Application Of Disulfide-trap Single-chain Trimer Technology For Preparation Of PR1-HLA-A*0201Tetramer

Cytotoxic T lymphocyte (Cytotoxic T Lymphocytes, CTL) is the important immune effector cells to the body’s antitumor, anti graft and effective control of various infections, quantitative detection of antigen specific CTL has been one of the objectives of immunology field.Target cells or antigen-presenting cell surface MHC-I molecules that bind to specific epitope peptide complex are identified by T cell receptor, but the specific recognition with pMHC-I is low affinity, and dissociated rapidly. MHC-I Tetramer technology was based on the specific interaction between TCR and antigen peptide complex, in vitro by using the principle of binding biotin and avidin, MHC-I molecular and antigenic peptides were assembled into a MHC-I antigen peptide tetramer complex. With the increasing of T cell receptor affinity, specific T cells can be identified by MHC-I tetramer though flow cytometry, then separated. But later found that preparation process of traditional tetramer is complex, and high cost, but the renaturation efficiency is low. It is very difficult to form a stable pMHC-I monomer, especially for the antigenic peptide had alow affinity to MHC-I Molecular. So limited the application of these MHC-I tetramer technology.The single chain trimer technology has solved the above problem. it is the major histocompatibility complex class Ⅰ molecules as single polypeptides wherein the antigenic peptide, β2m, and MHC-I heavy chain are attached by flexible linkers, only one time cloning and expression, one time purification, greatly simplifies the preparation process. And the rate of tetramer is much higher than the traditional method, and greatly reduces the cost. More importantly, the MHC-I SCT tetramer has a higher stability, expand to some low affinity antigen peptide with MHC-I molecules. However, if the binding capacity between peptide antigen and SCT antigen grooves in the low to a certain extent, low affinity InsB15-23antigenic peptide will be replaced. Out of this consideration, we improved the SCT by the introduction of "two disulfide bonds", make the antigen peptide more firmly anchored in the antigen-binding groove.Chronic myeloid leukemia (CML) is a kind of myeloid hematopoietic stem cell malignant hyperplastic diseases, there is still no effective treatment, adoptive cellular immunotherapy will become an ideal method. PR1is the HLA-A*0201restricted9peptide(VLQELNVTV)from CML related antigen protease3, which induces CTLs can kill leukemia cells, but has no toxicity to the bone marrow cell. PRl-hβ2m-A2Y84C-SCT tetramer used in this experiment aims to improve rate of detection with PRl specific CTL, expect to be a powerful tool for research of CML.In this study, we are using gene engineering technology to transform the existed PRl-SCT fusion gene, in the basis of PR1peptide, β2m and HLA*0201within two flexible linker, A2molecular "peptide C terminal binding region" eighty-fourth amino acid from tyrosine (Tyr) transformed into cysteine (Cys), at the same time, the introduce a Cys into linkerl. Two cysteine residues form a two disulfide bonds, the antigen peptide C end is anchored to antigen binding groove. Then the transformation of PRl-HLA-A*0201-dtSCT fusion gene, was cloned into pET22b vector for prokaryotic expression. A lot of transformed PR1-dtSCT inclusion body protein is gained by washing. Then the transformed PRl-SCT inclusion body protein after purificated, refolded, ultrafiltrated, intercepted, and biotinylated. According to the biotinylated efficiency, with PE-labeled streptavidin by the ratio of4.5:1, the fluorescent marker’s PRl-HLA-A*0201-dtSCT tetrame is Prepared.Clinical specimens of Chronic myeloid leukemia were detected by flow cytometry, found PRl-dtSCT tetramer in the detection rate of PR1-CTL is better than the traditional PRl-Pentamer obviously, and the PRl-CTL frequency of treatment in CML patients is higher than untreated patients, there is significant difference between them, In PRl-CTL group,both CD45RO+T cells and IFN-γ+T cells are exist.