Screening,preparation And Identification Of Human CD20 Specific Single Domain Antibody

CD20 molecule is a specific membrane protein present in a non-glycosylated form on the surface of B-lymphocytes,and this antigen is expressed in B-lymphocytes but not in haematopoietic stem cells,plasma cells.CD20 molecules play an important role in the treatment of B-lymphoma because binding to antibodies does not lead to antigenic modulation but also to significant internalisation and shedding.The experiment began with immunisation of adult Bactrian camels with a human CD20 extracellular region peptide and the CD20-specific single domain antibody gene obtained was transformed into E.coli TG1,thereby constructing a phage antibody library.Three rounds of enrichment and screening were performed with commercial human CD20 protein,and recombinant phages with high binding activity to human CD20 extracellular region polypeptide were identified by Phage ELISA.The positive clone was ligated into the prokaryotic expression vector p ET-25b-SBP and soluble expression was performed by low temperature induction.Soluble anti-CD20-specific single domain antibodies were obtained and their affinity activity was identified by ELISA and Western blot.The CD20-specific monodomain antibody was also investigated by Cellular immunofluorescence(CIF)assay,which demonstrated that it could also bind well in eukaryotic cells.The following are the results of the experimental studies.(1)CD20 protein was used as the antigen to immunize camels,and the serum antibody potency of 1:32,000 was measured,and the CD20 phage antibody library was successfully established.The antibody library capacity was 1.2×108 and the recombination rate was 91.7 %,and 54 positive recombinant phages were obtained after screening.(2)Six different sequences of VHH were successfully obtained,and the plasmids of these six single-domain antibodies were cloned into the prokaryotic expression vector to express and purify CD20single-domain antibodies.ELISA and Western-blot results showed that the purified single-domain antibody and CD20 antigen had some high binding activity.Cellular immunofluorescence confirmed the binding of single domain antibody to CD20 antigen on the cell surface.This experiment provides a better basis for the development of single-domain antibodies that could be useful in the immunotherapy of B-cell lymphoma.