Yeast Cell Surface Display IGRP_206-214H-2K^d Single Chain Trimer And Preliminary Identification

Yeast cell surface display system is an eukaryotic display system, which displays heterologous protein onto the surface of yeast, through insert exogenous gene into specific gene site of yeast. It has the advantages of posttranslational modification, promoting formation of disulfide bonds, processing target proteins into native conformations and displaying of the recombinant proteins onto the surface of yeast cell, which is beneficial for purification, via break off the disulfide bond between Agalp and Aga2p by deoxidizer. Furthermore, the expression levels of fusion protein can be improved significantly by high-cell-density fermentation in low-cost culture medium.Single chain trimer (single chain trimer, SCT) consists of MHC class I heavy chain,β2m chain, cytotoxic T cell (CTL) epitope peptides in molecular biology techniques, In cell-mediated immunity, SCT can avoid the complex processing and presentation of antigenic peptides, so that antigenic epitope can be securely linked to the antigen binding groove of MHC molecules, thereby, it increases the activation of specific T cell receptor (TCR).Type I diabetes (TID) is a kind of organ specific autoimmune disease, which expressed a variety of antigen epitope of specific CTL in the progression and development of the disease, such as InsBi5-23, GAD65p90, IGRP206-214, etc. Some of these epitopes play important roles in the progress of the TID. Recent studies show that toxin-coupled MHC class I tetramers can deplete CD8+T cells specific for IGRP206-214peptide and delay diabetes in nonobese diabetic mice. Base on the advantages of yeast display systems and single-chain trimer, to explore IGRP206-214single-chain trimer is able to be expressed on yeast cell surface or not and affect the NOD mouse IGRP specific CTL, we select former laboratory constructed IGRP206-214H-2Kd SCT for the study, using the yeast surface display systems, observing the influence of yeast surface expression of IGRP206-214H-2KdSCT single trimer.First, we used the former constructed eukaryotic expression plasmids. Then we constructed IGRP206-214H-2Kd SCT-pCT302yeast expression recombinant and transformed into yeast cells using electro transformation, then the yeast labeled with the H-2Kd antibody, the expression of IGRP206-214H-2KdSCT on yeast cells was detected by flow cytometry.To evaluate the effect of IGRP206-214H-2Kd SCT on IGRP specific CTL in mice, we prepared IGRP H-2Kd tetramer to detect the changes of specific CTL in NOD mice. With pET22B-IGRP206-214H-2Kd prokaryotic expression vector constructed early, the inclusion washing, purification and renaturation, ultrafiltration, interception, exchanging buffer, biotin, getting out of the free biotin, biotin efficiency detection, and with certain amount of PE labeled streptavidin were processed, we prepared the IGRP206-214-Tetramer-PE successfully. The we used BALB/c mice as negative control mice, proved that our preparation of the IGRP206-214-Tetramer-PE with correct function.Finally, we use self-made IGRP206-214-Tetramer-PE to assess the influence of the IGRP206-214SCT on was displayed on decreasing the number of IGRP206-214specific CTLs. However the data show that rates of IGRP206-214specific CTLs are increased after displayed on yeast surface compared with those without any treatment.In the study, we constructed engineered yeast that can secrete MHC I.It provides a reference for other recombinants expressed on yeast surface.Yeast cell surface display give a new way to overcome difficult purification problems appearing in yeast cells. Full text proved IGRP206-214H-2KdSCT can expressed on yeast cell surface, and displayed IGRP206-214H-2KdSCT have influence on the activation and proliferation of IGRP206-214specific CTL, which provides new ideas for further study of Type I diabetes.