CN111533788B

CN111533788B - Cell-penetrating antibacterial peptide targeting streptococcus agalactiae and preparation method and application thereof

Info

Publication number: CN111533788B
Application number: CN202010234605.2A
Authority: CN
Inventors: 单安山; 李家维
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-02-08
Anticipated expiration: 2040-03-30
Also published as: CN111533788A

Abstract

The invention provides a cell penetrating antibacterial peptide targeting streptococcus agalactiae, a preparation method and application thereof. The sequence of the antibacterial peptide L10 is shown in SEQ ID No. 1. The invention takes short-chain hydrophobic peptide in a streptococcus agalactiae group communication system as a targeting region of a targeting antibacterial peptide, takes a human extracellular superoxide dismutase 213-220 fragment rich in positive charges as a basic fragment, forms a cell penetrating structural domain after modification, and connects the two fragments to construct a novel antibacterial peptide L10 with the capabilities of targeting streptococcus agalactiae and penetrating cells, which shows better targeting effect and has high antibacterial activity on streptococcus agalactiae under the condition of less influence on other bacteria. The antibacterial peptide L10 has low cytotoxicity, can kill intracellular streptococcus agalactiae at the same time, and improves the development potential of replacing antibiotics.

Description

Cell-penetrating antibacterial peptide targeting streptococcus agalactiae and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cell penetrating antibacterial peptide targeting streptococcus agalactiae, and a preparation method and application thereof.

Background

As the abuse of antibiotics increases, the problem of resistance is becoming increasingly appreciated. Antibacterial peptides have also received attention from researchers as the most potential antibiotic alternatives. It is widely believed that the action mechanism of the antibacterial peptide is to utilize the positive charge residue to generate electrostatic interaction with the bacterial cell membrane, and then insert the hydrophobic new amino acid into the membrane to destroy the integrity of the membrane, and the content leaks out to kill the bacteria. It is due to this unique mechanism of action that bacteria are very difficult to develop resistance to antimicrobial peptides. However, the mechanism of action of membrane rupture also makes the antibacterial peptide have antibacterial action on pathogenic microorganisms or probiotics, and the balance of a microflora is damaged. Currently, over 7000 thousands of 16s RNAs are identified in humans, and microbial communities are closely related to the vital activities of the host. In addition, conventional antibiotics are unable to penetrate the cell membrane of mammalian cells, effectively killing pathogenic microorganisms that have invaded the cells. Therefore, the design of the antibacterial peptide with the targeting antibacterial ability and the cell penetrating function has very important significance.

Streptococcus agalactiae is a pathogenic bacterium that can survive inside cells and is infectious for both livestock and humans. Short chain hydrophobic peptides play an important role in the quorum sensing system of streptococcus agalactiae. The precursor peptide is transported to the outside of the cell through a receptor on the membrane, and is transported to the inside of the cell through oligopeptide transport protein on the membrane after enzyme digestion, and the precursor peptide is combined with a specific receptor in the cell to play a role in regulating the activity of a population. However, the short-chain hydrophobic peptide has high hydrophobicity, so that the short-chain hydrophobic peptide is difficult to be directly connected with the antibacterial peptide to play a role.

Disclosure of Invention

Based on the defects, the invention provides the cell penetrating antibacterial peptide targeting streptococcus agalactiae, the preparation method and the application thereof, has a targeted killing effect on streptococcus agalactiae, and has no influence on other microorganisms.

The technical scheme adopted by the invention is as follows: a cell penetrating antibacterial peptide L10 targeting Streptococcus agalactiae has a sequence shown in SEQ ID No. 1.

The invention also has the following technical characteristics:

1. the preparation method of the cell penetrating antibacterial peptide L10 targeting streptococcus agalactiae is characterized by comprising the following steps: selecting short-chain hydrophobic peptide of streptococcus agalactiae as a targeting region, modifying the 213-220 fragment of human extracellular superoxide dismutase rich in positive charges to form a cell penetrating domain, and connecting the cell penetrating domain and the cell penetrating domain to obtain the antibacterial peptide L10, wherein the sequence of the antibacterial peptide L10 is shown as SEQ ID No. 1.

2. The application of the cell penetrating antibacterial peptide L10 targeting streptococcus agalactiae in preparing the medicines for treating the streptococcus agalactiae infectious diseases is described.

The antibacterial peptide L10 obtained by the invention has a targeted killing effect on streptococcus agalactiae and has no influence on other microorganisms. Meanwhile, the antibacterial peptide L10 has low cytotoxicity, can kill intracellular streptococcus agalactiae, and proves that the antibacterial peptide L10 has the potential of becoming a targeted penetration resistant medicament for resisting streptococcus agalactiae. The cell-penetrating peptide can be transported into cells by an endocytosis mechanism and has a carrier transport function. The antibacterial drug is connected with the cell penetrating peptide, so that the drug can enter the cell to kill the intracellular bacteria, and the antibacterial peptide has a great application prospect.

Drawings

FIG. 1 is a cytotoxicity plot of L10;

FIG. 2 is an antibacterial potency profile of L10 against Streptococcus agalactiae in mouse macrophage cells.

Detailed Description

The invention is further illustrated by the following examples:

example 1

A preparation method of cell penetrating antibacterial peptide L10 targeting Streptococcus agalactiae comprises the following steps:

(1) selecting short-chain hydrophobic peptide of streptococcus agalactiae as a targeting region, modifying the 213-220 fragment of the human extracellular superoxide dismutase rich in positive charges to form a cell penetrating structural domain, and connecting the cell penetrating structural domain and the cell penetrating structural domain to obtain L10;

(2) obtaining peptide resin by a peptide synthesizer by adopting a solid-phase chemical synthesis method, and cutting the obtained peptide resin by TFA to obtain polypeptide;

(3) and (3) after reversed-phase high performance liquid chromatography purification and mass spectrum identification, the preparation of the antibacterial peptide L10 is completed, and the sequence of the antibacterial peptide L10 is shown as SEQ ID No. 1. The sequence and physicochemical parameters of antimicrobial peptide L10 are shown in table 1.

Sequence and physicochemical parameters of L10 in Table 1

Example 2

1. Bacteriostatic activity

Peptides were prepared as 2.56mM stock solutions for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined using the broth dilution method. Serial gradients of antimicrobial peptide solutions were prepared sequentially using a two-fold dilution method with 0.01% acetic acid (containing 0.2% BSA) as the diluent. Taking 100 mu L of the solution, placing the solution into a 96-hole cell culture plate, and then respectively adding the bacterial liquid to be detected (10-10) with the same volume⁵one/mL) in each well. 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, and determining the minimum inhibitory concentration when no turbidity is observed at the bottom of the well. The results are shown in tables 2 and 3.

TABLE 2 antibacterial Activity of L10 against gram-positive bacteria

TABLE 3 antibacterial Activity of L10 against gram-negative bacteria

2. Cytotoxicity

The cytotoxicity of the antibacterial peptide can be detected by a thiazole blue (MTT) colorimetric method, and the main method is as follows: adding 1.0-2.0X 10 per well in 96-well plate⁴The cells were then treated with antimicrobial peptides at concentrations varying from 1-128. mu.M. The cells were incubated at 37 ℃ for 18-24h, then incubated with MTT (50. mu.l, 0.5mg/ml) for 4h, and then centrifuged at 1,000 Xg for 5 min. The supernatant was discarded. Subsequently, 150. mu.l of dimethyl sulfoxide was used to dissolve formazan at the bottom of the well, and cytotoxicity was detected by an absorbance at 492nm using a microplate reader, as shown in FIG. 1, which showed that 80% and 9% of cells were maintained after L10 treatment at 64. mu.M, 32. mu.M, 16. mu.M, 8. mu.M, 4. mu.M and 2. mu.MCell viability of 9%, 107%, 114%, 116% and 123% indicated that L10 has good biocompatibility.

3. Intracellular bacteriostatic activity

The antibacterial ability of L10 is tested by the number of viable and dead bacteria in cells, and the main method is as follows: adding 1.0-2.0 × 10 of the mixture into each hole of a 24-hole plate⁵And (4) cells. After 24 hours of incubation, 2X 10 cells were added to the wells⁶CFU of Streptococcus agalactiae, 1 hour of infection. 100mg/mL gentamicin and 5. mu.g/mL penicillin were added to the wells and incubated for 1 hour to kill extracellular bacteria. After washing the antibiotics clean with PBS, different concentrations of L10 were added for 2, 6 and 8 hours of incubation. After the remaining L10 was washed with PBS, the cell membrane was disrupted with 0.025% Triton-100, and the number of intracellular bacteria was counted by plate dilution, as shown in fig. 2, it was revealed that the survival rates of intracellular streptococcus agalactiae were 4.7%, 0.8% and 0.3%, respectively, after L10-treated mouse macrophages that had been infected with streptococcus agalactiae for 4 hours, 6 hours and 8 hours, indicating that L10 has a good intracellular antibacterial ability.

Sequence listing

<110> northeast university of agriculture

<120> cell-penetrating antibacterial peptide targeting streptococcus agalactiae, and preparation method and application thereof

<140> 2020102346052

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Ile Leu Ile Ile Val Gly Gly Lys Arg Arg Arg-NH2

1 5 10

Claims

1. A cell-penetrating antimicrobial peptide L10 targeting streptococcus agalactiae, having the sequence shown in SEQ ID No. 1.

2. The method for preparing the cell penetrating antibacterial peptide L10 targeting Streptococcus agalactiae according to claim 1, wherein: selecting short-chain hydrophobic peptide of streptococcus agalactiae as a targeting region, modifying the 213-220 fragment of human extracellular superoxide dismutase rich in positive charges to form a cell penetrating domain, and connecting the cell penetrating domain and the cell penetrating domain to obtain the antibacterial peptide L10, wherein the sequence of the antibacterial peptide L10 is shown as SEQ ID No. 1.

3. The use of the cell-penetrating antimicrobial peptide L10 targeting Streptococcus agalactiae according to claim 1 for the preparation of a medicament for the treatment of an infectious disease caused by Streptococcus agalactiae.