| C-type lectins（CTLs）are expressed in various immune cells and are involved in the body’s immune defense,surveillance,regulation,and other responses.CTL antibodies are essential molecular tools for studying CTL function and are important for elucidating the role of CTLs in regulating the body’s self-stability,immune defense,and the development of preventative and therapeutic strategies for infectious diseases.Compared to natural antibodies,single-chain fragment variable（scFv）antibodies have the advantages of small molecular weight,strong tissue penetration,low immunogenicity,high specificity,high expression levels,and low production costs.They have broad applications in targeted therapy and diagnostic analysis.Establishing a rapid screening and preparation method for CTL-scFv will provide effective molecular tools for studying CTL function using immunological methods.In this study,we successfully constructed a phage-displayed scFv library using the total protein of East Asian migratory locust hemocytes and screened high-specificity CTL-scFvs from the library using recombinant CTL proteins as targets.The main research results are as follows:1.Construction of a phage-displayed scFv library using the total protein of East Asian migratory locust hemocytesMice were immunized with the total protein of East Asian migratory locust hemocytes,and the resulting antiserum had an efficacy of 1:128,000.Total RNA was extracted from the spleens of the immunized mice,and scFv fragments were obtained using RT-PCR and overlap PCR.The p CANTAB5E-scFv/TG1 vector was constructed using DNA recombination technology and then packaged into phages to produce the East Asian migratory locust hemocyte total protein scFv phage library.The titer of the library was3.48×10^7 pfu.Colony PCR detection and enzymatic digestion identification showed that the positive rate of recombinant clones containing inserted scFv was 100%,indicating the successful construction of the East Asian migratory locust hemocyte total protein scFv phage library.Gene sequence analysis of randomly selected clones showed that the homology between the clones ranged from 37.71%to 91.09%.2.Expression and purification of CTLp ET30a（+）-CTL was induced for expression in BL21（DE3）cells and the CTL recombinant protein was purified using a Ni-NTA column.SDS-PAGE analysis showed that the purity of the purified protein was 82%.3.Enrichment and screening of CTL-scFvTwo methods were used for the screening of CTL-scFv.The first method was magnetic bead conjugation.Carboxyl magnetic beads（MSP-COOH）were conjugated with the antigen.The enrichment screening was carried out in rounds with increasing concentrations of Tween-20（0%,0.1%,0.2%,0.3%）.The recovery rate for each round was 2.23×10-1,1.36×10-3,5.51×10-4,3.09×10-4,and 2.82×10-4respectively.The second method was antigen coating on a 96-well plate.The CTL recombinant protein was coated in a 96-well plate for phage screening.The enrichment screening was carried out in rounds with increasing concentrations of Tween-20（0-0.3%）.The recovery rate for each round was 1.28×10-1,3.23×10-1,7.25×10-2,3.16×10-2,and 2.56×10-1respectively.The average recovery rate for this method was 4.3 times higher than that of the magnetic bead conjugation method.4.Identification of CTL-scFvAfter screening,the monoclonal antibody positivity rate of the magnetic bead-coupling scFv library was 60%,and the monoclonal antibody positivity rate of the 96-well plate-coated screening method was 100%.It is worth noting that magnetic bead-coupling screening is prone to loss of scFv in the library,and the 96-well plate-coated screening method is more stable than magnetic bead-coupling screening.Analysis of the single clone plasmid sequences of the magnetic bead-coupling method showed that the homology between the sequences of each clone after screening was 77.73-98.84%,and analysis of the single clone plasmid sequences of the 96-well plate-coated screening method showed that the homology between the sequences of each clone after screening was 81.86-95.84%.The homology of both screening methods increased after screening,indicating that the common sequence of CTL-scFv was effectively enriched.SDS-PAGE electrophoresis showed successful expression of scFv in the CTL-scFv/HB2151 periplasmic space,with a molecular weight of 31 k Da.5.ELISA activity assay of CTL-scFvELISA assay was performed on the monoclonal periplasmic products of the fifth round of screening of the scFv library obtained by the 96-well plate antigen-coated screening method.The average activity of CTL-scFv obtained in the fifth round of screening was 2.2 times that of the initial scFv,with NT2 exhibiting an activity three times that of the initial scFv.6.Western blot specificity assay of CTL-scFvWestern blot detection of CTL-Sc FV（NT2）showed that CTL-Scf V（NT2）had good specificity for CTL recombinant protein,and could detect CTL protein in total locust blood cell protein,but the specificity needed to be improved. |
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