Delta4的功能性研究及与其他Notch配体的比较

Functional study of Delta4gene and the comparison with other Notch ligands

目的:探讨Delta4基因在Notch传导机制中的作用。方法:构建pTracer.CMV.Delta4.FLAG载体,瞬时转染COS7细胞、稳定转染CHO细胞,筛选高表达Delta4的稳定CHO细胞系。用Luciferase分析,观察并比较Delta4对Notch1-CHO及Notch2-CHO两个宿主细胞的信号功能活性,与Delta1、Jagged1及Jagged2比较,从而对此基因定性并获知其信号功能活性水平。结果:Western-blot证实pTracer.CMV.Delta4.FLAG可瞬时转染COS7细胞及稳定转染CHO细胞,并根据Delta4蛋白表达条带的强弱筛选高表达Delta4-CHO细胞系。Delta4对Notch1及Notch2均具有信号功能活性,且对后者的活性功能水平大于前者。对Notch1,Delta4的功能活性略低于Jagged2,但高于Delta1及Jagged1;对Notch2,Delta4的功能活性低于Delta1、Jagged1及Jagged2,但亦具有较强的活性水平。结论:建立的Delta4-CHO细胞系功能稳定,Delta4为Notch1及Notch2的配体,且对Notch2的活性水平高于Notch1。

Objective:To explore the function of Delta4in Notch signal transduction mecha-nism.Methods :The vector pTracer.CMV.Delta4.FLAG was constructed and transfected into COS7cells transiently,and then stably transfected into CHO cells.The high-expressing Delta4-CHO clones were set up and screened.Notch1-CHO and Notch2-CHO as the host cells,luciferase analysis was used to ob-serve and compare the signaling activity of Delta4.The signaling activity of Delta4was furthermore compared with Delta1,Jagged1and Jagged2to prove whether it is a ligand of the related host and get some knowledge of the level of its signaling activity.Results:After transiently transfecting COS7cells and stably transfecting CHO with pTracer.CMV.Delta4.FLAG,the existence of Delta4protein band was identified with Western-blot and CHO cell line expressing Delta4was set up.It was found that Delta4had signaling activity to both Notch1and Notch2.To Notch1,Delta4was slightly weaker than Jagged2,but stronger than Delta1and Jagged1.To Notch2,Delta4had a strong signaling activity,but weaker than Delta1,Jagged1and Jagged2.Conclusion:Delta4-CHO cell line is quite stable.Delta4is the lig-and to both Notch1and Notch2,with a stronger activity to Notch2than to Notch1.