Lactic acid bacteria source antibacterial peptide as well as database-based auxiliary construction method and application thereof

Abstract

The invention discloses a lactic acid bacteria source antibacterial peptide and a database-based auxiliary construction method and application thereof, and belongs to the field of bioengineering, and the amino acid sequence of the antibacterial peptide is shown as SEQ NO.1. Bioinformatics resources and calculation tools are used for aided design of peptide, and a database prediction model is used for predicting peptide activity; the optimal candidate peptide sequence can be screened out under the conditions of the shortest time and the lowest cost, the peptide is a non-natural peptide which is based on a lactic acid bacteria source antibacterial peptide database and can effectively inhibit gram-positive bacteria and gram-negative bacteria, good biological activity and low hemolytic toxicity are achieved, the therapeutic index reaches up to 168.98, and good application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a lactic acid bacteria-derived antimicrobial peptide and a database-assisted construction method and application thereof. Technical Background

[0002] The conformations of bacterial antimicrobial peptides are diverse, which can be α-helix, β-fold, linear extension or random curl, or cyclic peptide or a mixture of the above multiple conformations, but most antimicrobial peptides have an α-helix conformation. Although natural bacterial AMPs have shown superiority in overcoming multidrug resistance, their development lacks design principles and is not systematic. Therefore, this process becomes a "random" "black box trial and error" program. Under this view, the development of non-natural peptides is to provide a wider AMP library. At present, the most studied bacterial AMPs are lactic acid bacteria (LABs), and AMPs produced by LAB are generally considered safe. Among them, nisin produced by lactic streptococci is a type of food additive currently approved for use in many countries, but its antibacterial spectrum is narrow and only has a good antibacterial effect on Gram-positive bacteria. Therefore, it is critical to find or artificially design bacterial antimicrobial peptides with a wide antibacterial spectrum, strong stability and low cost. Traditional AMPs design methods are usually costly and time-consuming, so it is particularly important to find new design strategies to reduce iteration costs. With the gradual maturity of bioinformatics and computer algorithms, AMPs databases and prediction models have begun to appear, and the use of bioinformatics resources and computational tools to assist peptide design, predict peptide activity, and screen the best candidate peptides has gradually become a trend. Summary of the invention

[0003] Based on the above shortcomings, the present invention provides a lactic acid bacteria-derived antimicrobial peptide (LAB 0.4) to solve the problems of high artificial screening cost, low antibacterial efficiency and high hemolysis of existing lactic acid bacteria-derived antimicrobial peptides. It has good antibacterial activity against both Gram-negative and Gram-positive bacteria, but low antibacterial activity against probiotics.

[0004] The technical solution adopted by the present invention is as follows: a lactic acid bacteria-derived antimicrobial peptide, whose amino acid sequence is shown in SEQ NO.1.

[0005] Another object of the present invention is to provide a method for constructing a lactic acid bacteria-derived antimicrobial peptide as described above, which is based on database-assisted design, and the method is as follows: by searching the lactic acid bacteria-derived antimicrobial peptides in the two major databases of APD and DRAMP, a lactic acid bacteria-derived antimicrobial peptide library containing 129 sequences was established, as shown in Table 1 in this specification, and after peptide library analysis, it was determined that cationic amino acids: hydrophobic amino acids ≈ 1:4, cationic amino acids + hydrophobic amino acids accounted for 68.20%, and the average sequence length was 36 amino acids. The cationic amino acids and hydrophobic amino acids with the highest amino acid proportions were K: 8.23%, G: 14.00%, A: 11.09%, L: 6.25%, V: 6.05%. The sequence length of 25 was determined according to the amino acid ratio. 120 sequences were generated with (ABCDE)5 as a template. The 120 sequences were screened using activity prediction tools in the three databases of DRAMP, CAMP, and DBAASP to obtain the most likely peptide sequence with antimicrobial activity. Then, the sequence was mutated based on the genetic algorithm in the HeliQuest server to obtain ten sequences with hydrophobic moment = 0.4. The database activity prediction tool was used again to screen the best candidate polypeptide. The amino acid sequence of the polypeptide is shown in SEQ NO.1. The polypeptide was then synthesized by solid phase chemical synthesis, and then subjected to antibacterial activity assay, hemolytic activity assay, and stability assay, and finally named antimicrobial peptide LAB 0.4.

[0006] Another object of the present invention is to provide the use of the lactic acid bacteria-derived antimicrobial peptide as described above in the preparation of a drug for treating and / or preventing Gram-negative bacteria and / or infectious diseases caused by Gram-negative bacteria.

[0007] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii or Pseudomonas aeruginosa.

[0008] Furthermore, the Gram-positive bacteria mentioned above are Staphylococcus aureus, Enterococcus faecalis or Bacillus subtilis.

[0009] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the lactic acid bacteria-derived antimicrobial peptide as described above.

[0010] The present invention has the following advantages and beneficial effects: the present invention utilizes bioinformatics resources and computational tools to assist in designing peptides, and a database prediction model to predict peptide activity, and can screen out the best candidate peptide sequence under the conditions of the shortest time and the lowest cost. The antimicrobial peptide LAB 0.4 of the present invention has a stable structure, and the synthesized antimicrobial peptide is tested for antibacterial, hemolytic and stability activities. It is found that the antimicrobial peptide LAB 0.4 has a high inhibitory effect on Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, etc., has a low antibacterial activity on probiotics, and has very low hemolytic activity, and the therapeutic index is as high as 168.98. At the same time, the antimicrobial peptide LAB 0.4 still maintains a certain stability under serum and physiological salt ion conditions. In summary, the antimicrobial peptide LAB 0.4 is an antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the HPLC chromatogram of antimicrobial peptide (LAB 0.4);

[0012] Figure 2 This is the mass spectrum of antimicrobial peptide (LAB 0.4). DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1

[0015] Design of antimicrobial peptide (LAB 0.4)

[0016] By searching the lactic acid bacteria-derived antimicrobial peptides in the two major databases of APD and DRAMP, a lactic acid bacteria-derived antimicrobial peptide library containing 129 sequences was established, as shown in Table 1. After peptide library analysis, it was determined that cationic amino acids: hydrophobic amino acids ≈ 1:4, cationic amino acids + hydrophobic amino acids accounted for 68.20%, and the average sequence length was 36 amino acids. The cationic amino acids and hydrophobic amino acids with the highest amino acid ratios were K (8.23%), G (14.00%), A (11.09%), L (6.25%), and V (6.05), respectively. According to the amino acid ratio, the sequence length was determined to be 25, and 120 sequences were generated using (ABCDE) 5 as a template. The 120 sequences were screened using the activity prediction tools in the three databases of DRAMP, CAMP, and DBAASP to obtain the most likely peptide sequence with antimicrobial activity. The sequence was then mutated based on the genetic algorithm in the HeliQuest server to obtain ten sequences with a hydrophobic moment of 0.4. The best candidate peptide was obtained using the database activity prediction tool again. The final antimicrobial peptide amino acid sequence is shown in Table 2.

[0017] Table 1 Antimicrobial peptide library derived from lactic acid bacteria

[0018]

[0019]

[0020]

[0021]

[0022] Table 2 Antimicrobial peptide (LAB 0.4) amino acid sequence

[0023]

[0024]

[0025] The sequence length of the antimicrobial peptide (LAB 0.4) is 25, cationic amino acid: hydrophobic amino acid = 1:4, Lys provides a positive charge, and the charge number is + 5. The antimicrobial peptide designed by this method maximizes the antimicrobial activity while making the antimicrobial peptide have a lower hemolytic activity.

[0026] Example 2

[0027] 1. The peptides are synthesized one by one from the C-terminus to the N-terminus through a synthesis instrument. The first step is to connect Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) to Wang resin, and then remove the Fmoc group to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide), follow this process from the C-terminus to the N-terminus until the synthesis is completed, and obtain the resin with the side chain protection of the Fmoc group removed;

[0028] 2. Add a cutting agent to the polypeptide resin obtained above, react at 20°C in the dark for 2 hours, filter, wash the precipitate with FA (trifluoroacetic acid), mix the filtrate and washing solution evenly, concentrate using a rotary evaporator, then add 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, rinse the precipitate with anhydrous ether, dry under vacuum conditions, and finally obtain the polypeptide. The cutting agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0029] 3. Use 0.2 mol / L sodium sulfate (adjusted to pH = 7.5 with phosphoric acid) to balance the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and use C18 reverse phase atmospheric pressure column for gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1 mL / min, detection wave is 220 nm, and then collect the main peak and freeze-dry.

[0030] 4. Peptide identification: The obtained peptides were analyzed by electrospray mass spectrometry. The mass spectrum (see attached Figure 1 ) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide is greater than 95% (see Appendix Figure 2 ).

[0031] Example 3

[0032] Bioactivity assay of antimicrobial peptides

[0033] 1. Determination of antibacterial activity

[0034] The bacteria were grown in cation-adjusted MHB broth at 37°C and 220 g in a shaking incubator until the logarithmic growth phase and diluted to an OD 600nm =0.4(3×10 8 -9×10 8 CFU mL -1 ). The bacterial solution was diluted 1000 times before use. Equal volumes (50 μL) of bacterial suspension and solutions containing different concentrations of amphiphiles (0.25×10 -6 -128×10 -6 Bovine serum albumin solution (BSA, 0.2%; acetic acid, 0.01%) was added to a round-bottomed transparent polypropylene 96-well plate. MHB medium containing bacteria was used as a positive control, and MHB without bacteria was used as a negative control. The 96-well plate was incubated in a 37°C constant temperature incubator for 18-24 hours. The minimum inhibitory concentration was measured by naked eye and microplate reader OD 492 nm The minimum concentration at which no bacterial growth was observed was 1.38 Å. Each test was performed in three independent parallels, with each parallel repeated twice. The test results are shown in Table 3.

[0035] Table 3 Minimum inhibitory concentration of antimicrobial peptides (uM)

[0036]

[0037] It can be seen from Table 3 that the antimicrobial peptide (LAB 0.4) has good antibacterial activity against both Gram-negative and Gram-positive bacteria, but has low activity against probiotics.

[0038] 2. Hemolytic Activity Assay

[0039] (1) Collect 1 mL of healthy human blood and store it in a sodium heparin anticoagulant tube;

[0040] (2) Centrifuge at 3000 g for 5-10 min, discard the supernatant, and collect the red blood cells;

[0041] (3) Rinse the collected red blood cells three times with PBS solution, and then resuspend the red blood cells with 10 times the volume of PBS solution;

[0042] (4) Add 80 μL of PBS solution to the first well of each row of the 96-well plate, and add 50 μL of PBS solution to the remaining wells. Add 20 μL of antimicrobial peptide stock solution (2.56 mM) to the first well, mix thoroughly, then pipette 50 μL into the second well, mix thoroughly, and so on until the tenth well. After mixing, pipette 50 μL and discard;

[0043] (5) Add 50 μL of red blood cell suspension to wells 1 to 11 of the 96-well plate, and add 50 μL of 0.2% Triton X-100 to well 12, and mix well. Well 11 is used as a negative control, and well 12 is used as a positive control.

[0044] (6) Incubate in an incubator at 37°C for 1 h, then centrifuge at 3000 g for 5-10 min;

[0045] (7) Aspirate the supernatant and transfer it to a new 96-well plate. 570 The absorbance value was measured by an enzyme-labeled instrument under the following conditions;

[0046] (8) Hemolytic activity was calculated according to the following formula:

[0047] Hemolysis rate (%) = [(sample OD 570 - Negative control OD 570 ) / (positive control OD 570 - Negative control OD 570 )]×100%

[0048] The minimum hemolytic concentration is the concentration at which the antimicrobial peptide causes a 10% hemolytic rate.

[0049] Table 4 Determination of hemolytic activity of antimicrobial peptides

[0050]

[0051]

[0052] Table 4 shows that the antimicrobial peptide (LAB 0.4) had no hemolytic activity within the detection range. The ratio of the geometric mean of the minimum hemolytic concentration and the minimum inhibitory concentration was used to calculate the therapeutic index, which was 168.98.

[0053] 3. Stability determination: Each concentration (150×10 -3 M NaCl, 4.5 × 10 -3 M KCl, 2×10 -3 MCaCl2, 1×10 -3 M MgCl2, 6×10 -6 M NH4Cl, 8×10 -6 M ZnCl2, 4×10 -6 M FeCl3) was dissolved in BSA solution, and the subsequent test steps were consistent with the antibacterial activity determination method. To evaluate the effect of serum on antibacterial activity, the peptide was incubated with different concentrations of serum (100%, 50%) for 4 hours and then the antibacterial activity was determined. The test results are shown in Table 5.

[0054] Table 5 Antibacterial activity of antimicrobial peptides against Escherichia coli 25922 under physiological salt concentration and serum conditions (μM)

[0055]

[0056] According to the results in Table 5, it can be seen that the antimicrobial peptide (LAB 0.4) still maintains a certain antibacterial activity in the physiological concentration of salt ions, 50% and 100% serum environments.

[0057] Combining all the above results, the optimal parameters of the lactic acid bacteria-derived antimicrobial peptide library were obtained by bioinformatics analysis and combined with the database activity prediction and screening tools to obtain the antimicrobial peptide (LAB 0.4) with good antimicrobial activity, low hemolysis rate and stability. The therapeutic index of the antimicrobial peptide (LAB 0.4) is as high as 168.98, indicating that it has the potential to replace antibiotics and provides a feasible method for the design of antimicrobial peptides.

Claims

1. A lactic acid bacteria-derived antimicrobial peptide, characterized in that: Its amino acid sequence is shown in SEQ NO.

1.

2. The method for constructing a lactic acid bacteria-derived antimicrobial peptide according to claim 1, which is based on database-assisted design and is characterized in that: The method is as follows: by searching the lactic acid bacteria-derived antimicrobial peptides in the two major databases of APD and DRAMP, a lactic acid bacteria-derived antimicrobial peptide library containing 129 sequences was established, as shown in Table 1 in the manual, and after peptide library analysis, it was determined that cationic amino acids: hydrophobic amino acids ≈ 1:4, cationic amino acids + hydrophobic amino acids accounted for 68.20%, the average sequence length was 36 amino acids, and the cationic amino acids and hydrophobic amino acids with the highest amino acid proportions were K: 8.23%, G: 14.00%, A: 11.09%, L: 6.25%, and V: 6.05%, respectively. The sequence length was determined to be 25 according to the amino acid proportion, and 120 sequences were generated using (ABCDE)5 as a template. The 120 sequences were screened using activity prediction tools in the three databases of DRAMP, CAMP, and DBAASP to obtain the most likely peptide sequence with antimicrobial activity, and then the sequences were mutated based on the genetic algorithm in the HeliQuest server to obtain ten sequences with hydrophobic moment = 0.4, and the database activity prediction tool was used again to screen the best candidate polypeptide, and the amino acid sequence of the polypeptide was as shown in SEQ As shown in NO.1, the polypeptide was synthesized by solid phase chemical synthesis, and then subjected to antibacterial activity assay, hemolytic activity assay and stability assay, and finally named antimicrobial peptide LAB 0.

4.

3. Use of the lactic acid bacteria-derived antimicrobial peptide according to claim 1 in the preparation of a drug for treating and / or preventing Gram-negative bacteria and / or infectious diseases caused by Gram-negative bacteria.

4. The use according to claim 3, wherein the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Acinetobacter baumannii.

5. The use according to claim 3, wherein the Gram-positive bacteria is Staphylococcus aureus, Enterococcus faecalis or Bacillus subtilis.

6. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The medicine contains the lactic acid bacteria-derived antimicrobial peptide as claimed in claim 1.

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