| 兔抗猪CD163蛋白多克隆抗体制备及鉴定 |
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| 引用本文： | 郭娟,彭金美,安同庆,冷超良,吴江,宫大庆,陈家锃,杨永倩,田志军,张德礼.兔抗猪CD163蛋白多克隆抗体制备及鉴定[J].兽医大学学报,2012(11):1624-1628. |
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| 作者姓名： | 郭娟 彭金美 安同庆 冷超良 吴江 宫大庆 陈家锃 杨永倩 田志军 张德礼 |
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| 作者单位： | [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 |
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| 基金项目： | 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）. |
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| 摘 要： | 为制备兔抗猪CD163多克隆抗体，采用RT-PCR方法从猪PAMRNA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50000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10^5，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与受体一CDl63相互作用机制打下了基础。
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| 关 键 词： | CD163 抗体 鉴定 |
Generation of polyclonal antibodies specifically against porcine CD163 |
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| Authors: | GUO Juan-juan PENG Jin-mei AN Tong-qing LENG Chao-liang WU Jiang GONG Da-qing CHEN Jia-Zeng YANG Yong-qian TIAN Zhi-jun ZHANG De-li |
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| Institution: | 1. Investigation Group of Molecular Virology, Immunology, Oncology ＆ Systems Biology, Center for Bioinformatics ; Research Laboratory of Virology, Immunology ＆ Bioinformatics , College of Veterinary Medi- cine, Northwest A ＆ F University ,YangLing , Shaanxi 712100, China ; 2. State Key Laboratory of Veterinary Biotechnology Division of Swine Infectious Diseases, Harbin Veterinary Research In- stitute ,Chinese Academy of Agricultural Sciences, Harbin 150001 ,China) |
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| Abstract: | 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. |
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| Keywords: | CD163 antibody identification |
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