抗BVDV Erns蛋白单克隆抗体制备及生物学特性鉴定  

Preparation of Monoclonal Antibodies Against Erns Protein of Bovine Viral Diarrhea Virus and Characterization of Their Biological Characteristics

作　　者：陈文龙 米晓钰 张阳阳 张生英 张玉珺 邢小勇[1] 胡永浩[1] CHEN Wenlong;MI Xiaoyu;ZHANG Yangyang;ZHANG Shengying;ZHANG Yujun;XING Xiaoyong;HU Yonghao(Laboratory of Infectious Diseases,School of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院传染病学试验室,兰州730070

出　　处：《中国动物传染病学报》2022年第2期115-125,共11页Chinese Journal of Animal Infectious Diseases

基　　金：甘肃农业大学动物医学院教研产学创新基金资助项目(JYCX-KX008);抗病毒藏兽药的研发(GSAUXKJS-2018-075)。

摘　　要：为制备抗牛病毒性腹泻病毒(BVDV)Erns蛋白单克隆抗体,本试验用Erns重组蛋白免疫Balb/c鼠,刺激小鼠脾脏产生特异性免疫细胞;通过化学剂诱导剂PEG-4000诱导免疫细胞和骨髓瘤细胞融合,形成杂交瘤细胞;间接ELISA方法结合亚克隆方法筛选阳性细胞孔;鉴定为稳定分泌抗体的细胞株做扩大培养,细胞悬液计数并无菌注射KM小鼠腹腔,制备、收集腹水抗体;SDS-PAGE鉴定抗体大小、ELISA测定抗体效价、Western blot和IFA鉴定抗体特异性。本试验建立了3株稳定分泌抗体的细胞株;腹水抗体重链大小为55 kDa,轻链25 kDa,均为IgG1型,效价高于10^(4),抗体可特异性结合病毒抗原。结果证实,制备的单克隆抗体效价高、特异性好,为制备检测BVDV试剂盒奠定基础。In order to prepare monoclonal antibodies(mAbs)against bovine viral diarrhea virus(BVDV),Balb/c mice were immunized with the recombinant Erns protein to stimulate the production of specific immune cells in spleens,following by fusion of immune cells and myeloma cells with PEG-4000.The hybridoma cells were screened by indirect ELISA method and positive hybridomas were cloned by limited dilution method.The stable cell lines were propagated in large quantity and injected into the abdominal cavities of KM mice to prepare ascitic fluid.As a result,3 cell lines were established for their stably secreting mAbs with IgG1 subtype.These mAbs were visualized in SDS-PAGE as heavy chain of 55 kDa and light chain of 25 kDa and their specificity was demonstrated in Western blot and IFA.In addition,ELISA titers of these mAbs in ascitic fluid were titrated as over 10,000.The results showed that the MAbs specific for Erns protein were prepared for future study on development of BVDV diagnostic kit.

关 键 词：BVDV Erns蛋白 单克隆抗体 抗体生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]