非肌肉肌球蛋白Ⅱ型重链A单克隆抗体的制备及其靶位点区域的鉴定  

Preparation and characterization of monoclonal antibodies against non-muscle myosin heavy chain Ⅱ A

作　　者：吴海珍[1] 刘宁宁[1] 张璐[1] 吕军华[1] 肖一红[1] 周恩民[2] 

机构地区：[1]山东农业大学动物医学院,山东泰安271017 [2]西北农林科技大学动物医学院兽医免疫学研究所,陕西杨凌712100

出　　处：《中国预防兽医学报》2012年第10期815-818,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30871857;30901063;U0931003/L01);转基因生物新品种培育重大专项(2009ZX08009-147B)

摘　　要：为制备非肌肉肌球蛋白Ⅱ型重链A(NMHC-II A)的单克隆抗体(MAb),本研究将编码NMHC-Ⅱ A羧基端1 651～1 960氨基酸的表达重组质粒转化到大肠杆菌感受态细胞Rosseta中诱导表达,重组蛋白纯化后免疫BALB/c小鼠,分离小鼠脾细胞与SP2/0骨髓瘤细胞用PEG 4000介导融合。ELISA方法检测筛选阳性杂交瘤细胞,制备腹水并纯化MAb,以间接免疫荧光和免疫沉淀方法鉴定MAb的特异性,western blot检测MAb靶位点区域。结果表明,通过筛选得到1株稳定分泌抗体的杂交瘤细胞系,其抗体亚型为IgG1,细胞培养上清和腹水效价分别为1∶2 560和1∶1.0×106;该MAb能够特异性结合相应抗原,并结合Marc-145细胞;western blot结果显示该抗体与NMHC-Ⅱ A的1 812～1 894氨基酸位点结合。该抗体的制备为研究NMHC-Ⅱ A在猪繁殖与呼吸综合征病毒感染细胞中的作用奠定了基础。To study the role of non-muscle moysin heavy chain n A （NMHC-IIA） a in porcine reproductive and respiratory syndrome virus （PRRSV） infection, a monoclonal antibody （MAb） against NMHC-IIA was prepared. Recombinant plasmid containing the sequence encoding c-terminus of NMHC-IIA （aa1,651-aa1,960） was transformed into E. coil Rosseta competent cells and induced to express with IPTG. The purified recombinant protein was immunized into BALB/c mice. Cell fusion was carried out with induction of PEG 4000. The hybridomas secreting MAb were established and detected by ELISA. A MAb was obtained with IgG1 subtype. The titers of supematant and ascites were 1：2 560 and 1.0 × 10^6 respectively. The MAb reacted specifically with NMHC-IIA and Marc-145 cells and the epitope domain located between aa 1,812-aa 1,894. The preparation of this MAb would facilitate the study of NMHC-II A in PRRSV infection.

关 键 词：猪繁殖与呼吸综合征病毒 非肌肉肌球蛋白II型A重链羧基端 单克隆抗体 特性鉴定 

分 类 号：S852.4[农业科学—基础兽医学]