兔抗猪CD163蛋白多克隆抗体制备及鉴定  

Generation of polyclonal antibodies specifically against porcine CD163

作　　者：郭娟娟[1,2] 彭金美 安同庆[2] 冷超良[2] 吴江[1,2] 宫大庆[2] 陈家锃[2] 杨永倩[1,2] 田志军[2] 张德礼[1] 

机构地区：[1]西北农林科技大学、动物医学院病毒免疫与生物信息研究室、生物信息研究中心、分子病毒免疫与肿瘤系统生物学研究组,陕西杨凌712100 [2]中国农业科学院、哈尔滨兽医研究所兽医生物技术国家重点实验室、猪传染病研究室,黑龙江哈尔滨150001

出　　处：《中国兽医学报》2012年第11期1624-1628,共5页Chinese Journal of Veterinary Science

基　　金：兽医生物技术国家重点实验室开放基金重点资助项目(SKLVBF200905);国家自然科学基金资助项目(31072115;30270342);“十二五”国家科技计划农村领域基础研究类首批预备项目(NC2010CD0178)

摘　　要：为制备兔抗猪CD163多克隆抗体,采用RT-PCR方法从猪PAM RNA中扩增CD163胞质外区部分基因SRCR4-5,克隆至pGEX-6p-1原核表达载体,在IPTG诱导下获得约50 000以包涵体形式表达的重组蛋白。WesternBlotting分析表明,表达产物能够被GST单抗所识别。用该重组蛋白免疫兔制备多抗血清,经间接ELISA检测,多抗血清效价可达105,流式检测发现可与Marc-145和PAM上的天然CD163分子相互作用。抗体阻断试验证明制备的多抗血清可以部分阻断PRRSV感染。本试验为进一步研究PRRSV与受体-CD163相互作用机制打下了基础。The extracellular region of porcine CD163 gene,SRCR4-5,was amplified from total RNA of porcine alveolar macrophages and subcloned into prokaryotic expression vector pGEX-6p-1. Fol- lowed by inducing with IPTG,a 50 000 fusion protein which could be recognized by anti-GST mon- oclonal antibody was expressed. After purified,the fusion protein was employed to immunize rab- bits to generate rabbit anti-CD163 polyclonal antibodies. The polyclonal antibodies had a titer of 10s in GST-SRCR4-5 by indirect ELISA,and it also could partly block the infection of PRRSV to porcine alveolar macrophages in antibody-blocking assay. The results suggested that the polyclonal antibody was of biological function,and could be employed in the further studies on CD163 recep- tor.

关 键 词：CD163 抗体 鉴定 

分 类 号：S852[农业科学—基础兽医学]