A polypeptide and its preparation method and application in resisting Gardnerella

Abstract

The present invention discloses a polypeptide and a preparation method thereof and an application thereof in resisting Gardnerella. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1. The present invention provides a novel polypeptide having an excellent antibacterial effect specifically against Gardnerella, which is even better than the commonly used clinical antibiotic metronidazole, and has good safety and is substantially non-toxic to normal epidermal cells within a normal therapeutic dose, thereby providing a new technical option for solving the drug resistance problem that often occurs in current clinical antibiotic treatment schemes.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide drugs, and in particular to a polypeptide and a preparation method thereof and application thereof in resisting Gardnerella. Background Art

[0002] Bacterial vaginosis (BV) is a common vaginal infectious disease, the main manifestation of which is increased vaginal discharge accompanied by a fishy odor and occasional itching. It is directly related to a variety of gynecological and obstetric diseases, such as endometritis, salpingitis and pelvic inflammatory disease. In addition, it increases the risk of adverse pregnancy and sexually transmitted diseases, seriously affecting women's health.

[0003] The essence of BV is vaginal flora imbalance, lactobacillus reduction, and a mixed infection caused by the massive reproduction of pathogenic microorganisms mainly Gardnerella vaginalis (G. vaginalis). Therefore, effective inhibition of Gardnerella is the key to preventing and treating BV. The current standard clinical regimen is the combined treatment of metronidazole and clindamycin. Although the discomfort symptoms can be temporarily relieved, broad-spectrum antibiotics inhibit the growth of lactobacillus in the normal vaginal flora, aggravate the disorder of the vaginal microecology, and cause up to 50% of women to have recurrence after the initial successful treatment. The treatment of recurrent BV is more difficult. The existing measures are mainly to extend the course of antibiotics, which can easily lead to the emergence of drug-resistant strains and greatly reduce the treatment effect. Therefore, it is urgent to develop new anti-Gardnerella drugs that are highly specific and not easily resistant. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a polypeptide and a preparation method thereof and application thereof in resisting Gardnerella.

[0005] The first object of the present invention is to provide a polypeptide.

[0006] The second object of the present invention is to provide a method for preparing the polypeptide.

[0007] The third object of the present invention is to provide the use of the polypeptide in resisting Gardnerella.

[0008] The fourth object of the present invention is to provide the use of the polypeptide in preparing products against Gardnerella.

[0009] The fifth object of the present invention is to provide the use of the polypeptide in the preparation of a product for treating urogenital tract infections.

[0010] A sixth object of the present invention is to provide an anti-Gardnerella preparation.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] Biofilm is a membrane-like structure formed when bacteria attach to the surface of inorganic materials or organisms in order to adapt to changes in the living environment during their growth. The microorganisms themselves are wrapped in intercellular matrices such as polysaccharides and fibrin. There are two main reasons why biofilms are resistant to traditional antibiotics: first, the dense structure of the biofilm itself can effectively resist the attack of antibiotics and the immune system; second, the metabolic activity of bacteria in the special environment of the biofilm is low. Over-proliferation of Gardnerella in the vagina can produce clustered and dense biofilms that adhere tightly to the vaginal epithelium, continuously release cytotoxins, show stronger drug resistance, and are very likely to form persistent infections. Therefore, the formation of Gardnerella biofilm is the main reason for repeated attacks after antibiotic treatment.

[0013] The hemagglutinin fusion peptide is located at the N-terminus of the hemagglutinin protein 2 domain. It is a hydrophobic sequence of 20 amino acids. This sequence is the only sequence in the hemagglutinin protein that is inserted into the host cell membrane. The hemagglutinin fusion peptide sequence is very conservative. Although mutations in certain key amino acids do not affect the expression of hemagglutinin, enzymatic cleavage reactions, and conformational changes induced at low pH, they will change the membrane fusion function of hemagglutinin, thereby damaging the integrity of the cell membrane. This mechanism is different from that of most traditional antibiotics, which act on DNR, RNA, or enzymes. The hemagglutinin fusion peptide acts on the bacterial cell membrane and kills bacteria by changing the permeability or perforation of the membrane. Therefore, compared with traditional antibiotics, it has great potential in killing bacteria that have formed biofilms.

[0014] The present invention is mainly based on the structure of hemagglutinin fusion peptide, combines a scorpion venom peptide structure fragment with antibacterial activity with hemagglutinin fusion peptide, and further modifies and transforms the structure. Through rational molecular design and amino acid scanning substitution methods, a new antimicrobial peptide named IFG-22 is obtained. Under the premise of minimal damage to the microecology, it specifically inhibits the overgrowth of Gardnerella and inhibits the formation of mature biofilms of the bacteria. Compared with the traditional antibiotic metronidazole, it shows great potential. In addition, the polypeptide IFG-22 has low toxicity to normal cells.

[0015] Therefore, the present invention requests protection for the following contents:

[0016] A polypeptide, namely IFG-22, whose amino acid sequence is shown in SEQ ID NO.1.

[0017] The polypeptide IFG-22 of the present invention can be synthesized by solid phase synthesis, which has the characteristics of convenient and rapid synthesis, stability and controllability.

[0018] The method for preparing the polypeptide is to prepare it by solid phase synthesis according to the amino acid sequence shown in SEQ ID NO.1.

[0019] Preferably, the amino acids used to synthesize the polypeptide are all L-configuration amino acids.

[0020] Preferably, the solid phase synthesis method is Fmoc synthesis method.

[0021] More preferably, the Fmoc synthesis method comprises the following steps: preparing a resin peptide using a resin and an Fmoc-protected amino acid; cleaving the resin peptide to obtain a crude polypeptide; and purifying the crude polypeptide product by high performance liquid chromatography.

[0022] Further preferably, the Fmoc synthesis method comprises the following steps: preparing a resin peptide by a condensation reaction with an activated resin and a deprotected Fmoc amino acid, 1-hydroxybenzotriazole and N,N-dimethylformamide; cleaving the resin peptide with a cleavage solution, precipitating it with ice ether, washing and drying the resulting precipitate to obtain a crude polypeptide; purifying the crude polypeptide product by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography include: using 0.1 v / v% TFA aqueous solution as mobile phase A, using acetonitrile containing 0.1 v / v% TFA as mobile phase B, using a C18 chromatographic column, 40% to 60% acetonitrile concentration gradient elution, the detection wavelength is 220 nm, and the flow rate is adjusted to 40 mL / min.

[0023] More preferably, the resin is 2-chlorotrichloroethylene resin with a degree of substitution of 0.3 mmol / g.

[0024] Still more preferably, the resin is activated by dichloromethane.

[0025] More preferably, the Fmoc amino acid is deprotected by N,N-dimethylformamide, N,N-diisopropylethylamine, methanol and piperidine, and the successful deprotection is confirmed by ninhydrin colorimetric detection.

[0026] More preferably, the reaction endpoint of the condensation reaction is determined by ninhydrin colorimetric detection.

[0027] More preferably, the lysate is composed of TFA:H 2 O:EDT:Tis=95:1:2:2 composition.

[0028] More preferably, the cracking temperature is 20-25°C.

[0029] Specifically, the Fmoc synthesis method comprises the following steps:

[0030] Resin activation: weigh 0.3 g of 2-chlorotrichloroethylene resin (substitution degree 0.3 mmol / g) and add it to the chromatography column, add 3 mL of dichloromethane (DCM), stir and swell for 5 minutes, then stop, and remove the liquid by suction;

[0031] Amino acid deprotection: feed in an amount 1.2 times the molar number of the resin substitution degree, weigh each Fmoc-protected amino acid according to the amino acid sequence shown in SEQ ID NO.1, pour into the chromatography column in turn, add 5 mL of N,N-dimethylformamide to dissolve, add N,N-diisopropylethylamine dropwise after complete dissolution, and react for 1.5 hours; remove the liquid by suction filtration, then add a mixed solution of DCM, methanol and DIEA, the volume ratio of DCM: methanol: DIEA = 2: 1: 1, react for 30 minutes, and remove the liquid by suction filtration; add 20% piperidine-DMF solution (i.e., the volume ratio of piperidine to DMF is 20: 80) to the chromatography column, stir and react for 10 minutes, remove the liquid, then add 20% piperidine / DMF solution, stir and react for 5 minutes, remove the liquid, and wash with DMF 6 times; take a sample, and detect whether the deprotection is successful by ninhydrin color development, and record the detection color (whether it is blue-purple);

[0032] Amino acid connection: feed materials in an amount three times the molar number of the resin substitution degree, weigh Fmoc amino acid (the first amino acid at the C-terminus) and 1-hydroxybenzotriazole, add 5 mL of DMF to dissolve, add N,N'-diisopropylcarbodiimide dropwise to activate for 10 minutes, then add to the chromatography column, react for 1.5 hours (stop the reaction when the result of the ninhydrin colorimetric method is no color), filter out the liquid; wash the precipitate with DMF 5 times; continue to feed materials according to the amino acid sequence and repeat the above operation until the last amino acid is condensed; then wash with methanol 3 times, filter to dryness, and obtain the resin peptide;

[0033] Peptide cleavage: 15 mL of cleavage buffer (volume ratio, TFA:H 2 O:EDT:Tis=95:1:2:2) ratio, calculate the required amount of lysate; then, add each reagent of the lysate into the lysate reaction bottle in sequence, and control the temperature of the lysate reagent at 0~10℃; add the lysate reagent into the resin peptide under stirring, and after the temperature of the system is stable, control the temperature at 20~25℃, and react for 2 hours under stirring; then, filter out the lysate, precipitate it with 10 times the volume of the filtrate in ice ether, wash it by centrifugation 4 times, and dry the precipitate under reduced pressure at room temperature to obtain a crude polypeptide;

[0034] Purification of polypeptide: Purify the crude polypeptide by liquid chromatography, with 0.1v / v% TFA aqueous solution as mobile phase A, and acetonitrile containing 0.1v / v% TFA as mobile phase B; use 10μm reverse phase C18 (50×250mm) filler, set the detection wavelength of the UV detector to 220nm, adjust the flow rate to 40mL / min, with 40v / v% acetonitrile, balance for 10 minutes; take the crude IFG-22. Grind and dissolve it with 30% acetonitrile aqueous solution, microwave until completely dissolved, filter with 0.45μm filter membrane, and inject; use 40% to 60% acetonitrile concentration gradient elution at room temperature, pay attention to the change of absorption value, remove impurities, collect the target product, and analyze the purified solution by comparing the retention time of the purified component with the retention time of the component in the crude product; repeat this step twice, combine the freeze-dried target product, and obtain it.

[0035] The use of the polypeptide in resisting Gardnerella should also be within the protection scope of the present invention.

[0036] Preferably, the polypeptide inhibits biofilm formation of the Gardnerella.

[0037] Preferably, the polypeptide disrupts or degrades the biofilm of the Gardnerella.

[0038] More preferably, the polypeptide destroys or degrades a mature biofilm formed by the Gardnerella.

[0039] The use of the polypeptide in the preparation of anti-Gardnerella products should also be within the protection scope of the present invention.

[0040] Preferably, the polypeptide inhibits biofilm formation of the Gardnerella.

[0041] Preferably, the polypeptide disrupts or degrades the biofilm of the Gardnerella.

[0042] More preferably, the polypeptide destroys or degrades a mature biofilm formed by the Gardnerella.

[0043] Preferably, the anti-Gardnerella products include disinfectants and cleaners.

[0044] The use of the polypeptide in the preparation of a product for treating urogenital tract infection should also be within the protection scope of the present invention, and the pathogenic bacteria of the urogenital tract infection include Gardnerella.

[0045] Preferably, the polypeptide inhibits biofilm formation of the Gardnerella.

[0046] Preferably, the polypeptide disrupts or degrades the biofilm of the Gardnerella.

[0047] More preferably, the polypeptide destroys or disintegrates a mature biofilm formed by the Gardnerella.

[0048] Preferably, the urogenital tract infection is vaginitis.

[0049] More preferably, the vaginitis is bacterial vaginitis.

[0050] Preferably, the product for treating urogenital tract infection comprises an external wash.

[0051] An anti-Gardnerella preparation comprising the polypeptide.

[0052] Preferably, the preparation further comprises a pharmaceutically acceptable carrier and / or excipient.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention provides a novel polypeptide having an excellent antibacterial effect specifically against Gardnerella, which is even better than the commonly used clinical antibiotic metronidazole, and has good safety and is basically non-toxic to normal epidermal cells within a normal therapeutic dose. It provides a new technical option for solving the drug resistance problem that often occurs in current clinical antibiotic treatment programs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The identification results of IFG-22, A is the HPLC spectrum, B is the mass spectrum.

[0056] Figure 2 These are the evaluation results of the inhibitory effects of each peptide on the mature biofilm formed by Gardnerella.

[0057] Figure 3 These are the test results of the thermodynamic binding strength of each peptide with the Gardnerella biofilm, A is IFG-22; B is metronidazole; C is IKN-22; and D is LLG-22.

[0058] Figure 4 The results of CCK-8 assay of cytotoxicity of IFG-22 on human immortalized keratinocytes HaCaT. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified. Gardnerella vaginalis ATCC49145, Streptococcus mutans ATCC35668, Staphylococcus aureus ATCC25923 and Staphylococcus epidermidis ATCC49134 involved in the following examples were purchased from Guangdong Provincial Microbiological Culture Collection Center; human immortalized keratinocytes (HaCaT) were purchased from Nanjing Kebai Biotechnology Co., Ltd.; a full set of Fmoc-protected amino acids and solid phase resins and condensation agents (HBTU, HOBt) were purchased from Jier Biochemical Co., Ltd. (Shanghai, China); piperidine was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); ninhydrin, trifluoroacetic acid, 1,2-ethanedithiol, toluene thioether, chromatographic grade acetonitrile, chromatographic grade methanol and metronidazole (C 6 H 9 N 3 O 3 ) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); DMEM high glucose medium, 1640 medium, fetal bovine serum and trypsin were purchased from Gibco (USA); bacterial culture media (MH / LB / BHI medium, Columbia blood agar basal medium), defibrinated sheep blood and agar were purchased from Haibo Biotechnology Co., Ltd. (Qingdao, China); CCK-8 kit was purchased from Tongren Chemical Research Institute (Japan); MTT reagent was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0060] Example 1 Design, synthesis and identification of polypeptides

[0061] 1. Peptide design

[0062] The present invention is based on the combination of a scorpion venom peptide structure fragment with antibacterial activity and a hemagglutinin fusion peptide, and further structural modification and transformation are carried out through combinatorial chemistry approaches (such as specific amino acid replacement, mixed amino acid scanning, multi-position scanning, etc.), and a total of 4 polypeptides are designed as shown in Table 1.

[0063] Table 1 Peptide information

[0064]

[0065] Note: The amino acids in the table are all L-configuration amino acids.

[0066] 2. Synthesis of peptides

[0067] In this example, the solid phase synthesis method is used to synthesize each polypeptide. The following operation method can be referred to:

[0068] (1) Synthesis of IFG-22

[0069] Resin activation: Weigh 0.3 g of 2-chlorotrichloroethylene resin (2-Chlorotrityl Chloride, degree of substitution 0.3 mmol / g) and add it to a clean chromatography column, add 3 mL of dichloromethane (DCM), stir and swell for 5 minutes, then stop, and remove the liquid by suction.

[0070] Deprotection of amino acids: feed at 1.2 times the molar amount of the resin substitution degree, weigh each Fmoc-protected amino acid according to the amino acid sequence of IFG-22 (SEQ ID NO.1), pour into the chromatography column in turn, add 5mL N,N-dimethylformamide (DMF) to dissolve, add N,N-diisopropylethylamine (DIEA) dropwise after complete dissolution, react for 1.5h; remove the liquid by suction filtration, then add a mixed solution of DCM, methanol and DIEA, the volume ratio of DCM: methanol: DIEA = 2:1:1, react for 30 minutes, and remove the liquid by suction filtration. Add 20% piperidine-DMF solution (i.e., the volume ratio of piperidine and DMF is 20:80) to the chromatography column, stir and react for 10 minutes, remove the liquid, then add 20% piperidine / DMF solution, stir and react for 5 minutes, remove the liquid, and wash with DMF 6 times; take samples, and detect whether the deprotection is successful by ninhydrin color development, and record the detection color (whether it is blue-purple).

[0071] Amino acid connection: feed 3 times the molar amount of the resin substitution degree, weigh Fmoc amino acid (the first amino acid at the C-terminus) and 1-hydroxybenzotriazole (HoBt), add 5mL DMF to dissolve, add N,N'-diisopropylcarbodiimide (DIC) dropwise to activate for 10 minutes, then add to the chromatography column, react for 1.5h (stop the reaction when the result of the ninhydrin colorimetric method is no color), and remove the liquid by suction. Wash the precipitate with DMF 5 times. Continue to feed according to the amino acid sequence and repeat the above operation until the last amino acid is condensed. Then wash with methanol 3 times, filter and dry, and the resin peptide is obtained.

[0072] Peptide cleavage: 15 mL of cleavage buffer (volume ratio, TFA:H 2O:EDT:Tis=95:1:2:2) ratio, calculate the amount of lysate required. Then, add each reagent of the lysate into the lysate reaction bottle in sequence, and control the temperature of the lysate reagent at 0~10℃; add the lysate reagent into the resin peptide under stirring, and control the temperature at 20~25℃ after the system temperature stabilizes, and stir and react for 2 hours; then filter out the lysate, precipitate it with 10 times the volume of the filtrate in ice ether, wash it by centrifugation 4 times, and dry the precipitate under reduced pressure at room temperature to obtain IFG-22 crude product.

[0073] Purification of polypeptide: IFG-22 crude product was purified by liquid chromatography, with 0.1 v / v% TFA aqueous solution as mobile phase A and acetonitrile containing 0.1 v / v% TFA as mobile phase B; 10 μm reverse phase C18 (50×250 mm) filler was used, the UV detector was set to detect wavelength of 220 nm, the flow rate was adjusted to 40 mL / min, with 40 v / v% acetonitrile, and balanced for 10 minutes; IFG-22 crude product was taken. Grind and crush, dissolve with 30% acetonitrile aqueous solution, microwave until completely dissolved, filter with 0.45 μm filter membrane, and inject; 40% to 60% acetonitrile concentration gradient elution was used at room temperature, attention was paid to the change of absorption value, impurity peaks were removed, and the target product was collected. The retention time of the purified component and the retention time of the component in the crude product were compared to analyze and obtain the purified solution; repeat this step twice, combine the target product obtained by freeze-drying, and obtain the purified IFG-22 freeze-dried powder.

[0074] (2) Synthesis of IKN-22

[0075] According to the amino acid sequence of IKN-22 (SEQ ID NO. 2), the synthesis method of IFG-22 was followed to prepare IKN-22 lyophilized powder.

[0076] (3) Synthesis of NKI-22

[0077] According to the amino acid sequence of NKI-22 (SEQ ID NO. 3), the synthesis method of NKI-22 was followed to prepare IKN-22 lyophilized powder.

[0078] (4) Synthesis of LLG-22

[0079] According to the amino acid sequence of LLG-22 (SEQ ID NO. 4), the synthesis method of LLG-22 was followed to prepare LLG-22 lyophilized powder.

[0080] 3. Identification of peptides

[0081] IFG-22 (SEQ ID NO.1) contains 22 amino acid residues, with an average molecular weight of 2620.28 g / mol. Its extinction coefficient is calculated to be 12660 M -1cm -1 The theoretical isoelectric point is pH 10.63. The chromatogram of the purified IFG-22 identified by HPLC is as follows Figure 1 As shown in A, the mass spectrum is as Figure 1 As shown in B, it can be seen that the purity of IFG-22 is greater than 90%.

[0082] The same identification method was used, and the results showed that the purity of the purified IKN-22, NKI-22 and LLG-22 was also greater than 90%. The four polypeptides prepared above met the basic requirements of subsequent research.

[0083] Example 2 Evaluation of the antibacterial activity of polypeptides against Gardnerella

[0084] 1. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

[0085] Streptococcus mutans (S. mutans ATCC35668), Gardnerella vaginalis (G. vaginalis ATCC49145), Staphylococcus aureus (S. aureus ATCC25923) and Staphylococcus epidermidis (S. epidermidis) were cultured overnight at 37°C and then adjusted to a final concentration of 1.0 × 10 5 CFU / mL, inoculated into 96-well plates at a volume of 100 μL per well.

[0086] The four polypeptides synthesized in Example 1 were added to the culture medium of each bacteria, and the final concentration range was set to 0.5-128 mg / L. The antibiotic metronidazole was used as a positive control, and the sterile culture medium without the test bacteria was used as a negative control. The culture was continued, wherein Streptococcus mutans ATCC35668 was cultured at 37°C under anaerobic conditions for 16-24 hours; Gardnerella vaginalis ATCC49145 was cultured at 37°C under anaerobic conditions for 24-48 hours; and Staphylococcus aureus ATCC25923 and Staphylococcus epidermidis ATCC49134 were cultured at 37°C under aerobic conditions for 16-24 hours. The MIC of each bacteria was determined.

[0087] The supernatant (10 μL) was obtained from the wells showing no obvious bacterial growth in the MIC assay, incubated on agar plates at 37°C for 48 h, and observed. The lowest polypeptide concentration that inhibited bacterial growth on the plates was taken as the MBC.

[0088] The MIC and MBC determinations for all strains were repeated at least three times.

[0089] 2. Measurement results

[0090] Table 2 MIC and MBC test results of peptides against 4 strains (unit: μg / mL)

[0091]

[0092] Note: NT means that the result was out of the detection range and no result was obtained.

[0093] As shown in Table 2, among the four polypeptides in Example 1, IFG-22 (SEQ ID NO.1) has the lowest MIC (reaching 1.5 μg / mL) and the lowest MBC (reaching 3 μg / mL) against Gardnerella, even lower than metronidazole; however, IFG-22 (SEQ ID NO.1) has no obvious advantages in MIC and MBC against the other three bacteria. This indicates that IFG-22 (SEQ ID NO.1) has specificity for Gardnerella, and its antibacterial effect is significantly stronger than other polypeptides and commonly used bactericidal drugs in clinical practice.

[0094] Example 3 Evaluation of the inhibitory effect of peptides on mature biofilms formed by Gardnerella

[0095] 1. Experimental methods

[0096] Gardnerella vaginalis ATCC49145 in the logarithmic growth phase was added to a fresh culture medium containing 0.5% (w / v) sucrose, and the bacterial solution concentration was adjusted to 1.0×10 7 CFU / mL, inoculated into a 96-well plate at a volume of 100 μL per well. Next, the 96-well plate was centrifuged at 1300×g for 2 minutes to precipitate the cells, and cultured at 37°C under anaerobic conditions for 24 hours to form a biofilm. The supernatant was then removed, and the four polypeptides synthesized in Example 1 and metronidazole were dissolved in PBS, and adjusted to a final concentration of 2 μg / mL, 4 μg / mL and 8 μg / mL, and then added to the wells, with PBS as a negative control. After treatment for 30 minutes, the liquid was removed and PBS was added for washing. After MTT staining, the OD 570 The absorbance value at 100 nm was used to record the biofilm growth.

[0097] 2. Experimental results

[0098] like Figure 2 As shown, for Gardnerella that has formed a mature biofilm, the OD under treatment with different concentrations of metronidazole and NKI-22 (SEQ ID NO.3) 570 There was no significant difference in the OD values ​​under IFG-22 (SEQ ID NO.1), IKN-22 (SEQ ID NO.2) and LLG-22 (SEQ ID NO.4) treatments. 570The values ​​were dose-dependent and decreased with the increase of polypeptide concentration. The OD 570 The decrease in the value is the most significant. This shows that IFG-22 (SEQ ID NO.1) has a significantly stronger inhibitory effect on the mature biofilm formed by Gardnerella, destroys the biofilm, and kills Gardnerella by changing the permeability or perforation of the membrane, especially showing great potential compared with traditional antibiotics.

[0099] Example 4 Thermodynamic effects of peptide binding to Gardnerella biofilm

[0100] 1. Experimental methods

[0101] The logarithmic growth phase Gardnerella was collected and centrifuged (4000 rpm, 15 minutes), then repeatedly washed with PBS and the bacterial solution was suspended with PBS, followed by the addition of a mixed solvent of chloroform and methanol (1:2, volume ratio). After stirring for 90 minutes, chloroform-water (1:1, volume ratio) was added and further stirred for 30 minutes. The chloroform phase was separated by a separatory funnel, the solvent was removed by rotary evaporation, and then dried in a vacuum for 24 hours to obtain biomembrane lipids of Gardnerella.

[0102] 5% DMSO was added to the biomembrane lipids of Gardnerella to dissolve and adjust the concentration to 4 mg / mL. After 10 minutes of degassing, the heat changes of the interaction between IFG-22 (SEQ ID NO.1), IKN-22 (SEQ ID NO.2) and LLG-22 (SEQ ID NO.4) synthesized in Example 1 and metronidazole (all at a concentration of 4 μg / mL) and biomembrane lipids were measured by an isothermal titration calorimeter (ITC, MicroCal PEAQ-ITC, Malvern, UK). The ITC reaction conditions are as follows: temperature: 25°C; number of injections: 19; volume: 2 μL; interval: 100 seconds; reference power: 10 μcal / sec; stirring speed: 750 rpm. The model of thermodynamic parameters provided and analyzed by MicroCalPEAQ-ITC software is used to calculate the thermodynamic binding data of the test compound and the biomembrane.

[0103] 2. Experimental results

[0104] like Figure 3 As shown in A in FIG, as the titration number of IFG-22 increases, an obvious heat change trend curve is shown, indicating that the binding effect of IFG-22 (SEQ ID NO.1) and the Gardnerella biofilm is more significant, and the binding coefficient K dThe value was 60 μM, and the ITC thermodynamic curve showed that the ITC peak was negative, and ΔH = -2.79 (kJ / mol), -TΔS = -21.3 (kJ / mol), indicating that IFG-22 (SEQ ID NO.1) specifically targeted and bound to the Gardnerella biofilm, and this binding was mainly an exothermic reaction. Figure 3 As shown in Figures B to D, the heat change trend of metronidazole and the other two polypeptides after being added to the biofilm was not significant, indicating that the binding effect with the Gardnerella biofilm was weak.

[0105] Example 5 Evaluation of the toxic effect of IFG-22 on normal epidermal cells

[0106] 1. Experimental methods

[0107] To evaluate the toxicity of IFG-22 (SEQ ID NO.1) to normal epidermal cells, human immortalized keratinocytes HaCaT were used as experimental subjects and the CCK-8 method was used for determination. The method was as follows: HaCaT (about 5×10 4 / well) were inoculated in a 96-well plate, and after overnight growth, fresh culture medium containing different gradient concentrations of IFG-22 (SEQ ID NO.1) synthesized in Example 1 was added, and the culture was continued for 48 hours; then 10 μL of CCK-8 was added to each well, and the cells were incubated at 37°C for 2 hours; the absorbance was measured at 450 nm using an ELISA reader, and the cytotoxicity of IFG-22 (SEQ ID NO.1) was evaluated by comparing the growth inhibition rate of cells after sample treatment. The inhibition rate of the untreated control group cells was set to 0%.

[0108] 2. Experimental results

[0109] like Figure 4 As shown, IFG-22 (SEQ ID NO.1) acted on human immortalized keratinocytes HaCaT for 48 hours. When the working concentration was below 62.5 μg / mL, the growth of HaCaT cells was basically not inhibited. This concentration range is much higher than its half bactericidal dose (3 μg / mL), with a high safety index and good safety, which meets the toxicity safety requirements for the preparation of external vaginal wash preparations.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. 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 claims of the present invention.

Claims

1. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. The method for preparing the polypeptide according to claim 1, characterized in that: According to the amino acid sequence shown in SEQ ID NO.1, it was prepared by solid phase synthesis.

3. The preparation method according to claim 2, characterized in that The solid phase synthesis method is the Fmoc synthesis method.

4. Use of the polypeptide according to claim 1 in the preparation of products against Gardnerella.

5. Use of the polypeptide according to claim 1 in the preparation of a product for treating urogenital tract infections, characterized in that: The pathogenic bacteria of the urogenital tract infection include Gardnerella, and the urogenital tract infection is vaginitis.

6. The use according to claim 4 or 5, characterized in that: The polypeptide according to claim 1 inhibits the biofilm formation of the Gardnerella.

7. The use according to claim 4 or 5, characterized in that: The polypeptide of claim 1 degrades the biofilm of the Gardnerella.

8. An anti-Gardnerella preparation, characterized in that Comprising the polypeptide according to claim 1.

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