Antibacterial peptide RB3 and application thereof
By extracting and solid-phase synthesizing the antimicrobial peptide RB3 from the hemolymph of Penaeus vannamei, the problems of antibiotic resistance and dysbacteriosis in the treatment of endometritis were solved, and effective prevention and treatment of endometritis was achieved. It has significant antibacterial activity and potential for large-scale preparation.
Patent Information
- Application Number
- CN202510630155.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-09-30
AI Technical Summary
Existing antibiotics for the treatment of endometritis are prone to lead to drug resistance and disrupt the balance of beneficial bacteria in the uterine cavity. Existing antimicrobial peptides are insufficiently targeted against endometritis-related pathogens and are limited in large-scale production.
An antimicrobial peptide RB3 was developed by extracting it from the hemolymph of the shrimp Litopenaeus vannamei and preparing it by solid-phase synthesis. It has significant antibacterial activity against endometritis pathogens such as Escherichia coli, Klebsiella, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecium.
It significantly inhibits the growth and proliferation of endometritis pathogens, maintains the normal flora structure and immune microenvironment of the endometrium, and achieves effective prevention and treatment of endometritis. It is suitable for large-scale preparation and clinical application.
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Figure CN120718098A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to an antimicrobial peptide RB3 and applications thereof. Background Art
[0002] Endometritis is a common gynecological inflammatory disease, mainly caused by infection of the endometrium by pathogens such as bacteria, mycoplasma or chlamydia. Its occurrence and development are closely related to the imbalance of the uterine cavity microecology. The traditional view is that the uterine cavity is a relatively sterile environment, but recent studies have shown that it contains a low-biomass microbial community dominated by Lactobacillus. When the normal flora structure of the uterine cavity is disturbed, the excessive proliferation of pathogenic bacteria can cause a disorder of the immune microenvironment, leading to an inflammatory response of the endometrium. According to statistics from the World Health Organization (WHO), approximately 10% to 15% of women of childbearing age worldwide suffer from endometritis, of which about 30% of cases progress to chronic inflammation due to untimely treatment, leading to serious complications such as infertility and intrauterine adhesions.
[0003] Currently, the clinical treatment of endometritis is mainly based on broad-spectrum antibiotics (such as cephalosporins and metronidazole), but long-term use of broad-spectrum antibiotics can easily lead to the production of multidrug-resistant strains, and may disrupt the balance of beneficial bacteria in the uterine cavity and aggravate microecological disorders.
[0004] Antimicrobial peptides (AMPs), as an important component of the innate immune system, are considered potential alternatives to traditional antibiotics due to their broad-spectrum antimicrobial activity, resistance to drug resistance, and low host cell toxicity. They have become a research hotspot for anti-infective therapy. However, existing AMPs lack specificity against endometritis-associated pathogens, and the large-scale production of natural AMPs is limited by extraction sources and separation and purification efficiency, hindering their clinical application.
[0005] Therefore, the development of an antimicrobial peptide that is easy to synthesize and can specifically regulate intrauterine pathogens has important clinical value for the prevention and treatment of endometritis. Summary of the Invention
[0006] In order to solve the problems that endometritis is easily resistant to broad-spectrum antibiotics, resulting in decreased therapeutic efficacy, and long-term use easily destroys the balance of beneficial bacteria in the uterine cavity, the present invention provides an antimicrobial peptide RB3 and its application.
[0007] According to a first aspect of the present invention, an antimicrobial peptide RB3 is provided. The amino acid sequence of the antimicrobial peptide RB3 is shown in SEQ ID NO: 1.
[0008] The uterine cavity is not an absolutely sterile environment. Compared to the vagina, which is directly connected to the outside world, it is considered to be an environment with a small number of microorganisms, mainly Lactobacilli. Therefore, the development of endometritis may be related to changes in the immune microenvironment caused by the imbalance of normal flora in the uterine cavity. Common clinical pathogens of endometritis include mixed infections of Gram-negative and Gram-positive bacteria such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecium.
[0009] Through extensive experimental research, the inventors of this application obtained a short peptide with an amino acid sequence as shown in SEQ ID NO: 1 from the hemolymph of the shrimp Litopenaeus vannamei, which is the antimicrobial peptide RB3 provided by the present invention. Furthermore, based on the specific amino acid sequence of the antimicrobial peptide RB3, the antimicrobial peptide RB3 was synthesized by solid-phase synthesis, and antibacterial experiments demonstrated that the antimicrobial peptide RB3 has significant antibacterial activity against five endometritis pathogens: Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa), and Enterococcus faecium (E. faecium). The antimicrobial peptide RB3 provided by the present invention can be used in the prevention and / or treatment of endometritis. The antimicrobial peptide RB3 can significantly inhibit the growth and proliferation of endometritis pathogens, maintain the normal flora structure and immune microenvironment of the endometrium, thereby achieving effective prevention and treatment of endometritis, which is of great significance for the prevention and clinical treatment of endometritis. Furthermore, the antimicrobial peptide RB3 provided by the present invention can be directly obtained by solid-phase synthesis, which is conducive to the large-scale preparation and large-scale clinical application of the antimicrobial peptide RB3.
[0010] According to a second aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antimicrobial peptide RB3.
[0011] According to a third aspect of the present invention, a recombinant expression vector is provided, which comprises the above-mentioned nucleic acid molecule.
[0012] According to a fourth aspect of the present invention, a recombinant host cell is provided, wherein the recombinant host cell contains the above-mentioned recombinant expression vector.
[0013] According to a fifth aspect of the present invention, there is provided use of the antimicrobial peptide RB3 in the preparation of a medicament for preventing and / or treating uterine cavity-related diseases.
[0014] Preferably, the above-mentioned uterine cavity-related diseases include uterine cavity inflammation.
[0015] Preferably, the above-mentioned uterine cavity inflammation is endometritis.
[0016] Preferably, the drug has an inhibitory effect on at least two of Escherichia coli, Klebsiella, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecium.
[0017] The antimicrobial peptide RB3 provided by the present invention is used in the preparation of a drug for uterine cavity-related diseases. The drug can significantly inhibit the growth and proliferation of endometritis pathogens, maintain the normal flora structure and immune microenvironment of the endometrium, thereby achieving effective prevention and treatment of endometritis, which is of great significance for the prevention and clinical treatment of endometritis.
[0018] According to a sixth aspect of the present invention, a pharmaceutical composition for preventing and / or treating uterine cavity-related diseases is provided, wherein the pharmaceutical composition comprises the antimicrobial peptide RB3.
[0019] Preferably, the above-mentioned uterine cavity-related disease is endometritis. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is the high performance liquid chromatogram of the antimicrobial peptide RB3 obtained by solid phase synthesis provided in Example 1.
[0021] Figure 2 This is the mass spectrum of the antimicrobial peptide RB3 obtained by solid phase synthesis provided in Example 1.
[0022] Figure 3 This is the 3D structural model predicted for the antimicrobial peptide RB3 in Example 2.
[0023] Figure 4 This is a graph showing the calculated results of the inhibition rates of the antimicrobial peptide RB3 solutions of different concentrations against Escherichia coli and Staphylococcus aureus provided in Example 3.
[0024] Figure 5 This is a scanning electron micrograph of the antibacterial effect of the antimicrobial peptide RB3 solution with a concentration of 500 μg / mL provided in Example 3 on Escherichia coli.
[0025] Figure 6 This is a plate effect diagram of the antibacterial experiment of the antimicrobial peptide RB3 solution with a concentration of 0.5 mg / mL provided in Example 4 on five pathogenic bacteria.
[0026] Figure 7This is a graph showing the calculation results of the inhibition rate of the antimicrobial peptide RB3 solution with a concentration of 0.5 mg / mL provided in Example 4 against five pathogenic bacteria. DETAILED DESCRIPTION
[0027] The following is a further clear and complete description of the technical features of the technical solution provided by the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present invention.
[0028] Example 1
[0029] This embodiment provides an antimicrobial peptide RB3. The amino acid sequence of the antimicrobial peptide RB3 is shown in SEQ ID NO: 1. The specific amino acid sequence is as follows: FLLHKIYGNIR.
[0030] The antimicrobial peptide RB3 provided in this example was prepared by the following steps:
[0031] 1. Acquisition of antimicrobial peptide RB3
[0032] The inventors of the present application obtained a short peptide with an amino acid sequence as shown in SEQ ID NO: 1 from the hemolymph of Litopenaeus vannamei through a large number of experimental studies and named it antimicrobial peptide RB3.
[0033] 2. Solid-phase synthesis of antimicrobial peptide RB3
[0034] (1) Resin preparation
[0035] Fmoc-Ala-Wang resin with a functional group content of 0.33 mmol / g was selected as the resin used in the synthesis of antimicrobial peptide RB3.
[0036] (2) Reagent preparation
[0037] Dimethylformamide (DMF), various α-amino group Fmoc-protected amino acid solutions (concentration of 0.25 M), 0.33 M O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) solution, 0.33 M 1-hydroxybenzotriazole (HOBt) solution, 200 mL / L piperidine solution, 174.2 mL / L N,N-diisopropylethylamine (DIEA) solution, and dichloromethane (DCM).
[0038] (3) Synthesis of antimicrobial peptide RB3
[0039] a. Weigh 0.05 mmol Fmoc-Ala-Wang resin and place it in a solid phase reactor. At the same time, add 8 mL of DCM to the solid phase reactor to swell overnight, then remove the DCM under reduced pressure; add 8 mL of piperidine solution to the solid phase reactor containing the Fmoc-Ala-Wang resin and react at room temperature for 5 minutes, drain, continue to add 8 mL of piperidine solution to the solid phase reactor and react at room temperature for 20 minutes, drain; add 8 mL of DMF to the solid phase reactor to wash the Fmoc-Ala-Wang resin for a total of 3 washes, each for 1 minute; accurately measure 0.2 mmol of various α-amino Fmoc-protected amino acid solutions, 0.2 mmol of HBTU solution, and 0.195 mol of HOBt solution and add them to the solid phase reactor to react for 5 minutes, then add 0.4 mmol of DIEA solution and react under nitrogen bubble oscillation at room temperature for 2 hours; add 8 mL DMF was added for washing for 3 times, each time for 1 min; 8 mL of piperidine solution was added and reacted at room temperature for 5 min, then the mixture was drained; 8 mL of piperidine solution was added to the solid phase reactor and the mixture was reacted at room temperature for 20 min, then the mixture was drained; 8 mL of DMF was added for washing for 3 times, each time for 1 min;
[0040] b according to the above steps from the carbon end of the peptide chain to the nitrogen end one by one continuous synthesis of the antimicrobial peptide RB3, FLLHKIYGNIR;
[0041] c. After the synthesis of the antimicrobial peptide RB3 is completed, the resin with the short peptide chain is washed with DMF and DCM respectively, and then the resin is placed in a vacuum drying oven for drying;
[0042] d. Cleavage of short peptides: After the resin is dried, use a spatula to break up the resin as much as possible, transfer it to a chicken heart bottle, add a magnetic stirrer, and slowly add 10 mL of cutting solution for removing the resin (calculated according to the mass ratio, trifluoroacetic acid (TFA): pure water: anisole: phenol: dithiol = 82.5:5:5:5:2.5) in an ice-water bath, and react in an ice-water bath for 2 hours; after the reaction is completed, transfer the reaction solution together with the resin to a filter, filter it with a water pump, place the filtrate in a round-bottom flask, and connect a nitrogen tube to the round-bottom flask under nitrogen. Flow down and blow dry; when the sample in the round-bottom flask is blown to a viscous state, remove the nitrogen tube, pour about 20 mL of ice ether pre-cooled at 4 ° C into the round-bottom flask, mix and fully disperse, then place the obtained mixture in a refrigerated centrifuge, centrifuge at 4 ° C, 8000 r / min for 15 min, discard the supernatant, and disperse the obtained precipitate in 20 mL of ice ether (pre-cooled at 4 ° C in advance) and centrifuge. Repeat the above-mentioned dispersion and centrifugation operation 3 times, and the final precipitate is vacuum dried to obtain the crude antimicrobial peptide RB3 provided in this example.
[0043] 3. Purification and Identification of Antimicrobial Peptide RB3
[0044] The antimicrobial peptide RB3 obtained by solid phase synthesis was purified and identified by reversed-phase high performance liquid chromatography (HPLC). The HPLC parameters were as follows: a C18 semi-preparative column was used, the mobile phases included phase A (aqueous phase) and phase B (organic phase), wherein phase A was deionized water and phase B was 80% acetonitrile aqueous solution, and both phases A and B contained 0.1% TFA (m / v); the flow rate was 6 mL / min; and the elution was performed using a gradient of 1% per minute from 55% to 10% for phase A and 1% per minute from 45% to 90% for phase B. The final HPLC chromatogram is shown in FIG. Figure 1 shown.
[0045] according to Figure 1 The HPLC chromatogram shown is obtained by collecting the eluate from 10.4 to 11 minutes. The organic phase acetonitrile in the eluate is removed by a rotary evaporator. The remaining antimicrobial peptide RB3 aqueous solution is then placed in a refrigerator and frozen into ice cubes. Finally, the water is removed by a freeze dryer to obtain a fluffy solid powder, which is the pure antimicrobial peptide RB3 provided in this example.
[0046] In addition, the antimicrobial peptide RB3 pure product obtained above was identified by mass spectrometry, and the results were as follows: Figure 2 As shown. Figure 2 It can be seen that the molecular weight of the antimicrobial peptide RB3 is about 1417 Daltons (Da), and its triply charged ion ([M+3H] 3+ ) and doubly charged ions ([M+2H] 2+ ) showed good signal intensity and clear ion peaks at m / z = 472.5 and m / z = 708.5, respectively. These data indicate that the obtained antimicrobial peptide RB3 peptide sample is of high quality and suitable for further sequence analysis and identification.
[0047] Example 2
[0048] This example aims to identify the physicochemical properties of the antimicrobial peptide RB3 provided in Example 1. The physicochemical properties of the antimicrobial peptide RB3 are shown in Table 1. At the same time, the antimicrobial peptide RB3 was predicted by the AlphaFold3 (http: / / https: / / alphafoldserver.com / ) website, and a model was automatically established for this sequence. The 3D structural model of the antimicrobial peptide RB3 was then opened by PyMOL software. Figure 3 As shown, Figure 3 The 3D structure of the antimicrobial peptide RB3 at different positions is shown.
[0049] Table 1 Physicochemical properties of antimicrobial peptide RB3
[0050] Property Parameter Sequence FLLHKIYGNIR Molecular weigh 1414.73Da Total hydrophobic ratio 45％ Charge at pH 7 (total net charge at pH = 7) +2.25 Hydrophobicity <h>(Hydrophobicity) < / h> -1.41 Average hydropathy 0.236 Molar extinction coefficient 1490
[0051] Depend on Figure 3 It can be seen from the 3D structural model that there is an α-helix in the structure of the antimicrobial peptide RB3, and the 3rd to 10th amino acids of the antimicrobial peptide RB3 form an α-helix structure.
[0052] Example 3
[0053] This example aims to study the antibacterial activity of the antimicrobial peptide RB3 provided in Example 1 against Escherichia coli and Staphylococcus aureus to obtain the minimum inhibitory concentration (MIC) of the antimicrobial peptide RB3. The specific experimental steps are as follows:
[0054] 1. Materials and Reagent Preparation
[0055] (1) Synthesis of antimicrobial peptide RB3
[0056] The antimicrobial peptide RB3 was synthesized according to the steps of Example 1.
[0057] (2) Main reagents
[0058] Bovine trypsin, beef extract, agar powder, sodium chloride (NaCl), and sodium hydroxide (NaOH) were purchased from Guangzhou Weijia Technology Co., Ltd.
[0059] (3) Preparation of solutions and culture media
[0060] Antimicrobial peptide RB3 solutions of different concentrations: Take out the freeze-dried powdered antimicrobial peptide RB3 pure product from the -20°C refrigerator, equilibrate at room temperature for 1 hour, weigh the freeze-dried powdered antimicrobial peptide RB3 pure product, add sterile PBS buffer (0.01M, pH 7.4) to dissolve and mix, and prepare an antimicrobial peptide RB3 solution with a concentration of 500 μg / mL. Use gradient dilution to prepare antimicrobial peptide RB3 solutions with different concentration gradients, and store them in aliquots at -20°C for later use.
[0061] Broth medium (liquid): Weigh 10 g of peptone, 5 g of beef extract, and 5 g of NaCl, add 800 mL of distilled water, adjust the pH to 7.2 with 5 M NaOH, make up to 1000 mL, and autoclave at 121°C for 20–30 min. Store at room temperature.
[0062] 2. Antibacterial activity verification - minimum inhibitory concentration (MIC) determination
[0063] The antibacterial activity of the antimicrobial peptide RB3 provided in Example 1 was verified by referring to the following method, and the specific process is as follows:
[0064] (1) Two experimental endometritis pathogens (Escherichia coli and Staphylococcus aureus) stored at -80°C were first activated in small amounts. Specifically, 50 μL of Escherichia coli and Staphylococcus aureus were inoculated into 1 mL of broth liquid culture medium and cultured in a constant temperature shaker at 37°C for 6 to 12 h until the logarithmic growth phase. The bacterial solutions were then diluted with sterile PBS buffer (0.01 M, pH 7.4) to a concentration of 10 6 CFU / mL;
[0065] (2) Take 50 μL of the diluted bacterial solution and mix it with equal volumes of antimicrobial peptide RB3 solution of different concentrations, and incubate it in a constant temperature water bath at 30°C for 2 h to obtain a mixed solution;
[0066] (3) 10 μL of the above mixture was added to 100 μL of LB liquid medium, with two replicates per group, and cultured at 37°C for 12 h. Sterile PBS buffer (0.01 M, pH 7.4) was used as a negative control.
[0067] (4) The minimum inhibitory concentration (MIC) was measured on a 96-well plate reader (Biotek Epoch) at a wavelength of 600 nm. The lowest concentration of the antimicrobial peptide RB3 solution that inhibited 80% bacterial growth was defined as the MIC.
[0068] (5) The mixture obtained by mixing the diluted bacterial solution in step (2) with a 500 μg / mL antimicrobial peptide RB3 solution was centrifuged and washed twice with a sterile PBS buffer solution having a concentration of 0.01 M and a pH value of 7.4. The mixture was then fixed with 4% formaldehyde for 30 min and dehydrated with ethanol solutions of different concentration gradients (35%, 50%, 75%, 90%, 95% and 100%). The mixture was fixed on a copper tape and dried in air for 2 days. The effect of the antimicrobial peptide RB3 on the surface morphology of Escherichia coli was observed using a scanning electron microscope.
[0069] 3. Results Analysis
[0070] The calculation results of the inhibition rate of different concentrations of antimicrobial peptide RB3 solution on Escherichia coli and Staphylococcus aureus are as follows Figure 4 As shown in the scanning electron microscopy image, the antibacterial effect of the antimicrobial peptide RB3 solution with a concentration of 500 μg / mL on Escherichia coli is shown in the figure. Figure 5 As shown, in which Figure 4 In the figure, the horizontal axis Concentration of RB3 represents the concentration of the antimicrobial peptide RB3 solution (μg / mL), and the vertical axis Bacteria Inhibition represents the inhibition rate (%). Figure 5In the table, Control refers to sterile PBS buffer as a negative control.
[0071] Depend on Figure 4 It can be seen that the minimum inhibitory concentration of the antimicrobial peptide RB3 solution against Escherichia coli is 250 μg / mL, and the minimum inhibitory concentration against Staphylococcus aureus is 125 μg / mL.
[0072] Depend on Figure 5 It can be seen that for E. coli cells not treated with the antimicrobial peptide RB3 solution, the internal and surface structural integrity of the cells was intact. However, after treatment with the antimicrobial peptide RB3 solution, the cell membrane structure began to blur as the cells vacuolated, the cell shape became irregular, the cell membrane completely collapsed, and the cytoplasmic contents leaked out. These results indicate that the antimicrobial peptide RB3 has a destructive effect on the cell membrane and internal structure of E. coli, thereby inhibiting the growth and proliferation of E. coli.
[0073] Example 4
[0074] This example aims to study the antibacterial activity of the antimicrobial peptide RB3 provided in Example 1 against different types of pathogens. The specific experimental steps are as follows:
[0075] 1. Materials and Reagent Preparation
[0076] (1) Synthesis of antimicrobial peptide RB3
[0077] The antimicrobial peptide RB3 was synthesized according to the steps of Example 1.
[0078] (2) Bacterial strains
[0079] The five pathogenic bacteria used in this example include Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecium (E. faecium).
[0080] (3) Main reagents
[0081] Bovine trypsin, beef extract, agar powder, sodium chloride, and sodium hydroxide were purchased from Guangzhou Weijia Technology Co., Ltd.
[0082] (4) Preparation of solutions and culture media
[0083] 0.5 mg / mL antimicrobial peptide RB3 solution: Take out the lyophilized powdered antimicrobial peptide RB3 pure product from the -20°C refrigerator and equilibrate it at room temperature for 1 hour. Weigh the lyophilized powdered antimicrobial peptide RB3 pure product and add 1 mL of sterile PBS buffer with a concentration of 0.01 M and a pH value of 7.4 to dissolve and mix to obtain an antimicrobial peptide RB3 solution with a concentration of 0.5 mg / mL. Aliquot and store at -20°C for later use.
[0084] Broth medium (liquid): Weigh 10 g of peptone, 5 g of beef extract, and 5 g of NaCl, add 800 mL of distilled water, adjust the pH to 7.2 with 5 M NaOH, make up to 1000 mL, and autoclave at 121°C for 20–30 min. Store at room temperature.
[0085] Broth medium (solid): Weigh 10 g of peptone, 5 g of beef extract, 5 g of NaCl, and 15 g of agar powder, add 800 mL of distilled water, adjust the pH to 7.2 with 5 M NaOH, make up to 1000 mL, and autoclave at 121°C for 20–30 min. Store at room temperature.
[0086] 2. Antibacterial activity verification - plate colony counting method
[0087] The antibacterial activity of the antimicrobial peptide RB3 provided in Example 1 was verified by referring to the following method, and the specific process is as follows:
[0088] (1) First, activate a small amount of the above five pathogenic bacteria. Specifically, 50 μL of pathogenic bacteria liquid was inoculated into 1 mL of broth liquid culture medium and cultured in a constant temperature shaker at 37°C for 6-12 h until the logarithmic growth phase;
[0089] (2) Prepare bacterial suspension from the activated bacterial solution and dilute the bacterial solution to a concentration of 5 × 10 3 CFU / mL, take 50 μL of the diluted bacterial suspension of the five pathogens and mix them with 50 μL of the antimicrobial peptide RB3 solution with a concentration of 0.5 mg / mL, place them in a constant temperature shaker at 37°C for 2 h to obtain a mixed solution, take 30 μL of the mixed solution to spread on the plate, invert and culture at 37°C for 12 to 24 h, then count the colonies and take pictures, and at the same time, use sterile PBS buffer with a concentration of 0.01 M and a pH value of 7.4 as a negative control (Control).
[0090] (3) Calculate the antibacterial rate (%) = (number of colonies in the negative control - number of colonies in the experimental group) / number of colonies in the negative control × 100.
[0091] 3. Results Analysis
[0092] The plate effect diagram of the antibacterial experiment of the antimicrobial peptide RB3 solution with a concentration of 0.5 mg / mL against five pathogenic bacteria, namely Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecium (E. faecium) is shown in the figure. Figure 6 As shown in the figure, the calculation results of Bacteria Inhibition (inhibition rate) are as follows Figure 7 As shown, in which Figure 6 In the table, Control refers to the negative control of sterile PBS buffer with a concentration of 0.01 M and a pH of 7.4. Figure 7 In the figure, the vertical axis Bacteria Inhibition represents the inhibition rate (%).
[0093] Depend on Figure 6 and Figure 7 It can be seen that the antimicrobial peptide RB3 has significant antibacterial activity against five pathogenic bacteria, including Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecium (E. faecium). Among the five pathogenic bacteria, the antimicrobial peptide RB3 has the highest antibacterial activity against Escherichia coli, with an inhibition rate of up to 90%.
[0094] Comparative Example 1
[0095] This comparative example provides an antimicrobial peptide A. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 2. The specific amino acid sequence is as follows: DVNFLLHKIYGNIRY.
[0096] Comparative Example 2
[0097] This comparative example provides an antimicrobial peptide B. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 3, and the specific amino acid sequence is as follows: VNFLLHKIYGNIRYS.
[0098] Comparative Example 3
[0099] Referring to the solid-phase synthesis method of the antimicrobial peptide RB3 provided in Example 1, the antimicrobial peptide A of Comparative Example 1 and the antimicrobial peptide B of Comparative Example 2 were synthesized respectively. At the same time, referring to the experimental steps of Example 4, the antibacterial activity of the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2 against different types of pathogens was studied. The results are shown in Table 2.
[0100] Table 2 Antibacterial activity test results of antimicrobial peptide A and antimicrobial peptide B against different types of pathogens
[0101]
[0102] The antibacterial activity test results of the antimicrobial peptide A of Comparative Example 1 and the antimicrobial peptide B of Comparative Example 2 against five pathogenic bacteria, namely Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecium (E. faecium), are shown in Table 2.
[0103] The sequences of the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2 are both 15 amino acids, while the sequence of the antimicrobial peptide RB3 provided in Example 1 is 11 amino acids. Compared with the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2, the antimicrobial peptide RB3 peptide sequence provided in Example 1 is shorter, which makes it more conducive to application in production practice.
[0104] Furthermore, as shown in Table 2, the antimicrobial peptide RB3 provided in Example 1 had higher antibacterial rates against five pathogenic bacteria, namely Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa), and Enterococcus faecium (E. faecium), than the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2. The reason for the above results is that the α-helical structure in the tertiary structure of the antimicrobial peptide RB3 provided in Example 1 is more complete than that of the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2. The integrity of the α-helical structure is conducive to the antimicrobial activity of the peptide segment. Therefore, the antimicrobial activity of the antimicrobial peptide RB3 provided in Example 1 is theoretically superior to that of the antimicrobial peptide A provided in Comparative Example 1 and the antimicrobial peptide B provided in Comparative Example 2.
[0105] In summary, the antimicrobial peptide RB3 provided in the present application has significant antibacterial activity against five endometritis pathogens, namely Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabills), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecium (E. faecium). The antimicrobial peptide RB3 provided by the present invention can be used in the prevention and / or treatment of endometritis. The antimicrobial peptide RB3 can significantly inhibit the growth and proliferation of endometritis pathogens, maintain the normal flora structure and immune microenvironment of the endometrium, thereby achieving effective prevention and treatment of endometritis, which is of great significance for the prevention and clinical treatment of endometritis. In addition, the antimicrobial peptide RB3 provided by the present invention can be directly obtained by solid-phase synthesis, which is conducive to the large-scale preparation and large-scale clinical application of the antimicrobial peptide RB3.
[0106] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.
Claims
1. An antimicrobial peptide RB3, characterized in that: The amino acid sequence of the antimicrobial peptide RB3 is shown in SEQ ID NO:
1.
2. A nucleic acid molecule, characterized in that: The nucleic acid molecule comprises a nucleotide sequence encoding the antimicrobial peptide RB3 according to claim 1.
3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to claim 2.
4. A recombinant host cell, characterized in that: The recombinant host cell contains the recombinant expression vector according to claim 3.
5. Use of the antimicrobial peptide RB3 according to claim 1 in the preparation of a medicament for preventing and / or treating uterine cavity-related diseases.
6. Use of the antimicrobial peptide RB3 according to claim 5 in the preparation of a medicament for preventing and / or treating uterine cavity-related diseases, characterized in that: The uterine cavity related diseases include uterine cavity inflammation.
7. Use of the antimicrobial peptide RB3 according to claim 6 in the preparation of a medicament for preventing and / or treating uterine cavity-related diseases, characterized in that: The uterine cavity inflammation is endometritis.
8. The antimicrobial peptide RB3 according to claim 5, characterized in that: The drug has an inhibitory effect on at least two of Escherichia coli, Klebsiella, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecium.
9. A pharmaceutical composition for preventing and / or treating uterine cavity-related diseases, characterized in that: The pharmaceutical composition comprises the antimicrobial peptide RB3 according to claim 1.
10. The pharmaceutical composition for preventing and / or treating uterine cavity-related diseases according to claim 9, characterized in that: The uterine cavity related disease is endometritis.