Polypeptide for inhibiting helicobacter pylori

Abstract

The invention relates to the technical field of biology, in particular to a polypeptide for inhibiting helicobacter pylori. The polypeptide can significantly inhibit the growth of helicobacter pylori and reduce the urease activity of the helicobacter pylori, not only can effectively reduce the colonization number of the helicobacter pylori in the stomach, but also can inhibit the generation of proinflammatory cytokine interleukin 1 beta and tumor necrosis factor alpha, promote the secretion of anti-inflammatory cytokine interleukin 10, inhibit the growth of the helicobacter pylori and inhibit the growth of the helicobacter pylori. The preparation has huge potential in preventing and treating damage of helicobacter pylori to gastric tissues, and has practical application and popularization values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide for inhibiting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (H.pylori, Hp) is a common Gram-negative bacterium with a width of 0.5-1 μm and a length of 2-4 μm. It is usually spiral-shaped and sometimes rod-shaped and grows in a microaerophilic environment. Hp infection usually occurs in early childhood, and the prevalence increases with age. It now infects about 4.4 billion people worldwide. If left untreated, it may not only cause local complications such as peptic ulcer disease, gastric adenocarcinoma, or mucosa-associated lymphoid tissue lymphoma in adulthood, but also systemic complications associated with clinical inflammation, including iron deficiency anemia, thrombocytopenic purpura, growth retardation, vitamin B12 deficiency, and even cardiovascular, metabolic complications or autoimmune diseases. However, it is difficult to eradicate Hp infection. In clinical practice, only a few antibiotics, such as amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and rifabutin, can be used to effectively eradicate HP, and these antibiotics are usually given to patients in combination therapy. Combination therapy includes two or three of these antibiotics in combination with a proton pump inhibitor. With the widespread use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, and the success rate of treating Helicobacter pylori infection continues to decline.

[0003] Antimicrobial peptides have broad-spectrum antimicrobial activity and can effectively inhibit a variety of bacteria, including Helicobacter pylori. This broad spectrum makes them perform well against multi-drug resistant strains. At the same time, the bactericidal effect of antimicrobial peptides is very rapid and can significantly reduce the number of bacteria in a short period of time. For example, the Epinecidin-1 antimicrobial peptide (FIFHIKGLFHAGKMIHGLVTRRRH) extracted and isolated from grouper can reduce the bacterial count of Helicobacter pylori by 99% after 1 hour and 12 hours of culture in culture medium. However, these antimicrobial peptides are composed of ten or dozens of amino acids. Whether they are extracted from animals or plants or synthesized, the cost is high and cannot be promoted and applied in subjects. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a polypeptide consisting of only three amino acids having excellent Helicobacter pylori inhibitory activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a polypeptide for inhibiting Helicobacter pylori, and the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0007] The present invention also provides a use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an anti-Helicobacter pylori drug.

[0008] Furthermore, the drug is a drug that inhibits the growth of Helicobacter pylori.

[0009] Furthermore, the drug is a drug that reduces the urease activity of Helicobacter pylori.

[0010] Furthermore, the medicine is a medicine for improving gastritis caused by Helicobacter pylori infection.

[0011] Furthermore, the drug has the effect of reducing the levels of pro-inflammatory factors interleukin 1β and tumor necrosis factor α, and increasing the level of anti-inflammatory factor interleukin 10.

[0012] Furthermore, the drug is a drug for improving gastric damage caused by Helicobacter pylori infection.

[0013] Furthermore, the drug has the effect of reducing the infiltration of inflammatory cells in the gastric submucosal layer and the accumulation of blood vessels.

[0014] The present invention also provides a drug for resisting Helicobacter pylori infection, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 and pharmaceutically acceptable excipients.

[0015] Furthermore, it is in the form of solution, powder, paste, granule, tablet or pill.

[0016] The sequence of the polypeptide of the present invention is shown in SEQ ID NO: 1. It has been proved through experiments that the polypeptide can significantly inhibit the growth activity of Helicobacter pylori and reduce the urease activity of Helicobacter pylori. It can not only effectively reduce the number of Helicobacter pylori colonization in the stomach of mice, but also inhibit the production of pro-inflammatory cytokines interleukin 1β and tumor necrosis factor α, and promote the secretion of anti-inflammatory cytokine interleukin 10. Mouse gastric tissue pathological analysis showed that the polypeptide reduced the infiltration of inflammatory cells in the stomach of mice, eliminated gastric vascular congestion, and improved gastritis in mice. The polypeptide is easy to synthesize artificially, has great potential for preventing and treating Helicobacter pylori damage to gastric tissue, and has practical application and promotion value.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HPLC chart of the functional polypeptide of the present invention having the function of inhibiting the growth of Helicobacter pylori;

[0020] Figure 2 The MS mass spectrum of the bifunctional polypeptide capable of inhibiting the growth of Helicobacter pylori of the present invention;

[0021] Figure 3 This is the antibacterial graph of the functional polypeptide of the present invention. Note: The arrow indicates the antibacterial circle;

[0022] Figure 4 This is the antibacterial rate diagram of the functional polypeptide of the present invention, note: *P<0.05, ***P<0.001;

[0023] Figure 5 This is a graph showing the effect of the functional polypeptide of the present invention on the urease activity of Helicobacter pylori. Note:

[0024] **P<0.01, ***P<0.001;

[0025] Figure 6 The rapid urease test of each group, A is the Helicobacter pylori group, B is the 0.25 mg / g (body weight) polypeptide group; C is the 1 mg / g (body weight) polypeptide group; D is the quadruple drug group, and E is the normal saline group;

[0026] Figure 7 The inflammatory factor content of each group of mice, A is interleukin 1β, B is tumor necrosis factor α, C is interleukin 10

[0027] Figure 8 The following are the pathological sections of mice in each group. A is the Helicobacter pylori group, B is the 0.25 mg / g (body weight) polypeptide group, C is the 1 mg / g (body weight) polypeptide group, D is the quadruple drug group, and E is the normal saline group. DETAILED DESCRIPTION

[0028] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, the polypeptide of the present invention can be purchased or synthesized according to the following Fmoc synthesis method. The specific synthesis preparation steps are:

[0029] Under the catalytic action of HOBt / DCC condensation, 2-CTC-Resin resin was selected as the solid phase carrier to extend the peptide chain from the carboxyl end to the amino end. Triisopropylsilane: trifluoroacetic acid: anisole: water was prepared into a mixed solution in a volume ratio of 5:4:88:3, and it was separated and cut from the resin. After repeated precipitation with ether, it was purified by preparative RP-HPLC. A Kromasil C18 column (4.6mm X250 mm, 5μm) was used; the mobile phase was 0.1‰ trifluoroacetic acid, 10%-35% (volume percentage) acetonitrile as the mobile phase, the detection wavelength was 220nm, and the flow rate was 1.0mL / min for gradient elution. The purity was >95% (detected by RP-HPLC). Figure 1 ), and after freeze-drying, the polypeptide was obtained, and its amino acid sequence was SEQ ID NO: 1: Gly-Tyr-Pro.

[0030] The molecular weight of the product was identified by MS spectrometry, which was consistent with the theoretical molecular weight of the peptide intermediate to be prepared, m / z 333.6 [M+H] + ,m / z 671.25[2M+H] + ( Figure 2 ).

[0031] The preparation technology of the solution, powder, paste, granule, tablet and pill made from the polypeptide of the present invention is mature and can be prepared by referring to the conventional method in the prior art.

[0032] Example 1 Preparation of an aqueous extract containing the polypeptide of the present invention

[0033] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is taken, deionized water is added and mixed, and then filtered and sterilized to obtain a solution.

[0034] Example 2 Preparation of powder containing polypeptide of the present invention

[0035] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is taken, and a pharmaceutically acceptable excipient is added and mixed evenly, and the mixture is pulverized by dry method or wet method according to the dryness and wetness of the material, and the powder is sieved to obtain a powder.

[0036] Example 3 Preparation of an oil containing the polypeptide of the present invention

[0037] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is taken, and a pharmaceutically acceptable excipient is added, mixed, and compressed into tablets to prepare tablets.

[0038] Example 4 Preparation of an emulsion containing the polypeptide of the present invention

[0039] A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 is taken, and a pharmaceutically acceptable excipient is added, mixed, and granulated to obtain a granule.

[0040] The beneficial effects of the present invention are further illustrated by test examples below.

[0041] Experimental Example 1 Study on the in vitro antibacterial activity of the polypeptide of the present invention against Helicobacter pylori

[0042] 1. Helicobacter pylori inhibition zone test

[0043] 1.1 Methods

[0044] The Helicobacter pylori culture stored in an ultra-low temperature refrigerator at -80°C was thawed at 4°C, 50 μL of the Helicobacter pylori culture was spread on a Columbia blood agar plate in an ultra-clean workbench, and cultured at 37°C for 48 h in a microaerobic environment (7% oxygen, 10% carbon dioxide, 83% nitrogen). The colonies were picked and resuspended in tryptone soy broth medium. After solidification in the plate, the polypeptide diluted with sterile water was inoculated. At the same time, sterile water was used as a negative control and amoxicillin was used as a positive control. After culturing at 37°C for 24 h in a microaerobic environment, the inhibition zone was observed.

[0045] 1.2 Results

[0046] For specific results, see Figure 3 ,from Figure 3 From the results, compared with the negative control (lower left corner) and the positive control (lower right corner), the polypeptide solutions in the upper left and upper right corners of the plate produced inhibition zones, indicating that the polypeptide of the present invention can inhibit the growth of Helicobacter pylori.

[0047] 2. Helicobacter pylori inhibition rate experiment

[0048] 2.1 Methods

[0049] The inhibitory effect of peptides on Helicobacter pylori was determined by the doubling dilution method. The concentration of Helicobacter pylori solution was adjusted to 1×10 8CFU / mL, add 200 μL of 20 mg / mL polypeptide solution of the present invention to well A1 of the 96-well plate, add 100 μL of tryptone soy broth to wells A2-A7, then aspirate 100 μL of polypeptide solution in well A1 to well A2, mix well, take 100 μL of polypeptide solution in well A2 to well A3, and so on, dilute to well A6, discard the solution, add 100 μL of diluted bacterial solution to wells A1-A7, and perform three biological replicates per well. The working concentrations of the polypeptide in the experiment were 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, and 0 mg / mL, with 3 biological replicates per well. The 96-well plate was placed in a microaerobic environment (7% oxygen, 10% carbon dioxide, 83% nitrogen). After culturing at 37°C for 24 hours, each group of suspensions was spread on Columbia blood agar plates for colony counting.

[0050] 2.2 Results

[0051] For specific results, see Figure 4 . Figure 4 The results showed that when Helicobacter pylori culture was co-cultured with different concentrations of polypeptides for 24 hours, it was found that the minimum bactericidal concentration of the polypeptide was 5 mg / mL.

[0052] 3. Urease activity test of Helicobacter pylori

[0053] 3.1 Methods

[0054] The peptide solutions with working concentrations of 5, 2.5, 1.25, and 0.625 mg / mL and amoxicillin solution with a concentration of 1×10 8 The suspension of Helicobacter pylori with a volume of 1 mL CFU / mL was incubated in tryptone soy broth for 24 h and then the suspension was collected and the number of bacteria (10 4 Urease activity detection reagent was added in a ratio of 500 to 1000:1 in the extract volume (mL), followed by ultrasonic disruption in an ice bath, power 300w, ultrasonic 3s, interval 7s, total time 3min; then centrifuged in a high-speed refrigerated centrifuge at 4°C, 12000rpm for 15min, and finally the supernatant was placed on ice. Each group had 3 biological replicates, and the urease activity detection kit (provided by Beijing Solebow Technology Co., Ltd.) was used for the operation instructions, and the light absorption value of each well at 630nm was measured using an ELISA reader. At the same time, the concentration was 1×10 8 CFU / mL, and a volume of 1 mL of Helicobacter pylori liquid was used as the control, specifically named as the Hp group.

[0055] 3.2 Results

[0056] For specific results, see Figure 5.Depend on Figure 5 It can be seen that compared with the Hp group, the urease activity in the polypeptide experimental group was significantly lower than that in the Hp group. When the concentration of the polypeptide solution was greater than 2.5 mg / mL, the effect of reducing urease activity was equivalent to that of the amoxicillin group, ***P < 0.001, indicating that the polypeptide can significantly reduce the urease activity of Helicobacter pylori.

[0057] 4. Effect of peptides on improving gastritis in mice caused by Helicobacter pylori

[0058] 4.1 Methods

[0059] 40 BALB / c mice were divided into 5 groups, 8 mice in each group, and the groups were as follows:

[0060] Helicobacter pylori group: intragastric administration of Helicobacter pylori liquid for 1-4 weeks and intragastric administration of normal saline for 5-6 weeks;

[0061] 0.25 mg / g (body weight) peptide group: intragastric administration of Helicobacter pylori liquid for 1-4 weeks, intragastric administration of peptide for 5-6 weeks;

[0062] 1mg / g (body weight) peptide group: intragastric administration of Helicobacter pylori liquid for 1-4 weeks, intragastric administration of peptide for 5-6 weeks;

[0063] Quadruple drug group: intragastric administration of Helicobacter pylori solution for 1-4 weeks, and intragastric administration of quadruple drugs for 5-6 weeks;

[0064] Normal saline group: normal saline was gavaged intragastrically for 1-4 weeks and normal saline was gavaged intragastricly for 5-6 weeks.

[0065] The quadruple drug group selected the human recommended dosage of 20mg / 60kg (body weight) esomeprazole, 1000mg / 60kg (body weight) amoxicillin, 500mg / 60kg (body weight) clarithromycin, and 300mg / 60kg (body weight) colloidal pectin bismuth, which is converted into the mouse dosage of 3.03ug / g (body weight) esomeprazole, 151.67ug / g (body weight) amoxicillin, 75.83ug / g (body weight), and 45.5ug / g (body weight) colloidal pectin bismuth.

[0066] The weight and mental state of the experimental mice were recorded during the experiment. The stomach tissues of the mice were taken out 6 hours after the last intragastric administration for rapid urease test to test the eradication rate of Helicobacter pylori and pathological section test; specifically:

[0067] The mouse stomach tissue was added to the rapid urease reaction solution. The rapid urease reaction solution itself is yellow. When urease is present, urease decomposes the urea in the solution to produce ammonia, which increases the Ph value of the solution and changes the solution from yellow to purple-red. The depth of purple-red is proportional to the urease content. At the same time, the stomach tissue of each group of mice was taken for pathological sectioning.

[0068] After administration, blood was collected from the mouse orbits, and the levels of serum interleukin-1β, tumor necrosis factor-α, and interleukin-10 in each group of mice were detected by ELISA.

[0069] 4.2 Results

[0070] The results are as follows Figures 6 to 8 As shown. Figure 6 It can be seen that the solution of the saline group (E) is all yellow, indicating that none of the mice are infected with Helicobacter pylori. The solution of the Helicobacter pylori group (A) is all purple-red, indicating that all 8 mice are infected with Helicobacter pylori, 3 mice in the 0.25 mg / g (body weight) polypeptide group are infected with Helicobacter pylori, 2 mice in the 1 mg / g (body weight) polypeptide group and the quadruple drug group are purple-red, and the rest are yellow. It can be seen that the 1 mg / g (body weight) polypeptide group has a better eradication rate for Helicobacter pylori fixed in the mouse stomach than the 0.25 mg / g polypeptide group.

[0071] like Figure 7 As shown, compared with the Helicobacter pylori group, after the mice were treated with the polypeptide and quadruple drug, the levels of pro-inflammatory factors interleukin 1β and tumor necrosis factor α in the three groups of mice decreased, and the level of anti-inflammatory factor interleukin 10 increased. This shows that the polypeptide of the present invention can reduce the inflammatory response by reducing the pro-inflammatory factors in mice infected with Helicobacter pylori and increasing the release of anti-inflammatory factors, thereby protecting the gastric tissue of mice from the inflammatory response caused by Helicobacter pylori infection.

[0072] Depend on Figure 8 It can be seen that the gastric mucosal layer, submucosal layer, muscular layer and serosa structure of the five groups of mice are relatively intact, but the gastric submucosal layer of the mice in the Helicobacter pylori group (A) has inflammatory cell infiltration of lymphocytes and neutrophils, and red blood cells gather in the blood vessels, with obvious congestion symptoms; after treatment with the polypeptide of the present invention and the quadruple drug, the 0.25 mg / g (body weight) polypeptide group (B) has a small amount of inflammatory cell infiltration of neutrophils in the gastric submucosal layer of the mice, but no congestion is gathered in the blood vessels, and no inflammatory cell infiltration is found in the gastric submucosal layer of the 1 mg / g (body weight) polypeptide group (C), the quadruple drug group (D), and the normal saline group (E), and no congestion is gathered in the blood vessels, indicating that the polypeptide can improve the damage caused by Helicobacter pylori stimulating the gastric tissue of mice, and the effect is equivalent to that of the quadruple drug.

[0073] In summary, the polypeptide provided by the present invention can inhibit the growth activity of Helicobacter pylori and reduce the urease activity of Helicobacter pylori. In vivo animal experiments show that the polypeptide can not only effectively reduce the number of Helicobacter pylori colonization in the stomach of mice, but also inhibit the production of pro-inflammatory cytokines interleukin 1β and tumor necrosis factor α, promote the secretion of anti-inflammatory cytokine interleukin 10, and improve the damage caused by Helicobacter pylori to the gastric tissue of mice. It can be used to prevent or treat gastritis, gastric ulcer and other diseases caused by Helicobacter pylori, and has broad prospects.

Claims

1. A polypeptide for inhibiting Helicobacter pylori, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an anti-Helicobacter pylori drug.

3. The use according to claim 2, characterized in that: The drug is a drug that inhibits the growth of Helicobacter pylori.

4. The use according to claim 2, characterized in that: The drug is a drug that reduces the urease activity of Helicobacter pylori.

5. The use according to claim 2, characterized in that: The medicine is a medicine for improving gastritis caused by Helicobacter pylori infection.

6. The use according to claim 5, characterized in that: The drug has the effects of reducing the levels of pro-inflammatory factors interleukin 1β and tumor necrosis factor α, and increasing the level of anti-inflammatory factor interleukin 10.

7. The use according to claim 2, characterized in that: The medicine is a medicine for improving gastric damage caused by Helicobacter pylori infection.

8. The use according to claim 7, characterized in that: The drug has the effects of reducing the infiltration of inflammatory cells in the gastric submucosal layer and the congestion and accumulation of blood vessels.

9. A drug for resisting Helicobacter pylori infection, characterized in that: The invention comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 and pharmaceutically acceptable excipients.

10. The drug according to claim 9, characterized in that It is in the form of solution, powder, paste, granules, tablet or pill.

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