非泛素化蛋白降解体系靶向敲减KRAS癌蛋白的初步研究*

目的:尝试构建能与KRAS癌蛋白相互作用的“RBD+ODC”融合蛋白，经由“鸟苷酸脱羧酶/ 抗酶”系统（onithine decarboxylase/antizyme，ODC/AZ）在蛋白水平直接实现对KRAS癌蛋白的降解。方法:应用分子克隆的方法构建AZ、ODC 、“ RBD+ODC”、突变体“RBD+ODC”、突变体KRAS等真核表达质粒，分别瞬时转染人HEK 293T 和胰腺癌细胞PANC-1，应用Westernblot法在蛋白水平直接检测外源性和内源性KRAS表达水平的改变，应用Co-IP 法检测到“RBD+ODC”能够与KRAS癌蛋白相互作用。结果:成功构建了AZ、ODC 、“ RBD+ODC”、突变体“RBD+ODC”、突变体KRAS等多个真核表达质粒，相较于对照组，“ RBD+ODC”能够在蛋白水平直接实现对外源性和内源性KRAS癌蛋白的“敲减”，并且能够与KRAS有效结合。结论:靶向敲减KRAS癌蛋白的非泛素化蛋白降解体系的构建，为下一步功能试验奠定了基础。 

A preliminary study on targeted degradation of KRAS oncoprotein by using a ubiqui-tin-independent,proteasome-mediated degradation pathway"ODC/AZ"

Objective:To construct chimeric fusion proteins,"RBD+ODC,"which can bind to KRAS oncoprotein, and then"knockdown"the KRAS oncoprotein in PANC-1 cells via the"ODC/AZ"pathway. Methods:Five eukaryotic expression plasmids, including AZ, ODC,"RBD+ODC,"mutant"RBD+ODC,"and mutant KRAS, were constructed by molecular cloning and then transfected into HEK293T and PANC-1 cell lines transiently. The protein levels of KRAS in HEK293T and PANC-1 cel s were detected by Western blot. The interaction of KRAS and"RBD+ODC"was detected by Co-IP. Results:Five eukaryotic expression plasmids were constructed successful y. Compared with the controls,"RBD+ODC"can"knockdown"the endogenous and ectogenous KRAS at the posttranslational level in HEK293T and PANC-1 cel s separately. Furthermore,"RBD+ODC"can bind to KRAS effectively. Conclusion:The construction of ubiquitin-independent, proteasome-mediated degradation system targeted KRAS oncoprotein established the foundation for future studies.