pIRES-IL-24-PTEN双表达载体的构建及鉴定

目的构建携带人IL-24和10号染色体缺失的磷酸脂酶与PTEN双基因的真核表达载体,为研究其表达产物的抑癌机制提供基础。方法通过PCR方法获得PTEN、IL-24基因片段,进行T-A克隆,再酶切亚克隆到pIRES载体,酶切、测序鉴定正确后命名为pIRES-IL-24-PTEN载体。脂质体转染A549细胞,提取细胞的总蛋白,Western blot检测IL-24和PTEN蛋白的表达。结果酶切、测序显示pIRES-IL-24-PTEN载体序列正确,Western blot检测到IL-24和PTEN蛋白表达。结论成功构建pIRES-IL-24-PTEN载体,为后续研究奠定基础。

Construction and identification of pIRES-IL-24-PTEN bicistronic eukaryotic expression vector

Objective To clone IL-24 and PTEN genes and construct a eukaryotic co-expression vector pIRES-IL-24PTEN to provide research basis for investigation of the association between these two genes.Methods cDNA of IL-24 and PTEN were amplified by RT-PCR,then inserted into vector pIRES.The positive clones were verified by enzyme digestion and sequencing.Then the vector was transfected to A549 lung cancer cells by lipofectamine.Total cellular protein was extracted and western blot analysis was performed to detect the expression of IL-24 and PTEN.Results Results of enzyme digestion and sequencing analysis demonstrated that the recombinant plasmid was constructed successfully.Expression of IL-24 and PTEN were detected after transfection of the recombinant plasmid into A549 cells.Conclusion We have sucessfully constructed pIRES-IL-24-PTEN vector,which provides a basis for combined gene threapy of cancers.