CN107759664B

CN107759664B - Small molecular polypeptide AKK10 and application thereof

Info

Publication number: CN107759664B
Application number: CN201711090152.5A
Authority: CN
Inventors: 阿布力米提·伊力; 阿提坎·吾布力喀斯木; 阿吉艾克拜尔·艾萨
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Priority date: 2017-11-08
Filing date: 2017-11-08
Publication date: 2021-02-12
Anticipated expiration: 2037-11-08
Also published as: CN107759664A

Abstract

The invention discloses a small molecular polypeptide AKK10 and application thereof, wherein the small molecular polypeptide comprises 10 amino acid residues, the molecular weight is 1376.67, the isoelectric point is 12.02, and the amino acid sequence is shown as SEQ ID: 1: alanine-arginine-tryptophan-arginine-phenylalanine-lysine-tryptophan-alanine-lysine (Ala-Arg-Trp-Arg-Phe-Lys-Trp-Ala-Lys-Lys). The small molecular polypeptide AKK10 is artificially synthesized, has small molecular weight, convenient artificial synthesis and obvious bacteriostatic effect, and can be applied to the preparation of anti-infective medicaments.

Description

Small molecular polypeptide AKK10 and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of a small molecular polypeptide AKK10 in preparation of an anti-infective medicament and research on patent medicament properties.

Background

The antibacterial peptide is a micromolecular polypeptide with biological activity generated by induction in a living body, has the molecular weight of about 1000-7000 and consists of 10-60 amino acid residues. The antibacterial peptide is a high-efficiency and spectral antibacterial peptide molecule which is rapidly generated by an organism after the organism is invaded by microorganisms and participates in the immune reaction of the organism. Antimicrobial peptides are widely available as defense molecules for the body, and thousands of antimicrobial peptides have been identified in microorganisms, plants, insects, arthropods, amphibians, mammals, and even humans. The antibacterial mechanism of the antibacterial peptide is complex, but most theories believe that the mechanism involves the action of the cationic property and the hydrophobic property of the antibacterial peptide and the negative charge of microbial cell membranes, and after the antibacterial peptide is contacted with the bacterial cell membranes, the antibacterial peptide causes the membrane permeability to change or transmembrane pores are formed on the bacterial cell membranes, and finally the bacterial contents leak out and die. Therefore, the antibacterial peptide is far higher in the speed of killing bacteria than the traditional antibiotics, and unlike the antibiotics which inhibit the growth of bacteria at low concentration, the effect of the antibacterial peptide on the bacteria is almost lethal. The research shows that: compared with the traditional antibiotics, the antibacterial peptide is not easy to induce the generation of drug-resistant strains; has antibacterial spectrum and is effective on bacteria, fungi, viruses, protozoa and cancer cells. 3 families of antimicrobial peptides have been found in poultry, including cathelicidin, live-expressed antimicrobial peptide (LEAP), and β -defensin. The antibacterial peptides have a crucial effect on poultry resisting bacterial and viral diseases, and mutation or deletion of related genes has a remarkable influence on the capability of poultry resisting microbial infection; the antibacterial peptides have spectrum antibacterial activity, and also have high-efficiency antifungal, antiviral, antiprotozoal and/or antitumor activity, such as Bat5 and IMc7 can kill leptospira, and have good killing effect on Candida albicans, cryptococcus, cytovirus and parasite; some antibacterial peptides have obvious killing power on herpesvirus, influenza virus and HIV enveloped virus; in addition, some antibacterial peptides also have various other regulation functions, for example, Cathelicidin also has the functions of promoting wound healing, repairing tissue injury, chemochemotaxis, promoting angiogenesis, resisting parasites and the like, and has important biological activity on regulating the immunity of an animal body.

Disclosure of Invention

The invention aims to provide a small molecule polypeptide AKK10, which comprises 10 amino acid residues, has the molecular weight of 1376.67 and the isoelectric point of 12.02, and has the amino acid sequence of alanine-arginine-tryptophan-arginine-phenylalanine-lysine-tryptophan-alanine-lysine (Ala-Arg-Trp-Arg-Phe-Lys-Trp-Ala-Lys-Lys).

The invention also aims to provide application of the small molecule polypeptide AKK10 in preparing anti-infective medicaments.

The small molecular polypeptide AKK10 of the invention consists of 10 amino acid residues, has a molecular weight of 1376.67Da and an isoelectric point of 12.02, and has an amino acid sequence shown as SEQ ID: 1:

Ala-Arg-Trp-Arg-Phe-Lys-Trp-Ala-Lys-Lys

1 5 10

the small molecular polypeptide AKK10 is applied to the preparation of medicines for resisting bacterial or fungal infection.

The small molecule polypeptide AKK10 of the present invention includes AKK10 with A as alanine, KK as the last two lysines and 10 as amino acids.

The micromolecule polypeptide AKK10 and the application thereof have the beneficial effects that: the polypeptide AKK10 is artificially synthesized, and has the advantages of small molecular weight, convenient artificial synthesis and obvious antibacterial effect; the small molecular polypeptide AKK10 can be applied to the preparation of anti-infective drugs.

Drawings

Fig. 1 is a schematic diagram of the bacteriostatic activity of a small molecule polypeptide AKK 10.

FIG. 2 is a schematic diagram of the hemolytic activity of the small molecule polypeptide AKK 10.

FIG. 3 is a diagram showing the serum stability of the small molecule polypeptide AKK 10.

Detailed Description

The invention will be further described with reference to the drawings, to which the scope of the invention is not limited.

Example 1

The antibacterial activity of the micromolecular polypeptide AKK10 is detected by a bacteriostatic circle method:

the antibacterial activity is detected by a bacteriostatic loop method, firstly, microorganisms of candida albicans and bacillus subtilis which are respectively prepared and preserved by activated glycerol are taken, 10 mu l of glycerol containing bacterial liquid is inoculated into a sterilized LB liquid culture medium and cultured overnight in a constant temperature incubator at 37 ℃;

inoculating activated bacteria on an LB solid culture medium, inverting at the constant temperature of 37 ℃ for overnight culture, picking out a single colony to be uniformly coated on the surface of the LB solid culture medium after the colony grows, placing a sterilized circular filter paper sheet on the surface of the culture medium, and dripping 10 mu l of a sample to be detected in the center of the filter paper sheet;

when the sample is completely absorbed, carrying out inversion culture at the constant temperature of 37 ℃ for 18-20h, observing whether a bacteriostatic zone is formed, wherein the formation of the bacteriostatic zone indicates that the sample has bacteriostatic activity, and the strength of the bacteriostatic activity can be qualitatively measured by the size of the bacteriostatic zone;

the experimental results show that: AKK10 showed inhibitory activity against Bacillus subtilis and Candida albicans, Staphylococcus aureus, as observed by formation of zone of inhibition in Bacillus subtilis and Staphylococcus aureus, and water and ampicillin as control group (FIG. 1).

Example 2

And (3) detecting the minimum inhibitory concentration of the small molecular polypeptide AKK 10:

the Minimum Inhibitory Concentration (MIC) is the lowest sample concentration at which no bacterial growth can be detected;

selecting single colony, inoculating into LB liquid culture medium, shake culturing at 37 deg.C to logarithmic phase, detecting light absorption (OD600) of bacterial liquid at 600nm with ultraviolet spectrophotometer, and diluting to 2 × 10 with liquid LB culture medium⁵CFU/ml(1OD600＝1×10⁹CFU/ml)；

Adding 100 mul LB liquid culture medium into a sterile 96-well plate in advance, diluting an antibacterial peptide sample to 200 mul/ml concentration, filtering and sterilizing by using a 0.22 mu m microporous filter, then adding 100 mul samples into a first hole, uniformly mixing, adding 100 mul samples into a second hole, sequentially diluting in multiple times to a 12 th hole, uniformly mixing, discarding 100 mul, then respectively adding 100 mul diluted bacterial liquid into each hole, slowly shaking and culturing for 16h at 37 ℃ after uniform mixing, and detecting the light absorption value (OD600) of the bacterial liquid under 600nm by using an ultraviolet spectrophotometer;

the results show that: the micromolecular polypeptide AKK-10 has obvious antibacterial activity on bacillus subtilis, candida albicans and staphylococcus aureus standard strains, the MIC values are 75 mug/ml and 75 mug/ml respectively (table 1), and the negative control result is not given:

TABLE 1

Bacterial strain	AKK10(μg/ml)	Ampicillin (mu g/ml)
			Bacillus subtilis	75	1.46
Staphylococcus aureus (Staphylococcus aureus)	＞100	1.46
			Candida albicans ATCC10231	75	1.46
Candida albicans 08022710	＞100	-
			Candida albicans 08030401	＞100	-

Example 3

Detection of hemolytic activity of the small molecule polypeptide AKK 10:

firstly, preparing sterile donkey-hide gelatin solution 8.0g of sodium citrate, 0.55g of citric acid, 20.5g of glucose and 4.2g of sodium chloride, dissolving in 1L of deionized water, adjusting the pH value to 6.1, sterilizing at 115 ℃ under high pressure, and storing at 4 ℃;

mixing collected human blood and Ashi solution at volume ratio of 1:1, centrifuging at 1000rpm for 5min, discarding supernatant, washing erythrocyte in precipitate with sterilized normal saline for 3 times, and diluting washed erythrocyte with normal saline to 10 concentration⁷-10⁸The suspension of (a);

dissolving the sample with normal saline to the concentration of 80 mug/ml, 40 mug/ml, 20 mug/ml, 10 mug/ml and 5 mug/ml, mixing the diluted erythrocyte suspension and the sample according to the volume ratio of 1:1, keeping the temperature at 37 ℃ for 30min, centrifuging at 1000rpm for 5min after the temperature is over, and measuring the OD value of the supernatant at 540 nm;

physiological saline is used as a negative control of the experiment, 1% Triton X-100 is used as a positive control, and the hemolytic activity of the positive control is set to be 100% in the system;

the calculation formula is as follows: (AA-AB)/(AC-AB) × 100, where I%: sample hemolytic activity, AA: sample set at 540nm light absorption, AB: negative set 540nm light absorption, AC: 540nm light absorption value of the positive control group;

the results of the hemolytic activity assay (fig. 2) show that: the hemolytic activity of the AKK10 sample was only 2.15% when the sample concentration reached 80. mu.g/ml (above its MIC value), and the sample was considered to have substantially no hemolytic activity.

Example 4

And (3) performing bactericidal kinetics detection on the small molecular polypeptide AKK 10:

inoculating Candida albicans on a conventional Sabouraud's solid culture medium, carrying out inversion constant-temperature culture at 37 ℃ until colonies grow out, picking a single colony, transferring the single colony to a Sabouraud's liquid culture medium, and carrying out shake culture at 37 ℃ to a logarithmic phase;

the broth was diluted to 1X 10 with fresh conventional Sabouraud's broth⁶CFU/ml, adding the sample into diluted bacterial liquid to make the final concentration 2 x, ampicillin as positive control of 4 xMIC and normal saline as negative control;

placing the treated bacterial liquid into a shaking incubator at 37 ℃, shaking and culturing at 150rpm, diluting 10 mu l of the bacterial liquid with sterilized normal saline by 1 × 102 times for 0min, 0.1min, 1min, 10min, 30min, 1h, 3h and 6h respectively, and taking 50 mu l of a coated Husky plate;

then placing the Sha's plate into an incubator at 37 ℃ for inverted culture for 16h, counting the number of colonies, describing the result, and giving a conclusion that the unit of the count in the table is shown in table 2;

TABLE 2

Time of day	Physiological saline	2*MIC	4*MIC	Ampicillin
					0	50	48	53	52
0.1	52	45	42	49
					1	60	39	39	40
10	81	27	14	7
					30	101	22	6	3
60	152	10	0	22
					180	335	306	0	42
360	＞1000	943	0	189

From the results table 2, it can be seen that: the sterilization speed of the polypeptide AKK10 is general, and the polypeptide AKK10 can show obvious bacteriostasis within 60min at the concentration of 2 XMIC, while the effect on bacteria is lethal at the concentration of 4 XMIC, and the number of bacteria can not be continuously increased within 3 h. In contrast, ampicillin only had a temporary bacteriostatic effect on candida albicans at a concentration of 1 × MIC, and a large amount of candida albicans still recovered at 3 h.

Example 5

Serum stability experiment of small molecule polypeptide AKK 10:

preparation of serum: extracting 5ml of human blood by using an aseptic syringe, subpackaging by using an aseptic centrifuge tube, standing at room temperature for 1h, standing overnight at 4 ℃, centrifuging the blood which is placed overnight at 1000rpm for 10min the next day, wherein the supernatant light yellow liquid is serum, slightly sucking the serum by using a pipette gun, subpackaging into the aseptic centrifuge tube, and storing at-20 ℃ in a refrigerator for later use;

extracting 5ml of human blood, standing at room temperature for 2h, then standing in a refrigerator at the temperature of 4 ℃ overnight, centrifuging at 2000rpm for 10min to obtain yellowish supernatant as human serum, enabling the final concentration of AKK-10 to be 10mg/ml and the serum concentration to be 90%, placing the mixture in an incubator at the temperature of 37 ℃ for incubation, respectively taking 10 mu l of the mixture in the time periods of 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24h, and detecting the antibacterial activity on Candida albicans by using a bacteriostatic circle method;

AKK-10 was tested for stability after incubation with serum for various periods of time and the results (FIG. 3) showed that: AKK-10 (10. mu.g/ml) and serum (90%) still have antibacterial activity after 6h incubation at 37 ℃ and no antibacterial activity is obvious after 12 h.

Example 6

Influence of salt ions on the small molecule polypeptide AKK 10:

small molecule polypeptide AKK10 was dissolved into 200. mu.g/ml sample solutions using sterilized ultrapure water, 150mM NaCl solution and 150mM Phosphate Buffer Solution (PBS) buffer (0.2g KCl, 0.2g KH2PO4, 8.0g NaCl, 2.88g Na2HPO4.12H2O, 1L H2O), respectively, and then the antibacterial activities of the three sample solutions were examined by the MIC method as shown in Table 3;

TABLE 3

The results (Table 3) show that AKK10 retains its Candida albicans stabilizing antibacterial effect in both ultrapure water and 150mM Phosphate Buffer (PBS) buffer, while it is stronger in 150mM NaCl solution.

Sequence listing

<110> institute of physical and chemical technology in Xinjiang, China academy of sciences

<120> small molecular polypeptide AKK10 and application thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Small molecule polypeptide (AKK10 Artificial sequence)

<400> 1

Ala Arg Trp Arg Phe Lys Trp Ala Lys Lys

1 5 10

Claims

1. A small molecular polypeptide AKK10 is characterized by consisting of 10 amino acid residues, having a molecular weight of 1376.67Da and an isoelectric point of 12.02, and having an amino acid sequence shown as SEQ ID: 1:

Ala-Arg-Trp- Arg- Phe- Lys- Trp- Ala- Lys- Lys。

2. the use of the small molecule polypeptide AKK10 of claim 1 in the preparation of a medicament for resisting bacterial or fungal infection, wherein the bacteria is Bacillus subtilis or Staphylococcus aureus; the fungus is Candida albicans.