CN110156875B - Antibacterial peptide H5-p5 and its preparation method and application - Google Patents

Abstract

An antimicrobial peptide H5-p5, the antimicrobial peptide H5-p5 is composed of 18 amino acid residues, has a molecular weight of 2423.12 Da, a net charge of +10, an isoelectric point of 11.78, and its amino acid sequence is Tyr-Ile-Arg-Lys ‑Ile‑Arg‑Arg‑Phe‑Phe‑Lys‑Lys‑Leu‑Lys‑Lys‑Ile‑Leu‑Lys‑Lys‑NH ₂ . The antibacterial peptide H5-p5 has the advantages of small molecular weight, simple artificial synthesis, significant antibacterial effect, strong inhibitory effect on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, and low hemolytic activity, and has wide application prospects.

Description

Antibacterial peptide H5-p5 and its preparation method and application

technical field

The invention belongs to the technical field of biochemistry, and particularly relates to the preparation of antibacterial peptide H5-p5 and the application of the antibacterial peptide in the preparation of antibacterial drugs.

Background technique

Since the discovery of antibiotics such as penicillin, the treatment of bacterial infectious diseases has fundamentally improved, and the use of antibiotics has saved the lives of countless patients and extended the average human lifespan. However, the large-scale use or abuse of antibiotics, especially in developing countries, has led to the emergence and spread of some drug-resistant bacteria, including some highly virulent pathogens, such as methicillin-resistant staphylococcus and pneumonia Streptococcus etc. Therefore, the search for safe and effective antibacterial drugs that are not prone to drug resistance has become the direction of competition and efforts of scientists all over the world.

Antimicrobial peptides are highly efficient and broad-spectrum immune molecules that are rapidly produced by the body when organisms are invaded by microorganisms, generally consisting of 12 to 100 amino acids. Antimicrobial peptides widely exist in different types of organisms, and thousands of antimicrobial peptides have been identified from microorganisms, plants, amphibians, marine vertebrates, mammals and even humans. The antibacterial mechanism of antimicrobial peptides is complex, but most theories believe that the mechanism involves the interaction of the cationic and hydrophobic properties of antimicrobial peptides with negatively charged microbial cell membranes. Or the formation of transmembrane pores in the bacterial cell membrane, which eventually leads to the leakage of bacterial contents and death. The effect of antimicrobial peptides on bacteria is almost lethal, and it is not easy to develop drug resistance. Therefore, antimicrobial peptides are far more efficient at killing bacteria than traditional antibiotics.

The discovery and rapid development of antimicrobial peptides have provided a huge resource pool for the development of new antimicrobial drugs, and also provided a great possibility to solve the problem of clinical drug-resistant strains. application prospects. With the further understanding of the antibacterial mechanism of antimicrobial peptides and the discovery of new antimicrobial peptides, people can not only directly isolate and purify antimicrobial peptides from living organisms, but also use genetic engineering methods to recombine them to obtain antimicrobial peptides, and they can directly use chemical synthesis methods. A large number of small-molecule antimicrobial peptides are synthesized in a short time.

Antimicrobial peptides from natural sources are immunogenic due to their large molecular weight, have low antibacterial activity, have cytotoxic effects on host cells, or cause hemolysis, which limit the promotion and application of antimicrobial peptides as antibacterial drugs. Small, stronger antibacterial activity, especially antibacterial peptides that do not cause hemolysis or cytotoxicity are the most critical factors for the promotion of antibacterial peptides as antibacterial drugs. In recent years, while searching for new antimicrobial peptides, scientists have also begun to work on structural modification or redesign of the original natural antimicrobial peptides, such as replacing certain amino acid residues or directly designing the primary amino acids of antimicrobial peptides as needed. structure, in order to obtain antimicrobial peptides with higher activity, more specificity and non-toxic effect on host cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new antibacterial peptide H5-p5 with medicinal value and its preparation.

Another object of the present invention is to provide the use of the above antibacterial peptides in the preparation of antibacterial drugs or health care products or mouthwashes.

An antimicrobial peptide H5-p5, the amino acid sequence of the antimicrobial peptide H5-p5 is: Tyr-Ile-Arg-Lys-Ile-Arg-Arg-Phe-Phe-Lys-Lys-Leu-Lys-Lys-Ile -Leu-Lys-Lys-NH ₂

(Tyrosine-Isoleucine-Arginine-Lysine-Isoleucine-Arginine-Arginine-Phenylalanine-Phenylalanine-Lysine-Lysine-Leucine amino acid-lysine-lysine-isoleucine-leucine-lysine-lysine- _NH2 ). The C-terminal amidation of the full sequence of the antimicrobial peptide H5-p5.

The specific feature of this scheme is that it contains 18 amino acid residues, the molecular weight is 2423.12 Da, and the isoelectric point is 11.78.

A preparation method of the above-mentioned antibacterial peptide H5-p5, according to its amino acid sequence, using an automatic polypeptide synthesizer to synthesize its full sequence, and HPLC reverse phase column chromatography for desalting and purification.

An application of the above-mentioned antibacterial peptide H5-p5, in the preparation of anti-methicillin-resistant Staphylococcus aureus medicines or health products or mouthwashes.

The beneficial effects of the present invention are: the antibacterial peptide H5-p5 in the present invention is artificially synthesized, and has the advantages of small molecular weight, convenient artificial synthesis, strong bactericidal effect, etc. In addition, the antibacterial peptide H5-p5 also has extremely low hemolytic activity and eukaryotic cells. characteristics of toxicity. By aligning with 3072 antimicrobial peptide sequences in the antimicrobial peptide APD database, no sequence completely repeated with the H5-p5 sequence was found. H5-p5 has strong bacteriostatic effect on Staphylococcus aureus and drug-resistant Staphylococcus aureus, but has no strong bacteriostatic effect on beneficial bacteria such as Streptococcus sanguis, and has weak hemolysis and low cytotoxicity. H5-p5 has good antibacterial selectivity and low toxicity and side effects. In addition, H5-p5 can inhibit the formation of Staphylococcus aureus biofilm, affect the integrity of bacterial cell membrane, downregulate the expression of bacterial virulence factor-related genes, and exert antibacterial effects in multiple ways. There is no report on the multi-path antibacterial mechanism of the antibacterial peptides with similar sequences to H5-p5.

Description of drawings

Figure 1 is a high performance liquid chromatography HPLC purity chart. Figure 2 is an electrospray mass spectrum. Figure 3 is an analysis of the hemolytic activity of antimicrobial peptide H5-p5 (mouse blood). The above experimental results are the average of three independent experiments. Figure 4 is the cytotoxicity analysis of antimicrobial peptide H5-p5 (embryonic kidney cells HEK). The above experimental results are the average of three independent experiments. Figure 5 is an experiment of the effect of antimicrobial peptide H5-p5 on bacterial biofilm formation. Figure 6 is an experiment of the effect of antimicrobial peptide H5-p5 on bacterial cell membrane integrity. Figure 7 is an experiment of the effect of antimicrobial peptide H5-p5 on bacterial resistance-related gene spA. Figure 8 is an experiment of the effect of antimicrobial peptide H5-p5 on bacterial resistance-related gene hld.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the contents described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

Example 1: Preparation of antimicrobial peptide H5-p5

Ⅰ. Chemical synthesis method of antimicrobial peptide H5-p5: According to the amino acid sequence described in the summary of the invention, use the corresponding L-amino acid (purchased from Merck Sigma) and automatic peptide synthesizer (433A, Applied Biosystems, USA) Its full sequence was synthesized. Desalting and purification by HPLC reverse phase column (WelchXB C18 4.6×150 mm), the mobile phase is pure water (0.1% trifluoroacetic acid)-60% acetonitrile (0.1% trifluoroacetic acid), the flow rate is 1 mL/min, the wavelength of 220 nm The eluted peaks were collected and lyophilized. L-amino acids do not need to be deposited.

Ⅱ. The molecular weight of the synthesized antimicrobial peptide H5-p5 was identified by electrospray mass spectrometry. The sample was injected using a liquid phase system, the mobile phase was 50% H2O/50% CAN, the flow rate was 0.2ml/min, the protective gas flow rate was 1.5L/min, and the collision energy was 4.5kV.

Ⅲ. The purity of the purified antimicrobial peptide H5-p5 was identified by high performance liquid chromatography (Welch XB C18 4.6*250 mm), the molecular weight was determined by electrospray mass spectrometry, the isoelectric point was determined by isoelectric focusing electrophoresis, and the automatic amino acid sequencer was used Determine the amino acid sequence structure.

The results of high performance liquid chromatography (HPLC) purity identification are shown in Figure 1: the antimicrobial peptide H5-p5 showed a single symmetrical peak at 14.277min.

The identification results of electrospray mass spectrometry are shown in Figure 2: the molecular weight of antimicrobial peptide H5-p5 is 2406.16Da.

After HPLC and mass spectrometry identification, antimicrobial peptide H5-p5 contains 18 amino acid residues, molecular weight 2423.12 Da, isoelectric point 11.78, and its full sequence is:

Tyr-Ile-Arg-Lys-Ile-Arg-Arg-Phe-Phe-Lys-Lys-Leu-Lys-Lys-Ile-Leu-Lys-Lys-NH ₂

(Tyrosine-Isoleucine-Arginine-Lysine-Isoleucine-Arginine-Arginine-Phenylalanine-Phenylalanine-Lysine-Lysine-Leucine amino acid-lysine-lysine-isoleucine-leucine-lysine-lysine-NH2).

Example 2: Antibacterial experiment of antimicrobial peptide H5-p5:

Minimum inhibitory concentration (MIC): the lowest concentration of the sample at which no bacterial growth can be detected. The double dilution method is adopted, and the specific method is as follows:

Bacteria were inoculated on Luria-Bertani (LB) solid medium and cultured upside down in a 37°C incubator. After colonies grow, single colonies are picked with an inoculation loop and transferred to LB liquid medium, and cultured in a 37°C incubator with shaking to logarithmic growth phase. Detect the OD ₆₀₀ of the bacterial liquid on a UV spectrophotometer, and dilute the bacterial liquid with liquid LB medium to 2×10 ⁵ CFU/ml according to OD ₆₀₀ =1×10 ⁸ CFU/ml. Pre-add 100 μL of LB liquid medium to each well of a sterile 96-well plate, and then add 100 μL of the antimicrobial peptide sample diluted to a certain concentration and filtered through a 0.22 μm microporous membrane to the first well, mix well, and take 100 μL was added to the second well, and then folded in sequence, and 100 μL was aspirated from the 12th well and discarded. So far, the two-fold concentration gradient sample was prepared.

Add 100 μL of the diluted bacterial solution to each well, mix well and incubate at 37° C. for 16 h with slow shaking, and measure the light absorption value at 600 nm. Calculation of results: The average value of the sum of the sample concentrations of the wells with no bacterial growth and the adjacent wells with bacterial growth was taken as the minimum inhibitory concentration of the sample.

In addition, Candida albicans is a fungus, and the medium used for culture is PDA medium, and other conditions are similar.

The results are shown in Table 1. Table 1 is a list of antibacterial activity test data of antimicrobial peptide H5-p5. Among them, H5-p5 MIC: the minimum inhibitory concentration of antimicrobial peptide H5-p5, and the above results are the average of three independent repeated experiments.

It can be seen from Table 1 that the antimicrobial peptide H5-p5 has a very strong killing effect on the tested strains. For example, the MIC value of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus can reach as low as 8 μg/mL, indicating that antibacterial Peptide H5-p5 inhibits the growth of staphylococci at very low concentrations. At the same time, antimicrobial peptide H5-p5 also has a good bactericidal effect on Candida albicans isolated in oral clinic, and the minimum concentration can reach 32 μg/mL; while the minimum inhibitory concentration of Streptococcus sanguis, a beneficial oral bacteria, is greater than 64 μg/mL. The antimicrobial peptide H5-p5 has a significant effect on Gram-positive staphylococci with good selectivity.

Example 3: Hemolytic activity test of antimicrobial peptide H5-p5:

Blood was collected from the mouse heart, and the collected blood was mixed with Alsever Solution (Alsever Solution, 8.0g sodium citrate, 0.55g citric acid, 20.5g glucose, 4.2g sodium chloride, deionized water was added to 1L, pH was adjusted to 6.1, (stored at 4°C after autoclaving) mixed in a 1:1 ratio and placed in a centrifuge tube, centrifuged at 1000 rpm for 5 min, and washed with normal saline until the supernatant was no longer red. The above washed red blood cells were diluted with physiological saline into a suspension of 10 ⁸ concentration. The above-mentioned diluted red blood cell suspension was incubated with samples of different concentrations dissolved in physiological saline at 37° C. for 30 min, and then centrifuged at 1000 rpm for 5 min, and the supernatant was measured for absorbance at 540 nm. Physiological saline was used as a negative control, and Triton X-100 was used as a positive control. The hemolytic activity is proportional to the absorbance at 540 nm.

The results are shown in Figure 3.

It can be seen from Figure 3 that the antimicrobial peptide H5-p5 does not cause hemolysis in mouse blood even at a high concentration of 320 μg/ml.

Example 4: Cytotoxicity test of antimicrobial peptide H5-p5:

Human normal cell embryonic kidney cells HEK293 were cultured to log phase in Dulbecco's modified (DMEM) medium containing 10% fetal bovine serum and double antibody (100 U/mL each of penicillin and streptomycin), and the cells were washed three times with PBS buffer. After passage, digest with 0.25% trypsin, suspend the cells in fresh DMEM medium, adjust the cell density to 1×10 ⁶ cells/ml, and spread the cells in a 96-well plate with a volume of 200 μL per well. Samples with different concentrations were co-cultured for 24 hours at 37°C and 5% carbon dioxide. After the incubation, 20 μL of 5 mg/ml MTT solution (prepared with cell culture PBS buffer) was added to each well of the 96-well cell culture plate, and the culture was continued for 4 hours. , suck out the liquid in the well with a pipette, add 100 μL of dimethyl sulfoxide (DMSO) to each well, shake gently for 10 min at room temperature, and detect the light absorption at a wavelength of 490 nm with a microplate reader.

The results are shown in Figure 4.

It can be seen from Figure 4 that even at a concentration of 1000 μg/mL, antimicrobial peptide H5-p5 has a very low cytotoxic effect of about 10% on human normal cell embryonic kidney cells HEK293.

Example 5: Exploration of the mechanism of action of antimicrobial peptide H5-p5:

I. Staphylococcus aureus and MRSA bacteria were cultured with Tryptic Soy Broth (TSB) and diluted to about 10 ⁸ CFU/mL. 1 ml of bacterial broth was added to a 12-well plate and H5-p5 or physiological saline at an MIC concentration was added, after which the plate was incubated at 37°C for 24 hours. Samples were immersed overnight in PBS containing 2.5% glutaraldehyde. After rinsing three times with PBS, samples were dehydrated using 30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100% ethanol for 5-15 minutes consecutively. Finally, the samples were rinsed with 100% hexamethyldisilazane and placed on glass petri dishes to dry overnight before imaging using field emission scanning electron microscopy.

The results are shown in Figures 5 and 6.

It can be seen from Figure 5 that the antimicrobial peptide H5-p5 can reduce the aggregation of Staphylococcus aureus and MRSA bacterial community, and inhibit the formation of Staphylococcus aureus and MRSA bacterial biofilm. It can be seen from Figure 6 that the antimicrobial peptide H5-p5 can affect the integrity of the bacterial cell membrane, causing the bacterial cell membrane to rupture and the contents to flow out, thereby killing the bacteria.

II. Dilute overnight Staphylococcus aureus and MRSA bacteria to 10 ⁸ CFU/mL, and then incubate in H5-p5 at MIC concentration or in normal saline for 1 hour. Bacterial RNA was extracted from 5 mL of bacterial culture. Bacterial cultures were harvested by centrifugation at 12,000 x g for 2 min at 4°C. The pellet was rinsed with 1 ml of TRIzol and lysed with 0.5 ml of zirconia-silica beads (diameter, 0.1 mm) in a high-speed homogenizer. Extract with DNA/RNA extraction reagent (chloroform:isoamyl alcohol = 24:1), and isolate RNA sequentially with isopropanol and 70% ethanol, and the final RNA is dissolved in diethyl pyrocarbonate (DEPC) water.

Genomic DNA contained in RNA was degraded and reverse transcribed to generate cDNA using PrimeScript™ RT kit and gDNA Eraser (Perfect Real Time, Takara). This was followed by quantitative reverse transcription-PCR (qRT-PCR) for the detection of S. aureus using the LightCycler 480II system (Forrenstrasse 2,6343 Rotkreuz, Switzerland) and TB GreenTM Premix Ex TaqTMII (Tli RNaseH Plus, Takara) according to the manufacturer's instructions Virulence factor-related genes spA and hld. The expression level of 16S rRNA was used as a control to normalize the expression levels of other genes. All experiments were repeated at least three times.

The results are shown in Figures 7 and 8.

It can be seen from Figures 7 and 8 that the antimicrobial peptide H5-p5 can down-regulate the drug resistance-related genes spA and hld in Staphylococcus aureus and MRSA bacteria. The spA gene encodes Staphylococcus aureus protein A, which is a virulence factor of Staphylococcus aureus; the hld gene encodes a delta exotoxin, a hemolytic toxin. H5-p5 can not only play an antibacterial effect by inhibiting the formation of bacterial biofilms and affect the integrity of bacterial cell membranes, but also play an attenuating antibacterial effect by downregulating the expression levels of virulence factor-related genes.

To sum up, the antimicrobial peptide H5-p5 in the present invention has the advantages of small molecular weight, simple artificial synthesis, efficient killing of methicillin-resistant Staphylococcus aureus, and low hemolytic activity. The composition can not only be used as an anti-methicillin-resistant Staphylococcus aureus drug, but also can be used in the fields of health care products and mouthwashes.

sequence listing

<110> Institute of Biology, Shandong Academy of Sciences

<120> Antibacterial peptide H5-p5 and its preparation method and application

<141> 2019-05-21

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<221> AMIDATION

<222> (1)..(18)

<223> C-terminal amidation of the complete sequence of antimicrobial peptide H5-p5

<400> 1

Tyr Ile Arg Lys Ile Arg Arg Phe Phe Lys Lys Leu Lys Lys Ile Leu

1 5 10 15

Lys Lys

sequence listing

<110> Institute of Biology, Shandong Academy of Sciences

<120> Antibacterial peptide H5-p5 and its preparation method and application

<141> 2019-05-21

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<221> AMIDATION

<222> (1)..(18)

<223> C-terminal amidation of the complete sequence of antimicrobial peptide H5-p5

<400> 1

Tyr Ile Arg Lys Ile Arg Arg Phe Phe Lys Lys Leu Lys Lys Ile Leu

1 5 10 15

Lys Lys

Claims (4)

1. An antibacterial peptide H5-p5, which is characterized in that the amino acid sequence of the antibacterial peptide H5-p5 is as follows: Tyr-Ile-Arg-Lys-Ile-Arg-Arg-Phe-Phe-Lys-Lys-Leu-Lys-Lys-Ile-Leu-Lys-Lys-NH₂。

2. The antibacterial peptide H5-p5 of claim 1, which comprises 18 amino acid residues, has a molecular weight of 2423.12 Da and an isoelectric point of 11.78.

3. The method for preparing the antibacterial peptide H5-p5 as claimed in claim 1, wherein the full sequence of the antibacterial peptide is synthesized by an automatic polypeptide synthesizer according to the amino acid sequence of the antibacterial peptide, and the antibacterial peptide is desalted and purified by HPLC reverse phase column chromatography.

4. The use of the antimicrobial peptide H5-p5 as claimed in claim 1, which is used in the preparation of a methicillin-resistant Staphylococcus aureus (MRSA) resistant medicament or health product or mouthwash.

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