Expression And Purification Of The Recombinant Human CTLA-4 Extracellular Domain And Screening And Functional Identification Of Its Nanobody

Objective:The aim of this study was to screen Nanobodies binding to CTLA-4 through a large-capacity phage Nanobody library,laying the material foundation for its subsequent application and development.Methods and Results:（1）The pCMV-CTLA-4 ECD recombinant protein expression plasmid was obtained by looking up the CTLA-4 extracellular domain（CTLA-4 ECD）sequence in the Uniprot protein database and synthesizing it into the pCMV eukaryotic expression vector.The pcDNA3.4-CTLA-4 ECD recombinant protein expression plasmid was subcloned by PCR amplification,digestion,ligation,transformation and other techniques.（2）Comparing the expression levels of the two recombinant plasmids with a small amount of transient transformation,confirming that a large amount of pCMV-CTLA-4 ECD was transiently transferred into HEK 293F cells,and purified by Ni-NTA affinity chromatography column,and finally obtained with CTLA-4 mAb has a CTLA-4 ECD protein with binding activity and protein purity greater than 90%.（3）Firstly,the helper phage VCSM13 was amplified,and the helper phage titer was 5×1012cfu/mL.The purified CTLA-4 ECD protein was used for encapsμLation,and the large-volume natural phage Nanobody library was used to expand the first round.Increase 3 rounds of panning,and finally screening.Finally,three Nanobodies with differences in the nanobody sequences were obtained,which were designated as Nb_{CTLA-4 ECD} and named as F2,E3,and G4,respectively.The physicochemical properties of Nb_{CTLA-4 ECD} were analyzed by software.The molecμLar mass was in the range of 17.4-18.1 kDa,and the theoretical isoelectric point was in the range of 6.27-9.3.Because the GRAVY of all three antibodies was less than 0,it was a hydrophilic protein..（4）A small amount of pMECS-Nb_{CTLA-4 ECD} was extracted and electroporated into E.coli WK6,and the expression of the strain was induced by IPTG at a final concentration of 1 mM,and then the nano-antibody was extracted by high osmotic pressure difference method,and then Ni-NTA column was used for protein purification,finally obtaining Nb_{CTLA-4 ECD} protein purity greater than 93%.The protein yield of the three strains of Nb_{CTLA-4 ECD} WK6 ranged from 4.66 to5.84 mg/L,with the highest yield being G4,reaching 5.84 mg/L.（5）The specificity and thermal stability of Nb_{CTLA-4 ECD} were determined by ELISA.Specific experiments showed that all three Nb_{CTLA-4 ECD} had strong specific binding ability to CTLA-4 ECD,and had weaker binding ability to other six control antigens,among which G4had the strongest binding ability.Thermal stability experiments have shown that Nb_{CTLA-4 ECD} has a significant advantage over the positive control CTLA-4 mAb at 60°C.（6）Using A375 melanoma cells to study the specific binding activity of three Nanobodies to CTLA-4 on the cell membrane.Flow cytometry showed that all three Nanobodies could recognize CTLA-4 expressed on the surface of cell membrane,and G4 The highest binding activity.Conclusions:In this paper,the eukaryotic expression plasmid was successfully constructed and purified by fermentation to obtain the recombinant protein of CTLA-4 ECD with binding activity and purity greater than 90%,and the obtained antigenic protein was obtained.Three strains were obtained from the phage Nanobody library.Nanobody that binds to CTLA-4 ECD.It has the characteristics of small molecular weight,high thermal stability and strong specificity,and has certain binding activity to CTLA-4 protein on A375 cell membrane.In summary,the three selected Nanobodies have been developed to block the inhibitory potential of CTLA-4,laying a material foundation for its subsequent application development.