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| 猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立 | |
| 引用本文： | 唐青海,杨海,黎露,陈果亮,何丽芳,刘最,王芳宇.猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立[J].中国畜牧兽医,2017,44(8):2261-2268. |
| 作者姓名： | 唐青海 杨海 黎露 陈果亮 何丽芳 刘最 王芳宇 |
| 作者单位： | 1. 衡阳师范学院生命科学与环境学院, 衡阳 421008; 2. 衡阳师范学院生物药物研究所, 衡阳 421008; 3. 衡阳师范学院湖南益豚生态农业有限责任公司联合研究中心, 衡阳 421008 |
| 基金项目： | 国家自然科学基金青年基金（31101837）；衡阳师范学院引进人才专项项目（16D20）；湖南省教育厅优秀青年项目（17B038） |
| 摘 要： | 本研究旨在建立猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV） CH-HuN1301株N蛋白稳定表达细胞系。采用RT-PCR扩增PEDV新流行毒株CH-HuN1301株N基因，分别克隆至原核表达载体pET28a和真核表达载体pEGFP-C1构建重组表达载体，所测定的序列采用Mega 5.0软件进行进化分析。将原核表达的N蛋白免疫家兔制备多克隆抗体，重组真核表达载体pEGFP-PEDV-N转染HEK293细胞，经用G418筛选获得稳定的表达细胞系。荧光倒置显微镜观察细胞荧光，免疫过氧化物酶单层细胞染色法（IPMA）检测N蛋白的表达。结果表明，PEDV HuN1301-14毒株N基因大小为1 323 bp，原核表达重组N蛋白分子质量约60 ku，其多克隆抗体与PEDV呈特异性反应，荧光倒置显微镜观察和IPMA检测结果显示N蛋白在HEK293细胞中稳定表达。该细胞系的建立为进一步研究PEDV的诊断方法提供了基础材料。 |
| 关 键 词： | 猪流行性腹泻病毒 CH-HuN1301毒株 N基因 稳定细胞系 |
| 收稿时间： | 2017-01-19 |
Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301 |
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| TANG Qing-hai,YANG Hai,LI Lu,CHEN Guo-liang,HE Li-fang,LIU Zui,WANG Fang-yu.Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2261-2268. | |
| Authors: | TANG Qing-hai YANG Hai LI Lu CHEN Guo-liang HE Li-fang LIU Zui WANG Fang-yu |
| Institution: | 1. College of Life Sciences and Environment, Hengyang Normal University, Hengyang 421008, China; 2. Institute of Biological Medicine, Hengyang Normal University, Hengyang 421008, China; 3. Joint Research Centre of Hunan Yi-tun Ecological Agriculture LLC and Hengyang Normal University, Hengyang 421008, China |
| Abstract: | This study was aimed to construct stable cell line expressing N protein coded by porcine epidemic diarrhea virus (PEDV) strain CH-HuN1301. N gene of this virus strain was amplified by RT-PCR, which was then cloned into prokaryotic expression vector pET28a and eukaryotic expression vector pEGFP-C1,respectively. Recombinant plasmids were sequenced and analyzed by software Mega 5.0. To prepare polyclonal antibody of N protein, the prokaryotic expression recombinant N protein was used as antigen to immunize rabbits. HEK293 transfected with recombinant eukaryotic expression plasmids pEGFP-PEDV-N was selected by G418 to produce a stable cell line which then was identified by immunoperoxidase monolayer assay (IPMA) and fluorescence observation. Results showed that, the N gene of PEDV strain CH-HuN1301 was 1 323 bp, molecular weight of recombinant prokaryotic expressing N protein was about 60 ku, prepared polyclonal antibody against N protein showed wonderful reactivity with PEDV propagated in Vero cells, recombinant eukaryotic expressing N protein was positively detected in the established stable cell line by IPMA and fluorescence observation. This stable cell line provided foundation for the research in PEDV diagnosis methods. |
| Keywords: | porcine epidemic diarrhea virus (PEDV) CH-HuN1301 N gene stable cell line |
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