鸡新城疫病毒(NDV-XX08)单克隆抗体的制备与鉴定  

Preparation and identification of the monoclonal antibody against Newcastle disease virus(NDV-XX08)

作　　者：银梅[1] 孔令芸 张秋雨[1] 丰兰竹 周洁[1] 岳锋[3] 王选年[3] 

机构地区：[1]河南科技学院动物科学学院,河南新乡453003 [2]新乡市红旗区动物疫病预防控制中心,河南新乡453003 [3]新乡学院生命科学技术学院,河南新乡453003

出　　处：《中国兽医杂志》2016年第2期3-5,8,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31272539)

摘　　要：本研究采用鸡胚繁殖NDV-XX08,差速离心法纯化NDV,用纯化的病毒免疫BALB/c小鼠。取免疫小鼠的脾细胞与NSO细胞进行融合,用间接ELISA和血凝抑制试验（HJ）进行筛选,挑选效价高的杂交瘤细胞进行克隆和亚克隆,建立阳性杂交瘤细胞株,最终得到一株稳定分泌抗体的杂交瘤细胞,命名为4F12。采用体外细胞培养和体内诱生腹水的方法制备了其McAb。对获得的单克隆抗体采用ELISA,HI,Western-Blotting方法进行鉴定。杂交瘤细胞培养上清和腹水的ELISA效价分别为1：160和1：3.2×10~4。获得的单克隆抗体与NDV具有良好的特异性反应,与鸡胚阴性尿囊液、鸡正常组织、鸡[gG、鸡传染性囊病病毒、鸡传染性支气管炎病毒、禽流感检测抗原均无交叉反应性。HI试验表明,制备的单抗没有血凝抑制活性。Western-Blotting证明获得的单抗与新城疫病毒蛋白有特异性反应。经连续传代20次,冻存、复苏3次,4F12杂交瘤细胞株仍能稳定分泌抗体。In this study,the Newcastle Disease viruses were replicated to high titer in embryonated eggs and purified by differential centrifugation.Then,Balb/C mice were immunized with the purified NDV particles.The spleen cells of immunized Balb/C mice were fused with NSO murine myeloma cells after the titer of antiserum was analyzed.After being screened by indirect ELISA and HI assay,and then cloned by limiting dilution,a hybridoma named 4F12,which produces monoclonal antibody specifically against NDV,was successfully obtained.The McAb was characterized by ELISA,HI and westem-blotting.The McAb ELISA titers from cell culture supernatant and ascites were 1：160 and 1：3.2× 10~4,respectively.There were no cross-reactivity on negative allantoic fluid and other avian virus antigens including AIV,IBV and IBDV.The HI assay had proved that the McAb were no HI activity.And western-blotting analysis showed that the McAb specifically bound NDV.The hybridoma cell can stably secrete McAb against NDV after serial passages 20 times and freezing-revival three times in four months.In conclusion,a hybridoma which can secrete specific monoclonal antibody（McAb） against Newcastle Disease viruses（NDV） was obtained by hybridoma technique.The prepared McAb was specific.

关 键 词：鸡 新城疫病毒 单克隆抗体 Western-Blotting 

分 类 号：S852.659.5[农业科学—基础兽医学]