血清4型禽腺病毒penton蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against penton protein of fowl adenovirus serotype 4

【目的】制备和鉴定血清4型禽腺病毒(FAdV-4)五邻体(penton)蛋白单克隆抗体,为深入探究penton蛋白在FAdV-4感染致病机制中的作用及为抗原检测试剂研发提供抗体材料。【方法】以原核表达的FAdV-4 penton重组蛋白免疫6~8周雌性BALB/c小白鼠,加强免疫3次后,取鼠脾脏细胞与骨髓瘤细胞SP2/0进行细胞融合,通过penton-酶联免疫吸附剂测定(ELISA)和FAdV4 virus-ELISA方法分别筛选阳性杂交瘤细胞株,对筛选出的单克隆抗体杂交瘤细胞株进行亚类鉴定、特异性和广谱性鉴定及间接免疫荧光和稳定性试验。【结果】亚类鉴定结果表明,筛选获得7株能稳定分泌抗FAdV-4 penton蛋白的杂交瘤细胞株(1D11、2B3、2C4、2C9、6B3、8F12和8G11),其轻链均为Kappa链,重链3株为IgG1,4株为IgG2b;特异性检测结果表明,7株单克隆抗体只与FAdV-4反应,不与其他11种血清型FAdV及NDV、ARV、EDSV和IBV等常见禽病病原体发生交叉反应;广谱性鉴定结果表明,7株单克隆抗体均可与19株FAdV-4分离株结合,广谱性好;间接免疫荧光试验结果表明,7株单克隆抗体均与感染FAdV-4的鸡胚肝细胞(CEL)结合,出现明亮的绿色荧光。对筛选出的3株单克隆抗体(6B3、8F12和8G11)进行稳定性检测,发现其稳定性良好。【结论】通过单克隆抗体制备和鉴定获得3株与FAdV-4蛋白结合效果较好并具有特异性和广谱性的FAdV-4 penton蛋白杂交瘤细胞株(6B3、8F12和8G11),能稳定分泌penton蛋白单克隆抗体,为深入研究penton蛋白在FAdV-4感染致病机制中的作用提供单抗,为FAdV-4抗原检测试剂的研发打下基础。

【Objective】This study prepared and indentified monoclonal antibodies(mAbs)against penton protein of fowl adenovirus serotype 4(FAdV-4),in order to further study the function of penton protein in FAdV-4 infection patho-genesis and provide antibody materials for the research and development of antigen detection reagent.【Method】Female 6-8 weeks old BALB/c mice were immunized with the penton protein of FAdV-4 in prokaryotic expression.After three times of booster immunization,cell fusion was performed with spleen cells and SP2/0 myeloma cells of mice.Positive hy-bridoma cell lines were screened by penton-enzyme linked immunosorbent assay(ELISA)and FAdV4 virus-ELISA re-spectively.Subclass identification,specific detection,broad-spectrum detection,indirect immunofluorescence and stability tests were performed for the screened hybridoma cell lines against monoclonal antibodies.【Result】Results of subclass identification showed that seven hybridoma cell lines that could stably secrete penton protein against FAdV-4(1D11,2B3,2C4,2C9,6B3,8F12 and 8G11)have been screened.Light chains of seven hybridoma cell lines belonged to Kappa chain,three IgG1 heavy chains and four IgG2b heavy chains.The results showed that seven monoclonal antibodies only reacted with FAdV-4,and did not cross-react with other 11 serotypes FAdV,NDV,ARV,EDSV,IBV and other common avian pathogens.The results of broad spectrum identification showed that all seven monoclonal antibodies could bind to 19 FAdV-4 isolates with good broad spectrum.Indirect immunofluorescence assay showed that seven monoclonal antibodies were bound to FAdV-4 infected chick embryonic hepatocytes(CEL)and showed bright green fluorescence.The stability test results showed that three strains of monoclonal antibodies(6B3,8F12 and 8G11)were stable.【Conclu-sion】The three FAdV-4 penton hybridoma cell lines(6B3,8F12 and 8G11)obtained by monoclonal antibody preparation and identification,which have good binding effect on FAdV-4 protein and have specific and broad spectrum characteris-tics,can steadily secrete penton monoclonal antibody.It can provide monoclonal antibody for further study on the role of penton protein in the pathogenesis of FAdV-4 infection,and lay a foundation for the development of FAdV-4 antigen de-tection reagents.