CN114478736A - Potent antibacterial peptide and application thereof in skin injury repair - Google Patents

Abstract

According to the invention, through bioinformatics analysis, the American cockroach defense peptide is subjected to mutation of special sites, the physicochemical properties of the mutant are analyzed in a modeling mode, the mutant antibacterial peptide is synthesized by preferentially adopting a solid-phase synthesis method, and mass spectrum identification and bacteriostatic activity detection are carried out, so that the mutant antibacterial peptide with improved stability and antibacterial function is obtained. Wherein, both PD-1m1 and PD-1m8 have broad-spectrum antibacterial activity, and PD-1m7 has extremely strong antibacterial activity to staphylococcus aureus and staphylococcus epidermidis. The series of mutant polypeptides are expected to replace antibiotics to be used for preparing skin repair drugs, and have good medicinal value prospects.

Description

Potent antibacterial peptide and application thereof in skin injury repair

Technical Field

The invention relates to the field of polypeptides, in particular to a small-molecule antibacterial peptide and application thereof.

Background

Antibacterial peptides (also called defensive peptides) are coded by specific genes of various cells of organisms, and are the first defense line of organisms against bacteria, viruses, fungi and protozoa and inhibiting and killing tumor cells. The antibacterial peptide is expected to replace antibiotics for clinical use due to the characteristics of difficult drug resistance generation, broad-spectrum antibacterial property, low cytotoxicity, good thermal stability and the like. Insects are the largest species of organisms and the number of antimicrobial peptides is difficult to estimate. At present, more than 200 kinds of insect antibacterial peptide substances are found in 8 insects such as lepidoptera, diptera, coleoptera and dragonflies, and only 40 antibacterial peptide genes are obtained from one insect such as silkworm. The antibacterial peptide has broad-spectrum antibacterial activity and strong killing effect on bacteria, and particularly the killing effect on some drug-resistant pathogenic bacteria draws more attention. In addition, it has been found that certain antimicrobial peptides have a killing effect on some viruses, fungi, protozoa, cancer cells, etc., and even can improve immunity and accelerate wound healing. The wide biological activity of the antibacterial peptide shows good application prospect in medicine. The American cockroach has bad natural living conditions, and researches show that the American cockroach has highly diversified antibacterial peptides, but the separation and the function research of the antibacterial peptides in the American cockroach are far from sufficient at present.

In earlier studies, a plurality of defense peptides from periplaneta americana have been found, but most of them cannot be used for medicinal purposes due to poor stability, so that molecular structure modification, chemical modification and redesign of natural antimicrobial peptides are an important research direction for the excavation and development of novel antimicrobial peptides.

Disclosure of Invention

In order to fill the blank of the prior art, the invention aims to improve the stability and the bacteriostatic activity of the natural periplaneta americana antibacterial peptide by modifying the molecular structure of the natural periplaneta americana antibacterial peptide. Therefore, the invention provides the following technical scheme:

in a first aspect of the present invention, there is provided an antimicrobial peptide having improved stability, which is obtained by mutating lysine at one or more positions in an amino acid sequence shown in SEQ ID No.1 to histidine.

In a preferred embodiment, the positions are at positions 44 and 90.

In a second aspect of the invention, there is provided the use of an antimicrobial peptide as described above in the manufacture of an anti-infective medicament, said infection being caused by staphylococcus aureus and/or staphylococcus epidermidis.

In a third aspect of the present invention, there is provided the use of the broad-spectrum antimicrobial peptide described above in the manufacture of a medicament for the treatment of skin damage, wherein said skin damage is caused by infection with staphylococcus aureus and/or staphylococcus epidermidis.

In one embodiment, the medicament is an external preparation.

Preferably, the external preparation is selected from spray and liniment.

In one embodiment, the external preparation is an emulsion, a paste, a suspension, or the like.

Compared with the prior art, the invention has the following beneficial technical effects：

According to the invention, through bioinformatics analysis, the American cockroach defense peptide is subjected to mutation of special sites, the physicochemical properties of the mutant are analyzed in a modeling mode, the mutant antibacterial peptide is synthesized by preferentially adopting a solid-phase synthesis method, and mass spectrum identification and bacteriostatic activity detection are carried out, so that the mutant antibacterial peptide with improved stability and antibacterial function is obtained. Wherein, both PD-1m1 and PD-1m8 have broad-spectrum antibacterial activity, and PD-1m7 has extremely strong antibacterial activity to staphylococcus aureus and staphylococcus epidermidis. The series of mutant polypeptides are expected to replace antibiotics to be used for preparing skin repair drugs, and have good medicinal value prospects.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

EXAMPLE 1 design of the PD-1m series of antimicrobial peptides

The PD-1m series of antibacterial peptides is designed based on the amino acid sequence (SEQ ID NO.1) of natural antibacterial peptides from Periplaneta americana. In order to improve the stability of the antimicrobial peptides, the amino acid sequence composition, charge and hydrophobicity of the natural antimicrobial peptides were analyzed, and it was attempted to replace a part or all of 8 lysines (Lys) in the natural antimicrobial peptides with histidine (His), and the analysis of the specific substitution sites and physicochemical properties of each mutant is shown in table 1.

TABLE 1

As shown in Table 1, the stability of five polypeptide sequences of the mutant polypeptide PD-1m1, PD-1m7, PD-1m8, PD-1m9 and PD-1m10 is obviously improved by mutating lysine sites of a natural antibacterial peptide sequence.

Example 2 Synthesis of antibacterial peptide mutants PD-1m1, PD-1m7, PD-1m8, PD-1m9, PD-1m10

Weighing 0.23g of Rink Amide MBHA resin, placing the Rink Amide MBHA resin in a PSI300 type polypeptide synthesizer reactor, adding 10mL of DMF, soaking for 2h, then adding 10mL of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DMF for 7 times, then adding Fmoc-L-3- (2-Thienyl) L-alanine-OH, equimolar coupling reagents DIC (0.3mol/L) and HOBt (0.3mol/L) into the reactor for reaction, wherein the reaction temperature is room temperature, the reaction process is monitored by ninhydrin reaction, and the reaction is completed when monitoring is colorless, and the resin is washed with DMF for 5 times. After the first amino acid is coupled to the resin, the coupling reaction of the next amino acid is continued according to the method, and the process is circulated until all the amino acids are coupled. After the synthesis was complete, the resin was dried in vacuo and weighed. The cleavage reagent was added at a ratio of 1mL of the cleavage reagent to 100mg of the resin, the reagent ratio was TFA, thioanisole, 75% phenol, and water, 85:5:5:5, and the mixture was stirred at room temperature for reaction for 3 hours, followed by suction filtration. And then adding 10 times of volume of glacial ethyl ether into the cracking suction filtration liquid to precipitate the polypeptide, centrifuging, discarding the supernatant, repeatedly washing the precipitate for 4-5 times by using the glacial ethyl ether, drying in vacuum, and weighing the crude peptide.

The fractions (target peptides) with a purity of more than 95% were purified and collected using a Waters600E chromatography C18 reverse phase column and identified by ESI-MS.

The results of this series of mutant polypeptides are shown in Table 2, as determined by ESI-M3S:

TABLE 2

Mutants	Molecular weight (theoretical value)	Molecular weight (ESI-MS measured value)
			PD-1m1	16650.8	16648
PD-1m7	16659.7	16658
			PD-1m8	16668.6	16667
PD-1m9	16677.5	16675
			PD-1m10	16713.1	16712

Example 3 detection of minimum inhibitory concentration (MIC detection) of series of mutant antimicrobial peptides

In the test, a microdilution method is adopted for detecting the minimum inhibitory concentration, and the detection bacteria are escherichia coli (ATCC25922), staphylococcus aureus (ATCC25923), methicillin-resistant staphylococcus aureus (ATCC6538), staphylococcus epidermidis (ATCC12228), bacillus subtilis (ATCC6638) and klebsiella pneumoniae (ATCC 700603). The specific method comprises the following steps: recovering each strain, coating on MH solid culture medium, placing in a constant temperature incubator at 37 ℃ overnight, selecting a single colony, inoculating in a fresh liquid LB culture medium, and placing in the constant temperature incubator at 37 ℃ again overnight; the 5 bacterial suspensions were then diluted to 105CFU/m L with fresh LB broth by means of a Mach turbidimeter for use. The purified AMP-1 of known concentration was filtered through a 0.22 μm filter, and then the AMP-1 was diluted 2-fold with fresh liquid LB medium to adjusted concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μ g/mL in this order. And dropwise adding 100 mu L of diluted AMP-1 solution and 100 mu L of bacteria liquid into a 96-well plate, making 3 multiple wells respectively, and setting an action group only containing the culture medium as a negative control and an action group only containing the bacteria liquid as a positive control. And (3) putting the 96-hole culture plate into a constant-temperature incubator, culturing for 16-18h at 37 ℃, and detecting the light absorption value at 600nm by using a microplate reader. When the ratio of the light absorption value of the action group containing the antibacterial peptide to the light absorption value of the positive control group is less than 10%, the antibacterial peptide sample with the concentration is considered to have an inhibition effect on bacteria, the minimum concentration (mu g/ml) of the polypeptide sample capable of inhibiting the growth of the bacteria is an MIC value, and each group of experiments are repeated for three times to obtain an average value. The results are shown in table 3 below:

TABLE 3

ND represents a MIC value of greater than 100. mu.g/ml

EXAMPLE 4 cytotoxicity test of series of mutant antibacterial peptides

MTT method detects toxicity of series mutant antibacterial peptide and ceftriaxone sodium on human epidermal cells (HaCat immortalized epidermal cells). MTT can be reduced to insoluble blue-violet crystalline formazan by succinate dehydrogenase in mitochondria of living cells, deposited in cells, while dead cells do not. DMSO can dissolve blue-purple formazan crystal in cells, and the number of living cells can be indirectly reflected by the absorbance of the solution after the dissolution. Within a certain range of cell number, the amount of crystal formation is directly proportional to the number of cells.

HaCat cells were cultured in MEM complete medium at 37 ℃ under 5% CO2 and saturated humidity. Cells were blown on a pipette, suspended in MEM in complete medium, and cultured at 2.5 × l0⁵cells/mL density were seeded in 96-well plates at 100. mu.L per well in 3 replicates. After 24h the medium was removed and 100. mu.L of sample peptide at 1, 2, 4, 8, 16, 32, 64, 128, 256. mu.g/mL was added to each well in a concentration gradient and an equal amount of PBS solution was added to the control wells. After a further 24h incubation, the medium was removed, washed twice with PBS and 100. mu.L of MTT at a concentration of 5mg/mL was added to each well (protected from light). The 96-well plate was moved to an incubator for further 4 h. And (3) discarding the MTT solution, adding 150 mu L DMSO into each well, oscillating for 10min by an oscillator, and measuring the absorbance of each well at the wavelength of 570nm after the crystals at the bottom of the well are completely dissolved. Cell viability was calculated according to the following formula:

survival (%). ratio of OD value of treated group/OD value of control group X100%

The result of a cytotoxicity experiment shows that the survival rate of Hacat cells is over 90 percent under the concentration of 128 mu g/mL of the PD-1m series mutant antibacterial peptide, which indicates that the Hacat cell has no toxic effect on normal animal tissue cells. The viability of cells was slightly reduced at higher concentrations of 256 μ g/mL for PD-1m1 and PD-1m10 (79.1% and 77.4%, respectively), but the toxicity was not strong.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. The strong antibacterial peptide with improved stability is characterized in that the antibacterial peptide is obtained by mutating lysine at one or more positions in an amino acid sequence shown in SEQ ID No.1 into histidine.

2. The potent antimicrobial peptide of claim 1, wherein the antimicrobial peptide is obtained by mutating lysines at positions 44 and 90 of the amino acid sequence shown in SEQ ID No.1 to histidines.

3. Use of a potent antimicrobial peptide according to any one of claims 1-2 in the manufacture of an anti-infective medicament.

4. The use of claim 3, wherein the infection is an infection caused by a staphylococcal bacterium.

5. The use of claim 4, wherein the Staphylococcus is Staphylococcus aureus and Staphylococcus epidermidis.

6. Use of an antimicrobial peptide according to any one of claims 1 to 2 in the manufacture of a medicament for the treatment of skin damage.

7. The use of claim 6, wherein the skin lesion is a lesion caused by a staphylococcal infection.

8. The use according to any one of claims 3 to 7, wherein the medicament is a topical formulation.

9. The use of any one of claims 3 to 7, wherein the external preparation is selected from a spray or a wipe.

10. The use according to any one of claims 3 to 7, wherein the external preparation is an emulsion, a paste, a suspension or the like.