Preparation And Identification Of EGFRvⅢ Intracellular Protein Specific Single Domain Antibodies

Epidermal growth factor recetor variant Ⅲ(EGFRvⅢ),is a transmembrane glycoprotein with tyrosine kinase activity.EGFRv Ⅲ is highly expressed on the cell surface of glioblastoma,and intracellular signaling pathways are constantly activated,thus stimulating the abnormal proliferation of tumor cells.Moreover,EGFRv Ⅲ is not expressed in normal tissues,so the EGFRv Ⅲ intracellular region is used as an ideal target for intervention in the treatment of glioblastoma tumors.In this test,the EGFRvⅢ intracellular region recombinant adenovirus was used to immunize the one-year-old healthy bimodal camel,collected peripheral blood lymphocytes to extract total RNA,and then reverse transcribed cDNA.After PCR,the desired single-domain antibody gene was amplified,and it with the same vector p MECS was connected to construct the recombinant phage antibody display library.The commercial human EGFRv Ⅲ protein was screened by phage display technology,and then the screening results were identified by Phage ELISA,yielding positive clones with high binding force to the human EGFRv Ⅲ intracellular region protein.The p ET-22b vector was linked to the screened positive cloned gene,and after cold induction and soluble expression,the protein-specific single-domain antibodies against the EGFRv Ⅲ intracellular region were obtained,and the affinity activity was detected by ELISA.The test results are as follows:(1)After immunization with recombinant virus in EGFRvⅢ intracellular region,the serum antibody titer reached 1:800 was obtained,and then the EGFRv Ⅲ phage antibody library with storage capacity of 9.7×10~8and 95.8%was established,and 5 positive recombinant phages were selected.(2)Two VHH genes with different sequences were successfully obtained,and the plasmids of the two single-domain antibodies were cloned on the prokaryotic expression vector,and the EGFRvⅢ intracellular region-specific single-domain antibodies were expressed and purified.According to the ELISA results showed a high binding viability of the purified single-domain antibody and the EGFRv Ⅲ intracellular region proteins.Given the important role of the EGFR intracellular region genes in the signaling pathway,the protein-specific single-domain antibodies of the EGFRv Ⅲ intracellular region were successfully screened and prepared,providing a new direction for immunotherapy in glioblastoma.