Design,Expression And Pharmacological Activity Of Recombinant Cholera Toxin And Epidermal Growth Factor-Like Molecules Against Type 1 Diabetes Mellitus

Type 1 diabetes is a disease that threatens human health.Aluminum adjuvant,glutamic acid decarboxylase 65（GAD65）and epidermal growth factor（EGF）in combination with gastrin for type 1 diabetes have been conducted to phase III clinical trials.This thesis further investigates cholera toxin B subunit（CTB）and GAD65 antigenic epitope peptide fusion protein nasal drops for the treatment of type 1 diabetes,cholera toxin（CT）-like recombinant epidermal growth factor chimeric protein gavage combined with gastrin injection for the treatment of type 1 diabetes and the construction and expression of a recombinant protein pellet to extend the stability of EGF.New therapeutic ideas for clinical studies of these drugs are provided in terms of drug delivery formulations and structural modifications of the drugs.Chapter 1 Cholera Toxin B Subunit（CTB）and Glutamate Decarboxylase 65（GAD65）Epitope Peptide Fusion Protein Intranasal Treatment of Type 1 DiabetesAntigenic epitope peptide vaccines can prevent type 1 diabetes（T1D）,but few can treat T1D.GAD65 is a target of autoimmune attack in T1D.Our earlier study found that a fusion protein（CTB-GADIII）of CTB sequentially tandem with the antigenic epitope peptides p217-236,p524-538,and p290-306 of GAD65 could delay T1D after the onset of pancreatitis in non-obese diabetic（NOD）mice.To further explore the use of this fusion protein in the treatment of T1D,in this study we evaluated the efficacy of this fusion protein in the treatment of streptozotocin（STZ）in an animal model of T1D obtained by multiple low-dose injections（50 mg/kg of STZ administered intraperitoneally for 5 consecutive days）.Fasting blood glucose of approximately 18 mmol/L in mice after moulding indicates successful moulding.The results showed that CTB-GADIII had a therapeutic effect on type 1diabetic（T1D）mice after multiple low-dose moulding of STZ,as evidenced by an average reduction of fasting blood glucose to 12.8 mmol/L.However,the glucose tolerance data was not as good as expected.Intranasal administration of CTB-GADIII fusion protein resulted in an improved insulin secretion,together with a decrease in the inflammatory cytokines TNF-αand IFN-γand an increase in the anti-inflammatory cytokine IL-4.Histopathology showed that the fusion protein could play a role in repairing damaged pancreatic tissue in TID mice.To further explore the function of CTB-GADIII in anti-apoptosis of islet cells,we performed assays for apoptosis-related indicators.In conclusion,this study investigated the role of autoantigenic epitope peptides in the treatment of T1D.Chapter 2 Gavage CT-like recombinant epidermal growth factor chimeric protein combined with injectable gastrin for the treatment of type 1 diabetesAs a mucosal adjuvant,oral CTB could bind to ganglioside receptors in the intestinal mucosa and help the absorption of drugs.EGF can promote wound healing and tissue regeneration,and has been widely used in tissue engineering research and clinical practice;EGF combined with gastrin can induce pancreatic regeneration by injection.In this study,we examined the proliferation of cells in mouse pancreatic tissue by intraperitoneal injection of Brdu after gavage of mice with the CT-like recombinant epidermal growth factor chimeric protein EGF-CTA₂-TAT/（CTB）₅previously developed in our laboratory in combination with gastrin.The results of Brdu immunohistochemistry showed that the EGF-CTA₂-TAT/（CTB）₅gavage combined with gastrin intraperitoneal injection group had some pro-proliferative effect on pancreatic cells.In addition,to determine whether the proliferating cells were islet cells,we performed immunofluorescence co-localization assays with anti-Brdu and anti-insulin（INS）antibodies and found that some of the proliferating cells in the EGF-CTA₂-TAT/（CTB）₅gavage combined with gastrin intraperitoneal injection group were islet-functional,while others were not.To determine whether the chimeric protein could reach the pancreas by gavage,we used the CT-like recombinant green fluorescent protein chimeric protein EGFP-CTA₂-TAT/（CTB）₅,which was already available in the laboratory,to gavage mice to assess the metabolic distribution of the drug and found that the protein was able to partially enter the pancreas to exert its pro-proliferative effects.Chapter 3 Preliminary study on the construction and expression of a recombinant protein pellet to extend the stability of EGFEGF is a small molecule peptide,oral exogenous EGF is highly susceptible to degradation by gastric acid,which greatly reduces the bioavailability of EGF,and further research is needed to make oral EGF dosage forms.In this study,EGF recombinant protein was designed by tandemly associating a polyamino acid（PAS）modified peptide（Pastag200）with an pancreas targeting peptide（CHVLWSTRC）obtained by phage display technology and EGF.The aim of the study is to prolong the stability of EGF,increase the targeting of the recombinant protein and improve the bioavailability of the protein after oral administration.Two recombinant proteins,EGF-Pastag200-pancreas targeting peptide and pancreas targeting peptide-Pastag200-EGF,were designed expressed and purified,and can be used in subsequent oral treatment studies for type 1 diabetes.