CN116103212A

CN116103212A - Recombinant bacteria co-expressing GLP-1 and OXM

Info

Publication number: CN116103212A
Application number: CN202211309796.XA
Authority: CN
Inventors: 李芳红; 梁志成; 赵子建
Original assignee: Hainan Huasong Pharmaceutical Technology Co ltd
Current assignee: Hainan Huasong Pharmaceutical Technology Co ltd
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2023-05-12

Abstract

The invention relates to the field of microbial engineering, in particular to recombinant bacteria co-expressing GLP-1 and OXM. The recombinant bacterium comprises a GLP-1 gene expression cassette and an OXM gene expression cassette in series; wherein the GLP-1 gene expression cassette comprises a P32 promoter, a first terminator and a GLP-1 gene transcribed by the two promoters; the OXM expression cassette comprises a P8 promoter, a second terminator and an OXM gene of which transcription is regulated and controlled by the two promoters; the N-terminal of the GLP-1 gene and the OXM gene is also provided with a signal peptide. The recombinant bacterial medicament can be used for treating metabolic diseases, neurodegenerative diseases, memory and learning disorders, irritable bowel syndrome, stroke, postoperative catabolic changes, functional dyspepsia, ischemia reperfusion tissue injury and other various diseases.

Description

Recombinant bacteria co-expressing GLP-1 and OXM

Technical Field

The invention relates to the field of microbial engineering, in particular to recombinant bacteria co-expressing GLP-1 and OXM.

Background

GLP-1 (glucon-like peptide-1) is a polypeptide hormone synthesized and secreted by intestinal L cells, is composed of 29 amino acids in total, and has the effects of inhibiting Glucagon secretion and improving insulin level in blood. GLP-1 also inhibits food-induced gastric acid secretion, promotes gastric distension, delays gastric emptying, and produces satiety. GLP-1 is secreted by L cells of the small intestine and colon by post-translational processing of glucagon.

A basic feature of maintaining glucose tolerance in healthy humans, which is the ability of the endocrine pancreas to secrete insulin rapidly and accurately, is the adaptation of the amount to the size of the food, to maintain a relatively narrow range of fluctuations in the blood glucose. GLP-1 release is divided into two phases, first 10-15 minutes after feeding and then 30-60 minutes later again. Since most L cells are located in the distal ileum and colon, early stages may be caused by nerve signals, intestinal peptides or neurotransmitters, and the second stage may be caused by direct stimulation of L cells by digested nutrients. Regulating the rate of gastric emptying can affect nutrient entry into the small intestine, while one of the effects of GLP-1 is to inhibit gastric emptying, which slows the level of secretion upon postprandial activation.

GLP-1 is extremely rapidly cleaved by the enzyme dipeptidyl peptidase IV (DPP-IV), resulting in its half-life of only about 2 minutes, and therefore, GLP-1 concentration is highest in the submucosa of the intestine and is reduced in the hepatic portal vein and in the systemic circulation. GLP-1receptor (GLP-1R) is located on intestinal neurons, which increase action potential upon addition of GLP-1 in primary culture. Receptors on sensory afferent fibers of the node ganglia may also be activated, transmitting signals to central regions important for energy homeostasis. Nakagawa et al demonstrate the expression of GLP-1receptor in ganglion neuron cells and demonstrate that GLP-1 injection can increase the afferent signal of the rat hepatic vagus nerve, providing evidence for chemical reception of GLP-1 at the peripheral vagus nerve. Interestingly, intraperitoneal injection of GLP-1 was insensitive to the anorexic effects of the subdiaphragmatic vagal degenerative heart failure rats, whereas the effects of GLP-1 injection into the vena cava and hepatic portal were unaffected over a large dose range. This suggests that intraperitoneal GLP-1 requires intraperitoneal vagal afferent signals to exert its anorexic effect.

GLP-1 promotes insulin secretion from islet cells and inhibits glucagon secretion, and has the potential to reverse type 2 diabetes, and thus can be used for the treatment of type II diabetes. The research result of adopting GLP-1R antagonist exendin shows that the change of blood sugar after the oral glucose intake of human body is large, thus indicating the physiological importance of endogenous GLP-1. Notably, GLP-1 still has insulinotropic properties in subjects with type II diabetes who are not refractory to sulfonylurea treatment. However, the mechanism by which islet beta cells recover glucose sensitivity in diabetic patients via GLP-1R signaling is not completely understood, and may involve interactions between membrane ion channels, cAMP dependent signaling and intracellular glucose metabolism.

In the search for safe and effective treatment of obesity and related diseases-Type 2 diabetes (Type 2 diabetes mellitus T2D), the intestinal hormone human glucagon-like peptide 1 has been successfully used in therapy. Other corresponding GLP-1 analogue formulations, such as Liraglutide, exenatide, abilutide, etc., have been applied in clinical treatment. However, these GLP-1 analogues all need to be injected regularly to control the therapeutic effect of controlling blood sugar, which brings great inconvenience to long-term patients, not only is expensive, but also the risk of drug effect reduction caused by increased antibody when long-term use is met due to the fact that the amino acid sequence is different from that of natural GLP-1.

Oxyntomodulin (OXM), a peptide hormone comprising 37 amino acids found in the colon, was previously called intestinal glucagon, and is synthesized by the small intestinal mucosal L cells and secreted following nutrient intake. OXM was originally isolated from the porcine intestinal tract and found to be a potent inhibitor of gastric acid secretion and gastric emptying in rodents and humans, inhibiting appetite and increasing energy expenditure, and promoting lipolysis. In vitro experiments show that OXM binds to and activates the GLP-1receptor (GLP-1R) and the glucagon receptor (glucagon receptor, GCGR), and that the affinities for these two receptors are approximately the same, with the receptors being widely distributed in body organ tissue. The study by Bloom et al demonstrated that OXM could suppress appetite, but unlike GLP-1, OXM could also increase energy expenditure, suggesting that both GLP-1R and GCGR activation are involved in metabolic processes. Natural OXM has a short half-life, similar to GLP-1, and has a half-life of about 12 minutes in human blood and a shorter half-life of about 6 minutes in rat blood, and degradation products are excreted via renal clearance.

OXM inhibits food intake in rodents and humans and stimulates energy expenditure on regulating body weight. Anorexia effects caused by OXM can be eliminated by GLP-1R antagonist exendin, which was not observed in GLP-1R (-/-) mice, suggesting that the effect of OXM on food intake is mediated by GLP-1R. Other effects of OXM, including stimulation of heart rate and energy expenditure, appear to be independent of GLP-1R in vivo. Since OXM antagonizes GLP-1R and GCGR in vitro and it is reported that heart rate and energy expenditure increase after GCGR activation, the differential effect of OXM and GLP-1 may be mediated through GCGR activation.

Studies have shown that OXM significantly improves glucose metabolism in mice, and in mouse experiments OXM reduces GLP-1R (-/-), GCGR (-/-) and wild-type mice by the amount of exogenous glucose required to maintain high blood glucose levels. These data indicate that activating GCGR contributes to the hypoglycemic effect of OXM. In addition to GLP-1R and GCGR, OXM may also activate the weight controlling and hypoglycemic effects of other receptors, OXM. An ini et al study showed that OXM can inhibit pancreatic secretion in rats via the nervous system. Although glucagon can inhibit pancreatic exocrine in normal animals and humans, it is not known how glucagon can exert this effect. Intravenous administration of OXM and glucagon has also been reported to increase intestinal glucose uptake in rats. While in vivo data may be affected by differences in circulating insulin levels, OXM is considered a more potent regulator than glucagon. Since GLP-1 does not stimulate Glucose absorption, and Glucagon-like peptide-2 (GLP-2) and Glucose-dependent insulinotropic peptide (GIP) were found to increase hexose transport, it was shown that OXM could bind to the two G-protein-coupled receptors (G-protein-coupled receptors, GPCRs). After addition of OXM culture to GLP-1 or GCGR expressing rat BHK cells, cAMP accumulation was significantly increased. Taken together, these data suggest that OXM physiological effects are associated with other receptors, but that other downstream pathways besides cAMP may be involved. Since the current research on OXM is still in the preliminary stage, no related drugs have been marketed yet.

GLP-1 and OXM secreted by intestinal L cells have important physiological functions, can enhance glucose-induced insulin response, improve survival rate of beta cells, reduce appetite, improve body fat ratio, protect nerve tissue, prevent heart lesions, reduce inflammatory response and the like. However, GLP-1, OXM, etc. secretion or efficacy was reduced in type 2 diabetics, showing an impaired L cell. In some diabetics, patients taking GLP-1 analogues and Dipeptidylpeptidase 4 (Dipeptidylpeptidase 4) develop resistance, and the pathological mechanism behind this may be a decrease in secretion of GLP-1 by intestinal L cells, resulting in a decrease in GLP-1 plasma concentration, with or without decreased action of GLP-1 in beta cells, so-called GLP-1 resistance. Therefore, restoring endogenous production of intestinal hormone such as GLP-1, for example, simulating production of intestinal hormone by using engineering bacteria, is an effective measure for treating type 2 diabetes and obesity.

Lactococcus lactis (Lactococcus lactis) has long been widely used in the food industry, particularly dairy products, and is therefore recognized by the Food and Drug Administration (FDA) as a safety (GRAS) certificate, a model of the genus Lactobacillus. Because of its clear genetic background (at least four lactic acid bacteria strains have been fully sequenced) and ready-to-use expression systems, lactococcus lactis is a widely used microbial expression host. Various constitutive and inducible expression systems have been developed for lactococcus lactis, and P8 and P32 are commonly used as constitutive lactococcus lactis promoters. Since secreted proteins are easier to purify, production of heterologous proteins is more prone to secretory expression. Lactococcus lactis has an advantage in secreting proteins, which have a monolayer cell wall, allowing direct secretion into the extracellular environment, in comparison to E.coli, which proteins are mainly retained in the periplasm. In addition, lactococcus lactis only has a single extracellular tubular protease HrtA, thereby reducing the chance of degradation of heterologous proteins.

The viability of lactobacilli in the gastrointestinal tract (Gastrointestinal tract) is of interest because of their immunomodulatory properties, and thus lactobacillus has been used as a carrier for delivering therapeutic agents (e.g. cytokines) into the human body. Steidler et al utilized human Lactobacillus to express Interleukin 10 (IL-10) for the treatment of inflammatory bowel disease in colitis-induced mice (Inflammatory bowel disease). Therefore, the safe transgenic organism (Genetically modified organism, GMO) strain can directly secrete protein in intestinal tract, and has good therapeutic prospect. These factors make it an ideal food-safe model of intestinal therapeutic protein expression.

Disclosure of Invention

Modern eating habits tend to take a large amount of high-sugar and high-fat foods and eat more and less food, so that the amount of food intake is more than the amount of body consumption, the excess heat is stored in the body in the form of fat, so that the body becomes obese, obesity is a main factor inducing diabetes, hyperlipidemia, nonalcoholic fatty liver diseases and the like, and two proteins of OXM and GLP-1 secreted by intestinal L cells can promote gastrectasia, delay gastric emptying, generate satiety, inhibit appetite and increase energy consumption, and promote lipolysis. Thus dual agonists of GCGR and GLP-1R represent a new approach to the treatment of diabetes and obesity with the potential to suppress appetite, enhance weight loss and improve glycemic control, beyond single agonists. Animal experiments prove that the recombinant engineering bacteria have the potential of treating obesity, diabetes, hyperlipidemia and nonalcoholic fatty liver disease.

The invention aims at providing a recombinant bacterium which comprises a GLP-1 gene expression cassette and an OXM gene expression cassette in series;

wherein the GLP-1 gene expression cassette comprises a P32 promoter, a first terminator and a GLP-1 gene transcribed by the two promoters; the OXM expression cassette comprises a P8 promoter, a second terminator and an OXM gene of which transcription is regulated and controlled by the two promoters;

the N-terminal of the GLP-1 gene and the OXM gene is also provided with a signal peptide.

The invention also relates to a plasmid comprising the GLP-1 gene expression cassette and an OXM gene expression cassette.

The invention also relates to a composition comprising the recombinant bacterium and medical application of the recombinant bacterium.

The recombinant bacteria provided by the invention can efficiently and correctly secrete and express two proteins of recombinant human OXM and GLP-1. The secreted GLP-1 can effectively control blood sugar and reduce food intake, and the secreted OXM can increase heat consumption through glucagon activity, so that after the recombinant engineering bacterium is used for feeding a mouse, the mouse can keep constant energy consumption while eating, and long-term oral administration can effectively consume fat to reduce weight, so that the recombinant engineering bacterium has the potential of treating one or more metabolic disease indications of obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver disease, and can also be used for treating neuron degenerative diseases such as hypertension, heart failure, alzheimer disease and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

Fig. 1 is a schematic diagram of a pLD carrier structure according to an embodiment of the present invention.

FIG. 2 shows the construction of the vector pLD, pLD-GO provided by one embodiment of the present invention;

(a) M: DNA ladder (0.1-5 kb); 1, a CAF DNA fragment of repA, repC, lacF cut by BamHI and SacII double enzymes; 2, synthesizing an expression element PT DNA fragment by BamHI and SacII double enzyme digestion; 3:pld plasmid; 4, pLD plasmid subjected to BamHI and SacII double enzyme digestion; (b) M: DNA ladder (0.1-5 kb); 1, a CAF DNA fragment of repA, repC, lacF cut by BamHI and SacII double enzymes; 2, synthesizing an expression element GO DNA fragment through BamHI and SacII double enzyme digestion; 3:pld-GO plasmid; 4-pLD-GO plasmid digested with BamHI and SacII.

FIG. 3 shows the Western Blotting identification result of pLD-GO recombinant engineering bacteria expressing GLP-1 and OXM;

(a) M rainbow 245plus broad-spectrum protein Marker;1 lactococcus lactis NZ3900 control bacteria culture medium supernatant; 2pLD empty vector transformation positive clone culture medium supernatant; 3pLD-GO transformed positive clone culture medium supernatant. (b) M rainbow 245plus broad spectrum protein Marker;1 lactococcus lactis NZ3900 control bacteria culture supernatant; 2pLD empty vector transformation positive clone culture medium supernatant; 3pLD-GO transformed positive clone culture medium supernatant.

FIG. 4 shows the growth curve of pLD-GO engineering bacteria; * The experimental data are all expressed as mean ± SE.

FIG. 5 is an ELISA fitted curve and regression equation for OXM and GLP-1. ELISA fitting curve and regression equation (a) OXM; (b) GLP-1

FIG. 6 is a graph showing the change in body weight of mice; the experimental data are all expressed as mean ± SE; * P <0.0001, P <0.001, P <0.01 data were treated with one-way ANOVA.

FIG. 7 is 24 hour feeding of a gavage mouse; the experimental data are all expressed as mean ± SE; * P <0.01 data were treated with one-way ANOVA.

FIG. 8 is a graph of blood glucose curve and AUC of gastric lavage mice; the experimental data are all expressed as mean ± SE; * P <0.001, P <0.01, P <0.05, data were treated with one-way ANOVA.

FIG. 9 shows the results of metabolism cage experiments for each group of mice;

(a)Food consumption，(b)Water consumption，(c)energy expenditure，(d)O ₂ consumption，(e)CO ₂ the data of the experiment are expressed in the form of average value + -SE; * P (P)<Data 0.05 were treated with one-way ANOVA.

FIG. 10 shows the results of the concentration measurement of OXM and GLP-1 in the serum of mice;

(a) OXM, (b) GLP-1, data from experiments are all expressed as mean ± SE; * P <0.001, P <0.01, P <0.05 data were treated with one-way ANOVA.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "first" and "second" are used only to distinguish different kinds of terminators, and no additional limitation is imposed otherwise.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

The present invention relates to a recombinant bacterium comprising a GLP-1 gene expression cassette and an OXM gene expression cassette in tandem;

As used herein, the term "recombinant bacterium (recombinant bacteria)", or "recombinant engineering bacterium", refers to a bacterium that has been genetically modified from its natural state. For example, a recombinant bacterium may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into its DNA. These genetic modifications may be present in the chromosome of the bacterium or bacterial cell, or on a plasmid present in the bacterium or bacterial cell. The recombinant bacterial cells of the present disclosure may comprise an exogenous nucleotide sequence on a plasmid. Alternatively, the recombinant bacterial cell may comprise an exogenous nucleotide sequence stably incorporated into its chromosome. In some embodiments, the recombinant bacterial cells of the present disclosure are lactococcus lactis bacterial cells comprising an exogenous nucleotide sequence on a plasmid. In some embodiments, the recombinant bacterial cells of the present disclosure are lactococcus lactis bacterial cells having nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. In a further embodiment, the recombinant bacterial cell of the present disclosure is a genetically engineered lactococcus lactis bacterial cell.

In some embodiments, the recombinant bacterium is a nonpathogenic bacterium. "nonpathogenic bacteria" refers to bacteria that are incapable of causing a disease or adverse reaction in a host. In some embodiments, the non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to, bacillus, bacteroides, bifidobacteria, breve bacilli, clostridia, enterococci, escherichia coli, lactobacillus, lactococcus, yeast, and staphylococci, e.g., bacillus coagulans, bacillus subtilis (Bacillus subtilis), bacteroides fragilis, bacillus subtilis (Bacteroides subtilis), bacteroides thetaiotaomicron, bifidobacteria infantis, bifidobacterium lactis, bifidobacterium longum, clostridium butyricum, enterococcus faecalis, lactobacillus acidophilus, lactobacillus bulgaricus, lactobacillus casei, lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus paracasei, lactobacillus plantarum, lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus, and lactococcus lactis. In some embodiments, naturally pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity.

In some embodiments, the recombinant bacterium is a probiotic. "probiotic" is used to refer to a viable, non-pathogenic microorganism, such as a bacterium, that may bring a health benefit to a host organism containing an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Certain species, strains and/or subtypes of non-pathogenic bacteria are currently considered probiotics. Examples of probiotics include, but are not limited to, bacillus, bacteroides, bifidobacteria, breve bacillus, clostridium, enterococcus, escherichia coli, lactobacillus, lactococcus, yeast, and staphylococci, e.g., bacillus coagulans, bacillus subtilis (Bacillus subtilis), bacteroides fragilis, bacillus subtilis (Bacteroides subtilis), bacteroides thetaiotaomicron, bifidobacteria, streptococcus thermophilus, bifidobacterium infantis, bifidobacterium lactis, bifidobacterium longum, clostridium butyricum, enterococcus faecalis, lactobacillus acidophilus, lactobacillus bulgaricus, lactobacillus casei, lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus paracasei, lactobacillus plantarum, lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus, and lactococcus lactis (sonnenborne et al, 2009; dinley i et al, 2014; U.S. Pat. No. 6,835,376; U.S. 6,203,797; U.S. Pat. No. 5,589,168; and U.S. No. 7,731,976). The probiotics may be variants or mutant strains of bacteria (Arthur et al 2012; cuevas-Ramos et al 2010; olier et al 2012; nougayde et al 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, such as survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotics may be genetically engineered to enhance or improve the probiotic properties. In some specific embodiments, the recombinant bacterium is selected from the group consisting of: bacteroides, bifidobacteria, clostridia, escherichia, eubacteria, lactobacillus, lactococcus and ross.

In some embodiments, the recombinant bacterium is lactococcus lactis. In some specific embodiments, the recombinant bacterium is a lactococcus lactis NZ3900 strain.

In some embodiments, the nucleotide sequences of the GLP-1 gene are respectively shown in SEQ ID NO: 1.

In some embodiments, the OXM gene has the nucleotide sequence set forth in SEQ id no: 2.

In some embodiments, the GLP-1 gene expression cassette and the OXM gene expression cassette are expressed in reverse, in opposite, or in the same direction.

Reverse expression can be achieved in the manner schematically shown in FIG. 1, which reduces the interaction of the P32 promoter and the P8 promoter.

Opposite expression, i.e., the opposite direction of expression of the two gene expression cassettes, is opposite, with their direct terminators closest on the plasmid.

In some embodiments, a spacer DNA of 20bp or more is provided between the GLP-1 gene expression cassette and the OXM gene expression cassette; preferably, the DNA spacing is greater than or equal to 100bp, more preferably greater than or equal to 200bp, more preferably greater than or equal to 300bp, and most preferably greater than or equal to 500bp. The spacer DNA is also effective in reducing the interaction of the P32 promoter and the P8 promoter.

In some specific embodiments, the nucleotide sequence of the P32 promoter is set forth in SEQ ID NO: 3.

In some specific embodiments, the nucleotide sequence of the P8 promoter is set forth in SEQ ID NO: 4.

In some embodiments, the first terminator is T-usp45; in some embodiments, the nucleotide sequence of the T-usp45 terminator is set forth in SEQ ID NO: shown at 5.

In some embodiments, the second terminator is T-pepN; in some embodiments, the nucleotide sequence of the T-pepN terminator is set forth in SEQ ID NO: shown at 6.

One skilled in the art knows, SEQ ID NO:1 and 2 is codon optimized for lactococcus lactis, suitable nucleotide sequences for the GLP-1 gene and OXM gene, but also include nucleotide sequences that are identical to SEQ ID NO:1 or 2 to at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.

Likewise, SEQ ID NO:3 to 6, and SEQ ID NOs employed hereinafter: 7 and 8, variants of nucleotide sequences up to at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% homologous to their respective nucleotide sequences, as long as they are capable of achieving similar efficiency of initiation/termination; such modifications will be readily apparent to those skilled in the art.

Furthermore, it will be appreciated by those skilled in the art that constitutive promoters similar to the P32 or P8 promoter capability are also within the scope of the present invention, e.g., P1, P2,P 3,P 4,P 5,P 6,P 7,P lacA, etc.

In one embodiment, the BLAST algorithm is used to determine the percentage of "homology" between two sequences using the BLASTP algorithm using default parameters (Altschul et al, J. Nucleic acid research, 1997, month 1; 25 (17): 3389-402) can be known online by the following web sites: https:// blast.ncbi.nlm.nih.gov/blast.cgi. In another embodiment, the percentage of homology between two sequences is determined by the EMBOSS Needle algorithm using default parameters in order to develop a global alignment of the sequences. The EMBOSS Needle algorithm can be known online through the following websites: https:// www.ebi.ac.uk/Tools/psa/emboss_needle/.

Unless otherwise indicated, the term "homology" may be used interchangeably with the term "identity" in this specification.

"Signal sequence" (also referred to as "presequence)", "Signal peptide)", "leader sequence" or "leader peptide") refers to an amino acid sequence located at the N-terminus of a nascent protein, and which can facilitate secretion of the protein from a cell. The mature form of the resulting extracellular protein lacks the signal sequence, which is cleaved off during secretion. In some preferred embodiments, the signal peptide is a Usp45 signal peptide. In some specific embodiments, the nucleotide sequence of the signal peptide is as set forth in SEQ ID NO: shown at 7.

Any method may be used to introduce the exogenous nucleic acid molecule into the cell. Indeed, many methods for introducing nucleic acids into bacteria are known, including, for example, heat shock, lipofection, electroporation, conjugation, protoplast fusion, and gene gun delivery.

The exogenous nucleic acid molecule contained within the host cell may be maintained within the host cell in any form. For example, the exogenous nucleic acid molecule may be integrated into the genome of the host cell or maintained in an episomal state. In other words, the host cell may be a stable or transient transformant. The host cells described herein can contain a single copy or multiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100, or 150 copies) of a particular exogenous nucleic acid molecule as described herein. In some preferred embodiments, the GLP-1 gene expression cassette and OXM gene expression cassette are located on a plasmid.

In some preferred embodiments, the plasmid further comprises a replicon and a selection gene.

In some embodiments, the replicons include repA and repC.

In some embodiments, the recombinant bacterium is a lacF-deficient strain and the selection gene is lacF.

In some embodiments, the plasmid is derived from pNZ8149.

In some embodiments, the plasmid comprises SEQ ID NO:8.SEQ ID NO:8 comprises LacF, repA, repC three DNA elements.

In some embodiments, the expression direction of the other elements in the plasmid is consistent with the expression direction of the OXM expression cassette.

The invention also relates to a plasmid as defined above.

The invention also relates to a composition comprising a recombinant bacterium as described above.

In some embodiments, it is a pharmaceutical composition, preferably further comprising a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, as known to those of ordinary skill in the art, and combinations thereof (see, e.g., remington's Pharmaceutical Sciences, 18 th edition Mack Printing Company,1990, pages 1289-1329, which is incorporated herein by reference).

The pharmaceutical compositions may be administered by a variety of routes, including oral, rectal and intranasal. The compositions are formulated as injectable or oral compositions, or as ointments, lotions or patches, depending on the intended route of delivery. Compositions for oral administration may take the form of a bulk liquid solution or suspension or a bulk powder. More commonly, however, the compositions are presented in unit dosage form to facilitate accurate administration. Typical unit dosage forms comprise pre-filled, pre-measured ampoules or syringes of liquid compositions, or in the case of solid compositions, pills, tablets, capsules, etc. The components of the compositions described above for oral administration or injection administration are merely representative. Other substances and processing techniques are described in, inter alia, leimdons: pharmaceutical science and practice (Remington "s The Science and Practice of Pharmacy), 21 st edition, 2005, publishing company: section 8 of LiPinkot Williams (Lippincott Williams) & Wilkins, incorporated herein by reference. For oral administration, particular use is made of pills, tablets, capsules, suppositories, liquids or liquid suspensions.

In other embodiments, the composition is an edible composition. The edible composition may be, for example, yoghurt, cheese, milk, meat, cream or chocolate. Such edible compositions may be considered edible foods, meaning that they have been approved for human or animal consumption.

The term "food product" is intended to cover all edible products that are solid, gelatinous or liquid. Suitable foods may include, for example, functional foods, food compositions, pet foods, livestock feeds, health foods, feeds, and the like. In some embodiments, the food product is a specified health food product.

As used herein, the term "functional food" means a food that is not only capable of providing a nutritional effect, but is also capable of delivering further benefits to the consumer. Thus, a functional food is a component or ingredient having incorporated therein a particular functionality (e.g., medical or physiological benefit) imparted to the food.

Examples of specific foods suitable for use in the present disclosure include dairy products, ready-to-eat desserts, powders reconstituted with, for example, milk or water, chocolate milk drinks, malt beverages, ready-to-eat dishes or beverages for humans, or food compositions equivalent to a complete or partial diet intended for humans, pets, or livestock.

In some embodiments, the composition according to the present disclosure is a food intended for primates (preferably humans), pets or livestock. The composition may be intended for an animal selected from the group consisting of: non-human primates, dogs, cats, pigs, cows, horses, goats, sheep, or poultry. In another embodiment, the composition is a food or pharmaceutical product intended for adult species, in particular adults.

According to a further aspect of the invention, it also relates to the use of a recombinant bacterium as described above for the preparation of a medicament for the treatment of one or more of metabolic disorders, neurodegenerative disorders (such as Alzheimer's disease), memory and learning disorders, irritable bowel syndrome, stroke, post-operative catabolic changes (preferably islet cell transplantation), functional dyspepsia and ischemia-reperfusion tissue damage.

For the purposes of the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures of disease. Patients in need of treatment include those already with the disease, those prone to the disease, or those in need of prophylaxis of the disease.

In some embodiments, the metabolic disease is selected from one or more of diabetes, hypertension, hypoglycemia, dyslipidemia (e.g., hyperlipidemia), cardiovascular disease (e.g., atherosclerosis, coronary artery disease, heart failure), abnormal clotting, obesity, diabetic complications (e.g., diabetic retinopathy), hepatobiliary disease (e.g., alcoholic or non-alcoholic fatty liver), chronic kidney disease, insulin resistance, and glucose tolerance abnormality.

The compositions of the invention may be used in combination with other agents for use in the treatment or prevention of diseases involving GLP 1-R. Specific compounds for use in combination with the compositions of the present invention include: simvastatin, mevastatin, ezetimibe, atorvastatin, sitagliptin, metformin, orlistat, qnexa, topiramate, naltrexone, bupropion (buproprion), phentermine, and losartan, losartan+hydrochlorothiazide.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Examples

Materials and methods

1 Strain and plasmid

Coli JM109, lactococcus lactis L.lactis NZ3900 was kept by the laboratory; pNZ8149 is available from MoBiTec GmbH, germany; adding nucleic acid sequences of USP45 protein signal Peptide to N-terminal of Glucoago-Like Peptide 1 and Oxyntomodulin, optimizing codon of lactococcus lactis by jcat on-line tool, and synthesizing the DNA sequences by Shanghai chemical company; primers used in this experiment (Table 1) were synthesized from BGI Group (shenzhen).

2 PCR primer used in chapter

The primers for PCR amplification in this chapter are shown in Table 1.

TABLE 1 primers used in construction of pLD

3 database and biological software

The database GenBank (www.ncbi.nlm.nih.gov) employed herein; PCR primer design software: primer premier 5; gene and protein sequence processing software: dnasist (v 3.0); codon optimized JCat online tool: http:// www.jcat.de/; the ELISA standard Curve is drawn by Curve Expert1.4; image processing software: imageJ.

4 main culture medium and pharmaceutical agent

4.1 principal drugs and reagents

The drugs and reagents mainly used in this chapter are shown in table 2.

Table 2 contains the main drugs and reagents

4.2 Main Medium

1) GM17 medium: m17 Medium 45g,5g glucose, adding deionized water 950mL, dissolving thoroughly, fixing volume to 1000mL (solid medium requires adding 1.5% agar again), and autoclaving (121 ℃,15min, all following parameters).

2) Electrotransformation competent SM17 medium: m17 Medium 45g,0.5M sucrose, 25g glycine, 5g glucose, 950mL deionized water was added for complete dissolution, the volume was set to 1L, and autoclaved for 15 min.

3) Elliker medium, tryptone 20g,Yeast extract 10g, sodium chloride 4g, anhydrous sodium acetate 1.5g,Ascorbic acid 0.5g, lactose 5g, and deionized water 950mL were added for complete dissolution, the volume was set to 1L (solid medium required to be added with 1.5% agar), and the medium was autoclaved.

4) Sucrose-glycerol solution: 0.5M sucrose, 100mL glycerol, 950mL deionized water was added for sufficient dissolution, the volume was set to 1000mL, and the mixture was autoclaved.

5) Electrokinetic recovery medium: 45g of M17 culture medium, 0.5M sucrose, 25g of glycine, 5g of glucose, 20mM magnesium chloride and 2mM calcium chloride, 950mL of deionized water are added for complete dissolution, the volume is fixed to 1000mL, and the mixture is subjected to high-pressure sterilization.

4.3 reagents for molecular experiments

1)RIPA lysis buffer：50mM Tris(pH＝7.4)，150mM NaCl，1％TritonX-100，1mM EDTA，0.1％ SDS，10mM NaF，1mM PMSF。

2) 50×TAE: 242g of Tris, 37.2g of Na2 EDTA.2H2O, 800mL of deionized water and 57mL of glacial acetic acid are weighed and the volume is fixed to 1L.

3) 1M Tris-HCl (pH 6.8): tris 12.1g was dissolved in 90mL deionized water, pH adjusted to 6.8, and the volume was set to 100mL.

4) 1.5M Tris-HCl (pH 8.8): 18.17g Tris was dissolved in 90mL deionized water, pH adjusted to 8.8, and the volume was set to 100mL.

5) 10% SDS (W/V): 1g of SDS was weighed, added with deionized water to a volume of 10mL and stored at room temperature.

6) 10% APS: 1.0g of APS was weighed, added with deionized water to a volume of 10mL and stored at-20 ℃.

7) 30% acr/Bic: 29g of acrylamide and 1g of N, N' -methyleneacrylamide were dissolved in ultrapure water to a volume of 100mL and stored at 4 ℃.

8) 12% of separation gel: 1.6mL of deionized water, 2.0mL of 30% acrylamide, 1.3mL of 1.5mol/L Tris-HCl,0.05mL of 10% SDS,0.05mL of 10% APS, and 0.002mL of TEMED were taken.

9) 5% of concentrated glue: 0.68mL of deionized water, 0.17mL of 30% acrylamide, 0.13mL of 1.0mol/L Tris-HCl,0.01mL of 10% SDS,0.01mL of 10% APS,0.001mL of TEMED were taken.

10 5 XTris-glycine running buffer: 94g Gly,15.1g Tris and 5g SDS were weighed, 900mL deionized water was added, dissolved and the volume was set to 1L, and stored at room temperature.

11 Washing liquid: 20mM Tris-HCl,500mM Na Cl,1mM EDTA,0.5%Trition X-100, pH 8.5.

12 5×loading buffer): 1.25mL of 1M Tris-HCl (pH=6.8), 5mL of 10% SDS,25mg of bromophenol blue, 2.5mL of glycerol, 250ul of beta-mercaptoethanol, and ultrapure water was added to 5mL. Filtering with 0.22 μm filter membrane, and storing at-20deg.C.

13 Dyeing liquid: coomassie brilliant blue R-250 g, methanol 450mL, glacial acetic acid 450mL, adding ultrapure water to a volume of 1L, mixing uniformly, and preserving at room temperature.

14 Decolorized solution): 200mL of ethanol, 100mL of glacial acetic acid and 700mL of deionized water are taken, uniformly mixed and stored at room temperature for standby.

Reagents for extracting plasmids by alkaline lysis method:

a) Solution p1, 50mmol/L glucose, 10mmol/L EDTA,25mmol/L Tris-HCl,100mg/L RNase.

b) Solution p 2-0.2 mol/L NaOH,1% SDS.

c) Solution p3:2mol/L acetic acid, 3mol/L potassium acetate (pH=5.5).

5 instrument and apparatus

The instruments and equipment used in this chapter are shown in table 3.

Table 3 major instruments and apparatus

6 grouping of laboratory animals

20+ -1 g male C57 mice (6 weeks old) purchased from Hunan Stokes Levoda laboratory animal Limited (laboratory animal production license: SYXK (Guangdong) 2017-0125, laboratory animal ethical number: gddpulacspf 2017060). Mice were kept under constant temperature (22.+ -. 2 ℃) and humidity (50-70% relative humidity) in SPF class animal houses at Guangdong university of Pharmacology, and animals of different groups were kept in 4 animals/cage, alternately light and dark (12 hours/12 hours) throughout the test period, provided sufficient food and water, and observed at regular intervals daily for animal condition 1 time per week.

Preparation of 7 electric transfer competent lactococcus lactis NZ3900

Lactococcus lactis strain NZ3900 (lacF ^- pepN:: nisRK) is a food grade strain derived from a lacF deletion mutant of lactococcus lactis MG5267 which lacks lactase lacF and thus cannot grow in lactose-sole carbon source medium, whereas if the transferred plasmid contains the lacF gene, it can be allowed to resume growth in lactose-sole carbon source medium.

Preparation of competent lactococcus lactis NZ 3900:

a) NZ3900 glycerol frozen stock was thawed, streaked on G17 plates, and then allowed to stand at 30℃for 2 days.

b) Monoclonal colonies were picked from G17 plates and inoculated into 50mL SG17 medium, and cultured at 30℃in a static manner.

c) When the OD of the culture medium ₆₀₀ When 0.3 was reached, the medium was centrifuged at 3000 Xg for 5min at room temperature.

d) The pellet was resuspended in 5mL sucrose-glycerol solution (pre-chilled on ice) and washed and centrifuged at 6000 Xg for 3min at 4℃and the supernatant discarded to leave a bacterial pellet.

e) The bacterial pellet was resuspended in 5mL sucrose-glycerol solution (pre-chilled on ice), the bacterial pellet was placed on ice for 15min, then centrifuged at 6000 Xg for 3min at 4℃and the supernatant was discarded to leave the bacterial pellet.

f) Re-suspending thallus sediment by 1mL sucrose-glycerol solution (precooled on ice), at this time, the thallus sediment is electrotransformation competent, and the thallus sediment is respectively packaged into 80 mu L by a new EP pipe and stored in a refrigerator at-80 ℃ for standby.

8 preparation of DNA fragments required for constructing vectors

The expression element of the vector pLD consists of a P32 promoter and a P8 promoter, the terminators are T-usp45 and T-pepN respectively, the whole expression element sequence is named PT, and the DNA sequence is delivered to Shanghai biochemical synthesis; the target genes Glucago-Like Peptide 1 and oxynodomdulin with the secretion signal Peptide of USP45 protein added at the N end (shown as SEQ ID NO: 7) are subjected to codon optimization (shown as SEQ ID NO:1 and 2) and then placed after P32 and P8 promoters (shown as SEQ ID NO:3 and 5) respectively, and the terminators are T-USP45 and T-pepN (shown as SEQ ID NO:4 and 6 respectively), so that the whole GLP-1-OXM target gene expression cassette (hereinafter referred to as GO) DNA sequence is delivered to Shanghai biological engineering for synthesis.

The fragment (shown in SEQ ID NO: 8) comprising three DNA elements LacF, repA, repC (hereinafter referred to as CAF) is derived from a vector pNZ8149, wherein replicon genes repA and repC can make the vector perform replication and amplification on lactococcus lactis (L.lactis), and LacF gene expresses lactase F, so that the recombinant strain can obtain lactose utilization capacity to become a screening marker, and all three genes are derived from the lactococcus lactis genome. CAF-R and CAF-F amplify the whole DNA sequence of CAF, the template is the DNA of pNZ8149 plasmid, the target fragment size is about 2100bp, and the PCR amplification conditions are: pre-denaturation at 94℃for 5min; the product was subjected to 25 cycles of 94℃for 30s,55℃for 50s,72℃for 2min for 30s and extended at 72℃for 15min.

The PCR product DNA of PT and GO synthesized by Shanghai and the PCR product DNA of CAF are respectively subjected to double enzyme digestion reaction, and the enzyme digestion system is as follows: 2. Mu.L of restriction enzymes BamHI and SacII each; 10 Xenzyme cutting buffer 5L; 10. Mu.g of DNA fragment or PCR product, respectively; sterile water (up to 50 μl); water bath at 37 ℃ for 2h. And (3) respectively carrying out agarose gel electrophoresis on the enzyme digestion reaction products, and then cutting and recycling.

9 construction of recombinant vector

The BamHI and SacII double digested and purified PT and GO are respectively connected with the DNA fragment containing LacF, repA, repC element CAF after the same digestion and purification by T4 ligase to respectively generate pLD and pLD-GO recombinant vectors, and the connection reaction system is as follows (10 mu L system): 200ng of DNA fragments of CAF, and 500ng of 2 DNA fragments such as PT and GO; mixing well 10 Xbuffer 1.0 μL on ice; 1. Mu.L of T4 ligase was added with sterile water to 10. Mu.L, and after mixing well, the mixture was centrifuged briefly for 10sec and was connected in a water bath at 16℃overnight. The 2 recombinant vectors pLD and pLD-GO were respectively electrotransformed into L.lactis NZ3900 cells. 60. Mu.L of the electrocompetent lactococcus lactis NZ3900 melted on ice was placed in an electroconvulsive cup pre-cooled on ice, and 1. Mu.g of DNA to be transformed was added. Setting electric conversion parameters: 2000v, 5ms. Immediately after electric shock, 1mL of the resuspension broth of the electrotransport recovery medium was added, the electrotransport cup was placed on ice for 5min, incubated at 30℃for 1.5h, centrifuged at 3000 Xg for 5min, the supernatant was discarded and then resuspended in 200. Mu.L of physiological saline, plated on Elliker plates, and incubated at 30℃for 48 h.

After 48 hours of cultivation, the monoclonal colonies were then picked up and placed in Elliker medium at 30℃for resting cultivation for 48 hours, and the plasmids of interest were extracted from lactococcus lactis: 5mL of bacterial liquid is centrifuged for 1 min at 5000 Xg, the supernatant is removed by precipitation, 100 mu L of pre-cooled P1 solution on ice is added into the precipitate, shaking and uniformly mixing are carried out, 10 mu L of lysozyme solution (working concentration is 100 mg/mL) is added into the precipitate, uniformly mixing is carried out, cell walls are subjected to enzymolysis by shaking table standing at 37 ℃ for 60min, then 200 mu L P2 solution is added, 5 times of ice bath is rapidly reversed for 5min, 150 mu L of pre-cooled P3 solution on ice is added, 5min of ice bath is carried out after several times of inversion, 12 Xg of ice bath is carried out, 10 min is carried out at 000 Xg, supernatant is collected after centrifugation, equal amount of phenol chloroform extract is added into the mixture, and the mixture is fully mixed, and then 12,000Xg of the mixture is centrifuged for 3 minSucking the supernatant into another EP tube, adding 1 times volume of precooled isopropanol, mixing, standing at-20deg.C for 20min, centrifuging at 12,000Xg for 5min, discarding supernatant, adding 1mL of 70% ethanol, washing with shaking thoroughly for 3 times, centrifuging at 12,000Xg for 1 min, discarding supernatant, naturally airing at room temperature for 15min, and adding 30 μL of ddH ₂ O is fully dissolved and then frozen at the temperature of minus 20 ℃ for storage for standby.

The extracted plasmid DNA is subjected to BamHI and SacII double enzyme digestion identification, and the enzyme digestion system is as follows: 1 μl of each of the restriction enzymes BamHI and SacII; 2. Mu.L of 10 Xrestriction buffer; 3. Mu.g of plasmid DNA; sterile water (up to 20. Mu.L); water bath at 37 ℃ for 2h. After the reaction was completed, 5. Mu.L of the amplified product was subjected to 1% gel nucleic acid electrophoresis for identification, and a part of the plasmid DNA was sent for sequencing.

Identification of 10 recombinant engineering Strain Western Blotting

preparation of pLD-GO recombinant engineering strain protein sample (target protein secretion expression): 1mL of the bacterial liquid is centrifuged for 1 min at 5000 Xg, bacterial precipitate is discarded, a supernatant is reserved, a proper amount of 5X SDS loading Buffer (4:1 v/v) is added, the mixture is heated to 100 ℃ in water bath for 10min, and the supernatant is obtained after centrifugation at 5000 Xg for 3 min, thus obtaining the SDS-PAGE sample. protein samples were prepared in the same manner as in the pLD empty vector transformation control group and the control group of the parent strain NZ 3900.

Western Blotting detects the expression of the target protein:

1) SDS-PAGE gel (SDS polyacrylamide gel: concentrated gum 5%, isolated gum 10%).

2) mu.L of protein was loaded per well and electrophoresed in Tris-SDS-PAGE buffer at 80V for 2 hours.

3) Transferring membrane, spreading foam-rubber cushion, wet filter paper, PVDF membrane (pre-soaked in methanol), gel after electrophoresis, wet filter paper and foam-rubber cushion on polar plate in order, avoiding air bubble. And (3) electrophoresis is carried out for 100min at 90 milliamps in precooled glycine film transferring liquid.

4) Blocking, the transferred membranes were placed in 5% skim milk (formulated with TBST) and blocked for 1 hour.

5) Adding primary antibody diluted with blocking solution (pLD-GO recombinant engineering strain protein sample is respectively Anti-XM antibody-Human OXM Ig G and Anti-GLP-1ab200474 primary antibody of GLP-1), and incubating at 4deg.C for overnight.

6) Membranes were washed 3 times with TBST to remove unbound primary antibody, anti-rabbit IgG (Cell Signaling, # 7074) diluted with TBST was added and incubated with gentle shaking for 2 hours.

7) The membrane was washed 5 times with TBST to remove unbound secondary antibody.

8) Exposing, namely dripping ECL luminous liquid on a PVDF film, putting the PVDF film into a BIO-RAD multifunctional imager, selecting proper exposure time according to conditions, and taking a picture.

9) And analyzing the image result.

11 recombinant OXM expressed by recombinant engineering bacteria and GLP-1 expression quantity measurement

pLD-GO recombinant engineering bacteria are used for preparing 1 multiplied by 10 ⁴ Inoculating CFU into 100mL Elliker culture medium, standing at 30deg.C, culturing, taking culture solution once every 3 hr, adding the culture solution sample into 96-well plate with 200 μl of each well, setting 5 wells as biological repetition, using Elliker culture medium as blank control, placing in enzyme-labeled instrument, measuring absorbance (OD) of culture solution in each well with 600nm wavelength ₆₀₀ Value), samples were taken continuously for 70 hours, and data were analyzed for 70 hours.

And (3) taking 1mL of bacterial liquid when the pLD-GO recombinant engineering bacteria are cultured to reach the highest OD600 value, centrifuging at 5000 Xg for 1 minute, discarding bacterial precipitate to leave supernatant, freezing the supernatant in a refrigerator at the temperature of minus 80 ℃ for standby, and detecting the recombinant OXM and GLP-1 expression quantity of the supernatant of the culture medium by adopting an enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows.

OXM ELISA kit:

a) And taking out the aluminum foil bag, standing at room temperature for at least 20 minutes, taking out the required enzyme label strips, sealing the rest strips by using a self-sealing bag, and storing at the temperature of 4 ℃.

b) Preparing a standard substance: each experiment was tested by preparing a dilution of the standard within 15 minutes prior to the experiment. The protein standard is diluted in a gradient manner by taking a standard diluent, and the protein standard is diluted respectively at the concentration of 20, 10, 5, 2.5, 1.25, 0.63, 0.31 and 0 ng/mL.

c) A standard curve is set, 50 μl of standard substances with different concentrations are added to each standard substance hole, and two parallel holes are arranged for each concentration.

d) Adding 50 mu L of a culture medium supernatant sample into a sample hole; blank wells were filled with sample dilutions.

e) 50. Mu.L of antibody A was added to each well, and the reaction wells were sealed with a non-aerated membrane and incubated in an incubator at 37℃for 60 minutes. Then the liquid in the holes is discarded, after the water absorbing paper is reversely buckled and patted to be dried, each hole is filled with the washing liquid (about 350 mu L), the washing liquid is shaken for 1 minute, the washing liquid is thrown away, the water absorbing paper is patted to be dried, and the plate is repeatedly washed for 3 times.

f) Antibody B solution was added at 100. Mu.L each to each well and incubated at 37℃for 45 minutes.

g) Then the liquid in the holes is discarded, after the water absorbing paper is reversely buckled and patted to be dried, each hole is filled with the washing liquid (about 350 mu L), the washing liquid is shaken for 1 minute, the washing liquid is thrown away, the water absorbing paper is patted to be dried, and the plate washing is repeated for 5 times.

h) 100. Mu.L of TMB reaction solution was added to each well, and incubated at 37℃for 10-20 minutes in the absence of light.

i) The OD of each well was measured at a wavelength of 450nm within 15 minutes of adding 50. Mu.L of stop solution to each well.

j) And (3) establishing a standard curve according to the absorbance of the standard substances with different concentrations, and calculating the recombinant OXM expression quantity of the culture medium supernatant according to a regression equation of the curve.

GLP-1ELISA kit:

b) Preparing a standard substance: each experiment was tested by preparing a dilution of the standard within 15 minutes prior to the experiment. The protein standards were diluted in a gradient with the standard dilutions at concentrations of 1000, 500, 250, 125, 63, 31, 15, 0pg/mL, respectively.

e) 50. Mu.L of HRP-labeled cocktail antibody was added to each well, the reaction wells were sealed with a non-aerated membrane and incubated in an incubator at 37℃for 60 minutes. The liquid in the holes is discarded, after the water absorbing paper is reversely buckled and patted dry, each hole is filled with washing liquid (about 350 mu L), the washing liquid is placed for 1 minute, the washing liquid is thrown away, the water absorbing paper is patted dry, and the plate is repeatedly washed for 5 times.

f) 100. Mu.L of TMB reaction solution was added to each well, and incubated at 37℃for 10-20 minutes in the absence of light.

g) The OD of each well was measured at a wavelength of 450nm within 15 minutes of adding 100. Mu.L of stop solution to each well.

h) And (3) establishing a standard curve according to the absorbance of the standard substances with different concentrations, and calculating the recombinant GLP-1 expression quantity of the culture medium supernatant according to a regression equation of the curve.

12 recombinant engineering bacteria oral gastric lavage mouse assay

The 18 mice were divided into 3 groups of 6 (n=6) each, respectively, a control group for normal saline gastric lavage, an NZ3900-vector empty vector recombinant engineering strain gastric lavage group and a pLD-GO recombinant engineering strain gastric lavage group, the NZ3900-vector empty vector recombinant engineering strain was a recombinant strain for pLD empty vector conversion NZ3900, the pLD-GO recombinant engineering strain was a recombinant strain for vector pLD-GO conversion NZ3900, the recombinant engineering strain was picked up from a streak plate, inoculated into 100mL of Elliker medium for standing culture at 30℃for 48 hours, the culture solution was centrifuged for 5 minutes at 5000 Xg, the supernatant was removed to leave a bacterial pellet, washed with 10mL of normal saline for resuspension, centrifuged again for 5 minutes at 5000 Xg, the supernatant was removed to leave a bacterial pellet, and then the bacterial pellet was diluted to 2.5X 10 with a proper amount of normal saline solution for resuspension ¹⁰ CFU/mL. The mice are subjected to gastric lavage once every two days, the gastric lavage dosage of the normal saline gastric lavage control group is 0.2mL of normal saline, and the gastric lavage dosage of the NZ3900-vector empty vector recombinant engineering strain gastric lavage group is 5 multiplied by 10 ⁹ CFU/volume of 0.2mL and gastric lavage dose of 5×10 for pLD-GO recombinant engineering strain ⁹ CFU/volume is 0.2mL, oral administration of gastric lavage recombinant engineering bacteria is continuously carried out for 6 months, and the weight change condition of each group of mice is recorded in each month.

The method comprises the following steps of measuring the feeding amount of recombinant engineering bacteria after oral administration of gastric lavage mice:

a) According to the configuration of a squirrel cage corresponding to each mouse, a brand new padding is put into the squirrel cage, enough drinking water is supplied, and after accurate weighing, the squirrel grains are put into the squirrel cage.

b) The method comprises the steps of respectively performing stomach irrigation on normal saline, NZ3900-vector empty vector recombinant engineering bacteria and pLD-GO empty vector recombinant engineering bacteria of mice of the normal saline stomach irrigation group, NZ3900-vector empty vector recombinant engineering bacteria and pLD-GO recombinant engineering bacteria of the pLD-GO recombinant engineering bacteria stomach irrigation group, wherein the stomach irrigation doses are respectively 0.2mL of normal saline and 5 multiplied by 10 of the NZ3900-vector empty vector recombinant engineering bacteria ⁹ CFU/volume is 0.2mL, pLD-GO recombinant engineering bacteria is 5 multiplied by 10 ⁹ CFU/volume was 0.2mL. Immediately after stomach lavage, the mice are placed in a prepared new mouse cage, so that the mice can freely move and eat in the new mouse cage.

c) After 24 hours, the mice were removed and the remaining mouse grains in the cages were accurately weighed to calculate the mouse grains consumed by the mice over 24 hours. And then analyzed.

The recombinant engineering bacteria oral gavage mice random blood sugar test steps:

a) The day before blood glucose measurement, mice were fasted for 12 hours without water control, and cages and brand-new pads were replaced.

b) After 12 hours of no water forbidden, the mice are weighed, the corresponding physiological saline, NZ3900-vector empty vector recombinant engineering bacteria and pLD-GO recombinant engineering bacteria are respectively subjected to gastric lavage, wherein the gastric lavage doses are respectively 0.2mL of physiological saline, 5X 109 CFU/volume of NZ3900-vector empty vector recombinant engineering bacteria is 0.2mL, and 5X 109 CFU/volume of pLD-GO recombinant engineering bacteria is 0.2mL.

c) The mice after stomach filling are fasted for 2 hours without water inhibition, each cage of mice is supplemented with enough feed, the mice are taken out of the cage, the tail end of the tail of the mice is carefully cut off by about 5mm, the tail of the mice is gently extruded, blood is extruded into one drop, blood sugar is measured by a glucometer, the operation is careful, the mice are prevented from being frightened, and the mice are put back into the cage after blood sugar measurement. Blood glucose values were measured for 0 hours (fasting blood glucose), 1, 2, 4, 6, 8, 10, and 12 hours, respectively.

d) After the experiment is finished, recording and analyzing the blood glucose, observing the trend of blood glucose change, and then analyzing.

Metabolism cage analysis and determination after 13 mice are filled with stomach oral recombinant engineering bacteria

Male C57 BL/6 mice (8 weeks old) are randomly divided into 3 groups, 5 (n=5) in each group, namely a control group for normal saline gastric lavage, a NZ3900-vector empty vector recombinant engineering strain gastric lavage group and a pLD-GO recombinant engineering strain gastric lavage group, wherein the NZ3900-vector empty vector recombinant engineering strain is a recombinant strain for pLD empty vector transformation NZ3900, and the pLD-GO recombinant engineering strain is a recombinant strain for vector pLD-GO transformation NZ 3900. Starting from the week before the experiment, the mice are subjected to gastric lavage once every two days, the gastric lavage dosage of the normal saline gastric lavage control group is 0.2mL of normal saline, and the gastric lavage dosage of the NZ3900-vector air vector recombinant engineering strain gastric lavage group is 5 multiplied by 10 ⁹ CFU/volume of 0.2mL and gastric lavage dose of 5×10 for pLD-GO recombinant engineering strain ⁹ CFU/volume was 0.2mL.

The operation of the metabolism cage is automatically completed according to a computer control system of the metabolism cage, and the experiment comprises the following specific steps:

a) The experimental animals are 3 groups, namely a control group for filling the stomach with normal saline, a NZ3900-vector recombinant engineering strain gastric lavage group and a pLD-GO recombinant engineering strain gastric lavage group, and before the experiment, all devices of a metabolism cage are checked and corrected, and new padding, new feed and clean drinking water are put in.

b) The metabolism cage is started in advance for 2 hours, the mice are weighed, then the corresponding normal saline, NZ3900-vector empty vector recombinant engineering bacteria and pLD-GO recombinant engineering bacteria are respectively filled into the normal saline, NZ3900-vector empty vector recombinant engineering bacteria and pLD-GO recombinant engineering bacteria of the normal saline gastric lavage group, and the gastric lavage doses are respectively 0.2mL normal saline and 5 multiplied by 10 of the NZ3900-vector empty vector recombinant engineering bacteria ⁹ CFU/volume is 0.2mL, pLD-GO recombinant engineering bacteria is 5 multiplied by 10 ⁹ CFU/volume was 0.2mL and each group of mice was placed in a metabolic cage for initial recording after gastric lavage.

c) Then continuously collecting oxygen consumption (VO) of the mice under the normal water feeding state for 24 hours ₂ ) Carbon dioxide (VCO) ₂ ) The production and feeding, water intake, energy consumption and metabolism data, and the rest and proper temperature and humidity are maintained during the experiment.

d) After the experiment is finished, the mice are put back into the original cage position, and the automatically recorded metabolism cage data are analyzed and arranged by using ExpeData software.

After the metabolism cage experiment is finished, the mice are allowed to rest for one day, then the cages and brand new padding are replaced, the mice are fasted without water inhibition for 12 hours, then the mice are subjected to gastric lavage, the gastric lavage dosage of a normal saline gastric lavage control group is 0.2mL of normal saline, and the gastric lavage dosage of a NZ3900-vector empty carrier recombinant engineering strain gastric lavage group is 5 multiplied by 10 ⁹ CFU/volume of 0.2mL and gastric lavage dose of 5×10 for pLD-GO recombinant engineering strain ⁹ CFU/volume was 0.2mL. After 2 hours of gastric lavage, the orbit of the mouse is taken, the blood sample is placed in an EDTA anticoagulation tube, and the blood sample is centrifuged for 5min at 2000 Xg at room temperature, the supernatant plasma is taken, and the blood sample is put into a temperature of minus 80 ℃ for preservation to be measured.

Serum samples were tested for OXM, GLP-1 concentration using an enzyme-linked immunosorbent assay (ELISA).

14 data statistics

All data are presented as mean ± Standard Error (SE), statistical software using GraphPad Prism version 5.0.5.0 (CA, USA). Statistical analysis of the differences p < 0.05 was considered to be significantly different using the one-way ANOVA test.

Second, result

1 construction and identification of recombinant vectors

In order to construct a constitutive double expression vector and meet the requirements of FDA food grade expression, and to amplify and express in lactococcus lactis strain NZ3900 deficient in lacF, the DNA element repA, repC, lacF of pNZ8149 plasmid was selected, wherein repA, repC functions are maintained and amplified in lactococcus lactis, lacF is lactase gene, and the ability of lactococcus lactis NZ3900 to resume lactose utilization was used as a selection marker for positive clones. P32 and P8 are constitutive promoters, matched with terminator T-usp45 and T-pepN respectively, all of which are derived from lactic acid bacteria genome DNA, and the schematic diagram of the vector construction is shown in FIG. 1.

The CAF DNA fragment of repA, repC, lacF from pNZ8149 plasmid is successfully obtained through PCR amplification, the size is about 2100bp, after the PCR product is purified, bamHI and SacII are respectively used for double digestion, after the digestion product is purified, the digestion product is respectively connected with 2 DNA fragments of synthetic expression cassettes PT, GO and the like which are subjected to the same digestion, the connection product is electrically transferred to lactococcus lactis NZ3900, the Elliker culture medium is cultured for 48 hours, then the plasmid is extracted for digestion, the electrophoresis result is shown in figure 2, (a) the figure is a vector pLD, the size of which is about 2816bp, and two fragments of about 2100 and 700 sizes can be cut through BamHI and SacII double digestion, the two fragments are respectively consistent with the sizes of the CAF DNA fragment and the synthetic expression cassette PT DNA fragment, and the sequencing result shows that the recombinant vector is successfully constructed. (b) The vector pLD-GO has the size of about 3100bp, two fragments which can be cut into about 2100 and 1000 sizes after BamHI and SacII double digestion are respectively consistent with the sizes of the CAF DNA fragment and the synthetic expression cassette GO DNA fragment, and the sequencing result shows that the recombinant vector is successfully constructed.

In order to enable secretory expression of the target proteins OXM and GLP-1, the authors added a Usp45 protein secretion signal peptide of lactococcus lactis to the N-terminus of OXM and GLP-1, and the Usp45 protein is an extracellular secretion protein expressed by lactococcus lactis in a constitutive secretion manner, and the secretion amount is the highest among all extracellular secretion proteins of lactococcus lactis, so that many researches use the Usp45 secretion signal peptide to express secreted recombinant proteins.

The Western Blotting results are shown in FIG. 3, and FIG. (a) shows that the culture medium supernatant protein samples of the control group of the starting strain lactococcus lactis NZ3900 and the control group of the pLD empty vector transformation are detected by using an antibody Rabbit Anti-Human OXM Ig G, no bands exist, the pLD-GO recombinant engineering strain has a specific band at the position of 4.7KD, and the pLD-GO recombinant engineering strain is proved to be capable of secreting and expressing the OXM according to expectations.

Panel (b) shows that the detection of the culture medium supernatant protein samples of the control group of the starting bacterium lactococcus lactis NZ3900 and the control group of the pLD empty vector transformation by using the antibody Anti-GLP-1ab20047 has no band, and the pLD-GO recombinant engineering strain has a specific band at the 4.2Kd position, which accords with the expectation, thus proving that the pLD-GO recombinant engineering strain can secrete and express GLP-1.

2 recombinant OXM expressed by recombinant engineering bacteria and GLP-1 expression quantity determination

Recombinant engineering bacteria pLD-GO are subjected to static culture in an Elliker culture medium at 30 DEG CAs shown in FIG. 4, the growth curve for 70 hours of cultivation shows that the growth of the recombinant engineering bacteria pLD-GO shows an exponential increase in the first 24 hours of cultivation, reaching the peak at 24 hours, and the OD at that time ₆₀₀ The value is 0.3935, and then the quantity of the recombinant engineering bacteria pLD-GO is not increased and slightly reduced any more because of the consumption of nutrient components in the culture medium and the reduction of the pH value of the culture medium, so that the recombinant engineering bacteria pLD-GO is harvested after 24 hours of culture for the optimal time.

And (3) performing enzyme-linked immunosorbent assay (ELISA) on the supernatant obtained after 24 hours of culture and centrifugation of the recombinant engineering bacteria pLD-GO bacterial liquid, detecting the recombinant OXM and GLP-1 expression levels of the culture medium supernatant, wherein the experimental results are shown in figure 5, the fitted curve shows good experimental results, and the expression level of the recombinant OXM of the culture medium supernatant is 6.85ng/mL and the expression level of the recombinant GLP-1 is 47.21ng/mL through a regression equation.

3 determination of recombinant engineering bacteria oral gastric lavage mice

The weight change graphs of the mice of the control group, NZ3900-vector empty vector recombinant engineering strain gastric lavage group and the pLD-GO recombinant engineering strain gastric lavage group of physiological saline gastric lavage group are shown in FIG. 6, from which it can be seen that the weight increase amplitude of the mice of the first 4 months three groups is very close, without obvious difference, the weight of the mice of the control group which starts to be perfused with physiological saline gastric lavage after the 5 th month of gastric lavage still continues to increase, while the weight increase of the mice of the NZ3900-vector empty vector engineering strain gastric lavage group is slowed, the weight of the mice of the pLD-GO recombinant engineering strain gastric lavage group stops the trend of the rise and slightly decreases (at this time the mice are 7 months old), which shows that the recombinant OXM and GLP-1 can significantly control the weight of the mice with the increase of the age, and the physiological activities of the OXM and GLP-1 are closely related to the blood glucose level with the increase of the age. When the mice were gavaged for 6 months (at this time 8 months of age), the mice in the pLD-GO recombinant engineering strain gavage group had a very significant decrease in weight (P < 0.0001) compared to the control group, and the average weight was nearly 5 g less than the control group, and the weight of the mice in the NZ3900-vector empty vector engineering strain gavage group also had a very significant decrease in weight (P < 0.01) compared to the control group, and the average weight was nearly 3.4 g less than the control group, indicating that the mice could also have a weight decrease by oral lactococcus lactis over a long period of time. Compared with the NZ3900-vector group, the mice in the stomach-filled group of the pLD-GO recombinant engineering strain have a weight which is 1.75 g less, and have a very significant reduction (P < 0.01), and the result shows that the long-term oral administration of the pLD-GO recombinant engineering strain can more effectively reduce the weight of the mice, and the NZ3900-vector empty carrier engineering strain group has a significant weight reducing effect as compared with the pLD-GO recombinant engineering strain.

The 24 hour feeding rate of mice after gastric lavage is shown in FIG. 7, and the results show that after gastric lavage, the feeding rate of mice in the control group, NZ3900-vector recombinant engineering strain gastric lavage group and the feeding rate of mice in the pLD-GO recombinant engineering strain gastric lavage group are different, the average feeding rate of the control group is 2.32 g, the average feeding rate of mice in the NZ3900-vector group is 1.99 g, the average feeding rate of mice in the pLD-GO group is only 1.13 g, and the study shows that the pLD-GO group and the control group, the pLD-GO group and the NZ3900-vector group are quite different (P < 0.01), and the control group and the NZ3900-vector group have no statistical difference. The result shows that the pLD-GO recombinant engineering bacteria can effectively inhibit the appetite of mice.

The results of the random blood glucose curves and AUC of the mice after gastric lavage are shown in fig. 8, and the results show that the mice immediately begin to eat after 14 hours of fasting, the blood glucose of the control group and the NZ3900-vector empty vector engineering strain gastric lavage group mice in normal saline gastric lavage rise rapidly and have substantially the same amplitude, the area under the blood glucose AUC curve shows no difference between groups, the two groups of mice are very good in appetite and eat a lot of food to cause blood glucose rise, the blood glucose of the mice in the pLD-GO recombinant engineering strain gastric lavage group rises slower, the groups of mice are poor in appetite and eat a little, the area under the blood glucose AUC curve is smaller than that of the control group and the NZ3900-vector group (P < 0.05), the blood glucose curves of the 3 groups of mice tend to be consistent after 4 hours, the pLD-GO recombinant engineering strain in the gastric lavage gradually discharges the feces (lactococcus) along with the time, and the appetite of the mice becomes less in the intestinal tract becomes less affected.

Analysis and determination of metabolic cage after 4 mice are filled with recombinant engineering bacteria orally taken

As shown in figure 9, the results of the metabolism cage measurement of 24 hours after the gastric lavage of the control group, the NZ3900-vector empty vector recombinant engineering strain gastric lavage group and the pLD-GO recombinant engineering strain gastric lavage group 3 mice are shown, and comparing the ingestion of the mice in the 3 groups, the ingestion of the mice in the pLD-GO recombinant engineering strain gastric lavage group is 1.3329g, while the gastric lavage of the control group and the NZ3900-vector empty vector recombinant engineering strain gastric lavage group are 2.1443g and 2.0217g respectively, the gastric lavage of the pLD-GO recombinant engineering strain is obviously reduced (P < 0.05) compared with the gastric lavage of the other two groups, the reduction ranges are 37.83% and 34.07% respectively, and the pLD-GO recombinant engineering strain is proved to be capable of effectively reducing the ingestion of the mice.

The oxygen consumption rate, the carbon dioxide generation rate, the water intake and the energy consumption of the mice in the 3 groups are very similar, and the groups have no obvious difference, so that the pLD-GO recombinant engineering bacteria do not obviously influence the oxygen consumption rate, the carbon dioxide generation rate, the water intake and the energy consumption of the mice. It is therefore speculated that its regulatory mechanism for mouse body weight may be to maintain normal energy metabolism in mice while reducing their food intake, and thus long-term oral administration may lead to mice consuming body fat and losing body weight.

As shown in FIG. 10, the results of enzyme-linked immunosorbent assay (ELISA) after the gastric lavage of mice in the control group, NZ3900-vector empty vector recombinant engineering strain gastric lavage group and pLD-GO recombinant engineering strain gastric lavage group detect the concentration of OXM and GLP-1 in serum, and it can be known from the results that the concentration of OXM in the serum after the gastric lavage of mice is 0.3412ng/mL in the control group, 0.3639ng/mL in the NZ3900-vector group, 0.5002ng/mL in the pLD-GO group, and significant statistical difference (P < 0.05) exists between the concentration of OXM in the serum of the pLD-GO group and the other two groups, and the concentration of the recombinant OXM in the blood of the mice is increased due to the fact that the recombinant OXM can be absorbed by intestinal tracts after the pLD-GO recombinant engineering strain gastric lavage mice.

The GLP-1 concentration in the serum after the mice are 0.2176ng/mL of the control group, 0.2987ng/mL of the NZ3900-vector group and 0.4365ng/mL of the pLD-GO group respectively, and compared with the other two groups, the GLP-1 concentration in the serum of the pLD-GO group is obviously higher, and the serum of the pLD-GO group has quite obvious statistical difference (P < 0.0001) with the other two groups, so that the recombinant GLP-1 can be absorbed by the intestinal tract after the mice are filled with the pLD-GO recombinant engineering strain, and the GLP-1 concentration in the blood of the mice is improved. Of note is also a significant statistical difference (P < 0.01) between the control and NZ3900-vector groups, indicating that lactococcus lactis may stimulate GLP-1 secretion by intestinal L cells.

In the embodiment, the food-grade lactobacillus is taken as a transformation target, the genome DNA element of the food-grade microorganism lactobacillus is utilized to construct a food-grade expression vector pLD, the feasibility of co-expressing two polypeptide hormones of the food-grade human glucagon-like peptide 1 and the human oxyntomodulin is studied, the two polypeptides are successfully expressed in L.lactis through genetic engineering operation, and the Western Blotting result proves that the two polypeptides can be accurately secreted and expressed. Oral gastric lavage experiments on mice prove that the recombinant engineering strain can effectively control appetite and blood sugar of the mice, and can reduce the weight of the mice after long-term oral administration. An enzyme-linked immunosorbent assay (ELISA) experiment proves that the recombinant OXM and GLP-1 expressed by the oral recombinant engineering bacteria can be absorbed by the intestinal tract of a mouse to obviously improve the concentration of the recombinant OXM and GLP-1 in blood. The experiment result shows that the ingestion of the mice is reduced, the consumption of oxygen and energy is not obviously reduced, the mice can consume fat in bodies after long-term oral administration, the experiment result shows that the pLD-GO recombinant engineering bacteria have partial functions similar to L cells, the secreted GLP-1 activity can effectively control blood sugar and reduce food intake, the secreted OXM can increase heat consumption through glucagon activity, so that the mice can reduce food consumption and keep energy consumption unchanged after the recombinant engineering bacteria are used for filling the stomach of the mice, the long-term oral administration can effectively consume fat to reduce weight, and the recombinant engineering bacteria have the potential of treating one or more metabolic disease indications in obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver diseases and the like and can also be used for treating neuron degenerative diseases such as hypertension, heart failure, alzheimer disease and the like.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Claims

1. A recombinant bacterium comprising a GLP-1 gene expression cassette and an OXM gene expression cassette in tandem;

2. The recombinant bacterium of claim 1, which is a nonpathogenic bacterium.

3. The recombinant bacterium of claim 2, which is a probiotic.

4. A recombinant bacterium according to claim 3, selected from the group consisting of: bacteroides, bifidobacteria, streptococcus thermophilus, clostridia, escherichia, eubacteria, lactobacillus, lactococcus and ross bacteria.

5. The recombinant bacterium of claim 4, which is lactococcus lactis.

6. The recombinant bacterium of claim 5, which is a lactococcus lactis NZ3900 strain.

7. The recombinant bacterium of claim 5, wherein the nucleotide sequences of the GLP-1 gene are respectively set forth in SEQ ID NOs: 1 is shown in the specification; and/or; the nucleotide sequences of the OXM genes are respectively shown in SEQ ID NO: 2.

8. The recombinant bacterium of claim 1, wherein the GLP-1 gene expression cassette and the OXM gene expression cassette are expressed in reverse, in opposite or in the same direction.

9. The recombinant bacterium of claim 8, wherein a spacer DNA is provided between the GLP-1 gene expression cassette and the OXM gene expression cassette.

10. The recombinant bacterium of any one of claims 1-9, wherein the first terminator is T-usp45; and/or; the second terminator is T-pepN.

11. The recombinant bacterium of any one of claims 1-9, wherein the signal peptide is a Usp45 signal peptide.

12. The recombinant bacterium according to any one of claims 1 to 9, wherein the GLP-1 gene expression cassette and OXM gene expression cassette are located on a plasmid, which plasmid further comprises a replicon and a selection gene.

13. The recombinant bacterium of claim 12, wherein the replicon comprises repA and repC.

14. The recombinant bacterium of claim 12, which is a lacF-deficient strain, the screening gene being lacF.

15. The recombinant bacterium of claim 13 or 14, wherein the plasmid is derived from pNZ8149.

16. A plasmid as defined in any one of claims 12 to 15.

17. A composition, which is a pharmaceutical or edible composition, comprising the recombinant bacterium of any one of claims 1-15.

18. The composition of claim 17, which is a pharmaceutical composition or further comprising a pharmaceutically acceptable carrier.

19. Use of a recombinant bacterium according to any one of claims 1 to 15 in the manufacture of a medicament for the treatment of one or more of metabolic diseases, neurodegenerative diseases, memory and learning disorders, irritable bowel syndrome, stroke, post-operative catabolic changes, functional dyspepsia and ischemia reperfusion tissue damage.

20. The use according to claim 19, wherein the metabolic disorder is selected from one or more of diabetes, hypertension, hypoglycemia, dyslipidemia, cardiovascular disease, abnormal clotting, obesity, diabetic complications, liver and gall disease, chronic kidney disease, abnormal insulin resistance and glucose tolerance.