CN109867710B - Novel broad-spectrum antibacterial peptide SAMP1-A3 and preparation method thereof - Google Patents

Abstract

The invention discloses a novel broad-spectrum antibacterial peptide SAMP1-A3 and a preparation method thereof. The amino acid sequence of the antibacterial peptide is as follows: NH₂-RKKKVLLI-COOH. The preparation method adopts a solid-phase polypeptide synthesis method protected by amino acid nitrogen Fmoc. The invention overcomes the defects of poor biological safety, easy hemolysis or toxicity to mammalian cells of common antibacterial peptides, and is a novel antibacterial peptide which has broad-spectrum inhibition effect on non-filamentous fungi and bacteria, does not have hemolysis and has no toxicity to normal mammalian cells. The polypeptide provides a novel guide substance for clinical antifungal drug research.

Description

Novel broad-spectrum antibacterial peptide SAMP1-A3 and preparation method thereof

Technical Field

The invention belongs to biological agents, and particularly relates to a novel antibacterial peptide and a preparation method thereof.

Background

In recent years, due to antibiotic abuse, there has been an increasing number of isolated drug-resistant strains, particularly drug-resistant fungi, in infectious diseases. The emergence of super drug-resistant bacteria has increasingly urgent need for novel clinical antibiotics with less drug resistance to bacteria and fungi. Therefore, the method has important significance in researching and developing novel, efficient and low-toxicity antibacterial drugs to which pathogenic bacteria are not easy to generate drug resistance.

The antibacterial peptide is an important component of the natural immune system of an animal body. Unlike traditional antibiotics, which act primarily by inhibiting a biosynthetic pathway (e.g., cell wall, protein), most antimicrobial peptides inhibit or kill pathogenic bacteria via a multi-pathway, multi-target mechanism of action. The unique mechanism of action of antibacterial peptides renders microorganisms less susceptible to drug resistance (Forde E, et al. Molecules, 2015, 20(1): 1210-. Therefore, the antibacterial peptide has great potential clinically for treating human and animal diseases, and is probably the best choice for solving the problems of pathogenic bacteria drug resistance and drug toxic and side effects. Currently, antibacterial peptides have been the focus of research in various fields.

The polypeptide is expressed by utilizing the genetic engineering technology, and the expression product is unstable, easy to degrade and difficult to purify. The polypeptide with more than 30 amino acids is chemically synthesized, and the product yield and the purity are low. Therefore, the chemical synthesis of the antibacterial peptide lays a theoretical foundation for the development of antibacterial peptide medicaments.

Disclosure of Invention

The invention aims to prepare novel antibacterial peptide SAMP1-A3 with high antibacterial activity, wide antibacterial spectrum and high biological safety by adopting a solid-phase chemical synthesis technology.

The purpose of the invention is realized by the following technical scheme.

The polypeptide SAMP1-A3 is synthesized by using a solid phase chemical synthesis technology. The SAMP1-A3 antibacterial spectrum is measured by adopting a minimum bacteriostatic concentration method; SAMP1-A3 bactericidal and bacteriostatic activity is researched by adopting a plate counting method; the hemolytic activity of SAMP1-A3 was investigated by hemolytic activity experiments; the inhibition effect of SAMP1-A3 on the growth of umbilical vein endothelial cells is researched by adopting a cytotoxicity experimental method. The solid phase synthesis of the antibacterial peptide SAMP1-A3 is not reported at home and abroad at present. The antibacterial peptide SAMP1-A3 obtained by the method is expected to provide a good candidate drug for clinical pathogenic microorganism infection.

The antibacterial peptide SAMP1-A3 has the following advantages:

(1) has broad-spectrum antibacterial activity;

(2) has antifungal and antibacterial effects.

(3) Has no hemolytic activity.

(4) Has no toxicity to normal mammalian cells.

Therefore, the invention is a broad-spectrum, high-efficiency and safe candida-killing polypeptide, and the preparation thereof is not reported.

Drawings

FIG. 1 is a graph of the bactericidal and bacteriostatic activity results of the antimicrobial peptide SAMP 1-A3. FIG. 1A is Candida tropicalis; FIG. 1B shows Listeria monocytogenes.

FIG. 2 is a chart showing the results of hemolysis experiments using SAMP1-A3 as antibacterial peptide.

FIG. 3 is a graph showing the effect of antimicrobial peptide SAMP1-A3 on HUVEC growth of human umbilical vein endothelial cells. FIG. 3A is a control; FIG. 3B SAMP1-A3 at a concentration of 78 μ g/mL; FIG. 3C SAMP1-A3 at a concentration of 156. mu.g/mL; FIG. 3D SAMP1-A3 at a concentration of 234. mu.g/mL; FIG. 3E SAMP1-A3 at a concentration of 312. mu.g/mL.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings.

EXAMPLE 1 preparation of the antimicrobial peptides SAMP1-A3

Synthesizing a target peptide segment by adopting a solid phase Fmoc method, and synthesizing from a C end to an N end. Activating the carboxyl of the first Fmoc-Ile-OH at the C end of the polypeptide, then combining the activated carboxyl with resin, removing the Fmoc-protecting group, combining the activated carboxyl with the Fmoc-Leu-OH of the second carboxyl, then removing the Fmoc-protecting group, and repeating the steps until all amino acids are added. Cutting the peptide on the resin by using cutting fluid, collecting the cutting fluid, drying in a centrifuge tube, washing the precipitate for 2-3 times by using ethyl acetate, centrifuging to remove supernatant, and drying the precipitate. The resulting crude product was purified by HPLC.

EXAMPLE 2 antimicrobial peptide SAMP1-A3 antimicrobial Spectrometry

1) The protein was weighed and dissolved. SAMP1-A3 phosphate solution was prepared with an initial concentration of 500. mu.g/mL in the first column and a final concentration of 0.975. mu.g/mL. Then 2mg/mL of SAMP1-A3 stock solution is prepared. SAMP1-A3 was dissolved in 20 mmol/L pH6.0 sodium phosphate buffer and the solution was sterile filtered through a 0.22 μm sterile filter.

2) Using a pipette gun, 100. mu.L of 20 mmol/L sodium phosphate buffer pH6.0 was added to each well of a 96-well plate to dilute SAMP 1-A3.

3) Pipette 100. mu.L of 2mg/mL SAMP1-A3 stock solution into each well of the first column of a 96 well plate.

4) And repeating 6-8 times of the solution in the first row in the blowing and sucking plate, and uniformly mixing without splashing.

5) Sucking 100 mu L from the first row, adding the mixture into the second row, repeatedly blowing, sucking and mixing for 6-8 times, and sucking 100 mu L to the third row. This step is repeated through the tenth column.

6) The 100. mu.L aspirated in the tenth column was discarded without addition of column 11, and column 11 was a negative control well.

7) And respectively adding 100 mu L of bacterial suspension into the 1 st row to the 11 th row of the 96-well plate in sequence, repeatedly blowing and sucking for 6-8 times, and uniformly mixing. Note that no bacterial suspension was added to column 12. Column 12 is a blank control well.

8) The 96-well plate was incubated at rest. If the fungus is fungus, culturing at 30 deg.C for 48 hr; if the bacteria are bacteria, the bacteria are cultured for 16 h at 37 ℃.

9) mu.L of 5 mg/mL MTT solution was added to each well and incubation was continued for 4 h. The supernatant was aspirated off, 100. mu.L of dimethyl sulfoxide (DMSO) was added thereto, the mixture was left at 37 ℃ for 20 min without shaking, and after the crystals were completely dissolved, the OD value was measured at 490 nm using a microplate reader.

The bacteriostatic rate (%) [ (OD 570 (sample) -OD 570 (blank) ]/[ OD570 (negative) -OD 570 (blank) ] × 100.

MIC₁₀₀Is defined as: the lowest concentration of SAMP1-A3 when the bacteriostasis rate reaches 99.9 percent.

Each experiment was repeated three times.

The results are shown in Table 1. The results show that SAMP1-A3 has the strongest activity on cryptococcus neoformans, bacillus subtilis and listeria monocytogenes, and the 99.9% bacteriostatic concentration is 3.9 mug/mL; the 99.9% inhibitory concentration was 7.8. mu.g/mL, the secondary activity to E.coli and Candida tropicalis.

TABLE 1 SAMP1-A3 antibacterial Activity

Bacterial strains	MIC100 (μg/mL)
		Candida tropicalis	7.8
Candida albicans	15.6
		Candida krusei	15.6
Candida glabrata	31.2
		Candida parapsilosis	15.6
Cryptococcus neoformans	3.9
		Escherichia coli	7.8
Pseudomonas aeruginosa	62.5
		Bacillus subtilis	3.9
Listeria monocytogenes	3.9
		Staphylococcus aureus	62.5

The results showed that SAMP1-A3 has a strong inhibitory effect on fungi represented by Candida tropicalis, Cryptococcus neoformans and the like, gram-positive bacteria represented by Bacillus subtilis and Listeria monocytogenes and gram-negative bacteria represented by Escherichia coli.

Example 3 antimicrobial peptide SAMP1-A3 Bactericidal and bacteriostatic Activity assay

The sterilization and bacteriostasis activity of SAMP1-A3 is detected by measuring the number of live bacteria of bacteria/fungi after the action of the antibacterial peptide. Candida tropicalis is taken as a representative fungus, and Listeria monocytogenes is taken as a representative bacterial strain.

Candida tropicalis: culturing Candida tropicalis to OD at 28 deg.C with SD liquid culture medium₆₀₀0.6-0.8. Inoculating the bacterial suspension containing 1 × MIC₁₀₀In SD liquid medium of SAMP1-A3, the bacterial concentration is about 1X 10⁵CFU/mL. A blank SD medium without SAMP1-A3 was also included as a control. Culturing in a shaking table at 28 ℃ for 4h at constant temperature, sucking 100 mu L of bacterial liquid, diluting, spreading on an SD agar plate, culturing overnight at 28 ℃, and counting colonies.

Listeria monocytogenes: culturing Listeria monocytogenes to OD at 37 deg.C with TSB-YE liquid medium₆₀₀0.6-0.8. Inoculating the bacterial suspension to contain 1 × MIC₁₀₀TSB-YE broth of SAMP1-A3 to a bacterial concentration of about 1X 10⁵CFU/mL. A blank TSB-YE medium without SAMP1-A3 was also included as a control. Culturing in a shaking table at 37 ℃ at constant temperature, sucking 100 mu L of bacterial liquid after culturing for 4h, coating on an LB agar plate after diluting, culturing overnight at 37 ℃, and counting colonies.

The results are shown in FIG. 1. 1 × MIC₁₀₀After the SAMP1-A3 is treated for 4 hours, Candida tropicalis can not culture a single colony on an SD (SD) plate, and Listeria monocytogenes can grow a single colony, which shows that SAMP1-A3 has bactericidal activity on Candida tropicalis and has inhibitory activity on Listeria monocytogenes.

Example 4 determination of hemolysis of antimicrobial peptides SAMP1-A3

A sterile 96-well plate was taken and 100. mu.l of a 2% suspension of human red blood cells was pipetted into the 96-well plate. To each well was added 100. mu.l of SAMP1-A3 solution to give final concentrations of SAMP1-A3 of 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, 0.45 and 0.23. mu.g/mL, respectively, and the mixing was performed in triplicate for each concentration. After incubation at 37 ℃ for 30 min, the supernatants from each well were removed and transferred to a new 96-well microtiter plate, and the absorbance A of the samples was measured at 570 nm using a microplate reader. A negative control (0% hemolysis) was set up with a suspension of human erythrocytes without SAMP1-A3 added, and a positive control (100% hemolysis) was set up with a suspension of human erythrocytes with 1% Triton X-100 added. The hemolysis rate was calculated.

Hemolysis rate calculation formula:

percent hemolysis (%) = [ (sample a 570-negative control a 570)/(positive control a 570-negative control a570) ] × 100%

As shown in FIG. 2, the SAMP1-A3 concentration at 5% hemolysis rate was 140. mu.g/mL, which is much higher than the MICs measured for Candida tropicalis, Cryptococcus neoformans, Escherichia coli, Bacillus subtilis and Listeria monocytogenes₁₀₀36 times and 18 times, respectively. As a result, SAMP1-A3 was found to be highly safe.

Example 5 determination of cytotoxicity of antimicrobial peptides SAMP1-A3 on Normal mammals

HUVEC (human umbilical vein endothelial cells) are treated at 5X 10⁴Passaging to 24-well plates per well at 5% CO₂And culturing HUVEC cells in MCDB culture medium containing 10% FBS at 37 deg.C in a constant temperature and humidity cell culture chamber. After the cells had grown to confluency, they were treated with SAMP1-A3 at various concentrations (78. mu.g/mL, 156. mu.g/mL, 234. mu.g/mL, 312. mu.g/mL), removed after 24 hours, the old medium was aspirated, washed once with 1 XPBS, and observed under an inverted microscope for changes in cell morphology and dead cell number. The results are shown in FIG. 3.

The results show that the MIC of Candida tropicalis is 40 times higher₁₀₀At concentrations of SAMP1-A3 (312. mu.g/mL), the effect of SAMP1-A3 on umbilical vein endothelial cell growth was not detected. SAMP1-A3 has no toxic effect on mammalian cells.

Sequence listing

<110> industrial university of Henan

<120> novel broad-spectrum antibacterial peptide SAMP1-A3 and preparation method thereof

<140> 201811564473.9

<141> 2018-12-20

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial

<400> 1

Arg Lys Lys Lys Val Leu Leu Ile

1 5

Claims (4)

1. A novel broad-spectrum antibacterial peptide is characterized in that: the amino acid sequence of the antibacterial peptide is NH₂-RKKKVLLI-COOH。

2. The method for preparing the antibacterial peptide of claim 1, wherein: is prepared by adopting the conventional solid-phase polypeptide synthesis technology.

3. The method for preparing an antibacterial peptide of claim 2, characterized in that: in the solid-phase synthesis of an Fmoc protection system, Rinkamide-AM Resin is firstly arranged in a reactor, and 20% piperidine solution is used for deprotection; mixing the first amino acid Fmoc-Ile-OH at the C terminal with the deprotected Rinkamide-AM Resin in a reactor, and combining the mixture on a solid phase carrier under the catalysis of HOBt/DIC; coupling Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH and Fmoc-Arg (Pbf) -OH in sequence according to a given amino acid sequence, extending from the C end to the N end one by one, and finally cutting the polypeptide from the solid phase carrier by using a cutting fluid;

the cutting fluid comprises the following components: 87.5% trifluoroacetic acid, 2.5% 1, 3-dimethoxybenzene, 2.5% triisopropylsilane, 2.5% water, 5% methyl phenyl sulfide.

4. The method for preparing an antimicrobial peptide according to claim 2, wherein: various amino acid residues may be coupled using various coupling agents and coupling methods known in the art of peptide chemical synthesis, either by direct coupling using Diisopropylcarbodiimide (DIC), Dicyclohexylcarbodiimide (DCC), or by activating amino acids using hydroxybenzotriazole (HOBt) or 7-azahydroxybenzotriazole (HOAt).

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