Application Of MHC-peptide Tetramer Technology In Epitope Identification

Peptide-MHC tetramer is the key technology of Immunology and Clinical diagnosis, and has been widely used in immunology research and immunological disease clinical research. The principle is to directly detect specific T cell through enhancing the affinity between T cell receptor and MHC-peptide complex. The technology can help to detect specific T cell rapidly, directly, sensitively and specifically. However, it has a low rate of renaturation and cumbersome experimental steps. Although great improvements have been made by the researchers in MHC-peptide tetramer preparation, these problems haven't been solved.MHC-I molecule restricted CTL epitope identification has been the focus of Immunology research, because it is a crucial part of immunodiagnostics,immunotherapy and vaccine development. The traditional method of CTL epitope identification is to select CTL epitope through lymphocyte experiments follow-up after having synthesized many overlapping peptid. The method is time-consuming,laborious,costly and inefficient.Other researchers used TAP defected T2 cell in peptide affinity experiments to identify CTL epitope and achieved good results. But the cumbersome process of labeling and staining,together with the damage which might be caused during repeated cell washing, are the main reasons that this method couldnot achieve wide popularity.As we all know, DsRed and ZsGreen are expressed as fluorescent protein of tetramer structure.So we envisaged to fusion express MHC-Ⅰand ZsGreen in eukaryotic cells to get MHC-tetramer directly.This method could simplify the cumbersome process of prokaryotic expression,folding,biotinylating and fluorescent labeling of traditional method.And the MHC-tetramer could load any interested peptide in theory,it could make the inspection process more flexible.Using the principle,we coule fusion expressβ2M and ZsGreen to getβ2M-tetramer.The tetramer could bind with MHC-Ⅰand peptide epitope to generate peptide-MHC tetramer. The principle applied in the peptides binding affinity assay,we could improve themethod of epitope identification greatly. Objective To obtain MHC-tetramer directly by molecular cloning,eukaryotic expression, nickel affinity chromatography,and other methods. To optimize the technology of Peptide-MHC tetramer preparation. And apply this technology to improve the method of Identificating CTL epitopes.Methods1.To get the DNA sequence of the soluble part of mouse MHC-Ⅰmolecule(sKb),and to construct the eukaryotic expression vector(FUsKbZsGreenGGGS) of a fusion protein by linking the sequence of the sKb-4×GGGS-ZsGreen-6×His Tag through molecular cloning technique;2.Transfect FUsKbZsGreenGGGS into 293FT to fusion express the protein sKbZsGreenGGGS by cell transfection technology;3. Crack the 293FT cell that expresses sKbZsGreenGGGS,and then purify the protein sKbZsGreenGGGS by Ni-affinity chromatography;4. Cause he fusion protein sKbZsGreenGGGS to react withβ2M and peptide OVA to produce peptide OVA-tetramer,and the tetramer react with B3Z cell, then detect Cell staining by Flow cytometry.5. To get the DNA sequence ofβ2M,and to construct the eukaryotic expression vector(FUβ2MZsGreenGGGS) of a fusion protein by linking the sequence of theβ2M-4×GGGS-ZsGreen-6×His Tag through molecular cloning technique;6. Transfect FUβ2MZsGreenGGGS into 293FT to fusion expression the proteinsKbZsGreenGGGS by cell transfection technology;7. Crack the 293FT cell that expressesβ2MZsGreenGGGS,and then purify the proteinβ2MZsGreenGGGS by Ni-affinity chromatography;8. The fusion proteinβ2MZsGreenGGGS and peptide epitopes react with the T2 cell, then we can identify epitope by Flow cytometry.Mechanism investigation1.Optimization of MHC-peptide tetramer technology: Fusion express MHC-Ⅰand ZsGreen to get MHC-tetramer that carrying green fluorescent.The tetramer binded to specific peptide epitope,and then reacted with Specific hybridoma cells.We could prove this method is feasible by FACS.2. The application of MHC-peptide tetramer technology in epitope identification: Fusion expressβ2M and ZsGreen to getβ2M-tetramer that carrying green fluorescent. The tetramer reacted with different peptide epitope and T2 cell,then identificate epitopes by FACS.In the experiment,we use the feature of T2 cell that lack of TAP,and the feature ofβ2M that promote the combination of MHC-Ⅰand peptide epitope.Positive peptides could mark green fluorescent protein on the surface of T2 cells through the way of binding toβ2M-tetramer and MHC-Ⅰ.Then it is easy to identificate epitopes by FACS.Conclusion1.The MHC-Ⅰ-fluorescin tetramer is produced and purified. Proved by Flow Cytometry,the tetramer can be recognized by B3Z after bound toβ2 microglobulin and peptide OVA.It provides a convenient method for the detection of Antigenic Specificity T cells.2.Construct an eukaryotic expression vector that expresses Beta2- microglobulin- ZsGreen ,then express and purify the fusion protein,and use the fusion protein to identify epitope by MHC-Ⅰpeptides binding affinity assay.The research shows that it is technically feasible to prepare MHC-peptide tetramer by fusion expressing MHC-Ⅰmolecule and ZsGreen. With this method we can also prepareβ2M tetramer and identify CTL epitope in the peptide affinity experiments. The CTL epitope identification is greatly simplified and optimized, therefore might achieve great popularity.