应用SOEing方法构建SOD1-G93A的真核表达载体

【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。

Construction of a SOD1-G93A Eukaryotic Expression Vector by SOEing

[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.