SEM1 protein, recombinant plasmid expressing SEM1 protein, recombinant probiotics expressing SEM1 protein and applications thereof

Abstract

The present invention belongs to the field of biopharmaceuticals and specifically discloses a SEM1 protein, a recombinant plasmid expressing the SEM1 protein, a recombinant probiotic expressing the SEM1 protein, and applications thereof. The present invention provides the use of the SEM1 protein in the preparation of a drug for preventing and / or treating inflammatory diseases, such as diabetes and gastrointestinal diseases. Furthermore, the present invention prepares the SEM1 protein into a probiotic, changing the conventional administration method (intramuscular or intravenous injection) of protein drugs to an oral administration method for the treatment of gastrointestinal diseases and diabetes, thereby improving patient compliance and reducing production costs.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to the use of a SEM1 protein in preparing a drug for preventing and / or treating inflammatory diseases, wherein the inflammatory diseases are one or more of diabetes and gastrointestinal diseases. Technical Background

[0002] SEM1 (Split Hand / Foot Malformation (Ectrodactyly) Type 1) is a protein-coding gene that encodes a subunit of the 26S proteasome involved in the ATP-dependent degradation of ubiquitinated proteins. It participates in the cellular senescence pathway and the RET signaling pathway, which is associated with cell survival, migration, and proliferation. The SEM1 protein removes misfolded or damaged proteins that impair cellular function, as well as proteins that have fulfilled their physiological functions, playing a key role in maintaining protein homeostasis. Therefore, the SEM1 protein is involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair. The SEM1 protein is a multifunctional eukaryotic protein with a highly conserved sequence. In addition to being a subunit of the 26S proteasome and acting as a receptor for the ubiquitination system, SEM1 participates in protein clearance and maintains protein homeostasis. It also associates with various protein complexes, including the BRCA2–RPA complex involved in homologous recombination; the Csn12–Thp3 complex involved in RNA splicing; and the TREX-2 complex involved in mRNA nuclear export and transcription elongation. SEM1 participates in many important biological and cellular processes by binding to these complexes, including genome stability, homologous recombination and DNA repair, cell proliferation and tumor transformation, protein degradation, histone modification, and mRNA splicing, metabolism, and export. However, there are currently no reports that the protein encoded by SEM1 can treat inflammation-related digestive tract diseases and inflammation-related diabetes.

[0003] Diabetes mellitus, a multifactorial, inflammatory metabolic disease, is one of the major health threats of the 21st century. It is characterized by chronic hyperglycemia caused by deficient insulin secretion and / or peripheral insulin resistance. While insulin resistance and insufficient insulin secretion have long been considered the primary causes of diabetes, their specific pathogenesis remains largely unknown. In recent years, a growing number of clinical epidemiological studies have linked the development of type 2 diabetes and its complications to inflammatory factors. Some studies have even shown that patients with type 2 diabetes maintain a chronic hypoinflammatory state, and that anti-inflammatory medications can alleviate diabetic symptoms. Studies have reported that gut microbial homeostasis plays a crucial role in the development and progression of diabetes. When beneficial bacteria decrease and harmful bacteria increase in the gut, levels of metabolic endotoxins increase, leading to chronic inflammation. This in turn triggers the release of inflammatory factors, which in turn induces oxidative stress, ultimately leading to pancreatic islet cell damage and the development of diabetes. Probiotics have the ability to secrete antibacterial substances, compete with other pathogens, strengthen the intestinal barrier and regulate the immune system, mainly by regulating intestinal pH, balancing intestinal flora, stimulating the immune system, and reducing serum cholesterol and tumor risk; at the same time, studies have shown that probiotics (Lactobacillus bulgaricus, Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Lactobacillus acidophilus and Lactobacillus casei) can improve metabolism and have preventive, alleviating and therapeutic effects on diabetes.

[0004] Inflammatory-related gastrointestinal diseases cover diseases of the esophagus, stomach, small intestine, colon and rectum. Common main symptoms include rhythmic and cyclical upper abdominal pain, diarrhea, hunger abdominal pain, acid reflux, fever, black stools and bloody stools, gastrointestinal bleeding and intestinal obstruction. Gastrointestinal diseases are one of the most common diseases in humans, the most common of which include dysphagia, gastric ulcer, peptic ulcer, gastroparesis, delayed gastric emptying, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Peptic ulcer mainly includes gastric ulcer, duodenal ulcer and combined ulcer. It is a common and frequently occurring disease, also known as gastric and duodenal ulcer. Inflammatory bowel disease can be caused by organisms such as bacteria, fungi, viruses, parasites, protozoa, and can also be caused by allergic reactions and physical and chemical factors. Inflammatory bowel disease is listed by the World Health Organization as one of the modern intractable diseases, and its incidence rate is on the rise worldwide. The pathogenesis of colitis has not yet been fully elucidated. Commonly used drugs for clinical treatment include aminosalicylic acid preparations, glucocorticoids and immunosuppressants. Short-term use can control the symptoms of colitis, but the cure rate is extremely low. Long-term use can induce a variety of adverse reactions, and discontinuation of the drug can cause recurrence. Severe cases can cause cancer. Therefore, the development of a new drug for the treatment of gastrointestinal diseases is a technical problem that urgently needs to be solved. The drugs currently on the market for the treatment of inflammatory-related diseases mainly relieve symptoms and cannot cure them. They are symptomatic treatments. Long-term use can induce a variety of adverse reactions, and severe cases can cause cancer. Discontinuation of the drug is prone to recurrence. Therefore, there is still a need to develop new drugs for the treatment of inflammatory-related diseases to better serve patients.

[0005] Peptide and protein drugs are widely used to treat various diseases due to their high specificity and safety. However, due to the structure and physiological functions of the gastrointestinal tract, protein drugs have low bioavailability and short half-life after oral administration. Therefore, to improve the bioavailability of protein drugs, protein drugs are often administered clinically by injection, which reduces patient compliance. However, injection administration is not convenient for patients, requiring them to go to the hospital for injection, which increases the cost of drug storage and use.

[0006] The present invention discovered for the first time that SEM1 protein can treat inflammation-related gastrointestinal diseases and inflammation-related diabetes; secondly, the present invention constructed a recombinant probiotic expressing SEM1 protein, which can solve the problem of easy degradation of orally administered protein drugs, play a role in treating inflammation-related gastrointestinal diseases and inflammation-related metabolic diseases, and achieved good therapeutic effects. Summary of the Invention

[0007] In response to the above technical problems, the primary purpose of the present invention is to provide a use of SEM1 protein in the preparation of a drug for preventing and / or treating inflammatory diseases, wherein the amino acid sequence of the SEM1 protein is shown in SEQ ID No. 1. The gene sequence encoding the SEM1 protein is shown in SEQ ID No. 2.

[0008] Preferably, the inflammatory disease is one or more of diabetes and gastrointestinal diseases.

[0009] Preferably, the gastrointestinal diseases are inflammatory bowel disease and peptic ulcer, the inflammatory bowel disease includes one or more of ulcerative colitis and Crohn's disease; the peptic ulcer includes one or more of gastric ulcer, duodenal ulcer, postbulbar ulcer, pyloric ulcer, complex ulcer and kissing ulcer.

[0010] A second object of the present invention is to provide a recombinant plasmid expressing SEM1 protein, wherein the recombinant plasmid is obtained by inserting a gene encoding the SEM1 protein into an expression vector, wherein the expression vector includes pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45, and the amino acid sequence of the SEM1 protein is shown in SEQ ID No. 1.

[0011] Preferably, the expression vector includes pET-28a, the recombinant plasmid is named pET-28a-SEM1, and the gene sequence of the pET-28a-SEM1 is shown in SEQ ID No. 3.

[0012] The third object of the present invention is to provide a recombinant probiotic expressing SEM1 protein, wherein the recombinant probiotic is obtained by transforming the prepared recombinant plasmid into a probiotic, or integrating the gene encoding the SEM1 protein into the probiotic; the probiotic includes Escherichia coli Nissle 1917, probiotic Bacillus, Lactococcus, Butyric acid Bacillus, Lactobacillus, Bifidobacterium, Actinomycetes, and Pichia pastoris.

[0013] Preferably, the probiotic is Escherichia coli Nissle 1917.

[0014] The fourth object of the present invention is to provide the use of the recombinant probiotics in the preparation of a drug for preventing and / or treating inflammatory diseases, wherein the inflammatory diseases are one or more of diabetes and gastrointestinal diseases.

[0015] The fifth object of the present invention is to provide the use of the recombinant probiotics in the preparation of a drug for preventing and / or treating diabetic complications, characterized in that the diabetic complications are one or more of gestational diabetes, diabetic nephropathy, retinopathy, cataracts, diabetes-related uveitis, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, diabetic neuropathy and periodontitis.

[0016] The sixth object of the present invention is to provide the use of the recombinant probiotics in the preparation of health products with auxiliary blood sugar lowering effects.

[0017] Preferably, the medicine or health product is an oral preparation, specifically including any one of oral liquid, tablet, capsule, granule, powder, suspension, emulsion, pill, and powder.

[0018] The seventh object of the present invention is to provide a probiotic tablet, which is prepared by adding the recombinant probiotics to a pharmaceutically acceptable excipient.

[0019] Preferably, the content of recombinant probiotics in the probiotic tablet is 2×10 9 CFU.

[0020] The beneficial effects of the present invention are as follows: ① The present invention provides a new use of SEM1 protein for treating diabetes and gastrointestinal diseases; ② The present invention constructs a human SEM1 gene recombinant plasmid pET-28a-SEM1 and transfers it into probiotics to obtain recombinant probiotics, and the SEM1 gene is efficiently expressed in the recombinant probiotics; ③ The recombinant probiotics efficiently expressing SEM1 protein of the present invention can significantly reduce the weight loss percentage of colitis mice, reduce disease activity improvement, alleviate colon tissue pathological damage, and improve colon shortening, indicating that the recombinant probiotics efficiently expressing SEM1 protein of the present invention can be used as a drug for treating or alleviating ulcerative colitis, providing a new option for the clinical treatment of ulcerative colitis. ④ The recombinant probiotics efficiently expressing SEM1 protein of the present invention can significantly lower blood sugar in diabetic mice, indicating that the recombinant probiotics efficiently expressing SEM1 protein of the present invention can be used as a drug for treating or alleviating diabetes, providing a new option for the clinical treatment of diabetes. ⑤ The present invention changes the conventional administration mode of protein drugs (intramuscular injection or intravenous injection) and applies protein drugs to the treatment of gastrointestinal diseases and diabetes in the form of oral administration, thereby improving patient compliance and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the results of PCR reaction of EcN transformed with pET-28a-SEM1 plasmid and pET-28a-EGFP;

[0022] Figure 2 Planar diagram of gastric mucosal damage caused by recombinant probiotics in mice with gastric ulcer;

[0023] Figure 3 Effects of recombinant probiotics on ulcer index, ulcer area and ulcer inhibition rate in mice with gastric ulcer, ### p<0.001vs.Control group; * p<0.05, ** p < 0.01 and *** p<0.001 vs. Ethanol group.

[0024] Figure 4 Effects of recombinant probiotics on DAI score of ulcerative colitis, including ### p<0.001vs.control group; * p<0.05vs.SASP group.

[0025] Figure 5 Schematic diagram of the effect of recombinant probiotics on colon damage in mice with ulcerative colitis;

[0026] Figure 6 Effects of recombinant probiotics on colon length in ulcerative colitis ### p<0.001vs.control group; ** p < 0.01 and *** p<0.001vs.DSS group.

[0027] Figure 7 Effects of recombinant probiotics on fasting blood glucose in diabetic mice, including * p < 0.05 and *** p<0.001vs.Model+EcN-EGFP group.

[0028] Figure 8 Effects of recombinant probiotics on fasting body weight in diabetic mice.

[0029] Figure 9 Line graph of the effect of recombinant probiotics on glucose tolerance test in diabetic mice.

[0030] Figure 10 Histogram of the area under the blood glucose-time curve of the glucose tolerance test of diabetic mice treated with recombinant probiotics.

[0031] Figure 11 Detection results of the effects of recombinant probiotics on the 24-hour urine protein and urinary nitrogen levels in diabetic mice. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clear, the following examples are further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The reagents and materials used in the following examples are all commercially available unless otherwise specified.

[0033] In the following examples, Escherichia coli DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd. and is hereinafter abbreviated as Escherichia coli DH5α; Escherichia coli Nissle 1917 was purchased from Germany (trademark name: Mutaflor) and is hereinafter abbreviated as Escherichia coli EcN.

[0034] The C57BL / 6N mouse described in the present invention is one of the most widely used strains of mice and is also the mother of transgenic or gene knockout mice most commonly used in genetic engineering.

[0035] The KM mice described in the present invention refer to Kunming mice.

[0036] Cimetidine, also known as cimetidine, in the following embodiments of the present invention is a histamine H2 receptor antagonist, which is mainly used to inhibit gastric acid secretion. It can significantly inhibit basal and nocturnal gastric acid secretion, and can also inhibit gastric acid secretion caused by stimulation such as histamine, gastrin, insulin and food, and reduce its acidity. It has a preventive and protective effect on corrosive gastritis caused by chemical stimulation, and also has a significant therapeutic effect on stress gastric ulcers and upper gastrointestinal bleeding. Example 2 of the present invention uses cimetidine as a positive drug.

[0037] The following examples of the present invention use dextran sulfate sodium for the establishment of a colitis model in mice.

[0038] In the following examples of the present invention, the indications of sulfasalazine enteric-coated tablets are (1) ulcerative colitis for the treatment of mild to moderate ulcerative colitis; it can be used as an adjuvant therapy in severe ulcerative colitis. It can also be used for maintenance treatment of ulcerative colitis in the remission phase; (2) Crohn's disease for the treatment of active Crohn's disease, especially those involving the colon; (3) rheumatoid arthritis for rheumatoid arthritis and juvenile rheumatoid arthritis (polyarticular type) that are not significantly responsive to salicylates or other nonsteroidal anti-inflammatory drugs. In Example 3 of the present invention, sulfasalazine enteric-coated tablets are used as the positive drug.

[0039] The metformin hydrochloride in the following embodiments of the present invention, whose chemical name is 1.1-dimethylbiguanide hydrochloride, is used for patients with type 2 diabetes who are unsatisfied with simple dietary control, especially those who are obese and have hyperinsulinemia. This drug not only has a blood sugar-lowering effect, but also may reduce weight and have hyperinsulinemia effects. It can be effective for patients with poor efficacy of certain sulfonylureas. For example, when used in combination with sulfonylureas, intestinal glucosidase inhibitors, or thiazolidinediones hypoglycemic drugs, the effect is better than using them alone. It can also be used for patients treated with insulin to reduce insulin dosage. However, metformin hydrochloride cannot cure type 2 diabetes, and the dosage is large, the medication cycle is long, the side effects are large, and it has no therapeutic effect on diabetic complications. In Example 5 of the present invention, metformin hydrochloride is used as a positive drug.

[0040] Streptozocin, also known as streptozocin (STZ), used in the following examples of the present invention, has a chemical name of 2-deoxy-2-[[(methylnitrosamino)carbonyl]-amino]-D-glucopyranose, a molecular formula of C8H15N3O7, a molecular weight of 265.22100, and a pale yellow crystalline powder. It is readily soluble in water, but its aqueous solution is extremely unstable at room temperature and can decompose into gas and evaporate after half an hour, so it must be prepared immediately before use. It is soluble in relatively low-alcohol and ketones but insoluble in polar organic solvents. Streptozocin can damage animal pancreatic beta cells, reducing insulin secretion, and can therefore be used to induce diabetic animal models. Generally, a single high-dose (150 mg / kg / day) injection can induce type 1 diabetes, while a low-dose (40-60 mg / kg / day) injection over 3-5 days combined with a high-fat diet can induce type 2 diabetes.

[0041] Example 1 Preparation of recombinant plasmids and recombinant probiotics

[0042] 1. Experimental Reagents

[0043] The PCR primers used were purchased from Sangon Biotech (Shanghai) Co., Ltd., and the corresponding restriction sites were introduced.

[0044] EGFP (enhanced green fluorescent protein) is a mutant of GFP. The fluorescence intensity it emits is more than 6 times greater than that of GFP. It is more suitable than GFP as a reporter gene to study gene expression, regulation, cell differentiation, and protein localization and transport in organisms.

[0045] Bacterial plasmid DNA extraction kit was purchased from AXYGEN.

[0046] PrimeSTAR HS (Premix, 2X) and T4 DNA ligase were purchased from Takara.

[0047] DNA endonucleases BamHI and EcoRII were purchased from Beijing NEB Company.

[0048] The formula of LB (Luria-Bertani) liquid culture medium is: 1% tryptone, 0.5% yeast extract, and 0.5% NaCl.

[0049] The LB solid culture medium formula is: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 2% agar.

[0050] The recombinant plasmid pET-28a-SEM1 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the recombinant plasmid pET-28a-EGFP was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0051] 2. Primer Information Synthesis

[0052] T7 and T7ter primers were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. The primers were dissolved in sterile deionized water to a concentration of 10 μmol / L.

[0053] 3. Construction of recombinant strains

[0054] (1) Extraction of recombinant plasmid DNA: Escherichia coli DH5α containing the pET-28a-SEM1 plasmid and pET-28a-EGFP plasmid was transferred to LB liquid medium containing 30 μg / mL kanamycin for amplification and culture at 200 rpm and 37°C. The plasmid was extracted using the AXYGEN plasmid miniprep kit.

[0055] (2) Preparation of chemically competent E. coli EcN cells: Take out the preserved E. coli EcN from the -80℃ refrigerator, pick up a small amount of preservation solution with a sterilized pipette tip, streak it on LB solid culture medium, and culture it in a 37℃ incubator overnight: Pick up a moist, smooth single colony from the LB solid culture medium, put it into LB culture medium, and culture it at 37℃ for 12h; when the OD600 value of the bacterial solution is 0.3-0.5, transfer the bacterial solution to a sterilized centrifuge tube pre-cooled on ice, ice bath for 30min, and centrifuge at 1520g for 10min at 4℃; discard the supernatant and use 500μL pre-cooled 0.1mol / L Gently resuspend the cells in sterile-filtered CaCl2 solution and centrifuge at 1520g for 10 minutes at 4°C. Repeat the previous step once. Discard the supernatant and add 500 μL of pre-cooled 0.1 mol / L CaCl2 solution (sterilized-filtered). Carefully resuspend the cells to create a competent cell suspension. Use this suspension directly in transformation experiments. Add an equal volume of sterile 20% glycerol to any remaining competent cells, mix thoroughly, and aliquot into 1.5 mL centrifuge tubes (100 μL per tube). Store in a -80°C freezer.

[0056] (3) The recombinant plasmids pET-28a-SEM1 and pET-28a-EGFP were respectively transformed into Escherichia coli EcN: Take out the Escherichia coli EcN chemically competent cells from the -80℃ refrigerator and place them on an ice box for 10-20 minutes to thaw. Take out 50μL of Escherichia coli EcN competent cells and add 5μL of plasmid. Mix it by gently stirring several times with your fingers. Place the mixture on ice for 25 minutes. After heat shock at 42℃ for 45 seconds, quickly return it to ice and let it stand for 2 minutes. Add 0.5mL of room temperature LB liquid medium (without antibiotics) and culture it at 37℃ with shaking at 200rpm for 1 hour. Take 200μL of the above bacterial solution and spread it on an agar plate containing 30μg / mL kanamycin. After the bacterial solution is completely absorbed by the culture medium, invert the culture dish and culture it at 37℃ for 12 hours.

[0057] (4) Verification of successful colonies after heat shock transformation: Single clones were picked and amplified in liquid culture medium containing 30 μg / mL kanamycin.

[0058] (5) PCR verification: Using the monoclonal bacterial solution as a template, PCR amplification was performed using the pET-28a universal primers T7 and T7ter. The PCR amplification system was:

[0059]

[0060]

[0061] The PCR reaction conditions were: initial denaturation at 98.0°C for 5 minutes, followed by 30 cycles of denaturation, annealing, and extension (denaturation at 98.0°C for 10 seconds, annealing at 58.0°C for 5 seconds, extension I at 72.0°C for 30 seconds, and extension II at 72.0°C for 10 minutes). The PCR products were subjected to 1% agarose gel electrophoresis at 50V for 45 minutes and then stained with 3X Gel Green.

[0062] PCR verification results Figure 1 As shown, the PCR reaction of bacteria successfully transformed with pET-28a-SEM1 should contain the SEM1 target band (573 bp). The arrow in the figure points to the target band, indicating that the pET-28a-SEM1 plasmid was successfully transformed into E. coli EcN. The PCR reaction of bacteria successfully transformed with the pET-28a-EGFP plasmid should contain the EGFP target band (1080 bp). The presence of the target band in the figure indicates that the pET-28a-EGFP plasmid was successfully transformed into E. coli EcN.

[0063] Example 2 Therapeutic Effect of Recombinant Probiotics Containing Recombinant Plasmids on Mice with Gastric Ulcer

[0064] 1. Experimental Animals, Materials, and Sources

[0065] Experimental animals: 8-week-old SPF male Kunming (KM) mice weighing 18-22 g were used. No drugs were used before the experiment. The license number is SCXK(Gan)-2020-0002. The animals were adaptively raised for one week and given a diet and free access to distilled water. Then, they were divided into groups for the experiment.

[0066] Drugs and reagents: Probiotics containing the recombinant plasmid pET-28a-SEM1 (EcN-SEM1) and the probiotics containing the recombinant plasmid pET-28a-EGFP (EcN-EGFP) were obtained according to the method described in Example 1, wherein the recombinant plasmid pET-28a-EGFP was purchased from Miaoling Plasmid Platform; anhydrous ethanol was purchased from Tianjin Damao Chemical Reagent Company; cimetidine tablets (CIM) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; β-lactose (containing 70% β-lactose and 30% α-lactose) was purchased from MacLean Biotechnology Co., Ltd.

[0067] 2. Preparation of KM Mouse Alcoholic Gastric Ulcer Model

[0068] 48 KM male mice (20-25 g) were housed in the experimental animal room of Lanzhou University. After one week of adaptive feeding, the mice were randomly divided into 6 groups, with 8 mice in each group. The grouping and dosage are as follows:

[0069] Control group: 0.25 mL of 11.1 mg / mL (m / v) β-lactose solution was administered orally;

[0070] Ethanol group: 0.25 mL of anhydrous ethanol was administered by gavage;

[0071] CIM group: cimetidine, 80 mg / kg / day, administered by gavage;

[0072] EcN group: 2×10 9 cfu Escherichia coli Nissle 1917 original bacteria;

[0073] EcN-EGFP group: 2×10 9 cfu containing the recombinant plasmid pET-28a-EGFP (EcN-EGFP);

[0074] EcN-SEM1 group: 2×10 9 cfu recombinant bacteria (EcN-SEM1) containing the recombinant plasmid pET-28a-SEM1.

[0075] The mice in the above groups were pre-administered for 5 days (the number of bacteria was 2×10 9 CFU, all prepared with 11.1 mg / mL β-lactose solution), the drug was given again 2 hours before modeling, and the model was established by gavage with anhydrous ethanol 2 hours later. The experiment was ended 2 hours after modeling, and serum and gastric tissue were separated to measure various indicators.

[0076] 3. Ulcer area, ulcer index and inhibition rate

[0077] The stomach was taken out and cut open along the greater curvature of the stomach. The contents were rinsed clean and the gastric mucosal ulcer was observed. The horizontal and vertical diameters of the ulcer were measured with a ruler. The product of the two was the ulcer area (mm 2 ), as shown in Formula 1; the ulcer area of ​​the entire gastric tissue was then calculated, and the ulcer inhibition rate (%) was calculated, as shown in Formula 2. The mean of the sum of the ulcer points in each group of mice was used as the ulcer index (healing was scored as 0, superficial mucosal erosion was scored as 1, deep ulcer or transmural necrosis was scored as 2, and perforation or penetrating ulcer was scored as 3).

[0078] Ulcer area (mm2) = maximum length of ulcer * maximum width perpendicular to the maximum length (1)

[0079]

[0080] 4. Data Processing

[0081] The experimental data were statistically analyzed using SPSS 23.0 software. Data are expressed as (x ± s). One-way ANOVA and LSD-t method were used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0082] 5. Effects of recombinant probiotics on gastric tissue morphology, ulcer index, ulcer area and ulcer inhibition rate in mice with gastric ulcer

[0083] After the mice in each group were killed, their gastric tissues were quickly taken to observe their morphological changes and ulcer damage, and their ulcer areas and ulcer indices were statistically analyzed. The experimental results are shown in Table 2. Figure 2 and Figure 3 As shown. Figure 2The gastric tissue of mice in the control group showed normal macroscopic morphology and no obvious hemorrhagic lesions, while the gastric tissue of mice in the ethanol group showed obvious hemorrhagic lesions and the most severe ulcers, indicating that the ethanol gastric ulcer model was successfully established. Compared with the ethanol group, the EcN group significantly improved the degree of gastric mucosal damage and reduced the ulcer area (p < 0.01), with an ulcer inhibition rate of 51.91%, which was stronger than the ulcer inhibition rate of the clinical drug cimetidine (46.67%), indicating that E. coli EcN itself has a protective effect against gastric ulcers. Furthermore, compared with the EcN group, the ulcer inhibition rate of the EcN-SEM1 group was significantly higher than that of the EcN group, while the EcN-EGFP group showed no significant ulcer protection, indicating that the transfer of the ineffective EGFP protein into E. coli EcN did not enhance the preventive and therapeutic effects of E. coli EcN on gastric ulcers. Compared with the positive control group, the EcN-SEM1 group had a smaller ulcer area, a lower ulcer index, and a higher inhibition rate, all of which were significant differences. In summary, the recombinant probiotics capable of expressing SEM1 protein described in the present invention have a better effect in treating gastric ulcers.

[0084] Example 3 Therapeutic Effect of Recombinant Probiotics Containing Recombinant Plasmids on Mice with Ulcerative Colitis

[0085] 1. Animal feeding test

[0086] SPF-grade healthy male KM mice, weighing 18–22 g, had not received any drugs before the experiment and were provided by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with license number SCXK(Gan)-2020-0002. They were acclimated for one week and given diet and free access to water before being grouped for the experiment.

[0087] Drugs and reagents: Probiotics containing the recombinant plasmid pET-28a-SEM1 and the probiotics containing the recombinant plasmid pET-28a-EGFP were obtained according to the method described in Example 1 and pET-28a-EGFP was purchased from Miaoling Plasmid Platform; β-lactose (containing 70% β-lactose and 30% α-lactose) was purchased from MacLean Biotechnology Co., Ltd.; Salazosulfapyridine enteric-coated tablets (SASP) were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.; Dextran Sulfate sodium (DSS), MW: 40000, was purchased from Aladdin Biotechnology Co., Ltd.

[0088] 2. Experimental Methods

[0089] 2.1 Experimental groups and oral administration doses:

[0090] Eight-week-old male KM mice were randomly divided into six groups according to body weight, with 8 mice in each group. The grouping and dosage were as follows:

[0091] Normal control group (Control, oral administration of an equal volume of 11 mg / mL β-lactose solution);

[0092] DSS model control group (DSS, oral administration of an equal volume of 11 mg / mL β-lactose solution);

[0093] positive drug group (SASP, oral administration of sulfasalazine, 80 mg / kg / day);

[0094] EcN group: 2×10 9 cfu Escherichia coli Nissle 1917 original bacteria;

[0095] EcN-EGFP group: 2×10 9 cfu recombinant bacteria containing the recombinant plasmid pET-28a-EGFP;

[0096] EcN-SEM1 group: 2×10 9 cfu of recombinant bacteria containing the recombinant plasmid pET-28a-SEM1.

[0097] The above-mentioned drug-dosing groups were all prepared with 11 mg / mL β-lactose solution.

[0098] 2.2 Preparation of ulcerative colitis model

[0099] Prepare 4% DSS distilled water solution. The mice in the DSS model control group freely drink the DSS solution to establish the model. The mice in the DSS+bacteria treatment group freely drink the DSS solution to establish the model. At the same time, 0.25 mL of bacterial solution is given. The bacterial density is 2×10 9 CFU; mice in the DSS+SASP group drank DSS aqueous solution freely to establish the model and were given 80 mg / kg / day of SASP at the same time; mice in the blank control group drank distilled water freely, and all mice were fed with regular feed. When bloody diarrhea or bloody stool occurred, the DSS-containing aqueous solution was replaced with regular drinking water, and drug treatment was continued. Two hours after the last dose on the eighth day, serum and colon tissue were separated for later use.

[0100] 3. Ulcerative colitis detection indicators

[0101] 3.1 Disease Activity Index (DAI) score

[0102] The weight loss rate, stool characteristics and fecal occult blood in each group of mice were scored according to the literature methods.

[0103] DAI = (weight loss + stool characteristics + fecal occult blood) / 3

[0104] 3.2 Colonic injury evaluation

[0105] After each group of mice were killed, the colon was separated. The ileum was cut at the junction of the ileum and colon, and then the colon was cut near the anus. The fascia outside the colon was separated to fully stretch the colon. The length of the mouse colon from the ileum to the anus was measured with a ruler and photographed for record.

[0106] 4. Data Processing

[0107] The experimental data were statistically analyzed using SPSS 23.0 software. Data are expressed as (x ± s). One-way ANOVA and the LSD-t method were used for pairwise comparisons between groups. p < 0.05 was considered statistically significant.

[0108] 5. Results Analysis

[0109] 5.1 Effect of recombinant probiotics on the disease activity index of ulcerative colitis mice

[0110] During the experiment, the weight, stool viscosity, blood in stool and occult blood of the above mice were monitored, and the data were statistically analyzed by one-way analysis of variance using SPSS23.0 software.

[0111] like Figure 4 As shown in Figure 2, compared with the Control group, the DAI score in the DSS model group showed a significant upward trend ( ### p<0.001), indicating that the DSS model was successfully established; compared with the model control group, the SASP group, EcN group, EcN-EGFP group and EcN-SEM1 group could significantly reduce the DAI score ( * p<0.05), which preliminarily indicated that E. coli EcN and recombinant probiotics expressing SEM1 had therapeutic effects on ulcerative colitis.

[0112] 5.2 Effects of recombinant probiotics on colon morphology and length in colitis mice

[0113] After the mice in each group were killed, their colon tissues were quickly removed to observe their morphological changes and measure their lengths. The data of each group were statistically analyzed. The experimental results are shown in the figure below. Figure 5 and Figure 6 Compared with the Control group, the colon length of the DSS model group was significantly shortened ( ###p<0.001), indicating that the DSS model was successfully prepared; compared with the DSS model control group, the EcN group and the negative control EcN-EGFP group were able to improve colon edema and shortening, with an upward trend but no significant difference, which is consistent with literature reports; at the same time, it was found that the effect of the EcN-EGFP group was no different from that of the original bacteria, indicating that the transgenic EGFP gene has no therapeutic effect on ulcerative colitis. Compared with the EcN group, the EcN-SEM1 group was able to significantly improve colon edema and shortening, and there was a statistical difference ( * p<0.001), indicating that recombinant probiotics expressing SEM1 have a therapeutic effect on ulcerative colitis and SEM1 is a new target for the treatment of ulcerative colitis.

[0114] Example 4 Therapeutic effect of recombinant probiotics on diabetic mice

[0115] 1. Animal feeding test

[0116] SPF-grade healthy male C57BL / 6N mice, weighing 18-22 g, had not received any drugs before the experiment and were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences with license number SCXK(Gan)-2020-0002. They were acclimated for one week and given diet and free access to water before being grouped for the experiment.

[0117] Drugs and reagents: Probiotics containing the recombinant plasmid pET-28a-SEM1 and the probiotics containing the recombinant plasmid pET-28a-EGFP were obtained according to the method described in Example 1; pET-28a-EGFP was purchased from Miaoling Plasmid Platform; β-lactose (containing 70% β-lactose and 30% α-lactose) was purchased from MacLean Biotechnology Co., Ltd.; Metformin hydrochloride (Met) was prepared with 0.9% saline, and streptozocin (STZ) was prepared with 0.1 mol / L sodium citrate buffer at pH 4.

[0118] Preparation of citric acid buffer solution: Add 2.1g of citric acid to 100mL of distilled water to prepare Solution A. Add 2.94g of sodium citrate to 100mL of distilled water to prepare Solution B. Mix Solution A and Solution B in a ratio of 1:1.32 or 1:1, adjust the pH to 4, and filter the mixed solution through a 2.22μm filter for sterilization.

[0119] 2. Establishment of C57BL / 6N diabetic mouse model

[0120] Thirty-two C57BL / 6N mice were randomly divided into three groups, namely, high-fat diet + STZ group (32 mice).

[0121] Mice in the normal diet group: After being fed with normal diet for three weeks, mice were injected with an equal volume of citric acid buffer solution for 5 consecutive days, and then continued to be fed with normal diet until the model was established;

[0122] Mice in the high-fat diet group: After being fed with a high-fat diet for three weeks, mice were injected with an equal volume of citric acid buffer solution for 5 consecutive days, and then continued to be fed with a high-fat diet until the model was established;

[0123] Mice in the high-fat diet + STZ group: After three weeks of high-fat diet feeding, STZ solution was injected intraperitoneally at a dose of 45 mg / kg (fasting for 12 hours before STZ injection, but not water, and injections were performed at the same time between 9:00 am and 10:00 am. After STZ injection, fasting for another 2 hours before feeding high-fat diet was allowed). The injections were given once a day for five consecutive days, and the newly prepared STZ solution was injected within 30 minutes to prevent STZ from becoming ineffective. After the STZ solution injection (5 days), the mice were monitored weekly for changes in body weight and blood sugar. They were fed a high-fat diet for two weeks and then a normal diet for one week. If blood sugar remained basically unchanged (blood sugar ≥ 15.0 mmol / L), the type 2 diabetes model was considered stable and the model was successfully established. Mice were given ample food and water during the experiment.

[0124] 3. Grouping

[0125] A successful type 2 diabetes model was established when the fasting blood glucose level (blood glucose was measured between 7:30 pm and 9:00 pm, after fasting for 12 hours with no water or food) in the high-fat diet + STZ group was above 15 mmol / L. After the experiment officially began, all mice were fed a standard diet and divided into four groups of eight mice each, as follows:

[0126] a. Model group (Model+saline): gavage with 0.2 mL of normal saline daily, serving as the negative control group;

[0127] b. Model+EcN-EGFP group: 2×10 9 cfu recombinant bacteria containing the recombinant plasmid pET-28a-EGFP;

[0128] c. Model+EcN-SEM1 group: 2×10 9 cfu recombinant bacteria containing the recombinant plasmid pET-28a-SEM1;

[0129] d. Normal diet group (SD): gavage with an equal volume of normal saline daily;

[0130] All groups of mice were treated with the drug for a total of 12 weeks. During the treatment period, the growth of the mice (including fur color, motor ability, etc.) was monitored daily, and blood sugar and body weight were measured once a week.

[0131] 4. Detection indicators

[0132] 4.1 Fasting Blood Glucose (FBG) Monitoring

[0133] Before and after administration, fasting blood glucose was monitored at the same time period (7:30 pm - 9:00 pm) every Tuesday after fasting for 12 hours but not water. The animals were weighed with a balance and the weight values ​​were recorded. An appropriate amount of blood was collected by tail clipping and the blood glucose content was measured with a blood glucose meter (glucose oxidase method). The blood glucose values ​​were read and recorded.

[0134] 4.2 24-hour urine volume, urine protein, and urea nitrogen testing

[0135] In the sixth week of administration, the 24-hour urine volume, urine protein, and urea nitrogen content of the mice were measured according to the instructions of the corresponding assay kits.

[0136] 4.3 Intraperitoneal Glucose Tolerance Test (IPGTT)

[0137] In the fifth week of administration, a glucose tolerance test was performed. The mice in each group were fasted for 12 h but not water. The blood glucose level was measured with a blood glucose meter the next day as the blood glucose level at 0 min. 1 g / kg glucose was injected intraperitoneally, and the blood glucose levels were measured and recorded at 15, 30, 60, and 120 min after the injection. The IPGTT curve was drawn using Graphpad Prism 8 software, and the total area under the curve for the blood collection period of 0-120 min was calculated.

[0138] 5. Experimental Results

[0139] like Figure 7 As shown, the blood glucose levels of mice in the SD group remained within the normal range (<7 mmol / L); the blood glucose levels of mice in the Model + Saline group remained significantly higher than those in the SD group. From the first to the third week of treatment, the blood glucose levels of mice in the Model + EcN-SEM1 group were significantly lower than those in the Model + EcN-EGFP group. Glucose tolerance tests showed that Model + EcN-SEM1 mice had a better ability to regulate blood glucose than Model + EcN-EGFP mice. Urinary protein and urea nitrogen assays indicated that renal function in Model + EcN-SEM1 mice was better than that in Model + EcN-EGFP mice. These results indicate that recombinant probiotics expressing SEM1 can lower blood glucose in diabetic mice and even have a protective effect against diabetic nephropathy. This suggests that recombinant probiotics expressing SEM1 have a blood glucose-lowering effect and can be used to prevent or treat diabetes.

[0140] like Figure 8As shown in the figure, the weight of the SD group mice showed an overall slow upward trend, while the weight of the mice in the Model+Saline group, Model+EcN-EGFP group and Model+EcN-SEM1 group showed a slow downward trend, indicating that EcN-SEM1 had no obvious effect on the fasting weight of mice. Moreover, it can be seen from the figure that the weight of the diabetic group mice was significantly lower than that of the normal group mice.

[0141] Results of glucose tolerance test of diabetic mice using EcN-SEM1 Figure 9 and Figure 10 As shown in the figure, after intraperitoneal injection of glucose, the blood glucose levels of the mice in the Model+EcN-SEM1 group were significantly lower than those in the Model+Saline group at 15, 30, 60, and 120 minutes. The area under the curve (AUC) of the glucose tolerance test was also lower than that in the Model+Saline group. This indicates that the recombinant probiotic expressing K7 of the present invention can improve the glucose metabolism ability of diabetic mice.

[0142] The results of the recombinant probiotics on the 24-hour urine protein and urine nitrogen content in diabetic mice are as follows Figure 11 As shown in the results, the urine protein content and urea nitrogen content of the SD group mice were significantly lower than those of the Model+Saline group, indicating that the renal function of diabetic mice was impaired, that is, the reabsorption of urine protein was weakened and the filtration effect of urea nitrogen was reduced; the urine protein content and urea nitrogen content of the Model+EcN-SEM1 group mice were slightly decreased compared with the Model+Saline group, indicating that the recombinant probiotics described in the present invention can enhance the reabsorption of urine protein and the filtration effect of urea nitrogen by the kidney, thereby improving renal function.

[0143] In summary, the present invention constructs a human SEM1 gene recombinant plasmid pET-28a-SEM1 and transfers it into probiotics to obtain recombinant probiotics. The SEM1 gene is efficiently expressed in the recombinant probiotics. Experimental results show that the recombinant probiotics that efficiently express the SEM1 protein can significantly reduce the percentage of weight loss in colitis mice, reduce disease activity, alleviate colon tissue pathological damage, and improve colon shortening. This indicates that the recombinant probiotics that efficiently express the SEM1 protein of the present invention can be used as a drug to treat or alleviate ulcerative colitis, providing a new option for the clinical treatment of ulcerative colitis. At the same time, the recombinant probiotics that efficiently express the SEM1 protein can significantly lower blood sugar in diabetic mice, indicating that the recombinant probiotics that efficiently express the SEM1 protein of the present invention can be used as a drug to treat or alleviate diabetes, providing a new option for the clinical treatment of diabetes. The present invention changes the conventional administration method of protein drugs (intramuscular injection or intravenous injection) and applies protein drugs to the treatment of gastrointestinal diseases and diabetes through oral administration, improving patient compliance and reducing production costs.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Sequence Listing <110> Lanzhou University <120> SEM1 protein, recombinant plasmid expressing SEM1 protein, recombinant probiotics expressing SEM1 protein and applications thereof <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 70 <212> PRT <213> People <400> 1 Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp 1 5 10 15 Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu 20 25 30 Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val 35 40 45 Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Glu Leu Glu Lys His Gly 50 55 60 Tyr Lys Met Glu Thr Ser 65 70 <210> 2 <211> 213 <212> DNA <213> People <400> 2 atgtcagaga aaaagcagcc ggtagactta ggtctgttag aggaagacga cgagtttgaa 60 gagttccctg ccgaagactg ggctggctta gatgaagatg aagatgcaca tgtctgggag 120 gataattggg atgatgacaa tgtagaggat gacttctcta atcagttacg agctgaacta 180 gagaaacatg gttataagat ggagacttca tag 213 <210> 3 <211> 5582 <212> DNA <213> Artificial Sequence <400> 3 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180 cgacggagct cgaattcatg tcagagaaaa agcagccggt agacttaggt ctgttagagg 240 aagacgacga gtttgaagag ttccctgccg aagactgggc tggcttagat gaagatgaag 300 atgcacatgt ctgggaggat aattgggatg atgacaatgt agaggatgac ttctctaatc 360 agttacgagc tgaactagag aaacatggtt ataagatgga gacttcatag ggatccgcga 420 cccatttgct gtccaccagt catgctagcc atatggctgc cgcgcggcac caggccgctg 480 ctgtgatgat gatgatgatg gctgctgccc atggtatatc tccttcttaa agttaaacaa aattattct agggggat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctc gccggacgca tcgtggccgg catcaccggc 660 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct 720 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc 780 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac 840 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga 900 gatcccggac accatcgat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga 960 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgac caggccagcc acgtttctgc 1080 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 1200 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 1260 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 1320 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 1380 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 1440 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 1500 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 1560 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 1620 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 1680 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 1740 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg 1800 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 1860 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc 1920 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc 1980 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 2040 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc 2100 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc 2160 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 2220 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 2280 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 2340 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 2400 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 2460 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 2520 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 2580 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 2640 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 2700 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 2760 aacgttccag taaccgggca tgttcatcat cattaacccg tatcgtgagc atcctctctc 2820 gtttcatcgg tatcattacc cccatgaaca gaaatcccccc ttacacggag gcatcagtga 2880 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc 2940 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc 3000 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa 3060 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 3120 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 3180 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 3240 tgtactgaga gtgcaccata tatgcggtgt gaataccgc acagatgcgt aagagaaaa 3300 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3360 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3420 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3480 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3540 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 3600 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 3660 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 3720 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 3780 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 3840 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 3900 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 3960 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4020 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4080 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gacaataa actgtctgct tacataaca gtaacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat taattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 4620 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 4800 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 4860 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 4920 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 4980 gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg 5040 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 5100 gaaaagtgcc acctgaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt 5160 gttaaatcag ctcattttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 5220 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 5280 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 5340 gtgaaccatc accctaatca agtttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 5400 accctaaagg gagccccccga tttagagctt gacgggggaaa gccggcgaac gtggcgagaa 5460 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 5520 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc 5580 ca 5582

Claims

1. Use of recombinant probiotics expressing SEM1 protein in the preparation of a drug for preventing gastrointestinal diseases, characterized in that: The recombinant probiotic is obtained by transforming a recombinant plasmid into a probiotic or integrating a gene encoding a SEM1 protein into a probiotic. The probiotic is Escherichia coli EcN. The recombinant plasmid is obtained by inserting a gene encoding a SEM1 protein into an expression vector. The expression vector is pET-28a. The amino acid sequence of the SEM1 protein is shown in SEQ ID No.

1. The gastrointestinal disease is gastric ulcer.

2. Use of recombinant probiotics expressing SEM1 protein in the preparation of a drug for treating gastrointestinal diseases, characterized in that: The recombinant probiotic is obtained by transforming a recombinant plasmid into a probiotic or integrating a gene encoding a SEM1 protein into a probiotic. The probiotic is Escherichia coli EcN. The recombinant plasmid is obtained by inserting a gene encoding a SEM1 protein into an expression vector. The expression vector is pET-28a. The amino acid sequence of the SEM1 protein is shown in SEQ ID No.

1. The gastrointestinal disease is ulcerative colitis.

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