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目的构建能在酵母菌AH109中表达的mSOD1cDNA的穿梭表达质粒pGBKT7-mSOD1cDNA,研究突变SOD1编码蛋白的蛋白质-蛋白质相互作用。方法通过PCR法以真核表达载pEGFP-mSOD1cDNA为模板,扩增获得了120bp的mSOD1基因的末端及DNA结合域的片段,经过SalⅠ、EcoRⅠ双酶切后,基因重组的方法定向亚克隆于酵母双杂交载体pGBKT7中,构建形成pGBKT7-mSOD1真核表达载体。结果经转化,提取质粒双酶切,核苷酸测序,BALST比对鉴定表明构建成功。结论成功构建穿梭质粒pGBKT7-mSOD1cDNA,为深入研究突变的SOD1基因编码蛋白的蛋白质-蛋白质相互作用奠定了基础。
Objective To construct a shuttle expression plasmid pGBKT7-mSOD1 cDNA of mSOD1 cDNA expressed in yeast AH109 to study the protein-protein interaction of mutant SOD1 protein. Methods The eukaryotic expression vector pEGFP-mSOD1 cDNA was used as a template to amplify a 120 bp fragment of the mSOD1 gene and its DNA binding domain. After digestion with Sal Ⅰ and EcoR Ⅰ, the recombinant plasmid was subcloned into yeast Two-hybrid vector pGBKT7 was constructed to construct pGBKT7-mSOD1 eukaryotic expression vector. The results of transformation, double digestion of plasmid extraction, nucleotide sequencing, BALST identification showed that the construction was successful. Conclusion The shuttle plasmid pGBKT7-mSOD1 cDNA was successfully constructed, which lays the foundation for further study on the protein-protein interaction of mutated SOD1 gene.