title: Construction of chimeric E3s expression plasmids targeting oncoprotein ras.
abstract: OBJECTIVE
  To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.
authors.affiliation: Department of Pathology, PUMC Hospital, CAMS and PUMC, Beijing, China.
authors.forename: Yi-hui
authors.initials: YH
authors.lastname: Ma
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yu-mei
authors.initials: YM
authors.lastname: Gu
authors.forename: Chao-hui
authors.initials: CH
authors.lastname: Lu
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
mesh_terms: D003001:Cloning, Molecular; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D020726:ral Guanine Nucleotide Exchange Factor / Q000235:genetics; D018631:ras Proteins / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D020726:ral Guanine Nucleotide Exchange Factor; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D018631:ras Proteins