Targeted antibacterial peptide TLH13 and application thereof
By using deep learning and transfer learning techniques to mine the targeted antimicrobial peptide TLH13 from the animal gut microbiome, the problem of Clostridium perfringens infection has been solved, achieving a highly efficient and low-toxicity antimicrobial effect, which is suitable for the preparation of drugs and feed additives.
Patent Information
- Application Number
- CN202511409872.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2026-01-23
AI Technical Summary
Existing technologies are insufficient to effectively inhibit Clostridium perfringens infection, and antibiotic use leads to increased drug resistance. The development of targeted antimicrobial peptides is time-consuming and labor-intensive.
The targeted antimicrobial peptide TLH13 was extracted from the genome of animal gut microbiota using deep learning and transfer learning technologies. It was prepared and purified to a purity of over 95% by solid-phase chemical synthesis, and its biological activity was analyzed by high-performance liquid chromatography.
TLH13 exhibits targeted antibacterial activity against Clostridium perfringens, is resistant to high temperatures and physiological salinity environments, has low cytotoxicity, and can bind to DNA and disrupt cell walls, demonstrating good biocompatibility and antibacterial effects.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a targeted antibacterial peptide TLH13 and application thereof. BACKGROUND
[0002] With the long-term use of antibiotics in the fields of global medical treatment and livestock breeding, bacterial drug resistance is seriously threatening human health. In the livestock breeding industry, the use of antibiotics can not only prevent the occurrence of livestock diseases and promote animal growth, but also lead to the spread of drug-resistant genes and drug-resistant bacteria. The number of deaths caused by drug-resistant bacteria infection continues to rise globally every year, becoming one of the global challenges. Under this background, it is urgent to develop new antibacterial drugs to cope with bacterial drug resistance.
[0003] Clostridium perfringens is an anaerobic, spore-forming, gram-positive pathogenic bacterium, which widely exists in soil, sewage and human and animal intestines. For example, in the livestock industry, it causes diseases such as necrotic enteritis and enterotoxemia every year, resulting in huge economic losses to the breeding industry. Although the use of antibiotics can effectively inhibit Clostridium perfringens infection, it can disrupt the balance of intestinal flora and accelerate the production of drug-resistant bacteria. Targeted drugs can accurately recognize and act on the unique pathogenic target of Clostridium perfringens, block the synthesis and release of toxins, reduce the dosage of drugs and side effects, and avoid damaging other beneficial microorganisms, effectively delaying the drug resistance process.
[0004] Antibacterial peptides (AMPs) are a kind of natural immune molecules with antibacterial activity, which can play a role by destroying bacterial cell membranes, interfering with protein synthesis and DNA replication, etc., and are not easy to induce drug resistance. Compared with broad-spectrum antibacterial peptides, targeted antibacterial peptides are a kind of antibacterial peptides that can target and inhibit specific microorganisms, which can kill target microorganisms while reducing damage to normal flora of the body. However, the development of targeted antibacterial peptides requires the recombination of existing active antibacterial peptides based on protein interaction, which is time-consuming and laborious. SUMMARY
[0005] The technical problem to be solved by the present application is how to inhibit Clostridium perfringens and / or how to develop or prepare antibacterial peptide drugs that target and inhibit Clostridium perfringens.
[0006] In order to solve the above technical problems, the present application first provides a polypeptide or a (pharmaceutically) salt or a derivative thereof, which can be any of the following:
[0007] A1) a polypeptide with an amino acid sequence of SEQ ID No. 1 in the sequence listing;
[0008] A2) the polypeptide derived from A1) by substitution of one or several amino acid residues and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID No. 1 in the sequence listing and having the same function, or a polypeptide having more than 90% identity with the polypeptide shown in A1) and having the same function;
[0009] A3) a fusion polypeptide obtained by linking a protein tag to the N terminus or / and C terminus of A1) or A2).
[0010] The amino terminal end of the polypeptide described above can contain an amino terminal protection group, which can be acetyl, amino, maleoyl, succinoyl, tert-butyloxycarbonyl or benzoyloxy or any of other hydrophobic groups or macromolecular carrier groups; the carboxyl terminal end of the cyclic polypeptide of the present application can contain a carboxyl terminal protection group, which can be amino, amide, carboxyl, or tert-butyloxycarbonyl or any of other hydrophobic groups or macromolecular carrier groups.
[0011] The pharmaceutical salts of the polypeptide of the present application include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate bromomethane, bromide, nitromethide, calcium edetate, methylsulfate, dextrocamphosulfonate, mucate, acid, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium, dihydrochloride, oleate, ethylenediaminetetraacetate, oxalate, edisylate, pamoate (embonate), propionate laurylsulfate, palmitate, ethanesulfonate, pantothenate, fumarate, phosphate / diphosphate, glucoheptonate, polygalacturonate, gluconate salicylate, glutamate, stearate, p-hydroxyethyIamino-arsenical acid, sulfate, hydroxybenzoate, subacetate, heparin, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, 8-chlorotheophyllinate, iodide, tosylate, triethyl iodide, lactic acid, valerate, etc. Depending on the use, the pharmaceutical salt can be formed by a cation such as sodium, potassium, aluminum, calcium, lithium, manganese and zinc, bismuth, etc., or by a base such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanol, procaine, diethylamine, piperazine, tris-hydroxymethylaminomethane, and hydroxytetramethylammonium, etc. These salts can be prepared using standard methods, for example, by reacting a free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, an acidic salt such as hydrochloride, hydrobromide, acetate, pamoate, etc. can be used as a dosage form; in the presence of an acidic group or an alcoholic group, a pharmaceutically acceptable ester such as acetate, maleate, chloromethyltrimethylacetate, etc., and an ester known in the literature for improving solubility and hydrolysis can be used as a sustained release and prodrug preparation.
[0012] To solve the above technical problems, the present application also provides a nucleic acid molecule encoding the polypeptide described above.
[0013] To solve the above technical problems, the present application also provides the use of the polypeptide described above or its salt or its derivative or in the development and / or preparation of a medicament for preventing and / or treating Clostridium perfringens infection.
[0014] To solve the above technical problems, the present application also provides the use of the polypeptide described above or its salt or its derivative in the development and / or preparation of a feed additive.
[0015] To solve the above technical problems, the present application also provides the use of any one of the nucleic acid molecules described above:
[0016] C1) in the development and / or preparation of a medicament for preventing and / or treating Clostridium perfringens infection;
[0017] C2) in the development and / or preparation of a feed additive.
[0018] To solve the above technical problems, the present application also provides a medicament for inhibiting Clostridium perfringens, which contains the polypeptide described above or its salt or its derivative.
[0019] The medicament described above can have at least one of the following properties:
[0020] B1) inhibiting the growth of Clostridium perfringens;
[0021] B2) increasing the cell wall permeability of Clostridium perfringens;
[0022] B3) destroying the cell wall of Clostridium perfringens;
[0023] B4) inhibiting the synthesis of genomic DNA of Clostridium perfringens.
[0024] The concentration of the polypeptide or its salt or its derivative in the medicament can be 4 μg / mL to 128 μg / mL.
[0025] To solve the above technical problems, the present application also provides a feed additive containing the polypeptide described above or its salt or its derivative.
[0026] The concentration of the polypeptide or its salt or its derivative in the feed additive can be 4 μg / mL to 128 μg / mL.
[0027] The present application combines deep learning technology and transfer learning technology to search for target antibacterial peptides in the candidate protein sequence space.
[0028] In order to solve the shortcomings of traditional antibiotics, the purpose of the present application is to provide an antibacterial peptide against Clostridium perfringens infection. The amino acid sequence of the antibacterial peptide is SEQ ID NO. 1: MFVKDVVVQNQVGLHARPATFFIQK.
[0029] The antibacterial peptide disclosed in the present application is obtained by using deep learning and transfer learning technology to analyze and mine from the intestinal microbial genome of animals. Due to the niche competition relationship of the intestinal microorganisms of animals, the intestinal microorganisms can code antibacterial peptides that can target and inhibit other microorganisms, which is a potential antibacterial peptide resource library. Using deep learning to mine antibacterial peptides from the intestinal microbial genome can effectively avoid the saprophytic nature of intestinal microorganisms and realize high-throughput screening of antibacterial peptides. The present application uses a method combining deep learning and transfer learning technology, and a new antibacterial peptide is found in the intestinal microbial genome by constructing a deep learning prediction model. Then the antibacterial peptide is synthesized by solid-phase chemical synthesis, further purified by high performance liquid chromatography (HPLC) to a purity of more than 95%, and its biological activity is determined.
[0030] In one aspect, the peptide provided by the present application has a targeted antibacterial activity against Clostridium perfringens.
[0031] In one aspect, the peptide provided by the present application is resistant to high temperature and physiological salt environment.
[0032] In one aspect, the peptide provided by the present application has good biocompatibility.
[0033] In one aspect, the present application provides the antibacterial mechanism of the peptide.
[0034] The present application mines a new type of targeted antibacterial peptide from the intestinal microbial genome by bioinformatics methods, which is named TLH13. The minimum inhibitory concentration of the peptide TLH13 against the pathogenic bacteria Clostridium perfringens ATCC 13124 is 4 μg / mL. The peptide TLH13 has low cytotoxicity and maintains antibacterial activity in salt ions and high temperature environment. The peptide TLH13 can kill Clostridium perfringens by binding to the DNA of Clostridium perfringens and destroying the cell wall. BRIEF DESCRIPTION OF DRAWINGS
[0035] Figure 1 is a high performance liquid chromatogram.
[0036] Figure 2 is an antibacterial peptide mass spectrum.
[0037] Figure 3 is the result of antibacterial peptide cytotoxicity determination, the horizontal coordinate is the antibacterial peptide concentration, and the vertical coordinate is the cell survival rate.
[0038] Figure 4 is the gel electrophoresis of antibacterial peptide and Clostridium perfringens genome DNA.
[0039] Figure 5 The results of the influence of the antibacterial peptide on the permeability of Clostridium perfringens cell wall are shown in the figure. The abscissa represents the antibacterial peptide concentration; the ordinate represents the permeability.
[0040] Figure 6 The results of the hemolytic activity determination of the antibacterial peptide are shown in the figure. The abscissa represents the antibacterial peptide concentration; the ordinate represents the hemolysis rate. DETAILED DESCRIPTION
[0041] The application will be further described in detail below with specific embodiments. The examples provided below are only intended to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.
[0042] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.
[0043] In the following examples, the data were processed using SPSS 26 software, and the experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used, and P<0.05 (*) indicates a significant difference, P<0.01 (**) indicates a highly significant difference, and P<0.001 (***) indicates a highly significant difference.
[0044] In the following examples, Clostridium perfringens was obtained from the American Type Culture Collection (ATCC) with the accession number ATCC 13124.
[0045] Example 1. Mining, synthesis and property analysis of antibacterial peptide TLH13
[0046] The present application uses transfer learning technology to perform transfer learning on the existing three publicly available source code antibacterial peptide deep learning models. By inputting the verified sequences with high activity against Clostridium perfringens, the model is optimized to increase the likelihood of having significant inhibition of Clostridium perfringens activity in the predicted sequence. Based on the constructed model, by predicting the intestinal metagenome assembled genomes of nearly 100,000 humans, pigs, chickens, ruminants and rodents and a series of bioinformatics methods for screening, a new antibacterial peptide was found, which was named TLH13.
[0047] 1. Antibacterial peptide synthesis
[0048] TLH13 (SEQ ID NO. 1: MFVKDVVVQNQVGLHARPATFFIQK) was synthesized by solid phase chemical synthesis (AAPPTec synthesizer). The synthesized peptide was further purified by high performance liquid chromatography (HPLC) (Waters 2695) to a purity of more than 95% ( Figure 1 ). Finally, the molecular weight shown in the mass spectrum ( Figure 2 ) was basically consistent with the theoretical molecular weight 2873.33, that is, it was determined to be successfully synthesized.
[0049] Specifically, gradient elution was used, the ESI ion source was in positive mode, the data acquisition mode was data-dependent acquisition mode, and the detailed chromatographic and mass spectrometric parameters were as follows:
[0050] High performance liquid chromatograph: Waters 2695: Waters (USA);
[0051] Single quadrupole mass spectrometer ZQ2000: Waters (USA);
[0052] Liquid chromatography conditions: XBridge BEH C18 column (100 mm x 2.1 mm, 2.5 μm, Waters, USA), column temperature 40℃, injection volume 10 μL;
[0053] Mobile phase: A is ultrapure water, B is acetonitrile, both have a volume fraction of 0.1% formic acid, and the flow rate is 0.4 mL min -1 ;
[0054] Gradient elution conditions: 0-0.5 min, 30% B; 0.5-2 min, 30%-99% B; 2.0-4.0 min, 99% B; 4.0-4.1 min, 99%-30% B; 4.1-6.0 min, 30% B;
[0055] Mass spectrometry conditions: heated electrospray ion source (HESI) temperature 300℃; capillary voltage 1.5 kV; ion transmission tube temperature 320℃; sheath gas 35 unit, auxiliary gas 10 unit. Full scan / ddms2 scan mode: acquisition range 50-1000 Da, negative ion acquisition.
[0056] 2. TLH13 antibacterial activity determination
[0057] The minimum inhibitory concentration of the synthesized peptide (TLH13) on bacteria was determined by microdilution method. The experiment set treatment group, positive control group and negative control group:
[0058] Treatment group: six kinds of bacterial pathogens (Clostridium perfringens ATCC 13124, Escherichia coli K88 (China Veterinary Culture Collection Center, Accession No. CVCC-192), Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 13311, Salmonella typhimurium SL1344 (China Veterinary Culture Collection Center) and Salmonella gallinarum CVCC534) were cultured in LB medium to the logarithmic growth phase, washed twice with PBS and diluted to 0.5-1×10 6 CFU / mL with MHB medium. The antibacterial peptide powder synthesized in step 1 was diluted by 2-fold using a microdilution method with 0.2% bovine serum albumin solution containing 0.01% acetic acid in a 96-well plate to obtain 4-256 μg / mL (including 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL and 256 μg / mL) antibacterial peptide solutions, and the liquid volume in each well was 50 μL. Then 50 μL of the bacteria solution to be tested diluted with MHB medium (0.5-1×10 6 CFU / mL) was added to each well;
[0059] Positive control group: containing six kinds of bacteria solution with a concentration of 0.5-1×10 6 CFU / mL without antibacterial peptide solution;
[0060] Negative control group: without six kinds of bacteria solution and antibacterial peptide solution.
[0061] The 96-well plates of the treatment group, the positive control group and the negative control group were placed in a 37℃ constant temperature incubator or a 37℃ anaerobic incubator (Clostridium perfringens is an anaerobic bacteria that needs to grow in an anaerobic environment) for 18h. MIC (minimum inhibitory concentration) is the lowest peptide concentration that completely inhibits bacterial growth (minimum inhibitory concentration in Table 1), and the lowest antibacterial peptide solution concentration without visible bacterial growth is MIC.
[0062] The results are shown in Table 1. The peptide TLH13 synthesized in the present application has good antibacterial effect on Clostridium perfringens ATCC 13124, and does not show antibacterial activity on other pathogenic bacteria, indicating that the peptide TLH13 has targeted antibacterial activity on Clostridium perfringens.
[0063] Table 1 Antimicrobial activity of antibacterial peptide TLH13 on bacteria
[0064] Strains Minimum inhibitory concentration (pg / mL) Clostridium perfringens ATCC 13124 4 Escherichia coli K88 ＞128 Escherichia coli ATCC 25922 ＞128 Salmonella typhimurium ATCC 13311 ＞128 Salmonella typhimurium SL1344 ＞128 Salmonella pullorum CVCC 534 ＞128
[0065] 3. TLH13 stability determination
[0066] 3.1 Thermal stability detection
[0067] The dry powder of the antibacterial peptide synthesized in Step 1 was diluted with deionized water to 1 mg / mL, and the antibacterial peptide solution was treated in a metal bath at 80°C for 30 min. The heat-treated antibacterial peptide solution was diluted to 2-128 μg / mL (including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL) using the microdilution method of Step 2 above, and the minimum inhibitory concentration of the peptide against C. perfringens was determined. The control group was an antibacterial peptide solution that was not treated at 80°C (also at a concentration of 2-128 μg / mL, including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL), and the results are shown in Table 2.
[0068] The results in Table 2 show that the antibacterial peptide has high stability. The minimum inhibitory concentration of TLH13 against C. perfringens at 80°C is 32 μg / mL, and the activity of the peptide is not completely lost, retaining certain antibacterial activity, indicating that the peptide still has antibacterial activity under high temperature conditions.
[0069] Table 2 Thermal stability of antibacterial peptide TLH13
[0070] Temperature Minimum inhibitory concentration (pg / mL) Control 4 80℃ 32
[0071] 3.2 Stability detection under different salt ion concentration treatment conditions
[0072] NaCl, KCl, NH4Cl, CaCl2 and FeCl3 were added to the MH medium, respectively, to obtain five kinds of MH medium containing different salt ions (final concentrations: 150 nM NaCl, 4.5 mM KCl, 6 μM NH4Cl, 4 mM CaCl2 and 4 μM FeCl3), and the C. perfringens bacterial solution was resuspended using the five kinds of MH medium containing different salt ions, and then the antibacterial activity and minimum inhibitory concentration of TLH13 peptide against C. perfringens under different salt ion treatment conditions were determined using the microdilution method of Step 2 above, and the results are shown in Table 3.
[0073] The results in Table 3 show that under different salt ion concentration treatment conditions, the activity of TLH13 peptide is not completely lost, retaining certain antibacterial activity; under different salt ion treatment conditions, the minimum inhibitory concentration of TLH13 against C. perfringens is less than 128 μg / mL.
[0074] Table 3 Salt ion stability of antibacterial peptide TLH13
[0075] Salt ions Minimum inhibitory concentration (pg / mL) Control 4 150 nm NaCl 32 4.5 mM KCl 128 6 μM NH4Cl 16 4 mM CaCl2 16 4 μM FeCl3 64
[0076] 4. TLH13 cytotoxicity determination
[0077] CCK8 colorimetric method was used to determine the cytotoxicity of TLH13. After the cryopreserved human colorectal adenocarcinoma cells Caco-2 (North China Biology, BNCC350769) were recovered, they were inoculated in DMEM medium containing 10% fetal bovine serum and subcultured at 37°C, 5% CO2. When the cells entered the rapid growth phase, the cells were digested with 0.25% trypsin. The cell concentration was adjusted to about 10 4 cells / well by using 10% fetal bovine serum medium to obtain a cell suspension, and 100 μL of the cell suspension was added to each well of a 96-well plate.
[0078] Three treatments were set up: a treatment group, a negative control group, and a blank control group.
[0079] Treatment group: the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After the culture was completed, the culture medium was discarded, and the cells were washed with PBS three times. Then 100 μL of an antibacterial peptide solution containing different concentrations (antibacterial peptides were diluted by 2-128 μg / mL in serum-free DMEM medium, including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL) was added to each well.
[0080] Negative control group: the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After the culture was completed, the culture medium was discarded, and the cells were washed with PBS three times. Then 100 μL of DMEM medium was added to each well.
[0081] Blank control group: the cell-free wells with only 100 μL of DMEM medium were blank controls.
[0082] The 96-well plates of the above three treatments were placed in a 37°C, 5% CO2 incubator for 12 hours. After the culture was completed, the original culture medium was discarded, and the cells were washed twice with DMEM medium containing 10% fetal bovine serum. Then DMEM medium containing 10% fetal bovine serum was added, and the 96-well plate was placed in a cell incubator (37°C, 5% CO2) for 2 hours. After the incubation was completed, the absorbance at 450 nm of each well was measured using a microplate reader. The cell survival rate was calculated according to the following formula (1):
[0083] Cell survival rate (%) = (treatment group OD 450 nm value - negative control OD 450 nm value) / (negative control OD 450 nm value - blank control OD 450 nm value) x 100% formula (1).
[0084] Figure 3The results showed that after treatment with different concentrations of antimicrobial peptides, the survival rate of Caco-2 cells was over 95%, indicating that TLH13 has low cytotoxicity and certain clinical application potential.
[0085] 5. Interaction between TLH13 and bacterial DNA
[0086] After overnight culture of *Clostridium perfringens* ATCC 13124 on FTG medium, DNA was extracted from *Clostridium perfringens* ATCC 13124 using a bacterial genomic DNA extraction kit (Kangwei, DN1101). The DNA was then analyzed using bacterial OD... 260nm and OD 280 nm Optical density ratio (OD) 260nm / OD 280nm The purity of the extracted genomic DNA was evaluated using a ≥1.90 ppm index. 600 ng of Clostridium perfringens DNA was mixed with 10 μL of TLH13 peptide at different concentrations (2–128 μg / mL, specifically 0 μg / mL (negative control), 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL), incubated at 37°C for 1 h, and then loaded with 10% loading buffer. Gel electrophoresis was performed, and gel imaging was used to analyze the binding of the peptide to the DNA.
[0087] like Figure 4 As shown, at the highest dose (TLH13 peptide concentration of 128 μg / mL), no DNA bands were observed in the lanes, but remained in the sample wells. As the TLH13 concentration decreased, blurred DNA bands appeared in the lanes, and the amount of DNA remaining in the sample wells decreased. The clarity and brightness of the bands in the lanes increased with decreasing TLH13 concentration. This indicates that the antimicrobial peptide TLH13 can bind to the genomic DNA of Clostridium perfringens, thereby mediating its antimicrobial activity.
[0088] 6. Effects of TLH13 on cell wall permeability
[0089] Clostridium perfringens ATCC 13124 was cultured in MHB medium to the logarithmic growth phase. After culture, the bacterial cells were collected, centrifuged at 6000 rpm for 3 min, and the supernatant was discarded. The cells were washed twice with 5 mM HEPES buffer (pH 7.4, containing 5 mM glucose) and resuspended at OD200. 600nm =0.2. The bacterial suspension was mixed with N-phenyl-1-naphthylpropanol (Aladdin, P110559) at a final concentration of 10 μM and incubated at 37°C in the dark for 30 min to obtain the bacterial mixture. Treatment groups, negative control groups, and positive control groups were set up:
[0090] Treatment group: Equal volume of bacteria mixture and different concentrations of TLH13 polypeptide (2-128 μg / mL, including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL) were mixed in a 96-well plate. The fluorescence intensity was detected by a microplate reader under the conditions of excitation wavelength 350 nm and emission wavelength 420 nm.
[0091] Negative control group: The bacteria mixture (untreated bacterial suspension) was used as the negative control;
[0092] Positive control group: Equal volume of bacteria mixture and polymyxin B solution (Macklin, P914153, CAS No.: 1404-26-8) were mixed as the positive control.
[0093] The permeability calculation formula is as follows formula (2):
[0094] Permeability (%) = (treatment group fluorescence value-negative control fluorescence value) / (positive control fluorescence value-negative control fluorescence value) x 100% formula (2).
[0095] The results are shown in Figure 5 TLH13 can destroy Clostridium perfringens cell wall and increase its permeability, N-phenyl-1-naphthylamine enters and contacts the hydrophobic environment, and the fluorescence intensity increases. The effect of the peptide on the cell wall of Clostridium perfringens shows a dose-dependent effect, and the higher the concentration, the stronger the damage to the cell wall.
[0096] In summary, the antibacterial peptide TLH13 has targeted antibacterial activity against Clostridium perfringens, can retain certain antibacterial activity under high temperature and physiological salt environment, and has low cytotoxicity. The antibacterial peptide TLH13 of the present application can bind to bacterial genomic DNA, inhibit the synthesis of bacterial DNA, thereby causing bacterial death, and can also dose-dependently destroy the cell wall to cause bacterial death. The antibacterial peptide has high application potential.
[0097] 7. Hemolytic activity
[0098] Fresh 10% chicken red blood cells (Hongquan Biotechnology, HQ80071-S010) were washed twice with PBS, and then resuspended to obtain a 2% red blood cell suspension. The antibacterial peptide solution (2-128 μg / mL, including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL) was diluted by PBS in a 96-well plate, and the liquid volume in each well was 50 μL. 50 μL of red blood cell suspension was added to the 96-well plate. The treatment group, negative control group and positive control group were set as follows:
[0099] Treatment group: Equal volume of erythrocyte suspension and antibacterial peptide solution (2-128 μg / mL, including: 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL) were mixed in 96-well plates.
[0100] Positive control: erythrocyte suspension treated with 0.1% triton X-100 (Solebo, T8200).
[0101] Negative control: untreated erythrocyte suspension.
[0102] Subsequently, the 96-well plates were incubated in a 37°C cell incubator for 1 h. After incubation, centrifugation was performed at 3500 rpm for 5 min, the supernatant was aspirated into a new 96-well plate, and the absorbance value was determined at 570 nm by an enzyme-labeled instrument.
[0103] Hemolysis rate was calculated by the following formula (3):
[0104] Hemolysis rate (%) = (treatment group OD 570 nm value - negative control OD 570 nm value) / (positive control OD 570 nm value - negative control OD 570 nm value) x 100% formula (3).
[0105] The results are shown in Table 1. Figure 6 TLH13 did not produce hemolytic activity on chicken erythrocytes at low concentration doses (2 μg / mL, 4 μg / mL, 8 μg / mL), but produced certain hemolytic activity at high doses (16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL), which suggests that attention should be paid to whether the amount of THL13 is reasonable when using THL13.
[0106] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application.
Claims
1. A polypeptide, or a salt thereof, or a derivative thereof, characterized in that: The polypeptide is any one of the following: A1) a polypeptide having an amino acid sequence of SEQ ID No. 1 in the sequence listing; A2) a polypeptide derived from A1) or having more than 90% identity to the polypeptide shown in A1) and having the same function, obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 1 in the sequence listing and having the same function; A3) a fusion polypeptide obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).
2. A nucleic acid molecule encoding the polypeptide as claimed in claim 1.
3. Use of the polypeptide as claimed in claim 1 or a salt or derivative thereof or in the development and / or preparation of a medicament for preventing and / or treating Clostridium perfringens infection.
4. Use of the polypeptide as claimed in claim 1 or a salt or derivative thereof in the development and / or preparation of a feed additive.
5. Use of the nucleic acid molecule as claimed in claim 2 in any one of the following: C1) the development and / or preparation of a medicament for preventing and / or treating Clostridium perfringens infection; C2) the development and / or preparation of a feed additive.
6. A medicament for inhibiting Clostridium perfringens, characterized by: The medicament contains the polypeptide as claimed in claim 1 or a salt or derivative thereof.
7. The medicament according to claim 6, having at least one of the following properties: B1) inhibiting the growth of Clostridium perfringens; B2) increasing the cell wall permeability of Clostridium perfringens; B3) destroying the cell wall of Clostridium perfringens; B4) inhibiting the synthesis of genomic DNA of Clostridium perfringens.
8. Feed additive, characterized in that: The feed additive contains the polypeptide as claimed in claim 1 or a salt or derivative thereof.
9. The feed additive according to claim 8, characterized in that: The concentration of the polypeptide or a salt or derivative thereof in the feed additive is 4 μg / mL to 128 μg / mL.