Construction of chimeric E3s expression plasmids targeting oncoprotein ras / 中国医学科学院学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown.</p><p><b>METHODS</b>We chose the binding domain of Raf-1, PI3K, RalGDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double enzyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression efficiency of the plasmids in eukaryotic cells was detected by Western blot analysis.</p><p><b>RESULTS</b>Five of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T cells, and (RBD+CRD)(Raf-1)- U-Box-pcDNA3.1 can knocked down the protein level of Ras in PANC-1 cells.</p><p><b>CONCLUSIONS</b>We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.</p>