A recombinant strain and its use in the treatment of diabetes

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a recombinant bacterial strain and its use in the treatment of diabetes. The invention provides a recombinant Bacillus subtilis strain displaying gastric inhibitory peptides and membrane-penetrating peptides on its surface. This recombinant strain shows great promise in the preparation of drugs for the prevention, treatment, mitigation, and / or improvement of diabetes, and has potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant bacterial strain and its use in the treatment of diabetes. Background Technology

[0002] As a country with a high burden of type 2 diabetes mellitus (T2DM), my country is experiencing a rapid increase in the prevalence of diabetes. However, the current treatment and control rates for T2DM are both at a low level. As one of the three most common chronic diseases, type 2 diabetes mellitus (T2DM) is also closely linked to microvascular and macrovascular complications and increased cardiovascular mortality, thus posing a serious public health problem. Currently, T2DM management measures include dietary control, lifestyle modifications to promote weight loss, antihyperglycemic drug therapy (such as oral metformin hydrochloride tablets, insulin injections), and even metabolic surgery. Insulin, as the only hormone in the body that lowers blood sugar, has made insulin therapy an extremely important treatment method.

[0003] With the continuous deepening and development of research on blood glucose lowering, the types of insulin are also constantly increasing. Among them, the application of incretin drugs for lowering blood glucose has gradually occupied an extremely important position in the treatment of type 2 diabetes mellitus (T2DM). Incretins, as a polypeptide hormone secreted by intestinal secretory cells, are mainly composed of two major classes: glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP). Both are novel drugs for regulating blood glucose.

[0004] Glucose-dependent insulinotropic polypeptide (GIP) is an incretin synthesized and secreted by small intestinal endocrine cells. It is a 42-amino acid polypeptide with a glucose-dependent effect on promoting insulin secretion. GIP not only helps improve insulin secretion function and glucose tolerance, and promotes the proliferation and presence of pancreatic β-cells, but also inhibits the breakdown of hepatic glycogen, promotes the digestion of glucose into lipids by adipocytes, and increases the activity of lipoprotein lipase. Therefore, GIP is a potential drug for regulating diabetes and related metabolic disorders.

[0005] Insulin is typically administered subcutaneously to lower blood sugar. However, long-term subcutaneous insulin therapy can have negative effects, such as weight gain, fluctuations in blood sugar levels, and high treatment costs. Incretins, a newer type of blood sugar-lowering drug, can effectively address these shortcomings when administered orally. However, oral medications for blood sugar regulation still need to overcome the attack of enzymes in the gastrointestinal tract and tolerate the acidic environment of the stomach. Therefore, it is necessary to select a transport vehicle with relevant resilience to ensure successful delivery to the digestive site.

[0006] In view of this, the present invention provides a product that can withstand extreme physiological environments, thereby precisely delivering gastric inhibitory peptides to the site of action to effectively exert the effect of blood glucose regulation. Summary of the Invention

[0007] This invention aims to construct a recombinant plasmid containing a membrane-penetrating peptide and a gastric inhibitory peptide, and then transform it into Bacillus subtilis to obtain a fusion-type CPPS-GIP recombinant Bacillus subtilis. The recombinant Bacillus subtilis is then identified and its activity is analyzed, enabling Bacillus subtilis to carry the gastric inhibitory peptide through the acidic environment of the stomach and be successfully digested in the small intestine, allowing the gastric inhibitory peptide to reach the intestine and exert its hypoglycemic effect.

[0008] Cell penetrating peptides (CPPs), as a known class of short peptides (approximately 5-30 aa) capable of crossing the cell membrane, can efficiently deliver various bioactive molecules across the cell membrane into the cytoplasm via protein transduction to exert their functions. Because CPPs can efficiently transport various bioactive conjugates into cells, their non-toxic and highly efficient transcellular properties make them uniquely advantageous as delivery vectors. Therefore, this invention selects to fuse the expression of cell penetrating peptides and gastric inhibitory peptides, integrating them into a recombinant plasmid.

[0009] In a first aspect, the present invention provides a recombinant plasmid comprising a nucleic acid molecule encoding a glucose-dependent insulinotropic polypeptide (GIP) and a nucleic acid molecule encoding a cell penetrating peptide (CPP), a backbone plasmid, and an acceptable expression element.

[0010] In some embodiments, the amino acid sequence of the gastric inhibitory peptide is shown in SEQ ID NO: 1.

[0011] SEQ ID NO: 1: ‌YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ.

[0012] In some embodiments, the amino acid sequence of the transmembrane peptide is shown in SEQ ID NO: 2.

[0013] SEQ ID NO: 2: YGRKKRRQRRR.

[0014] In some implementations, the acceptable expression elements include, but are not limited to, one or more of promoters, enhancers, and terminators.

[0015] In some implementations, the backbone plasmid includes a replication origin, selection markers, etc.

[0016] In some embodiments, the backbone plasmid includes shuttle plasmids and integrative plasmids.

[0017] In some preferred embodiments, the backbone plasmid is an integrative plasmid.

[0018] In some preferred embodiments, the backbone plasmid is pJS700.

[0019] In a second aspect, the present invention provides a recombinant strain that is transformed and / or transfected with the aforementioned recombinant plasmid.

[0020] In some embodiments, the chassis strains transformed and / or transfected include one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris.

[0021] In some preferred embodiments, the chassis strain being transformed and / or transfected is Bacillus subtilis.

[0022] In some embodiments, the recombinant strain stably expresses biologically active membrane-penetrating peptides and gastric-inhibiting peptides.

[0023] In a third aspect, the present invention provides a method for constructing a recombinant bacterial strain, the method comprising: (1) Integrate the nucleic acid molecules encoding gastric inhibitory peptide and the nucleic acid molecules encoding membrane-penetrating peptide into the integration site of the backbone plasmid to obtain the recombinant plasmid; (2) The recombinant plasmid is transformed and / or transfected into the chassis strain to obtain the recombinant strain.

[0024] In some implementations, the backbone plasmid includes a replication origin, selection markers, etc.

[0025] In some embodiments, the backbone plasmid includes shuttle plasmids and integrative plasmids.

[0026] In some preferred embodiments, the backbone plasmid is an integrative plasmid.

[0027] In some preferred embodiments, the backbone plasmid is pJS700.

[0028] In some embodiments, the chassis strains transformed and / or transfected include one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris.

[0029] In some preferred embodiments, the chassis strain being transformed and / or transfected is Bacillus subtilis.

[0030] In some embodiments, the recombinant strain stably expresses biologically active membrane-penetrating peptides and gastric-inhibiting peptides.

[0031] In a fourth aspect, the present invention provides a culture obtained by culturing the recombinant strain under conditions suitable for growth and / or proliferation.

[0032] In a fifth aspect, the present invention provides the use of the aforementioned recombinant plasmids, recombinant strains, recombinant strains and / or cultures obtained by the aforementioned construction methods in the preparation of medicaments or pharmaceutical compositions for the prevention, treatment, mitigation and / or improvement of diabetes.

[0033] In some implementations, the prevention, treatment, mitigation, and / or improvement of diabetes are achieved by regulating blood glucose levels.

[0034] In some implementations, the diabetes is type 2 diabetes.

[0035] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent for the prevention, treatment, mitigation, and / or improvement of diabetes.

[0036] In some preferred embodiments, the second therapeutic agent includes, but is not limited to, one or more of: metformin, glibenclamide, glipizide, gliclazide, glimepiride, repaglinide, nateglinide, acarbose, voglibose, dapagliflozin, empagliflozin, canagliflozin, insulin, liraglutide, exenatide, smegglutide, dulaglutide, lixisenatide, benaglutide, and polyethylene glycol loxenatide.

[0037] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0038] In some preferred embodiments, the excipients include, but are not limited to, one or more of the following: absorption promoters, anti-sticking agents, antifoaming agents, antioxidants, adhesives, buffers, carriers, coating agents, pigments, delivery promoters, delivery polymers, glucosan, glucose, diluents, disintegrants, emulsifiers, swelling agents, fillers, flavorings, flow aids, humectants, lubricants, oils, polymers, preservatives, brine, salt, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotensins, carriers, waterproofing agents, and wetting agents.

[0039] Bacillus subtilis, a species of Bacillus, is abundant in soil and decaying organic matter. It gets its name from its ease of reproduction in hay infusion. Under suitable conditions of light, temperature, water, and nutrients, young cells form numerous spores. Bacillus subtilis maintains its biological activity in the acidic environment of the stomach and possesses the ability to withstand various enzymes in saliva and bile, thus effectively reaching the intestines. Therefore, it has gradually become an effective agent for various heterologous proteins in extreme environments such as the gastrointestinal tract, and this ability is also a crucial factor in maintaining the activity of the strain, surviving in the intestines, and exerting its probiotic effects.

[0040] To ensure the stable absorption and efficacy of oral hypoglycemic drugs, not only is a stable transport carrier required, but also the ability of Bacillus subtilis carrying GIP to effectively cross the cell membrane and enter the cell for effective expression.

[0041] Therefore, this invention combines transmembrane peptides, gastric inhibitory peptides, and Bacillus subtilis to achieve precise and effective delivery of gastric inhibitory peptides.

[0042] This invention constructs a fusion plasmid pJS700-CPPs-GIP and confirms through PCR and amylase plate experiments that the target gene is successfully fused on the surface of Bacillus subtilis, effectively improving the stability and bioavailability of GIP, enabling GIP to cross the extreme environment of the stomach and reach the digestive site to exert its effects.

[0043] This invention successfully binds and displays the gastric inhibitory peptide GIP, which incorporates cell-penetrating peptides (CPPs), on the surface of Bacillus subtilis, thereby obtaining a recombinant Bacillus subtilis strain with blood glucose-regulating effects. Therefore, this recombinant strain shows great promise in the preparation of drugs for the prevention, treatment, mitigation, and / or improvement of diabetes, and has potential clinical application value. Attached Figure Description

[0044] Figure 1 The identification results of plasmid 30a-CPPs-GIP are shown. This experiment used 1% agarose gel electrophoresis with a 2K bp DNA marker. In diagram A, the electrophoresis image of the 30a-CPPs-GIP plasmid is shown, with lanes 1 and 2 showing the electrophoresis of the fusion plasmid; in diagram B, the electrophoresis image of the 30a-CPPs-GIP amplification is shown, with lanes 1 and 2 showing the amplified CPPs and GIP products, respectively.

[0045] Figure 2 The identification results of plasmid pJS700-CPPs-GIP are shown. This experiment used 1% agarose gel electrophoresis with a 2Kbp DNA marker. In the diagram, A is the electrophoresis image of the pJS700 plasmid, lane 1 is the pJS700 plasmid; B is the electrophoresis image of the pJS700-CPPs-GIP plasmid, lane 1 is the pJS700-CPPs-GIP plasmid; and C is the electrophoresis image of the CPPs-GIP plasmid, lane 1 is the CPPs-GIP plasmid.

[0046] Figure 3 A flowchart of the pJS700-CPPs-GIP plasmid construction is shown.

[0047] Figure 4 The results of the identification of recombinant Bacillus subtilis amylase plates are shown. Among them, Wild type Bacillus subtilis is wild-type Bacillus subtilis, and Transgenic Bacillus subtilis / pJS700-CPPs-GIP is recombinant Bacillus subtilis.

[0048] Figure 5 The results of PCR amplification and identification of the strains are shown. The WT group is wild-type Bacillus subtilis, and the TG group is recombinant Bacillus subtilis.

[0049] Figure 6 Fasting blood glucose and fasting body weight levels in diabetic mice are shown. Here, a represents the fasting blood glucose level in the normal group and diabetic mice, and b represents the fasting body weight in the normal group and diabetic mice. Data are expressed as mean ± SEM. Statistical analysis was performed using a t-test. p <0.05, *** p <0.01.

[0050] Figure 7 Fasting blood glucose levels in diabetic mice under different treatment groups are shown. The BS-GIP group represents diabetic mice treated with recombinant Bacillus subtilis; the BS group represents diabetic mice treated with wild-type Bacillus subtilis; and the PBS group represents diabetic mice treated with PBS. Data are presented as mean SEM (n=10), and statistical analysis was performed using a t-test. * indicates... p <0.05, ** represents p <0.01.

[0051] Figure 8 Results of the intraperitoneal glucose tolerance test are shown. Data are presented as mean ± SEM (n=8). Statistical analysis was performed using a t-test. p <0.01 and * p <0.05. Detailed Implementation

[0052] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0053] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0054] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0055] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0056] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0057] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0058] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0059] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0060] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0061] Materials and Methods 1. Strains and plasmids Escherichia coli ( Escherichia coli, E. coli Bacillus subtilis ( Bacillus subtilisThe expression vector DH5-30a, CPPs-GIP plasmid, and plasmid vector pJS700 were all provided by the School of Biological and Food Engineering, Suzhou University.

[0062] The amino acid sequence of the gastric inhibitory peptide GIP is shown in SEQ ID NO: 1; the amino acid sequence of the membrane-penetrating peptide CPPs is shown in SEQ ID NO: 2.

[0063] SEQ ID NO: 1: ‌YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ.

[0064] SEQ ID NO: 2: YGRKKRRQRRR.

[0065] 2. Experimental equipment Shaker (Shanghai Zhichu Instrument Co., Ltd.), PCR instrument (BIO-RAD), ice maker (Jiangsu Green Electric Appliance Co., Ltd.), electronic balance (Ohaus Instruments (Shanghai) Co., Ltd.), benchtop high-speed low-temperature centrifuge (Likang Development Co., Ltd.), electrophoresis apparatus (BIO-RAD), ultrapure water system (Likang Development Co., Ltd.), HH digital display constant temperature water bath (Changzhou Guoyu Instrument Manufacturing Co., Ltd.), gel imaging system (Thermo Fisher Scientific China Co., Ltd.), vertical automatic autoclave (Zhiwei Xiamen Instrument Co., Ltd.), micro spectrophotometer (Thermo Fisher Scientific China Co., Ltd.), clean bench (Suzhou Purification Equipment Co., Ltd.). The above experimental equipment was provided by the School of Biological and Food Engineering, Suzhou University.

[0066] 3. Experimental reagents Disodium ethylenediaminetetraacetate, trihydroxymethylaminomethane, sodium citrate, agar, calcium chloride, glycerol, 10× minimum salt solution, PCR reagents, restriction endonucleases ( Nco I, Sac I, Kpn I), T4 DNA ligase, upstream and downstream primers, DNA Ladder Marker, 100 g / mL ampicillin solution, 0.4 g / mL erythromycin solution, GM I and GM II solutions, LB medium, etc. All reagents were provided by the School of Biological and Food Engineering, Suzhou University. The primer sequences used in PCR are shown in Table 1 below.

[0067] Table 1 Primer sequence listing Among them, primers F and R are primer pairs for amplifying the gastric inhibitory peptide GIP; primers Amy EF and Amy ER are primers for amplifying the amylase gene.

[0068] 4. Preparation of the main solution (1) 10% CaCl2 solution. Take 10 g of calcium chloride and dilute to 90 g of deionized water. Sterilize by autoclaving at 121°C for 15 min and store at 4°C.

[0069] (2) 5TBE buffer. Take 54 g of Tris-base, 27.5 g of boric acid, and 20 mL of 0.5 mol / L EDTA, and make up to 1 L of deionized water. Store at 4℃.

[0070] (3) 10× minimum salt solution. Take 14 g of dipotassium hydrogen phosphate, 6 g of potassium dihydrogen phosphate, 2 g of ammonium sulfate, 1 g of sodium citrate dihydrate, and 1.4 g of magnesium sulfate heptahydrate, dissolve them in distilled water and bring the volume to 100 mL. Sterilize the solution by autoclaving at 121℃ for 15 min to obtain the mother liquor. Store at 4℃.

[0071] (4) GMⅠ solution. 95.6 mL of 1× minimum salt solution, 2.5 mL of 20% glucose solution, 0.4 mL of 5% hydrolyzed casein solution, 1 mL of 10% yeast extract, and 0.5 mL of 10 mg / mL L-tryptophan solution.

[0072] (5) GMⅡ solution. 96.98 mL of 1× minimum salt solution, 2.5 mL of 20% glucose solution, 0.04 mL of 10% yeast, 0.1 mL of 10 mg / mL L-tryptophan solution, 0.08 mL of 5% hydrolyzed casein, 0.25 mL of 1M MgCl2, and 0.25 mL of 1M CaCl2.

[0073] 6. Preparation of culture medium (1) LB liquid culture medium. 1.0 g yeast powder, 2.0 g tryptone, 2.0 g sodium chloride, 200 mL distilled water, adjust pH to about 7.0, autoclave at 121℃ for 20 min, and store at 4℃.

[0074] (2) LB solid medium. 2.0 g yeast powder, 4.0 g tryptone, 4.0 g sodium chloride, 4.0 g agar powder, 400 mL distilled water, adjust pH to about 7.0, autoclave at 121℃ for 20 min, and store at 4℃.

[0075] 7. Preparation of recombinant strains (1) Preparation of competent Escherichia coli cells. Take 1 mL of Escherichia coli and culture it in 200 mL of sterile LB liquid medium at 180 rpm and 37℃ for 6 h on a shaker. Take 50 mL of the culture medium and put it into two EP tubes and incubate on ice for 30 min. Centrifuge at 4000 rpm for 10 min and discard the supernatant. Add 10 mL of pre-cooled 10% CaCl2 to each tube to resuspend the precipitate and immediately incubate on ice for 30 min. Centrifuge again at 4000 rpm for 10 min and discard the supernatant. Add 2 mL of 10% glycerol CaCl2 to each tube to resuspend the precipitate. Aliquot into EP tubes and store at -80℃.

[0076] (2) Preparation of competent Bacillus subtilis cells. First, Bacillus subtilis strains were inoculated onto LB solid medium and cultured overnight at 37°C. On the same day, positive single colonies were selected and cultured overnight in 5 mL of GMⅠ solution at 30°C and 130 rpm in a shaker. The next day, 2 mL of the culture was transferred to 8 mL of freshly prepared GMⅠ solution and cultured at 37°C and 250 rpm in a shaker for 3.5 h. Then, 5 mL of the culture medium from the above step was transferred to 45 mL of GMⅡ solution and cultured at 37°C and 250 rpm for 2 h. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded. Finally, the bacterial pellet was resuspended in 5 mL of the original culture medium. The resuspended bacterial cells were competent cells. 30% sterile glycerol was added to a final concentration of 10% and then dispensed into EP tubes and stored at -80°C.

[0077] (3) Transformation of CPPs-GIP recombinant plasmid into Bacillus subtilis. The CPPs-GIP plasmid purchased from Shanghai Sangon Biotech Co., Ltd. was integrated with the DH5α-30a plasmid to obtain the fusion type 30a-CPPs-GIP plasmid, which was then transformed into Escherichia coli again. The plasmid was extracted and subjected to double enzyme digestion with the pJS700 vector to obtain the recombinant plasmid pJS700-CPPs-GIP. 2 L of the plasmid was added to competent cells of Bacillus subtilis and cultured in a shaker at 37℃ and 200 rpm for 60 min, 75 min, 90 min and 105 min respectively. 100 L of the culture medium at each time point was spread on erythromycin-resistant plates in the dark and cultured overnight in a constant temperature incubator at 37℃ in the dark. Several single colonies were selected on the same day and cultured in LB liquid medium at 37℃ and 180 rpm until the logarithmic growth phase.

[0078] 8. Screening and identification of recombinant strains Screening and identification of recombinant Bacillus subtilis. Since the plasmid pJS700-CPPs-GIP retains the biological characteristics of erythromycin resistance after recombination, it will express erythromycin resistance when transduced into Bacillus subtilis. Based on these characteristics, erythromycin resistance can be verified by plate culture or PCR. First, multiple single colonies after overnight incubation on erythromycin-resistant plates were picked and expanded on LB liquid medium. Then, a suitable amount of bacterial culture was lightly spotted onto LB starch solid plates and incubated overnight at 37°C. The next day, an appropriate amount of I-KI solution was evenly added to the plate, and the results were observed after several minutes in the dark. PCR experiments were performed using appropriate primers. The plasmid fragment size was determined to be consistent with the expected results by agarose gel electrophoresis and PCR experiments.

[0079] Example 1: Construction and identification of integrative plasmid 30a-CPPs-GIP The intermediate bridging plasmid 30a-CPPs-GIP was constructed by double enzyme digestion, combining the DH5α-30a plasmid with the CPPs-GIP plasmid. Sac I and Nco I. Restriction endonucleases were integrated, and the enzyme digestion system is shown in Table 2. First, the digested fragments were subjected to gel electrophoresis. After gel recovery, two target fragments were obtained. The target fragments were ligated using T4 DNA ligase at 16°C overnight to form an integration vector, and the ligation system is shown in Table 3. PCR was then performed, and the system is shown in Table 4. The results of the 1% agarose gel electrophoresis experiment are presented. Sac I and Nco The target fragment was successfully inserted between the I restriction sites, meaning the bridge plasmid was successfully constructed. Figure 1 ).

[0080] Table 2 Double enzyme digestion system Table 3 Enzyme ligation system Table 4 PCR System PCR conditions: 94℃, 1 min; 95℃, 30 s, 50℃, 45 s; 72℃, 30 s, 30 cycles, 72℃, 10 min.

[0081] Example 2 Construction and identification of fusion plasmid pJS700-CPPs-GIP The method for constructing the integrative vector pJS700-CPPs-GIP is the same as in Example 1. However, due to differences in the restriction enzyme sites, the restriction endonuclease used is [specific enzyme name missing]. Sac I and Kpn The enzyme I digestion system is shown in Table 5, and the ligation system is shown in Table 6. The PCR and gel electrophoresis systems are shown in Table 7. Figure 2 The results showed that the pJS700-CPP-GIP vector was successfully constructed. The plasmid construction procedure is as follows: Figure 3 .

[0082] Table 5 Double enzyme digestion system Table 6 Connection System Table 7 PCR System Example 3 Screening and Identification of Recombinant Bacillus subtilis Validation results using LB solid plates containing 1% starch showed that wild-type Bacillus subtilis exhibited a transparent halo around its periphery, while recombinant Bacillus subtilis did not. Figure 4 PCR cross-validation using amylase primers and GIP primers confirmed that the target fragment was successfully integrated into Bacillus subtilis. Figure 5 ).

[0083] Example 4: Application of recombinant Bacillus subtilis in the treatment of diabetes (1) Construction of a diabetic mouse model Preparation of streptozotocin solution and intraperitoneal injection in mice: 2.1 g of citric acid was dissolved in 100 mL of deionized water to prepare a 0.1 mol / L citric acid solution. 2.94 g of trisodium citrate was dissolved in 100 mL of deionized water to prepare a sodium citrate solution. The two solutions were then mixed in a 1:1 volume ratio and the pH was adjusted to 4.5. This was the required STZ citrate buffer solution. After filtration and sterilization, the solution was stored at 4°C for later use. 0.01 g of STZ powder was dissolved in the citrate buffer solution to prepare a 1% STZ citrate buffer solution. After feeding mice a high-fat, high-sugar diet for 30 days, all mice were fasted overnight. Mice in the model group were intraperitoneally injected with 30 mg / kg of STZ citrate buffer solution, while mice in the normal group were injected with the same dose of citrate buffer solution. Simultaneously, the high-fat, high-sugar diet was replaced with a normal mouse diet to avoid hyperglycemia and ketosis.

[0084] 72 hours after STZ injection, 30 out of 35 mice showed a blood glucose level greater than 16.7 mmol / L (indicating successful modeling), resulting in a modeling rate of 85.7%. Observation and comparison between the normal and model groups revealed that the model group mice were less active, more lethargic, and had significantly duller, grayish-yellow fur. Their urine output, food intake, and water consumption gradually increased compared to the normal group. The body weight of mice in both groups was measured, and the data were statistically analyzed using a t-test with SPSS 16.0 software. The results showed that the body weight of the model group mice was significantly lower than that of the normal group mice, and their blood glucose levels were significantly higher. Figure 6 This indicates that the diabetic mouse model was successfully constructed.

[0085] (2) Regulation of blood glucose in diabetic mice by recombinant Bacillus subtilis Fasting blood glucose levels in diabetic mice were measured weekly at 12 h, and a one-sample t-test was performed to statistically analyze the blood glucose values ​​of each group. Mice receiving BS-GIP (recombinant Bacillus subtilis with surface-displaying gastric inhibitory and membrane-penetrating peptides) by gavage showed a significant decrease in fasting blood glucose from 25 mM to 17 mM by week 5 compared to mice orally administered PBS and wild-type Bacillus subtilis spores. Figure 7 At the beginning of the sixth week of gavage, an intraperitoneal glucose tolerance test (IPGTT) was performed. Mice orally administered BS-GIP recombinant Bacillus subtilis showed significantly lower blood glucose levels at 30 and 120 minutes after intraperitoneal glucose injection compared to mice orally administered PBS and wild-type Bacillus subtilis, indicating that oral administration of recombinant Bacillus subtilis improved the ability of diabetic mice to regulate blood glucose. Figure 8 ).

[0086] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A recombinant plasmid, characterized in that, The recombinant plasmid contains a nucleic acid molecule encoding a gastric inhibitory peptide and a nucleic acid molecule encoding a membrane-penetrating peptide, a backbone plasmid, and acceptable expression elements.

2. The recombinant plasmid according to claim 1, characterized in that, The amino acid sequence of the gastric inhibitory peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID NO:

2.

3. The recombinant plasmid according to claim 2, characterized in that, The acceptable expression elements include one or more of promoters, enhancers, and terminators.

4. The recombinant plasmid according to claim 3, characterized in that, The backbone plasmid is an integrative plasmid.

5. The recombinant plasmid according to claim 4, characterized in that, The backbone plasmid is pJS700.

6. A recombinant bacterial strain, characterized in that, The recombinant strain is transformed and / or transfected with any of the recombinant plasmids according to claims 1-5.

7. The recombinant strain according to claim 6, characterized in that, The chassis strain used for transformation and / or transfection is Bacillus subtilis.

8. The method for constructing the recombinant strain according to claim 7, characterized in that, The construction method includes: (1) Integrate the nucleic acid molecules encoding gastric inhibitory peptide and the nucleic acid molecules encoding membrane-penetrating peptide into the integration site of the backbone plasmid to obtain a recombinant plasmid; (2) Transform and / or transfect the recombinant plasmid into the chassis strain to obtain the recombinant strain.

9. A culture, characterized in that, The culture is obtained by culturing the recombinant strain of claim 7 or the recombinant strain obtained by the construction method of claim 8 under suitable growth and / or proliferation conditions.

10. The use of the recombinant plasmid according to claim 5, the recombinant strain according to claim 7, the recombinant strain obtained by the construction method according to claim 8, or the culture according to claim 9 in the preparation of a medicament or pharmaceutical composition for the prevention, treatment, mitigation, and / or improvement of diabetes, characterized in that, The prevention, treatment, mitigation, and / or improvement of diabetes are achieved by regulating blood glucose levels.

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