Construction Of Human Immunoglobulin Combinatorial Library And Screening Of Phage Antibodies To Platelet

Molecular cloning of antibody repertoire in combinatorial phage display vectors has turned out to be a powerful technique for obtaining human monoclonal antibody genes and probing the antibody immunoresponse against a given antigen.This study aims to construct a human immunoglobulin combinatorial library by using phage surface display expression system and to screen the autoantibodies against platelet, preparing for the further screening of antibodies directed against the GPâ…¡b/â…¢a which have been regarded as potential agents to diagnose or treat acute coronary syndromes.In this study, we produced a combinatorial library of Fab Fragment from B cells of patients with idiopathic thrombocytopenic purpura(ITP). PBMC were separated from three ITP patients. Total RNA was extracted routinely, and cDNA was synthesized by reverse transcriptase. Half-nested PCR was employed to amplify the heavy chain Fd and light chain k. Firstly, the k genes were cloned into phagemid pComb3 and electrotransformed into E.Coli to construct a K-chain library which contained 2.88×10⁶ separate clones,the k gene positive ratio is 60%. Then we cloned heavy chain Fd genes into k library. After 20 electroporations,we got a Fab-phage display library with the volume of 2.3×10⁶cfu.The recombination rate reached 40%.Under the super-infection of helper phage M13KO7, the phage antibody titer was about 5×10¹²pfu/ml.To evaluate the quality of the antibody library, we panned it with activated coated platelet. 3 rounds of biopanning were conducted and 5 specific antibodies were selected with ELISA from the enriched phages, which can bind to platelet strongly. Three antibodies with the highest binding ability were sent to be sequenced and the result showed the genes belong to one number of the antibody genes. In conclusion, the constructed antibody library may be useful to further screening of antibodies against the GPâ…¡b/ma.