The Construction And Evaluation Of Lactic Acid Bacteria For Recombinant Express HGLP-1 And Hleptin

The increasing obesity and diabetes at present.The effective methods in clinical for obesity and diabetes are mostly through drug treatment and surgery.However,they often have large side effects.Today,more research is focuse on intestinal peptide drugs.Glucagon-like peptide-1(GLP-1)is a hormone secreted by intestinal cells that can promote insulin synthesis and delay gastric emptying.Leptin is encoded by the ob gene and secreted by fat cells to control food intake and fat synthesis.As a kind of chemical synthetic drug,They can only be administered by injection and are expensive,which makes them unacceptable for most patients.Lactic acid bacteria are considered to be a good delivery vehicle for oral protein drugs depend it's benefits.The GLP-1 and Leptin expressed by a food-grade expression system for Lactococcus lactis.the food-grade expression system of Lactococcus lactis was first used to express GLP-1 and Leptin,a strain of L.paracasei with potential hypoglycemic effect was selected,and an animal model with obesity and diabetes was constructed,The following are the research content and results.In this study,the electrotrans formation efficiency of Lactococcus lactis were invested after the recombinant plasmids pNZ8148-glp-1 and pNZ8148-ob were constructed.The results showed that the highest efficiency,1.6×108 CFU/?g was obtained by culture medium with 2.5%glycine,recipient cells growing at 0.3 OD,formula I washing solution,0.2 ?g plasmid,2.5 kV,0.2 cm Cuvette,and 1.5 h gentle shaking before plating.The protein was detected by SDS-PAGE and Western Blot After transfer to Lactococcus lactis,The sizes of GLP-1 and Leptin were 18.9kDa and 6.3kDa respectively.In addition,it was also found that the recombinant protein could continue to secrete for 7 hours without nisin.The results set a stage for further work.Lactobacillus play a role on regulating blood sugar based on ?-glucosidase inhibitory activity.In this study,three strains of Lactobacillus were isolated from fermented yogurt.The purpose was to screen a strain of Lactobacillus with potential hypoglycemic effect.In this test,the ?-glucosidase inhibitory rate and simulated gastrointestinal tolerance were used as the screening conditions.Finally,it was found that the inhibition rate of ?-glucosidase by Lactobacillus paracasei was higher than other isolates,reaching 35.6%;The survival rate of this strain in the simulated gastrointestinal tract was also higher than others.The recombinant Lactococcus lactis and Lactobacillus paracasei isolated can enhance the biological function of the Lactococcus lactis in reducing blood glucose and treating obesity and diabetes theoretically.This study constructed a high-fat diet-induced obese mouse model successfully,its body weight,blood glucose and blood lipids were significantly higher than normal diet mice.thus providing the experimental animal materials for the further construction of the biological activity analysis of recombinant lactic acid bacteria and Lactobacillus paracasei in vivo.