CN115873092A - Antibacterial polypeptide from banded snake, preparation method and application thereof - Google Patents

Abstract

The invention discloses Cathelicidin family antibacterial polypeptide TS-CATH from a bungarus fasciatus as well as a preparation method and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO:1 or an amino acid sequence having more than 90% homology therewith. According to the invention, a Cathelicidin family mature peptide segment is intercepted by carrying out sequence comparison and excavation on a banded snake protein database to obtain the polypeptide TS-CATH (KRFKKFFKKIKKSVKKRVKKLFKKPRVIPISIPF). The antibacterial polypeptide TS-CATH designed and synthesized by the invention has wide antibacterial spectrum to gram-negative bacteria, high sterilization speed, low hemolysis to sheep red blood cells and small mammalian cytotoxicity, can be applied to the treatment of common bacterial infection and drug-resistant bacterial infectious diseases, and is an excellent substitute drug or adjuvant therapy drug for the existing anti-infection treatment.

Description

A kind of antibacterial polypeptide derived from garter snake and its preparation method and use

technical field

The invention relates to the technical field of polypeptide drugs, in particular to polypeptides derived from the Cathelicidin family of snake garters and their preparation methods and applications.

Background technique

Most traditional antibiotics are designed for a specific target in bacteria. Although they can exert their efficacy more effectively, they also narrow the antibacterial spectrum of the drug. When bacteria evolve or obtain compensatory drug-resistant genes from some unknown hosts, they will quickly cause bacteria to be resistant to this type of antibiotics, and the drug-resistant genes can quickly spread in parallel through plasmids, which will lead to drug-resistant bacteria in a short period of time. Outburst. However, in the case of continuous emergence of drug-resistant bacteria, the development of new antibiotics appears to be slow.

Antimicrobial peptides are a class of small molecule polypeptides. Most antimicrobial peptides are rich in positively charged and hydrophobic amino acids, and generally only have secondary structures. Antimicrobial peptides exist in almost all organisms in various forms and are an important part of biological innate immunity, so they are also called host defense peptides. Antimicrobial peptides are not easy to produce drug resistance due to their unique membrane-breaking bactericidal mechanism, coupled with their non-immunogenicity and low toxicity to eukaryotic cells, making antimicrobial peptides have great potential as a new type of therapeutic drug.

However, the potential toxicity of antimicrobial peptides is the most important problem hindering their clinical application. The antimicrobial peptides currently under clinical experimental research are only used for the treatment of skin infections due to toxicity, and most antimicrobial peptides have also been eliminated in clinical trials due to excessive toxicity, and most of the currently reported antimicrobial peptides cannot meet the effectiveness and safety of antibacterial activity sexual unity. The linear structure of native polypeptides marks the enzymatic instability of the polypeptides. Antimicrobial peptides are sensitive to certain proteases in the wound or plasma, are easily degraded, and have a short half-life in vivo. Therefore, the stability of antimicrobial peptides is another important reason that limits their development.

Currently, there is an urgent need to obtain peptides that are both safe and have good antibacterial activity.

Invention content:

Purpose of the invention: The technical problem to be solved by the present invention is to provide a polypeptide derived from the Cathelicidin family of garter snakes. The polypeptide has a wide antibacterial spectrum for Gram-negative bacteria, a fast bactericidal speed, low hemolyticity to red blood cells, and low toxicity to mammalian cells. .

The technical problem to be solved by the present invention is to provide the preparation method of the above polypeptide.

The technical problem to be solved by the present invention is to provide the application of the polypeptide in the field of antibacterial drugs.

Technical solution: In order to solve the above technical problems, the present invention provides the polypeptide TS-CATH derived from the Cathelicidin family of snake garter, the amino acid sequence of the polypeptide is:

SEQ ID NO: 1:

LYS-ARG-PHE-LYS-LYS-PHE-PHE-LYS-LYS-ILE-LYS-LYS-SER-VAL-LYS-LYS-ARG-VAL-LYS-LYS-LEU-PHE-LYS-LYS-PRO- ARG-VAL-ILE-PRO-ILE-SER-ILE-PRO-PHE;

Or an amino acid sequence having more than 90% homology therewith.

The content of the present invention also includes the preparation method of the polypeptide TS-CATH, and the preparation method includes the polypeptide solid-phase synthesis method.

The content of the present invention also includes the use of the polypeptide TS-CATH derived from the Cathelicidin family of snake garters in the preparation of products for preventing and/or treating diseases caused by bacterial infections.

Wherein, the bacteria include Gram-negative bacteria and/or Gram-positive bacteria.

Wherein, the bacteria include antibiotic-sensitive and/or drug-resistant bacteria, and the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii Several, the Gram-positive bacteria include one or more of Staphylococcus epidermidis and Staphylococcus aureus.

Wherein, the bacterial concentration is 1×10 ⁵ CFU/ml˜1×10 ⁹ CFU/ml.

Wherein, the products include bactericidal, bacteriostatic or antibacterial products.

The content of the present invention also includes the application of the Cathelicidin family polypeptide TS-CATH derived from snake garter in the preparation of medicines for preventing and/or treating sepsis, sepsis and related diseases.

Wherein, the concentration of the polypeptide is 2-1250 μg/ml, preferably, the concentration of the polypeptide is 4-64 μg/ml.

The research on the bacteremia model constructed by intraperitoneal infection of mice with drug-resistant bacteria shows that the antibacterial polypeptide TS-CATH prepared by the present invention can significantly improve the survival rate of mice infected with ceftazidime-resistant E. Protective effect, reducing bacterial load in blood and lung, kidney, spleen and liver tissues of mice.

Beneficial effects: Compared with the prior art, the present invention has the following advantages: the antibacterial polypeptide TS-CATH designed and synthesized in the present invention has a broad antibacterial spectrum, fast bactericidal speed, low hemolysis to red blood cells, and low toxicity to mammalian cells. The antibacterial polypeptide TS-CATH prepared by the invention can be used in the preparation of anti-pathogen infection drugs, has good anti-bacterial activity in vivo and in vitro, and is an excellent substitute drug or auxiliary therapeutic drug for existing anti-infection treatments.

Description of drawings

The HPLC pattern of Fig. 1 polypeptide TS-CATH;

The mass spectrogram of Fig. 2 polypeptide TS-CATH;

The bactericidal curve of Fig. 3 polypeptide TS-CATH to Escherichia coli;

The bactericidal curve of Fig. 4 polypeptide TS-CATH to Klebsiella pneumoniae;

The bactericidal curve of Fig. 5 polypeptide TS-CATH to Staphylococcus aureus;

The hemolytic property of Fig. 6 polypeptide TS-CATH to sheep erythrocyte;

The cytotoxicity of Fig. 7 polypeptide TS-CATH to mammalian cell;

Figure 8 The survival protection effect of polypeptide TS-CATH on bacteremia mice;

The body weight protective effect of Fig. 9 polypeptide TS-CATH on bacteremia mice;

The effect of Fig. 10 polypeptide TS-CATH on the number of bacteria in the blood of bacteremia mice;

Figure 11 The effect of polypeptide TS-CATH on the number of bacteria in the lung tissue of bacteremia mice;

Figure 12 The effect of polypeptide TS-CATH on the number of bacteria in the liver tissue of bacteremia mice;

The effect of Fig. 13 polypeptide TS-CATH on the number of bacteria in the spleen tissue of bacteremia mice;

Fig. 14 Effect of polypeptide TS-CATH on the number of bacteria in kidney tissue of mice with bacteremia.

Detailed ways

Embodiment 1: Design and synthesis of polypeptide TS-CATH

(1) Using the classic snake-derived Cathelicidins family antibacterial peptide OH-CATH (GenBank: ACF21002.1) as a template, use the online "BLAST" tool on the NCBI website (https://www.ncbi.nlm.nih.gov) , perform sequence alignment on the protein database of garter snakes, and mine the amino acid sequences of Cathelicidins in the protein database.

(2) Download the Cathelicidin family antibacterial polypeptide precursor sequences from different species such as human, mouse, pig, sheep, and krait from the UniPort protein database (https://www.uniprot.org), which are consistent with the sequences obtained by BLAST before Import MEGA 7 software for multiple sequence alignment, verify the conserved sequence in the cathlin region, predict the amino acid residue cleavage site of elastase, determine and intercept the mature peptide sequence, and obtain the antibacterial polypeptide TS-CATH sequence of the present invention (KRFKKFFKKIKKSVKKRVKKLFKKPRVIPIPF), As shown in SEQ ID NO:1.

(3) The antibacterial polypeptide TS-CATH was synthesized in vitro by conventional solid-phase polypeptide synthesis method.

The preparation of embodiment 2 polypeptide TS-CATH:

The amino acid sequence of the prepared TS-CATH is: lysine-arginine-phenylalanine-lysine-lysine-phenylalanine-phenylalanine-lysine-lysine-iso Leucine-Lysine-Lysine-Serine-Valine-Lysine-Lysine-Arginine-Valine-Lysine-Lysine-Leucine-Phenylalanine - Lysine - Lysine - Proline - Arginine - Valine - Isoleucine - Proline - Isoleucine - Serine - Isoleucine - Proline - Phenylalanine

Polypeptide synthesis: The synthesis of the polypeptide TS-CATH proceeds one by one from the C-terminus to the N-terminus. Soak Fmoc-Val-Wang Resin with dichloromethane for 15 minutes, until the resin swells, remove the dichloromethane; add hexahydropyridine/dimethylformamide (DMF) solution with a volume ratio of 1:4 (per gram of resin 10ml), use nitrogen agitation, react 2 times, the time is 5 minutes and 15 minutes, wash resin 9 times with DMF after the reaction finishes. Take 20-40g of resin and add 2-3 drops of each color test agent ABC (A solution: ninhydrin/absolute ethanol solution; B solution: pyridine; C solution: phenol/absolute ethanol solution, A, B, and C solutions are all Just add 2-3 drops) and heat at 100°C for 3 minutes, the color of the solution and resin will turn blue to remove the protection of the amino group. Add Fmoc-VaI-OH and HOBT with twice the molar excess, dissolve with 10ml of DMF per gram of resin, add twice the molar DIC and Collidine, blow with nitrogen, and react for 1h. After the reaction, the resin was washed with DMF for 6 times, the condensation reaction was repeated, and each Fmoc-protected amino acid was connected in sequence to complete the synthesis of the linear sequence. The resin was soaked in dichloromethane and ether and then drained. Add TFA, react in a constant temperature shaker for 2 hours, the shaker speed is 110r/min, and the temperature is 25°C. Filter off the resin, add anhydrous ether to the filtrate, centrifuge to obtain a solid, add anhydrous ether to wash, then centrifuge, repeat several times, and then dry to obtain the crude polypeptide.

Purification of peptides: Weigh a certain amount of crude product, add appropriate amount of acetonitrile, sonicate until clarification, and use a filter to remove large particles of impurities. At the same time, samples were collected in sections by a preparative liquid chromatograph. Do gradient analysis with an analytical chromatograph, and retain the samples that have reached the required purity. Then freeze-drying is carried out.

The synthesis and purification of the polypeptide TS-CATH were completed by Jill Biochemical (Shanghai) Co., Ltd., and the purity of the obtained product reached 95.75%.

The purity measurement (HPLC method) and mass spectrometry analysis result of embodiment 3 polypeptide TS-CATH

The polypeptide TS-CATH of Example 1 and Example 2 was synthesized and then purified to obtain the finished product, which was identified by high performance liquid chromatography and mass spectrometry.

Liquid chromatography analysis conditions: C18 chromatographic column (4.6×250mm, 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 220nm; the flow rate is 1.0ml/min; the injection volume is 10μl, and gradient elution is performed. See Table 1 for gradient elution conditions. See Figure 1 for the HPLC chromatogram and Figure 2 for the mass spectrum.

Table 1 Gradient elution conditions of polypeptide TS-CATH

MIC value and MBC value of embodiment 4 polypeptide TS-CATH to Gram-negative bacteria and Gram-positive bacteria

Peptide TS-CATH was diluted to different concentration gradients with MH liquid medium (Beijing Sanyao Science and Technology Development Company, product number: 11E01), and 100 μl was taken in a 96-well plate, and then inoculated with an equal volume and diluted with liquid medium to 2×10 ⁵ CFU/ml bacterial suspension, so that the final drug concentration was 128, 64, 32, 16, 8, 4, 2, 1 μg/ml, and a blank control group without drug was set. Culture the bacteria at 37°C for 18-22 hours, observe and record the experimental results. After confirming the normal growth of bacteria without drug treatment, the lowest drug concentration that does not grow bacteria was taken as the minimum inhibitory concentration (MIC), and the MIC value that could inhibit the growth of 50% of clinical strains was calculated at the same time, that is, MIC ₅₀ . The results are shown in Table 2.

Take 100 μl of cultures without colony growth in the 96-well plate measured by the above MIC, add 900 μl of sterile medium to dilute, transfer all the diluted solution to a sterile plate, and pour about 10 ml of corresponding agar medium (temperature 50 ° C ~ 55°C), culture the bacteria at 37°C for 24 hours, observe and record the number of colonies in the plate, and the lowest drug concentration that makes the number of colonies less than 5 is the minimum bactericidal concentration (MBC) value. The results are shown in Table 3.

Table 2 The MIC value of polypeptide TS-CATH to Gram-negative bacteria and Gram-positive bacteria

IS: clinical isolate; MRSA: methicillin-resistant Staphylococcus aureus

The MBC value of table 3 polypeptide TS-CATH to Gram-negative bacteria and Gram-positive bacteria

IS: clinical isolate; MRSA: methicillin-resistant Staphylococcus aureus

From the results in Table 2 and Table 3, it can be seen that the antibacterial polypeptide TS-CATH of the present invention has a broad antibacterial spectrum, and has good bacteriostatic and bactericidal activities against standard strains and clinical drug-resistant strains. The MIC of antibacterial peptides to Gram-negative bacteria is in the range of 4-32 μg/ml, and the MBC of partially drug-resistant Gram-negative bacteria is in the range of 4-16 μg/ml; MIC was in the range of 8-16 μg/ml, and MBC was in the range of 8-32 μg/ml.

The bactericidal curve of embodiment 5 polypeptide TS-CATH to standard bacterial strain and clinical drug-resistant bacterial strain

About 10 ⁵ CFU/ml concentration of the test bacteria liquid (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and its drug-resistant clinical strains) were grown in the MHB medium of 4 times the MIC drug concentration. At 0, 0.5, 1, 2, 4, 8, 12, and 24 hours after co-incubation, samples of the co-culture were serially diluted to count live bacteria. Taking time as the abscissa and the logarithmic value of the number of bacterial cells as the ordinate, a bactericidal curve is drawn, and the results are shown in Figures 3 to 5.

The bactericidal curve result shows that, for Klebsiella pneumoniae and Pseudomonas aeruginosa, the antibacterial polypeptide TS-CATH of the present invention is at 4 times the MIC drug concentration of 16 μ g/ml, and Klebsiella pneumoniae and Pseudomonas aeruginosa standard bacterial strain And its clinical drug-resistant strains were completely killed within 0.5h. For Staphylococcus aureus, the antimicrobial polypeptide TS-CATH of the present invention can be completely eliminated within 1h and 4h respectively at 4 times the MIC drug concentration of 64μg/ml. kill.

Embodiment 6 The hemolytic property of polypeptide TS-CATH to sheep erythrocyte

The centrifuged sheep erythrocytes were diluted to 3% (v/v) with sterile PBS (0.01mol/L, pH 7.4), and diluted with the polypeptide TS-CATH synthesized in Example 1 so that the final concentration of the drug was 512, 256, 128, 64, 32, 16, 8, and 0 μg/ml were mixed and inoculated in a 96-well plate, and a PBS negative control and a final concentration of 0.1% Triton X-100 positive control were set up. Incubate for 30 minutes in a CO ₂ incubator. After centrifugation, the supernatant was measured with a microplate reader for the absorbance OD value at a wavelength of 450 nm. The hemolysis rate of red blood cells treated with Triton X-100 was 100%, and the red blood cells treated with PBS was 0%. The hemolysis rate of the drug on sheep red blood cells was calculated, and the results are shown in Figure 6.

The results of hemolysis showed that even if the concentration of the antibacterial polypeptide TS-CATH reported in the present invention reaches 512 μg/ml, the hemolysis rate is lower than 10%, and has very low hemolysis.

Cytotoxicity of Example 7 Polypeptide TS-CATH to Mammalian Cells

Human hepatocyte L02, human umbilical vein endothelial cell Huvec and mouse fibroblast L929 were cultured to a cell density of about 80% to 90%. After digestion and centrifugation, the supernatant was removed, and the cells were resuspended and diluted with DMEM complete medium. The concentration is 5-10×10 ⁴ cells/ml, inoculated in 96-well plates, placed in an incubator for 12 hours, and administered after the cells adhere to the wall. Polypeptide TS-CATH was dissolved in DMEM complete medium and diluted to 128, 64, 32, 16, 8 μg/ml administration group and a non-administration blank group, with 3 replicate wells for each group, and incubated at 37°C for 48h. Add 10 μl of MTT solution (thiazolium blue, 5 mg/ml, ie 0.5% MTT) to each well, and continue culturing for 4 h. Carefully suck off the supernatant, add 100 μl dimethyl sulfoxide to each well, shake on a shaker at low speed for 10 minutes, and fully dissolve the crystals. The absorbance value of each well was measured at OD490 of a microplate reader, and the cell viability was counted. The results are shown in FIG. 7 .

It can be seen from the results of cytotoxicity that the antibacterial polypeptide TS-CATH of the present invention has lower cytotoxicity to mammalian cells at both bacteriostatic and bactericidal concentrations. In particular, the 48h survival rate of human hepatocyte L02, human endothelial cell Huvec and mouse fibroblast L929 is higher than 80% at a concentration of 64 μg/ml.

The effect of embodiment 8 serum on the antibacterial activity of polypeptide TS-CATH

The polypeptide TS-CATH synthesized in Example 1 was formulated with PBS (0.01mol/L, pH 7.4) to a solution with a concentration of 2048 μg/ml, mixed with human serum at a volume ratio of 3:1, and co-incubated in an incubator at 37°C. At the same time, serum-free PBS was set as a control. Samples were taken at 0.5h, 1h, and 2h at different time points, and 15% trichloroacetic acid was added at a ratio of 6:1, incubated at 4°C for 30 minutes, centrifuged at 13,000rpm for 10 minutes to remove the precipitate, and diluted with MHB medium, according to the example The method of 4 was used to measure the MIC change of the polypeptide, and the results are shown in Table 4.

The impact of table 4 serum on the antibacterial activity of polypeptide TS-CATH

IS: Clinical isolates

It can be known from the results that the polypeptide TS-CATH still has certain antibacterial activity after serum treatment. The peptide TS-CATH still has antibacterial activity against clinical drug-resistant strains even after being treated with serum for 2 hours.

Example 9 Protective Effect of Polypeptide TS-CATH on Bacteremia Model Mice

ICR female mice with a healthy body weight of 20-22 g were randomly divided into 6 groups with 10 mice in each group. Five groups of mice were intraperitoneally inoculated with the bacterial suspension of the lowest lethal concentration (ceftazidime-resistant Escherichia coli clinical strain, 1×10 ⁹ CFU/ml, 0.5ml/mouse) for infection to establish a bacteremia model, and intraperitoneal injection etc. The mice with normal saline were the blank control group. The remaining modeling components were 10mg/kg TS-CATH (high-dose group), 5.0mg/kg TS-CATH (middle-dose group), 2.5mg/kg TS-CATH (low-dose group), 5mg/kg ceftazidime (control group). group), normal saline (model control). The polypeptide TS-CATH administration group and the normal saline group were intraperitoneally administered twice 0.5h and 2h after infection respectively, and the ceftazidime group was administered once by tail vein 2h after infection. The mice were observed continuously for 7 days and the death and body weight changes of the mice were recorded.

Eight hours after the mice were infected with bacteria, 3 mice were taken from each group, the eyeballs were peeled off, and the blood was taken into a sterile EP tube, mixed slightly, and 200 μl was taken into a sterile EP tube containing heparin sodium (1% heparin saline solution, 50 μl To a sterile EP tube, dried at 60°C), 10-fold gradient dilution of blood, 1ml of diluted blood was taken from each tube, added to nutrient agar medium and poured onto a plate to count viable bacteria. The abdomen of each group of mice was soaked in 75% ethanol, and after being dissected on the ultra-clean workbench, the lungs, liver, spleen, and kidneys were taken, and the tissues were placed in 5ml EP tubes, according to the ratio of V _tissue : V _{normal saline} = 1:9 Add sterile normal saline, cut it into pieces with sterile scissors, grind it with a homogenizer, and dilute it 10 times in a gradient. Take 1ml of the diluted solution and add it to the nutrient agar medium to count the viable bacteria. Place it upside down in a 37°C incubator and observe And record the number of colonies in the organs of infected mice.

It can be seen from Figure 8 that ceftazidime-resistant Escherichia coli can cause most of the mice in the ceftazidime control group to die, with a mortality rate as high as 83.3%. There is a substantial increase, and the administration of low, medium and high doses of the polypeptide has a dose-dependent increase in the survival rate of mice, which proves that the polypeptide TS-CATH has a certain protective effect on mice infected with drug-resistant bacteria. It can be seen from FIG. 9 that, one day after the bacterial infection, the body weight of the mice in the polypeptide administration group and the ceftazidime group decreased to a certain extent. On the seventh day after bacterial infection, the weight of the mice in the high-dose polypeptide group returned to the weight of the mice in the blank control group, and compared with the mice in the low-dose polypeptide group, the mice in the high-dose group recovered faster, indicating that the polypeptide TS -CATH also had a protective effect on body weight in mice.

It can be seen from Figures 10-14 that after 8 hours of bacterial infection, compared with the model group, each dose of polypeptide administration can significantly reduce the number of bacteria in the mouse blood, lung tissue, liver tissue, spleen tissue, and kidney tissue, and Bacterial clearance was dose-dependent, and the number of bacteria in each tissue of the ceftazidime administration group was comparable to that of the model group. The polypeptide TS-CATH exhibited a strong ability to clear bacteria, which could significantly improve the survival rate of mice infected with drug-resistant bacteria.

Claims (8)

1. The Cathelicidin family polypeptide TS-CATH derived from the garter snake, is characterized in that the amino acid sequence of the polypeptide is: SEQ ID NO: 1: LYS-ARG-PHE-LYS-LYS-PHE-PHE-LYS-LYS-ILE-LYS-LYS-SER-VAL-LYS-LYS-ARG-VAL-LYS-LYS-LEU-PHE-LYS-LYS-PRO- ARG-VAL-ILE-PRO-ILE-SER-ILE-PRO-PHE; Or an amino acid sequence with more than 90% homology to it. 2. The preparation method of the polypeptide TS-CATH according to claim 1, characterized in that the preparation method comprises a polypeptide solid-phase synthesis method. 3. The use of the Cathelicidin family polypeptide TS-CATH derived from snake garters according to claim 1 in the preparation of products for preventing and/or treating diseases caused by bacterial infections. 4. The use according to claim 3, characterized in that the bacteria comprise Gram-negative bacteria and/or Gram-positive bacteria. 5. purposes according to claim 3, is characterized in that, described bacterium comprises antibiotic sensitive and/or drug-resistant bacterium, and described Gram-negative bacterium comprises Escherichia coli, Pseudomonas aeruginosa, Klein pneumoniae one or more of Burger bacteria and Acinetobacter baumannii, and the Gram-positive bacteria include one or more of Staphylococcus epidermidis and Staphylococcus aureus. 6. The use according to claim 3, wherein the product comprises a bactericidal, bacteriostatic or antibacterial product. 7. The application of the Cathelicidin family polypeptide TS-CATH derived from snake garter according to claim 1 in the preparation of medicines for preventing and/or treating sepsis, sepsis and related diseases. 8. The application according to any one of claims 3-7, wherein the concentration of the polypeptide is 2-512 μg/ml.

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