| 甲型H1N1流感病毒血凝素HA1蛋白的真核表达与纯化 |
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| 引用本文： | 李燕,迟莹,卞倩,温恬,张文帅,焦永军.甲型H1N1流感病毒血凝素HA1蛋白的真核表达与纯化[J].实用老年医学,2011,25(5):378-381. |
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| 作者姓名： | 李燕 迟莹 卞倩 温恬 张文帅 焦永军 |
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| 作者单位： | 江苏省疾病预防控制中心病原微生物研究所,江苏省南京市,210009 |
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| 基金项目： | 江苏省自然科学基金资助项目，江苏省卫生厅资助项目 |
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| 摘 要： | 目的构建甲型SWH1N1流感病毒血凝素HA1蛋白的杆状病毒真核表达载体,转染sf9细胞表达并纯化其编码蛋白。方法从江苏甲型H1N1流感病毒毒株A/Jiangsu/1/2009（H1N1）]提取病毒RNA,采用RT-PCR技术扩增HA1基因,将其克隆至pMD18-T Simple Vector中构建pMD18-T/HA1质粒,双酶切pMD18-T/HA1与p-fast后,连接构建真核表达载体p-fast-HA1,转化DH10Bac细胞,转座构建杆状病毒HA1-Bacmid,经酶切及测序鉴定后将HA1-Bacmid转染到sf9细胞中,通过免疫印迹法鉴定HA1蛋白的表达,采用亲和层析法纯化HA1蛋白。结果经双酶切、测序鉴定证实HA1基因的真核表达载体构建成功;免疫印迹法证实HA1基因的表达;纯化获得了高纯度的HA1蛋白。结论成功克隆了HA1基因,并构建了其杆状病毒真核表达载体,该表达载体的构建为后期的流感快速检测以及基因工程疫苗制备奠定了良好的基础。
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| 关 键 词： | 甲型SWH1N1流感病毒 HA1基因 克隆 杆状病毒真核表达系统 蛋白纯化 |
Eukaryotic expression and purification of haemagglutinin HA1 protein of influenza A virus subtype H1N1 |
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| LI Yan,CHI Ying,BIAN Qian,WEN Tian,ZHANG Wen-shuai,JIAO Yong-jun.Eukaryotic expression and purification of haemagglutinin HA1 protein of influenza A virus subtype H1N1[J].Practical Geriatrics,2011,25(5):378-381. |
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| Authors: | LI Yan CHI Ying BIAN Qian WEN Tian ZHANG Wen-shuai JIAO Yong-jun |
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| Affiliation: | LI Yan,CHI Ying,BIAN Qian,WEN Tian,ZHANG Wen-shuai,JIAO Yong-jun.Key Laboratory of Enteric Pathogenic Micro-biology,Ministry of Health,Institute of Pathogenic Microbiology,Jiangsu Provincial Center for Disease Prevention and Control,Nanjing 210009,China |
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| Abstract: | Objective To construct type A SWH1N1 influenza virus haemagglutinin HA1gene in Baculovirus expression system,and to express and purify its encoded peptide.Methods The HA1 gene of influenza A (SWH1N1) was amplified by RT-PCR from a Jiangsu type A H1N1 influenza virus strainA/ Jiangsu/1/2009(H1N1) ].After T-A cloning and sequencing validation,HA1 gene was cloned into p-fast vector.Following the transposition of pFastBac-HA1 into the bacmid in DH10Bac E.coli competent cells,the recombinant bacmid (rBacmid-HA1... |
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| Keywords: | influenza A (SWH1N1) HA1 gene clone Baculovirus expression system protein purify |
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