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应用SOEing方法构建SOD1-G93A的真核表达载体

陆玉成1,于继徐2   

  1. 1. 临沂市人民医院脑科医院神经病学实验室
    2. 临沂市人民医院脑科医院神经内科
  • 收稿日期:2013-06-07 修回日期:2013-07-26 出版日期:2013-12-15 发布日期:2013-12-15
  • 通讯作者: 于继徐

Construction of a SOD1-G93A Eukaryotic Expression Vector by SOEing

  • Received:2013-06-07 Revised:2013-07-26 Published:2013-12-15 Online:2013-12-15

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摘要：

【摘要】目的  构建铜/锌超氧化物歧化酶（Cu/Zn superoxide dismutase, SOD1）-G93A的真核表达载体。方法  应用重叠区扩增基因拼接法（SOEing）分别扩增前段M1基因与包含突变位点的后段M2基因，然后将2段基因拼接起来，得到SOD1-G93A基因，并克隆至pcDNA3.1(-)真核表达载体上。  结果  成功扩增出SOD1-G93A基因，测序结果与GenBank完全一致；对重组质粒SOD1-G93A-pcDNA3.1(-)进行双酶切鉴定，测序结果与预期完全一致。  结论    成功构建了SOD1-G93A-pcDNA3.1(-)真核表达载体。

关键词: 超氧化物歧化酶, 点突变, 质粒, DNA, 重组, 肌萎缩侧索硬化, 重叠区扩增基因拼接法

Abstract: [Abstract] Objective To construct the eukaryotic expression vector of SOD1-G93A gene. Methods The M1 and including mutation gene of M2 were amplified by PCR. The two amplified gene sequence was annealed to form a chimeric SOD1-G93A gene with gene splicing by overlap extension. The resulting chimera was cloned in pcDNA3.1(-) vector and verified by sequencing analysis. Results The result showed that SOD1-G93A gene was successfully amplified with gene splicing by overlap extension, and sequencing was not changed except one base compared with the GeneBank. The recombinant plasmid SOD1-G93A-pcDNA3.1(-) was identified by double digestion and sequencing results entirely consistent with the expected. Conclusion The eukaryotic expression vector of SOD1-G93A-pcDNA3.1(-) was successfully constructed.