Project Epidermal Growth Factor Receptor Tyrosine Protein Kinase Inhibitor Screen Model

Epidermal Growth Factor receptor(EGFR)Belongs to Tyrosine Protein Kinase(TPK)and catalyzes tyrosine phosphorylation. EGFR adjusts cell proliferation,apoptosis and differatiation by cell signal transduction. EGFR's over-expression is in many cancer cells. The over-expression enhances the TPK activity and it is correlated with many tumors. It is necessary to project EGFR/TPK assay model to screen its inhibitor to explore and discover anticancer drug. Firstly, 1)prepared the tumor cell plasma membrane that high-expressed EGFR to supply EGFR, and constructed the fusion protein-GST-C25 as substrate. Added the EGFR/TPK inhibitor-AG1478 and assayed EGFR catalyzed GST-C25 phosphorylation by Western blot. The results displayed EGFR that came from cell plasma membrane catalyzed GST-C25 phosphorlation and the phosphorylation was blocked by AG1478. Then, 2) based on the western blot to project EGFR/TPK time resolved fluorescence Immunological assay (TRFIA). Prepared the minim EGFR/TPK reaction system and added it to the plate that has coated the anti-GST antibody. The phosphorylated GST-C25 was fixed the plate by special binding of anti-GST and GST-C25, added the Eu-antiPY20 to bind the phosphorylated GST-C25 and washed out the remainder Eu-antiPY20. Measured the Eu3+ fluorescence intensity. The results displayed the fluorescence intensity increased by activated EGFR/TPK catalysed GST-C25 phosphorylation and decreased by added special EGFR/TPK inhibitor—AG1478. The more of the intensity, the more of EGFR activity and the less of the drug inhibition. Finally 3)applied the EGFR/TPK TRFIA to screen about 100 Chinese medicine purified compounds, and found the inhibition ratio of four compounds was high. The assay model is helpful to research and discover the EGFR/TPK inhibitors for Chinese medicines. Compared the common EGFR activity assay, such as radioactive method or ELISA,TRFIA is more safe and simple. Because of preparing the cell plasma membrane for supplying EGFR, it is cost-low. The sensitivity is increased by applying Eu3+ and the enhancement solution. What's more, The TRFIA is more special for constructing GST-C25 as substrate that contains auto-phosphorylation site. The method is adapt to screen not only synthesized compounds but also Chinese medicine crude purified compounds.