| 牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备与鉴定 |
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| 引用本文： | 杨飞,黄小洁,刘丹,侯力丹,李建,张敏,郎洪武.牛传染性鼻气管炎病毒gD蛋白单克隆抗体的制备与鉴定[J].动物医学进展,2020(3):7-11. |
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| 作者姓名： | 杨飞 黄小洁 刘丹 侯力丹 李建 张敏 郎洪武 |
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| 作者单位： | 中国兽医药品监察所;国药集团动物保健股份有限公司 |
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| 基金项目： | 国家重点研发计划项目(2017YFD0500900)。 |
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| 摘 要： | 为制备牛传染性鼻气管炎病毒(IBRV)gD蛋白单克隆抗体,并对其免疫学特性进行分析与鉴定。用CHO细胞表达的IBRV-gD蛋白作为免疫原免疫8周龄的Balb/c小鼠,无菌取其脾细胞与SP2/0细胞进行细胞融合,筛选阳性杂交瘤细胞株,经小鼠腹腔注射,待小鼠腹腔膨胀后,收集腹水,纯化后进行单克隆抗体浓度、纯度、类及亚类、抗体效价、相对亲和常数、Western blot和间接免疫荧光测定。结果表明,筛选到2株阳性杂交瘤细胞株,分别命名为4G3D4和9D7A7。4G3D4和9D7A7这2株单抗纯化后的浓度分别为2.6 mg/mL、1.6 mg/mL;亲和常数分别为2.30E+10、1.88E+09;抗体亚类均为IgG1,轻链为kappa链;且均能与IBRV发生特异性反应,与接种IBRV的MDBK细胞发生反应,产生特异性荧光;间接ELISA测定腹水效价分别为1∶204800、1∶12800。利用CHO细胞表达的IBRV-gD蛋白成功制备了2株单克隆抗体,为下一步建立特异性的IBRV检测方法奠定了基础。
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| 关 键 词： | 牛传染性鼻气管炎病毒 gD蛋白 单克隆抗体 |
Preparation and Identification of Monoclonal Antibodies Against gD Protein of Infectious Bovine Rhinotracheitis Virus |
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| YANG Fei,HUANG Xiao-jie,LIU Dan,HOU Li-dan,LI Jian,ZHANG Min,LANG Hong-wu.Preparation and Identification of Monoclonal Antibodies Against gD Protein of Infectious Bovine Rhinotracheitis Virus[J].Progress In Veterinary Medicine,2020(3):7-11. |
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| Authors: | YANG Fei HUANG Xiao-jie LIU Dan HOU Li-dan LI Jian ZHANG Min LANG Hong-wu |
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| Institution: | (China Institute of Veterinary Drug Control,Beijing,100081,China;Sinopharm Animal Health Corporation LTD,Wuhan,Hubei,430070,China) |
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| Abstract: | To prepare and identify the monoclonal antibody(McAb)against gD protein of infectious bovine rhinotracheitis,the recombinant IBRV-gD protein expressed by CHO cells was used to immunize 8-weekold Balb/c mice.The splenocytes of immunized mice were fused with SP2/0 cells,and the positive hybridoma cells were screened and injected i.p.into mice,of which the ascites were collected.McAbs were purified from the ascites and subclassed,and identified by Western blot,indirect enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence assay(IFA).Two hybridoma cell strains stably secreting McAbs were screened and named as 4 G3 D4,9 D7 A7,which belonged to IgG1 subgroup with kappa light chain,and the concentrations of obtained McAbs were 2.6 mg/mL,1.6 mg/mL.The affinity constants were 2.30 E+10,1.88 E+09.The titers of ascites by indirect ELISA were 1∶204800,1∶12800,respectively.Both McAbs could specifically react with IBRV,and showed specific fluorescence in MDBK cells infected with IBRV.The study successfully prepared two McAbs using IBRV-gD protein expressed by CHO cells,which laid a foundation for the establishment of specific IBRV diagnostic methods. |
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| Keywords: | Infectious bovine rhinotracheitis virus gD protein monoclonal antibody |
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