The Screening And Identification Of The Single Chain Fragment Variable Of Protein EGFR's Mutation At Locus L858R

Objective:To construct a phage display library of humanized single chain fragment variable(SCFV),and then conduct titer test and multivariate analysis on the antibody to eventually obtain the sequence of the SCFV that mutates at locus L858R of the targeted epidermal growth factor receptor(EGFR),which shows higher affinity,through affinity selection.Methods:1.Collect 10 samples of venous blood from different healthy donors;extract lymphocytes from the samples to get total RNA;obtain cDNA through reverse transcription;increase,via the method of PCR,the quantity of the genes from the SCFV's variable of heavy chain(VH)and of light chain(VL)to form the SCFV;and then connects it with the phage carrier of pComb3 enzyme and inject them into the escherichia coli TG1 after electro-transformation,eventually constructing a phage display library of natural humanized SCFV by assisting the role of phages and the function of packaging;2.Take EGFR-L858R as the targeted antigen,and by adopting the method of acid elution to conduct several rounds of panning through steps of combination,elution,and increase;3.Conduct enzyme linked immunosorbent assay(ELISA)on the monoclonal phages that have been selected from previous steps,and send those that are positive to sequencing company for further analysis.Results:1.A phage display library of natural humanized SCFV that has a capacity of 1×10~9 is successfully constructed,while the library's positive rate and the performance of the multivariate analysis on it meet the standard.2.Take EGFR-L858R protein as the target antigen,10 positive monoclones were obtained through 2 rounds of enrichment screening and preliminary identification by pickling and stripping.3.By analyzing the 10 strains of monoclonal phages,6 sequences of the SCFV that mutates at the locus L858R of protein EGFR will be obtained.Conclusion:In this study,the whole human SCFV phage antibody library was successfully constructed.After enrichment screening and preliminary identification,the sequence of single-chain antibody targeting EGFR protein L858R site mutation was obtained.It can be further applied to the immunotherapy of related malignant tumors,providing a new idea and material basis for the treatment of solid tumors with CAR-T therapy.