| Diabetes mellitus is one of the complex metabolic diseases prevalent in the world,which is associated with hyperglycemia or hyperglycemia state,mainly due to insulin resistance or/and insufficient insulin secretion.Worldwide,537 million(10.5%)adults aged 20-79 had diabetes in 2021,equivalent to 1 in 10 people with diabetes,and this number is expected to increase to 643 million by 2030.In 2021,there are 140 million Chinese adults aged 20-79 with diabetes,accounting for 26.1%of the world’s diabetes patients,and this number is expected to increase to 164 million by 2030.At present,the treatment of type 2 diabetes is mainly based on hypoglycemic drugs,supplemented by diet regulation and strengthening exercise,hypoglycemic drugs include oral drugs and subcutaneous injections.Lactic acid bacteria(LAB)are a general term for a class of bacteria that can decompose carbohydrates and produce a large amount of lactic acid.They have been recognized as safe by the U.S.Food and Drug Administration(FDA)due to their long-term safe use in human nutrition and are commonly used as leavening agents in the food industry.Lactic acid bacteria,as probiotics widely present in the intestinal tract of humans and animals,have adhesion and colonization functions,and are often used as engineered bacteria as a mucosal drug delivery carrier.Glucagon-like peptide 1(GLP-1)is a short-acting incretin primarily synthesized and secreted by enteroendocrine L cells in the proximal small intestine.The mature form of GLP-1 consists of 30 amino acids,and GLP-1 has pleiotropic therapeutic effects on β-cell recovery and proliferation.However,its very short plasma half-life(about 2 min in the circulatory system)limits its drug therapy research and clinical application.Therefore,the design of a recombinant Lactococcus lactis containing a human long-acting GLP-1 analog(LGLP)could be explored as a potential vector for mucosal delivery,and the gene could secrete the biologically active form of LGLP extracellularly.The contents and results of this study are as follows:(1)Construction and identification of transformed long-acting GLP-1 analog Lactococcus:The plasmid pLN-LGLP containing the LGLP gene was constructed by molecular cloning,and the target plasmid was introduced into competent lactic acid bacteria by electroporation.The recombinant LGLP lactic acid bacteria(L-pLN-LGLP)synthesizing LGLP protein was successfully obtained and identified by Gram staining,plasmid PCR and Western blotting.(2)In vitro and in vivo viability study of transformed long-acting GLP-1 analog lactic acid bacteria:OD value detected by microplate reader,plate counting method,in vitro simulated gastrointestinal fluid culture and small animal live imaging to explore the in vitro and in vivo viability and correlation of recombinant LGLP lactic acid bacteria.The results show that the LGLP gene can be stably inherited in the recombinant lactic acid bacteria,the recombinant LGLP lactic acid bacteria can tolerate the extreme environment of gastrointestinal fluid,and colonize the intestinal tract of mice for at least 72 hours.(3)In vivo safety analysis of transformed long-acting GLP-1 analog lactic acid bacteria:Acute(14 days)and subacute(28 days)oral toxicity tests were conducted to evaluate the safety and possible pathogenesis of the recombinant bacteria.The results showed that short-term oral administration of recombinant LGLP lactic acid bacteria did not cause significant adverse effects on host health.(4)Pharmacokinetic study of LGLP in mice:Liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was used to determine the levels of GLP-1 and LGLP in mouse plasma after intraperitoneal administration.According to the fitting results,LGLP was rapidly absorbed in mice,the plasma concentration reached the highest 3 minutes after administration,and the half-life was about 20 minutes.(5)In vivo and in vitro effects of transformed long-acting GLP-1 analog lactic acid bacteria:In vitro and in vivo experiments were conducted to further verify whether LGLP has the function of GLP-1,and whether gavage of LGLP recombinant bacteria can achieve the effect of LGLP administration.The results show that,recombinant LGLP lactic acid bacteria can be colonized in the intestinal tract of mice,and can persist in a short period of time,and the LGLP secreted by the recombinant bacteria can play the relevant role of GLP-1.In conclusion,this study successfully constructed recombinant Lactococcus lactis expressing LGLP protein,and verified that LGLP has a longer half-life and better stability than GLP-1.Expression of LGLP protein by recombinant LGLP lactic acid bacteria can regulate blood sugar by regulating insulin secretion,promoting islet cell proliferation,and reducing body weight. |
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