Genetically engineered bacteria secreting glp-1 and preparation method and application thereof

Abstract

The present application relates to a kind of genetically engineered bacteria of GLP-1 secretion and its preparation method and application, the genetically engineered bacteria CB-pMTL007-GLP-1 is prepared by integrating GLP-1 gene sequence into the genome of Clostridium butyricum NCU-02 (CGMCC No.25504), the genetically engineered bacteria has the effect of treating type 2 diabetes, reducing high blood pressure and improving Parkinson's disease.The present application uses Clostridium butyricum NCU-02 (CGMCC No.25504) as the host of recombinant expression vector for expressing GLP-1, so that the recombinant plasmid in the protein expression system of the GLP-1 secretion has good genetic stability, also has the same acid resistance, bile salt resistance, antioxidant capacity as wild-type Clostridium butyricum NCU-02 (CGMCC No.25504).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a genetically engineered bacterium that secretes GLP-1, its preparation method, and its application. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1), the second incretin discovered in 1983, is encoded and synthesized by the proglucagon gene. As an intestinal-derived hormone, GLP-1 can bind to receptors in multiple tissues throughout the body to exert various physiological functions. However, endogenous GLP-1 contains a site in its amino acid sequence that can be recognized by DPP-IV, making it easily degraded by DPP-IV, resulting in an extremely short half-life that severely limits its physiological efficacy. Currently, most research focuses on two strategies to extend its half-life: optimizing the natural GLP-1 sequence and developing DPP-IV inhibitors. Although GLP-1 analogs and DPP-IV inhibitors have shown good therapeutic effects, their primary source is chemical synthesis, leading to high costs and prices. Furthermore, they require long-term subcutaneous injection, and the significant economic burden and treatment discomfort limit the widespread clinical application of GLP-1-related drugs.

[0003] Clostridium butyricum, also known as butyric acid clostridium, is a normal part of the human gut microbiota. It is a strictly anaerobic, Gram-positive bacterium that can tolerate gastric acid and enter the intestines. It secretes butyric acid, an important nutrient for intestinal mucosal regeneration and repair, and promotes the growth of beneficial intestinal bacteria such as Bifidobacteria while inhibiting the growth of harmful intestinal bacteria such as Shigella dysenteriae. This restores intestinal flora balance, reduces the production of intestinal toxins such as amines, ammonia, and indole, and their toxicity to the intestinal mucosa. It also restores intestinal immune function and normal physiological functions, corrects intestinal immune disorders, restores intestinal immune tolerance, and eliminates inflammation and ulcers. It can effectively treat hypertension, lower blood sugar, and improve Parkinson's disease. Simultaneously, Clostridium butyricum produces beneficial enzymes and vitamins in the intestines, promoting the digestion and absorption of nutrients.

[0004] With technological advancements, the feasibility and applicability of engineered bacteria have gained increasing authoritative recognition. The use of genetically modified bacteria for disease treatment is receiving growing attention, as they can sustainably release target gene proteins, overcoming the challenge of rapid degradation of target gene proteins such as GLP-1. Combining Clostridium butyricum with GLP-1 may be a viable approach; through genetic engineering, Clostridium butyricum can serve as a drug delivery carrier, expressing specific products to achieve diagnostic and therapeutic purposes.

[0005] The challenges of using Clostridium butyricum as a genetically engineered bacterium:

[0006] 1. Clostridium has a very strict restriction modification system, and foreign DNA is quickly degraded after entering Clostridium.

[0007] 2. Combined use with GLP-1 resulted in lower extracellular GLP-1 expression levels. Summary of the Invention

[0008] The purpose of this invention is to provide a genetically engineered bacterium that can solve the above-mentioned technical problems, and to provide a method for preparing the genetically engineered bacterium.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a genetically engineered bacterium that secretes GLP-1. The genetically engineered bacterium CB-pMTL007-GLP-1 is prepared by integrating the GLP-1 gene sequence into the genome of Clostridium butyricum NCU-02 (CGMCC No. 25504). The genetically engineered bacterium has therapeutic effects on type 2 diabetes, lowers hypertension, and improves Parkinson's disease. The Clostridium butyricum NCU-02 has the accession number CGMCC No. 25504, the accession date is August 8, 2022, the accession location is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, and the classification name is Clostridium butyricum.

[0011] This invention also provides a method for preparing genetically engineered bacteria that secrete GLP-1, the steps of which are as follows:

[0012] (1) Preparation of E. coli CA434 competent cells;

[0013] (2) The nucleic acid fragment encoding human GLP-1 was inserted into the empty vector pMTL007 to obtain a recombinant plasmid, and the recombinant plasmid was heat-shocked into E. coli CA434;

[0014] (3) The recombinant plasmid was transformed into Clostridium butyricum NCU-02 (CGMCC No.25504) to obtain the genetically engineered bacterium CB-pMTL007-GLP-1;

[0015] The nucleotide sequence of the nucleic acid fragment encoding human GLP-1 is shown in SEQ ID NO.1; the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.2.

[0016] The genetically engineered bacteria provided by this invention can be used as fermentation strains, to prepare probiotic tablets with hypoglycemic function, and also for use in the preparation of drugs for treating type 2 diabetes, lowering hypertension, and improving Parkinson's disease.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) In this invention, Clostridium butyricum NCU-02 (CGMCC No. 25504) is used as the host of the recombinant expression vector for GLP-1, so that the recombinant plasmid in the protein expression system that secretes GLP-1 has good genetic stability and also has the same acid resistance, bile salt resistance and antioxidant capacity as wild-type Clostridium butyricum NCU-02 (CGMCC No. 25504). The GLP-1 expression level secreted by this genetically engineered bacterium is higher than that of other genetically engineered bacteria.

[0019] (2) The genetically engineered bacteria that secrete GLP-1 provided by this invention can significantly reduce blood glucose in type 2 diabetic (T2DM) mice, inhibit weight gain in T2DM mice, and improve glucose tolerance in T2DM mice, and can be used in the preparation of drugs for treating type 2 diabetes. Furthermore, in the treatment of Parkinson's disease, it improves dopaminergic neuron damage and motor dysfunction in Parkinson's disease (PD) mice; in hypertension, the above-mentioned GLP-1-secreting protein expression system can significantly reduce blood pressure in spontaneously hypertensive rats (SHR) and improve their symptoms of myocardial hypertrophy. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the in vitro expression detection results of GLP-1 in Example 1; where: CB: Clostridium butyricum; CBG: Clostridium butyricum-pMTL007-GLP-1 engineered bacteria; MG1363-GLP-1: Lactococcus lactis GLP-1 engineered bacteria; EcN-GLP-1: Escherichia coli-GLP-1 engineered bacteria;

[0021] Figure 2 This is a schematic diagram of the acid resistance evaluation test results in Example 2;

[0022] Figure 3 This is a schematic diagram of the bile salt tolerance test evaluation results in Example 2;

[0023] Figure 4 This is a schematic diagram of the antioxidant test results in Example 2;

[0024] Figure 5 This is a schematic diagram of the fasting blood glucose change curves of mice in each group in Example 3;

[0025] Figure 6 This is a schematic diagram of the weight change curves of mice in each group in Example 3;

[0026] Figure 7 This is a schematic diagram of the changes in glucose tolerance in each group of mice in Example 3;

[0027] Figure 8 This is a schematic diagram of the area under the glucose curve for each group of mice in Example 3;

[0028] Figure 9 This is a schematic diagram illustrating the results of the pole climbing test performed on PD mice in Example 4.

[0029] Figure 10 This is a schematic diagram illustrating the results of the suspension test on PD mice in Example 4;

[0030] Figure 11 This is a schematic diagram illustrating the results of the open field test on PD mice in Example 4;

[0031] Figure 12 The results of immunohistochemical analysis of TH in PD mice in Example 4 are shown, magnified 200×. Scale bar = 100 μm;

[0032] Figure 13 The image shows the immunohistochemical analysis results of α-syn in PD mice in Example 4, magnified 200×. Scale bar = 100 μm;

[0033] Figure 14 This is a schematic diagram of the examination results of SBP in SHR mice in Example 5;

[0034] Figure 15 This is a schematic diagram of the examination results of DBP in SHR mice in Example 5;

[0035] Figure 16 This is a schematic diagram of the HE staining and Masson staining results of the heart of SHR mice in Example 5. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be provided below. The invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be more thorough and complete.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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.

[0038] Example 1: Construction of engineered bacteria CB-pMTL007-GLP-1

[0039] 1. Preparation of E. coli CA434 competent cells

[0040] (1) Pick a single colony of Escherichia coli and inoculate it into 20 mL of LB medium. Incubate overnight at 37°C with shaking.

[0041] (2) Inoculate 1 mL of bacterial culture into 500 mL of LB medium and incubate at 37°C with shaking at 150 rpm for 2-3 hours until the OD value (600 nm) reaches 0.5-0.6, preferably close to 0.6 and not exceeding 0.6. (The optimal bacterial count is 5 × 10^7 cfu / mL.)

[0042] (3) Place 500 mL of culture in an ice bath for 30 min, centrifuge at 4℃ and 4000 rpm / min for 3 min, and remove the supernatant.

[0043] (4) Add 5 mL of ice-cold 0.1 mol / L CaCl2 solution to resuspend the bacterial precipitate, centrifuge at 4000 rpm for 3 min at 4℃, and discard the supernatant.

[0044] (5) Add 5 mL of ice-cold 0.1 mol / L CaCl2 solution to resuspend the bacterial precipitate, centrifuge at 4000 rpm for 3 min at 4℃, and discard the supernatant.

[0045] (6) Resuspend the bacterial precipitate in 300 μL of ice-cold 0.1 mol / L CaCl2 solution and incubate on ice for 3-24 hours to obtain competent E. coli cells. They can be stored at -80°C.

[0046] 2. Extraction of plasmid DNA

[0047] (1) Escherichia coli carrying recombinant plasmid pMTL007-hGLP-1 was inoculated into 10 mL LB chloramphenicol-containing culture medium and cultured overnight at 37°C in a shaker.

[0048] (2) Centrifuge at 8000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacterial cells;

[0049] (3) Add 250 μL of Solution I / RNase A mixture and vortex to completely dissolve the cells;

[0050] (4) Add 250 μL Solution II, gently invert to mix, let stand for about 2 minutes to obtain a clear lysate;

[0051] (5) Add 350 μL Solution Ⅲ and immediately invert to mix until a white flocculent substance is formed;

[0052] (6) Centrifuge at 8000 rpm for 15 min at room temperature;

[0053] (7) Take the supernatant into the HiBind DNA binding column, collect the waste liquid in the 2mL collection tube, centrifuge at 8000rpm for 2min, and discard the waste liquid.

[0054] (8) Add 500 μL HBC Buffer, centrifuge at 8000 rpm for 2 min, and discard the waste liquid;

[0055] (9) Add 700 μL DNA Wash Buffer, centrifuge at 8000 rpm for 2 min, and discard the waste liquid;

[0056] (10) Repeat step (9);

[0057] (11) Centrifuge at 8000 rpm for 4 min and remove the residual ethanol;

[0058] (12) Let stand at room temperature for about 5 minutes until all the ethanol has evaporated;

[0059] (13) Load the spin column into a clean 1.5mL EP tube, add 50μL ELution Buffer, let stand for 1-2min, centrifuge at 8000rpm for 2min to elute plasmid DNA and measure the concentration, and store at -20℃.

[0060] 3. The recombinant plasmid was heat-shocked and transformed into E. coli CA434.

[0061] (1) Take 2 μL of pMTL007-hGLP-1 recombinant plasmid with a concentration of not less than 100 ng / μL, and gently mix it with 100 μL of ice-cold E. coli CA434 competent cell suspension prepared by the above method. Place it on ice for 30 min. (The volume of plasmid should not exceed 10% of the competent cells).

[0062] (2) Heat shock at 42℃ for 60s, then quickly place on ice for 2min, add 800μL of LB medium, incubate at 37℃ and 180rpm for 1h, take 100μL to resuspend the transformation product and spread it on LB plate medium containing resistance, incubate upside down at 37℃ for 16h, and screen positive clones.

[0063] 4. Recombinant plasmid was transformed into Clostridium butyricum NCU-02 (CGMCC No. 25504).

[0064] (1) After heat shock transformation of E. coli CA434 with the recombinant plasmid that was verified to be correct by sequencing, positive colonies were picked onto liquid LB medium containing chloramphenicol (30 μg / mL) and cultured at 37℃ and 200 rpm until OD600 reached 1.5.

[0065] (2) Take 3 mL of E. coli CA434 bacterial culture, centrifuge at 4000×g for 2 min to collect the bacterial cells, wash away the residual liquid with sterile PBS buffer, centrifuge again and resuspend the E. coli CA434 bacterial culture in 400 μL of Clostridium butyricum NCU-02 (CGMCC No.25504) bacterial culture (OD600 to 2.0);

[0066] (3) Spread the resuspension onto a non-resistant TSA solid plate, and then place the plate in an anaerobic incubator at 37°C for incubation;

[0067] (4) After 24 hours of conjugation culture, scrape a small amount of bacterial cells from the TSA solid plate and streak them on the surface of the TSA solid plate containing 15 μg / mL thiamphenicol and 250 μg / mL D-cycloserine. Incubate at 37°C for 36-48 hours.

[0068] (5) Pick the transformant colonies from the re-stripe plate and inoculate them into 1 mL of anaerobic TSB supplemented with 15 μg / mL thiamphenicol. After incubation at 37°C for 36-48 h, colony PCR is performed for identification. If the identification is correct, the CB-pMTL007-GLP-1 expression system is obtained.

[0069] 5. ELISA detection of GLP-1 secretion expression in the culture supernatant of engineered bacteria.

[0070] (1) Coating process: Dilute the antigen to an appropriate concentration with coating diluent, add 100 μL of antigen to each well, incubate at 37℃ for 4 h, and discard the liquid in the well; (2) Blocking enzyme-labeled reaction wells: Add 5% fetal bovine serum to the reaction wells and block at 37℃ for 40 min. When blocking, fill each reaction well with blocking solution and remove air bubbles from each well. After completion, wash 3 times, 3 min each time; (3) Adding the sample to be tested (establishing an appropriate concentration gradient): Use a dilution of 1:50-1:400 for detection. Add 100 μL of the diluted sample to the enzyme-labeled reaction wells and incubate at 37℃ for 60 min. Fill with washing solution and wash 3 times, 3 min each time; (4) Adding enzyme-labeled antibody: Add enzyme-labeled antibody according to the reference working dilution provided by the company. 100 μL per well, incubate at 37℃ for 30-60 min, then wash 3 times for 3 min each time; (5) Add substrate solution (prepare fresh for use): 100 μL per well, incubate at 37℃ in the dark for 3-5 min, then add stop solution for color development; (6) Terminate the reaction: add 50 μL of stop solution to each well to terminate the reaction, and measure the experimental results within 20 min. The results are as follows: Figure 1 As shown.

[0071] Quantification was performed using an ELISA kit to detect GLP-1. For example... Figure 1 As shown, the expression level of GLP-1 protein in the culture supernatant of CB-GLP-1 bacteria was significantly higher than that in MG1363-GLP-1 and EcN-GLP-1.

[0072] For the specific preparation method of MG1363-GLP-1, please refer to the article: Engineered commensal bacteria prevent systemic inflammation-induced memory impairment and amyloidogenesis via producing GLP-1.

[0073] For the specific preparation method of EcN-GLP-1, please refer to the article: Neuroprotective effects of anengineered Escherichia coli Nissle 1917 on Parkinson's disease in mice by delivering GLP-1 and modulating gut microbiota.

[0074] Example 2: Evaluation of the genetic stability and functionality of engineered bacteria

[0075] 1. Evaluation of genetic stability of recombinant plasmids

[0076] (1) Activation of strains: Take 1 mL of TSB medium containing thiamphenicol (final antibiotic concentration of 15 μg / mL) and add it to a sterilized EP tube, then add 10 μL of bacterial solution, place it in a shaker at 37°C and incubate overnight.

[0077] (2) Divide the activated engineered bacteria into two portions and culture them overnight in liquid culture medium with and without antibiotics (first generation). After culture, subculture once a day for one month.

[0078] (3) Take 100 μL of bacterial culture and place it into an EP tube sterilized with 900 μL of PBS (phosphate buffered solution). Mix well and take another 100 μL of the bacterial culture from this tube into another new EP tube sterilized with 900 μL of PBS. Dilute using this method to the appropriate dilution factor (generally to 10⁶ to 10⁷ times). Streak the petri dish to divide it into sections. Add the diluted bacterial culture to both antibiotic-containing and antibiotic-free culture media. Incubate overnight at 37°C. Perform plate counting the next day. Perform plate counting every three days. For strains cultured on antibiotic-containing media, do not perform further counting after the first count.

[0079] 2. Acid resistance test

[0080] The engineered strain CB-pMTL007-GLP-1 and Clostridium butyricum NCU-02 (CGMCC No. 25504) were cultured in TSB medium in an anaerobic incubator at 37°C for 24 h. After 24 h, 100 μL of the bacterial culture was taken out and added to PBS buffer at pH = 2, 4, 5, 6 and 7 respectively. After anaerobic culture for 4 h, the bacterial culture was counted by plate spot.

[0081] The results are as follows Figure 2 As shown, the results indicate that both wild-type and engineered Clostridium butyricum exhibit good tolerance to high concentrations of acid, and the difference in acid tolerance between the two strains is not significant.

[0082] 3. Bile salt tolerance test

[0083] The engineered strain CB-pMTL007-GLP-1 and Clostridium butyricum NCU-02 (CGMCC No. 25504) were cultured in TSB medium containing 0.1%–0.3% ox bile salts (bile salt contents of 0%, 0.1%, 0.2%, and 0.3%, respectively) in a 37°C incubator for 24 h, and then plate counts were performed.

[0084] The results are as follows Figure 3 As shown, the results indicate that both wild-type and engineered Clostridium butyricum exhibit good tolerance to different concentrations of bile salts, and the difference in bile salt tolerance between wild-type and engineered Clostridium butyricum is not significant.

[0085] 4. Antioxidant test

[0086] (1) Determination of DPPH free radical scavenging ability:

[0087] The engineered strain CB-pMTL007-GLP-1 and Clostridium butyricum NCU-02 (CGMCC No.25504) were cultured in TSB medium in an anaerobic incubator at 37℃ for 24 h. After 24 h, the bacterial culture was centrifuged at 8000 rpm for 3 min and the supernatant was collected.

[0088] Take 1 mL of supernatant and mix it with 1 mL of prepared methanol solution of DPPH free radicals. After shaking and mixing, react at room temperature for 30 min in the dark and measure its OD value at a wavelength of 517 nm (deionized water is used as blank control).

[0089] DPPH radical scavenging rate: [1 - A517(sample) / A517(blank)] × 100%

[0090] (2) Determination of ability to scavenge hydroxyl radicals:

[0091] The engineered strain CB-pMTL007-GLP-1 and Clostridium butyricum NCU-02 (CGMCC No.25504) were cultured in TSB medium in an anaerobic incubator at 37℃ for 24 h. After 24 h, the bacterial culture was centrifuged at 8000 rpm for 3 min and the supernatant was collected.

[0092] Add 1 mL of supernatant to a glass test tube (sample tube), add 1 mL of ddH2O to a blank tube, and then add 1 mL of 3 mmol / L salicylic acid, 1 mL of 1 mmol / L FeSO4, and 1 mL of 3 mmol / L H2O2 respectively. Mix well and react in a water bath at 37°C for 15 min. Measure the absorbance at 510 nm using a spectrophotometer.

[0093] Hydroxyl radical scavenging rate (%) = [1 - A510(sample) / A510(blank)] × 100%

[0094] Antioxidant test results as follows Figure 4 As shown, the results indicate that both wild-type (CB) and engineered (CB-GLP-1) Clostridium butyricum exhibit good antioxidant capacity, with low DPPH and O2 levels. - The engineered bacteria have better OH- reduction and scavenging abilities than the wild type.

[0095] Example 3: Therapeutic effect of engineered bacteria on T2DM mice

[0096] 1. Establishment of a T2DM mouse model

[0097] Eight-week-old (20-22g) male C57BL / 6 mice were purchased from Hunan Slack Jingda Experimental Animal Company. After one week of acclimatization feeding with a normal diet, blood glucose and body weight were measured. These mice were then fed a high-fat diet for 12 weeks. Twelve mice were randomly selected as the control group, while the remaining mice received intraperitoneal injections of 40 mg / kg / day of STZ (dissolved in 0.1 mol / L citrate buffer, pH 4.5) for five consecutive days. Mice were fasted for 12 hours before STZ injection. Blood glucose levels were monitored every three days. Mice with stable blood glucose levels above 11.1 mmol / L were included in the study. Diabetic mice were then randomly divided into a model group, a positive control group treated with exenatide, a Clostridium butyricum treatment group, and a GLP-1 engineered bacteria treatment group.

[0098] Model evaluation: Gross observation showed that the normal group mice were of moderate size, in good spirits, moved freely, reacted quickly, and had smooth, glossy fur. In contrast, the type 2 diabetic mice were lighter, lethargic, sluggish, had dull, frizzy fur, moved slowly, arched backs, curled bodies, and had significantly increased urine output. The diabetic mice showed significantly less weight than the normal group, and their cardiac and liver coefficients were significantly increased, with statistical significance.

[0099] 2. Grouping and Treatment of T2DM Experimental Mice

[0100] (1) Control group (C): 100 μL of bacterial coating solution (0.9% physiological saline containing 0.01% gelatin) was administered by gavage once a day for 9 weeks;

[0101] (2) Model group (M): 100 μL of bacterial coating solution (0.9% physiological saline containing 0.01% gelatin) was administered by gavage once a day for 9 weeks;

[0102] (3) Exenatide group (E): GLP-1 receptor agonist exenatide 24 nmol / kg / d was injected intraperitoneally once a day for 9 weeks.

[0103] (4) Clostridium butyricum treatment group (Cb): 100 μL of Clostridium butyricum was administered by gavage, with a bacterial concentration of 10. 9 cfu / mL, once daily, for 9 weeks;

[0104] (5) GLP-1 engineered bacteria treatment group (Cb-G): 100 μL CCB-pMTL007-GLP-1 engineered strain was administered by gavage, with a bacterial concentration of 109 CFU / mL, once a day for 9 weeks;

[0105] (6) EcN-GLP-1 engineered bacteria treatment group (EcN-G): 100 μL EcN-GLP-1 engineered bacteria were administered by gavage, with a bacterial concentration of 109 cfu / mL, once a day for 9 weeks.

[0106] (7) MG1363-GLP-1 engineered bacteria treatment group (MG1363-G): 100 μL of MG1363-GLP-1 engineered bacteria was administered by gavage, with a bacterial concentration of 109 cfu / mL, once a day for 9 weeks.

[0107] 3. The therapeutic effect of engineered strains on type 2 diabetes

[0108] (1) Blood glucose and weight: Treatment began after successful modeling. Blood glucose was measured by taking blood from the tail vein. Weight was measured the day before blood glucose measurement. Blood glucose and weight were measured once a week to observe the treatment effect.

[0109] (2) Food and water intake: The weekly food and water intake of each group of mice was recorded. The extent to which the polydipsia and polyphagia symptoms of T2DM were relieved was determined.

[0110] (3) Nausea and vomiting: Exenatide is a hypoglycemic drug, which may cause some damage to the gastrointestinal tract after injection. Patients often experience symptoms such as decreased appetite, nausea and vomiting, and indigestion. Mice do not have a vomiting reflex, but their pica behavior can be used as an indicator of vomiting. Kaolin and gum arabic powder were mixed in a ratio of 50:1, and water was added to form a cylindrical shape similar to that of standard mouse feed. The mixture was then dried and stored for later use. Mouse feed and the prepared kaolin were weighed and placed in mouse cages. The percentage of kaolin intake to feed intake was calculated based on the kaolin intake and feed intake of each mouse within 24 hours. The data were expressed as mean ± standard deviation (M ± SD) and analyzed using STAT software.

[0111] 1) Oral glucose tolerance test (OPGTT): This test was conducted two weeks before the end of the experiment. Mice were starved for 16 hours and then given oral glucose at a dose of 2 g / kg. Blood glucose levels were measured by tail vein sampling at 0, 15, 30, 60, 90, and 120 minutes, and the area under the glucose tolerance curve (AUC) was calculated.

[0112] 2) Insulin Tolerance Test (ITT): Conducted one week before the end of the experiment. Mice were starved for 6 hours and then injected intraperitoneally with human insulin at 1 U / kg. Blood glucose changes were measured by tail vein sampling at 0, 15, 30, 60, 90, and 120 minutes, and the area under the insulin tolerance curve (AUC) was calculated.

[0113] Calculate the area under the curve

[0114] = 0.5*(Bg0min+Bg30min) / 2+0.5*(Bg30min+Bg60min) / 2+1*(Bg120min) / 2, (BloodGlucose is the blood glucose value at each time point).

[0115] To investigate the ameliorative effect of engineered bacteria CB-pMTL007-GLP-1 on type 2 diabetes mellitus (T2DM), we constructed a T2DM mouse model using HFD feeding combined with STZ injection. After modeling, we monitored changes in blood glucose and body weight in the mice. Figure 5 Except for group C, the random blood glucose levels of mice in all other groups reached above 16.7 mmol / L, indicating successful model establishment. After 4 weeks of treatment, the blood glucose levels of the treated mice began to show a downward trend compared to the model group. Continued treatment until week 6 resulted in a significant decrease in blood glucose levels in the Cb-G group, achieving an effect similar to that of the exenatide positive control group (group E), and demonstrating a superior hypoglycemic effect compared to the EcN-G and MG1363-G groups. Figure 5 One of the typical symptoms of type 2 diabetes mellitus (T2DM) is weight loss, such as... Figure 6As shown, the body weight of mice in the normal group remained relatively stable over 10 weeks (Group C), while the body weight of mice in the other groups treated with STZ showed a decreasing trend, proving the successful establishment of the T2DM model. The results showed that the EcN-G and MG1363-G groups had no significant change in body weight compared to Group M, while the Cb and Cb-G groups showed a mitigating effect on the weight loss in mice. Furthermore, OGTT results indicated that, compared to Group M, treatment with the engineered bacteria Cb-G group significantly improved glucose tolerance in mice. Figure 7 The area under the glucose curve (AUC) analysis also confirmed this result, and the engineered bacteria Cb-G group performed better than the EcN-G and MG1363-G groups. Figure 8 The above results indicate that treatment with engineered bacteria CB-pMTL007-GLP-1 can alleviate hyperglycemia and weight loss symptoms in T2DM mice, providing fundamental data support for further elucidating the relevant molecular mechanisms.

[0116] Example 4: The therapeutic effect of engineered bacteria on PD

[0117] 1. Establishment of PD mouse model

[0118] Eight-week-old male C57BL / 6 mice were purchased from Hunan Silek Jingda Company and divided into four groups: normal control group (C group), model group (M group), model group + Clostridium butyricum (CB group), model group + GLP-1 drug group (A group), model group + Clostridium butyricum-GLP-1 group (G group), model group + EcN-GLP-1 engineered bacteria group (EcN-G group), and model group + MG1363-GLP-1 engineered bacteria group (MG1363-G group), with 12 mice in each group. A mouse PD model was established by intraperitoneal injection of MPTP (20 mg / kg / day) for one week.

[0119] 2. Grouping of experimental animals

[0120] (1) Control group (C): 100 μL of gelatin saline solution (0.9% saline solution containing 0.1% gelatin) was administered by gavage once a day for 8 days.

[0121] (2) MPTP model group (M): MPTP (20 mg / kg / d) was injected for 7 days; 10 g / 100 μL

[0122] (3) MPTP model group + Clostridium butyricum (CB): administered at a concentration of 10 9 CFU / 100μL, the usual oral gavage dose is 100μL / 10g / day.

[0123] (4) MPTP model group + GLP-1 drug group (A): liraglutide (0.4 mg / (kg·d)

[0124] (5) MPTP model group + Clostridium butyricum-GLP-1 group (G): 100 μL CB-pMTL007-GLP-1 was administered by gavage, with a bacterial concentration of 10. 9 cfu / mL, once daily, for 8 days.

[0125] (6) MPTP model group + EcN-GLP-1 engineered bacteria group (EcN-G): 100 μL EcN-GLP-1 engineered bacteria were administered by gavage, with a bacterial concentration of 10. 9 cfu / mL, once daily, for 8 days.

[0126] (7) MPTP model group + MG1363-GLP-1 engineered bacteria group (MG1363-G): 100 μL of MG1363-GLP-1 engineered bacteria was administered by gavage, with a bacterial concentration of 10. 9 cfu / mL, once daily, for 8 days.

[0127] 3. Evaluation Indicators

[0128] (1) Pole Climbing Experiment

[0129] Mice were placed atop a rough wooden pole, 10 mm in diameter and 55 cm high. Each mouse was trained for 3 days prior to the experiment (3-5 minutes / session / day), and only those mice demonstrating a time of <18 seconds in pre-experiment trials (usually 2.5 hours before the experiment) were used. Mice were placed headfirst on the pole, and the time required to reach the bottom was recorded. The mice underwent three trials at one-minute intervals, and the average time was analyzed. The mice's motor coordination was evaluated.

[0130] (2) Open field experiment

[0131] The test box was a 50cm×50cm×40cm plastic box with a black bottom and white walls. The experimental parameters of the ANY-MAZE data acquisition software were set. The data acquisition time was 5 minutes. The data acquisition indicators were the total number of times the mouse traversed the grid and the total distance it moved. At the start of the experiment, each mouse was placed facing the box wall in any corner of the test box, and its spontaneous activity was recorded over the next 5 minutes. After each test, mouse excrement was removed, and the bottom and walls of the box were wiped with 70% alcohol. The next mouse was tested after the alcohol had evaporated. Each experiment was repeated 3 times. This method evaluates the spontaneous behavior, exploratory behavior, and stress levels of experimental animals in unfamiliar environments.

[0132] (3) Suspension test

[0133] The suspension test used a horizontal rope (1.5 mm in diameter) to evaluate muscle strength and balance. Simply put, the mouse's forepaws were placed on the rope, and the mouse was observed for 10 seconds. Then, the hind limbs were positioned according to the following criteria: 4 points (mouse gripping the rope with both forepaws and hind paws); 3 points (mouse gripping the rope with both forepaws and one hind paw); 2 points (mouse gripping the rope with both forepaws); 1 point (mouse gripping the rope with only one forepaw). The average score for each mouse was calculated, and the test was repeated three times before euthanasia. This test evaluated the mouse's muscle strength and balance.

[0134] (4) HE staining to detect pathological changes in brain tissue

[0135] 1) Dehydration: The mouse head was cut off along its neck, and the brain tissue was carefully removed using ophthalmic surgical scissors and forceps. Three mice from each group were immersed in 10 mL tubes containing 4% paraformaldehyde and stored at room temperature. The paraformaldehyde-fixed brain tissue was removed, rinsed with PBS, and then dehydrated with ethanol. Dehydration was performed for 180 min each with 70%, 80%, 90%, and 95% ethanol; then dehydrated for 60 min with 100% ethanol I and 60 min with 100% ethanol II.

[0136] 2) Xylene is transparent. Xylene I: 10 min; Xylene II: 10 min;

[0137] 3) Paraffin impregnation and embedding. Dehydrated tissues treated with xylene are embedded in paraffin wax at 56-58℃. The melting point of the wax should be suitable for the hardness of the embedded tissue.

[0138] 4) Sectioning and mounting. Sections were 5 μm thick and continuous. The wax slides were flattened on poly-L-lysine-treated glass slides in a slide spreader water bath (45℃). After blotting with absorbent paper, the slides were placed on a slide baker (60℃) and baked for 4 hours.

[0139] 5) Dewaxing the sections. Dewax with xylene I and II for 10 min each, then treat with anhydrous ethanol I and II for 3-5 min, followed by treatment with ethanol solutions of decreasing concentration: 95% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 3-5 min each, and rinse with tap water for 2 min.

[0140] 6) Staining. Harris hematoxylin staining for 5-10 min, rinse with tap water for 1 min, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with 0.6% ammonia water, rinse with running water, stain with eosin for 3-5 min, rinse with tap water for 1 min;

[0141] 7) Dehydration, clearing, and sealing. Treat with ethanol solutions of increasing concentration: 70% ethanol and 80% ethanol for 10-20 seconds, 95% ethanol I and II for 3-5 minutes, and 100% ethanol I and II for 3-5 minutes. Clear in xylene I, II, and III for 3-5 minutes each. Mount with neutral resin.

[0142] 8) Observation. Place the mounting slide under an optical microscope and take pictures under 200x and 400x magnification respectively, and perform pathophysiological analysis.

[0143] To observe the effect of CB-GLP-1 on the motor coordination ability of PD mice, each mouse underwent a pole climbing test. Figure 9 In the pole climbing test, the descent time of group M was significantly longer than that of the control group (p<0.0001). However, the MG1363-GLP-1, EcN-GLP-1, and CB-GLP-1 groups significantly alleviated the prolonged time of group M, especially the CB-GLP-1 group (p<0.0001), where the improvement was the most significant. In the open field test, the exploration ability of mice in group M after MPTP injection was lower than that in group C, as can be seen from the total movement distance (p<0.0001). Figure 10 After treatment with MG1363-GLP-1, EcN-GLP-1, and CB-GLP-1 groups, the total movement distance within 10 minutes was significantly increased compared to the M group (p<0.0001), especially the CB-GLP-1 group, which showed performance more similar to the C group. Further, the distance moved in the central region ( Figure 11 We found that the movement distance in the central region of mice in the MG1363-GLP-1 group, EcN-GLP-1 group, and CB-GLP-1 group showed a similar trend. In conclusion, CB-GLP-1 treatment can significantly restore motor dysfunction induced by MPTP in PD mice.

[0144] To evaluate the effect of CB-pMTL007-GLP-1 treatment on MPTP-induced dopaminergic neuron damage, we performed immunohistochemical analysis on TH. Figure 12 As shown, compared with the control group, the number of TH-positive dopamine neurons in the substantia nigra and striatum of the MPTP group was sharply reduced. However, treatment with liraglutide, CB, and CB-GLP-1 significantly prevented these changes. To evaluate the effect of CB-GLP-1 on the expression of α-syn, a characteristic protein of mouse PD, immunohistochemical analysis was performed on α-syn. We found that compared with group C ( Figure 13 In group M mice, the expression of α-syn in the substantia nigra was significantly increased, while the expression of α-syn in the substantia nigra was inhibited to some extent in the liraglutide, CB, and CB-GLP-1 groups, with the CBG group showing the most significant effect. These results indicate that CB-GLP-1 can significantly inhibit neuronal damage in PD mice.

[0145] Example 5: Therapeutic effect of engineered bacteria on hypertensive mice

[0146] 1. Establishment and treatment of a mouse model of hypertension

[0147] (1) Eight-week-old Wistar rats and SHR rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed under standard conditions (humidity 50±15%, temperature 22±2℃, 12 / 12 light-dark cycle) in the Specific Pathogen Free (SPF) laboratory animal barrier system of the Institute of Translational Medicine, Nanchang University, and fed a standard diet.

[0148] (2) Six Wistar birds were assigned to group C (n=6, normal drinking water), and the remaining 24 SHR birds were randomly divided into group M (n=6, normal drinking water) and group CB (n=6, 10...). 8 -10 9 CFU / L Clostridium butyricum was administered via gavage every two days until the end of the experiment; CB-GLP group (n=6, 10) 8 -10 9 CFU / L engineered bacteria were administered via gavage every two days until the end of the experiment; EX group (0.4 mg / kg exenatine was injected intraperitoneally every two days until the end of the experiment); EcN-GLP-1 group (n=6, 10) 8 -10 9 CFU / L EcN-GLP-1 engineered bacteria were administered by gavage every two days until the end of the experiment; MG1363-GLP-1 group (n=6, 10) 8 -10 9 CFU / LMG1363-GLP-1 engineered bacteria were administered via gavage every two days until the end of the experiment.

[0149] 2. Mouse experiment

[0150] (1) Blood pressure measurement via tail vein

[0151] After fasting overnight, tail artery blood pressure was measured using a non-invasive tail blood pressure monitor. Normal mice showed successful blood pressure reduction with systolic pressure (95–125 mmHg) and diastolic pressure (67–90 mmHg). Blood pressure was monitored weekly to observe the antihypertensive effect.

[0152] (2)Weight

[0153] The weight of each mouse was measured individually, and the weight of the mice was measured once a week after administration.

[0154] (3) Feces

[0155] Two to three fecal pellets were collected from each mouse before drug administration and at the point of blood pressure reduction. The pellets were then stored at -80°C for 30 minutes. 16S rRNA sequencing and high-throughput sequencing were used to determine the bacterial genus and species in the feces.

[0156] (4) Quantitatively detect the amount of food and water consumed by mice in each group.

[0157] The weekly food and water intake of each group of mice was recorded, and the weekly food and water intake of each mouse was statistically analyzed.

[0158] (5) Organ collection and calculation of organ index

[0159] After dissection, organs such as the heart, kidneys, and intestines were removed, organ indices were calculated, and the organs were cryopreserved in liquid nitrogen and paraformaldehyde for future use.

[0160] Organ coefficient = (organ weight / mouse body weight) × 100%

[0161] (6) Serum sample collection and testing

[0162] Before execution, the animals were fasted overnight, and about 1 ml of blood was drawn from their eye sockets. The blood was centrifuged at 3500 rpm for 15 minutes, and the upper serum sample was collected and frozen.

[0163] 3. Serum test samples and indicators

[0164] (1) Sampling: Before the execution, the patient fasted overnight, and about 1 ml of blood was taken from the eye socket. The blood was centrifuged at 3500 rpm for 15 min, and the upper serum sample was frozen.

[0165] (2) Enzyme-linked immunosorbent assay (ELISA) was used to detect: endothelial nitric oxide synthase (eNOS); serum angiotensin II (Ang II) level; and serum GLP-1 content.

[0166] 4. Pathological staining

[0167] (1) Heart: HE staining was used to observe the size and cross-section of left ventricular cardiomyocytes; transmission electron microscopy was used to observe changes in the microstructure of cardiomyocytes, such as the arrangement of mitochondria and the state of myofibrils. Long-term stress load can lead to a reduction in mitochondria and fiber breakage; Masson staining was used to assess collagen deposition.

[0168] CB-GLP-1 reduced systolic and diastolic blood pressure in SHR mice. After overnight fasting, tail artery blood pressure was measured in different groups of mice using a non-invasive tail blood pressure monitor. We monitored blood pressure changes after treatment with the engineered bacteria for 6 consecutive weeks. We found that in the SHR group, after 6 weeks of intervention with the bacteria and liraglutide, both systolic and diastolic blood pressure were significantly reduced (P<0.05), with a particularly significant effect on systolic blood pressure.

[0169] HE staining revealed that the cross-sectional area of ​​cardiomyocytes in group M was larger than that in the control group, and this improved after treatment with engineered bacteria and intervention with liraglutide in both the CB-GLP-1 and EX groups. Masson staining was used to assess collagen deposition, and it was found that ventricular wall fibrosis and a large amount of collagen deposition in the myocardium and around blood vessels were observed in group M, which also improved after treatment with engineered bacteria and intervention with liraglutide in both the CB-GLP-1 and EX groups.

[0170] In summary, this study conducted multi-faceted mechanistic analyses using various techniques in T2DM, PD, and SHR animal models, providing a reference for the development and clinical application of novel GLP-1 drugs. Probiotics, as drug carriers, not only alleviate the suffering caused by intravenous injection of existing protein peptides, but also, through the combined use of probiotics and effector proteins, can directly (through effector protein action) and indirectly (through probiotics restoring intestinal flora homeostasis), thus amplifying the therapeutic effect.

Claims

1. A genetically engineered GLP-1 secreting bacteria CB-pMTL007-GLP-1, characterized by, The genetically engineered bacteria are prepared by integrating a GLP-1 gene sequence into a genome of Clostridium butyricum (NCU-02). Clostridium butyricum The genetically engineered bacteria have the effects of treating type 2 diabetes, reducing high blood pressure or improving Parkinson's disease. The Clostridium butyricum NCU-02 has a preservation number of CGMCC No. 25504, a preservation date of August 8, 2022, and a preservation location of the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Xili, Beichen, Chaoyang District, Beijing.

2. A method for preparing a genetically engineered bacterium secreting GLP-1, characterized by, The steps are as follows: (1) E. coli CA434 competence preparation; (2) The nucleic acid fragment encoding human GLP-1 is inserted into the empty vector pMTL007 to obtain a recombinant plasmid, and the recombinant plasmid is transformed into E. coli JM109 by heat shock E. coli CA434; (3) The recombinant plasmid is transformed into Clostridium butyricum NCU-02 to obtain a genetically engineered bacterium CB-pMTL007-GLP-1. The nucleotide sequence of the nucleic acid fragment encoding human GLP-1 is shown in SEQ ID NO. 1; the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO. 2; the Clostridium butyricum NCU-02 has a preservation number of CGMCC No. 25504, a preservation date of August 8, 2022, and a preservation location of the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Xili, Beichen, Chaoyang District, Beijing.

3. Use of the genetically engineered bacteria according to claim 1, characterized in that, The genetically engineered bacterium is applied in fermentation strains.

4. Use of the genetically engineered bacteria according to claim 1, characterized in that, The genetically engineered bacterium is applied in the preparation of probiotic tablets with hypoglycemic function.

5. Use of the genetically engineered bacteria according to claim 1, characterized in that, The genetically engineered bacterium is applied in the preparation of drugs for treating type 2 diabetes, lowering blood pressure, or improving Parkinson's disease.

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