Preparation And Identification Of EV71Humanized Genetically Engineered Antibody

Antibody technology is an integral part of modern life science technology, whichplays a vital role in the gene-researching, protein structure and function research andimmunological diagnosis. In1975， Kohler and Milstein etc. using hybridomatechnology to make a fushion of myeloma cells and antibody-producing Blymphocyte cells, successfully established the monoclonal antibody preparationtechniques.Then genetically engineered antibody technology,another importantmonoclonal antibody technology, shows great potential,represented by the phageantibody library technology which was established by Winter in1994. Geneticallyengineered antibody mainly includes two types, the transformation of human geneticengineering antibody of monoclonal antibodies, and the fully human antibody byphage antibody library technology. Currently, genetically engineered antibodies havebeen widely used in disease prevention, diagnosis and treatment.This experiment will sort the specific secretory P24antibody B cells by couplingP24antigen bead,after the limiting dilution and extraction of total mRNA, then usingreverse transcription and nested PCR to amplify the immunoglobulin heavy and lightchain genes.Then we sequence and clone the sequence into the eukaryotic expressionCloning vector, to obtain recombinant antibody through the instantaneoustransmission of293T cells.Then Use the protein affinity chromatography purificationto obtain purified antibody.Finally we can establish a new humanized monoclonalantibody preparation technology. Then this method can be used in preparation of theanti-EV71VP1humanized monoclonal antibody, and then coupled EV71VP1antigenwith Nano-Beads to select B cells from the blood of HFMD patients which arecapable of secreting specific EV71VP1antibodies.Similarly,after limited dilution andextraction of total mRNA, amplification of immunoglobulin heavy and light chaingenes by reverse transcription and nested PCR, I sequenced and cloned the sequenceinto the eukaryotic expression vector Cloning vector AbVec-hIgG1、Cloning vectorAbVec-hIgKappa、Cloning vector AbVec-hIgLambda,and finally obtain rebombinantantibody by transiently transfection293T cells.Resultly, I sucessfuly screened a pairof human monoclonal antibody gene against EV71VP1.This study preliminarily and successfully established humanized monoclonal antibody screening method, laid afoundation for the early diagnosis of EV71and selection of other humanizedmonoclonal antibodies.