超氧化物歧化酶(Val16Ala)基因的克隆及其真核表达载体的构建

Cloning of manganese superoxide dismutase gene (Val16Ala) and the construction of its eukaryotic expression vector

目的获得锰超氧化物歧化酶(MnSOD)基因并进行Val16Ala(GTT→GCT)位点的定点突变,构建MnSOD(Val)及MnSOD(Ala)基因的真核表达载体。方法采用RT-PCR法从人乳腺癌细胞MCF-7中扩增MnSOD(Val)基因;PCR引物延伸法对Val16Ala进行定点突变以获得MnSOD(Ala)基因;通过EcoRⅠ+XhoⅠ双酶切PCR产物与相应质粒pEGFP-N1连接构建两基因的真核表达载体pEGFP-N1-MnSOD(Val)和pEGFP-N1-MnSOD(Ala)。结果突变位点经测序证明正确,重组质粒经双酶切及测序鉴定证明正确。结论本实验成功获得了MnSOD(Val)及MnSOD(Ala)基因并成功构建了两基因的真核表达载体。

Objective To clone manganese superoxide dismutase gene （MnSOD） and the site-directed mu- tation for Vall6Ala （GTT→GCT）, then construct eukaryotic effective expression vector with the two genotypes. Methods Firstly, we cloned gene MnSOD（Val） from the total RNA of human breast carcinoma cells MCF-7 by RT-PCR and the site-directed mutated mutation for Vail 6Ala （GTT→GCT） by overlap extension using PCR to acquire genotype MnSOD（Ala）. Secondly, the two genotypes digested by EcoR I and Xho I, and then ligated to expression vector pEGFP-N1 to construct the plasmid pEGFP-N1-MnSOD（Val） and pEGFP-N1-MnSOD（AIa）. Results Muta- tion site were proved correctly by sequencing, and the recombinant vectors were proved to contain the expected inserts by double digestion with restriction enzymes and sequencing. Conclusion Two genotypes MnSOD（Val） and MnSOD （Ala） have been cloned, and two eukaryotic expression vectors containing the two genes have been constructed suecessfully.