带Flag标签的Sirt2真核表达载体的构建及其与PKM2的相互作用

Construction of Sirt2 eukaryotic expression vector with Flag tag and its interaction with PKM2

目的构建带Flag标签pcDNA3.0载体的沉默信息调节因子2(Sirt2)真核表达载体,验证其表达蛋白与M2型丙酮酸激酶(PKM2)的相互结合。方法利用聚合酶链反应(PCR)从人乳腺文库中扩增出人Sirt2的CDS编码序列,将扩增出的Sirt2基因片段插入双酶切后pcDNA3.0-Flag载体上,转化大肠杆菌感受态细胞DH5α后提取质粒,验证表达,测序正确后,分别转染pcDNA3.0-Flag/myc-PKM2和Flag-Sirt2/myc-PKM2共转染至人胚肾细胞(293T),通过免疫共沉淀实验证明Sirt2蛋白表与PKM2蛋白有相互结合。结果重组质粒Flag-Sirt2的基因序列与目的序列完全一致,Western印迹检测蛋白表达成功;免疫共沉淀实验证实Sirt2蛋白与PKM2蛋白有相互结合。结论成功构建了Flag-Sirt2真核表达载体,证实Sirt2蛋白与PKM2蛋白有相互结合,为进一步研究Sirt2在糖酵解中发挥的功能提供了基础。

Objective To construct a eukaryotic expression vector of silent information regulator 2(Sirt2)with Flag label pcDNA3.0,and to verify the binding of its expression protein with PKM2.Methods The CDS sequence of human Sirt2 was amplified from the human mammary gland library by polymerase chain reaction(PCR).The Sirt2 gene fragment was inserted into the pcDNA3.0-Flag vector after double enzyme digestion.The constructed gene was transformed into E.coli DH5α.The plasmid was extracted,verified,and the sequence was correct.pcDNA3.0-Flag/myc-PKM2 and FlagSirt2/myc-PKM2 were transfected into human embryonic kidney cells(293 T)respectively.The results of immunocoprecipitation showed that Sirt2 protein was bound to PKM2 protein.Results The gene sequence of the recombinant plasmid Flag-Sirt2 was identical with the target sequence,and Western blotting showed that the protein was expressed successfully.Immunocoprecipitation showed that Sirt2 protein and PKM2 protein were bound to each other.Conclusion The gene sequence of the recombinant plasmid Flag-Sirt2 is identical with the target sequence,and Western blotting shows that the protein is expressed successfully.Immunocoprecipitation shows that Sirt2 protein and PKM2 protein are bound to each other.