鸭坦布苏病毒囊膜糖蛋白结构域3(ED3)单克隆抗体的制备及其中和活性分析  

Preparation of monoclonal antibody against E domain Ⅲ(ED3)protein of duck Tembusu virus with neutralizing activity

作　　者：张淼 吕炫 张冲 阚莹 王若洁 于胜祖 卢奇 祝萌 方天 刘丹 朱英奇[1] 曹守林[1,2] 王蓓 殷冬冬[1,2] 王晴[1] 王桂军 ZHANG Miao;LYU Xuan;ZHANG Chong;KAN Ying;WANG Ruojie;YU Shengzu;LU Qi;ZHU Meng;FANG Tian;LIU Dan;ZHU Yingqi;CAO Shoulin;WANG Bei;YIN Dongdong;WANG Qing;WANG Guijun(Department of Preventive Veterinary Medicine,College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036;Anhui Key Laboratory of Veterinary Pathobiology and Disease Control,Hefei 230036,China)

机构地区：[1]安徽农业大学动物科技学院预防兽医学科,安徽合肥230036 [2]兽医病理生物学与疫病防控安徽省重点实验室,安徽合肥230036

出　　处：《细胞与分子免疫学杂志》2022年第10期931-938,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(32072874);科技部国家重点研发计划(2018YFD0500100,2016YFD0500805)。

摘　　要：目的以纯化的重组鸭坦布苏病毒(DTMUV)囊膜糖蛋白结构域3(ED3)蛋白作为免疫原,制备ED3蛋白的单克隆抗体并研究其中和活性。方法利用反转录PCR扩增DTMUV AH-F10株ED3基因,构建重组表达载体pET32a-ED3并转化至表达菌株Rosetta,诱导表达后利用镍柱亲和层析法纯化目的蛋白。以纯化的目的蛋白为抗原免疫BALB/c小鼠,取免疫后小鼠的脾细胞与Sp2/0细胞进行细胞融合,采取有限稀释法筛选杂交瘤细胞并大量制备单克隆抗体。利用Western blot法、间接免疫荧光法、ELISA及分型试剂盒对单克隆抗体进行鉴定并通过病毒中和试验检测抗体中和效价。结果成功表达DTMUV ED3蛋白,并制备3株ED3蛋白的鼠源单克隆抗体B9D10C7、B9D7B8G10、B9D7B8F11,其亚型分别为IgG1、IgG2a、IgG2b,腹水间接ELISA检测抗体效价均达到1∶51200,且能够识别DTMUV的E蛋白,具有一定的中和活性。结论获得3株特异性强、敏感性高的DTMUV ED3的单克隆抗体。Objective To prepare the specific monoclonal antibody(mAb)against E domain Ⅲ(ED3)of duck Tembusu virus(DTMUV)and explore its neutralization activity.Methods The ED3 gene was amplified by using reverse transcription PCR according to the genome of the DTMUV AH-F10 strain.Then,the recombinant expression vector pET32a-ED3 was constructed and transformed into E.coli Rosetta.The ED3 protein was expressed and purified by nickel column affinity chromatography.After the recombinant ED3 protein was identified by SDS-PAGE and Western blot analysis,the BALB/c mice were immunized subcutaneously three times.The splenocytes of the immunized mice were hybridized with Sp2/0 myeloma cells,and the hybridization was screened by the limiting dilution method.The specificity and sensitivity of the antibody were identified by indirect immuno-fluorescent assay and Western blot analysis.Subsequently,antibody titers were determined by ELISA.Finally,this study titrated the neutralization titers of the antibodies on DTMUV-infected BHK-21 cells.Results The ED3 protein was successfully prepared and purified using the prokaryotic expression system.Three strains of monoclonal antibodies named B9D10C7、B9D7B8G10 and B9D7B8F11 were prepared.Their subtypes were IgG1,IgG2a and IgG2b,respectively.The titers of monoclonal antibody ascites can reach 1∶51200,and they could specifically recognize the E protein of DTMUV.Neutralization test showed that they had a certain neutralizing activities.Conclusion The monoclonal antibodies against ED3 protein of DTMUV are successfully prepared.

关 键 词：鸭坦布苏病毒(DTMUV) 囊膜糖蛋白结构域3(ED3) 原核表达 单克隆抗体 

分 类 号：S718.85[农业科学—林学] S852.43[医药卫生—免疫学] R392[医药卫生—基础医学]