| 高致病性猪繁殖与呼吸综合征病毒Nsp1蛋白锌指结构1(ZF1)突变体的构建及活性鉴定 |
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| 引用本文： | 赵鹏伟,吴家强,杜以军,李俊,孙文博,丛晓燕,时建立,彭军,于江,刘月月,王金宝.高致病性猪繁殖与呼吸综合征病毒Nsp1蛋白锌指结构1(ZF1)突变体的构建及活性鉴定[J].中国畜牧兽医,2013,40(6):1-6. |
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| 作者姓名： | 赵鹏伟 吴家强 杜以军 李俊 孙文博 丛晓燕 时建立 彭军 于江 刘月月 王金宝 |
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| 作者单位： | 青岛农业大学动物科技学院;山东省畜禽疫病防治与繁育重点实验室;山东省农业科学院畜牧兽医研究所 |
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| 基金项目： | 国家自然科学基金(31170146);山东省自主创新成果转化重大专项(2010ZHZX1A0417);山东省优秀中青年科学家科研奖励基金(BS2009NY010);山东省现代农业产业技术体系 |
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| 摘 要： | 本试验旨在构建高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)SX-1株非结构蛋白Nsp1的真核表达质粒,并对Nsp1蛋白锌指结构1(ZF1)进行定点突变,检测其在真核细胞中的表达活性。利用TRIzol法提取总RNA,RT-PCR扩增Nsp1目的基因,经双酶切构建pCI-neo-Nsp1真核表达质粒后对Nsp1蛋白锌指结构1的8C、10C、25C和28C的4个位点进行定点突变,成功获得分别突变为A和S的8个突变体。转染HeLa细胞,通过间接免疫荧光方法(IFA)和Western blotting检测Nsp1蛋白的活性。IFA结果显示,Flag抗体检测时蛋白质主要分布在细胞质和细胞核中,Myc抗体检测时蛋白质主要分布在细胞核中。Western blotting结果显示,Flag抗体检测时,S突变体出现大小约为44和20ku条带,A突变体出现约为44ku条带;Myc抗体检测时,S突变体出现大小约为44和27ku条带,A突变体出现大小约为44ku条带。试验结果表明,本试验成功构建了Nsp1突变体的真核表达质粒,证实其能在真核细胞中表达,为进一步研究Nsp1蛋白的锌指结构是否对机体Ⅰ型IFN产生起下调作用提供基础。
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| 关 键 词： | 高致病性猪繁殖与呼吸综合征病毒SX-1株 Nsp1 突变体构建 活性鉴定 |
| 收稿时间： | 2012-11-20 |
Construction of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp1 Protein Zinc Finger 1 (ZF1) Mutants and Identification of its Activity |
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| ZHAO Peng-wei,WU Jia-qiang,DU Yi-jun,LI Jun,SUN Wen-bo,CONG Xiao-yan,SHI Jian-li,PENG Jun,YU Jiang,LIU Yue-yue,WANG Jin-bao.Construction of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp1 Protein Zinc Finger 1 (ZF1) Mutants and Identification of its Activity[J].China Animal Husbandry & Veterinary Medicine,2013,40(6):1-6. |
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| Authors: | ZHAO Peng-wei WU Jia-qiang DU Yi-jun LI Jun SUN Wen-bo CONG Xiao-yan SHI Jian-li PENG Jun YU Jiang LIU Yue-yue WANG Jin-bao |
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| Institution: | 1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China;2. Shandong Key Laboratory of Animal Disease Control and Breeding, Jinan 250100, China;3. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China |
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| Abstract: | The eukaryotic expression plasmid of nonstructural protein Nsp1 of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) strain SX-1 was constructed, and the mutants of Nsp1 protein zinc finger 1 (ZF1) were obtained by site-directed mutagenesis, their expression activities in eukaryotic cells were examined. The Nsp1 target gene was amplified by RT-PCR from the total RNA of SX-1 strain, inserted into the eukaryotic expression plasmid of pCI-neo, and the positive plasmid was designated as pCI-neo-Nsp1. 8C, 10C, 25C and 28C in the Nsp1 protein ZF1 were mutated to A or S respectively by site-directed mutagenesis using pCI-neo-Nsp1 as template and 8 mutant plasmids were obtained. The plasmids were transfected into HeLa cells, the activity of Nsp1 protein was detected by indirect immunofluorescence assay (IFA) and Western blotting. The results showed that Nsp1 protein distributed in the cytoplasm and the nucleus using Flag antibody and Nsp1 protein mainly distributed in the nucleus using Myc antibody. Western blotting analysis showed S mutant proteins were about 44 and 20 ku and A mutant proteins were about 44 ku when detected by Flag antibody, while S mutant proteins were about 44 and 27 ku and A mutant proteins were about 44 ku when detected by Myc antibody. These results showed the eukaryotic expression plasmid of Nsp1 mutant were constructed successfully by site-directed mutagenesis and could be successfully expressed in HeLa cells, which provided the foundation for further researching whether ZF1 of Nsp1 protein downregulated the type Ⅰ IFN. |
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| Keywords: | HP-PRRSV SX-1 strain Nsp1 mutant construct activity identification |
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