| H3N2亚型猪流感病毒核蛋白基因的优化表达及抗原性分析 |
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| 引用本文： | 郭利敏,乔传玲,陈艳,杨焕良,张健,辛晓光,陈化兰.H3N2亚型猪流感病毒核蛋白基因的优化表达及抗原性分析[J].中国兽医科学,2010,40(3). |
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| 作者姓名： | 郭利敏 乔传玲 陈艳 杨焕良 张健 辛晓光 陈化兰 |
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| 作者单位： | 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,农业部动物流感重点开放实验室,黑龙江,哈尔滨,150001 |
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| 基金项目： | 哈尔滨市科技攻关计划项目 |
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| 摘 要： | 根据已获得的猪流感病毒(SIV)分离株A/Swine/Guangdong/9/05(H3N2)NP基因的核苷酸序列设计合成了1对带有特定酶切位点的引物,经RT-PCR扩增NP基因,将其克隆到pMD19-Simple-T载体,鉴定并测序正确后,构建pET32a-NP表达质粒,并转化大肠杆菌Rosetta(DE3),经IPTG诱导表达,SDS-PAGE电泳分析结果显示,表达的融合蛋白分子质量约为73ku,在25℃条件下主要以可溶性形式存在。将重组NP蛋白纯化后进行Western-blotting和间接免疫荧光试验,结果显示,表达蛋白可分别与H1N1、H3N2及H9N2亚型SIV阳性血清发生特异性反应,表明该蛋白具有良好的反应原性和交叉反应性;用纯化的重组NP蛋白免疫小鼠制备的多克隆血清也可与H1N1、H3N2及H9N2亚型SIV发生特异性反应,进一步证实所表达的重组NP蛋白具有良好的免疫原性。
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| 关 键 词： | 猪流感病毒 核蛋白基因 原核表达 抗原性 |
Optimal expression and antigenic analysis of nucleoprotein of swine influenza virus subtype H3N2 |
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| GUO Li-min,QIAO Chuan-ling,CHEN Yan,YANG Huan-liang,ZHANG Jian,XIN Xiao-guang,CHEN Hua-lan.Optimal expression and antigenic analysis of nucleoprotein of swine influenza virus subtype H3N2[J].Veterinary Science in China,2010,40(3). |
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| Authors: | GUO Li-min QIAO Chuan-ling CHEN Yan YANG Huan-liang ZHANG Jian XIN Xiao-guang CHEN Hua-lan |
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| Institution: | GUO Li-min,QIAO Chuan-ling,CHEN Yan,YANG Huan-liang,ZHANG Jian,XIN Xiao-guang,CHEN Hua-lan(Animal Influenza Laboratory of the Ministry of Agriculture/State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China) |
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| Abstract: | The complete NP gene was amplified from the RNA of swine influenza virus(SIV) subtype H3N2 by RT-PCR using a pair of primers with specific enzyme digestion site and was cloned into pMD19-Simple-T vector.It was confirmed by restriction enzyme and sequencing,then the NP gene was subcloned into pET32a vector after being digested with specific enzyme.Rosetta(DE3) transformed with recombinant plasmid pET32a-NP was induced by IPTG.The optimal expressing condition was established by adjusting the concentration of ... |
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| Keywords: | swine influenza virus NP gene prokaryotic expression antigenicity |
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