Preparation And Characterization Of Shark Single Domain Antibody Against Transmembrane Activator And Calcium-Modulating Cyclophilin Ligand Interactor

Transmembrane activator and calcium-modulating cyclophilin ligand interactor(TACI)is a member of the tumor necrosis factor receptor(TNFR)superfamily and one of the receptors of B cell activation factor(BAFF).TACI is a regulator of immune response.It is one of the main targets for the treatment of autoimmune diseases.Single domain antibody derived from the serum of camelidae and cartilaginous fish.They have the advantages of small size,high stability,strong antigen-binding affinity,high water solubility,and easy production and expression.Single domain antibodies are the next generation of biological drugs with great potential.Chiloscyllium plagiosum has a high yield in Fujian and is an important economic species in China The preparation of single domain antibody by using this kind of fish can provide reference for the further development and application of its function.To prepare shark single-domain antibody targeting TACI,the human TACI extracellular domain coding region fragment was amplified by PCR with human c DNA as template.The gene fragment size was 126 bp.It was then ligated into a digested V29H vector to construct a prokaryotic expression vector.The recombinant prokaryotic expression strain was induced to express the soluble protein at 16°C for 16 h.After nickel column purification,the recombinant TACI protein with a molecular weight of about 8 k Da was obtained and it was further identified by mass spectrometry.The results were consistent with the target protein sequence.The recombinant TACI-V29H protein was used as an antigen to immunize C.plagiosum with three times subcutaneous injection of pectoral fin and two times tail vein injection.At the end of the immune cycle,the blood was collected from the tail vein of the shark to extract the total RNA of PBMC,which was reverse transcribed into c DNA.The 360 bp variable region of the shark antibody was cloned with c DNA as a template.After connecting with p R2 phage,it was electrically transformed into TG1 competent cell,and the library capacity was 2.2×10~6 CFU phage library and then panned the prepared library to prepare soluble monoclonal antibody fragments for monoclonal phage ELISA identification.The positive monoclonal phage was sequenced and analyzed to obtain the variable region sequences of two different shark-derived monoclonal antibodies targeting TACI.Then,the shark antibody variable region was re-amplified from the positive monoclonal phage plasmid,and ligated into the eukaryotic expression vector.The 293F mammalian cells were used to express the shark-derived single domain antibody protein with Fc tag with a molecular weight of about 40 k Da,they are named as T-35 and T-39.Finally,the binding activity of the two antibody proteins to TACI was identified by size exclusion chromatography.Non competitive ELISA and blitz biomolecular interaction analysis were used to evaluate the affinity.It was found that T-35 antibody had specificity for TACI,and the dissociation constant Kd was 2.36?mol/L.In conclusion,the extracellular domain protein of human TACI was obtained by prokaryotic expression,which was used as an antigen to immunize C.plagiosum.Antibody proteins with different sequences of two variable domain against TACI were prepared.It was found that T-35antibodies had a certain affinity for TACI,which provided a theoretical basis for the study of shark derived single domain antibodies.In this study,C.plagiosum was used as the object to explore the preparation process of shark monoclonal antibody and identify its activity,affinity,and other related properties.It provided a theoretical basis for the subsequent research and development of therapeutic drugs for autoimmune diseases,and the establishment of high-sensitivity monitoring methods for agricultural and veterinary drug residues in foods.