鸡不同组织嗜性IBV毒株单克隆抗体的筛选研究
发布时间：2018-08-28 17:17
【摘要】：鸡传染性支气管炎（Infectious bronchitis, IB）是由IBV感染引起的急性炎症疾病，具有高度传染性。IBV血清型众多，变异频率高，，IBV的组织嗜性也在不断变化，目前国际公认的有呼吸型、肾型、产蛋下降型、腺胃型及肠型。最近几年国内外都有对IBV组织嗜性的研究报道，但结论各异。本研究尝试性地通过比较呼吸型与肾型不同致病性毒株S1蛋白的差异，来研究能否筛出有特异性中和活性的单克隆抗体，以探讨不同IBV毒株间致病性与组织嗜性的差异性。 本实验利用浓缩的IBV H120和K136病毒分别免疫6周龄的雌性BALB/c小鼠，采用传统杂交瘤技术，建立间接ELISA检测方法，经筛选克隆后共获得了三株单克隆抗体杂交瘤细胞系，分别为抗IBV H120的3F6G7株和抗IBV K136的1E9A3株、4F5E8株。单克隆抗体亚型鉴定结果显示3F6G7株为IgG2b亚型，1E9A3和4F5E8株为IgM亚型，轻链均为Kappa。ELISA结果表明3F6G7与H120的反应比与K136的反应强烈，而1E9A3、4F5E8则没有这种差异；这三株单抗都可与其他IBV毒株发生反应，说明其针对的抗原表位比较保守，具有群特异性。Western blot试验结果表明这三株单抗都特异性识别IBV S1蛋白。病毒中和试验结果显示，仅3F6G7株单抗具有中和活性，但3F6G7没有表现出ELISA试验中的差异性。 本研究虽未能成功筛选出鉴别性单克隆抗体，仍可为以后IBV组织嗜性的研究提供参考与借鉴。另外，获得的三株单抗仍可作为防治、检测与诊断IBV的候选工具以及研究S1蛋白抗原表位的有力素材，具有实践与研究价值。
[Abstract]:Avian infectious bronchitis (Infectious bronchitis, IB) is an acute inflammatory disease caused by IBV infection. Egg-laying type, glandular stomach type and intestinal type. In recent years, there have been reports of IBV tissue tropism at home and abroad, but the conclusions are different. The purpose of this study was to study whether monoclonal antibodies with specific neutralizing activity could be screened by comparing the differences of S1 protein between respiratory and renal virulence strains in order to explore the differences of pathogenicity and tissue tropism among different IBV strains. In this study, female BALB/c mice aged 6 weeks were immunized with concentrated IBV H120 and K136 viruses respectively. Using traditional hybridoma technique, indirect ELISA detection method was established, and three monoclonal antibody hybridoma cell lines were obtained after screening and cloning. 3F6G7 strains resistant to IBV H120 and 1E9A3 strains resistant to IBV K136 were strain 4F5E8, respectively. The results of monoclonal antibody subtype identification showed that 3F6G7 strain was IgG2b subtype 1 E9A3 and 4F5E8 strain was IgM subtype. The results of light chain Kappa.ELISA showed that the reaction ratio of 3F6G7 to H120 was stronger than that of K136, but 1E9A3O4F5E8 had no such difference. All of the three McAbs could react with other IBV strains, indicating that the antigenic epitopes were conserved. The results of group specificity. Western blot test showed that all three McAbs specifically recognized IBV S1 protein. The results of neutralization test showed that only 3F6G7 McAb had neutralization activity, but 3F6G7 showed no difference in ELISA test. Although this study failed to screen the discriminant monoclonal antibody successfully, it can be used as reference for the study of IBV tissue tropism in the future. In addition, the obtained McAbs can be used as a candidate tool for the prevention and treatment, detection and diagnosis of IBV, as well as a powerful material for the study of S1 antigen epitopes, which has practical and research value.
【学位授予单位】：天津农学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
本文编号：2210052
[Abstract]:Avian infectious bronchitis (Infectious bronchitis, IB) is an acute inflammatory disease caused by IBV infection. Egg-laying type, glandular stomach type and intestinal type. In recent years, there have been reports of IBV tissue tropism at home and abroad, but the conclusions are different. The purpose of this study was to study whether monoclonal antibodies with specific neutralizing activity could be screened by comparing the differences of S1 protein between respiratory and renal virulence strains in order to explore the differences of pathogenicity and tissue tropism among different IBV strains. In this study, female BALB/c mice aged 6 weeks were immunized with concentrated IBV H120 and K136 viruses respectively. Using traditional hybridoma technique, indirect ELISA detection method was established, and three monoclonal antibody hybridoma cell lines were obtained after screening and cloning. 3F6G7 strains resistant to IBV H120 and 1E9A3 strains resistant to IBV K136 were strain 4F5E8, respectively. The results of monoclonal antibody subtype identification showed that 3F6G7 strain was IgG2b subtype 1 E9A3 and 4F5E8 strain was IgM subtype. The results of light chain Kappa.ELISA showed that the reaction ratio of 3F6G7 to H120 was stronger than that of K136, but 1E9A3O4F5E8 had no such difference. All of the three McAbs could react with other IBV strains, indicating that the antigenic epitopes were conserved. The results of group specificity. Western blot test showed that all three McAbs specifically recognized IBV S1 protein. The results of neutralization test showed that only 3F6G7 McAb had neutralization activity, but 3F6G7 showed no difference in ELISA test. Although this study failed to screen the discriminant monoclonal antibody successfully, it can be used as reference for the study of IBV tissue tropism in the future. In addition, the obtained McAbs can be used as a candidate tool for the prevention and treatment, detection and diagnosis of IBV, as well as a powerful material for the study of S1 antigen epitopes, which has practical and research value.
【学位授予单位】：天津农学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
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