CN112778401B - Caprylic acid acylation modified antibacterial peptide and application thereof - Google Patents
Caprylic acid acylation modified antibacterial peptide and application thereof Download PDFInfo
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of genetic engineering and biological agents, and particularly discloses an octanoic acid acylation modified antibacterial peptide and application thereof. The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1. The method simulates the arrangement rule of alpha-helix secondary structure amino acids in natural protein, leucine and arginine are used for respectively providing hydrophobicity and cationicity, a heptapeptide repeat sequence 'abcdefg' is used as a template, the sequence is repeated twice, then octanoic acid acylation modification is carried out on the N terminal of the sequence, and the C terminal is amidated. The peptide of the invention has broad-spectrum antibacterial activity on fungi and bacteria and has wide clinical application potential.
Description
Technical Field
The invention relates to the technical field of genetic engineering and biological agents, in particular to caprylic acid acylation modified antibacterial peptide and application thereof.
Background
Abuse of antibiotics has led to the emergence of drug-resistant fungi and bacteria, especially hyper-fungi and the like, which have become resistant to a variety of antibiotics and seriously threaten human health. Although the traditional antifungal medicines such as ketoconazole, fluconazole and the like have certain treatment effect, the traditional antifungal medicines are easy to induce fungi to generate drug resistance and generate certain side effect on human bodies. Therefore, the development of a novel antifungal drug with low toxicity and high efficacy is urgently needed.
The antibacterial peptide exists in almost all animals and forms the first line of defense of animal bodies. Generally, the antibacterial peptide exerts antibacterial activity by disturbing the membrane stability of bacteria to cause leakage of bacteria content, the secondary structure of the antibacterial peptide has a crucial influence on biological activity, the antibacterial peptide is in a random conformation in a water environment, and when the antibacterial peptide enters the membrane environment, the bacterial membrane is ruptured along with the change of the secondary structure, and then the bacterial strain dies. Compared with antibiotics, the physical membrane rupture mode is not easy to cause the thalli to generate drug resistance. Therefore, antimicrobial peptides are also ideal antibiotic substitutes. Although a large number of antibacterial peptides are isolated from animals at present, natural antibacterial peptides have the disadvantages of poor activity and high toxicity, especially insufficient antibacterial activity against fungi. Therefore, artificial peptide design studies are necessary to solve the drawbacks of natural antibacterial peptides.
Disclosure of Invention
Based on the disadvantages of natural antibacterial peptides, it is an object of the present invention to provide a peptide which is effective against fungi and bacteria. So as to solve the problem of drug resistance of fungi caused by antibiotics.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
an amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1.
The secondary structure plays an important role in the exertion of the activity of the antimicrobial peptide, and among all the known secondary structures, the α -helical structure is dominant. The heptad repeat is a basic and simplified form of the alpha-helical structure, commonly abbreviated (abcdefg) n The structure is stabilized by the hydrogen bonding of the hydrophobic amino acid side chains at the a and d positions. This repetitive pattern has important effects on the balance of activity performance and toxicity of the antimicrobial peptide. The peptide is caprylic acid acylation modified antibacterial peptide, which simulates the arrangement rule of alpha-helix secondary structure amino acid in natural protein, leucine and arginine are respectively used for providing hydrophobicity and cationicity, a heptad repetitive sequence 'abcdefg' is used as a template, the sequence is repeated twice to ensure sufficient charge quantity, then caprylic acid acylation modification and C terminal amidation are carried out on the N terminal of the sequence to obtain the antibacterial peptide (C8 LR) with the sequence of C8-LRRLRRLRRR-NH 2 (C8-Leu Arg Arg Leu Arg Arg Arg Leu Arg Arg Leu Arg Arg Arg-NH 2 ) And the molecular weight is 2157.76. The charge of the C8LR is 11, 10 arginines and the C-terminal amidation provides sufficient charge for the peptide to ensure that the peptide is in sufficient contact with the pellicle. 4 leucine and caprylic acid acylated N-terminal modification provide enough driving force for the peptide, and ensure that the peptide can be inserted into a mycoderm to destroy a strain. The method can make polypeptide have strong antibacterial and antifungal activity, and low hemolytic activity.
According to the invention, the leucine is placed at the a and d positions to serve as the hydrophobic core of the peptide, so that a hydrogen bond is formed between the leucine at the a and d positions, the alpha-helix structure of the peptide is ensured, and the strong alpha-helix tendency and strong hydrophobicity of the leucine are reasonably utilized.
Since the cation is a necessary condition for the peptide to have antibacterial activity. The present invention selects arginine and places arginine at the b, c, e, f and g positions. The guanidino side chain of the antibacterial peptide and the phosphate ester parts of the two lipid head groups form strong bidentate hydrogen bonds, so that the antibacterial peptide is promoted to have deeper membrane insertion capacity. In addition, the heptapeptide sequence mode consisting of arginine and leucine is repeated twice, so that the peptide sequence has 10 positive charges, the sequence has sufficient cationic property, the heptapeptide is favorably and fully combined with fungus and bacterial biofilms through electrostatic interaction, and the killing effect is improved.
The N end of the peptide sequence is modified by caprylic acid, so that the hydrophobicity and the stability of the peptide are further enhanced, and the C end of the peptide sequence is amidated, so that the cationic property and the stability of the peptide are further enhanced. Finally, the peptide with simple structure and ideal antifungal and bacterial effects is formed.
The invention considers the helicity, integral hydrophobicity, amphipathy and charge number of the peptide at the same time, balances a plurality of parameters, creates an artificial peptide with broad-spectrum antibacterial activity, especially antifungal activity, has good biocompatibility and good antibacterial effect, can solve the problem of drug resistance of germs, has short amino acid sequence, is easy to create and is suitable for large-scale popularization.
The present invention also provides:
the application of the antibacterial peptide in preparing antifungal preparations.
The application of the antibacterial peptide in preparing antibacterial preparations.
Wherein the preparation is a medicament or a health-care product.
The application of the antibacterial peptide in preparing disinfectants.
The application of the antibacterial peptide in preparing a cleaning agent.
The antibacterial peptide is applied to the preparation of preservatives.
A product which is a medicine, a health product, a disinfectant, a cleaning agent or an antiseptic is characterized by comprising the antibacterial peptide.
The invention has the beneficial effects that:
the peptide chain is short, the chemical method is simple to synthesize, the preparation cost is low, the obtained caprylic acid acylation modified peptide is subjected to antibacterial activity detection, and the peptide is found to have antifungal and antibacterial activity, is not easy to induce bacteria and fungi to generate drug resistance, and has higher application value for treating fungi and bacterial infection. In addition, hemolytic activity tests are carried out on the peptide, and the peptide is found to have low hemolytic activity, so that the peptide has good biocompatibility, the therapeutic index reaches 145.45, and the peptide has great clinical application potential.
Drawings
FIG. 1 is a schematic structural diagram of the antimicrobial peptide of the present invention.
FIG. 2 is a high performance liquid chromatogram of the antimicrobial peptide of the present invention. In the figure, the peak-off time of the highest peak was 14.860 minutes.
FIG. 3 is a mass spectrum of the antibacterial peptide of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
This example used solid phase chemical synthesis to synthesize the antibacterial peptide of the present invention (see SEQ ID No. 1), which has the structure shown in fig. 1:
1. the preparation of the antibacterial peptide is carried out one by one from the C end to the N end and is completed by a polypeptide synthesizer. Firstly, fmoc-X (X is the first amino acid at the C end of each antibacterial peptide) is grafted to Wang resin, and then an Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C end of each antibacterial peptide); synthesizing the resin from the C end to the N end in sequence according to the procedure until the synthesis is finished to obtain the resin with the side chain protection of the Fmoc group removed;
2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing precipitate TFA (trifluoroacetic acid), mixing washing liquor with the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times, precipitating for 3 hours at-20 ℃, separating out white powder, centrifuging for 10min at 2500g, collecting precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain polypeptide, wherein a cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 95;
3. performing column equilibrium for 30min by using 0.2mol/L sodium sulfate (adjusting the pH value to 7.5 by using phosphoric acid), dissolving the polypeptide by using a 90% acetonitrile aqueous solution, filtering, performing C18 reverse phase atmospheric column, performing gradient elution (an eluent is a mixture of methanol and a sodium sulfate aqueous solution according to a volume ratio of 30-70); further purification by reverse phase C18 column, eluent A was 0.1% TFA/aqueous solution; the elution B was 0.1% by weight of a TFA/acetonitrile solution, the elution concentration was 25% by weight of B-40% by weight, the elution time was 12min, the flow rate was 1mL/min, and the main peak was collected as above and lyophilized;
4. identification of antibacterial peptides: the antibacterial peptide obtained by the method is analyzed by an electrospray mass spectrometry, the molecular weight (shown in a mass spectrogram of the antibacterial peptide shown in figure 3) shown in the mass spectrogram is basically consistent with the theoretical molecular weight of 2157.76, the purity of the antibacterial peptide is more than 95 percent (see a high-efficiency liquid chromatogram of the antibacterial peptide shown in figure 2, a chromatographic column is Kromasil C18-5 (4.6 x 250mm,220nm,10 mu L), and a nonlinear gradient of water/acetonitrile (containing 0.1 percent of trifluoroacetic acid) is used, and the flow rate is 1.0 mL/min).
Example 2
Determination of the antifungal Activity of the peptides: the minimum inhibitory concentration of the peptide (C8 LR) prepared in example 1 against fungi was determined by microdilution. With 0.01% acetic acid0.2% fetal bovine serum albumin was added as a diluent to a 96-well plate, and a series of gradients of the antimicrobial peptide solution obtained in example 1 of the present invention were sequentially prepared using a two-fold dilution method so that the volume of the solution in each well was 50. Mu.L. Then respectively adding 50 mu L of bacterial liquid (10) to be detected 3 CFU/mL) in each well, RPMI 1640 with MOPS (pH = 7.0). Positive controls (containing the bacterial solution but not the antimicrobial peptide) and negative controls (containing neither the bacterial solution nor the peptide) were set separately. Culturing at 28 deg.C for 48h, measuring light absorption value at 492nm with enzyme labeling instrument, and determining minimum inhibitory concentration of peptide on fungi by using value greater than 0.1 as determination standard of strain growth. The experiments were set up in two replicates and repeated three times. The results are shown in Table 1. In table 1, c.albicans 10231 was obtained from bekka bio-technology ltd, beijing, and c.albicans CMCC (F) 98001 was obtained from shanghai, ltd.
TABLE 1 antibacterial activity of the antibacterial peptide C8LR against fungi (μ M)
As can be seen from Table 1, the peptide C8LR showed very strong inhibitory effect on Candida albicans, and could completely inhibit the growth of Candida albicans at a concentration of 2. Mu.M.
Example 3
Determination of antibacterial activity of peptides: the minimum inhibitory concentration of the peptide (C8 LR) prepared in example 1 against bacteria was determined by microdilution. 0.2% bovine serum albumin containing 0.01% acetic acid was added as a diluent to a 96-well plate, and a series of gradients of the antimicrobial peptide solution were sequentially prepared by a double dilution method so that the volume of the solution in each well was 50. Mu.L. Then respectively adding 50 mu L of bacterial liquid (10) to be detected 5 CFU/mL) in each well, MHB (pH = 7.0) medium. Positive controls (containing the bacterial solution but not the antimicrobial peptide) and negative controls (containing neither the bacterial solution nor the peptide) were set separately. Culturing at 37 deg.C for 18h, measuring light absorption value at 492nm with enzyme labeling instrument, and determining the minimum inhibitory concentration of peptide on bacteria with the value greater than 0.1 as the determination standard of strain growth. Set two levels for the testLine, repeat three times. The results are shown in Table 2. In table 2, e.coli 25922, s.aureus 1882, s.aureus 6538, s.epidermidis 49134, s.typhimurium sl1344, c.amolonticus 51459 were obtained from bekko bovich biotechnology limited, beijing, e.coli K88, e.coli K99, s.aureus 43300 were from the chinese veterinary microbial cultures collection management center.
TABLE 2 antibacterial activity (μ M) of the antibacterial peptide C8LR against bacteria
As can be seen from table 2, the peptide C8LR retained antibacterial activity against gram-negative and gram-positive bacteria, exhibited broad-spectrum antibacterial activity, and also exhibited bacteriostatic activity against methicillin-resistant staphylococcus aureus (s.aureus 43300), indicating that the peptide C8LR has potential to kill drug-resistant bacteria.
Example 4
Determination of the hemolytic activity of the peptide: 1mL of fresh porcine red blood cells were taken and diluted 10-fold with PBS for use. PBS was added as a diluent to a 96-well plate, and a serial gradient of the antimicrobial peptide (peptide C8LR prepared in example 1) solution was sequentially prepared using a double dilution method so that the volume of the solution in each well was 50. Mu.L. 50 μ L of the red blood cell suspension was added to the wells containing the peptide, and the red blood cell suspension treated with 0.1% triton was used as a positive control, and the untreated red blood cell suspension was used as a negative control. The 96-well plate was then placed in a 37 ℃ incubator for 1 hour. After centrifugation at 1000g for 5min, 50. Mu.L of the supernatant was aspirated from each well of a 96-well plate, transferred to a new 96-well plate, and then the absorbance was measured at 570nm with a microplate reader to calculate the hemolysis rate by the following equation:
hemolysis rate (%) = [ (sample OD 570-negative control OD 570)/(positive control OD 570-negative control OD 570) ] × 100%. The experiments were set up in two replicates and repeated three times.
Antibacterial activity, hemolytic activity and therapeutic index of antibacterial peptide C8 LR. The results are shown in Table 3.
TABLE 3 antibacterial Activity, hemolytic Activity and therapeutic index of the antibacterial peptide C8LR
a The minimum hemolytic concentration represents the concentration that causes 10% hemolysis of porcine red blood cells.
b Therapeutic index = minimum hemolytic concentration/total geometric mean, minimum hemolytic concentration>128, take 256 for calculation.
As can be seen from table 3, the peptide C8LR had no significant damaging effect on erythrocytes at a concentration of 128 μ M, indicating that the peptide has good biocompatibility. The therapeutic index was calculated using the ratio of the geometric mean of the minimum hemolytic concentration and the minimum inhibitory concentration, and the therapeutic index of the peptide C8LR reached 145.45 (table 3), indicating that it has good biocompatibility.
The results are combined to show that the peptide C8LR shows strong bacteriostatic activity to all tested fungi and bacteria and has low hemolytic activity, and the peptide C8LR has certain clinical application potential in the aspect of treating the infection caused by the fungi and the bacteria.
Sequence listing
<110> university of agriculture in China
<120> octanoic acid acylation modified antibacterial peptide and application thereof
<130> KHP201118825.8
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Leu Arg Arg Leu Arg Arg Arg Leu Arg Arg Leu Arg Arg Arg
1 5 10
Claims (8)
1. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.1, octanoic acid acylation modification is carried out at the N terminal of the amino acid sequence, and amidation modification is carried out at the C terminal of the amino acid sequence.
2. Use of the antimicrobial peptide of claim 1 for the preparation of an antifungal formulation, wherein the fungus is candida albicans.
3. Use of the antimicrobial peptide of claim 1 for the preparation of an antimicrobial formulation, said bacteria being escherichia coli, salmonella typhimurium, staphylococcus aureus and/or staphylococcus epidermidis.
4. The use of claim 2 or 3, wherein the formulation is a medicament.
5. Use of the antimicrobial peptide of claim 1 for the preparation of a disinfectant.
6. Use of the antimicrobial peptide of claim 1 for the preparation of a cleaning agent.
7. Use of the antimicrobial peptide of claim 1 for the preparation of a preservative.
8. A product which is a pharmaceutical, disinfectant, cleanser or antiseptic comprising the antimicrobial peptide of claim 1.
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