Cloning And Expression Of Human Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Screening Model To Build

The Epidermal growth factor receptor(EGFR) is a member of the receptor tyrosine kinase family(PTK).After binding with the ligand(EGF),the process of receptor dimerization,active tyrosine kinase and autophosphorylation has been induced in the intracellular domain.The genetic locus of human EGFR encoding the protein consisted of 1210 amino acid residues,located on the chromosome 7p12.3-p12.1.As a transmembrane glycoprotein with Mw 170 kDa,the tyrosine kinase domain situated on between 712 and 979 amino acid.The molecular structure of EGFR consists of an amino-terminal extracellular domain with 621 amino acid residues,a single transmembrane-anchoring region containing 23 amino acid residues,and a carboxylterminal intracellular domain with tyrosine kinase(TK) activity consisting of 542 amino acid residues.EGFR plays an essential role in the regulation of signal transduction in the cells.Due to the abnormal expression of EGFR-TK,the certain type cancers may developed and progressed.Based on that,the inhibitors of EGFR-TK could be effective medicines for the treatment of cancer.In this study,the gene encoding the catalyzing domain of EGFR-TK was amplified by RT-PCR with the RNA of HUVCEs cells as the template,which was cloned and expressed in E.coli BL21(DE3) using pET30a as vector.The recombinant protein EGFR-TK was purified with an affinity chromatography(Ni-NTA),which was identified to be the tyrosine kinase catalyzing the substrate phosphorylated with ATP in the enzymatic reaction.With the EGFR-TK as target,the screening model of enzymatic inhibitors was constructed,and some positive strains has been selected from more than 400 microbe strains.It will lay the foundation studying novel medicine for the treatment of cancer originated from microbe.