Construction Of The Attenuated Salmonella Typhimurium Strain Carrying GLP-1Gene

Glucagon like peptide-1(GLP-1) derived from the glucagon raw(proglucagon, PG), which contains two kinds of glucagon-like peptide,namely GLP-1and GLP-2. Glucagon raw molecule containing160amino acids, the mammalian glucagon gene transcription of glucagonRNA in brain, pancreas and intestine in different translation process.Glucagon raw molecules in the gut and pancreas were differentenzyme processing into the effects of different hormone, pancreaticmain glucagon raw fragment accounted for71%, GLP-1(1-36) NH224%, GLP-1(1-37) NH2accounted for5%, while in the intestinal GLP-1(1-36) NH280%, GLP-1(1-37) NH220%. GLP-1has protective effect ofGLP-1β cell effect on pancreatic islet beta cell, promote the insulingene transcription, the synthesis and secretion of insulin, and maystimulate the islet β cell proliferation and differentiation, inhibition ofpancreatic islet beta cell apoptosis, increased pancreatic beta cellvolume. Because GLP-1shorter half-life, clinical treatment using multiplesubcutaneous injection therapy, together with recombinant proteinGLP-1is expensive, limiting its clinical application. Therefore, this studyusing attenuated Salmonella as carrier, observation of GLP-1genemedicine for disease treatment effect.In Cytology test, the recombinant expression vectorGFP-GCG-GLP-1, GFP-NT4-GLP-1, GFP-EX4-GLP-1was constructed and transfected in HEK293Tcell with Vigofect. The mRNA expression of GLP-1was analysed by RT-PCR in HEK293T cell and the protein secretion ofGLP-1was tested by ELISA in HEK293T cells supernatant. Results RT-PCRresults show that there were no significant differences in the expressionlevels of GLP-1among the constructs at24h after transfection. ELISAresult show that the GLP-1protein secretion of GFP-GCG-GLP-1group,GFP-NT4-GLP-1group and GFP-EX4-GLP-1group was (104.12±3.11pmol/L),(117.65±5.98pmol/L) and (156.2±3.2pmol/L) at24h aftertransfection. The GLP-1protein secretion of GFP-EX4-GLP-1group wassignificantly higher than other group(P<0.01). Conclusion The EX4signal sequence could be effective on promoting the secretion of otherheterologous proteins in HEK293T cells.In four of alloxan induced diabetic rats, oral contraceptivecontaining carrying GLP-1of attenuated Salmonella typhimurium canobviously alleviate the symptoms of high blood sugar, lower bloodglucose, increased serum GLP-1, insulin, C peptide, pathologicalsections showed hyperplasia of islet cell clusters, promoting damagedpancreatic beta cell repair. That carry the GLP-1attenuatedSalmonella may become a new effective method for treatment ofdiabetes.