Preparation And Identification Of EGFRvⅢ Extracellular Domain Specific Single Domain Antibody

Epidermal growth factor receptor(EGFR)is a transmembrane tyrosine kinase receptor in the ErbB receptor family.It is only expressed on the surface of epithelial cells in normal human tissues.EGFR is significantly expressed in most glioblastoma(GBM).When EGFR is activated,downstream signaling pathways and protein kinases are activated successively,and initiating the regulation of related genes and regulating the proliferation and death of tumor cells.Epidermal growth factor receptor TypeⅢ mutant(EGFRvⅢ)are the most common in GBM.The studies indicated that EGFRvⅢ is not expressed in normal cells and is only expressed in cancer cells makes it an ideal biomarker for cancer therapy.With the deepening of the research on EGFRvⅢ and its signaling pathway,it is gradually confirmed that the abnormal expression of EGFRvⅢ in malignant tumor tissues is directly related to the degree of malignancy and the signaling pathway.Single domain antibody(sd Ab),with a molecular weight of 12-15 kDa.It’s a special dimer antibody.Compared with the traditional monoclonal antibody(150 kDa),sdAb has the characteristics of small size,high affinity,easy expression,good permeability,high stability(heat,pH and fluid free medium),and low production cost.Based on this,we immunized healthy bactrian camels and measured the antibody titer of EGFRvⅢ extracellular region protein.Blood was used to separate lymphocytes and reverse transcription cDNA.EGFRvⅢ extracellular region protein single domain antibody library was successfully established by linking the VHH fragment with pMECS vector.In the subsequent tests,EGFRvⅢ extracellular region protein was used as screening antigen to screen EGFRvⅢ extracellular region protein single domain antibody library,and a positive clone with relatively strong binding ability to EGFRvⅢ extracellular region protein was selected from Phage ELISA.Through plasmid recombinantion,the single domain antibody gene fragment was ligated into the expression vector pET-22 b,and transformed into E.coli BL21(DE3)receptor cells for expression and purification,so as to obtain high binding activity of EGFRvⅢ extracellular region protein-specific single domain antibody.The single domain antibody specific for EGFRvⅢ extracellular region was prepare in this study provides a new choice for the diagnosis and treatment of GBM with EGFRvⅢ as the target.