| 禽偏肺病毒C型核衣壳N蛋白的哺乳动物细胞表达系统表达及鉴定 |
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| 引用本文： | 李子璇,韦莉,王菁,朱姗姗,张春燕,柳舒航,全荣,阎旭,陈武.禽偏肺病毒C型核衣壳N蛋白的哺乳动物细胞表达系统表达及鉴定[J].北京农学院学报,2014,29(4). |
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| 作者姓名： | 李子璇 韦莉 王菁 朱姗姗 张春燕 柳舒航 全荣 阎旭 陈武 |
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| 作者单位： | 北京农学院动物科学技术学院,北京102206;北京市农林科学院畜牧兽医研究所,北京100097;北京市农林科学院畜牧兽医研究所,北京,100097;北京农学院动物科学技术学院,北京,102206 |
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| 基金项目： | 国家现代农业产业技术体系 |
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| 摘 要： | 利用哺乳动物细胞表达系统对禽偏肺病毒C型核衣壳N蛋白进行表达,并检测所表达融合蛋白的抗原性。首先将N基因克隆到真核表达载体pEGFP-C1中,构建重组真核表达载体,然后将阳性重组质粒转染BHK-21细胞,通过倒置荧光显微镜和共聚焦显微镜检测N蛋白的表达,并用Western blot检测表达蛋白的抗原性。结果显示,禽偏肺病毒核衣壳N蛋白主要表达在细胞浆中,在转染后48h后表达量最高;与抗N蛋白抗体有抗原性,所表达核衣壳N融合蛋白大小为72kDa左右,与预期结果一致。结论,本试验成功应用哺乳动物表达系统表达了禽偏肺病毒C型核衣壳N蛋白,表达蛋白具有抗原性,为与禽偏肺病毒核衣壳N蛋白的相关研究奠定了基础。
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| 关 键 词： | 禽偏肺病毒核衣壳N蛋白 哺乳动物细胞表达系统 显微镜观察 Western blotting |
Mammalian expression and identification of avian metapneu movirus subgroup C nucleocapsid protein |
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| Abstract: | The aims of this paper were to express subgroup C avian metapneumovirus virus(aMPV)nucleocapsid(N)protein by Eukaryotic expression system and to further analyze the antigenicity of the recombinant protein.The N gene was cloned into eukaryotic expression pEGFP-C1 vector to obtain the recombinant expression vector.The positive recombinant expression vector was transfected into BHK-21 cells.The recombinant N protein expression was analyzed by confocal microscopy and inverted fluorescence microscope and the antigenicity of the recombinant protein by western-blotting.The results demonstrated that the N protein expressed mainly in the cytoplasm and exhibited a specific and single band with a size of about 72 kDa when reacted with a monoclonal antibody raised against subgroup C aMPV N protein.In conclusion,this study expressed aMPV N protein in mammalian expression system with a good antigenicity,which made the foundation work for the relatied study of aMPV N protein. |
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| Keywords: | avian metapneumovirus nucleocapsid (N) protein mammalian expression microscopic observation Western blotting |
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