鸡Mx蛋白单克隆抗体的制备及鉴定

摘要

Mx蛋白为GTP酶动态蛋白家族成员,已被证明具有抗禽流感病毒、Thogoto病毒和水泡性口膜炎病毒等的作用.为制备鸡Mx蛋白的单克隆抗体(MAb),本研究以原核表达并纯化的鸡Mx蛋白为免疫原,免疫BALB/c小鼠,分离脾细胞与SP2/0细胞融合,采用间接ELISA方法筛选阳性克隆,经3次细胞克隆纯化后获得4株稳定分泌抗鸡Mx蛋白抗体的杂交瘤细胞株.间接ELISA测定4株MAb纯化后腹水的效价介于1∶3.2×104～1∶2.5×105之间.Westem blot结果证实4株MAb均具有良好的反应性；间接免疫荧光试验表明其中一株MAb与真核表达的鸡Mx蛋白发生反应.本研究为鸡Mx蛋白功能及表达鸡Mx蛋白的转基因动物等研究奠定了基础.%Mx protein is a member of large GTPases family and proven to have antivirus activities to avain influenza virus (AIV),Thogoto virus and Vesicular stomatitis virus.To prepare the monoclonal antibodies (MAbs) against chicken Mx protein,the splenocytes of BALB/c mice immunized with purified chicken Mx protein expressed in E.coli were fused with SP2/0 cells and 4 hybridomas secreting MAbs were scanned by ELISA.Titers of the MAbs in ascites were ranged from 1∶3.2×104 to 1∶2.5×105assayed by ELISA.Western blot analysis showed that all the MAbs reacted positively with chicken Mx protein expressed by E.coli,but only 1 of which reacted with Mx protein expressed in MDCK cells transfected with pcDNA3.1-Mx detected by indirect immunofluorescence assay.These MAbs would facilitate the further studies on the function of the transgenic animals expressing chicken Mx protein.