| 摘要: |
| 【目的】 构建甲型流感病毒H1N1血凝素基因HA/HA1的真核表达载体,并检测其在BHK-21细胞中的表达。【方法】 按照人的偏爱密码子,对H1N1流感病毒的HA/HA1基因进行优化改造，以提高其表达量，经全基因合成后插入到真核表达载体pSNAV中，构建了真核表达质粒pSNAV-Mod.HA与pSNAV Mod.HA1；质粒转染BHK-21细胞，G418筛选后，用免疫荧光及Western blot技术检测其表达情况。【结果】 成功构建了血凝素基因HA/HA1的真核表达载体。经G418筛选得到单克隆，免疫荧光技术和Western blot检测10代，显示Mod.HA和Mod.HA1皆可在BHK-21细胞中稳定表达。【结论】 成功构建的流感病毒HA和HA1真核表达载体，可为今后开展多基因表达的基因工程疫苗及H1N1检测试剂奠定基础。 |
| 关键词: 流感病毒 HA基因；HA1基因 密码子优化 真核表达 |
| DOI： |
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| 基金项目:国家“十一五”科技支撑计划项目（2006BAD06A15） |
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| Construction and primary expression of eukaryotic expressing vectors of codon-optimized H1N1 influenza virus HA and HA1 gene |
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| Abstract: |
| 【Objective】 The study was done to construct influenza A virus H1N1-Mod.HA and H1N1-Mod.HA1 eukaryotic expressing plasmids and test their expression in BHK-21 cells.【Method】HA1 and HA1 DNA sequences of H1N1 influenza A virus were optimized in accordance with human codon preference so as to enhance its protein expression in eukaryotic expressing system.Synthetic HA and HA1 genes were inserted into the eukaryotic expressing vector pSNAV,which constructed Eukaryotic Expressing Plasmids of Mod.HA and Mod.HA1.Mod.HA and Mod.HA1 eukaryotic expressing plasmids were transfected into BHK-21 cells.G418 selection acquired monoclone.Immunofluorescence assay and Western blot detected expression in BHK-21 cells.【Result】 It was confirmed that the construction of Mod.HA and Mod.HA1 eukaryotic expressing plasmids was made successfully.The 10 generations BHK-21cells were tested with immunofluorescence and Western blot,which showed HA and HA1 were both stably and accurately expressed in BHK-21 cells.【Conclusion】 The experiment successfully constructed eukaryotic expressing plasmids for Mod.HA and Mod.HA1,which could provide a foundation for the further study on the development of genetic engineering vaccine and the tested reagent of H1N1 influenza. |
| Key words: influenza virus HA gene HA1 gene codon opitimization eukaryotic expression |
