A Cathelicidin family polypeptide derived from an aquatic mammal, and its preparation method and use

Abstract

The present invention discloses a Cathelicidin family polypeptide derived from aquatic mammals. The polypeptide is obtained by mining the proteome database of aquatic mammals. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, SEQ ID NO: 2, or an amino acid sequence having more than 90% homology with SEQ ID NO: 1 or 2. The present invention intercepts the mature peptide segments of the Cathelicidin family by sequence alignment and mining of the protein database of aquatic mammals, and obtains the polypeptides BM-CATH and LV-CATH. The antibacterial polypeptides BM-CATH and LV-CATH designed and synthesized by the present invention have a broad antibacterial spectrum, a fast bactericidal speed, low hemolytic activity against sheep red blood cells, and low cytotoxicity to mammalian cells. They can be applied to the treatment of common bacterial infections and drug-resistant bacterial infectious diseases, and are excellent alternative drugs or adjuvant drugs for existing anti-infection treatments.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drugs in biochemistry, and particularly relates to a Cathelicidin family polypeptide derived from aquatic mammals, a preparation method thereof, and uses thereof. Background Art

[0002] Cathelicidin family antimicrobial peptides are a class of antimicrobial peptide families with variable structures existing in mammals. There is a highly conserved cathlin fragment between the signal peptide and the mature peptide in the precursor peptide sequence. Cathelicidin family polypeptides have broad-spectrum antimicrobial activities and play important roles in the host innate immune defense system. Research reports in recent years have shown that Cathelicidin can induce chemotaxis of various immune cells, induce angiogenesis, induce apoptosis of mutant cells, promote cell differentiation, and promote tissue damage repair, etc. Cathelicidin has become a research hotspot in the fields of contemporary medicine, biology, and pharmacy at home and abroad.

[0003] So far, Cathelicidin family polypeptides have been found in almost all vertebrates, including mammals, birds, reptiles, amphibians, and lower fish. However, there are few reports on Cathelicidin family antimicrobial peptides derived from aquatic mammals. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide Cathelicidin family polypeptides BM-CATH and LV-CATH derived from aquatic mammals. The polypeptides are obtained by mining the proteome database of aquatic mammals. The polypeptides have a broad antibacterial spectrum, a fast bactericidal speed, low hemolytic activity against red blood cells, and low cytotoxicity to mammalian cells.

[0005] Another technical problem to be solved by the present invention is to provide a preparation method of the above polypeptides.

[0006] Another technical problem to be solved by the present invention is to provide the application of the polypeptides in the field of antibacterial drugs.

[0007] Technical Solution: To solve the above technical problems, the present invention provides a Cathelicidin family polypeptide derived from aquatic mammals. The amino acid sequence of the polypeptide is selected from:

[0008] SEQ ID NO: 1:

[0009] GLY-ARG-PHE-SER-ARG-LEU-ARG-LYS-ARG-ILE-ARG-LYS-VAL-TRP-ARG-LYS-ILE-GLY-PRO-ILE-ALA-GLY-PRO-ILE-ILE-GLY-HIS-PHE-GLY,

[0010] SEQ ID NO: 2:

[0011] GLY-ARG-PHE-ARG-ARG-LEU-ARG-ASN-ARG-ILE-ARG-ASN-ILE-TRP-ARG-LYS-ILE-GLY-PRO-ILE-ALA-GLY-PRO-LEU-ILE-SER-ARG-PHE-GLY,

[0012] or an amino acid sequence having more than 90% homology with SEQ ID NO: 1 or 2.

[0013] Among them, the preparation method of the polypeptide is characterized in that the preparation method includes the conventional solid-phase synthesis method of polypeptides.

[0014] The present invention also includes the use of the Cathelicidin family polypeptide derived from aquatic mammals in the preparation of products for preventing and / or treating diseases caused by bacterial infections.

[0015] Among them, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0016] Among them, the bacteria are drug-resistant bacteria. The Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, and the Gram-positive bacteria include one or more of Staphylococcus epidermidis and Staphylococcus aureus.

[0017] Among them, the concentration of the bacteria is 1×10 5 CFU / ml to 1×10 9 CFU / ml.

[0018] Among them, the products include bactericidal, bacteriostatic or antibacterial products.

[0019] The use of the Cathelicidin family polypeptide derived from aquatic mammals in the preparation of drugs for treating diseases related to anti-bacterial infections.

[0020] Among them, for the use, the concentration of the polypeptide is 2 to 512 μg / ml. Preferably, the concentration of the polypeptide is 4 to 64 μg / ml.

[0021] Advantages: Compared with the prior art, the present invention has the following advantages: The two antibacterial polypeptides BM-CATH and LV-CATH designed and synthesized in the present invention have a broad antibacterial spectrum, fast bactericidal speed, low hemolysis of red blood cells, and low cytotoxicity to mammalian cells. The antibacterial polypeptides BM-CATH and LV-CATH prepared by the present invention can be used in the preparation of anti-pathogen infection drugs, have good antibacterial activities in vivo and in vitro, and can be applied to various common infections and stubborn infectious diseases, and are excellent alternative drugs or adjuvant therapy drugs for existing anti-infection treatments. Description of the Drawings

[0022] Figure 1 HPLC spectrum of polypeptide BM-CATH;

[0023] Figure 2 Mass spectrum of polypeptide BM-CATH;

[0024] Figure 3 HPLC spectrum of polypeptide LV-CATH;

[0025] Figure 4 Mass spectrum of polypeptide LV-CATH;

[0026] Figure 5 Bactericidal curve of polypeptide BM-CATH against Escherichia coli;

[0027] Figure 6 Bactericidal curve of polypeptide BM-CATH against Klebsiella pneumoniae;

[0028] Figure 7 Bactericidal curve of polypeptide BM-CATH against Staphylococcus aureus;

[0029] Figure 8 Bactericidal curve of polypeptide LV-CATH against Escherichia coli;

[0030] Figure 9 Bactericidal curve of polypeptide LV-CATH against Klebsiella pneumoniae;

[0031] Figure 10 Bactericidal curve of polypeptide LV-CATH against Staphylococcus aureus;

[0032] Figure 11 Hemolysis of polypeptide on sheep red blood cells;

[0033] Figure 12 Cytotoxicity of polypeptide to human normal liver cells L02;

[0034] Figure 13 Cytotoxicity of polypeptide to human umbilical vein endothelial cells Huvec;

[0035] Figure 14Cytotoxicity of the polypeptide against mouse fibroblast L929 cells. Detailed implementation mode

[0036] Example 1 Design and synthesis of the polypeptide drug of the present invention

[0037] (1) Using the classic snake-derived Cathelicidins family antimicrobial polypeptide OH-CATH (GenBank: ACF21002.1) as a template, on the NCBI website (https: / / www.ncbi.nlm.nih.gov), the online tool "BLAST" was used to perform sequence alignment on the protein databases of blue whales and baiji dolphins respectively, and the Cathelicidins amino acid sequences in the biological proteome database were mined.

[0038] (2) Download the precursor sequences of Cathelicidin family antimicrobial polypeptides from different species such as humans, mice, pigs, sheep, and king cobras in the UniPort protein database (https: / / www.uniprot.org), and import them into the MEGA 7 software together with the sequences obtained by BLAST before for multiple sequence alignment to verify the conserved sequences in the cathlin region, and at the same time predict the amino acid residue cleavage sites of elastase, and intercept the mature peptide sequences to obtain the two antimicrobial polypeptides BM-CATH (SEQ ID NO: 1) and LV-CATH (SEQ ID NO: 2) reported in this patent. The amino acid sequences are shown in Table 1.

[0039] (3) The antimicrobial polypeptides BM-CATH and LV-CATH were synthesized in vitro by the conventional solid-phase synthesis method of polypeptides.

[0040] Table 1 Names and amino acid sequences of the two antimicrobial polypeptides

[0041]

[0042] Example 2 Preparation of antimicrobial polypeptides BM-CATH and LV-CATH:

[0043] The amino acid sequence of the prepared BM-CATH is: glycine-arginine-phenylalanine-serine-arginine-leucine-arginine-lysine-arginine-isoleucine-arginine-lysine-valine-tryptophan-arginine-lysine-isoleucine-glycine-proline-isoleucine-alanine-glycine-proline-isoleucine-isoleucine-glycine-histidine-phenylalanine-glycine.

[0044] The amino acid sequence of the prepared LV-CATH is: glycine-arginine-phenylalanine-arginine-arginine-leucine-arginine-asparagine-arginine-isoleucine-arginine-asparagine-isoleucine-tryptophan-arginine-lysine-isoleucine-glycine-proline-isoleucine-alanine-glycine-proline-leucine-isoleucine-serine-arginine-phenylalanine-glycine.

[0045] Synthesis of polypeptide: After the synthesis of polypeptide TS-CATH was carried out one by one from the C-terminus to the N-terminus. Fmoc-Val-Wang Resin was soaked in dichloromethane for 15 minutes. After the resin swelled, the dichloromethane was pumped out, and a solution of hexahydropyridine / dimethylformamide (DMF) with a volume ratio of 1:4 (10 ml per gram of resin) was added. Nitrogen was used for agitation, and the reaction was carried out twice for 5 minutes and 15 minutes. After the reaction, the resin was washed with DMF 9 times. Take 20 - 40 resin particles and add 2 - 3 drops of each of the color reagent ABC (Solution A: ninhydrin / absolute ethanol solution; Solution B: pyridine; Solution C: phenol / absolute ethanol solution, 2 - 3 drops of each of Solution A, B, and C are sufficient). Heat together at 100 °C for 3 minutes, and the color of the solution and the resin turns blue to remove the amino protection. Excess Fmoc-VaI-OH and HOBT with twice the molar amount of reaction were added, dissolved in 10 ml of DMF per gram of resin, twice the molar amount of DIC and collidine were added, and nitrogen was used for agitation. The reaction was carried out for 1 h. After the reaction, the resin was washed with DMF 6 times, and the condensation reaction was repeated successively to connect each Fmoc-protected amino acid to complete the synthesis of the linear sequence. The resin was soaked in dichloromethane and ether and then dried by suction. TFA was added, and the reaction was carried out in a constant temperature shaker for 2 h, with the shaker speed of 110 r / min and the temperature of 25 °C. The resin was filtered off, anhydrous ether was added to the filtrate, and after centrifugation, a solid was obtained. It was washed with anhydrous ether and centrifuged again. After repeating several times, it was dried to obtain the crude polypeptide.

[0046] Purification of polypeptide: Weigh a certain amount of the crude product, add an appropriate amount of acetonitrile, ultrasonicate until clear, and remove large particle impurities with a filter. At the same time, pass through a preparative liquid chromatograph, and collect samples in segments. Perform gradient analysis with an analytical chromatograph, and retain the samples that reach the required purity. Then carry out freeze-drying treatment.

[0047] The synthesis and purification of the polypeptide were both completed by Gil Biochemical (Shanghai) Co., Ltd. The purity of the obtained polypeptide BM-CATH reached 95.81%, and the purity of the polypeptide BM-CATH reached 95.80%.

[0048] Example 3 Purity determination (HPLC method) and mass spectrometry analysis results of the polypeptide

[0049] After the polypeptide in Example 1 was synthesized and purified, the finished product was identified by high performance liquid chromatography and mass spectrometry.

[0050] Liquid chromatography analysis conditions: C18 chromatographic column (4.6×250 mm, 5 μm); mobile phase A is acetonitrile solution containing 0.1% trifluoroacetic acid; mobile phase B is pure water containing 0.1% trifluoroacetic acid. The detection wavelength is 220 nm, the flow rate is 1.0 ml / min, the injection volume is 100 μl, and gradient elution is performed. The gradient elution conditions are shown in Table 2-3. The HPLC chromatogram and mass spectrometry results are shown in Figure 1-4 .

[0051] Table 2 Gradient elution conditions of polypeptide BM-CATH

[0052]

[0053] Table 3 Gradient elution conditions of polypeptide LV-CATH

[0054]

[0055] Example 4 MIC values and MBC values of the polypeptide of the present invention against Gram-negative bacteria and Gram-positive bacteria

[0056] The polypeptide was serially diluted into different concentration gradients with MHB medium (Beijing Sanyao Science and Technology Development Company, product number: 11E01). Then, 100 μl was taken and placed in a 96-well plate respectively. After that, an equal volume of bacterial suspension diluted to 2×10 5 CFU / ml was inoculated, so that the final drug concentration was 128, 64, 32, 16, 8, 4, 2, 1 μg / ml. At the same time, a blank control group without drug was set up. The bacteria were cultured at 37 °C for 16-18 h, and the test results were observed and recorded. The lowest drug concentration without bacterial growth was the minimum inhibitory concentration (MIC). At the same time, the MIC value that could inhibit the growth of 50% of clinical strains was calculated, namely MIC 50 . The results are shown in Table 4.

[0057] 100 μl of the culture without colony growth in the 96-well plate for the above MIC determination was taken, diluted with 900 μl of sterile medium, and then transferred to a sterile petri dish. About 10 ml of the corresponding agar medium (temperature 50 °C - 55 °C) was poured. The bacteria were cultured at 37 °C for 24 h, and the number of colonies in the petri dish was observed and recorded. The lowest drug concentration with the number of colonies less than 5 was the MBC value. The results are shown in Table 5.

[0058] Table 4 MIC values of the polypeptide against Gram-negative bacteria and Gram-positive bacteria

[0059]

[0060] IS: Clinically isolated strain; MRSA: Methicillin-resistant Staphylococcus aureus

[0061] Table 5 MBC values of the polypeptide against Gram-negative bacteria and Gram-positive bacteria

[0062]

[0063]

[0064] IS: Clinically isolated strains; MRSA: Methicillin-resistant Staphylococcus aureus

[0065] As can be seen from the results in Table 2, the two antibacterial polypeptides reported in this patent have a broad antibacterial spectrum and show good antibacterial and bactericidal activities against standard strains and clinically drug-resistant strains. The MIC of these two antibacterial polypeptides against Gram-negative bacteria is in the range of 2 - 8 μg / ml, and the MBC is in the range of 4 - 16 μg / ml; while for Gram-positive bacteria, the MIC of these two antibacterial polypeptides is in the range of 4 - 16 μg / ml, and the MBC is in the range of 4 - 32 μg / ml. At the same time, it can be found that the two polypeptides reported in this patent still show good antibacterial and bactericidal activities against methicillin-resistant Staphylococcus aureus.

[0066] Example 5 Bactericidal curves of the polypeptides of the present invention against standard strains and clinically drug-resistant strains

[0067] Approximately 10 5 CFU / ml of the test bacterial suspension (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and their clinically drug-resistant strains) grows in MHB medium at 4 times the MIC drug concentration. Samples of the co-culture are taken at 0, 0.5, 1, 2, 4, 8, 12, 24 h after co-incubation, gradient diluted and viable cell counts are performed. Taking time as the abscissa and the logarithm of the number of bacterial cells as the ordinate, a bactericidal curve graph is made, and the results are as Figures 5 to 10 shown.

[0068] The results of the bactericidal curves show that the two antibacterial polypeptides reported in the present invention have rapid bactericidal activities against a variety of clinically drug-resistant strains at 4 times the MIC drug concentration, and most of the test strains are completely killed within half an hour.

[0069] Example 6 Hemolytic activity of the polypeptides of the present invention against sheep red blood cells

[0070] The centrifuged sheep red blood cells were diluted to 3% with sterile PBS (0.01 mol / L, pH 7.4). The polypeptides were diluted with PBS at concentration gradients of 512, 256, 128, 64, 32, 16, 8, 0 μg / ml and mixed and inoculated into 96-well plates. A PBS negative control and a positive control with a final concentration of 0.1% Triton X-100 were established. They were cultured in a CO2 incubator for 30 min. After centrifugation, the absorbance OD value at a wavelength of 450 nm was measured for the supernatant using an enzyme-linked immunosorbent assay (ELISA) reader. Taking Triton X-100 as 100% hemolysis rate and PBS as 0% hemolysis rate, the hemolysis rate of the drug on sheep red blood cells was calculated. The results are as Figure 11 shown.

[0071] The hemolytic results showed that even when the concentration of the two antibacterial polypeptides reported in the present invention reached 512 μg / ml, the hemolysis rate was lower than 10%, indicating very low hemolytic activity.

[0072] Example 7 Cytotoxicity of the polypeptides of the present invention on mammalian cells

[0073] Human hepatocytes L02, human umbilical vein endothelial cells Huvec, and mouse fibroblasts L929 were cultured until the cell density reached approximately 80% - 90%. After digestion and centrifugation to collect the cells, the supernatant was removed, and the cells were resuspended and diluted with complete DMEM medium to a concentration of 5 - 10×10 4 cells / ml, inoculated into 96-well plates, and cultured in an incubator for 12 h. After the cells adhered to the wells, the drugs were administered. The concentrations of the two polypeptides synthesized in Example 1 were set as dosing groups at 128, 64, 32, 16, 8 μg / ml respectively, and a blank group without drug administration was set. Each group had 3 replicates. They were incubated at 37°C for 48 h. 10 μl of MTT solution (thiazolyl blue, 5 mg / ml) was added to each well, and incubation continued for 4 h. The supernatant was carefully aspirated, 100 μl of dimethyl sulfoxide was added to each well, and the plate was placed on a shaker and shaken at low speed for 10 min to fully dissolve the crystals. The absorbance values of each well were measured at OD 490 of the ELISA reader, and the cell survival rate was counted. The results are as Figures 12 to 14 shown.

[0074] From the cytotoxicity results, it can be seen that the two antibacterial polypeptides reported in the present invention have low cytotoxicity on mammalian cells at effective antibacterial and bactericidal concentrations.

Claims

1. A Cathelicidin family polypeptide derived from an aquatic mammal, characterized in that, The amino acid sequence of the polypeptide is selected from: SEQ ID NO: 1: GLY-ARG-PHE-SER-ARG-LEU-ARG-LYS-ARG-ILE-ARG-LYS-VAL-TRP-ARG-LYS-ILE-GLY-PRO-ILE-ALA-GLY-PRO-ILE-ILE-GLY-HIS-PHE-GLY or SEQ ID NO: 2: GLY-ARG-PHE-ARG-ARG-LEU-ARG-ASN-ARG-ILE-ARG-ASN-ILE-TRP-ARG-LYS-ILE-GLY-PRO-ILE-ALA-GLY-PRO-LEU-ILE-SER-ARG-PHE-GLY.

2. Use of the Cathelicidin family polypeptide derived from aquatic mammals according to claim 1 in the preparation of an antibacterial agent for treating diseases caused by bacterial infections; the bacteria are Gram-negative bacteria and / or Gram-positive bacteria; the Gram-negative bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and the Gram-positive bacteria are one or more of Staphylococcus epidermidis, Staphylococcus aureus.

3. The use according to claim 2, characterized in that, The antibacterial agent includes bactericidal, bacteriostatic or antibacterial drugs.

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