人DR5真核表达载体的构建及稳定转染NS-1细胞系的建立

Construction of eukaryotic expression vector of human DR5 and establishment of its stable transfected NS-1 cell line

目的:构建人死亡受体5(DR5)真核表达载体,转染NS-1细胞,建立稳定转染的NS-1细胞系。方法:采用RT-PCR方法,以人Jurkat细胞cDNA为模板扩增人DR5基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.0,经酶切和测序鉴定后,用脂质体转染法转染NS-1细胞,通过G418筛选,建立稳定转染的NS-1细胞系,用FACS法检测DR5的表达。结果:成功构建了pcDNA3.0/DR5真核表达载体,并建立了稳定转染的NS-1细胞系,成功地表达目的基因。结论:真核表达载体成功构建和稳定转染NS-1细胞系的建立为进一步进行基因治疗研究奠定良好的实验基础。

Objective： To construct eukaryotic expression vector of human death receptor（DR5） and transfect NS-1 cells to establish stable NS-1 cell line. Methods： The full-length DR5 cDNA fragment was amplified by RT-PCR from the human Jurkat cells and was inserted into eukaryotic expression vector pcDNA3.0.After the identification by digestion and sequencing on the recombinant eukaryotic expression vector pcDNA3.0/DR5,the recombinant was transfected into NS-1 cell by lipofectamine 2000.After screening culture by G418,stable transfected NS-1 cell line was established,and the transcription and expression of DR5 were identified by FACS. Results： The eukaryotic expression vector pcDNA3.0/DR5 was constructed successfully.The stable transfected NS-1 cell line was established.The DR5 protein was expressed successfully. Conclusion： The construction of the eukaryotic expression vector pcDNA3.0/DR5 and the establishment of stable transfected NS-1 cell line provide solid foundation for further experimental studies.