CN116675757A - A narrow-spectrum antimicrobial peptide AWK14 synthesized based on large yellow croaker CENPW and its application - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a narrow-spectrum antibacterial peptide AWK14 synthesized based on a large yellow croaker CENPW and application thereof. The narrow-spectrum antibacterial peptide AWK14 has a complete sequence of Ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys-Lys, a molecular weight of 1724Da and an isoelectric point of 12.22, and belongs to alkaline polypeptides. The narrow-spectrum antibacterial peptide AWK14 has narrow-spectrum bactericidal activity, can specifically kill pseudomonas deformans at low concentration, and has weak antibacterial activity on other gram-positive bacteria and gram-negative bacteria. Meanwhile, the narrow-spectrum antibacterial peptide AWK14 has weak hemolytic activity and small cytotoxicity, and has high stability in different temperatures, PH and salt ions. The narrow-spectrum antibacterial peptide AWK14 can replace antibiotics and is applied to the prevention and treatment of fish visceral ichthyophthiriasis caused by pseudomonas deformans.

Description

A narrow-spectrum antimicrobial peptide AWK14 synthesized based on large yellow croaker CENPW and its application

technical field

The invention relates to the field of biotechnology, in particular to a narrow-spectrum antimicrobial peptide AWK14 synthesized based on large yellow croaker CENPW and its application.

Background technique

Antimicrobial peptides (AMPs), also known as host defense peptides, are widely distributed in nature and are an important part of the innate immune system of organisms. Antimicrobial peptides play an important role in the prevention of infections such as bacteria, fungi, and parasites. They can help the host reduce the invasion of various microorganisms and provide a strong defense for the host against infection. Antimicrobial peptides also have immunomodulatory effects. Studies have shown that antimicrobial peptides can quickly kill invading microorganisms through non-specific membrane penetration, and have the characteristics of a broad antibacterial spectrum, while traditional antibiotics mostly interact with specific targets to play a killing role. This unique mechanism of antimicrobial peptides makes it difficult for bacteria to develop resistance to them. In addition, antimicrobial peptides also exhibit good bactericidal activity against antibiotic-resistant strains, and can even reverse the emergence of bacterial resistance through mechanisms related to membrane permeability or efflux pumps. Therefore, antimicrobial peptides have the potential to replace antibiotics and be used in the prevention and control of bacterial diseases, especially bacterial infections caused by drug-resistant strains.

However, the clinical application of antimicrobial peptides is affected by many factors, such as high production costs, in vivo toxicity, and non-specific killing of host probiotics. Therefore, the design of small molecule antimicrobial peptides through structural optimization has gradually become a research hotspot. Studies have shown that by adjusting the sequence and composition of amino acids in the polypeptide chain through structural optimization, a symmetrical amino acid sequence is formed in the structure of the antimicrobial peptide, which is more likely to cause translocation that damages the cell membrane, thereby showing stronger bactericidal activity. In addition, regardless of the polypeptide of α-helix, β-sheet or other secondary structures, the existence of symmetrical structure is beneficial to reduce the toxicity of antimicrobial peptides to host cells. The application of database screening technology can determine the parameters affecting the bactericidal activity of antimicrobial peptides, such as amino acid composition, hydrophobic content, positive charge and sequence length, and finally obtain the best small molecule antimicrobial peptides with strong bactericidal activity, short polypeptide sequence and low cytotoxicity. Studies have shown that hydrophobicity and positive charge are critical for the biological activity of antimicrobial peptides. Arginine has a high isoelectric point and a specific guanidine side chain, which makes it easy to protonate and can form strong hydrogen bonds with the phosphate groups in the lipid head. Therefore, compared with lysine and histidine, the introduction of arginine is more likely to enhance the antibacterial activity of antimicrobial peptides.

Pseudomonas plecoglossicida is a Gram-negative bacterium with polar flagella, which can infect large yellow croaker, grouper, rainbow trout, sweetfish and zebrafish, etc., causing infection in the spleen, kidney and liver of fish White nodules appear, so the disease is called visceral white spot disease. Visceral white spot disease has caused huge economic losses to the aquaculture industry, seriously restricting the development of the industry. The development of antibacterial peptides that kill Pseudomonas mutans can provide potential therapeutic drugs for the prevention and treatment of visceral white spot disease in aquaculture animals.

Contents of the invention

In order to obtain an antimicrobial peptide with narrow-spectrum bactericidal activity and good biological safety, the present invention synthesized a narrow-spectrum antimicrobial peptide AWK14 based on the large yellow croaker CENPW, and clarified its application.

The object of the present invention is achieved through the following technologies:

The present invention firstly provides a narrow-spectrum antibacterial peptide AWK14, the amino acid sequence of the narrow-spectrum antibacterial peptide AWK14 is: Ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys -Lys.

The present invention further provides a preparation method of the above-mentioned narrow-spectrum antimicrobial peptide AWK14, specifically as follows:

(1) Using the CENPW protein sequence of large yellow croaker as a template, a linear polypeptide of 14 amino acids was intercepted and named ASK14. The amino acid sequence of the linear polypeptide ASK14 is: Ala-Gln-His-Leu-Lys-Ala-Val-Ser-Lys- Lys-Val-Leu-Lys-Lys;

(2) Replace the second glutamine (Gln) and the third histidine (His) in the amino acid sequence of the linear polypeptide ASK14 with arginine (Arg), and replace the eighth serine (Ser) with tryptophan (Trp) to obtain the narrow-spectrum antimicrobial peptide AWK14, the amino acid sequence of the narrow-spectrum antimicrobial peptide AWK14 is Ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys-Lys;

(3) The entire sequence of the narrow-spectrum antimicrobial peptide AWK14 was synthesized by solid-phase chemical synthesis.

The present invention also provides the application of the above-mentioned antibacterial peptide AWK14 in the preparation of antibacterial drugs, and the antibacterial drugs are anti-Pseudomonas mutans drugs.

The present invention also provides the application of the above antimicrobial peptide AWK14 in the preparation of feed additives.

The antimicrobial peptide AWK14 of the present invention has the following advantages and beneficial effects:

The antimicrobial peptide AWK14 prepared by the invention has narrow-spectrum bactericidal activity, can specifically kill Pseudomonas mutans at a low concentration, and only has weak antibacterial activity to other Gram-positive bacteria and Gram-negative bacteria. At the same time, the antimicrobial peptide AWK14 also has low hemolytic activity, low cytotoxicity, and high stability in different temperatures, pH and salt ions.

Description of drawings

Figure 1 is the mass spectrum of the antimicrobial peptide AWK14.

Fig. 2 is a graph showing the bactericidal activity and bactericidal kinetics of the antimicrobial peptide AWK14 against Pseudomonas mutans.

Fig. 3 is a graph showing the growth curves of Gram-negative bacteria and Gram-positive bacteria after treatment with the antimicrobial peptide AWK14.

Fig. 4 is a graph showing the cytotoxicity and erythrocyte hemolysis activity of the antimicrobial peptide AWK14 on LYC-FM.

Figure 5 is a diagram showing the influence of temperature, pH and salt ions on the bactericidal activity of the antimicrobial peptide AWK14.

Detailed ways

Below in conjunction with specific embodiment the present invention is described in further detail:

Embodiment 1: Design and synthesis of the antimicrobial peptide AWK14, the specific steps are:

(1) Using the CENPW protein sequence of large yellow croaker as a template, a linear polypeptide of 14 amino acids was intercepted, named ASK14, by adding the second glutamine (Gln) and the third histidine ( His) is replaced by arginine (Arg), and the eighth serine (Ser) is replaced by tryptophan (Trp) to obtain the antimicrobial peptide AWK14;

The amino acid sequence of the linear polypeptide ASK14 is:

Ala-Gln-His-Leu-Lys-Ala-Val-Ser-Lys-Lys-Val-Leu-Lys-Lys.

The amino acid sequence of the antimicrobial peptide AWK14 is:

Ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys-Lys.

Compared with ASK14, the theoretical molecular weight of AWK14 changed from 1577Da to 1724Da, the net charge increased from +5 to +7, and the isoelectric point changed from 11.29 to 12.22.

The mass spectrum of the antimicrobial peptide AWK14 is shown in Figure 1.

(2) AWK14 was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. by solid-phase chemical synthesis.

Embodiment 2: the minimum inhibitory concentration (MIC) of antimicrobial peptide AWK14 is measured, concrete steps are:

(1) Pick the test strains to the liquid culture medium, culture with shaking until the logarithmic growth phase, and adjust the concentration of the bacterial liquid to 2×10 ⁵ CFU/mL with the medium;

(2) Dissolve the antimicrobial peptide AWK14 into 1×PBS (pH=7.4) to make the concentration 256 μM, then pipette 50 μL of the diluted antimicrobial peptide AWK14 and serially dilute to the final concentration of 2, 4, 8, 16, 32 , 64, 128μM;

(3) Add 50 μL of the antimicrobial peptide AWK14 solution (2-256 μM) of different concentrations in the 96-well plate in sequence, and then add 50 μL of the test bacteria solution in sequence, and add 50 μL of the test bacteria solution and 50 μL of 1×PBS (pH=7.4), add 50 μL of medium and 50 μL of 1×PBS (pH=7.4) to the negative control in the 10th column, set 3 repetitions, and complete the above process within 15 minutes;

(4) Place the 96-well plate in an incubator for 12 hours. After taking it out, the minimum concentration of antimicrobial peptide AWK14 with no turbidity at the bottom of the well is the minimum inhibitory concentration;

(5) Place the 96-well plate in a spectrophotometer, read the absorbance value at 600nm, take the concentration of antimicrobial peptide AWK14 as the abscissa, and the absorbance value as the ordinate, and draw the growth curves of different test bacteria.

As shown in Figure 2A, the antimicrobial peptide AWK14 has strong antibacterial activity against Pseudomonas mutans, and the minimum inhibitory concentration and minimum bactericidal concentration are 8 μM and 16 μM, respectively. As shown in Figure 3, the antimicrobial peptide AWK14 showed weaker antibacterial activity to other tested Gram-positive bacteria and Gram-negative bacteria, and only had antibacterial activity to Salmonella pullorum (Salmonella pullorum) at the maximum concentration of 128 μ M, while It has only weak antibacterial activity against other tested bacteria. It shows that the antimicrobial peptide AWK14 of the present invention has narrow-spectrum bactericidal activity.

Embodiment 3: antimicrobial peptide AWK14 bactericidal kinetics assay, concrete steps are:

(1) Shake Pseudomonas mutans to the logarithmic growth phase, measure the OD value and convert it to the concentration of the bacterial solution, and adjust the concentration of the bacterial solution to 1.2×10 ⁵ CFU/ml with the medium;

(2) Determination of bactericidal kinetic curve: Mix 90 μL of the above-mentioned Pseudomonas mutans bacteria solution with 10 μL of antimicrobial peptide AWK14 (the final concentration of antimicrobial peptide AWK14 is 1×MBC), add 10 μL of PBS (pH=7.4) to the control group, and after co-incubation Samples were taken at different time points (0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h and 14h), spread on solid medium after doubling dilution, and then the bacterial survival rate at each time point was calculated. With PBS as the control, each time point was repeated three times.

As shown in Figure 2B, the antimicrobial peptide AWK14 showed a certain bactericidal effect when co-incubated with Pseudomonas mutans for 2 hours, and after 6 hours of co-incubation, Pseudomonas mutans could be completely killed.

Embodiment 4: Determination of the cytotoxicity of the antimicrobial peptide AWK14, the specific steps are:

(1) Adjust the large yellow croaker macrophage LYC-FM to 2×10 ⁵ cell/mL, add 100 μL of cell suspension to each well of a 96-well plate, and culture overnight at 28°C;

(2) Remove the medium, add 100 μL of fresh medium containing antimicrobial peptide AWK14 to each well, so that the final concentration of antimicrobial peptide AWK14 is 2, 4, 8, 16, 32, 64, 128 μM, mix 60 μL of blank medium with 40 μL of 5% TritonX-100 mixed medium, 60 μL blank medium and 40 μL 1×PBS (pH=7.4) mixed medium were added to the control wells as 100% and 0% cytotoxicity controls, and each group was set with 3 replicates;

(3) After static culture at 28°C for 24 hours, add 10 μL CCK-8 solution to each well, mix well, continue to culture at 28°C for 4 hours, and read the absorbance value at 450 nm;

(4) Calculate the live cell ratio:

Viable cell ratio=[(A _peptide -A _100% lysis)/(A _0% lysis-A _100% lysis)]×100%, A is the absorbance at 450nm.

As shown in Figure 4A, the cell survival rate of 2-64 μM antimicrobial peptide AWK14 on large yellow croaker macrophage LYC-FM is higher than 90%, indicating that the antimicrobial peptide AWK14 of the present invention has little cytotoxicity.

Embodiment 5: Determination of the hemolytic activity of the antimicrobial peptide AWK14, the specific steps are:

(1) Add anticoagulant to the blood of large yellow croaker and centrifuge at 500g for 10min;

(2) Discard the supernatant, resuspend in 1×PBS (pH=7.4), centrifuge at 500g for 10min, repeat three times;

(3) Resuspend the cells in 1×PBS (pH=7.4), and adjust the cell concentration to 1.5×10 ⁸ cell/mL;

(4) Add 120 μL of cell suspension to the 96-well plate, add 80 μL of different concentrations of antibacterial peptide AWK14 to the final concentration of 2, 4, 8, 16, 32, 64, and 128 μM, and add 80 μL of 5% Triton-X100, 80 μL of 1× PBS (pH=7.4) was added to the control well, as the control of 100% and 0% hemolysis, and each group was set with 3 repetitions;

(5) Cells were incubated at 28°C for 1 hour, after centrifugation, 100 μL supernatant was transferred to a 96-well plate, and the absorbance value at 405 nm was read;

(6) Calculation of hemolytic activity:

Hemolytic activity=[(A _peptide -A _0% lysis)/(A _100% lysis-A _0% lysis)]×100%, where A is the absorbance at 405 nm.

As shown in Figure 4B, the hemolysis rate of the antimicrobial peptide AWK14 on red blood cells of large yellow croaker at each detected concentration was lower than 2%, indicating that the antimicrobial peptide AWK14 of the present invention has little hemolysis on red blood cells.

Embodiment 6: the mensuration of the influence of temperature on the bactericidal activity of antibacterial peptide AWK14, concrete steps are:

(1) Take 35 μL of antimicrobial peptide AWK14 with a concentration of 160 μM (10×MBC) into 1.5 mL centrifuge tubes, a total of 5 tubes, and place them at 20 ° C, 40 ° C, 60 ° C, 80 ° C and 100 ° C for 30 minutes. Place on ice immediately;

(2) Shake Pseudomonas mutans to the logarithmic growth phase, measure the OD value and convert it into the concentration of the bacterial solution, and adjust the concentration of the bacterial solution to 1×10 ⁵ CFU/ml with the medium;

(3) Add 90 μL of the above-mentioned Pseudomonas mutans bacteria solution to the 96-well plate, then add 10 μL of the above-mentioned temperature-treated antimicrobial peptide AWK14 solution to each well in turn, and add 10 μL of MilliQ water to the positive control, and set 3 replicates;

(4) Cultivate overnight at 28°C, plate and count the number of colonies after gradient dilution in each well;

(5) Take the temperature as the X-axis, and the antimicrobial peptide AWK14 bactericidal rate (Bactericidal activity) as the Y-axis drawing;

Antimicrobial peptide AWK14 bactericidal rate=(1-temperature treatment group CFU/positive control CFU)×100%

The test results are shown in FIG. 5A , the bactericidal rate of the antimicrobial peptide AWK14 at each test temperature was higher than 99%, indicating that the antimicrobial peptide AWK14 of the present invention has good temperature stability.

Embodiment 7: the mensuration of the influence of PH on the bactericidal activity of antibacterial peptide AWK14, concrete steps are:

(1) Take 30 μL of buffer solutions with different pHs into 1.5 mL centrifuge tubes, add 10 μL of antimicrobial peptide AWK14 to each tube, so that the final concentration of antimicrobial peptide AWK14 is 10×MBC, mix well and place at room temperature for 4 hours;

(2) Shake Pseudomonas mutans to the logarithmic growth phase, measure the OD value and convert it into the concentration of the bacterial solution, and adjust the concentration of the bacterial solution to 1×10 ⁵ CFU/ml with the medium;

(3) Add 90 μL of the above-mentioned Pseudomonas mutans bacteria solution to the 96-well plate, then add 10 μL of the above-mentioned antimicrobial peptide AWK14 solution after different pH treatments to each well, and add 10 μL of MilliQ water to the positive control, and set 3 replicates;

(4) Cultivate overnight at 28°C, plate and count the number of colonies after gradient dilution in each well;

(5) Taking pH as the X-axis, and antimicrobial peptide AWK14 bactericidal rate (Bactericidal activity) as the Y-axis drawing;

Bactericidal rate of antimicrobial peptide AWK14=(1-pH treatment group CFU/positive control CFU)×100%

The detection results are shown in Figure 5B. Under different pH treatments, the antimicrobial peptide AWK14 has a bactericidal rate of more than 99% against Pseudomonas mutans, indicating that the antimicrobial peptide AWK14 of the present invention has good pH stability.

Embodiment 8: the determination of the influence of salt ions on the bactericidal activity of antimicrobial peptide AWK14, the specific steps are:

(1) Solution preparation: prepare 1500 mM NaCI, 45 mM KCI, 60 μM NH4CI, 80 μM ZnCl ₂ , 10 mM MgCl ₂ , 20 mM CaCl ₂ and 40 μM FeCl ₃ with ultrapure water, adjust the pH to 7.4, and sterilize at high temperature;

(2) Shake Pseudomonas mutans to the logarithmic growth phase, measure the OD value and convert it into the concentration of the bacterial solution, and adjust the concentration of the bacterial solution to 1.3×10 ⁵ CFU/mL with the medium;

(3) Add 80 μL of the above-mentioned Pseudomonas mutans bacteria solution to the 96-well plate, then add 10 μL of the antimicrobial peptide AWK14 and 10 μL of the above-mentioned different salt solutions in sequence, so that the final concentration of the antimicrobial peptide AWK14 is 1×MBC, and add 10 μL of PBS (pH =7.4) and 10 μL of the above-mentioned different salt solutions, all set 3 repetitions;

(4) Cultivate overnight at 28°C, plate and count the number of colonies after gradient dilution in each well;

(5) Taking different salt solutions as the X-axis, and the antimicrobial peptide AWK14 bactericidal rate (Bactericidal activity) as the Y-axis drawing: antibacterial peptide AWK14 bactericidal rate=(1-salt solution treatment group CFU/positive control CFU)×100%

The test results are shown in FIG. 5C , the bactericidal rate of the antimicrobial peptide AWK14 after the action of each tested salt ion was greater than 99%, indicating that the antimicrobial peptide AWK14 of the present invention has good salt ion stability.

In summary, the antimicrobial peptide AWK14 of the present invention has a narrow-spectrum bactericidal activity, can kill Pseudomonas mutans at low concentrations, and has only weak antibacterial activity against other Gram-positive bacteria and Gram-negative bacteria. At the same time, the antimicrobial peptide AWK14 also has low hemolytic activity, low cytotoxicity, and good stability to temperature, pH, and salt ions. Therefore, the antimicrobial peptide AWK14 of the present invention is expected to be better applied in the preparation of Pseudomonas mutans antibacterial drugs and related feed additives.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (4)

1. A narrow-spectrum antibacterial peptide AWK14 synthesized based on large yellow croaker CENPW is characterized in that: the amino acid sequence of the narrow-spectrum antibacterial peptide AWK14 is as follows: ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys-Lys.

2. A method for preparing the narrow-spectrum antibacterial peptide AWK14 according to claim 1, wherein: 14 amino acids in the CENPW protein of the large yellow croaker are intercepted to obtain a linear polypeptide ASK14 with 5 positive charges and an isoelectric point of 11.29, and the amino acid sequence of the linear polypeptide ASK14 is Ala-Gln-His-Leu-Lys-Ala-Val-Ser-Lys-Lys-Val-Leu-Lys-Lys; replacing the second glutamine and the third histidine in the amino acid sequence of the linear polypeptide ASK14 with arginine, replacing the eighth serine with tryptophan to obtain a narrow-spectrum antibacterial peptide AWK14 with 7 positive charges and an isoelectric point of 12.22, wherein the amino acid sequence of the narrow-spectrum antibacterial peptide AWK14 is Ala-Arg-Arg-Leu-Lys-Ala-Val-Trp-Lys-Lys-Val-Leu-Lys-Lys; the whole sequence of the narrow-spectrum antibacterial peptide AWK14 is synthesized by adopting a solid-phase chemical synthesis method.

3. The use of the narrow-spectrum antimicrobial peptide AWK14 according to claim 1 for the preparation of an antimicrobial medicament, characterized in that: the antibacterial drug is a drug for resisting pseudomonas deformans.

4. Use of the antibacterial peptide AWK14 according to claim 1 for the preparation of a feed additive.