Recombinant vector and recombinant yeast expressing GLP-1 and their application

Abstract

The present invention provides a recombinant vector, recombinant yeast and application for expressing GLP-1, and relates to the field of biotechnology. The recombinant vector contains a HXT4 gene promoter sequence and a GLP-1 gene regulated by the HXT4 gene promoter. The recombinant vector can be regulated to express GLP-1 by changes in the glucose content in the environment to alleviate the defects of GLP-1 drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant vector and recombinant yeast for expressing GLP-1 and applications thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Diabetes is one of the most common complex metabolic diseases in the world, with high blood sugar as the main sign, most of which are caused by insulin resistance and / or insufficient insulin secretion. Many diabetic patients are improperly or insufficiently treated, which can lead to chronic morbidity and mortality.

[0004] At present, the treatment of diabetes is mainly based on hypoglycemic drugs, supplemented by diet regulation and increased exercise. GLP-1 is an endogenous incretin that can promote insulin secretion in a glucose-dependent manner, while enhancing the function of pancreatic β cells and delaying gastric emptying, thereby playing a role in lowering blood sugar. GLP-1 is secreted by L cells in the intestinal epithelium. The top of the L cell is in direct contact with the intestinal cavity. It can sense the glucose level in the intestine and then secrete GLP-1 into the blood to exert its physiological function. The plasma half-life of GLP-1 is extremely short, usually less than 2 minutes. It is produced and exerts its effect quickly after a meal, and is then rapidly degraded by DPP-4. This is the normal physiological process of GLP-1. At present, most GLP-1 drugs are long-acting dosage forms. By optimizing the natural GLP-1 sequence or developing DPP-4 inhibitors to extend its half-life, it can be maintained in the plasma for a longer time. However, the long-term presence of high concentrations of GLP-1 in the body may cause some side effects, such as decreased appetite, weight loss, injection site reactions, nausea, diarrhea, vomiting, indigestion and other gastrointestinal discomfort, as well as more serious hypoglycemia, pancreatitis, thyroid cancer and other problems. More and more studies have shown that long-acting GLP-1 drugs have a weight loss effect during use, but they also cause muscle loss, and the weight will rebound rapidly after discontinuation of the drug. In response to the above problems, there is an urgent need for a drug that can sense glucose and release GLP-1, which will provide a safer treatment option for diabetic patients. In addition, the GLP-1 drugs currently used in clinical practice are subcutaneous injections. Long-term subcutaneous injections can bring some potential hazards to patients, such as high dependence, subcutaneous fat damage and lesions, skin infections, etc. Improving the dosage form of GLP-1 drugs and avoiding long-term subcutaneous injections can help reduce drug side effects.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant vector for expressing GLP-1, so as to achieve that the expression of GLP-1 can be regulated by the glucose content in the environment.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a recombinant vector for expressing GLP-1 is provided, wherein the recombinant vector contains a HXT4 gene promoter sequence and a GLP-1 gene whose expression is regulated by the HXT4 gene promoter.

[0009] In a second aspect, a recombinant yeast is provided, wherein the recombinant yeast contains the recombinant vector expressing GLP-1 as described in the first aspect.

[0010] In a third aspect, there is provided a use of the recombinant vector described in the first aspect or the recombinant yeast described in the second aspect in any of the following:

[0011] (I) Preparation of drugs for the prevention and / or treatment of diabetes;

[0012] (II) Regulating blood glucose and / or insulin levels of subjects for non-diagnostic and non-therapeutic purposes;

[0013] (III) Preparation of drugs for lowering blood sugar;

[0014] (IV) Preparation of drugs for promoting insulin.

[0015] In a fourth aspect, a pharmaceutical composition is provided, wherein the pharmaceutical composition contains the recombinant vector described in the first aspect or the recombinant yeast described in the second aspect.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The recombinant vector expressing GLP-1 provided by the present invention integrates the HXT4 gene promoter sequence upstream of the GLP-1 gene, so that the HXT4 gene promoter regulates the expression of the GLP-1 gene. When the glucose level in the environment rises to about 1g / L, the HXT4 promoter added in the recombinant vector turns on the switch for expressing GLP-1, simulating the function of L cells in the body to secrete GLP-1; when blood sugar is reduced, the HXT4 promoter is inhibited and the expression of GLP-1 is stopped. When the recombinant yeast containing the recombinant vector is used to regulate blood sugar, when the intestinal glucose concentration increases, the recombinant yeast expresses GLP-1 to reduce blood sugar levels; when the blood sugar level is reduced or when no food is taken, the recombinant yeast does not express GLP-1, avoiding the long-term high concentration state of long-acting GLP-1 in the body. In the preferred method, the recombinant yeast is a recombinant Saccharomyces boulardii, and the release of GLP-1 is controlled by glucose concentration, so that Saccharomyces boulardii can release GLP-1 while producing a prebiotic effect, thereby realizing intelligent regulation of blood sugar, and the recombinant Saccharomyces boulardii can also be prepared as an oral preparation to avoid long-term subcutaneous injection of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the HXT4 promoter-mediated glucose sensing and transport activation pathway;

[0020] Figure 2 This is a schematic diagram of the expression of GLP-1 by the recombinant vector of the present invention in a glucose environment;

[0021] Figure 3 A schematic diagram of the plasmid map of the recombinant vector constructed in Example 1;

[0022] Figure 4 The restriction endonuclease gel electrophoresis diagram of the recombinant plasmid in Example 1;

[0023] Figure 5 This is a photo of the plate screened three times in Example 2;

[0024] Figure 6The results of the luciferase assay of each experimental group in Example 3 are shown in the upper figure for the measured values, and the lower figure for the values ​​expressed in a bar graph; BC group: saline control group, MG group: protoplast group not treated with lysis solution, TG group: protoplast group treated with lysis solution, compared with BC group and MG group, p < 0.001;

[0025] Figure 7 The results of SDS-PAGE electrophoresis of the recombinant Saccharomyces boulardii induced expression in Example 4, M: Marker; 1: 0h; 2: 4h; 3: 8h; 4: 24h; 5: 28h; 6: 32h; 7: 48h;

[0026] Figure 8 HPLC chromatograms of the culture supernatant of the recombinant Saccharomyces boulardii before induction and 4 hours after induction in Example 5;

[0027] Fig. 9 : is the growth curve of the empty vector strain of Saccharomyces boulardii and the recombinant Saccharomyces boulardii in Example 6;

[0028] Fig.10 is a bar graph of the survival rate of the recombinant Saccharomyces boulardii in simulated gastrointestinal fluid in vitro in Example 6;

[0029] Fig.11 is a line graph of the survival rate of the recombinant Saccharomyces boulardii in simulated gastrointestinal fluid in vitro in Example 6;

[0030] Fig.12 It is a line graph of the survival rate of the recombinant Saccharomyces boulardii in Example 6 in simulated small intestinal fluid in vitro. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between such entities or actions, such as numbering (I), (II)... (VII).

[0033] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0034] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0035] As used herein, the articles "a", "an", and "the" include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0036] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0037] The vector is well known to those skilled in the art, including but not limited to: plasmid, episomal plasmid, minicircle DNA, phagemid, cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. In some embodiments, the vector contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0038] In a first aspect, a recombinant vector for expressing GLP-1 is provided, wherein the recombinant vector contains a HXT4 gene promoter sequence and a GLP-1 gene whose expression is regulated by the HXT4 gene promoter.

[0039] The recombinant vector expressing GLP-1 is used in a yeast expression system, and the functional strength of the promoter element in the yeast expression system is closely related to the expression efficiency. The promoter elements commonly used in yeast expression systems are divided into two categories, namely constitutive promoters represented by strong promoters such as pPGK1, pTEF1, pTDH3 and pADH1, and inducible promoters represented by pGAL related to galactose metabolism and pHXT related to hexose metabolism. Among them, the hexose transporter family is composed of HXT1p-HXT17p and GAL2p. The HXT transporter in yeast is a protein closely related to glucose sensing, containing 12 putative transmembrane domains, which mediates glucose transport across the plasma membrane through a facilitated diffusion mechanism.

[0040] The HXT4 gene encodes a hexose transporter, and the expression of the HXT4 gene is regulated by the glucose concentration. The affinity is strongest at a glucose concentration of 1g / L, which can activate the transcription of the HXT4 gene and the subsequent production of the Hxt4p protein. The glucose sensing system and signal transduction pathway involved in regulating the expression of the gene encoding the hexose transporter in yeast include the Snf3p / Rgt2p-Rgt1p pathway and the Snf1p-Mig1p pathway, and the expression of the HXT4 gene is regulated by binding of each transcription factor to a specific DNA sequence in the promoter region of the HXT4 gene. The recombinant vector for expressing GLP-1 provided by the present invention integrates the HXT4 gene promoter sequence upstream of the GLP-1 gene, so that the HXT4 gene promoter regulates the expression of the GLP-1 gene.

[0041] The fasting glucose level of a normal adult is 0.7-1.1 g / L. When the blood glucose level in the body rises to 1 g / L after a meal, the HXT4 promoter added to the recombinant vector turns on the switch to express GLP-1, simulating the function of L cells in the body to secrete GLP-1; when blood glucose drops, the HXT4 promoter is inhibited and the expression of GLP-1 stops. Figure 1 As shown, HXT4 promoter is used as the promoter for regulating GLP-1 gene, and the schematic diagram of GLP-1 expression in glucose environment is shown in Figure 2 shown.

[0042] In an optional embodiment, the nucleotide sequence of the HXT4 gene promoter is shown in SEQ ID NO.1.

[0043] In an optional embodiment, the nucleotide sequence of the GLP-1 gene is as shown in SEQ ID NO.2.

[0044] In an optional embodiment, the recombinant vector further contains at least one of a resistance gene, a marker gene and a nucleotide sequence encoding a signal peptide.

[0045] In an optional embodiment, the resistance gene includes but is not limited to one or more of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a streptomycin resistance gene and a hygromycin resistance gene, preferably including an ampicillin resistance gene and a hygromycin resistance gene.

[0046] In an optional embodiment, the marker gene includes but is not limited to one or more of a fluorescent protein marker gene, a chloramphenicol acetyltransferase gene, a luciferase gene and a β-glucuronidase gene, preferably including a luciferase gene.

[0047] In an optional embodiment, the 5' end of the GLP-1 gene contains a nucleotide sequence encoding a signal peptide, and the signal peptide is a signal peptide of IL-2.

[0048] In an optional embodiment, the nucleotide sequence encoding the signal peptide of IL-2 is shown as SEQ ID NO.4.

[0049] In an optional embodiment, the recombinant vector is a recombinant plasmid.

[0050] In an optional embodiment, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO.3.

[0051] In a second aspect, a recombinant yeast is provided, wherein the recombinant yeast contains the recombinant vector expressing GLP-1 as described in the first aspect.

[0052] In an alternative embodiment, the recombinant yeast is recombinant Saccharomyces boulardii.

[0053] Saccharomyces boulardii ( Saccharomyces boulardii ) is a non-pathogenic, probiotic yeast that is genetically a group of Saccharomyces cerevisiae strains that share >99% genomic relatedness. As a microecological preparation, Saccharomyces boulardii can be used to prevent and treat infantile diarrhea, traveler's diarrhea, recovery after intestinal surgery, and regulation of intestinal environmental balance. Saccharomyces boulardii has been proven to be safe and effective in a large number of clinical applications. Saccharomyces boulardii tolerates various digestive enzymes and gastric acid secreted by the gastrointestinal tract, is naturally resistant to antibiotics, has an optimal growth temperature of 37°C, and can maintain a certain number of viable bacteria in the gastrointestinal tract. Pharmacokinetic studies have shown that Saccharomyces boulardii cannot be detected in the feces 5 to 7 days after oral administration of Saccharomyces boulardii. In summary, Saccharomyces boulardii is an ideal intestinal drug delivery carrier. In a preferred embodiment, the recombinant vector expressing GLP-1 of the first aspect is introduced into Saccharomyces boulardii to construct recombinant Saccharomyces boulardii, and a recombinant probiotic expressing GLP-1 is obtained. The probiotics are colonized in the intestine, and the expression of GLP-1 is achieved while exerting a probiotic effect, providing patients with a more convenient and comfortable administration method. By modifying Saccharomyces boulardii and adding the GLP-1 gene, which is regulated by the HXT4 gene promoter, the recombinant Saccharomyces boulardii can be colonized in the intestines through oral administration. After eating, the intestinal glucose concentration increases, and the recombinant Saccharomyces boulardii expresses GLP-1 to lower blood sugar levels; when the blood sugar level decreases or when no food is eaten, the recombinant Saccharomyces boulardii does not express GLP-1, avoiding the long-term high concentration state of long-acting GLP-1 in the body.

[0054] In the third aspect, there is also provided the use of the recombinant vector described in the first aspect or the recombinant yeast described in the second aspect in any of the following:

[0055] (I) Preparation of drugs for the prevention and / or treatment of diabetes;

[0056] (II) Regulating blood glucose and / or insulin levels of subjects for non-diagnostic and non-therapeutic purposes;

[0057] (III) Preparation of drugs for lowering blood sugar;

[0058] (IV) Preparation of drugs for promoting insulin.

[0059] In a fourth aspect, a pharmaceutical composition is also provided, wherein the pharmaceutical composition contains the recombinant vector described in the first aspect or the recombinant yeast described in the second aspect.

[0060] In an optional embodiment, the pharmaceutical composition contains pharmaceutically acceptable optional excipients, exemplary pharmaceutical excipients include, but are not limited to, solvents, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film-forming agents, fragrances, viscosity enhancers, anti-adhesive agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protective agents, humectants, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrices and drug carrier materials. One or more.

[0061] In an optional embodiment, the pharmaceutical composition contains the recombinant Saccharomyces boulardii, and the pharmaceutical composition is an oral preparation. The pharmaceutical composition uses the recombinant Saccharomyces boulardii as a therapeutic agent, and has glucose-activated and repeatable GLP-1 secretion functions. The recombinant Saccharomyces boulardii expresses GLP-1 and self-regulates according to the fluctuation of intestinal glucose concentration, simulating the natural physiological response of intestinal L cells to secrete GLP-1 after sensing changes in intestinal glucose concentration, thereby realizing intelligent regulation of blood sugar. Saccharomyces boulardii itself can regulate intestinal microbial homeostasis, and intestinal microbial homeostasis itself has an improvement effect on diabetes. Saccharomyces boulardii that conditionally induces the expression of GLP-1 is expected to bring a therapeutic effect of 1+1>2 for the treatment of diabetes.

[0062] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0063] Example 1 Selection of glucose-inducible promoter and construction of recombinant plasmid

[0064] The gene sequence of glucose affinity transporter HXT4 encoded by Saccharomyces boulardii was used as a promoter to construct a recombinant plasmid expressing GLP-1. The plasmid vector was pYEP-Hygro-{sce_HXT4pro-2000bp}>IL2-sig / GLP-1:{ERBV-1 2A}:Luciferase (vector ID: VB240101-1203mvs). Ampicillin antibiotic resistance gene and hygromycin antibiotic resistance gene were added for screening of positive clones; luciferase gene Luciferase was added for bioluminescent labeling. The recombinant plasmid was transformed into E. coli competent cells, and the Escherichia coli carrying the recombinant plasmid was cultured and collected in a culture medium containing resistance; positive clones were screened, and clone identification was performed to obtain a recombinant plasmid that met the requirements.

[0065] Results: The gene sequence of Saccharomyces boulardii encoding glucose transporter HXT4 with different affinity was constructed as the promoter, Ampicillin was selected as the resistance screening of Escherichia coli, Hygro B was selected as the resistance screening of recombinant bacteria, the signal peptide part of IL-2 was added upstream of the GLP-1 gene to ensure the secretory expression of the target protein, and luciferase was added for the identification of the transformed recombinant engineering bacteria ( Figure 3 ). The results showed that the protein encoded by the HXT4 gene can initiate transcription and translation switches at a glucose concentration of 1g / L, achieving high induction expression of GLP-1. Restriction endonucleases ApaLI+NcoI (3193,1246, 771, 230, 72, 2073) were used for digestion, and the insertion sequence and restriction endonuclease sites on the vector backbone were analyzed. The digested DNA was subjected to agarose gel electrophoresis, and the gel electrophoresis bands were consistent with the reference sequence ( Figure 4 ).

[0066] Example 2 Transformation and screening of episomal plasmids of Saccharomyces boulardii

[0067] The recombinant plasmid prepared in Example 1 was introduced into competent Saccharomyces boulardii, and the successfully transformed recombinant Saccharomyces boulardii was screened by resistance, and the recombinant bacteria were identified by PCR to obtain the recombinant engineered bacteria. The experimental method is as follows:

[0068] 1. Prepare competent Saccharomyces boulardii: Take 2mL of overnight culture of Saccharomyces boulardii, cool on ice and centrifuge at 3000×g for 3min to collect cells. Rinse cells with sterile water, centrifuge at 3000×g for 3min, and remove supernatant. Resuspend cells with 200μL 0.1M LiAc, incubate at 220rpm and 30℃ for 20min. Centrifuge at 3000×g for 3min, discard supernatant; add 1mL and 500μL 1M sorbitol to resuspend cells, centrifuge at 3000×g for 3min, and discard supernatant. Add 200μL 1M sorbitol to resuspend cells, and aliquot into 100μL to make competent cells.

[0069] 2. Add 5 μL of plasmid (130.5 ng / μL) to the competent cells and mix gently. Transfer to an electroporation cup and place on ice for 2 minutes. Set the following electroporation instrument parameters for electroporation: voltage 1500, capacitance 25, resistance 200. Take out the electroporation cup, add 1 mL of 1 M sorbitol, mix well, transfer to a clean epoxide tube, and let stand at 30°C for 30 minutes. Centrifuge at 4000 rpm for 5 minutes, discard the supernatant, add 1 mL of YPD liquid culture medium, mix well, and place in a shaker at 30°C, 220 rpm for 1 hour.

[0070] 3. Centrifuge at 4000rpm for 5min, discard part of the supernatant and mix well, and spread on a YPD solid plate containing 10μg / mL hygromycin antibiotics and Amp antibiotics. Place in a constant temperature incubator and culture at 30℃. When the single colony grows to about 2mm, further streak screening is performed, streaking on a YPD solid plate containing hygromycin. Place in a constant temperature incubator and culture at 30℃. Perform the third streak screening as above.

[0071] The results showed that the plasmid was transformed by the classical yeast transformation method, and after three screenings with 10 μg / mL Hygro B, a recombinant engineered bacterium containing the recombinant plasmid was obtained ( Figure 5 ).

[0072] Example 3 Lysis and fluorescence detection of recombinant Saccharomyces boulardii

[0073] The positive colonies screened in the last time were selected and inoculated into liquid culture medium, and then placed in a shaker at 30°C and 220 rpm for overnight culture. The groups were BC group: saline control group, MG group: protoplast group without lysis solution treatment, and TG group: protoplast group with lysis solution treatment.

[0074] Transfer 500μL of bacterial solution to a clean epoxies tube, centrifuge at 4000rpm for 5min, and discard the supernatant. Add 300μL 1.2M sorbitol buffer, add 10μL Lyticase, mix thoroughly, and treat on a shaker at 220rpm, 30℃ for 1h. Centrifuge at 4000rpm for 5min, and discard the supernatant. Add 100μL lysis buffer and mix well. Incubate on ice for 5min. After bringing to room temperature, add 20μL of each of the blank and the ELISA plate, add 100μL firefly luciferase detection reagent, and shake the plate to mix 2~3 times. Set the parameters, measurement time is 10s, measurement interval is 2s, shake for 2s, and measure RLU.

[0075] The results are as follows Figure 6 As shown, the colonies were selected for seed amplification and then the luciferase assay was performed. The value of the TG2 group measured by the microplate reader was as high as 1.01×10 8 ( Figure 6 Middle and upper figure), from the bar chart ( Figure 6 The fluorescence value was significantly higher than that of the BC group and the MG group, indicating that the recombinant plasmid was successfully transformed.

[0076] Example 4 Induced expression of recombinant Saccharomyces boulardii and SDS-PAGE electrophoresis detection

[0077] The recombinant Saccharomyces boulardii culture liquid of the TG2 group in Example 3 was inoculated into 50 mL YPD liquid culture medium at a ratio of 1:250 (volume ratio), and cultured in a shaker at 30°C and 200 rpm until OD600≈5. Before starting induction, take 1 mL of the bacterial liquid and transfer it to a clean ep tube, centrifuge it at 4000 rpm for 5 minutes, transfer the supernatant to a clean centrifuge tube, and store it at 4°C. Add glucose solution to the bacterial liquid so that the glucose concentration in the bacterial liquid is 1 g / L. Add glucose solution to the bacterial liquid every 1 hour to reach the initial concentration. Take a sample every 4 hours. Take 40 μL of each of the above supernatants and mix them with 10 μL of Buffer, and place them in a metal bath at 100°C for 5 minutes. Prepare 15% concentrated gel and 5% separation gel; perform ice bath electrophoresis (100V, 3.5 hours) with 10 μL of sample and 5 μL of Marker. Stain with Coomassie Brilliant Blue overnight. Decolorize with decolorizing solution 3 times, 40 minutes each time. The results are as follows Figure 7 shown.

[0078] Example 5 HPLC Identification of Recombinant Saccharomyces boulardii Induction Supernatant

[0079] The fermentation broth of Example 4 was centrifuged at 4000r / min for 5min, and the supernatant was taken. After filtering the supernatant through a 0.22μm filter membrane, it was detected by reverse HPLC. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. Gradient elution was performed with 5% to 100% mobile phase B within 60min, gradient elution was performed with 100% to 5% mobile phase B within 60-65min, and equilibrium was performed with 5% mobile phase B for 65-80min. The flow rate was 1.0mL / min, the detection wavelength was 214nm, the column temperature was 30°C, and the sample volume was 10μL. The results are shown in Figure 8 As shown, after 4 h of induction, the peak of GLP-1 expression appeared at 4 min.

[0080] Example 6 Study on the activity of recombinant GLP-1 Saccharomyces boulardii in vitro

[0081] To explore the growth cycle of recombinant Saccharomyces boulardii. The empty vector bacteria of Saccharomyces boulardii and the recombinant Saccharomyces boulardii were activated and cultured, and the OD600 absorbance was detected every 4-8 hours, and the detection was continued until the growth plateau phase. To observe whether the introduction of the recombinant plasmid would affect the growth of Saccharomyces boulardii, a bacterial growth curve was established, and whether the recombinant bacteria had the possibility of in vivo gavage experiments. To explore the tolerance of recombinant Saccharomyces boulardii to artificial gastrointestinal fluid, and to simulate the tolerance of the recombinant bacteria to gastrointestinal fluid when it was ingested with food (10% skim milk). The results showed that the introduction of the recombinant plasmid did not affect the growth of Saccharomyces boulardii, and the recombinant bacteria had the possibility of oral administration in vivo.

[0082] The OD600 absorbance of the empty vector bacteria and recombinant Saccharomyces boulardii at different times was detected by a multifunctional microplate reader to indirectly reflect whether the introduction of the recombinant plasmid would affect the growth of Saccharomyces boulardii. Fig. 9 As shown in the figure, it can be seen from the statistical results of the absorbance value that the growth states of the empty vector bacteria of Saccharomyces boulardii and the recombinant Saccharomyces boulardii strains are generally consistent, indicating that the introduction of the recombinant plasmid does not affect the growth of Saccharomyces boulardii. Among them, 0~20h is the delay period, 20~102h is the logarithmic growth period, and after 102h, the bacteria enter the stable period and the growth rate slows down.

[0083] By co-culturing recombinant Saccharomyces boulardii with artificial gastrointestinal fluid, we simulated the survival of the strain under extreme conditions of gastrointestinal fluid as much as possible. According to relevant literature, Saccharomyces boulardii is a cold-resistant, acid-resistant, and temperature-resistant fungal microorganism with good tolerance in gastric acid and small intestinal fluid. In addition, when Saccharomyces boulardii is ingested with food, the buffering capacity of the food matrix may increase its survival rate in gastrointestinal fluid. Therefore, we designed normal saline (NS) and 10% skim milk to resuspend recombinant Saccharomyces boulardii to verify whether it can improve the survival rate. The experimental grouping and treatment methods are as follows:

[0084] Experimental group 1 - normal saline (NS) group: resuspend the recombinant Saccharomyces boulardii in normal saline until the end of the experiment;

[0085] Experimental group 2: NS + gastric juice + small intestinal juice: recombinant Saccharomyces boulardii was resuspended in normal saline and treated with gastric juice for 4 h. After 4 h, it was replaced with small intestinal juice and continued to be treated until the end of the experiment;

[0086] Experimental group 3: 10% skim milk + gastric juice + small intestinal juice: recombinant Saccharomyces boulardii was resuspended in 10% skim milk and treated with gastric juice for 4 hours. After 4 hours, it was replaced with small intestinal juice and continued to be treated until the end of the experiment;

[0087] Experimental group 4—NS+small intestinal fluid: recombinant Saccharomyces boulardii was resuspended in normal saline and treated with small intestinal fluid until the end of the experiment;

[0088] Experimental group 5: 10% skim milk + small intestinal fluid: The recombinant Saccharomyces boulardii was resuspended in 10% skim milk and treated with small intestinal fluid until the end of the experiment.

[0089] The experimental results are as follows Fig.10 , Fig.11 and Fig.12 shown.

[0090] Recombinant bacteria group resuspended in normal saline: the survival rates after 2h and 4h of treatment with normal saline were 120% and 100%, respectively; the survival rates after 2h and 4h of treatment with artificial gastric juice were 40% and 27.6%, respectively; the survival rate of bacteria treated with artificial gastric juice was lower than that of normal saline, indicating that some of the recombinant Saccharomyces boulardii were killed by gastric juice; after 4h of treatment with artificial gastric juice, the artificial small intestinal juice was used for further treatment, and the survival rate of the recombinant Saccharomyces boulardii could be maintained between 30% and 40%; when cultured in the small intestinal juice for 192h, some recombinant bacteria still survived; the survival rates after 4h, 8h and 24h of treatment with artificial small intestinal juice alone were 80%, 33.3% and 43.3%, respectively. Compared with artificial gastric juice, the survival rate of the recombinant Saccharomyces boulardii in artificial small intestinal juice was higher.

[0091] 10% skim milk re-suspended group: the survival rates after 2h and 4h of treatment with artificial gastric juice were 60% and 33.3%, respectively, which were higher than those of the recombinant bacteria group resuspended in normal saline; the survival rates after 4h, 8h and 24h of treatment with artificial small intestinal fluid alone were 73.3%, 66.7% and 56.7%, respectively, including the survival rate within the following 192h, which was higher than that of the recombinant bacteria group resuspended in normal saline, indicating that 10% skim milk has a certain protective effect on Saccharomyces boulardii and can effectively increase its survival rate in gastric juice and small intestinal juice, indicating that the matrix buffering capacity of food has a certain protective effect on recombinant Saccharomyces boulardii.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant vector expressing GLP-1, characterized in that: It contains a HXT4 gene promoter sequence and a GLP-1 gene whose expression is regulated by the HXT4 gene promoter. The nucleotide sequence of the HXT4 gene promoter is shown in SEQ ID NO.

1.

2. The recombinant vector according to claim 1, characterized in that The nucleotide sequence of the GLP-1 gene is shown in SEQ ID NO.

2.

3. The recombinant vector according to claim 1, characterized in that The recombinant vector also contains at least one of a resistance gene, a marker gene and a nucleotide sequence encoding a signal peptide.

4. The recombinant vector according to claim 3, characterized in that The 5' end of the GLP-1 gene contains a nucleotide sequence encoding a signal peptide, and the signal peptide is a signal peptide of IL-2.

5. The recombinant vector according to any one of claims 1 to 4, characterized in that The recombinant vector is a recombinant plasmid, and the nucleotide sequence is shown in SEQ ID NO.

3.

6. Recombinant Saccharomyces boulardii, characterized in that The recombinant Saccharomyces boulardii contains the recombinant vector expressing GLP-1 according to any one of claims 1 to 5.

7. Use of the recombinant vector according to any one of claims 1 to 5 or the recombinant Saccharomyces boulardii according to claim 6 in the preparation of a medicament for preventing and / or treating diabetes, or a medicament for lowering blood sugar.

8. A pharmaceutical composition, characterized in that The method comprises the recombinant vector according to any one of claims 1 to 5, or the recombinant Saccharomyces boulardii according to claim 6.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition contains the recombinant Saccharomyces boulardii according to claim 6, and the pharmaceutical composition is an oral preparation.

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