Construction of chimeric E3s expression plasmids targeting oncoprotein ras.
- Author:
Yi-hui MA
1
;
Qiang ZHANG
;
Yu-mei GU
;
Chao-hui LU
;
Jie CHEN
Author Information
- Publication Type:Journal Article
- MeSH: Cloning, Molecular; Genetic Vectors; HEK293 Cells; Humans; Phosphatidylinositol 3-Kinases; genetics; Plasmids; Recombinant Fusion Proteins; genetics; metabolism; Transfection; Ubiquitin-Protein Ligases; genetics; ral Guanine Nucleotide Exchange Factor; genetics; ras Proteins; genetics
- From: Acta Academiae Medicinae Sinicae 2012;34(4):313-318
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown.
METHODSWe 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.
RESULTSFive 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.
CONCLUSIONSWe successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.
