CN114478742B

CN114478742B - Helicobacter pylori resistant active polypeptide and application thereof

Info

Publication number: CN114478742B
Application number: CN202210230514.0A
Authority: CN
Inventors: 张孝林; 熊友谊; 秦梅松; 窦金凤; 周国梁; 张钦元
Original assignee: Anhui University of Science and Technology
Current assignee: Anhui University of Science and Technology
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2023-07-04
Anticipated expiration: 2042-03-10
Also published as: CN114478742A

Abstract

The invention belongs to the technical field of medicines for treating bacterial infection diseases, and particularly relates to an anti-helicobacter pylori active polypeptide and application thereof. The amino acid sequence of the helicobacter pylori active polypeptide is shown as sequence NO. 1. The minimum antibacterial concentration of the polypeptide to helicobacter pylori in gastric acid or gastric protein is as low as 8 mug/mL; the polypeptide can not induce helicobacter pylori to generate drug resistance, but also resist helicobacter pylori strain with drug resistance to antibiotics; the polypeptide shows good selective toxicity to prokaryotes and little or no toxicity to eukaryotes.

Description

Helicobacter pylori resistant active polypeptide and application thereof

Technical Field

The invention belongs to the technical field of medicines for treating bacterial infection diseases, and particularly relates to an anti-helicobacter pylori active polypeptide and application thereof.

Background

Helicobacter pylori infection can lead to clinical symptoms of chronic gastritis, peptic and duodenal ulcers, gastric cancer, and mucosa-associated lymphomas. The world health organization has classified these bacteria as a class of carcinogens. People with high helicobacter pylori infection rates are generally at greater risk of gastric cancer than those without infection. In china, gastric cancer has become the second leading cause of death next to the second largest cancer of lung cancer. The above-mentioned conditions of helicobacter pylori infection are associated with long-term colonization of the stomach by helicobacter pylori. Currently, proton pump combined triple therapy with two antibiotics or quadruplex therapy with bismuth re-agent is the standard therapy for helicobacter pylori infection. However, helicobacter pylori is resistant to various antibiotics, and the cure rate of helicobacter pylori is decreasing. Thus, there is a need to develop new antibacterial compounds capable of killing helicobacter pylori strains that are resistant to antibiotics.

Among the known compounds, antibacterial peptide (AMPs) may be a suitable candidate, for example, LL-37 (a cationic antibacterial peptide) is an antibacterial protein derived from neutrophils and various epithelial cells, with a molecular weight of 18kDa. LL-37 is a member of the human Cathelicidin family, and is a polypeptide cleaved from the carboxy terminus of hCAP 18. LL-37 has potent antibacterial effects against both gram-positive and gram-negative bacteria. The prior researches find that the LL-37 not only has stronger bactericidal action on wild helicobacter pylori strain than antibiotics, but also has stronger bactericidal action on antibiotic-resistant helicobacter pylori strain. Unfortunately, the acidic environment of the stomach and pepsin have a great influence on its anti-helicobacter pylori activity. Mass spectrometry analysis showed that LL-37 became a short peptide after incubation in simulated gastric fluid. At the same time, its initial activity against helicobacter pylori is almost lost.

There is no disclosure of antimicrobial peptides or other antibiotics that have little or no effect on helicobacter pylori activity in the presence of gastric acid and pepsin.

Disclosure of Invention

In view of the above, the present invention aims to provide an anti-helicobacter pylori active polypeptide or a functional derivative thereof for the treatment of helicobacter pylori infection, which has an antibacterial activity against helicobacter pylori in gastric acid or pepsin, at a minimum concentration of as low as 8 μg/mL. The amino acid sequence of the anti-helicobacter pylori active polypeptide is shown as sequence No. 1.

Specifically, the helicobacter pylori active polypeptide was designed based on LL-37.

Specifically, the short peptide KRIVQRIKDFLR-NH2 (designated KR-12) of amino acid residues 18 to 29 of LL-37 exhibited stronger antimicrobial activity than LL-37, but this short peptide was degraded in the presence of gastric acid and pepsin, and the shorter peptide KRIVQRIKD-NH2 (designated KR-9) of 9 amino acids in the degraded composition was determined not to be degraded by pepsin at pH <1.3 and pH > 2. Based on-line study data https:// web. Expasy. Org/peptide_cutter/and mass spectrometric analysis, the study showed that KR-12 was the smallest LL-37-derived short peptide with antibacterial activity, KR-9 did not have antibacterial activity, including anti-helicobacter pylori activity, based on structural and activity relationship studies. Based on the above, the invention creatively adds 3 amino acid residues at the C-terminal of KR-9 to form peptides containing 12 amino acid residues in each short peptide, and 28 peptides are designed in the process of the invention.

Further, the present invention uses on-line work https:// web. Expasy. Org/peptide_cutter/and mass spectrometric technique for analysis and mass spectrometry in order to ensure that the 28 designed peptides are stable at low pH and in the presence of pepsin. The 28 polypeptides designed were then screened for the strongest antimicrobial activity and designated as SAMP-12aa. The bactericidal activity of the SAMP-12aa and the safety of clinical application thereof are further verified, and the result shows that the SAMP-12aa short peptide designed based on LL-37 maintains the bactericidal activity and the bactericidal kinetic activity in simulated gastric fluid; SAMP-12aa exerts an antibacterial effect while itself does not induce the production of drug-resistant strains, and also has antibacterial activity against antibiotic-resistant helicobacter pylori strains; and its bactericidal mechanism was found to be based on enhancing the permeability of the outer membrane and disrupting the integrity of the inner membrane; SAMP-12aa shows good selective toxicity to prokaryotic cells and little toxicity to eukaryotic cells.

The object of the present invention is also to provide a SAMP-12aa with very good helicobacter pylori killing kinetics in simulated gastric fluid.

Further, the effective dose is not less than the minimum concentration of the helicobacter pylori resistant activity of the helicobacter pylori resistant active polypeptide, namely, the minimum antibacterial concentration or the bactericidal concentration of the helicobacter pylori resistant active polypeptide is selected according to actual needs.

Further, the 8. Mu.g/mL is the minimum inhibitory concentration of the anti-helicobacter pylori polypeptide against helicobacter pylori, and the minimum inhibitory concentration of the anti-helicobacter pylori polypeptide against helicobacter pylori is 32. Mu.g/mL.

Further, the pharmaceutical may be formulated into a dosage form suitable for oral administration.

The invention aims to provide an application of the helicobacter pylori resistant active polypeptide in preparation of a preparation for inhibiting or killing helicobacter pylori.

Further, the helicobacter pylori is antibiotic-resistant. The anti-helicobacter pylori active polypeptide also has killing activity on helicobacter pylori with antibiotic resistance.

Further, the helicobacter pylori is resistant to antibiotics. Helicobacter pylori resistant to existing first-line helicobacter pylori antibiotics (such as metronidazole, amoxicillin, clarithromycin), and the helicobacter pylori resistant polypeptide also has bactericidal activity against it.

The invention also aims to provide the application of the anti-helicobacter pylori active polypeptide in serving as or preparing a preparation for increasing the permeability of the outer membrane of bacterial cells, in particular to helicobacter pylori.

The invention also aims to provide the application of the anti-helicobacter pylori active polypeptide in preparation of a preparation for destroying the integrity of bacterial cell inner membranes, in particular to helicobacter pylori.

The invention aims to provide the application of the anti-helicobacter pylori active polypeptide in preparing medicines for treating diseases caused by helicobacter pylori infection, wherein the diseases comprise, but are not limited to, chronic gastritis, peptic ulcer, duodenal ulcer, gastric cancer and mucosa-associated lymphomas.

In the present invention, the term "functional derivative" refers to a regulatory polypeptide in which the amino acid of the polypeptide having anti-helicobacter pylori activity is regulated but has the same function as the polypeptide having anti-helicobacter pylori activity.

The invention has the beneficial effects that: the anti-helicobacter pylori active polypeptide provided by the invention has the inhibition concentration of helicobacter pylori in gastric acid or gastric protein as low as 8 mug/mL; the anti-helicobacter pylori can not induce drug-resistant helicobacter pylori, and has the activity of resisting the antibiotic helicobacter pylori; it shows good selective toxicity to prokaryotes and little toxicity to eukaryotes.

Drawings

FIG. 1 shows the bactericidal kinetics of SAMP-12aa prepared with physiological saline against H.pylori ATCC43504 strain.

FIG. 2 shows the bactericidal kinetics of SAMP-12aa prepared with artificial gastric juice against H.pylori ATCC43504 strain.

FIG. 3 shows the effect of SAMP-12aa on H.pylori outer membrane permeability.

FIG. 4 shows the effect of SAMP-12aa on the permeability of H.pylori membrane.

FIG. 5 shows that in vitro antibiotic induced production of H.pylori resistant bacterial strain while the SAMP-12aa peptide itself did not induce production of resistant bacterial strain.

FIG. 6 shows that SAMP-12aa has little or no cytotoxicity on human gastric adenocarcinoma cells, demonstrating safety for oral administration.

Wherein, in FIG. 4, A is the condition of cells observed by fluorescence confocal microscopy after incubation of 1mL of helicobacter pylori suspension with 1mL of PBS (control) and 2mL of 100. Mu.M Propidium Iodide (PI) for 90 minutes at room temperature under microaerophilic conditions; b is 1mL of helicobacter pylori suspension with 2mL of 100. Mu.M Propidium Iodide (PI) and 1mL of 16. Mu.g/mL SAMP-12aa under microaerophilic conditions at room temperature for 90 minutes, and the cells were observed by fluorescence confocal microscopy.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

In the embodiment of the invention, the artificial gastric juice is prepared according to the following formula: the artificial gastric juice per liter contains 2.05 g of sodium chloride, 8.3 g of peptone, 0.6 g of potassium dihydrogen phosphate, 0.37 g of KCl, 3.5 g of D-glucose, 0.11 g of calcium chloride, 0.05 g of bile, 13.3 mg of pepsin and 0.1 g of lysozyme. In addition, 5.5g of urea was supplemented in artificial gastric juice to evaluate the effect of helicobacter pylori urease activity.

In the examples of the present invention, helicobacter pylori strains were used: ATCC43504 (NCTC 11637), helicobacter pylori strain ATCC700392 (26659), helicobacter pylori strain ATCC 63629 (NCTC 11639), helicobacter pylori strain SS1, helicobacter pylori gastric ulcer clinical strain and helicobacter pylori gastric cancer clinical strain were stored in the laboratory of the applicant.

In the example of the present invention, the liquid medium used was helicobacter pylori liquid medium (3.85 g, 93 ml distilled water 121 ℃ for 15 minutes, and fetal bovine serum or 7 ml of sterilized defibrinated sheep blood at 50-55 ℃ C.) was added (Qingdao Gaojingyuan Haibo biotechnology Co., ltd.).

In the implementation of the invention, the preparation of the helicobacter pylori solution is as follows: reviving helicobacter pylori ATCC43504 in the liquid culture medium prepared as described above; then, helicobacter pylori was inoculated into Columbia blood agar supplemented with 7% defibrinated sheep blood, and cultured under microaerophilic conditions at 37℃for 36 hours, and bacterial preparation in the logarithmic growth phase was collected for use.

In the embodiment of the invention, the method for measuring the number of helicobacter pylori cells comprises the following steps: viable bacterial cells were counted by plating appropriately diluted helicobacter pylori cells on a Columbia blood agar plate and grown into single colonies, followed by colony counting on a solid medium, and the number of viable helicobacter pylori cells was determined by cell colony counting.

Example 1 design and Synthesis of Polypeptides

In the practice of the present invention, fragments and sequencing and on-line tools are used in accordance with the hydrolysis of human host defensin LL-37 in acid and pepsinhttps://web.expasy.org/peptide_cutter/18-26 amino acid residues KRIVQRIKD-NH2 of the antibacterial peptide LL-37 were predicted and established by mass spectrometry to be stable in the presence of acid and pepsin, but not antibacterial activity, and the 12 amino acid residue peptide was found to be the peptide with the shortest antibacterial activity according to extensive experiments of the present invention in the early stages. The present example uses 9 amino acid residues to supplement 3 amino acid residues at the carboxy terminus (C-terminal) of the peptide to arrive at a polypeptide having finally 12 amino acid residues, 28 polypeptides were designed in total, as shown in Table 1, and by an on-line toolhttps://web.expasy.org/peptide_cutter/These polypeptides were analyzed and by mass spectrometry these short peptides were not degraded by pepsin in the simulated fluid.

TABLE 128 polypeptides contemplated by the present invention

In the embodiment of the invention, all polypeptides are synthesized by a biological biotechnology company of the Shanghai or a blaze biotechnology company, and the purity of the polypeptides is 95% through high performance liquid chromatography detection.

EXAMPLE 2 screening for the polypeptide having the strongest anti-helicobacter pylori Activity and designated as SAMP-12aa

In the embodiment of the invention, the specific steps for detecting the antimicrobial polypeptide with the strongest activity against helicobacter pylori ATCC43504 are as follows: (1) Bacteria in logarithmic growth phase were obtained by culturing helicobacter pylori under microaerophilic conditions as described above at 37℃for 36 hours, and then a bacterial solution (1X 10) ⁸ CFU/ml); (2) The polypeptide was diluted from 256. Mu.g/mL to 1. Mu.g/mL with liquid medium using double dilution (serial dilutions were made 9 times in 9 tubes, no polypeptide was used as a control in the 10 th tube), and then 10. Mu.l of bacterial solution was added to each tube to give a tube bacterial concentration of about 10 ⁶ CFU/ml; (3) The cell culture was then shaken at 200 rpm under microaerophilic conditions at 37℃for 36 hours, the optical transparency of the tube was observed, and the Minimum Inhibitory Concentration (MIC) was determined from the absence of visible turbidity or bacterial growth

In the examples of the present invention, the MICs of 28 polypeptides designed in example 1 against helicobacter pylori ATCC43504 were measured by a double dilution method, and the antimicrobial peptide having the strongest activity against helicobacter pylori ATCC43504 was established, and as shown in Table 2, there was a large difference between these polypeptides, among which, the antimicrobial peptide having the strongest activity against helicobacter pylori ATCC43504 was KRIVQRIKDVIR, the MIC of which was 8. Mu.g/mL, and KRIVQRIKDVIR was designated as SAMP-12aa.

Anti-helicobacter pylori ATCC43504 Strain Activity of the Polypeptides designed in Table 2

EXAMPLE 3 survival analysis of helicobacter pylori in artificial gastric juice

In the examples of the present invention, the method for the survival analysis of helicobacter pylori ATCC43504 in artificial gastric juice is as follows: (1) Logarithmic phase helicobacter pylori ATCC43504 cultured in a liquid medium at 37 ℃ for 24 hours in a microaerophilic atmosphere was centrifuged at 3000 rpm for 10 minutes to collect bacterial cell pellet; (2) Bacterial cell pellet was then split into two equal parts for survival experiments and used as a control, and then cells were suspended in physiological saline (PBS) as a control group and artificial gastric juice as a survival experiment, respectively, and their concentrations were adjusted to 10, respectively ⁸ CFU/ml; (3) The number of H.pylori ATCC43504 cells was then measured at various time points after 0 minutes, 30 minutes and 60 minutes of exposure to PBS or artificial gastric juice, and the results are shown in Table 3, in which the survival rate of H.pylori was decreased with time, H.pylori is a microaerophilic bacterium to which high concentration of oxygen had a certain toxic effect, and artificial gastric juice had little effect on the survival of H.pylori, i.e., it was shown that H.pylori was highly adapted to artificial gastric juice.

TABLE 3 survival results of H.pylori ATCC43504 strain in artificial gastric juice

EXAMPLE 4 minimum inhibitory and bactericidal concentration and bactericidal kinetics of SAMP-12aa against helicobacter pylori ATCC43504 in artificial gastric juice

In the examples of the present invention, to demonstrate the bactericidal activity of SAMP-12aa in artificial gastric juice, the Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC) and the bactericidal kinetics of SAMP-12aa were determined in liquid and solid media prepared with physiological saline and artificial gastric juice (prepared by the method of example 3).

(1) Minimum inhibitory concentration test of SAMP-12aa in Artificial gastric juice

In the examples of the present invention, the minimum inhibitory concentration of SAMP-12aa in artificial gastric juice is 8. Mu.g/mL as shown in example 2; the minimum inhibitory concentration in physiological saline was also 8. Mu.g/mL.

(2) Minimum bactericidal concentration test of SAMP-12aa in artificial gastric juice

In the examples of the present invention, 5. Mu.l of the mixed solution was taken out of the clarified test tube of the SAMP-12aa group of example 2 and inoculated on a solid agar medium, respectively; they were then incubated at 37℃for 72 hours under microaerophilic conditions. The minimum peptide concentration without colony growth is the Minimum Bactericidal Concentration (MBC) of the polypeptide SAMP-12aa.

As a result, it was found that SAMP-12aa exhibited the same antibacterial activity in a liquid medium prepared with physiological saline or artificial gastric juice, and that MIC and MBC thereof were 8. Mu.g/mL and 32. Mu.g/mL, respectively, i.e., it was revealed that the antibacterial activity of SAMP-12aa was not affected by artificial gastric juice, as shown in Table 4 below.

TABLE 4 minimum inhibitory concentration and minimum inhibitory concentration of helicobacter pylori ATCC43504 strain in different environments

Medium preparation	Minimum inhibitory concentration (μg/mL)	Minimum sterilizing concentration (μg/mL)
			Physiological saline	8	32
Artificial gastric juice	8	32

(3) Sterilization kinetics of SAMP-12aa in Artificial gastric juice

In the practice of the present invention, the method for determining the number of viable cells after exposure of H.pylori to a solution of SAMP-12aa is: handle 10 ⁶ After the helicobacter pylori of CFU/mL is incubated with the SAMP-12aa solution with different concentrations for a certain period of time, the viable Count (CFU) is measured, 100 mu L of the helicobacter pylori liquid at different exposure time points is taken for 10 times ratio serial dilution, and the solution is coated on a solid agar medium for CFU counting (the colony count is between 30 and 300 and is an effective colony count).

In the examples of the present invention, in order to further determine whether artificial gastric juice has an effect on the sterilization kinetics of SAMP-12aa, SAMP-12aa was prepared with physiological saline and artificial gastric juice having concentrations of 2 XMIC, 4 XMIC, 8 XMIC and 16 XMIC, respectively, and 10 ⁶ CFU/mL H.pylori incubation. The number of viable cells after exposure to SAMP-12aa solution prepared with physiological saline and artificial gastric juice, respectively, at 37 ℃ was determined for 0 min, 5 min, 15 min, 30 min, 45 min and 60 min. The results are shown in fig. 1 and 2:

SAMP-12aa solution in physiological saline was prepared with 16. Mu.g/mL (2 MIC) and exposure times of 5 minutes, 15 minutes, 30 minutes, 45 minutes and 60 minutes, helicobacter pylori survival number (CFU) 10 ^5.8±0.36 ,10 ^5.4±0.31 ,10 ^3.5±0.32 ,10 ^3.1±0.28 The method comprises the steps of carrying out a first treatment on the surface of the Preparation of SAMP-12aa solution from artificial gastric juice, helicobacter pylori survival number (CFU) 10 ^5.8±0.45 ,10 ^5.5±0.36 ,10 ^3.6±0.35 ,10 ^3.2±0.34 (P>0.05 without significant difference).

SAMP-12aa solution in physiological saline was prepared with 32. Mu.g/mL (4 MIC) and exposure times of 5 minutes, 15 minutes, 30 minutes, 45 minutes and 60 minutes, helicobacter pylori survival number (CFU) 10 ^5.2±0.37 ，10 ^3.5±0.33 ，10 ^1.7±0.31 ，10 ^0.3 ^±0.37 Preparation of SAMP-12aa solution from artificial gastric juice, helicobacter pylori survival number (CFU) 10 ^5.2±0.39 ，10 ^3.7±0.36 ，10 ^1.7 ^±0.38 ，10 ^0.4±0.34 (P>0.05 without significant difference).

In SAMP-12aa solution prepared with physiological saline at 64. Mu.g/mL (8MIC=2MBC) and exposure times of 5, 15, 30, 45 and 60 minutes, the viable bacteria Count (CFU) was detected at only 5 and 15 minutes for H.pylori survival (CFU) of 10 ^4.6±0.35 ，10 ^2.4±0.39 No viable bacteria were detected after 30 minutes; in the preparation of SAMP-12aa solution with artificial gastric juice, the viable count of H.pylori (CFU) was detected at only 5 minutes and 15 minutes to 10 ^4.7±0.37 ，10 ^4.5±0.31 No viable bacteria were detected after 30 minutes, (P>0.05 without significant difference).

In SAMP-12aa solution prepared with physiological saline at 128. Mu.g/mL (16 MIC=4 MBC) and 5, 15, 30, 45 and 60 minutes exposure time, the number of surviving helicobacter pylori (CFU) was detected only at 5 minutes for 10 ^3.3±0.37 No viable bacteria were detected after 15 minutes; in the preparation of SAMP-12aa solution with artificial gastric juice, the viable count of helicobacter pylori (CFU) was only 10 detected at 5 minutes ^3.4±0.38 No viable bacteria were detected after 15 minutes, (P>0.05 without significant difference).

The above results indicate that the effect of artificial gastric juice on the bactericidal kinetics of SAMP-12aa is not obvious, and that SAMP-12aa shows good anti-helicobacter pylori activity in gastric juice.

EXAMPLE 5 permeability test of SAMP-12aa to increase outer Membrane

In the implementation of the invention, according to the 'the antibacterial mechanism of the polypeptide is to enhance the outer membrane permeability, and when the integrity of a cell membrane is damaged, bacteria are not easy to generate drug resistance to the polypeptide'; and according to the fact that helicobacter pylori is a gram-negative bacterium having an outer membrane structure, the outer membrane of the bacterium has a certain preventing effect on the entry of drugs into cells, and if the antibacterial agent has the ability to penetrate the outer membrane, a better sterilizing effect is obtained than in the case of being unable to penetrate the outer membrane; 1-N-phenyl-naphthylamine (NPN) exhibits weaker fluorescence in aqueous solution than in hydrophobic environment, and NPN fluorescence intensity will increase if SAMP-12aa has the ability to enhance permeability of the outer membrane of helicobacter pylori, NPN probe enters the hydrophobic environment of bacterial cell wall (outer membrane) from solution. "principle design test.

In the examples of the present invention, the preparation method of the helicobacter pylori ATCC43504 cell suspension comprises: (1) Cell pellet was collected by centrifugation at 5000 Xg for 10 min with 50 ml of H.pylori cells in logarithmic growth phase; (2) Then after three washes with buffer, the cell pellet was suspended in HEPES sodium buffer (pH 7.2); (3) The optical density of the suspension cells was then set at OD ₆₀₀ Down-regulating to 0.5 for standby.

In the practice of the present invention, SAMP-12aa solutions were prepared at concentrations of 4, 8, 16, 32 and 64. Mu.g/mL; 4ml of each test sample at different concentrations contained: 1ml of SAMP-12aa solution, 2ml of helicobacter pylori ATCC43504 cell suspension and 1ml of 40mM NPN; while the 4ml control sample contained: 1ml of HEPES buffer without peptide, 2ml of helicobacter pylori ATCC43504 cell suspension and 1ml of 40mM NPN.

In the examples of the present invention, the effect of SAMP-12aa on the permeability of the outer membrane of H.pylori ATCC43504 was tested by using a fluorescence spectrophotometer (F4600, hitachi, tokyo, japan) using hydrophobic 1-N-phenyl naphthylamine (NPN) as a fluorescent probe, using a method of measuring the fluorescence intensity at 30 seconds intervals from the beginning without any increase in the fluorescence intensity by using an excitation wavelength of 350nm and an emission wavelength of 420 nm. As shown in FIG. 3, when the helicobacter pylori suspension contained SAMP-12aa, the fluorescence intensity of NPN was remarkably enhanced, and the enhancement of fluorescence exhibited dose dependence, and SAMP-12aa at different concentrations could increase the permeability of the membrane to different extents, as shown by the change of the fluorescence intensity with time (when NPN penetrated the hydrophobic environment of the helicobacter pylori outer membrane, the fluorescence intensity increased), i.e., it was demonstrated that SAMP-12aa had the ability to enhance the permeability of the helicobacter pylori outer membrane due to the effect of SAMP-12aa on the outer membrane.

EXAMPLE 6 integrity test of SAMP-12aa disrupting cell membranes

In the embodiments of the present invention, according to "Propidium Iodide (PI) can enter dead cells but cannot enter living cells; after PI enters cells, the PI is combined with DNA to emit fluorescence; that is, if SAMP-12aa breaks the integrity of the bacterial cell membrane, resulting in cell death, a "fluorescence" principle design test is generated, and the test results can be used to demonstrate whether SAMP-12aa breaks the integrity of the H.pylori cell inner membrane and causes bacterial death, and when the cells die, the absorption of PI is promoted, and fluorescence is generated.

In the examples of the present invention, to determine whether SAMP-12aa disrupts the integrity of the cell membrane of H.pylori ATCC43504, after thorough mixing, the following procedure was used: (1) 4mL of a test sample containing 1mL of H.pylori cell suspension (prepared according to the preparation method of example 5), 1mL of 16. Mu.g/mL of SAMP-12aa polypeptide and 2mL of 100. Mu.M Propidium Iodide (PI), and 4mL of a control sample containing 1mL of H.pylori cell suspension (prepared according to the preparation method of example 5), 1mL of PBS buffer (pH=7.2) and 2mL of 100. Mu.M Propidium Iodide (PI) were stored at room temperature in the absence of light for 90 minutes, respectively; (2) Fluorescence was observed using a fluorescence confocal microscope (LSM 710Meta, zeiss, jene, gemany) at 535nm excitation wavelength and 615nm emission wavelength. The results are shown in FIG. 4, in which H.pylori incubated with PBS as a control is shown as A, which showed no fluorescence spots observed, indicating that PI was not absorbed by the cells; however, when H.pylori was incubated with SAMP-12aa, the results are shown in B, which has a distinct fluorescent spot, indicating that PI can enter the cell and bind to DNA. Namely, SAMP-12aa has the ability to disrupt the integrity of bacterial cell membranes and thus has bactericidal efficacy. This bactericidal mechanism of SAMP-12aa does not readily induce resistance.

EXAMPLE 7 SAMP-12aa does not induce drug resistance test by itself when antibacterial

In the examples of the present invention, the use of H.pylori strain ATCC43504 explored in vitro that the SAMP-12aa peptide does not induce resistance while being antibacterial. Metronidazole, amoxicillin and clarithromycin are the conventional antibiotics used clinically to treat helicobacter pylori infection as positive controls. The Minimum Inhibitory Concentration (MIC) of SAMP-12aa and the antibiotic is determined according to the procedure of example 2.

In the implementation of the invention, the drug resistance analysis is carried out according to the following method: (1) Will be in exponential growth phaseHelicobacter pylori is incubated under microaerophilic conditions for 36 hours in a medium containing subtropical concentrations of anti-different antibacterial substances, and then cells exhibiting approximately 50% growth inhibitory activity are collected by centrifugation; (2) The cells were further diluted with fresh medium and adjusted to 10 ⁵ CFU/ml concentration, and culturing again until 15 similar continuous cultures are reached; the medium without antimicrobial agent was used as a negative control; (3) The change in MIC after continuous exposure of helicobacter pylori to sub-inhibitory concentrations of the antibacterial agent was evaluated and if the relative MIC per generation was increased, the results indicated that the agent induced the production of drug-resistant strains.

In the examples of the invention, the relative MIC for each passage is determined by calculating the ratio of MIC to the measured value for a given subculture, which is compared to the value obtained from the first exposure; that is, after 15 serial passages, the relative MIC is the ratio of the MIC obtained for a given subculture to the MIC obtained for the first exposure.

In the examples of the present invention, to determine whether antibiotic-resistant H.pylori strains are susceptible to SAMP-12aa, MIC of the resistant strain to SAMP-12aa was detected according to the method of the above-described resistance analysis, and statistical data were processed from the three analyses. As shown in FIG. 5, SAMP-12aa does not induce resistance to helicobacter pylori; the relative MIC of SAMP-12aa for H.pylori remained stable during 15 consecutive subcultures; however, the first-line antibiotics for the treatment of helicobacter pylori infection induce helicobacter pylori resistance to them to varying degrees. The resistance of H.pylori to metronidazole is particularly pronounced after repeated administration. Metronidazole has a 35-fold increase in MIC for H.pylori, followed by clarithromycin, which has a 16-fold increase in MIC for H.pylori. Helicobacter pylori induces resistance to amoxicillin, which increases the MIC against helicobacter pylori by a factor of 6. Furthermore, the helicobacter pylori strain which is resistant to the antibiotics is still susceptible to SAMP-12aa. The results indicate that SAMP-12aa is useful for the treatment of drug-resistant helicobacter pylori infection.

Example 8 therapeutic index test of SAMP-12aa

According to the embodiment of the invention, if the antibacterial polypeptide medicament has prokaryotic selectivity, shows optimal antibacterial activity and kills bacterial cells but not mammalian cells, the peptide medicament has good therapeutic potential and relative clinical safety. The Therapeutic Index (TI) reflects the prokaryotic selectivity of the polypeptide. "design test".

In the examples of the present invention, the Therapeutic Index (TI) of the polypeptide was determined by calculating the ratio of the Minimum Hemolysis Concentration (MHC) value obtained for six different helicobacter pylori strains (helicobacter pylori 43504 (NCTC 11637), helicobacter pylori 700392 (26659), helicobacter pylori SS1ATCC 63629 (NCTC 11639), helicobacter pylori SS1, helicobacter pylori gastric ulcer clinical strain, helicobacter pylori gastric cancer clinical strain) to the Geometric Mean (GM) of MIC value; minimum Hemolysis Concentration (MHC) is determined as the concentration at which a polypeptide can induce 10% hemolysis; the therapeutic index (PI) is calculated as the ratio of MHC (. Mu.g/mL) to GM (. Mu.g/mL).

In the examples of the present invention, the hemolytic activity of SAMP-12aa is determined according to the following procedure: (1) Fresh human blood was collected using a 1000 Xg centrifugation for 7 minutes to obtain human red blood cells (hRBCs) and then washed with PBS (43 mM Na ₂ HPO ₄ 、11mM KH ₂ PO ₄ And 80mM NaCl) carefully and thoroughly washing hRBCs 3 times; (2) 4% (w/v) hRBC suspension was prepared using the hRBC resuspended in PBS as described above; (3) Five different concentrations of SAMP-12aa were prepared with two-fold serial dilutions from 512. Mu.g/mL to 32. Mu.g/mL, then 250. Mu.L of the different concentrations of SAMP-12aa and 250. Mu.L of 4% w/v hRBC suspension were added to the same 0.5mL centrifuge tube and thoroughly mixed; in the same manner, 250. Mu.L of PBS and 250. Mu.L of 4% w/v hRBC suspension without SAMP-12aa were added to the same 0.5mL centrifuge tube as a negative control, and then 250. Mu.L of 0.1% Triton X-100 and 250. Mu.L of 4% w/v hRBC suspension were added to the same 0.5mL centrifuge tube as the positive control. (4) All mixtures were incubated for 1 hour in a 37 ℃ water bath, then centrifuged at 1000 x g for 5 minutes, 200 μl supernatant was collected from each centrifuge tube, and then added to the wells of a 96-well plate; (5) The absorbance of the supernatant was measured at 414nm using a microplate reader.

In the examples of the present invention, absorbance value (A _PBS ) Identified as zero hemolysis, supernatant of 0.1% (v/v) Triton X-100-lysed hRBCsAbsorbance value of the liquid (A _Triton ) Determined as 100% hemolysis; the hemolytic activity of SAMP-12aa was calculated as percent hemolysis according to the formula SAMP-12aa hemolysis (%) = (a) _sample -A _PBS )/(A _Triton -A _PBS ) X 100. The minimum hemolysis concentration value (minimal hemolytic concentration) is expressed as MHC.

In the examples of the present invention, in order to accurately determine the MIC of a peptide for six different H.pylori strains, SAMP-12aa was diluted from 4. Mu.g/mL to 12. Mu.g/mL in 1. Mu.g/mL increments, and the MIC was determined according to the method of example 2, and the Geometric Mean (GM) of MIC values for the different H.pylori strains were expressed as GM by SAMP-12aa.

In the examples of the present invention, the GM, MHC and TI values of SAMP-12aa were calculated as described above and are shown in Table 4. The results show that SAMP-12aa has stronger antibacterial specificity than the antibiotic evaluated; the hemolytic concentration of SAMP-12aa reaches 216. Mu.g/mL, and the concentration is low in hemolytic activity (high in MHC). MIC was 8.5. Mu.g/mL, indicating high antibacterial activity (GM low). Thus, the antimicrobial peptide SAMP-12aa is a desirable antimicrobial agent for the treatment of helicobacter pylori infection.

TABLE 5 Treatment Index (TI) of SAMP-12aa

EXAMPLE 9 cytotoxicity Studies of SAMP-12aa

In the practice of the present invention, human gastric adenocarcinoma cells (ATCC; CRL-1739) are used to determine the cytotoxicity of SAMP-12aa.

In the examples of the present invention, standard MTT proliferation assays were used to determine the cytotoxicity of SAMP-12aa on CRL-1739 cells, assessed by measuring insoluble blue-violet crystalline formazan, which is produced only in living cells where the succinate dehydrogenase in the mitochondria reduces exogenous MTT, whereas dead cells do not have this function; the method comprises the following specific steps:

(1) CRL-1739 cells were cultured in RPMI-1640 medium supplemented with 5% CO for 3-5 days ₂ 10% of the bovine serum.

(2) Adherent cells were treated by trypsinization to form dispersed single cells, and then cell pellet was collected after centrifugation at 1000×g for 4 min. Preparation of 1X 10 with medium containing 10% fetal bovine serum ⁵ cell/mL cell suspension.

(3) 200 μl of the cell suspension was then added to each well of a 96-well plate, and the plate was incubated at 37 ℃ with 5% co ₂ For 24 hours to form adherent cells.

(4) 40 microliters of SAMP-12aa solution was added to the wells, and the solution was serially diluted 2-fold from 256. Mu.g/mL to 16. Mu.g/mL with RPMI-1640 medium. Wells containing only 40 μl RPMI-1640 medium and no SAMP-12aa were used as controls. Plates were further incubated for 48 hours.

(5) mu.L of 5mg/mL MTT was then added to each well and carefully thoroughly mixed. Plates were incubated for 4 hours at 37 ℃. The supernatant from each well was carefully removed.

(6) Then, 150 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the insoluble blue-violet crystalline formazan produced by MTT. Microplate ELISA readers were used to measure absorbance at 550nm of the dissolved complete formazan crystals.

In the examples of the present invention, CRL-1739 cell viability was calculated using the formula survivin (%) = (A550 of SAMP-12aa-treated cells)/A550 of SAMP-12aa-untreated cells). Times.100.

In the examples of the present invention, the cytotoxicity of SAMP-12aa against human gastric adenocarcinoma cells (ATCC; CRL-1739) was evaluated as described above, and the results are shown in FIG. 6, which shows that SAMP-12aa is non-toxic to CRL-1739 cells at 128. Mu.g/mL and has more than 90% of living cells at this concentration. The results indicate that SAMP-12aa is a good candidate for development and application in the treatment of helicobacter pylori infection due to its low toxicity to eukaryotic cells.

EXAMPLE 10 test of clearance of SAMP-12aa on helicobacter pylori in the stomach of mice

In the implementation of the invention, the SAMP-12aa nanoparticle is prepared according to the technical scheme disclosed in patent 201410485607.3 (patent name: preparation method of anti-helicobacter pylori active antibacterial peptide gastric mucosa nanoparticle drug delivery system).

In the examples of the present invention, an animal model of helicobacter pylori infection was established using helicobacter pylori SS1 strain. After the mice were randomly divided into 10 groups (8 in each group) and fasted for 24 hours, 0.3 ml of the stomach was filled with 10 ⁸ BIH broth of CFU helicobacter pylori. 14 days after inoculation (2 per group, established by microscopic examination, oxidase test, catalase test and urease test, validated model), SAMP-12aa and SAMP-12aa nanoparticles were orally administered at a dose of 1, 3, 10 or 30mg/kg body weight, once daily for three consecutive days. Placebo mucoadhesive nanoparticles were orally administered in the same manner as a control. One day after the last dose, mice were euthanized under anesthesia and the stomach was homogenized in 1.5mL BHI broth using a tissue homogenizer. Eradication of H.pylori was determined using a microbiological culture method. The remaining homogenates were serially diluted 10-fold and spread evenly on BHI agar plates supplemented with 7% sheep blood and the above antibiotics. Plates were incubated under microaerophilic conditions for 4 days at 37 ℃. The number of H.pylori bacteria was determined by counting bacterial colonies on agar plates.

The bacteria were then further identified by microscopy, oxidase test, catalase test and urease test. Bacterial colony counts in each mouse stomach were calculated by counting colonies on each plate and expressed as log CFU per stomach sample. The results are shown in Table 6.

Table 6 compares the effects of mucosal delivery of SAMP-12aa and SAMP-12aa nanoparticles on H.pylori clearance in the stomach of mice

Note that: ND: not detected (no detection)

The average bacterial count in the stomach of the mice, the non-drug-receiving control mice, and an average of about 10 in the stomach of each mouse are shown in Table 6 ^7.57 (CFU/stomach) bacterial colonies. Average bacterial count in the stomach of mice treated with oral SAMP-12aa was followedThe dose was increased but decreased, but no complete clearance was observed, even at the highest dose of 30 mg/kg. However, the gastric mucosa nanoparticle prepared by taking SAMP-12aa has obviously enhanced helicobacter pylori clearance, and completely eliminates helicobacter pylori in the stomach when the dosage of 10mg/kg is taken orally.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Sequence listing

<110> applicant's Anhui academy of science and technology

<120> an anti-helicobacter pylori active polypeptide and application thereof

<130> 2022-3-3

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Lys Arg Ile Val Gln Arg Ile Lys Asp Val Ile Arg

1 5 10

Claims

1. Use of a polypeptide having anti-helicobacter pylori activity for the manufacture of a medicament for the treatment of a disease caused by helicobacter pylori infection, characterized in that the disease is chronic gastritis, peptic ulcer; the medicine is used for killing helicobacter pylori;

the amino acid sequence of the helicobacter pylori active polypeptide is shown as SEQ ID NO. 1.