Expression And Purification Of The Recombinant Human EGFR Extracellular Domain ? And Screening And Functional Identification Of Its Nanobody

Objective:The purpose of this study was to screen out the nanobodies against to EGFR-ECD₃ from the natural phage nanobody library for targeted therapy of cancer.Methods and Results:?1?Recombinant plasmid construction.Amplify the EGFR-ECD₃ gene fragment from the full length of the extracellular of EGFR using PCR technology,add different signal peptide sequences,and insert it to the eukaryotic expression vector p CMV.Recombination plasmids p CMV-EGFR-ECD₃ and p CMV-Gluc-EGFR-ECD₃were successfully constructed by colony PCR,double digestion and sequencing.?2?Extracellular segment expression.Transfect the recombinant plasmid into HEK293F cells transiently.The results show that the recombinant protein can be secreted and expressed in the supernatant.After large-scale transient transfer to HEK293F cells and subsequent purification using Ni-NTA affinity chromatography columns,high-purity EGFR-ECD₃ protein was finally obtained,with a purity greater than 90%.Using ELISA,CCK8 cell proliferation experiment and Western Blot experiments,it was proved that the obtained EGFR-ECD₃ has strong binding activity with EGF protein,its EC₅₀ is 0.034?mol/L,and it can effectively inhibit tumor cell A549proliferation caused by EGF?inhibition rate is 65%?and endogenous EGFR phosphorylation on its cell surface.?3?Nanobody screening.First,amplify the helper phage VCSM13,calculate its titer to 5×10¹² cfu/m L,use the purified EGFR-ECD₃ protein for encapsulation,and use the large-capacity natural phage nanobody library for the first round of amplification,after 3 rounds of scouring and enrichment verification,and then screening,a Nanobody was finally obtained and named Nb _H3.Bioinformatics analysis shows that Nb _H3 has a total of 122 amino acids,CDR1,CDR2,and CDR3contain 10,7,and 16 amino acids,respectively.Its molecular weight?Mw?is 13225.56,isoelectric point?PI?is 7.82,and the total average hydrophilicity?GRAVY?is-0.579,which is a hydrophilic protein.?4?Nanobody expression.Extract the p MECS-Nb _H3 plasmid and transfer it into E.coli WK6,induce its expression with IPTG at a final concentration of 1 m M,then extract Nb _H3 using osmotic shock method,purify it with Ni-NTA column.The Nb _H3 protein with a purity of 95%were verified by Western Blot experiment and grayscale scanning,and the protein yield was about 5 mg/L.?5?Nanobody biological and physical and chemical properties analysis.Nb _H3 affinity,binding specificity,and thermostability were detected by indirect ELISA;and its ability to compete with EGF for EGFR was tested by competitive ELISA.Affinity experiments show that Nb _H3can bind well with EGFR-ECD₃,and its EC₅₀ value is 0.486?mol/L;Specific experiments show that Nb _H3has a strong specific binding ability to EGFR-ECD₃,but has particularly weak ability to bind to other control antigens,especially it does not cross-bond with HER2 proteins of the same family;thermal stability experiments have proven that Nb _H3 has a clear advantage at a temperature of 60?and above for a long time;competitive ELISA shows that Nb _H3 can compete with EGF and bind to EGFR with a competition rate of about 25%.?6?Nanobody biological activity analysis.CCK8 experiments show that Nb _H3 can compete with EGF to bind to EGFR on the cell surface,thereby inhibition the proliferation of A549 cells caused by EGF,with an inhibition rate of 30%;phosphorylation experiments show that Nb _H3 can inhibit EGF-induced Phosphorylation of EGFR on the cell surface;using immunofluorescence and flow cytometry experiments,it was shown that Nb _H3 can better recognize EGFR on A549 cell membrane.Conclusions:In this paper,the EGFR-ECD₃ plasmid was successfully constructed and transfected into the eukaryotic expression system HEK293F.The EGFR-ECD₃ recombinant protein with high biological activity and high purity was finally purified.After three rounds of scouring from the phage nanobody library a nanobody was screened.It has the advantages of certain affinity,high thermal stability and strong specificity,and has binding ability with the EGFR protein on the A549cell membrane.In summary,the screened Nanobodies have the potential to develop into EGFR inhibitors,providing material for their subsequent application development.