小鼠Cdc25B基因的真核表达载体构建及鉴定

Construction and identification of eukaryotic expression vector of mouse Cdc25B gene

目的构建含有小鼠Cdc25B基因的真核表达载体pcDNA3.1-3xflag-Cdc25B,并观察和验证其在真核细胞中的表达。方法目的基因Cdc25B经化学合成后,定向克隆至pcDNA3.1(+)真核表达载体中,经过限制性内切酶KpnⅠ和ApaⅠ进行双酶切和测序鉴定,构建真核表达载体pcDNA3.1-3xflag-Cdc25B,应用脂质体法转染HEK293细胞,通过Western blot检测细胞内Cdc25B的表达。结果pcDNA3.1-3xflag-Cdc25B真核表达载体构建成功,将其转染HEK293细胞48 h,提取细胞蛋白,利用Western blot检测到细胞内Cdc25B的表达,并且测序结果与预期结果一致。结论成功构建了真核表达载体pcDNA3.1-3xflag-Cdc25B。

Objective To construct the eukaryotic expression vector of mouse pcDNA3.1-3xflag-Cdc25B gene,and observe and verify its expression in eukaryotic cells.Methods The target gene Cdc25B was synthesized by chemical synthesis and cloned into eukaryotic expression vector pcDNA3.1(+).After the restriction enzyme KpnⅠand ApaⅠdigestion and sequencing,the gene was transformed into HEK293 cells by lipid method,and the expression of Cdc25B in HEK293 cells was detected by Western blot.Results The eukaryotic expression vector pcDNA3.1-3xflag-Cdc25B was constructed successfully.The expression of Cdc25B in HEK293 cells was detected after transfection for 48 h by Western blot,and the sequencing results were consistent with the expected results.Conclusion Eukaryotic expression vector pcDNA3.1-3xflag-Cdc25B is successfully constructed.