Keratin 7 protein, recombinant plasmid expressing Keratin 7 protein, recombinant probiotics and applications thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a new use of Keratin 7 protein, and a recombinant plasmid expressing Keratin 7 protein, a recombinant probiotic expressing Keratin 7 protein, and a use for preparing a medicine for treating and / or preventing metabolic diseases. The present invention first inserts the Keratin 7 gene into the expression vector pET‑28a to obtain a recombinant plasmid expressing Keratin 7; secondly, the recombinant plasmid is introduced into Escherichia coli Nissle 1917 (EcN) tolerant bacteria to obtain a functional recombinant probiotic. The recombinant probiotics described in the present invention can effectively reduce blood sugar while efficiently expressing Keratin 7 protein, and has a significant therapeutic effect on diabetes and its complications, and the effect is higher than that of clinical drugs, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a new use of Keratin 7 protein, a recombinant plasmid expressing Keratin 7 protein, a recombinant probiotic expressing Keratin 7 protein, and uses thereof for preparing a medicine for treating and / or preventing metabolic diseases. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar, which is caused by defects in insulin secretion or its biological function. According to the latest global diabetes map published by the International Diabetes Federation (IDF), the number of diabetes patients in the world reached 425 million by the end of 2017. It is estimated that by 2045, the number of diabetes patients in the world will reach 629 million, and the number of diabetes patients in the world will continue to rise. Diabetes can be divided into two categories: type 1 diabetes and type 2 diabetes according to the patient's age of onset, symptoms and treatment drugs. Type 1 diabetes is insulin-dependent diabetes. Autoimmune reactions damage pancreatic B cells, and insulin secretion is absolutely insufficient. It is more common in children and adolescents and must be treated with exogenous insulin. Type 2 diabetes is non-insulin-dependent diabetes. Pancreatic B cells are defective, insulin secretion is relatively insufficient and insulin resistance is more common in obese adults. It can be controlled by diet control or oral hypoglycemic drugs. Continuous high blood sugar and long-term metabolic disorders can lead to damage to tissues and organs throughout the body, resulting in a series of complex complications, such as diabetic nephropathy, cardiovascular disease (atherosclerosis, etc.), retinopathy, gestational diabetes, diabetic foot disease, etc., which seriously endanger human health.

[0003] At present, the non-insulin drugs used in clinical treatment of type 2 diabetes and its complications mainly include biguanides, sulfonylureas, non-sulfonylurea insulin secretagogues, insulin sensitizers and α-glucosidase inhibitors. The common serious side effect of hypoglycemic drugs is hypoglycemia. α-glucosidase inhibitors, biguanides and insulin sensitizers generally do not cause hypoglycemia when used alone, but it may still occur when used in combination with other drugs. In addition, patients may experience adverse reactions such as a strong sense of hunger, nausea, cold sweats, palpitations, trembling hands and feet, and liver and kidney toxicity during medication. The cure rate of existing hypoglycemic drugs for diabetic complications is very low, and most patients need to take medication for life to control blood sugar.

[0004] In view of the limitations of existing therapeutic drugs, the present invention constructs a Keratin 7 gene recombinant plasmid pET-28a-K7 and transfers it into probiotics to obtain recombinant probiotics. Keratin 7 is efficiently expressed in the recombinant probiotics, thereby obtaining a recombinant probiotic expressing Keratin 7; the recombinant probiotic expressing Keratin 7 can lower blood sugar, improve glucose metabolism, improve insulin sensitivity, enhance pancreatic islet cell function, and reduce insulin resistance, and the effect is equivalent to or better than that of the clinically positive drug metformin, and does not affect body weight changes; the recombinant probiotic expressing Keratin 7 has the effect of significantly improving renal function, can reduce the urine protein, urine creatinine and urea nitrogen content of diabetic mice, protect the kidneys while lowering blood sugar, and reduce the risk of diabetic nephropathy, and its effect is significantly better than metformin; the recombinant probiotic expressing Keratin 7 has the effect of significantly improving renal function, can reduce the urine protein, urine creatinine and urea nitrogen content of diabetic mice, protect the kidneys while lowering blood sugar, and reduce the risk of diabetic nephropathy, and its effect is significantly better than metformin; The recombinant probiotics of 7 have a significant therapeutic effect on cardiovascular complications caused by diabetes, and its protective effect is significantly stronger than that of the positive drug metformin; the present invention changes the conventional administration method of protein drugs (muscular injection or intravenous injection) and applies protein drugs to the treatment of metabolic diseases in the form of oral administration, which solves the long-standing unresolved technical problems in this field and achieves significant results. Summary of the invention

[0005] In view of the above technical problems, the primary purpose of the present invention is to provide a use of Keratin 7 protein for preparing a drug for preventing and / or treating metabolic diseases. The Keratin 7 protein can be Keratin 7 protein, or recombinant Keratin 7 protein, or a recombinant plasmid expressing Keratin 7 protein, or a recombinant strain expressing Keratin 7 protein.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a use of Keratin 7 protein in the preparation of a drug for preventing and / or treating metabolic diseases.

[0008] Preferably, the amino acid sequence of the Keratin 7 protein is as shown in SEQ ID NO.1.

[0009] Preferably, the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, osteoporosis, and diabetic complications.

[0010] Preferably, the diabetic complications include diabetic nephropathy, cardiovascular complications caused by diabetes, diabetic foot disease, diabetic neuropathy, and periodontitis.

[0011] Preferably, the disease is diabetes.

[0012] Preferably, the Keratin 7 protein is added with pharmaceutically acceptable excipients or carriers to prepare any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form is an oral preparation.

[0014] Preferably, the oral preparation includes tablets, capsules, and granules.

[0015] In a second aspect, the present invention provides a use of a recombinant plasmid or a recombinant probiotic expressing Keratin 7 protein in the preparation of a drug for preventing and / or treating metabolic diseases.

[0016] Preferably, the amino acid sequence of the Keratin 7 protein is as shown in SEQ ID NO.1.

[0017] Preferably, the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, osteoporosis, and diabetic complications.

[0018] Preferably, the diabetic complications include diabetic nephropathy, cardiovascular complications caused by diabetes, diabetic foot disease, diabetic neuropathy, and periodontitis.

[0019] Preferably, the disease is diabetes.

[0020] Preferably, the recombinant plasmid or recombinant probiotic expressing Keratin 7 protein is added with a pharmaceutically acceptable excipient or carrier to prepare any pharmaceutically acceptable dosage form.

[0021] Preferably, the dosage form is an oral preparation.

[0022] Preferably, the oral preparation includes tablets, capsules, and granules.

[0023] In a third aspect, the present invention provides a recombinant plasmid for expressing Keratin 7 protein, wherein the recombinant plasmid is obtained by inserting a gene encoding Keratin 7 protein into an expression vector.

[0024] Preferably, the amino acid sequence of the Keratin 7 protein is as shown in SEQ ID NO.1.

[0025] Preferably, the gene sequence encoding Keratin 7 protein is shown as SEQ ID NO.2.

[0026] Preferably, the expression vector includes but is not limited to pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYA M75P, PNZ8149-usp45.

[0027] Preferably, the expression vector is pET-28a, the recombinant plasmid is named pET-28a-K7, and the gene sequence of pET-28a-K7 is shown in SEQ ID NO.3.

[0028] In a fourth aspect, the present invention provides a use of the recombinant plasmid expressing Keratin 7 protein described in the third aspect in the preparation of a drug for preventing and / or treating metabolic diseases.

[0029] Preferably, the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, osteoporosis, and diabetic complications.

[0030] Preferably, the diabetic complications include diabetic nephropathy, cardiovascular complications caused by diabetes, diabetic foot disease, diabetic neuropathy, and periodontitis.

[0031] Preferably, the disease is diabetes.

[0032] Preferably, the recombinant plasmid expressing Keratin 7 protein is added with a pharmaceutically acceptable excipient or carrier to prepare any pharmaceutically acceptable dosage form.

[0033] Preferably, the dosage form is an oral preparation.

[0034] Preferably, the oral preparation includes tablets, capsules, and granules.

[0035] In a fifth aspect, the present invention provides a recombinant probiotic expressing Keratin 7 protein, wherein the recombinant probiotic is obtained by transforming the probiotic with the recombinant plasmid described in the third aspect, or by integrating the gene encoding Keratin 7 protein into the probiotic.

[0036] Preferably, the amino acid sequence of the Keratin 7 protein is as shown in SEQ ID NO.1.

[0037] Preferably, the gene sequence encoding Keratin 7 protein is shown as SEQ ID NO.2.

[0038] Preferably, the probiotics include but are not limited to Escherichia coli Nissle 1917, probiotic Bacillus, Lactococcus, Butyric acid Bacillus, Lactobacillus, Bifidobacterium, Actinomycetes, Pichia pastoris.

[0039] Preferably, the probiotic is Escherichia coli Nissle 1917.

[0040] In a sixth aspect, the present invention provides a use of the recombinant probiotic expressing Keratin 7 protein described in the fifth aspect in the preparation of a drug for preventing and / or treating metabolic diseases.

[0041] Preferably, the metabolic diseases include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, osteoporosis, and diabetic complications.

[0042] Preferably, the diabetic complications include diabetic nephropathy, cardiovascular complications caused by diabetes, diabetic foot disease, diabetic neuropathy, and periodontitis.

[0043] Preferably, the disease is diabetes.

[0044] Preferably, the recombinant probiotics expressing Keratin 7 protein are added with pharmaceutically acceptable excipients or carriers to prepare any pharmaceutically acceptable dosage form.

[0045] Preferably, the dosage form is an oral preparation.

[0046] Preferably, the oral preparation includes tablets, capsules, and granules.

[0047] In a seventh aspect, the present invention provides a method for preparing the recombinant probiotics described in the fifth aspect, the method comprising: transforming the probiotics with the recombinant plasmid described in the third aspect, culturing, picking a single clone, culturing in a culture medium containing kanamycin, and screening to obtain the recombinant probiotics expressing the Keratin 7 protein.

[0048] The beneficial effects of the present invention are as follows: ① the present invention constructs a Keratin 7 gene recombinant plasmid pET-28a-K7 and transfers it into probiotics to obtain recombinant probiotics, Keratin 7 is efficiently expressed in the recombinant probiotics, and a recombinant probiotic expressing Keratin 7 is obtained; ② the recombinant probiotic expressing Keratin 7 of the present invention has an activity of lowering blood sugar that is equivalent to that of metformin and does not affect changes in body weight; it can improve the glucose metabolism ability of diabetic mice, and the effect is better than that of the clinically positive drug metformin; it can improve the sensitivity of diabetic mice to insulin, enhance the function of pancreatic islet cells, and reduce their resistance to insulin, and the effect is equivalent to that of the clinically positive drug metformin; it shows that the recombinant probiotic expressing Keratin 7 of the present invention can be used to treat diabetes, providing a new option for the clinical treatment of diabetes; ③ the recombinant probiotic expressing Ke ratin of the present invention The recombinant probiotics expressing Keratin 7 have the effect of significantly improving renal function, can reduce the urine protein, urine creatinine and urea nitrogen content of diabetic mice, protect the kidneys while lowering blood sugar, and reduce the risk of diabetic nephropathy, and its effect is significantly better than metformin; ④ The recombinant probiotics expressing Keratin 7 described in the present invention have a significant therapeutic effect on cardiovascular complications caused by diabetes, and its protective effect is significantly stronger than the positive drug metformin; ⑤ The present invention changes the conventional administration method of protein drugs (muscular injection or intravenous injection), and applies protein drugs to the treatment of metabolic diseases in the form of oral administration, which solves the long-standing unresolved technical problems in this field and achieves remarkable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The results of PCR reaction of recombinant plasmid transformed into Escherichia coli Nissle 1917 are shown in the figure;

[0050] Figure 2 Effects of recombinant probiotics expressing Keratin 7 protein on fasting blood glucose in diabetic mice;

[0051] Figure 3 Effects of recombinant probiotics expressing Keratin 7 protein on fasting body weight of diabetic mice;

[0052] Figure 4 Results of glucose tolerance test on diabetic mice using recombinant probiotics expressing Keratin 7 protein;

[0053] Figure 5 Results of the insulin resistance test on diabetic mice using recombinant probiotics expressing Keratin 7 protein;

[0054] Figure 6The results of the detection of the recombinant probiotics expressing Keratin 7 protein on the urinary protein content in the 24-hour urine of diabetic mice;

[0055] Figure 7 The results of the detection of creatinine content in 24-hour urine of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0056] Figure 8 The results of the detection of urea nitrogen content in 24-hour urine of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0057] Fig. 9 The results of the test on the low-density lipoprotein cholesterol content in the plasma of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0058] Fig.10 The results of the test on the triglyceride content in the plasma of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0059] Fig.11 The results of the detection of total cholesterol content in the plasma of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0060] Fig.12 The results of the detection of glycosylated hemoglobin in the plasma of diabetic mice by recombinant probiotics expressing Keratin 7 protein;

[0061] The abbreviations of the groups in the figure represent: Model is the high-fat diet + STZ combined induced diabetes model group; SD is Standard Diet, ordinary diet group; Model+Metformin is the model + metformin hydrochloride group, positive control group; Model+EcN is the model + Escherichia coli Nissle 1917 probiotic solution; Model+β-Lactose is the model + β-lactose solution; Model+EcN-EGFP is the model + recombinant Escherichia coli Nissle 1917 probiotic solution containing pET-28a-EGFP; Model+EcN-K7 is the recombinant Escherichia coli Nissle 1917 probiotic solution containing pET-28a-K7; Model vs.SD, ## p<0.01, ### p<0.001; Model vs. Model+Metformin, * p<0.05, ** p<0.01, ***p<0.001; Model vs.Model+EcN-K7, * p<0.05, ** p<0.01, *** p<0.001; Model+EcN-K7 vs.Model+EcN-EGFP, * p<0.05, ** p<0.01, *** p<0.001. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical scheme and advantages of the present invention clearer, the following examples further describe the present invention 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 conventional methods unless otherwise specified. The reagents and materials used in the following examples are commercially available unless otherwise specified.

[0063] The Escherichia coli DH5α described in the following examples was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the Escherichia coli Nissle 1917 was purchased from Germany (trademark name Mutaflor); the recombinant plasmid pET-28a-EGFP was purchased from Miaoling Plasmid Platform; anhydrous ethanol was purchased from Tianjin Damao Chemical Reagent Company; β-lactose (β-lactose, containing 70% β-lactose and 30% α-lactose) was purchased from MacLean Biotechnology Co., Ltd.; the PCR primers used were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.; the bacterial plasmid DNA extraction kit was purchased from AXYGEN; the PrimeSTAR HS (Premix, 2X) and T4 DNA ligase were purchased from Takara; the DNA endonucleases BamHI and HindIII were purchased from Beijing NEB;

[0064] The formula of the LB (Luria-Bertani) liquid culture medium is: 1% tryptone, 0.5% yeast extract, 0.5% NaCl; the formula of the LB solid culture medium is: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar.

[0065] The streptozocin (STZ) described in the following examples has a relative molecular mass of 265.22 and an appearance of small flakes or prismatic crystals; it is hygroscopic and soluble in water, low-carbon alcohols and ketones; STZ has a selective destructive effect on pancreatic β cells in mice and can induce diabetes in mice; the preparation method of the streptozocin solution is as follows: ① 2.1 g of citric acid (MW: 210.14) is added to 100 mL of double distilled water to prepare solution A; ② citric acid 2.94 g of sodium (MW: 294.10) was added to 100 mL of double distilled water to prepare liquid B; ③ When using, mix liquid A and liquid B in a certain ratio to prepare citric acid buffer (1:1.32, pH 4.2-4.5); ④ When injecting streptozotocin, dissolve streptozotocin in citric acid buffer at a concentration of 1%, and inject the corresponding streptozotocin according to the fasting body weight, and the injection should be completed within 30 minutes; STZ is easy to inactivate, so it should be weighed quickly and stored in dry aluminum foil (or tin foil) paper away from light for later use.

[0066] The reagents, kits and instruments described in the following examples are shown in Table 1:

[0067] Table 1 Experimental reagents and kits

[0068]

[0069] The names and abbreviations described in the following examples correspond to those shown in Table 2 below:

[0070] Table 2 Names and abbreviations

[0071]

[0072] Example 1 Preparation of recombinant plasmid and recombinant probiotics expressing Keratin 7 protein

[0073] 1. Primer Synthesis

[0074] According to the nucleotide sequence of Keratin 7 protein shown in SEQ ID NO.2, compared with the pET-28a plasmid map, specific primers were designed, and restriction endonuclease sites BamHI and HindIII were inserted into the primers. The specific information is shown in Table 3. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; the primers were dissolved in sterile deionized water to a concentration of 10 μmol / L.

[0075] Table 3 Primer information and synthesis

[0076]

[0077] 2. Construction of recombinant plasmid

[0078] The pET-28a-K7 plasmid was synthesized by Genewise Biotechnology Co., Ltd.

[0079] 3. Transformation of Escherichia coli DH5α with recombinant plasmid

[0080] Take out DH5α competent cells from the -80℃ refrigerator, place them on an ice box for 10-20 minutes to melt; take out 50μL competent cells and add 5μL plasmid, gently flick with your fingers several times to mix, and place the mixture on ice for 25min; after heat shock at 42℃ for 45s, quickly put it back on ice and let it stand for 2min, add 0.5mL of room temperature LB liquid culture medium (without antibiotics), shake and culture at 37℃ and 200rpm for 1h, take 200μL of the above bacterial solution and spread it on a plate containing 15μg / m kanamycin, after the bacterial solution is completely absorbed by the culture medium, invert the culture dish, and culture at 37℃ for 12h.

[0081] 4. Successful colony verification of heat shock transformation

[0082] Single clones were picked and cultured in liquid medium containing 15 μg / mL kanamycin for PCR verification. Figure 1 As shown, the molecular weight of the target band is 1640 bp, and the verification result is correct (the position indicated by the arrow in the figure is the target band).

[0083] 5. Extraction of recombinant plasmid DNA

[0084] The strain successfully transformed with the recombinant plasmid was transferred to LB medium for amplification culture at the following conditions: rotation speed 200 rpm, temperature 37° C. The plasmid was extracted using the plasmid mini-extraction kit of AXYGEN Company.

[0085] 6. Preparation of Escherichia coli Nissle 1917 Chemically Competent Cells

[0086] Take out the Nissle 1917 (Mutaflor) strain of Escherichia coli from the -80℃ freezer, pick up a small amount of the preservation solution with a sterilized pipette tip, streak it on LB solid medium, and culture it in a 37℃ incubator overnight: pick up a moist, smooth single colony from the LB solid medium, put it into LB culture medium, and culture it at 37℃ for 12h; when the OD of the culture solution is When the 600 value is 0.3-0.5, transfer the bacterial solution to a sterilized centrifuge tube precooled on ice, place in an ice bath for 30 minutes, and centrifuge at 1520g for 10 minutes at 4°C; discard the supernatant, gently suspend the cells with 500μL precooled CaCl2 solution (filtered and sterilized), and centrifuge at 1520g for 10 minutes at 4°C; repeat the previous step once; discard the supernatant, add 500μL precooled CaCl2 solution (filtered and sterilized), and carefully suspend the cells to prepare the competent cell suspension; the prepared competent cell suspension is directly used for transformation experiments. Add an equal volume of sterilized 20% glycerol to the unused competent cells, mix well, and dispense into 1.5mL centrifuge tubes (100μL per tube), and store in a -80°C refrigerator.

[0087] 7. Transform the recombinant plasmid into Escherichia coli Nissle 1917 strain

[0088] Take out Escherichia coli Nissle 1917 chemical competent cells from the -80℃ refrigerator and place them on an ice box for 10-20 minutes to thaw. Take out 50μL competent cells and add 5μL plasmid, gently stir with your fingers several times to mix, and place the mixture on ice for 25min; after heat shock at 42℃ for 45s, quickly put it back on ice and let it stand for 2min, add 0.5mL room temperature LB liquid culture medium (without antibiotics), shake and culture at 37℃, 200rpm for 1h, take 200μL of the above bacterial solution and spread it on a plate containing 15μg / mL kanamycin, wait for the bacterial solution to be completely absorbed by the culture medium, invert the culture dish, and culture at 37℃ for 12h.

[0089] 8. Successful colony verification of heat shock transformation

[0090] Single clones were picked and cultured in a liquid medium containing 15 μg / mL kanamycin, followed by PCR verification and enzyme digestion verification. Figure 1 As shown, it was verified that the recombinant Escherichia coli Nissle 1917 probiotic bacteria expressing Keratin 7 protein was correctly obtained.

[0091] It should be noted that the method described in the present invention is not the only limiting method for constructing recombinant probiotics expressing Keratin 7 protein. By conventional technical means, any recombinant plasmid expressing Keratin 7 protein can be constructed, and by conventional transformation operations, the recombinant plasmid expressing Keratin 7 protein can be transformed into Escherichia coli Nissle 1917 strain or other probiotics, and recombinant probiotics expressing Keratin 7 protein can be screened.

[0092] Example 2 Therapeutic effect of recombinant probiotics expressing Keratin 7 protein on diabetic mice

[0093] 1. Establishment and grouping of C57BL / 6N mouse diabetes model

[0094] 1.1 Model establishment

[0095] Forty-eight C57BL / 6N mice were randomly divided into two groups: a normal diet group (6 mice) and a high-fat diet + STZ group (42 mice).

[0096] 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;

[0097] 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 but not water for 12 hours before STZ injection, injection was performed at the same time period of 9:00am-10:00am, and high-fat diet could be fed after fasting for 2 hours after STZ injection), once a day for five consecutive days, and the newly prepared STZ solution was injected within 30 minutes to prevent STZ from failing. After the mice were injected with STZ solution (5 days), the weight and blood sugar of the mice were monitored weekly, and they continued to be fed with high-fat diet for two weeks, and then fed with ordinary diet for one week. If the blood sugar remained basically unchanged (blood sugar ≥ 15.0mmo l / L), the type 2 diabetes model was considered stable and the model was successfully established. During the experiment, the mice were given sufficient food and water.

[0098] 1.2 Grouping

[0099] When the fasting blood glucose (fasting but not water for 12 hours, blood glucose measured at 7:30pm-9:00pm) of mice in the high-fat diet + STZ group was higher than 15mmol / L, the type 2 diabetes model was considered to be successfully established. After the experiment officially began, the mice with successful modeling were divided into 6 groups (as shown in af below) plus a normal diet group (SD, as shown in g), with 6 mice in each group, and all mice were fed with normal diet, as follows:

[0100] a. Model group: gavage with 0.2 mL of normal saline daily, serving as the negative control group;

[0101] b. Model + Metformin group: Orally administered at a dose of 200 mg / kg / d, serving as the positive control group;

[0102] c. Model+EcN group: intragastrically administered with 0.2 mL of Escherichia coli Nissle1917 probiotic solution;

[0103] d. Model + β-Lactose: gavage with 0.2 mL of 11 mg / mL β-lactose solution prepared with normal saline;

[0104] e. Model+EcN-EGFP group: intragastrically administered with 0.2 mL of recombinant Escherichia coli Nissle 1917 probiotic solution containing pET-28a-EGFP;

[0105] f. Model+EcN-K7 group: gavage with 0.2 mL of recombinant Escherichia coli Nissle 1917 probiotic solution containing pET-28a-K7;

[0106] g. Normal diet group (SD): gavage with an equal volume of normal saline daily;

[0107] All groups of mice were administered the drug for a total of 10 weeks. During the administration period, the growth status of the mice (including fur color, motor ability, etc.) was monitored daily, and blood sugar and body weight were measured once a week.

[0108] 2. Detection indicators

[0109] 2.1 Body weight and fasting blood glucose (FBG) monitoring

[0110] Starting from the 0th week of medication, fasting blood sugar was measured at the same time period (7:30pm-9:00pm) after fasting for 12 hours every week. An appropriate amount of blood was collected by cutting the tail, and the blood sugar content was measured with a blood glucose meter. The blood sugar value was read and recorded; an electronic balance was used to weigh the blood sugar before measuring it every week.

[0111] 2.2 Detection of 24-hour urine volume, urine protein, urine creatinine and urea nitrogen

[0112] At the end of the 8th week of administration, the 24-hour urine volume, urine protein, urine creatinine and urea nitrogen content of the mice were measured according to the instructions of the corresponding assay kits.

[0113] 2.3 Intraperitoneal Glucose Tolerance Test (IPGTT) and Intraperitoneal Insulin Tolerance Test (IPITT)

[0114] At the end of the 9th week of administration, an insulin tolerance test was performed. The mice in each group were fasted but not deprived of water for 6 hours, and the blood glucose content was measured with a blood glucose meter as the blood glucose value at 0 min (BG0). Then 0.3 U / kg insulin was injected intraperitoneally, and the blood glucose levels were measured at 15, 30, 60 and 120 min after injection (BG15, BG30, BG60 and BG120), respectively. The IPITT curve was drawn using Graphpad prism 8 software, and the total area under the curve of the blood collection period of 0-120 min was calculated.

[0115] At the end of the 9th week of administration, a glucose tolerance test was conducted. 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 then the blood glucose levels were measured and recorded at 15, 30, 60 and 120 min after the injection. The IPGTT curve was drawn with Graphpad prism 8 software, and the total area under the curve of the blood collection period of 0-120 min was calculated.

[0116] 2.4 Detection of Glycated Hemoglobin

[0117] At the end of the 10th week of drug administration, blood was collected from all mice by removing their eyeballs before dissection. Before blood collection, in order to prevent blood coagulation, the centrifuge tube was soaked in EDTA-Na2 (15 mg / mL) solution overnight, and then placed in a 55°C constant temperature oven for 5 hours for use. About 500 μL of blood was collected from each mouse; the obtained blood sample was allowed to stand at 4°C for 48 hours, and then centrifuged at 4°C, 700-1,000xg, for 10 minutes to separate plasma (upper layer) and red blood cells (lower layer). The plasma and red blood cells were stored and processed separately for detection by relevant kits.

[0118] Red blood cells were made into hemolysate to measure glycosylated hemoglobin. Red blood cells were mixed with double distilled water at a ratio of 1:1.5 and mixed thoroughly with a vortex mixer for 1 minute to prepare the hemolysate. The hemolysate was stored at -20°C and could be stored for 70 days.

[0119] 2.5 TC, TG, and HDL content determination

[0120] The frozen plasma was thawed on ice and the contents of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in serum were determined according to the instructions of the kit.

[0121] 3. Data processing

[0122] The experimental data were statistically analyzed using SPSS23.0 software, and the data were expressed as (x±s). One-way ANOVA and LSD-t method were used for comparison between groups. p<0.05 was considered statistically significant.

[0123] 4. Experimental Results

[0124] 4.1 Fasting blood glucose monitoring results

[0125] Effects of recombinant probiotics expressing Keratin 7 protein on fasting blood glucose in diabetic mice Figure 2 As shown, the blood glucose of SD group mice was maintained within the normal range (≤7mmol / L); Model vs. SD, ### p<0.001, indicating that the type 2 diabetic mouse model was successfully established, and the fasting blood glucose of the Model group mice was maintained at about 17mmol / L for 10 weeks; the blood glucose of the Model+EcN-K7 group was significantly lower than that of the Model and Model+EcN-EGFP groups, that is, Model vs.Model+EcN-K7, ** p<0.01, *** p<0.001; Model+EcN-K7 vs.Model+EcN-EGFP, ** p<0.01, *** p<0.001; the trend of the Model+EcN-K7 group was consistent with that of the positive control drug metformin (Model+Metformin) group, indicating that the blood sugar-lowering activity of the recombinant probiotic expressing Keratin7 of the present invention is equivalent to that of metformin.

[0126] 4.2 Fasting weight monitoring results

[0127] Effects of recombinant probiotics expressing Keratin 7 on fasting body weight of diabetic mice Figure 3 As shown, the recombinant probiotics expressing Kerati n 7 had no significant effect on the fasting body weight of mice, and it can be seen from the figure that the body weight of the diabetic group mice was significantly lower than that of the normal group mice.

[0128] 4.3 Glucose tolerance test results

[0129] Results of glucose tolerance test of diabetic mice with recombinant probiotics expressing Keratin 7 Figure 4 As shown, after intraperitoneal injection of glucose, the blood glucose levels of mice in the Model+EcN-K7 group were significantly lower than those in the Model, Model+EcN-EGFP, and Model+Metformin groups at 15min, 30min, and 60min, and there were significant differences, Model vs.Model+EcN-K7, * p<0.05, ** p<0.01; the blood glucose of mice in the SD group slightly increased at 15 minutes and then quickly returned to normal. The area under the curve AUC of the glucose tolerance test was consistent with the trend of the blood glucose change graph, indicating that the recombinant probiotics expressing Keratin 7 described in the present invention can improve the glucose metabolism ability of diabetic mice, and the effect is better than the clinical positive drug metformin.

[0130] 4.4 Insulin resistance test results

[0131] The results of the test on insulin resistance of diabetic mice by recombinant probiotics expressing Keratin 7 are as follows Figure 5 As shown, after intraperitoneal injection of insulin, the blood glucose levels of mice in the Model+EcN-K7 group at 15min, 30min, 60min and 120min were significantly lower than those in the Model group, which was consistent with the trend of the Model+Metformin group; the area under the curve AUC of the insulin resistance test was consistent with the changes in blood glucose, Model vs.Model+EcN-K7, ** p<0.01; Model+EcN-K7 vs.Model+EcN-EGFP, ** p<0.01. Blood glucose of SD group mice dropped significantly at 30 minutes and then rose steadily. Model vs. SD, ### p<0.001; indicating that the recombinant probiotic expressing Keratin 7 described in the present invention can improve the sensitivity of diabetic mice to insulin, enhance the function of pancreatic islet cells, and reduce their resistance to insulin, and the effect is equivalent to that of the clinically positive drug metformin.

[0132] 4.5 24-hour urine protein test results

[0133] The results of the recombinant probiotics expressing Keratin 7 on the 24-hour urine protein content in diabetic mice are as follows Figure 6 As shown, the urine protein content of SD group mice was about 0.5mg / 24hours, Model vs. SD, ###p<0.001, indicating that the renal function of diabetic mice was impaired, that is, the reabsorption of urinary protein was weakened; the urine protein content of the mice in the Model+EcN-K7 group was significantly decreased compared with that in the Model group, indicating that the recombinant probiotics expressing Keratin 7 described in the present invention can enhance the reabsorption of urinary protein by the kidneys and improve renal function, and its effect is better than that of metformin.

[0134] 4.6 24-hour urine creatinine test results

[0135] The results of the detection of creatinine content in 24-hour urine of diabetic mice by recombinant probiotics expressing Keratin 7 are as follows Figure 7 As shown, the urine creatinine content of SD group mice was about 0.7μmol / 24hours, Model vs. SD, ### p<0.001, indicating that the filtration effect of diabetic mice on urine creatinine was reduced, that is, renal function was impaired; the urine creatinine content of mice in the Model+EcN-K7 group was significantly lower than that in the Model and Model+EcN-EGFP groups, * p<0.05, indicating that the recombinant probiotic expressing Keratin 7 of the present invention can enhance the filtering effect of kidney on urine creatinine, improve renal function, and reduce the risk of complications of diabetic nephropathy.

[0136] 4.7 Detection results of urea nitrogen content in 24-hour urine

[0137] The results of the detection of urea nitrogen content in 24-hour urine of diabetic mice by recombinant probiotics expressing Keratin 7 are as follows Figure 8 As shown, the urea nitrogen content of SD group mice was about 0.15mmol / 24hours, Model vs.SD, ### p<0.001, indicating that the filtration effect of diabetic mice on urea nitrogen is reduced, that is, renal function is impaired; the urine creatinine content of mice in the Model+EcN-K7 group showed a downward trend compared with the Model l and Model+EcN-EGFP groups, indicating that the recombinant probiotics expressing Keratin 7 described in the present invention can enhance the filtration effect of the kidney on urea nitrogen and improve renal function, and its effect is better than metformin.

[0138] comprehensive Figure 6-8 It can be seen that the recombinant probiotic expressing Keratin 7 described in the present invention has the effect of significantly improving renal function, can reduce the urine protein, urine creatinine and urea nitrogen content of diabetic mice, protect the kidneys while lowering blood sugar, and reduce the risk of diabetic nephropathy. Its effect is significantly better than metformin.

[0139] 4.8 Results of low-density lipoprotein cholesterol test

[0140] The results of the detection of recombinant probiotics expressing Keratin 7 on the content of low-density lipoprotein cholesterol in the plasma of diabetic mice are as follows Fig. 9 As shown, the LDL-C content of mice in the Model group was significantly higher than that in the SD group. ## p<0.01; indicating that the risk of type 2 diabetic mice suffering from cardiovascular disease is increased compared with normal mice. The LDL-C content of mice in the Model+EcN-K7 group was significantly lower than that in the Model and Model+EcN-EGFP groups, Model vs.Model+EcN-K7, ** p<0.01; Model+EcN-K7vs.Model+EcN-EGFP, *** p<0.001; that is, the recombinant probiotics expressing Keratin 7 can significantly reduce the LDL-C content in the plasma of type 2 diabetic mice and tend to normal values, reducing the risk of type 2 diabetic mice suffering from cardiovascular diseases (such as atherosclerosis) at the same time, and has a protective effect on cardiovascular.

[0141] 4.9 Test results of triglyceride content

[0142] The results of the detection of recombinant probiotics expressing Keratin 7 on the triglyceride content in the plasma of diabetic mice are as follows Fig.10 As shown, the TG content of mice in the Model group was significantly higher than that in the SD group. ## p<0.01; indicating that the risk of type 2 diabetic mice suffering from cardiovascular disease is higher than that of normal mice. The TG content of mice in the Model+EcN-K7 group was significantly lower than that in the Model and Model+EcN-EGFP groups, Model vs.Model+EcN-K7, * p<0.05; Model+EcN-K7 vs.Model+EcN-EGFP, *** p<0.001; that is, the recombinant probiotics expressing Keratin 7 can significantly reduce the TG content in the plasma of type 2 diabetic mice and tend to normal values, reducing the risk of type 2 diabetic mice suffering from cardiovascular diseases (such as atherosclerosis) at the same time, and has a protective effect on cardiovascular.

[0143] 4.10 Total cholesterol test results

[0144] The results of the detection of total cholesterol content in the plasma of diabetic mice by recombinant probiotics expressing Keratin 7 are as follows Fig.11 As shown, the TC content of the Model group mice was significantly higher than that of the SD group. ##p<0.01; indicating that the risk of type 2 diabetic mice suffering from cardiovascular disease is increased compared with normal mice. The TC content of mice in the Model+EcN-K7 group was significantly lower than that in the Model and Model+EcN-EGFP groups, Model vs.Model+EcN-K7, ** p<0.01; Model+EcN-K7 vs.Model+EcN-EGFP, ** p<0.01; that is, the recombinant probiotics expressing Keratin 7 can significantly reduce the TC content in the plasma of type 2 diabetic mice and tend to normal values, reducing the risk of type 2 diabetic mice suffering from cardiovascular diseases (such as atherosclerosis) at the same time, and has a protective effect on cardiovascular.

[0145] 4.11 Glycated hemoglobin test results

[0146] The results of the detection of the recombinant probiotics expressing Keratin 7 on the content of glycosylated hemoglobin in the plasma of diabetic mice are as follows Fig.12 As shown, the HbA1c content of mice in the Model group was significantly higher than that in the SD group. ### p<0.001; indicating that the glycated hemoglobin in type 2 diabetic mice increased compared with normal mice. The HbA1c content of mice in the Model+EcN-K7 group was significantly lower than that in the Model and Model+EcN-EGFP groups, Model vs.Model+EcN-K7, ** p<0.01; Model+EcN-K7 vs.Model+EcN-EGFP, *** p<0.001; that is, the recombinant probiotics expressing Keratin 7 can significantly reduce the content of glycosylated hemoglobin in the plasma of type 2 diabetic mice and tend to normal values, reducing the risk of type 2 diabetic mice suffering from cardiovascular diseases (especially heart disease) at the same time, and has a protective effect on cardiovascular.

[0147] comprehensive Figure 9-12 It can be seen that the recombinant probiotics expressing Keratin 7 of the present invention have a significant therapeutic effect on cardiovascular complications caused by diabetes, and its protective effect is significantly stronger than that of the positive drug metformin.

[0148] Metabolic diseases are disorders of the metabolic process of substances, mostly caused by enzyme or protein abnormalities. There are about a hundred kinds of human enzymes that are changed, which almost affect the functions of all tissues and organs in the body. Clinically, they can be divided into two categories: the first category is congenital deficiency, some of which have hereditary chromosomal abnormalities, with a wide range but low incidence, such as glycogen storage disease, familial hypercholesterolemia, familial hypertriglyceridemia, gout, etc. The second category is symptomatic metabolic disorders. Many other systemic diseases may cause metabolic abnormalities in the body at a certain stage, making clinical manifestations and diagnosis and treatment more complicated. For example, hypoglycemia and hyperglycemia, hyperinsulinemia may occur in cirrhosis and liver dysfunction, and electrolyte disorders and hypoproteinemia may occur in the late stage of renal disease; hypothyroidism is often accompanied by hyperlipidemia, and excessive adrenal glucocorticoids can cause negative nitrogen balance. Diabetes is an endocrine metabolic disease characterized by hyperglycemia. Its characteristics are metabolic disorders of carbohydrates, proteins, fats, electrolytes and water due to absolute or relative insufficiency of insulin and reduced sensitivity of target cells to insulin. When patients have severe hyperglycemia, they will experience typical "three more and one less" symptoms, which are more common in type 1 diabetes. The "three more and one less" symptoms are more obvious when ketosis or ketoacidosis occurs. Fatigue and obesity are more common in type 2 diabetes, and obesity is an important factor leading to diabetes. Obese people generally have irregular diets, love to drink, overeat, love to eat a lot of fish and meat, and lack exercise. This will lead to decreased insulin sensitivity, insulin resistance, and increased blood sugar.

[0149] Diabetic patients often have cerebrovascular disease, which may lead to cerebral hemorrhage, double stroke, etc. In addition, the incidence of diabetic nephropathy is 30-40%, the incidence of diabetic foot disease is as high as 35% or more, and neuropathy and blindness are also unavoidable. It can be seen that diabetes drugs should not only play their hypoglycemic effect, but also reduce the incidence of diabetic complications. The probiotics expressing Keratin 7 protein of the present invention have the above potential advantages.

[0150] It should be noted that although the present invention uses Escherichia coli Nissle 1917 as a probiotic to construct a recombinant probiotic expressing Keratin 7 protein, and has a significant effect of preventing and / or treating diabetes, the present invention is not limited to the probiotic Escherichia coli Nissle 1917. On the basis of the present invention, constructing other recombinant probiotics that can express Keratin 7 protein can also achieve the effect of preventing and / or treating diabetes.

[0151] 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 protection scope of the present invention. Sequence Listing <110> Lanzhou University <120> Keratin 7 protein, recombinant plasmid expressing Keratin 7 protein, recombinant probiotics and applications thereof <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 469 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Ile His Phe Ser Ser Pro Val Phe Thr Ser Arg Ser Ala Ala 1 5 10 15 Phe Ser Gly Arg Gly Ala Gln Val Arg Leu Ser Ser Ala Arg Pro Gly 20 25 30 Gly Leu Gly Ser Ser Ser Leu Tyr Gly Leu Gly Ala Ser Arg Pro Arg 35 40 45 Val Ala Val Arg Ser Ala Tyr Gly Gly Pro Val Gly Ala Gly Ile Arg 50 55 60 Glu Val Thr Ile Asn Gln Ser Leu Leu Ala Pro Leu Arg Leu Asp Ala 65 70 75 80 Asp Pro Ser Leu Gln Arg Val Arg Gln Glu Glu Ser Glu Gln Ile Lys 85 90 95 Thr Leu Asn Asn Lys Phe Ala Ser Phe Ile Asp Lys Val Arg Phe Leu 100 105 110 Glu Gln Gln Asn Lys Leu Leu Glu Thr Lys Trp Thr Leu Leu Gln Glu 115 120 125 Gln Lys Ser Ala Lys Ser Ser Arg Leu Pro Asp Ile Phe Glu Ala Gln 130 135 140 Ile Ala Gly Leu Arg Gly Gln Leu Glu Ala Leu Gln Val Asp Gly Gly 145 150 155 160 Arg Leu Glu Ala Glu Leu Arg Ser Met Gln Asp Val Val Glu Asp Phe 165 170 175 Lys Asn Lys Tyr Glu Asp Glu Ile Asn His Arg Thr Ala Ala Glu Asn 180 185 190 Glu Phe Val Val Leu Lys Lys Asp Val Asp Ala Ala Tyr Met Ser Lys 195 200 205 Val Glu Leu Glu Ala Lys Val Asp Ala Leu Asn Asp Glu Ile Asn Phe 210 215 220 Leu Arg Thr Leu Asn Glu Thr Glu Leu Thr Glu Leu Gln Ser Gln Ile 225 230 235 240 Ser Asp Thr Ser Val Val Leu Ser Met Asp Asn Ser Arg Ser Leu Asp 245 250 255 Leu Asp Gly Ile Ile Ala Glu Val Lys Ala Gln Tyr Glu Glu Met Ala 260 265 270 Lys Cys Ser Arg Ala Glu Ala Glu Ala Trp Tyr Gln Thr Lys Phe Glu 275 280 285 Thr Leu Gln Ala Gln Ala Gly Lys His Gly Asp Asp Leu Arg Asn Thr 290 295 300 Arg Asn Glu Ile Ser Glu Met Asn Arg Ala Ile Gln Arg Leu Gln Ala 305 310 315 320 Glu Ile Asp Asn Ile Lys Asn Gln Arg Ala Lys Leu Glu Ala Ala Ile 325 330 335 Ala Glu Ala Glu Glu Arg Gly Glu Leu Ala Leu Lys Asp Ala Arg Ala 340 345 350 Lys Gln Glu Glu Leu Glu Ala Ala Leu Gln Arg Gly Lys Gln Asp Met 355 360 365 Ala Arg Gln Leu Arg Glu Tyr Gln Glu Leu Met Ser Val Lys Leu Ala 370 375 380 Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu 385 390 395 400 Ser Arg Leu Ala Gly Asp Gly Val Gly Ala Val Asn Ile Ser Val Met 405 410 415 Asn Ser Thr Gly Gly Ser Ser Ser Gly Gly Gly Ile Gly Leu Thr Leu 420 425 430 Gly Gly Thr Met Gly Ser Asn Ala Leu Ser Phe Ser Ser Ser Ala Gly 435 440 445 Pro Gly Leu Leu Lys Ala Tyr Ser Ile Arg Thr Ala Ser Ala Ser Arg 450 455 460 Arg Ser Ala Arg Asp 465 <210> 2 <211> 1410 <212> DNA <213> Artificial Sequence <400> 2 atgtccatcc acttcagctc cccggtattc acctcgcgct cagccgcctt ctcgggccgc 60 ggcgcccagg tgcgcctgag ctccgctcgc cccggcggcc ttggcagcag cagcctctac 120 ggcctcggcg cctcacggcc gcgcgtggcc gtgcgctctg cctatggggg cccggtgggc 180 gccggcatcc gcgaggtcac cattaaccag agcctgctgg ccccgctgcg gctggacgcc 240 gacccctccc tccagcgggt gcgccaggag gagagcgagc agatcaagac cctcaacaac 300 aagtttgcct ccttcatcga caaggtgcgg tttctggagc agcagaacaa gctgctggag 360 accaagtgga cgctgctgca ggagcagaag tcggccaaga gcagccgcct cccagacatc 420 tttgaggccc agattgctgg ccttcggggt cagcttgagg cactgcaggt ggatgggggc 480 cgcctggagg cggagctgcg gagcatgcag gatgtggtgg aggacttcaa gaataagtac 540 gaagatgaaa ttaaccaccg cacagctgct gagaatgagt ttgtggtgct gaagaaggat 600 gtggatgctg cctacatgag caaggtggag ctggaggcca aggtggatgc cctgaatgat 660 gagatcaact tcctcaggac cctcaatgag acggagttga cagagctgca gtcccagatc 720 tccgacacat ctgtggtgct gtccatggac aacagtcgct ccctggacct ggacggcatc 780 atcgctgagg tcaaggcgca gtatgaggag atggccaaat gcagccgggc tgaggctgaa 840 gcctggtacc agaccaagtt tgagaccctc caggcccagg ctgggaagca tggggacgac 900 ctccggaata cccggaatga gatttcagag atgaaccggg ccatccagag gctgcaggct 960 gagatcgaca acatcaagaa ccagcgtgcc aagttggagg ccgccattgc cgaggctgag 1020 gagcgtgggg agctggcgct caaggatgct cgtgccaagc aggaggagct ggaagccgcc 1080 ctgcagcggg gcaagcagga tatggcacgg cagctgcgtg agtaccagga actcatgagc 1140 gtgaagctgg ccctggacat cgagatcgcc acctaccgca agctgctgga gggcgaggag 1200 agccggttgg ctggagatgg agtgggagcc gtgaatatct ctgtgatgaa ttccactggt 1260 ggcagtagca gtggcggtgg cattgggctg accctcgggg gaaccatggg cagcaatgcc 1320 ctgagcttct ccagcagtgc gggtcctggg ctcctgaagg cttattccat ccggaccgca 1380 tccgccagtc gcaggagtgc ccgcgactga 1410 <210> 3 <211> 6760 <212> DNA <213> Artificial Sequence <400> 3 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taaggatt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attackttt acatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttattttc taaatacatt caatatgta 540 tccgctcatg aattaatttct tagaaaact catcgagcat caatgaac tgcaatttat 600 tcatatcagg attatcaata ccatatttt gaaaaagccg ttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctgta tcggtctgcg attccgactc 720 gtccaacatc atacaacct attaatttcc cctcgtcaaa ataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gatccggtg agaatggcaa aagtttagc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaccaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 attackacaac aggaatcgaa tgcaccggc gcaggacc tgccagcgca tcacaat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgtttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860. accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggga acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gccttttc ggttcctggc cttttgctgg cttttgctc acatgttctt tcctgcgtta 2160 tccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggag cggaagagcg cctgatgcgg 2280. tattttctcc ttacgcatct gtgcggtatt tcacaccgca fatherggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980 attaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040 ttttgtttaa cttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100 agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160 ggtcgcggat ccatgtccat cacttcagc tccccggtat tcacctcgcg ctcagccgcc 5220 ttctcgggcc gcggcgccca ggtgcgcctg agctccgctc gccccggcgg cttggcagc 5280 agcagcctct acggcctcgg cgcctcacgg ccgcgcgtgg ccgtgcgctc tgcctatggg 5340 ggcccggtgg gcgccggcat ccgcgaggtc accattaacc agagcctgct ggccccgctg 5400 cggctggacg ccgacccctc cctccagcgg gtgcgccagg aggagagcga gcagatcaag 5460 accctcaaca acaagtttgc ctccttcatc gacaaggtgc gttttctgga gcagcagaac 5520 aagctgctgg agaccaagtg gacgctgctg caggagcaga agtcggccaa gagcagccgc 5580 ctcccagaca tctttgaggc ccagattgct ggccttcggg gtcagcttga ggcactgcag 5640 gtggatgggg gccgcctgga ggcggagctg cggagcatgc aggatgtggt ggaggacttc 5700 aagaataagt acgaagatga aattaaccac cgcacagctg ctgagaatga gttttgtggtg 5760 ctgaagaagg atgtggatgc tgcctacatg agcaaggtgg agctggaggc caaggtggat 5820 gccctgaatg atgagatcaa cttcctcagg accctcaatg agacggagtt gacagagctg 5880 cagtcccaga tctccgacac atctgtggtg ctgtccatgg acaacagtcg ctccctggac 5940 ctggacggca tcatcgctga ggtcaaggcg cagtatgagg agatggccaa atgcagccgg 6000 gctgaggctg aagcctggta ccagaccaag ttgagaccc tccaggccca ggctgggaag 6060 catggggacg acctccggaa tacccggaat gagattcag agatgaaccg ggccatccag 6120 aggctgcagg ctgagatcga caacatcaag aaccagcgtg ccaagttgga ggccgccatt gccgaggctg aggagcgtgg ggagctggcg ctcaaggatg ctcgtgccaa gcaggaggag 6240 ctggaagccg ccctgcagcg gggcaagcag gatatggcac ggcagctgcg tgagtaccag gaactcatga gcgtgaagct ggccctggac atcgagatcg ccacctaccg caagctgctg gagggcgagg agagccggtt ggctggagat ggagtggag ccgtgaat ctctgtgatg 6420. aattccactg gtggcagtag cagtggcggt ggcattgggc tgaccctcgg gggaaccatg 6480 ggcagcaatg ccctgagctt ctccagcagt gcgggtcctg ggctcctgaa ggcttattcc 6540. atccggaccg catccgccag tcgcaggagt gcccgcgact gaaagcttgc ggccgcactc 6600 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat

Claims

1. Application of recombinant probiotics expressing Keratin 7 protein in the preparation of drugs for the treatment of diabetes.

2. The use according to claim 1, characterized in that The recombinant probiotics expressing Keratin 7 protein are added with pharmaceutically acceptable excipients or carriers to prepare any pharmaceutically acceptable dosage form.

3. The use according to claim 2, characterized in that The dosage form is an oral preparation, which includes tablets, capsules, and granules.

Images