Abstract:
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 HEK 293T and PANC- 1 cells were detected by Western blot. The interaction of KRAS and "RBD+ODC" was detected by Co-IP. Results:Five eukaryotic expression plasmids were constructed successfully. Compared with the controls, "RBD+ODC" can "knockdown" the endogenous and ectogenous KRAS at the posttranslational level in HEK293T and PANC- 1 cells 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.