| 摘要: |
| 【目的】制备鸭瘟病毒（DPV）gB蛋白单克隆抗体，为建立DPV快速诊断方法及进一步鉴定DPV的抗原表位奠定基础。【方法】克隆DPV-gB基因，构建其表达载体并进行原核表达，纯化DPV gB蛋白，以其作为抗原免疫8周龄BALB/c小鼠，取其脾细胞与SP2/0骨髓瘤细胞进行融合，进行间接ELISA及亚克隆筛选，并对单抗亚类及抗原性进行鉴定。【结果】PCR扩增出915 bp的目的基因，成功连接于pET-28a载体，并转化大肠杆菌Rosetta进行诱导表达，获得4株稳定分泌抗DPV gB蛋白的杂交瘤细胞株，分别命名为A8D7、E6C3、H11F8、H6F6，间接ELISA检测其腹水效价分别为1∶103、1∶103、1∶105、1∶103，亚类鉴定结果分别为IgG2b、IgG2a、IgG2b、IgG1，轻链均为kappa链。Western blot结果显示4株单抗均能与DPV gB蛋白特异性结合，IFA结果表明4株单抗是针对DPV产生的。【结论】成功获得了特异性针对DPV gB蛋白的单克隆抗体。 |
| 关键词: 鸭瘟病毒 gB蛋白 单克隆抗体 |
| DOI： |
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| 基金项目:国家现代农业产业技术体系项目（CARS-43） |
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| Preparation and identification of monoclonal antibodies against gB protein of duck plague virus |
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ZHAO Dandan,DIAO Youxiang,YANG Guoping,et al
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| Abstract: |
| 【Objective】This study prepared monoclonal antibodies against gB protein of duck plague virus to provide basis for establishing diagnosis method of DPV infection and identifying antigen epitope of DPV.【Method】The DPV gB gene was cloned to establish its expression vector and conduct prokaryotic expression.DPV gB protein was purified and used to immunize 8 weeks old BALB/c mice,whose splenocytes were fused with SP2/0 myeloma cells for indirect ELISA and sub-clone screening.【Result】PCR amplification obtained the aim gene of 915 bp and it was connected to pET-28a vector before being transformed to Rosetta for expression.Four hybridoma cell lines that stably secreted monoclonal antibody against gB protein of DPV named A8D7,E6C3,H11F8,and H6F6 were obtained with indirect ELISA determined ascitic fluid titers of 1∶103,1∶103,1∶105,and 1∶103,respectively.The immunoglobulin subtypes of the monoclonal antibodies were IgG2b,IgG2a,IgG2b,and IgG1,with the light chain of kappa. Western blot showed the four monoclonal antibodies were able to specifically recognize gB protein of DPV and IFA showed that the four monoclonal antibodies were specific to DPV.【Conclusion】Specific monoclonal antibodies against gB protein of duck plague virus were obtained. |
| Key words: duck plague virus gB protein monoclonal antibody |
