Screening Of Single Chain Variable Fragments To EGFR Protein P.T790M

Purpose:To screen the human phage antibody library by subtractive competition screening method to obtain the single chain variable fragments(sc Fvs)against the biotinylated epidermal growth factor receptor(EGFR)p.T790M mutant antigen.The affinity and specificity of the antibodies screened were verified by indirect enzyme-linked immunosorbent assay(ELISA),and positive monoclonals with high affinity and specificity were selected for sequencing to obtain their corresponding gene sequence information.This information can provide a basis for the subsequent construction of CAR-like molecules.Methods:1.Design and synthesize a biotinylated EGFR p.T790M mutant protein containing Avi-tag and His-tag by querying the genetic information of human EGFR protein in Gen Bank.2.The fully human sc Fv phage antibody library,which has undergone titer determination and library diversity detection,is screened by subtractive competition screening method with the help of streptavidin-biotin affinity screening system to obtain the secondary phage antibody library targeting biotinylated EGFR p.T790M mutant protein.3.Apply part of the secondary phage antibody library targeting biotinylated EGFR p.T790M mutant protein on the 2YTAG plate to pick out monoclonal strains for indirect ELISA identification of monoclonal bacteria liquid.Compare the absorbance OD450 value of biotinylated EGFR p.T790M mutant protein and wild-type EGFR protease mark hole,pick the positive clones with absorbance≧2 times and send them for sequencing,analyze and obtain the gene sequence of the sc Fv.Result:1.The human EGFR p.T790M mutant protein containing Avi-tag and His-tag and biotinylated human EGFR p.T790M mutant protein was obtained.2.By using the biotinylated EGFR p.T790M mutant protein as the target antigen,the fully human sc Fv phage display library was panned using the subtractive competition screening method,and secondary phage antibody library targeting the EGFR p.T790M mutant protein was obtained.Storage capacity of secondary phage antibody library was about 1×10~4pfu.3.260 single clones from the phage culture plate screened by the subtractive competition screening method were picked randomly.The indirect ELISA verification of the monoclonal phage was carry out.By comparing the absorbance OD450 value of the enzyme-labeled hole coated biotinylated EGFR p.T790M mutant protein and wild-type EGFR protein(the OD450 value of the enzyme-labeled hole coated with the mutant antigen/the wild-type antigen≧2 times representing a positive clone),and 4 positive clones were obtained.By sequencing and analyzing the 4 positive monoclonals,4humanized sc Fv gene sequences targeting EGFR p.T790M mutant protein were obtained.Conclusion:The biotinylated EGFR p.T790M mutant protein was panned using the subtractive competition screening method,and the secondary phage antibody library targeting EGFR p.T790M mutant protein was obtained.Storage capacity of secondary phage antibody library was about 1×10~4pfu.Through indirect ELISA identification of phage antibodies in the secondary phage antibody library obtained by screening,4humanized sc Fvs targeting EGFR p.T790M mutant protein with high specificity were obtained.