．冯静兰．中国蓝舌病预防措施研究的进展．第一届东南亚太平洋地区蓝舌病学术讨论会论文汇编，1995，云南昆明
3．花群义，周晓黎，徐自忠，董俊．蓝舌病病毒单克隆抗体的制备及生物学特性鉴定．中国兽医科技，2000，30(9)：8-10
4．花群义，徐自忠，周晓黎，董俊．竞争酶联免疫吸附试验检测蓝舌病抗体的研究．动物医学进展，2002，23(2)：66-69
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6．李志华，张开礼，皱福中．绵羊蓝舌病(湖北毒株)的分离鉴定报告．云南畜牧兽医，1989，4：49-51
7．李朝君，刘际彬，茹亚光．我国北方五省(区)绵羊四种主要传染病的血清学调查．中国畜禽传染病，1998，20(1)：39-40
8．林理惠，黄巧玲，康长风．四川省蓝舌病调查和病原分离报告．云南畜牧兽医，1989，4：57-60
9．刘景利，孙建宏，管雪婷，曾祥伟．蓝舌病的研究进展．畜牧兽医科技信息，2005，9：19-21
10．卢炳魁，王桂生，李志成．绵羊蓝舌病传播途径和防制措施．内蒙古畜牧科学，1995，4：46-47
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13．王健伟，姜慧英，洪涛，李晓成，李云岗．抗蓝舌病毒VP7单克隆抗体的制备及其特性．畜牧兽医学报，2001，32(5)：458-462
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15．阎高峰．蓝舌病研究进展．甘肃畜牧兽医，2000，30(4)：32-34
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71. Murray PK, Eaton BT. Vaccines for bluetongue. Australian Veterinary Journal, 1996, 73 (6) : 207-210
72. Osburn B I, McGowan B, Heron B, Loomis E, Bushnell R, Stott J, Utterback W. Epizootiologic study of bluetongue: virologic and serologic results. Am J Vet Res, 1981,42(5): 884-887
73. Parker J, Herniman K A J, Gibbs E P J, Sellers R F. An experimental inactivated vaccine against bluetongue. Veterinary Record, 1975, 96 (13): 284-287
74. Parsonson I M. Pathology and pathogenesis of bluetongue infections. Curr Top Microbiol Immunol, 1990,162: 119-41
75. Pearson J E, Carbrey E A, Gustafson G A. Bluetongue and orbivirus diagnosis in the United States. Prog Clin Biol Res, 1985, 178 : 469-475
76. Pearson J E. Diagnosis bluetongue and epizootic hemorrhagic disease. In: walton TE, Osburn B 1, Bluetogue, African Horse Sickness and Related Orbiviruses. 1991. 533-546
77. Pearson J. E. .Protocol for the immunodiffusion test for bluetongue. Proc Amer Assoc Vet lab Diagn, 1979,22:463-471
78. Purdy M, Petre J, Roy P. Cloning of the bluetongue virus L3 gene. J Virol, 1984, 51 (3) : 754-759
79. Ritter D G, Roy P. Genetic relationships of bluetongue virus serotypes isolated from different parts of the world. Virus Research, 1988, 11(1): 33-47
80. Roy P, Marshall J J A, French T J. Structure of the bluetongue virus genome and its encoded proteins. Current Topics in Microbiology and Immunology, 1990, 162 :43-87
81. Roy P, Ritter G D, Akashi H, Collisson E, Inaba Y. A genetic probe for identifying bluetongue virus infections in vivo and in vitro. J Gen Virol, 1985, 66 (7): 1613-1619
82. Roy P. Bluetongue virus genetics and genome structure. Virus Research, 1989, 13 (3): 179-206
83. Sawyer M M, et al.The use of the competitive ELISA to detect neutralizing antibodies resuling from superinfection with ah eterotypic bluetongue virus serotype. In: walton T E, Osburn B I, Bluetongue, African Horse Sickness and Related Orbiviruses. 1991. 617-625
84. Spreull. Malarial catarrhal fever (bluetongue) of sheep in South Africa. Journal of Comp Path Therap, 1905, 18:321-37
85. Squire KRE, Chuang R Y, Chuang L F, Doi R H, Osbum B I. Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe. Journal of Virological Methods 1985, 10 (1): 59-68
86. St George T D, St George T D, Peng Kegao . A short history of the discovery of bluetongue in the world. Bluetongue Disease in Southeast Asia and the Pacific: Proceedings of the First Southeast Asia and Pacific Regional Bluetongue Symposium, 1996, Kunming, China
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88. Stott J L, Osburn B I, Barber T L. The current status of research on an experimental inactivated bluetongue virus vaccine. Proceedings of the United States Animal Health Association, 1979, USA
89. Verwoerd D, Huismans H. Studies on the in vitro and the in vivo transcription of the bluetongue virus genome. Onderstepoort Journal of Veterinary Research, 1972, 39 (4) : 185-191
90. Wade-Evans A M, Mertens P P, Bostock C J. Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples. J Virol Methods, 1990, 30 (1) : 15-24
91. Walton T E, Walton T E, and Osburn B I. Attenuated and inactivated orbiviral vaccines. Bluetongue, African Horse Sickness, and Related Orbiviruses: Proceedings of the Second International Symposium. 1992, USA
92. Wang L F, Hyatt A D, Whiteley P L, Andrew M, Li J K, Eaton B T. Topography and immunogenicity of bluetongue virus VP7 epitopes. Arch Virol, 1996, 141 (1): 111-123
93. Wang L, Kemp M C, Roy P, Collisson E W. Tissue tropism and target cells of bluetongue virus in the chicken embryo. J Virol, 1988, 62 (3): 887-893
94. Wechsler S J, Austin K J, Wilson W C. Limits of detection of bluetongue virus with different assay systems. J Vet Diagn Invest, 1990, 2 (2): 103-106
95. Wilson W C, Chase C C. Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis. J Virol Methods, 1993, 45 (1): 39-47
96. Wilson W C, Ma H C, Venter E H, van Djik A A, Seal B S, Mecham J O. Phylogenetic relationships of bluetongue viruses based on gene S7. Virus Res, 2000, 67 (2): 141-151
97. Xu G, Wilson W, Mecham J, Murphy K, Zhou E M, Tabachnick W. VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis. Journal of General Virology, 1997, 78,(7): 1617-1623
98. Yu Y, Fukusho A, Ritter DG, Roy P. Complete nucleotide sequence of the group-reactive antigen VP7 of Bluetongue virus. Nucleic Acids Research, 1988,16 (4): 16-20
99. Zhang Nianzu, Li Zihua, Peng Kegao. Bluetongue history ,serology and virus isolation in China, ACIAR proceedings, 1995, 66:43-50