CN106432513A - Efficient hybrid antibacterial peptide LI and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a high-efficiency hybrid antibacterial peptide LI and its preparation method and application. Its sequence is shown in the sequence table SEQ ID No.1; its preparation method includes the following steps: according to the fixed-point amino acid fragment interception method, intercepting human antibacterial peptide LL37 The 14-21st amino acid residue fragment and the 5-13th amino acid residue fragment of the bovine antimicrobial peptide Indolicidin were connected in sequence to obtain the LI antibacterial peptide; 2) The peptide resin was synthesized by solid-phase synthesis using a peptide synthesizer ; The peptide was obtained after TFA cleavage, and purified by reversed-phase high performance liquid chromatography. The antimicrobial peptide LI obtained in the present invention has strong antibacterial activity and weak hemolytic activity, high therapeutic index and great development potential.

Description

A kind of efficient hybrid antimicrobial peptide LI and its preparation method and application

technical field

The invention relates to a high-efficiency hybrid antibacterial peptide LI and its preparation method and application.

Background technique

Antimicrobial peptides are an animal's first line of defense against exogenous pathogens. It not only has the function of killing pathogenic bacteria, but also has biological functions such as anti-fungal, virus, parasite and anti-cancer cells. Antimicrobial peptides have a unique bactericidal mechanism, making them the most promising alternatives to antibiotics. Antimicrobial peptide molecules have positively charged amino acids, such as arginine, lysine, and histidine, and hydrophobic amino acids with hydrophobic residues, such as leucine, isoleucine, valine, tryptophan, Phenylalanine, alanine, etc. Antibacterial peptides with electrostatic positive charges and negatively charged bacterial cell membranes attract each other electrostatically, antibacterial peptides are close to bacterial cells, and hydrophobic residues penetrate into the lipid bilayer of bacterial cells, making bacterial cell membranes form micropores or pores structure, resulting in the outflow of intracellular material, resulting in bacterial death. At present, there are two reasons that hinder the application of antimicrobial peptides in medicine, cosmetics, food and animal husbandry and other industries: 1. The toxicity of antimicrobial peptides. Natural antimicrobial peptides have high cytotoxicity. It is imperative to find a design method to improve the cell selectivity of antimicrobial peptides. The cell selectivity of antimicrobial peptides means that antimicrobial peptides have the function of killing pathogenic bacteria but have no toxic and side effects on normal animal cells, showing selective killing effect on cells. The therapeutic index of antimicrobial peptides is a key index to evaluate their cell selectivity. Second, the production cost of antimicrobial peptides. At present, the preparation of antimicrobial peptides mainly involves two methods: chemical method and genetic engineering. Antimicrobial peptides produced by chemical methods are precisely synthesized, but the output is small and the cost is high. The method of genetic engineering to produce antimicrobial peptides is in the process of testing, and the screening of engineering bacteria and the condition control of gene expression process have always been the main factors restricting the production of antimicrobial peptides by this method. Blindly using genetic engineering methods to express antimicrobial peptides, without comprehensively exploring the structure and function of antimicrobial peptides and in vitro tests, resulting in most of the expressed antimicrobial peptides having low activity or cytotoxicity such as hemolysis.

Antimicrobial peptides have the smallest molecular weight, various types and complex structures. At present, there are many methods to study the relationship between the structure and function of antimicrobial peptides, for example, the modification of natural antimicrobial peptides and the new design of antimicrobial peptides and other research methods.

Contents of the invention

Based on the above shortcomings, the present invention provides a high-efficiency hybrid antibacterial peptide LI and its preparation method and application, which has high antibacterial activity and low cytotoxicity.

The technology adopted in the present invention is as follows: a high-efficiency hybrid antimicrobial peptide LI, the sequence of which is shown in SEQ ID No.1 in the sequence table.

The present invention also has the following technical features:

1, the preparation method of a kind of high-efficiency hybridization antimicrobial peptide LI as described above comprises the steps:

1) According to the fixed-point amino acid fragment interception method, the 14th-21st amino acid residues of the human antimicrobial peptide LL37 and the 5th-13th amino acid residues of the bovine antimicrobial peptide Indolicidin were intercepted, and the above two fragments were sequentially connected to obtain the LI antimicrobial peptide;

2) The peptide resin is obtained by using a solid-phase chemical synthesis method through a peptide synthesizer; after the peptide resin is cut by TFA, the peptide product is initially obtained; and then the antimicrobial peptide is completed after purification by reverse-phase high-performance liquid chromatography and identification by mass spectrometry. preparation.

2. The application of a high-efficiency hybrid antibacterial peptide LI as described above in the preparation of medicaments for treating Gram-positive or Gram-negative bacterial infectious diseases.

The present invention obtains a new antimicrobial peptide LI (GlyLysGluPheLysArgIleValLysTrpProTrpTrpProTrpArgArg-NH ₂ ) by intercepting the 14th-21st amino acid residues (GlyLysGluPheLysArgIleVal) of the natural antimicrobial peptide LL37 and the characteristic amino acid residue fragment (LysTrpProTrpTrpProTrpArgArg) of Indolicidin (In13), and hybridizing them. Antimicrobial peptide LL37 is an α-helical antimicrobial peptide derived from human hCAP18 protein, which has broad-spectrum antibacterial, fungal, and viral biological activities. Indolicidin is a small peptide of 13 amino acids rich in tryptophan and proline derived from bovine neutrophils. Tryptophan is a hydrophobic amino acid, which can interact with phospholipids expressed by bacteria to destroy the phospholipid membrane of bacteria, thereby producing biological activity. Both have certain hemolytic cytotoxicity. At home and abroad, there is no report on the study of the relationship between the structure and function of antimicrobial peptides by using the hybridization of the two characteristic fragments. The experimental technique of the antimicrobial peptide prepared by this method is simple, and the obtained antibacterial peptide is tested for antibacterial and hemolytic activity. The antibacterial activity is high and the cytotoxicity is low. It is found that LI is not only effective against E. , Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis, and eight strains have obvious inhibitory effects, and have very low hemolytic activity. Therefore, on the whole, LI is an antimicrobial peptide with high application value.

Description of drawings

Fig. 1 is the three-dimensional prediction diagram of the designed antimicrobial peptide;

Figure 2 is the mass spectrum of the designed antimicrobial peptide.

detailed description

Below according to the accompanying drawings of description, the present invention will be further described by way of example:

Example 1

The amino acid sequence of human antimicrobial peptide LL37 is:

The amino acid sequence of bovine antimicrobial peptide In13 is:

Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg-NH2

1 5 10 13

According to the site-specific amino acid fragment interception method, the amino acid residues 14-21 of human antimicrobial peptide LL37 (GlyLysGluPheLysArgIleVal) and the fragments of amino acid residues 5-13 of bovine antimicrobial peptide In13 (LysTrpProTrpTrpProTrpArgArg) were intercepted.

The above two fragments were sequentially connected to obtain LI (GlyLysGluPheLysArgIleValLysTrpProTrpTrpProTrpArgArg-NH ₂ ) antibacterial peptide.

Example 2

The above two antimicrobial peptides were synthesized using a peptide synthesizer. The method was solid-phase chemical synthesis, and the specific steps were:

1) The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminal of each antimicrobial peptide) is inserted into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9 -Fmoxy-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus until the synthesis is completed, and the side of the Fmoc group is removed chain protection resin;

Add cutting reagent to the peptide resin obtained above, react at 20°C for 2 hours in the dark, filter; precipitate TFA (trifluoroacetic acid) for washing, mix the washing liquid with the above filtrate, concentrate with a rotary evaporator, and then add about 10 times volume of pre-cooled anhydrous ether, precipitated at -20°C for 3h, and precipitated a white powder, centrifuged at 2500g for 10min, collected the precipitate, washed the precipitate with anhydrous ether, and dried in vacuum to obtain the polypeptide, in which the cleavage reagent consisted of TFA, water and TIS (Triisopropylchlorosilane) mixed according to the mass ratio of 95:2.5:2.5;

Use 0.2mol/L sodium sulfate (adjusted to pH7.5 with phosphoric acid) to equilibrate the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and use gradient elution (eluent is methanol and sodium sulfate) on a C18 reversed-phase normal pressure column The aqueous solution is mixed according to the volume ratio of 30:70 to 70:30), the flow rate is 1ml/min, the detection wave is 220nm, the main peak is collected, and freeze-dried; then further purified by reverse-phase C18 column, eluent A is 0.1% TFA/ Aqueous solution; eluent B is 0.1% TFA/acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12min, the flow rate is 1ml/min, and then the main peak is collected as above, and freeze-dried;

Identification of the antimicrobial peptide: the antimicrobial peptide obtained above was analyzed by electrospray mass spectrometry, and the molecular weight (see accompanying drawing) shown in the mass spectrogram was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide was greater than 95%.

Table 1 Design of antimicrobial peptides

Example 3

The designed and synthesized antimicrobial peptides were compared and detected by in vitro antibacterial and hemolytic activity tests;

Determination of antibacterial activity: Prepare the peptide as a stock solution for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. 100 μl of the above solution was taken and placed in a 96-well cell culture plate, and then an equal volume of the bacteria solution to be tested (~10 ⁵ cells/ml) was added to each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with the naked eye.

The test results are shown in Table 2. As can be seen from Table 2, LI shows strong antibacterial activity for Gram-negative and positive bacteria, and its antibacterial activity is greater than that of LL37 and In13. Bacteriostatic activity of antimicrobial peptides.

Bacteriostatic and hemolytic activity of table 2 antimicrobial peptides

Determination of hemolytic activity: Collect 1mL of fresh human blood, dissolve it in 2ml of PBS solution after anticoagulation with heparin, centrifuge at 1000g for 5min, collect red blood cells; wash with PBS for 3 times, then resuspend in 10ml of PBS; take 50μL of red blood cell suspension and 50μL for use Antimicrobial peptide solutions of different concentrations dissolved in PBS were mixed evenly, and incubated at a constant temperature in a 37°C incubator for 1h; after 1h, they were taken out, and centrifuged at 4°C and 1000g for 5min; the supernatant was taken out and measured at 570nm with a microplate reader; Group averages were taken and compared for analysis. Among them, 50 μL red blood cells plus 50 μl PBS were used as negative control; 50 μL red blood cells plus 50 μl 0.1% Tritonx-100 were used as positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration at which the antimicrobial peptide causes 10% hemolysis rate.

The test results are shown in Table 2. The greater the hemolytic concentration, the smaller the hemolytic activity; as can be seen from Table 2, LI has no hemolytic activity within the detection range, while LL37 and In13 both show certain hemolytic activity.

The above results showed that the antimicrobial peptide LI obtained by intercepting the two characteristic fragments of human antimicrobial peptide LL37 and bovine antimicrobial peptide Indolicidin and sequentially connecting them had higher antibacterial activity and was not toxic to blood cells. By comprehensively analyzing the antibacterial and hemolytic activities of antimicrobial peptides, the cell selectivity of each antimicrobial peptide can be more comprehensively evaluated through the therapeutic index (ratio of hemolytic concentration to antibacterial concentration). It can be seen from Table 2 that LI has a high therapeutic index, indicating that the designed LI antimicrobial peptide has a high development potential to replace antibiotics.

<110> Northeast Agricultural University

<120> A high-efficiency hybrid antimicrobial peptide LI and its preparation method and application

<160> 1

<210> 1

<211> 17

<212> PRT

<213> Artificial sequence

<400> 1

Gly Lys Glu Phe Lys Arg Ile Val Lys Trp

1 5 10

Pro Trp Trp Pro Trp Arg Arg-NH2

11 15 17

Claims (3)

1. a kind of antibacterial peptide LI that efficiently hybridizes is it is characterised in that its sequence is as shown in sequence table SEQ ID No.1.

2. according to claim require described in 1 a kind of efficiently hybridize antibacterial peptide LI it is characterised in that preparation method include as follows Step：

1) according to pinpoint amino acid fragment intercept method, people's derived antimicrobial peptide LL37 14-21 amino acids residue and Niu Yuan are intercepted Antibacterial peptide Indolicidin 5-13 amino acid residue segment, above-mentioned two fragment is sequentially connected, and obtains LI antibacterial peptide；

2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method；By the peptide resin obtaining after TFA cutting, Tentatively obtain polypeptide products；After being then passed through reversed-phase high-performance liquid chromatography purification and Mass Spectrometric Identification, that is, complete the system of this antibacterial peptide Standby.

3. a kind of efficiently hybridization antibacterial peptide LI according to claim 1 is it is characterised in that treat Gram-positive in preparation Application in bacterium or gram positive bacterial infection disease medicament.