In Vitro Generation Of Humanized Monoclonal Antibody With Single-cell PCR Technology And Antibody Repertoire-Sequencing

Objective:Antibodies are commonly used in biochemistry,molecular biology and clinical medicine.43 monoclonal antibodies have been approved by the U.S.FDA for clinical use in the treatment of tumors,autoimmune diseases and infectious diseases[1,3]Since the hybridoma technology was invented by Milstein and Kohler in 1975,the development of new monoclonal antibody preparation technology[2],such as hybridoma technology,phage display technology,ribosome display technology,mRNA display technology,etc.These techniques have been widely used for screening the epitope of antigen.While both hybridoma technology and new development of all kinds of surface display technologies still can not meet the needs of a large number of clinical research and monoclonal antibody for its operation is complex and time-consuming.With the development of the next-generation sequencing technology,more and more genetic information is decoded[4].In this study,the latest methods of next-generation were applied in the preparation of humanized monoclonal antibody to improve the process of the production of humanized monoclonal antibody.Method:In this study,we combined single cell RT-PCR and antibody sequencing to synthesize humanized monoclonal antibodies.Human p53 protein expressed in prokaryotic expression system as an immunogen to immune 2 New Zealand rabbits.Then the peripheral blood was taken from the ear vein for isolation of B cells.Specific memory B cells were identified by flow cytometry and then split in 96-well plant and were reverse transcription into cDNA.Using antibodies specially designed primers combine with nested PCR to amplify variable region of the antibody genes.Then the variable region was cloned into a new eukaryotic expression vector to transfect 293T cells.After a certain period of time,the cell culture supernatant was collected to isolate and detect the antibody.Result:In our study,we rapidly synthesis of humanized monoclonal antibody which with biological function..In addition,through the sequencing,comparison and analysis of the antibody gene sequence information,we found that the mutation rate of the light chain genes in the New Zealand rabbits was significantly higher than that of the heavy chain compared with in humans and mice.It is suggested that the rearrangement of the light chain genes may also play an important role in the formation and evolution of the antibody in New Zealand white rabbits.