Asymmetric antibacterial peptide taking PG as center as well as preparation method and application thereof

Abstract

The invention discloses an asymmetric antibacterial peptide taking PG as a center as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID No.1, and the C end of the antibacterial peptide is amidated by adopting-NH2; detection on antibacterial activity, hemolytic activity, cytotoxicity and salt ion stability of the antibacterial peptide shows that the antibacterial peptide has optimal cell selectivity (SI = 72.60), does not show obvious cytotoxicity when the test concentration is 32 mu M or below, and can keep ideal stability in salt ions with different physiological concentrations. In conclusion, the antibacterial peptide has the potential to become a broad-spectrum antibacterial drug for treating gram-positive bacterium and gram-negative bacterium infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an asymmetric antimicrobial peptide GPRW3Q centered on PG, and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of small polypeptides widely present in nature, usually composed of 10 - 60 amino acids, and are an important part of the body's innate immune system, having a wide range of inhibitory effects on bacteria, fungi, parasites and viruses. Due to their unique bactericidal mechanism and multiple biological activities, they are not easily resistant to drugs, and are regarded as a strategic solution in the post-antibiotic era. However, there are significant bottlenecks in the application of existing natural antimicrobial peptides in animal husbandry. For example, some natural antimicrobial peptides have defects such as high hemolysis and high toxicity, and the large number of amino acids in the polypeptide composition leads to a high cost of artificial synthesis, making it difficult to further apply them to animal husbandry production applications, which greatly limits the application of antimicrobial peptides in animal husbandry production. Although many design strategies have made progress in improving the biocompatibility of antimicrobial peptides, some of these strategies have also had some negative effects on AMPs, such as sacrificing a certain antibacterial activity when improving biocompatibility, selecting D-amino acids and other unnatural amino acids to maintain antibacterial activity while potentially increasing the cost of artificial synthesis and the obstacles of subsequent expression, so it is difficult to be used as an antibacterial drug. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide an asymmetric antimicrobial peptide GPRW3Q centered on PG, which solves the problems of high hemolytic activity, high cytotoxicity and instability of the polypeptide under physiological salt conditions during the application of antimicrobial peptides, and can be used as an antibacterial drug, thus ensuring the health of livestock and poultry and improving the quality of animal husbandry production.

[0004] The technical solution adopted by the present invention is as follows: An asymmetric antimicrobial short peptide GPRW3Q centered on PG, whose amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

[0005] Further, the molecular formula of the antimicrobial short peptide GPRW3Q as described above is shown in formula (I):

[0006]

[0007] The present invention also provides a preparation method of an asymmetric antimicrobial peptide GPRW3Q centered on PG as described above, and the steps are as follows:

[0008] Step 1: Select arginine (Arg) as the positively charged amino acid and place it at the 2nd, 3rd, 4th, and 13th positions of the polypeptide amino acid sequence. The arginine at the 13th position is placed at the C-terminus to enhance the positive charge effect of the polypeptide, jointly providing positive charge for the polypeptide. Select tryptophan (Trp) and isoleucine (Ile) as hydrophobic amino acids to provide hydrophobicity for the polypeptide. Place Trp at the 7th, 8th, and 9th positions of the polypeptide amino acid sequence, and place Ile at the 10th and 11th positions of the polypeptide amino acid sequence to assist Trp in providing the hydrophobicity of the polypeptide. Select uncharged glutamine (Gln) and place it at the 12th position of the polypeptide amino acid sequence to achieve a flexible balance between the positive charge and hydrophobicity of the polypeptide. Select phenylalanine (Phe) and glycine (Gly) and place them at the 5th and 6th positions of the polypeptide amino acid sequence respectively. Phe is expected to enhance the stability of Arg at the 4th position, and the structure containing PG has enhanced antibacterial properties and low cytotoxicity. The N-terminus of the polypeptide amino acid sequence is capped with glycine (Gly) to prevent the R group at the N-terminus from being directly exposed on the surface and reduce the electrostatic attraction to the surface neutral structure of human hemoglobin. Finally, amidation is carried out at the C-terminus of the polypeptide to construct a polypeptide with a net charge number of +5, such as the polypeptide shown in SEQ ID No.1.

[0009] Step 2: Synthesize the polypeptide by solid-phase chemical synthesis method. After purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry, perform antibacterial activity detection, hemolytic activity detection, cytotoxicity detection, and salt ion stability detection on the polypeptide, and finally name it antimicrobial peptide GPRW3Q.

[0010] Another object of the present invention is to provide an application of the PG-centered asymmetric antimicrobial peptide GPRW3Q as described above in the preparation of a drug for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

[0011] Further, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis.

[0012] Further, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Salmonella pullorum.

[0013] Another object of the present invention is to provide a drug suitable for treating and / or preventing infections caused by Gram-positive bacteria and / or Gram-negative bacteria, and the drug contains the PG-centered asymmetric antimicrobial peptide GPRW3Q as described above.

[0014] The present invention has the following advantages and beneficial effects: By means of the flexible balance of neutral amino acids and the N-terminal capped glycine lid, the present invention significantly reduces hemolytic activity and cytotoxicity without affecting antibacterial activity, and is expected to solve the problem that existing antibacterial peptides are difficult to be applied to maintaining the healthy development of animal husbandry. The antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability of the antibacterial peptide GPRW3Q centered on "PG" of the present invention were detected, and it was found that the antibacterial peptide GPRW3Q has high antibacterial activity against several measured Gram-negative and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis. At the same time, the antibacterial peptide GPRW3Q has a high hemolysis value, does not produce obvious hemolysis at 256 μM, and does not show obvious cytotoxicity at 32 μM. In addition, the antibacterial peptide GPRW3Q still maintains a low MIC in different physiological concentrations of salt ions and different concentrations of serum environments, and has high salt ion stability and serum stability. In summary, the antibacterial peptide GPRW3Q centered on "PG" has the potential to become a broad-spectrum antibacterial drug for the treatment of Gram-positive and Gram-negative bacterial infections, so as to achieve the purpose of maintaining the healthy development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the reverse high performance liquid chromatography chart of the antibacterial peptide GPRW3Q;

[0016] Figure 2 It is the mass spectrometry chart of the antibacterial peptide GPRW3Q;

[0017] Figure 3 It is the comparison chart of the hemolytic activities of the antibacterial peptides PRW3 and GPRW3Q;

[0018] Figure 4 It is the comparison chart of the cytotoxicities of the antibacterial peptides PRW3 and GPRW3Q. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The present invention will be further described in detail below in conjunction with the embodiments and the drawings.

[0020] Example 1

[0021] Design of Antibacterial Peptide

[0022] 1. Select arginine (Arg) as the positively charged amino acid and place Arg at positions 2, 3, 4, and 13 of the polypeptide amino acid sequence. At positions 2, 3, and 4, arginine forms an arginine cluster at the N-terminus, and the arginine at position 13 is placed at the C-terminus to enhance the positive charge effect of the polypeptide, ultimately providing a positive charge for the polypeptide together. Select tryptophan (Trp) and isoleucine (Ile) as hydrophobic amino acids to provide hydrophobicity for the polypeptide. Place Trp at positions 7, 8, and 9 to form a polytryptophan, and place Ile at positions 10 and 11 to provide hydrophobicity for the polypeptide as an auxiliary to tryptophan. Select the uncharged neutral amino acid glutamine (Gln) and place it at position 12 to achieve a reasonable flexible balance between the positive charge and hydrophobicity of the polypeptide. Select phenylalanine (Phe) and glycine (Gly) and place them at positions 5 and 6 respectively. Phe is used to enhance the stability of Arg at position 4, thereby enhancing the antibacterial performance and reducing cytotoxicity. The N-terminus is capped with glycine (Gly). This arrangement can prevent the R group at the N-terminus from being directly exposed on the surface, with the expectation of reducing the electrostatic attraction to the surface neutral structure of human hemoglobin. Finally, amidation is carried out at the C-terminus of the polypeptide to obtain the antibacterial peptide GPRW3Q with a net charge number of +5, and its amino acid sequence is shown in SEQ ID No.1. The sequences, molecular weights, and charge numbers of the antibacterial peptides are shown in Table 1.

[0023] Table 1 Sequences, molecular weights, and charge numbers of polypeptides PRW3 and GPRW3Q

[0024]

[0025] Example 2

[0026] Synthesis and identification of antibacterial peptides

[0027] The designed antibacterial peptide was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. (Synpeptide Co Ltd.) through solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain the target compound for subsequent determination of antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability.

[0028] The reverse-phase high-performance liquid chromatography chart of antibacterial peptide GPRW3Q is shown in the appendix Figure 1 .

[0029] The mass spectrometry chart of antibacterial peptide GPRW3Q is shown in the appendix Figure 2 .

[0030] Example 3

[0031] Antibacterial activity of polypeptides

[0032] The minimum inhibitory concentration of several antimicrobial peptides was determined by the microbroth dilution method. The antibacterial activity of the polypeptides was understood by measuring the minimum inhibitory concentration (MIC) of the polypeptides. The designed and successfully synthesized polypeptides were dissolved in sterile ultrapure water to a concentration of 2.56 mM in a sterile workbench to obtain a stock solution of AMPs for subsequent bioactivity assays. The bacteria were cultured overnight in a shaker at 37 °C and 220 rpm, and then transferred to fresh MHB until the logarithmic growth phase. The bacteria cultured to the logarithmic phase were adjusted to an OD 600nm = 0.38 - 0.40 for standby. The above-prepared bacteria were diluted 1000-fold, and 50 μL of the bacterial culture was added to 50 μL of a sterile 96-well plate containing BSA with different concentrations of antimicrobial peptides and incubated at 37 °C for 16 - 18 h. The 96-well plate was sealed tightly around the edges with a sealing film to prevent bacterial contamination and incubated in a 37 °C incubator for 16 - 18 h. After incubation, the negative control wells remained clear and transparent, indicating no contamination during the experiment. The turbidity of each well was observed visually and measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 492 nm. The lowest peptide concentration at which the turbidity did not increase compared to the negative control was defined as the MIC of the polypeptide. Three independent replicate experiments were performed, with two parallels for each replicate. The minimum inhibitory concentrations are shown in Table 2.

[0033] Table 2 Antibacterial activities of polypeptide PRW3 and antimicrobial peptide GPRW3Q (μM)

[0034]

[0035] As can be seen from the above table, the MIC value of polypeptide PRW3 against Gram-negative bacteria was 2 - 16 μM, and the MIC value against Gram-positive bacteria was 2 - 4 μM. The MIC value of antimicrobial peptide GPRW3Q against all tested bacteria was 2 - 4 μM, all showing good antibacterial activity, and the activity was better than that of polypeptide PRW3.

[0036] Example 4

[0037] Hemolytic activity of antimicrobial peptides

[0038] To evaluate the safety of antimicrobial peptides, the disruptive behavior of polypeptides in the concentration range of 2 - 128 μM on human red blood cells (hRBC) was studied. 1 mL of fresh blood from healthy volunteers was collected and stored in a heparin sodium anticoagulant tube. After centrifugation at 3000 r for 10 min at 4°C, the supernatant was discarded, and the cells were washed 2 - 3 times with sterile PBS buffer and resuspended in 10 mL of PBS for standby. 50 μL of the red blood cell suspension was mixed evenly with 50 μL of polypeptide solutions at different concentrations serially diluted with PBS and incubated at a constant temperature in an incubator at 37°C for 1 h. After incubation, the mixture was centrifuged at 1000 g for 10 min at 4°C. 70 μL of the supernatant was taken out and transferred to a new 96-well plate, and the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The average value of each group was taken and compared and analyzed. Among them, 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 was used as a positive control. This experiment was repeated three times. The test results are shown in the appendix of the instruction manual Figure 3 The minimum hemolytic concentration is the concentration of the antimicrobial peptide when it causes a 10% hemolysis rate. The test results are shown in Table 3. The biocompatibility of the antimicrobial peptide was evaluated by the lowest concentration (MHC) of the antimicrobial peptide when it caused 10% hemolysis of human red blood cells, and its therapeutic potential was further evaluated by calculating the therapeutic index (SI) of the antimicrobial peptide, as shown in Table 3.

[0039] Table 3 Hemolytic activities of polypeptide PRW3 and antimicrobial peptide GPRW3Q

[0040]

[0041] a The geometric mean (GM) of the minimum inhibitory concentration of the antimicrobial peptide against the tested bacteria. When no detectable antimicrobial activity was observed at 64 μM, 128 μM was used to calculate the therapeutic index;

[0042] b MHC is the lowest concentration of the antimicrobial peptide when it causes 10% hemolysis of human red blood cells (hRBC). When no detectable hemolytic activity was observed at 128 μM, 256 μM was used to calculate the therapeutic index;

[0043] c The calculation method of SI is MHC / GM. The larger the treatment value, the higher the therapeutic potential.

[0044] As shown in the appendix of the instruction manual Figure 3It can be seen that the antimicrobial peptide GPRW3Q did not cause hemolysis at the highest concentration tested. The minimum hemolytic concentration of the antimicrobial peptide GPRW3Q was much higher than its minimum inhibitory concentration, indicating that the antimicrobial peptide GPRW3Q has high biosafety while exerting its antibacterial activity. By calculating the therapeutic index (SI) of PRW3 and GPRW3Q, it was found that the SI value of the antimicrobial peptide GPRW3Q (72.60) was significantly higher than that of PRW3 (2.42). Based on the above results, the polypeptide of the antimicrobial peptide GPRW3Q has the best therapeutic potential.

[0045] Example 5

[0046] Cytotoxicity of Antimicrobial Peptides

[0047] The MTT method was used to determine the cytotoxicity of AMPs. The cells selected for determination were human embryonic kidney cells RAW264.7.

[0048] Prepare high-glucose RAMP-1640 complete medium containing 10% fetal bovine serum and 1% double antibiotics, place it at 4 °C for standby, and place it in a 37 °C constant temperature water bath to room temperature before use. Inoculate the cells frozen in liquid nitrogen into the corresponding water-bathed complete medium for resuscitation, and place them in a carbon dioxide incubator for culture. Observe under a microscope, and the next subculture can be carried out when the cells cover more than 80% of the bottom of the cell culture flask. After the cells are subcultured well, pour out the complete medium in the cell culture flask, and wash the cells 2-3 times with sterile PBS filtered through a 0.22 μM aqueous filter membrane. Add 1 mL of 0.25% trypsin solution to the cell culture flask and place it in a carbon dioxide incubator for 1 min to digest the adherent cells. After digestion, observe under a microscope. If there are still adherent cells, gently pat until they completely fall off, then add complete medium and pipette to form a cell suspension, and add 50 μL of the cell suspension to each well in columns 1-11 of a sterile 96-well culture plate, with 3-5×10 5 cells per well. Finally, place the 96-well plate in a carbon dioxide incubator and incubate overnight. Take a new sterile 96-well plate, and serially dilute AMPs with complete medium to the 10th column. Then aspirate 50 μL of each concentration of AMPs and add them to the corresponding columns 1-10 of the 96-well plate covered with cell suspension. Among them, 50 μL of complete medium is added to column 11 as a positive control, and 100 μL of complete medium is added to column 12 as a negative control. The 96-well plate is continued to be placed in a carbon dioxide incubator and incubated for 4 h. Add 50 μL of MTT solution with a concentration of 5 mg / mL to each well of the 96-well plate. Continue to incubate in a carbon dioxide incubator for 3 h, and then aspirate and discard all the liquid in each well. Finally, add 100 μL of DMSO to each well to fully dissolve the purple crystals, and measure the absorbance at 570 nm. This experiment was independently repeated three times. The test results are shown inFigure 4 。

[0049] From the attached instructions Figure 4 It can be seen that the cell survival rates of polypeptide PRW3 and antimicrobial peptide GPRW3Q are both higher than 80% in the concentration range of 1 - 32 μM, and no obvious cytotoxicity is shown.

[0050] Example 6

[0051] Salt ion stability of antimicrobial peptides

[0052] E. coli 25922 and S. aureus 29213 were selected as typical Gram - negative bacteria and typical Gram - positive bacteria respectively to determine the MIC values of antimicrobial peptides under salt ion conditions with different physiological concentrations. Different concentrations of salt ions were dissolved in a 0.2% BSA (containing 0.01% glacial acetic acid) solution filtered through a 0.22 μM aqueous filter membrane, and the initial concentrations of the configured salt ions were 300 mM NaCl, 9 mM KCl, 5 mM CaCl2, 2 mM MgCl2, 12 μM NH4Cl, 16 μM ZnCl2, and 8 μM FeCl3. Using the above different salt ions as diluents, the MIC of antimicrobial peptides in different salt ions was determined by the microbroth dilution method. This experiment was independently repeated three times. The test results are shown in Table 4.

[0053] Table 4 Salt ion stability of antimicrobial peptide GPRW3Q

[0054]

[0055] As can be seen from Table 4, the MIC value range of antimicrobial peptide GPRW3Q against S. aureus in the presence of physiological salt is 4 μM - 32 μM, showing strong salt ion stability. For the change in the MIC value of antimicrobial peptide GPRW3Q against E. coli in the presence of physiological salt, except that the presence of Mg 2+ and Ca 2+ will have a relatively large impact due to the strong charge interaction with the antimicrobial peptide, the MIC value range shown under the presence of the remaining salt ions is 4 μM - 8 μM, also showing strong salt ion stability. Considering the above results, antimicrobial peptide GPRW3Q shows more excellent performance.

Claims

1. An antibacterial peptide GPRW3Q that is asymmetric with PG as the center, characterized in that: Its amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

2. An antibacterial peptide GPRW3Q that is PG-centered and asymmetric as described in claim 1, characterized in that, Its molecular formula is shown in formula (I), 3. The preparation method of an antibacterial peptide GPRW3Q that is asymmetric with PG as the center as described in claim 1, wherein The steps are as follows: Step 1: Select arginine (Arg) as the positively charged amino acid and place it at the 2nd, 3rd, 4th, and 13th positions of the polypeptide amino acid sequence. The arginine at the 13th position is placed at the C-terminus to enhance the positive charge effect of the polypeptide, jointly providing positive charges for the polypeptide; select tryptophan (Trp) and isoleucine (Ile) as hydrophobic amino acids to provide hydrophobicity for the polypeptide. Place Trp at the 7th, 8th, and 9th positions of the polypeptide amino acid sequence, and place Ile at the 10th and 11th positions of the polypeptide amino acid sequence to assist Trp in providing the hydrophobicity of the polypeptide; select uncharged glutamine (Gln) and place it at the 12th position of the polypeptide amino acid sequence to achieve a flexible balance between the positive charge and hydrophobicity of the polypeptide; select phenylalanine (Phe) and glycine (Gly) and place them at the 5th and 6th positions of the polypeptide amino acid sequence respectively. Phe is expected to enhance the stability of Arg at the 4th position, and the structure containing PG has enhanced antibacterial properties and low cytotoxicity; the N-terminus of the polypeptide amino acid sequence is capped with glycine (Gly) to prevent the R group at the N-terminus from being directly exposed on the surface, reducing the electrostatic attraction to the surface neutral structure of human hemoglobin; finally, amidation is carried out at the C-terminus of the polypeptide to construct a polypeptide with a net charge number of +5 and an amino acid sequence shown in SEQ ID No.

1. Step 2: Synthesize the polypeptide by solid-phase chemical synthesis method. After purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry, perform antibacterial activity detection, hemolytic activity detection, cytotoxicity detection, and salt ion stability detection on the polypeptide, and finally name it antibacterial peptide GPRW3Q.

4. Use of an asymmetric antibacterial peptide GPRW3Q with PG as the center as described in claim 1 in the preparation of a drug for treating infectious diseases caused by Gram-positive bacteria or / and Gram-negative bacteria.

5. The application according to claim 4, wherein: The Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

6. The application according to claim 4, characterized in that: The Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Salmonella pullorum.

7. A drug suitable for the treatment and / or prevention of Gram-positive and / or Gram-negative bacterial infections, characterized in that, The drug contains an asymmetric antibacterial peptide GPRW3Q with PG as the center as described in claim 1.

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