| Type1diabetes (TID) is a kind of organ specific autoimmune disease.There will appear a variety of specific CTL to antigen epitope in the progression and development of the disease, such as InsB15-23, GAD65p90, IGRP206-214, etc. These epitopes plays a different role in the progress of the disease by antigen spreading. In the process of type I diabetes, InsB15-23expected to early antigen epitope which is important to other antigens exposure and key to the corresponding production of CTL;Toxin-coupled MHC class I tetramers can specifically ablate auto-reactive CD8+T cells and delay diabetes in nonobese diabetic mice. Therefore we choose the InsB15-23and IGRP206-214as research objects respectively in this study, observing the influence of membrane expression of InsB15-23H-2KdSCT and IGRP206-214H-2KdSCT single trimer on the process of type I diabetes in NOD mice.First, we made use of the recombinant plasmids former constructed transfecting the K562cells.Then,the mouse original H-2Kd antibody as primary antibody was used to label the H-2Kd molecular and the goat anti mouse antibody as the second antibody to label the H-2Kd antibody.We demonstrated that mInsB15-23-SCT can express in the eukaryotic cells using the fluorescence microscope. Then we immuned the3weeks’old NOD mice and7weeks’old NOD mice respectively using the mInsB15-23-SCT and mIGRP206-214-SCT to observe the effect of the two recombinant plasmids.The result showed that the mInsB15-23-SCT delayed the type1diabetes occurred significantly.However,mIGRP206-214-SCT accelerated the development of the disease compared to the control group.The tetramer technology is the gold standard in detecting specific CTLs.With pET22b-InsB15-23-β2mH-2Kd constructed early by our laboratory as the foundation using genetic engineering methods,we built the pET22-IGRP206-214-β2mH-2Kd prokaryotic expression vector successfully. Through the inclusion of washing,protein 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,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 two recombinant plasmids to the TID of the NOD mice from cellular level,we found that mInsB15-23-SCT had no effect on decreasing the number of IGRP206-214specific CTLs.However the mIGRP206-214-SCT had distinguished effect on increasing the IGRP206-214specific CTLs.This thesis shows that aiming at the early antigen (Ins) of the disease exposure, overexpression of mInsB15-23H-2Kd-SCT can significantly reduce the incidence NOD mice TID; but it is unable to determine whether it can reduce the amount of NOD mice IGRP206-214specific CTL;However, for the late antigen (IGRP) of the disease exposure, overexpression of mIGRP206-214H-2Kd-SCT can increase the NOD mice IGRP206-214specific CTL frequency, showed a slightly increased TID incidence; In general, there has little effect on the TID;... |
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