Construction And Screening Of Phage Display Single Chain Antibody Library Against Oriental Migratory Locust TLR7

TLR7 is a member of the TOLL family and has an important role in the specific detection of single-stranded viral RNA,induction of IFN-involved pro-inflammatory cytokine release and regulation of the body’s immunity.Single-chain fragment variable（scFv）is a genetically engineered antibody with smaller molecular weight,better tissue penetration,shorter half-life,easier metabolism,and less likely to accumulate in vivo than natural antibodies.In addition,scFv retains only the variable region of antigen recognition without Fc region,which makes it highly specific and low immunogenicity,and has wide application prospects as antibody drugs and immunoassay tools.The main work of this study includes:The results obtained provided a good basis for the subsequent exploration of TLR7 immune-related functions using the East Asian locust as a model.1.Construction of a TLR7-scFv phage display library of the East Asian flying locustTLR7 recombinant protein was purified from the cultured TLR7 genetically engineered strain,and mice were immunized at 100μg TLR7/each/each for four times between weeks.The serum was collected and the potency was measured to be greater than 64000.Total RNA was extracted from mouse spleen tissue,and scFv was obtained by RT-PCR amplification and fragment fusion.p CANTAB5E vector was ligated to the scFv gene,and TG1 cells were transformed by electrical excitation to construct the East Asian flying locust p CANTAB5E-scFv/TG1 library.The TLR7-scFv phage library was then phage packed and obtained with a titer of 8.9×10¹⁰ pfu.Randomly selected library transformants were identified by colony PCR and enzyme digestion,and the results showed that the recombination rate of the examined transformants was 100%,suggesting successful library construction.2.Enrichment and screening of TLR7-scFvThe ELISA method was used to encapsulate the purified TLR7 recombinant protein,and phage elution was performed at increasing concentrations of 0.1%,0.2%,and 0.3%Tween in that order.After three rounds of screening,the recovery rate stabilized.The results of the third round of screening were identified by colony PCR and enzyme digestion,and the positive rate of the tested monoclonal was 100%.The plasmids of 50 positive monoclonal strains were randomly selected for sequencing and five different sequences were obtained.Homology analysis showed that the homology of these five sequences ranged from 81.23%to 89.49%,with the highest percentage of plasmids having the same sequence being 53%.3.ELISA activity assay of TLR7-scFvThe monoclonal TLR7-scFv of five different sequences obtained from the third round of screening was subjected to small expression in the periplasmic space.TLR7-scFv activity was detected by sandwich ELISA using strain HB2151 as a negative control,and the highest scFv-9 activity was found.This was then validated with an indirect ELISA,and the results were consistent with the sandwich ELISA.4.Western blot specificity assay of TLR7-scFvWestern blot of scFv-9,which had the highest ELISA activity,showed that scFv-9 could bind specifically to TLR7 recombinant protein.