【摘要】 目的:构建真核表达载体pcDNA3.1(+)-RUNX3,并在乳腺癌T47D细胞株中表达。方法:应用基因重组技术和限制性内切酶EcoRI和XhoI酶切,构建并鉴定pcDNA3.1(+)-RUNX3真核表达载体,经脂质体Lipofectamine2000介导质粒转染T47D细胞后应用逆转录-聚合酶链反应(RT-PCR)和Western blot实验,检测RUNX3在T47D细胞中的表达。结果:重组真核表达载体pcDNA3.1(+)-RUNX3经限制性内切酶EcoRI和XhoI酶切,电泳后显示1.3kb的RUNX3目的片段和5.4kb的pcDNA3.1(+)载体片段。测序证实酶切片段与gene bank中登记的RUNX3序列相同,证实pcDNA3.1(+)-RUNX3真核表达载体构建成功。经RT-PCR和Western blot检测,表明转染pcDNA3.1(+)-RUNX3的T47D细胞RUNX3阳性表达。结论:重组真核表达载体构建正确,并建立稳定表达RUNX3的T47D细胞系,从而为后续研究提供有用的细胞研究模型。更多还原

【Abstract】 Objective: To construct the eukaryotic expression vector pcDNA3.1(+)-RUNX3 and to express it in human breast cancer T47D cell line.Methods:Eukaryotic expression vector for pcDNA3.1(+)-RUNX3 was constructed by use of recombinant DNA technique and was transfected into T47D cell by Lipofectamine 2000.The mRNA and protein expression of RUNX3 gene was detected by RT-PCR and Western blot methods.Results:The recombined eukaryotic expression vector for RUNX3 was digested with EcoRI and XhoI,and the electrophoresis of digested products showed two fragments: 1.3kb fragment for RUNX3 and 5.4kb fragment for pcDNA3.1(+).The sequence of DNA fragment was identified to that published in Genebank.The RUNX3 gene showed positive expression in transfected T47D cell line by RT-PCR and Western blot.Conclusion:Eukaryotic expression vector pcDNA3.1(+)-RUNX3 was constructed and RUNX3 could be expressed in human breast cancer T47D cell line.This provides useful cell model for further research.更多还原