禽腺病毒Hexon蛋白多克隆抗体制备及特异性鉴定
|
摘要
本研究旨在制备禽腺病毒(FAd V)Hexon蛋白多克隆抗体并对其特异性进行分析鉴定。通过构建重组的原核表达载体p ET-28a-Hexon,转入表达菌E. coli BL21 (DE3),IPTG诱导,并运用SDS-PAGE进行重组蛋白的鉴定,最后纯化蛋白。利用纯化的融合蛋白进行常规新西兰大白兔免疫,以制备Hexon蛋白的多克隆抗体。采用间接免疫荧光、免疫组织化学和Western blot的方法鉴定多克隆抗体的特异性。结果表明,Hexon蛋白的原核表达量较高,并且是以包涵体的形式存在,分子质量大小约为43 k Da;免疫兔后获得能与FAd V发生特异性反应且高效价的多克隆抗体。因此,本试验制备的FAd V Hexon蛋白的多克隆抗体证明特异性良好,为后期禽腺病毒病的诊断、致病机制研究及亚单位疫苗的研制提供了良好的物质基础。
The purpose of this study is to prepare polyclonal antibodies against fowl adenovirus(FAd V) Hexon protein and to identify its specificity. The recombinant prokaryotic expression plasmid p ET-28 a-Hexon was constructed and transformed into E.coli BL21(DE3), IPTG induction, the recombinant protein was identified by SDS-PAGE, and the protein was purified. The polyclonal antibodies of Hexon protein was prepared by routine immunity in New Zealand white rabbits with purified fusion protein. The specificity of polyclonal antibodies was identified by indirect immunofluorescence, immunohistochemistry and Western blot. The results showed the prokaryotic expression of Hexon protein was high and existed in the form of inclusion body, and its molecular weight was about 43 k Da;Polyclonal antibodies having specific reaction with FAd V and high potency could be obtained in the immunized rabbits. The above results indicated the polyclonal antibody of FAd V Hexon protein prepared in this study proved to be of good specificity and provided a good material basis for the diagnosis, pathogenesis study and subunit vaccine development of avian adenovirus diseases.
The purpose of this study is to prepare polyclonal antibodies against fowl adenovirus(FAd V) Hexon protein and to identify its specificity. The recombinant prokaryotic expression plasmid p ET-28 a-Hexon was constructed and transformed into E.coli BL21(DE3), IPTG induction, the recombinant protein was identified by SDS-PAGE, and the protein was purified. The polyclonal antibodies of Hexon protein was prepared by routine immunity in New Zealand white rabbits with purified fusion protein. The specificity of polyclonal antibodies was identified by indirect immunofluorescence, immunohistochemistry and Western blot. The results showed the prokaryotic expression of Hexon protein was high and existed in the form of inclusion body, and its molecular weight was about 43 k Da;Polyclonal antibodies having specific reaction with FAd V and high potency could be obtained in the immunized rabbits. The above results indicated the polyclonal antibody of FAd V Hexon protein prepared in this study proved to be of good specificity and provided a good material basis for the diagnosis, pathogenesis study and subunit vaccine development of avian adenovirus diseases.
引文
[1] Hess M. Detection and differentiation of avian adenoviruses:a review[J]. Avian Pathology, 2000,29(3):195-206
[2] Reece RL, Barr DA, Grix DC, et al. Observations on naturally occurring inclusion body hepatitis in Victorian chickens[J]. Australian Veterinary Journal, 1986,63(6):201-202
[3] AnjumAD, Sabri MA, Iqbal Z. Hydropericarditis syndrome in broiler chickens in Pakistan[J]. Veterinary Record, 1989,124(10):247-248
[4] Gowda SRN, Satyanarayana ML. Hydropericardium syndrome in poultry[J]. Indian Journal Veterinary Pathology, 1994,18:159-161
[5] Balamurugan V, Kataria JM. The Hydropericardium Syndrome in Poultry–A Current Scenario[J]. Veterinary Research Communications, 2004,28(2):127-148
[6] Roy P, Vairamuthu S, Sakthivelan SM, et al. Hydropericardium syndrome in Japanese Quails(Coturnix coturnix japonica)[J]. Veterinary Research, 2004,155(9):273-274
[7] Kim JN, Byun SH, Min JK, et al. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates[J]. Avian Diseases, 2008,52(3):526-530
[8] Boyle DB, Mcferran JB. Avian adenoviruses isolated from poultry in Queensland[J]. Australian Veterinary Journal,1976,52(12):587-589
[9]朱广柱,林钧安.鸡包涵体肝炎病理形态学观察[J].中国兽医杂志,1987,12(13):15-17
[10]刁有祥,李久芹.鸡包涵体肝炎的诊断[J].中国畜禽传染病,1996,1(3):40-41
[11]郝先谱,林曦.包涵体肝炎的病理学研究[J].中国兽医科技,1993,23(5):5-7
[12] Niu YJ, Sun W, Zhang GH, et al. Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015[J]. Journal of General Virology, 2016,97(10):2684-2690
[11] Norby E. The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological complexity of soluble antigens[J]. Virology, 1969,37(4):565-576
[12] Norby E, Wadell G. Immunological relationships between hexon of certain human adenoviruses[J]. Journal Virology, 1969,4(5):663-670
[13] Toogood CIA, Murali R, Burnett RM, et al. The adenovirus type 40 hexon:sequence, predicted structure and relationship to other adenovirus hexons[J]. Journal of General Virology, 1989,70(12):3203-3214
[14] Russell WC. Adenoviruses:update on structure and function[J]. Journal of General Virology, 2009,90(1):1-20
[15] Toogood CI, Crompton J, Hay RT. Antipeptide antisera define neutralizing epitopes on the adenovirus hexon[J]. Journal of General Virology, 1992,73(6):1429-1435
[16] Li C, Li H, Wang D, et al. Characterization of fowl adenoviruses isolated between 2007 and 2014 in China[J].Veterinary Microbiology, 2016,197:62-67
[2] Reece RL, Barr DA, Grix DC, et al. Observations on naturally occurring inclusion body hepatitis in Victorian chickens[J]. Australian Veterinary Journal, 1986,63(6):201-202
[3] AnjumAD, Sabri MA, Iqbal Z. Hydropericarditis syndrome in broiler chickens in Pakistan[J]. Veterinary Record, 1989,124(10):247-248
[4] Gowda SRN, Satyanarayana ML. Hydropericardium syndrome in poultry[J]. Indian Journal Veterinary Pathology, 1994,18:159-161
[5] Balamurugan V, Kataria JM. The Hydropericardium Syndrome in Poultry–A Current Scenario[J]. Veterinary Research Communications, 2004,28(2):127-148
[6] Roy P, Vairamuthu S, Sakthivelan SM, et al. Hydropericardium syndrome in Japanese Quails(Coturnix coturnix japonica)[J]. Veterinary Research, 2004,155(9):273-274
[7] Kim JN, Byun SH, Min JK, et al. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates[J]. Avian Diseases, 2008,52(3):526-530
[8] Boyle DB, Mcferran JB. Avian adenoviruses isolated from poultry in Queensland[J]. Australian Veterinary Journal,1976,52(12):587-589
[9]朱广柱,林钧安.鸡包涵体肝炎病理形态学观察[J].中国兽医杂志,1987,12(13):15-17
[10]刁有祥,李久芹.鸡包涵体肝炎的诊断[J].中国畜禽传染病,1996,1(3):40-41
[11]郝先谱,林曦.包涵体肝炎的病理学研究[J].中国兽医科技,1993,23(5):5-7
[12] Niu YJ, Sun W, Zhang GH, et al. Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015[J]. Journal of General Virology, 2016,97(10):2684-2690
[11] Norby E. The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological complexity of soluble antigens[J]. Virology, 1969,37(4):565-576
[12] Norby E, Wadell G. Immunological relationships between hexon of certain human adenoviruses[J]. Journal Virology, 1969,4(5):663-670
[13] Toogood CIA, Murali R, Burnett RM, et al. The adenovirus type 40 hexon:sequence, predicted structure and relationship to other adenovirus hexons[J]. Journal of General Virology, 1989,70(12):3203-3214
[14] Russell WC. Adenoviruses:update on structure and function[J]. Journal of General Virology, 2009,90(1):1-20
[15] Toogood CI, Crompton J, Hay RT. Antipeptide antisera define neutralizing epitopes on the adenovirus hexon[J]. Journal of General Virology, 1992,73(6):1429-1435
[16] Li C, Li H, Wang D, et al. Characterization of fowl adenoviruses isolated between 2007 and 2014 in China[J].Veterinary Microbiology, 2016,197:62-67