Study Of The Silkworm-expressed Green Fluorescent Protein Tagged Cholera Toxin B Subunit And Human Insulin Fusion Protein Protecting Against Tvpe1Diabetes

Type1diabetes (T1D) is an autoimmune disease whose target is the pancreas (3cells and causes the short of hormone insulin. Presence of self-reactive autoantibody and T lymphocytes; T cell-mediated T1D transfer and sensitivity of T1D to immunosuppressive treating are the main autoimmune traits. During the development of T1D, the primary autoantigens recognized by T cells are insulin (Ins) and its precursors and gintamic acid decarbonxylase.1. The study of oral administration of the green fluorescent protein (GFP) tagged cholera toxin B subunit (CTB) and human Insulin fusion protein produced in silkworm protecting against T1D.Oral administrating disease-specific autoantigen can induce specific oral immune tolerance to prevent or delay the processing of the autoimmune disease. The hint is to obtain enough autoantigen to make and keep the special oral tolerance. As mucosal carrier molecule, CTB has been utilized to conquere such difficulties by chemically or genetically conjugated autoantigens to induce oral tolerance. The application of CTB for oral tolerance may be a potential therapeutic strategy to prevent or treat autoimmune disease including T1D.Here we prescribe construction of the edible protein vaccine composed of CTB, insulin and GFP by Bac-to-bac baculovirus expression system. The corresponding48-kDa fusion protein that produced in the fifth silkworm larvae attained up to0.58mg/ml in hemolymph. This fusion protein vaccine produced by silkworm bioreactor was in a pentameric CTB-Ins-GFP form retaining the GMl-ganglioside binding affinity and the live antigenicity of CTB and insulin. Feeding microgram of the CTB-Ins-GFP fusion protein in hemolymph to Non-obese diabetic (NOD) mice prominently reduced the pancreatic islet inflammation and delayed the development of clinical diabetes symptoms. These results hint that the silkworm bioreactor is a effective production and delivery system to obtain oral protein vaccine designed to induce immunological tolerance for autoimmune diabetes cure. This study offers new point to prevent and treat TID by using our country's silkworm resource to explore oral vaccine.2. The study of the relationship between the function mechanisms of oral administrating silkworm produced CTB-Ins-GFP fusion protein against T1D and specific regulatory T (Treg) cells.Mucosal tolerance has a great prospect in treating autoimmune diseases. It has been reported that the special tolerances induced by nasal, sublingual or gut mucosal antigen administration are related with specific regulatory T cell. In the gut, special antigen protein is presented to immunocytes such as dendritic cells to make a corresponding environment for specific Treg induction. These special induced Treg cells play great parts in immunosuppressive action. The similar role of Treg cell is also verified in tumor metastasis。In our study, the CTB-Ins-GFP fusion protein orally administrated could introduce insulin specific immunotolerance the same as the previously obtained CTB-INS protein. We may detect that the fusion protein is specially combined with intestinal mucosa by GFP. Additionally, the anti-CTB, Insulin antibody subtype IgG1levels were observed elevated in the blood and the CD4+CD25+Foxp3+T cells in spleen, intestinal lymph node and blood increased contrasted to control groups. At the same time, the proliferation and migration abilities of the spleen lymphocyte were suppressed. It hinted the specific orally induced tolerance might relate with Treg cell increasing. The NOD/SCID mouse adoptive transfer experiment demonstrated that oral tolerance induced immunosuppressive lymphocyte. These results showed that the CTB-Ins-GFP fusion protein orally administrated induced antigen specific Treg cells in protecting against insulin dependent diabetes mellitus (IDDM) and the immunosuppressive cells might supress the biological abilities of lymphocytes to treat TID. These experimental results supported the hypothesis that orally administering the insulin protein may be a feasible way to treat TID patients, and oral tolerance might be a new powerful therapeutic strategy to treat autoimmune disease.