The Effects Of Single-stranded Trimer Of IGRP On The Specific CD8 ⁺ T Cells Were Investigated

T1DM (Type 1 diabetes mellitus), also known as insulin-dependent diabetes, is mediated by T lymphocytes, through direct or indirect effects on insulin-producing pancreatic β cells damaged, leading to life-long dependence on exogenous insulin as the main feature of organ-specific autoimmune disease. CD8+T cells (cytotoxic T lymphocytes, CTLs) has an important role in the course of T1DM, directly destroy islet β cells, and is indispensable in the occurrence of T1DM development.Studies have found that, NOD mice as a murine model of type I diabetes, their CD8+T cells recognize IGRP. IGRP is expressed in pancreatic islet β cell-specific protein, only a small part in a cells. Some researchers found that, In the earliest NOD insulitis, there is a population of cells capable of recognizing IGRP206-214 the same time, along with the development of the disease, the number of specific T cells increased, top to maximum value after 15 weeks.Cell surface display allows peptides or proteins to be expressed on the virus or cell surface, but still able to maintain its relatively independent spatial structure and biological activity. It is a powerful technique to study the nature of the polypeptide, mutual recognition and interaction. It can be used in protein engineering, environmental bioremediation, biocatalysis, as well as vaccine and therapeutics development and many other applied gene manipulation technologies. Yeast is a eukaryote, has a folding enzymes, endoplasmic reticulum chaperone, etc., compared with bacterial display technology, it is more similar to mammals in the mechanism of protein folding and secretion. Yeast surface display technology can be used to show the need glycosylation and disulfide isomerization modified eukaryotic proteins based on the above. What we used it dtSCT technique, which connect to antigenic peptide, β2-microglobulin and MHCI molecule covalently with two short flexible linkers and introduced a disulfide bond between the linkerl and MHC I molecule to trap antigenic peptide into the binding groove. Then exogenous protein expressed in yeast cell surface by connecting AGA2. Our previous experiments have been constructed expressing IGRP206-214H-2Kd-SCT and IGRP206-214H-2Kd-SCT is connected to the carrier, expressed in yeast cells surface.Thus, on this basis, we optimize the yeast expression system by induce the concentration, induction time, induction temperature and filter galactose concentration so that the yeast expression reached a higher level. In vitro, we found that with the NOD mouse spleen cells co cultured for 24 hours CD69 expression was the highest, accounting for 1.89% of CD8+T cells and co cultured for 96 hours, CD25 expression was the highest, accounting for 4.25% of CD8+T cells. It suggests that yeast display can activate specific CTLs, and promote their proliferation. Thus providing a basis for further research to extract IGRP206-214H-2Kd protein and study its molecular mechanism in the pathogenesis of T1D.