| Glucagon-like peptide-1(GLP-1)is a incretin derived by its cleavage from proglucagon,which increases insulin secretion in a glucose-dependent manner.Intestinal insulin is a hormone produced by gastrointestinal tract in response to nutrients and is necessary to maintain glucose homeostasis.GLP-1 exists in two circulating equipotent molecular forms,GLP-1(7-37)and GLP-1(7-36)amide,and GLP-1(7-36)amide is more abundant in the circulation.GLP-1(7-36)amide was rapidly degraded by enzyme after secretion to form GLP-1(9-36)amide and GLP-1(28-36)amide.GLP-1(28-36)amide is widely distributed in vascular smooth muscle cells,endothelial cells,cardiac cells and renal epithelial cells.GLP-1(28-36)amide has multiple beneficial effects in improving diabetes,such as improving liver metabolism,protecting beta cells,improving glucose treatment,inhibiting weight gain,and protecting the heart.Studies have shown that GLP-1(28-36)amide has an anti-diabetic effect involving Wnt and mitochondria-mediated signaling pathways.Bacillus subtilis spores are the most comprehensive surface display system in which several coat proteins have been successfully used as anchors for heterologous proteins.Spores remain active in a low pH gastric acid environment and have potential as an oral drug delivery vehicle.Therefore,Bacillus subtilis spores were selected as delivery carriers in this study.According to human intestinal environment,five GLP-1(28-36)(5*GLP-1(28-36)in series were displayed on the surface of spores after adding enterokinase sites.Then the recombinant spores were used to verify the efficacy of recombinant spores in the treatment of diabetes mellitus in type 2 diabetic mice.The main researches and results of this study are as follows:?Construction of recombinant plasmid pJS700-GLP-1(28-36)with enterokinase siteTo construct fusion expression CotC-GLP-1(28-36)integrated recombinant plasmid pJS700-GLP-1(28-36),the synthetic 5*GLP-1(28-36)was cloned into plasmid pJS700 and fused with spore capsid protein gene cotC.?Displaying GLP-1(28-36)on the surface of Bacillus subtilis sporesPlasmid pJS700-GLP-1(28-36)was transformed into Bacillus subtilis competent cells.A recombinant Bacillus subtilis strain,which displayed 5*GLP-1(28-36)on the spore surface,has been successfully constructed and identified by Western blot and immunofluorescence.?Construction of a mouse model of type II diabetesThe type 2 diabetes model mouse was constructed by a high-fat,high-sugar diet combined with intraperitoneal injection of STZ.Mice's body weight and blood glucose concentrations were monitored during this period.The results showed that the body weight of mice in the model group was significantly higher than that in the normal group after feeding with high fat and high sugar diet for 4 weeks,and the body weight of the mice in the model group was significantly lower than that in the normal group after 3 weeks of STZ injection.After the successful establishment of the model,the average fasting blood glucose of the model group increased from the initial 7mM to 24 mM.?Functional analysis of oral recombinant spores in miceThe efficacy of recombinant spores was detected after 13 weeks of oral administration of recombinant spores in diabetic model mice.The results showed that the average fasting blood glucose level in the experimental group decreased from 24 mM to 17 mm,which was significantly lower than that in the control group(P<0.05).After injection of pyruvate 30 min,the mean fasting blood glucose level in the experimental group was significantly lower than that in the control group.The blood glucose of the experimental group was significantly lower than that of the control group(P<0.01),which proved that the recombinant spores decreased the gluconeogenesis level of mice,and after intraperitoneal injection of glucose 30 min and 120 min,the blood glucose of the experimental group was significantly lower than that of the control group(P<0.05).The blood glucose of the experimental group was significantly lower than that of the control group(P<0.01),which proved that the recombinant spores improved the ability of regulating blood glucose in mice.The insulin sensitivity index was calculated by the steady-state model,which showed that the recombinant spores increased the insulin sensitivity of mice.The results of pathological observation showed that the recombinant spores also had a certain protective effect on the liver and islets of mice,and the content of GLP-1(28-36)in the pancreas of the experimental group was increased.?In vitro experimental identification of GLP-1(28-36)GLP-1(28-36)pentapeptide with FITC was added to colon cancer cells,and GLP-1(28-36)was found to enter colon cancer cells after 1 hour of rest,indicating that after the recombinant spores entered the intestinal tract,The 5*GLP-1(28-36)shown on its surface can enter colon cells after being cut by enterokinase. |
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