传染性囊病病毒(IBDV)VP3蛋白单克隆抗体的制备与鉴定

Development and identification of monoclonal antibodies against Infectious Bursal Disease Virus(IBDV) VP3

为制备抗IBDV VP3蛋白的单克隆抗体,以VP3-His蛋白免疫BALB/c小鼠,经4次免疫、融合、亚克隆,筛选出1株能够稳定分泌抗VP3蛋白单克隆抗体的细胞株,命名为4H8F7C10。经间接ELISA方法检测,小鼠的腹水效价为2.05×106,单克隆抗体的解离常数(k D)为9.67×10-8,此株抗体亚类为Ig G3。通过Western Blot和间接免疫荧光试验检测,该株单克隆抗体可以识别IBDV感染DF-1细胞产生的VP3蛋白。初步确定此株单克隆抗体的抗原识别区位于VP3蛋白N端的第50-90位氨基酸。此株单克隆抗体的制备为IBDV临床检测方法的建立以及致病分子机制的研究奠定了基础。

To develop antibodies against IBDV VP3,BALB/c mice were vaccinated with VP3-His.After four times immunization,fusion,and subcloning,a hybridoma cell clone that stably secreted Mc Abs against VP3 was obtained and named 4H8F7C10.Antibody titers were 2.05×106as examined by indirect ELISA in ascite fluid of mice inoculated intraperitoneally with monoclonal antibody-producing hybridoma cells.The dissociation constant（k D）of the monoclonal antibody was determined to be 9.67×10-8and the isotype was Ig G3.The Mc Ab specifically bound to VP3 in IBDV-infected DF-1 cells was validated by Western Blot and indirect Fluorescence Antibody assay.The epitope in VP3 that was recognized by 4H8F7C10 was located between 50 to 90 aa as demonstrated by Western Blot.The Mc Ab will be useful for the examination of IBDV antigen and to elucidate the molecular mechanism of IBDV infection.