Eukaryotic Expression,purification Of Human IgEC?2-4 Protein And Its Target Fishing

IgE is the major mediator of type I hypersensitivity.IgE is an immunoglobulin with the shortest half-life and the lowest amount in normal human serum.The IgE concentration in normal human serum is only 0.3 ?g/mL,and the half-life is about 2.5 days.However,in the serum of patients with IgE-mediated allergic diseases,the IgE level can be 1,000 to 10,000 times higher than normal,which lasts about 12 days.Reducing the level of IgE in the blood of patients makes a great significance to control allergic disease.The hemodialysis machine,mode and techniques of hemodialysis had been improved well,therefore,removing effector molecule IgE in the blood of severe allergy sufferers specifically to control symptoms by immunoadsorption techniques(IA)is possible.The core technology of immunoadsorption technology is the preparation of immune adsorbent.The purpose of our team is to study and prepare immunoadsorbents which bind to IgE with high affinity,while acquiring IgE molecules is the basis for preparing and screening its adsorbent.The binding between the heavy chain constant region and the high-affinity receptor(Fc?RI)on the surface of mast cells or basophils is a key step in the development of type I hypersensitivity.There are four regions in the IgE heavy chain constant region,and the affinity between IgE heavy chain 2-4 region(IgE C?2-4)and Fc?RIa is close to which between intact IgE molecule and Fc?RI?.Therefore,the recombined and expressed human IgE C?2-4 can be used to replace the intact IgE molecule.we used to prepared monoclonal antibodies by immunizing mice with human IgE Cs2-4 protein expressed in the prokaryotic expression system as an immunogen.The study found that most of ultimately screened monoclonal antibodies did not bind to natural IgE molecules.The reason for this result may be that there is no post-translational modification of the prokaryotic expressed immunogen,and the protein does not fold well,resulting in low affinity of the prepared antibody and natural IgE molecules.In contrast to prokaryotic expression systems,mammalian cell expression systems possess the spatial structure and modifications necessary for active proteins and provide the closest human translation of post-translational modifications to recombinant human proteins.Therefore,this study intends to secrete and express human IgEC?2-4 protein through the human embryonic kidney cell 293FT eukaryotic expression system,and use the surface plasmon resonance(SPR)to target fishing the protein interacted in the human sera.To lay the foundation for the further development and screening of IgE immunoadsorbent.The study is divided into two parts:In the first part,eukaryotic expression and purification of human IgEC?2-4 protein.Choose Eukaryotic expression vector pcDNA3.1(-),construct three kinds of recombinant eukaryotic expression vector of IgEC?2-4 containing different signal peptide,then transiently transfected into 293FT suspension cells respectively.Collect the cell culture supernatant and identify the expression level of different recombinant plasmids by Western blot.The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount,then the recombinant protein was purified by Ni-NTA affinity chromatography.The intereaction between high affinity IgE receptor(Fc?R I)of KU812 cells surface and IgEC?2-4 was identified by cell immunofluorescence.Western blot results showed that recombinant plasmid which containing the signal peptide III had the highest-level expression.Therefore,the recombinant plasmid containing signal peptide III was used to expand the expression of the recombinant protein IgEC?2-4 by an equal proportion.A plurality of batches of transiently transfected expression were used to obtain 5L of cell culture supernatant containing recombinant protein IgEC?2-4,and 31 mg of high purity recombinant human IgECs2-4 protein was obtained by nickel column affinity purification.The process of transient transfection consume less time and suitable for high-throughput,high-efficiency expression of proteins,meeting the needs of this experiment.Immunofluorescence showed that the recombinant protein IgEC?2-4 could combine to surface receptor of KU812 cell,which proof that the spatial structure of human IgEC?2-4 protein was correctly folded.In this experiment,recombinant human IgEC?2-4 protein was obtained through eukaryotic expression and purification,which laid the foundation for the subsequent target fishing of its interacted protein and preparation of IgE immunoadsorbent.In the second part,the unknown proteins that specifically interacted with IgEC?2-4 were captured from human serum by surface plasmon resonance(SPR)technique.SPR is an optic sensing technology that can analyze and detect bio-molecular interactions according to spectral changes on the dielectric layer attached to the surface of the sensor chip,such as the refractive index of reflected light caused by changes in the refractive index and thickness.Due to its advantages of no labeling,high specificity,high sensitivity,trace detection and fast analysis speed,SPR technology has been widely applied in multiple life-science fields,such as proteomics,cell signal transduction,receptor-ligand,antibody-antigen,molecular fishing and new drug screening.The recombinant human IgEC?2-4 protein was immobilized on 3D Dextran chips,and PlexArray HT A100,a Plexera biomolecular interaction instrument,was used to capture protein interacted with human IgEC epsilon 2-4 in human serum.After the protein on the chips was digested in situ,the captured protein was analyzed by Liquid Chromatography-Mass Spectrometry(LC-MS).Mass spectrometry analysis revealed that thirty-seven kinds of proteins which are likely to interact with IgEC?2-4 were captured from human serum by SPR technique.Based on the above experimental results,we conclude:In this study,we successfully constructed the eukaryotic expression vector of human IgECs2-4 protein,transiently transfected 293FT suspension cells to express the recombinant protein,and purified by affinity chromatography to obtain high-purity recombinant human IgEC?2-4.The results of immunofluorescence showed that the recombinant human IgECs2-4 protein obtained by eukaryotic expression could bind to the human leukocyte surface Fc?R1? receptor.The preparation of recombinant human IgE C?2-4 protein provides material for subsequent capture of target molecules and screening of IgE immunoadsorbent.Thirty-seven kinds of proteins that had direct or indirect binding with human IgEC?2-4 protein were catched in human serum by SPR technique.It provides the basis for the development of IgE immunoadsorbent,and then lays a firm foundation for the immunoadsorption therapy of IgE-mediated allergic diseases.