Cloning,in Vitro Expression,and Verification Of Monoclonal A Blood Group Antibody

Objective: in vitro expression and verification of our previously screened A blood group antibodies(including 12 heavy chains and 8 light chains)by high-throughput sequencing and mass spectrum,which will pave the way for A blood group antibody repertoire identification in ABOi-KT patients and provide new methods for immune accommodation research.Materials and methods:(1)construct vectors for antibody light chain and heavy expression: p VITRO1-Trastuzumab-Ig G1/??p VITRO1-102.1F10-Ig M/? were used as template,and PCR was performed and cyclized to generate four vectors p61883Hg?p61883Lk?p61880Hm?p61880La,which were used for Ig G heavy chain,kappa chain,Ig M heavy chain,lambda chain expression respectively.(2)Transfer previous synthesized 20 variable region of antibodies to above vectors: PCR was performed using designed primers to amplify vectors and variable regions.The amplified vector fragment and variable region were recombined with a kit.The combined product was transformed to DH5 alpha competent cells.PCR was performed to screen positive colonies.Sequencing was performed to make sure the plasmid is right.(3)Identification of antibodies.12 heavy chains and 8 light chains were paired to each other,which makes 96 combinations.The 96 combinations were transfected to four 24-well dishes with 293 cells.The supernatant which contain antibodies were collected.Slot blot was performed to identify real A antibodies.Results:(1)restriction enzyme double digestion was performed.Electrophoresis showed that there are predicted bands in all four vectors p61883 Hg,p61883Lk,p61880 Hm and p61880 La,indicating cloning success.(2)Several colonies grow out in each dish,PCR screened out positive colonies in each dish.Sanger sequencing of these colonies indicated right insertion.12 heavy chains and 8 light chains were all successfully cloned to expression vectors.(3)Slot blot screened out at least one specific A blood group antibodies,several others with reaction to KLH,the coupled matrix protein of A trisaccharide.Conclusion:Antibodies expression in 293 cells was performed successfully,and A blood group antibodies were identified,which proved the feasibility of constructing A blood group antibody repertoire in peripheral blood of B blood group individual.