CN113717247A

CN113717247A - Antibacterial polypeptide and application thereof

Info

Publication number: CN113717247A
Application number: CN202111000093.4A
Authority: CN
Inventors: 杨文龙; 冷宁; 王艳; 娄晶莹; 辜茂艳; 苏宏健; 余荣熹
Original assignee: Harbin Jixianglong Biological Technology Co ltd
Current assignee: Harbin Jixianglong Biological Technology Co ltd
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-11-30
Anticipated expiration: 2041-08-31
Also published as: CN113717247B

Abstract

The invention discloses an antibacterial polypeptide and application thereof, and belongs to the technical field of polypeptides. To provide an antibacterial polypeptide which has strong bactericidal activity, is easy to absorb and has no toxicity. The invention mutates at least one amino acid in the 1 st, 2 nd and 6 th positions of antifungal polypeptide to obtain 3 kinds of polypeptide with powerful bactericidal capacity, easy absorption and no toxicity. The invention provides an antibacterial polypeptide with the size of 1315Da, and experiments prove that the antibacterial polypeptide has strong sterilization capability, quick response, long antibacterial time, easy absorption and no toxicity in application. The antibacterial polypeptide disclosed by the invention can be applied to mouthwash.

Description

Antibacterial polypeptide and application thereof

Technical Field

The invention belongs to the technical field of polypeptides, and particularly relates to an antibacterial polypeptide and application thereof.

Background

The resistance to infectious diseases caused by bacterial micro-infection can be traced back to ancient civilizations. With the discovery of penicillin in 1924 by Alexander Fleming, a milestone was achieved in the treatment of bacterial infections. This forms the basis of modern antibiotic drugs. However, with repeated antibiotic treatments, bacteria can protect themselves from certain types of antibiotics due to their genetic mutations, thereby enabling them to grow at higher antibiotic concentrations. The bacterial population will adapt and eventually become resistant and tolerant to the drug. Resistance mechanisms include direct inactivation of the drug, alteration of drug targets to reduce binding affinity, reduction of uptake or increase efflux, redundant pathways bypassing the affected drug target, and the like.

In recent years, diseases caused by drug-resistant bacteria infection have become important and difficult points of clinical treatment due to antibiotic abuse. The antibacterial peptide is a small molecular polypeptide widely existing in organisms, and has the characteristics of wide antibacterial spectrum, high antibacterial efficiency, difficulty in generating drug resistance and the like. It can kill bacteria, has low toxicity to normal cells, and has high biological safety.

The current antibacterial peptide technology is characterized in that the polypeptide result contains a plurality of cations, the side chain property is mostly hydrophobic groups, and the polypeptide shows amphipathy. The cationic antibacterial peptide acts on the cell membrane of bacteria mainly through an amphiphilic structure, a transmembrane ion channel is formed by combining a negatively charged hydrophobic region and cell membrane lipid, and a positively charged hydrophilic region is combined with water or a negatively charged residue to perforate the cell membrane and destroy the integrity of the cell membrane. The antibacterial peptide causes flocculation reaction of intracellular substances after entering cells, inhibits cell wall generation, is combined with nucleic acid substances, inhibits nucleic acid or protein synthesis, inhibits enzyme activity, changes cell membranes and inhibits septum formation, and the mechanism of killing the cells is reported for many times, so that the aim of sterilization is fulfilled. Such as bufotalin (Magainin), Melittin (Melittin), etc., which have been reported in many publications. However, the antibacterial peptides are all found in animals, and the antibacterial peptides are found to be highly toxic to human bodies and have strong rejection to other species.

Disclosure of Invention

The invention aims to provide an antibacterial polypeptide with strong bactericidal ability, easy absorption and no toxicity, and solves the problem of how to specifically sterilize antibacterial peptide.

The invention provides an antibacterial polypeptide, which is obtained by mutating at least one amino acid in the 1 st, 2 nd and 6 th positions by taking an antifungal polypeptide with an amino acid sequence shown as SEQ ID NO.1 as an initial sequence.

Further defined, the antibacterial polypeptide is any one of the following (a) to (c):

(a) mutating the 6 th amino acid in the amino acid sequence shown in SEQ ID NO.1 into ornithine;

(b) mutating the 6 th amino acid in the amino acid sequence shown in SEQ ID NO.1 into histidine;

(c) the 1 st and 2 nd amino acids in the amino acid sequence shown in SEQ ID NO.1 are mutated into arginine, and the 6 th amino acid is mutated into histidine.

Further defined, the amino acid in the amino acid sequence of said antibacterial polypeptide is in the L-form or the D-form.

Further defined, the antibacterial polypeptide has an amino or hydroxyl fused to the C-terminus.

Further defined, the antibacterial polypeptide is chemically modified at the C-terminus, N-terminus, or amino acid side chain.

Further defined, the chemical modification is acetylation, formylation, fatty acid modification, trifluoroacetylation, benzoylation, 2-aminobenzoylation, chloroacetylation, bromoacetylation, DOTA modification, NOTA modification, TATE modification, amidation, esterification, AMC modification, AFC modification, pNA modification, CMK modification, FMK modification, hydroformylation, alcohol functionalization, AOMK modification, or PEG modification.

The invention also provides a gene encoding the antibacterial polypeptide.

The invention also provides a recombinant vector which carries the coding gene.

The invention also provides a recombinant microbial cell, wherein the recombinant cell carries the coding gene or the antibacterial polypeptide.

Further defining the microbial cell as a prokaryote or a eukaryote.

The invention also provides the application of the antibacterial polypeptide in preparing products for controlling microbial infection.

Has the advantages that: the invention provides an antibacterial polypeptide with the size of 1315Da, and experiments prove that the antibacterial polypeptide has strong sterilization capability, quick response, long antibacterial time, easy absorption and no toxicity in application.

Drawings

FIG. 1 is a Staphylococcus aureus inhibition curve in which the abscissa is time and the ordinate is colony count;

FIG. 2 is a Streptococcus mutans inhibition curve in which the abscissa is time and the ordinate is colony count;

FIG. 3 is a plot of Escherichia coli inhibition in which the abscissa is time and the ordinate is colony count;

FIG. 4 is a Candida albicans inhibition curve, wherein the abscissa is time and the ordinate is colony count;

FIG. 5 is a graph showing the results of the effect of the antibacterial polypeptide on the safety of human embryonic kidney cells 293T, wherein in the graph A, the inhibition of cells in a three-day period of 5K/well (96-well plate) is observed with the absorbance (survival rate by CCK8 method) as the ordinate and the K04 concentration as the abscissa. FIG. B is a graph in which inhibition of cells was observed within three days at a cell count of 15K/well (96-well plate) on the ordinate of absorbance (survival rate by CCK8 method) and on the abscissa of K04 concentration. In FIG. C, the inhibition of cells at a concentration of K04 of 5K/well (96-well plate) was observed on the ordinate of the absorbance (survival rate by CCK8 method) and on the abscissa of the date. FIG. D is a graph in which the inhibition of cells at a concentration of K04 of 15K/well (96-well plate) was observed on the ordinate of the absorbance (viability measured by CCK8 method) and on the abscissa of the date.

FIG. 6 is a graph showing the effect of the antibacterial polypeptide on the safety of human normal hepatocyte LO2, wherein, in the graph A, the inhibition of the cell in three days under the condition that the cell number is 5K/well (96-well plate) is observed with the absorbance (survival rate measured by CCK 8) as ordinate and the K04 concentration as abscissa. FIG. B is a graph in which inhibition of cells was observed within three days at a cell count of 15K/well (96-well plate) on the ordinate of absorbance (survival rate by CCK8 method) and on the abscissa of K04 concentration. In FIG. C, the inhibition of cells at a concentration of K04 of 5K/well (96-well plate) was observed on the ordinate of the absorbance (survival rate by CCK8 method) and on the abscissa of the date. FIG. D is a graph in which the inhibition of cells at a concentration of K04 of 15K/well (96-well plate) was observed on the ordinate of the absorbance (viability measured by CCK8 method) and on the abscissa of the date.

FIG. 7 is a graph showing the effect of antibacterial polypeptides on the safety of human immortalized epidermal cells HaCaT, wherein in the graph A, the inhibition of cells within three days is observed with the absorbance (survival rate by CCK8 method) as ordinate and the K04 concentration as abscissa, at a cell number of 5K/well (96-well plate). FIG. B is a graph in which inhibition of cells was observed within three days at a cell count of 15K/well (96-well plate) on the ordinate of absorbance (survival rate by CCK8 method) and on the abscissa of K04 concentration. In FIG. C, the inhibition of cells at a concentration of K04 of 5K/well (96-well plate) was observed on the ordinate of the absorbance (survival rate by CCK8 method) and on the abscissa of the date. FIG. D is a graph in which the inhibition of cells at a concentration of K04 of 15K/well (96-well plate) was observed on the ordinate of the absorbance (viability measured by CCK8 method) and on the abscissa of the date.

FIG. 8 is a graph showing the effect of antibacterial polypeptides on the safety of human embryonic lung fibroblast MRC-5 cells, wherein in the graph A, the inhibition of cells in a three-day period of 5K/well (96-well plate) is observed with the absorbance (survival rate by CCK8 method) as the ordinate and the K04 concentration as the abscissa. FIG. B is a graph in which inhibition of cells was observed within three days at a cell count of 15K/well (96-well plate) on the ordinate of absorbance (survival rate by CCK8 method) and on the abscissa of K04 concentration. In FIG. C, the inhibition of cells at a concentration of K04 of 5K/well (96-well plate) was observed on the ordinate of the absorbance (survival rate by CCK8 method) and on the abscissa of the date. FIG. D is a graph in which the inhibition of cells at a concentration of K04 of 15K/well (96-well plate) was observed on the ordinate of the absorbance (viability measured by CCK8 method) and on the abscissa of the date.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The sequence of the peptide chains is: from N-terminal to C-terminal.

Human embryonic kidney cells 293T cells, human normal liver cells LO2, human immortalized epidermal cells HaCaT and human embryonic lung fibroblasts MRC-5 were purchased commercially.

The antifungal polypeptide sequences disclosed are: Lys-Lys-Val-Val-Phe-Lys-Val-Lys-Phe-Lys, (SEQ ID NO.1) KKVVFKVKFK.

Example 1 Synthesis of an antibacterial polypeptide

1. Based on FMOC synthesis method, adopting solid-phase synthesis method, taking amide resin as solid-phase carrier (crosslinking degree is 11%, 0.97mmol/g), FMOC protected special amino acid, HOBT/DIC coupling agent, 10% piperidine/DMF (volume fraction) organic base, feeding amino acid according to 3 times of synthesis scale, and sequentially connecting Lys-Lys-Val-Val-Phe-Orn-Val-Lys-Phe-Lys-NH according to sequence₂。

2. Mixing the following components in percentage by weight of phenol: and (3) taking TA, EDT, water, TIS, trifluoroacetic acid and a shearing agent in a volume ratio of 5:5:5:4:1:80, and stirring and reacting in a dark place. And after the reaction is finished, carrying out suction filtration, precipitating the filtrate, carrying out suction filtration again, and taking the filter cake as the target peptide.

3. The synthesized polypeptide crude product is separated and purified by preparative reverse phase high performance liquid chromatography (50mm inner diameter preparative column) with C18 as filler, 5 μm particle size,

Pore size, acetonitrile for mobile phase A, and 1 ‰ TFA water for mobile phase BGradient elution, from A to B-5%: 95% to A, B-50%: 50%, run time 90 minutes.

4. Collecting the effluent at the peak of the highest peak in a segmented manner, wherein the standard is that the liquid phase purity is more than 90%, combining the qualified collected liquids, distilling under reduced pressure to obtain the final concentration of 5mg/ml, and finally preparing the freeze-dried powder which is the antibacterial peptide compound.

The amino acid sequence is KKVVF-ornithine-VKKFK-NH₂And detecting the molecular weight of the linear peptide by mass spectrum to be 1236 Da. Fusion of an NH at the N-terminus of SEQ ID NO.2₂，KKVVFOrnVKFK(SEQ ID NO.2)。

Example 2A Synthesis of an antibacterial polypeptide

1. Based on FMOC synthesis method, adopting solid-phase synthesis method, taking amide resin as solid-phase carrier (crosslinking degree is 11%, 0.97mmol/g), FMOC protected special amino acid, HOBT/DIC coupling agent, 30% piperidine/DMF (volume fraction) organic base, feeding amino acid according to 5 times of synthesis scale, and sequentially connecting Lys-Lys-Val-Val-Phe-His-Val-Lys-Phe-Lys-NH according to sequence₂。

The pore size is determined by using acetonitrile as a mobile phase A and 1 thousandth TFA water as a mobile phase B, and performing gradient elution on the components by using the ratio of A to B-5%: 95% to A, B-50%: 50%, run time 90 minutes.

4. Collecting the effluent at the peak of the highest peak in a segmented manner, wherein the standard is that the liquid phase purity is more than 90%, combining the qualified collected liquids, distilling under reduced pressure to obtain the final concentration of 30mg/ml, and finally preparing the freeze-dried powder which is the antibacterial peptide compound.

The amino acid sequence is Lys-Lys-Val-Val-Phe-His-Val-Lys-Phe-Lys-NH₂In SEQ ID NO.3, an NH is fused at the N-terminus₂，KKVVFHVFK (SEQ ID NO. 3). The molecular weight of the linear peptide is 1259Da through mass spectrum detection.

Example 3A Synthesis of an antibacterial polypeptide

1. Based on FMOC synthesis method, the method adopts solid-phase synthesis method, takes amide resin as solid-phase carrier (crosslinking degree is 11%, 0.97mmol/g), FMOC protected special amino acid, HOBT/DIC coupling agent and 30% piperidine/DMF (volume fraction) organic base, charges amino acid with 5 times of synthetic mass, and sequentially connects Arg-Arg-Val-Val-Phe-His-Val-Lys-Phe-Lys-NH according to sequence₂。

The amino acid sequence is Arg-Arg-Val-Val-Phe-His-Val-Lys-Phe-Lys-NH₂In SEQ ID NO.4, an NH is fused at the N-terminus₂RRVVFHVKFK (SEQ ID NO.4), the molecular weight of the linear peptide is 1315Da by mass spectrometry.

Example 4 preparation of products Using antibacterial Polypeptides

Mouthwash: adding antibacterial polypeptide into commercial available mouthwash according to the proportion of 10-50 mug/ml to obtain the mouthwash.

The experimental effect was verified using the following experiment:

1. drawing an antibacterial curve

The prepared 10 mu g/ml drug concentration is adopted³-10⁸CFU/mL of the suspension was mixed with the drug. And (3) sucking the culture at 0h, 4h, 8h, 12h, 16h, 20h and 24h, performing serial dilution, counting viable bacteria, performing three parallel experiments at each dilution, and calculating an average value. And finally, drawing a sterilization curve by taking the logarithm of the concentration of the bacteria as a vertical coordinate and the culture time as a horizontal coordinate.

Result 1, as shown in FIG. 1 (Staphylococcus aureus CMCC (B)26003 bacteriostasis curve), k04 (N end of SEQ ID NO.4 fused with an NH in Staphylococcus aureus bacteriostasis experiment₂) Fusion of an NH with K01 (N-terminus of SEQ ID NO.1)₂) In contrast, k04(SEQ ID NO.4) -NH₂Has quick effect and long bacteriostatic time.

Compared with K01, K04 in a streptococcus mutans bacteriostasis experiment has the advantages of quick response and lasting bacteriostasis time of K04.

As shown in FIG. 3 (Escherichia coli CMCC (B)44102 inhibition curve), k04 (N-terminal of SEQ ID NO.4 fused with an NH in Escherichia coli inhibition experiment₂) Fusion of an NH with K01 (N-terminus of SEQ ID NO.1)₂) In contrast, k04 (N-terminal of SEQ ID NO.4 fused to an NH)₂) Has quick effect and long bacteriostatic time.

As shown in FIG. 4 (Candida albicans CMCC (B)98001 bacteriostasis curve), k04 (N-terminal of SEQ ID NO.4 fused with an NH)₂) Fusion of an NH with K01 (N-terminus of SEQ ID NO.1)₂) In contrast, k04 (N-terminal of SEQ ID NO.4 fused to an NH)₂) Has quick effect and long bacteriostatic time.

2. Safety feature

(1) Anti-bacterial peptide cytotoxicity (effect on human embryonic kidney cell 293T):

and (3) respectively selecting human embryonic kidney cell 293T cells in the logarithmic growth phase, and respectively inoculating the cells to a 96-well plate according to 5 k/well and 15 k/well. Polypeptide drug (N end of SEQ ID NO.4 is fused with NH)₂) k04 was diluted in a gradient starting from 0.25mg/ml, each concentration being 1/4 of the previous concentration, to obtain 8 different groups of concentrations, and the absorbance of 293T cells at 450nm was measured for 4 consecutive days using the method of CCK 8.

Day0 is the absorbance of 293T without drug, and Day1-Day3 are the absorbance of 293T after 24h, 48h and 72h of drug treatment, respectively. The concentration and the number of days are plotted on the abscissa and the absorbance on the ordinate, respectively. The results are shown in FIG. 5.

(2) Antibacterial peptide cytotoxicity (effect on human normal hepatocytes LO 2):

human normal hepatocytes LO2 in logarithmic growth phase were selected and seeded in 96-well plates at 5 k/well and 15 k/well, respectively.

Polypeptide drug (N end of SEQ ID NO.4 is fused with NH)₂) k04 was diluted in a gradient starting from 0.25mg/ml, each concentration being 1/4 of the previous concentration, to obtain 8 different groups of concentrations, and the absorbance at 450nm of human normal hepatocytes LO2 was measured for 4 consecutive days using the method of CCK 8.

Day0 is the absorbance of 293T without drug, and Day1-Day3 are the absorbance of 293T after 24h, 48h and 72h of drug treatment, respectively. The concentration and the number of days are plotted on the abscissa and the absorbance on the ordinate, respectively. The results are shown in FIG. 6.

(3) Antibacterial peptide cytotoxicity (human immortalized epidermal cell HaCaT)

HaCaT cells in the logarithmic growth phase are selected and inoculated to a 96-well plate according to the ratio of 5 k/well to 15 k/well.

Polypeptide drug k04 (N end of SEQ ID NO.4 fused with one NH)₂) A gradient dilution was started at 0.5mg/ml, each at 1/4 of the previous concentration, to obtain 8 different concentrations. The absorbance of HaCaT cells at 450nm was measured for 4 consecutive days using the method of CCK 8. Day0 is the absorbance of HaCaT without drug, and Day1-Day3 are the absorbance of HaCaT after 24h, 48h and 72h of drug treatment, respectively. The concentration and the number of days are plotted on the abscissa and the absorbance on the ordinate, respectively. The results are shown in FIG. 7.

(4) Cytotoxicity of antibacterial peptides (Effect on human embryonic lung fibroblasts MRC-5)

MRC-5 cells in the logarithmic growth phase were selected and seeded in 96-well plates at 5 k/well and 15 k/well, respectively.

Polypeptide drug (N end of SEQ ID NO.4 is fused with NH)₂) k04 gradient dilutions were started from 0.5mg/ml, each concentration being 1/4 of the previous concentration, giving 8 different groupsThe concentration of (c). The absorbance of MRC-5 cells at 450nm was measured for 4 consecutive days using the method of CCK 8. Absorbance of MRC-5 after 48h, 72 h. The concentration and the number of days are plotted on the abscissa and the absorbance on the ordinate, respectively. The results are shown in FIG. 8.

As a result: (fusion of an NH to the N-terminus of SEQ ID NO.4)₂) The data for the effect of the pharmaceutical polypeptide k04 on human cells show that it has a slight inhibitory effect on human hepatocytes at the highest concentration of 0.5mg/ml, and no effect on other cells. The safety to human body is very high.

3. Stability: stability in mouthwash

(1) Bacterial passage dilution: diluting Microforium of Staphylococcus aureus and Escherichia coli with 1ml broth culture medium, inoculating to LB plate culture medium and Microforium of Escherichia coli, culturing at 37 deg.C for 48 hr, collecting Staphylococcus aureus and Escherichia coli with logarithmic growth period, preparing into 1 × 10 by McLeeb turbidimetric method with broth culture medium as diluent⁶CFU/ml bacterial fluid.

(2) Stability test

The test conditions are as follows: the mixture was left at 37 ℃ for 90 days.

Positive control: staphylococcus aureus and Escherichia coli.

Negative control: and (4) a culture medium.

And (3) testing the sample: the mouthwash of 7 formulas and the solution of each formula after being diluted 2 times with the culture medium.

And (3) test operation: taking a 96-well plate, and adding 100 mul of mouthwash and bacterial liquid into each well of a sample group; adding 100 mul of broth culture medium and bacterial liquid into each hole of the positive control; negative controls 200. mu.l of broth per well were added. Cultured at 37 ℃ for 24 h. The negative control should be clear (-), the positive control should be turbid (+), the test hole for clarifying the test sample group is bacteriostatic for the mouthwash, and the test hole for turbid the test sample group is non-bacteriostatic for the mouthwash.

TABLE 1 concentration of polypeptide of the formulation in mouthwash

Name (R)	Formulation 1	Formulation 2	Formulation 3	Formulation 4	Formulation 5
						Concentration of polypeptide	10μg/ml	15μg/ml	20μg/ml	10μg/ml	10μg/ml

TABLE 2 stability results

As a result: as shown in tables 1 and 2, the

formulas

1, 2 and 3 have inhibitory effects on Staphylococcus aureus and Escherichia coli after being placed at 37 ℃ for 90 days; formulas 5 and 6 have inhibitory effect on Staphylococcus aureus, and have no inhibitory effect on Escherichia coli.

SEQUENCE LISTING

<110> Harbin Gilfuron Biotech Ltd

<120> an antibacterial polypeptide and uses thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Artificial Synthesis

<400> 1

Lys Lys Val Val Phe Lys Val Lys Phe Lys

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial Synthesis

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa can be Orn

<400> 2

Lys Lys Val Val Phe Xaa Ala Ala Val Lys Phe Lys

1 5 10

<210> 3

<211> 10

<212> PRT

<213> Artificial Synthesis

<400> 3

Lys Lys Val Val Phe His Val Lys Phe Lys

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Artificial Synthesis

<400> 4

Arg Arg Val Val Phe His Val Lys Phe Lys

1 5 10

Claims

1. An antibacterial polypeptide is characterized in that the antibacterial polypeptide is obtained by taking an antifungal polypeptide with an amino acid sequence shown as SEQ ID NO.1 as an initial sequence and mutating at least one amino acid in the 1 st, 2 nd and 6 th positions.

2. The antibacterial polypeptide according to claim 1, wherein the antibacterial polypeptide is any one of the following (a) to (c):

3. The antibacterial polypeptide of claim 1, wherein the amino acid in the amino acid sequence of the antibacterial polypeptide is in the L-form or D-form.

4. The antibacterial polypeptide of claim 1, wherein the C-terminus of the antibacterial polypeptide is fused to an amino group or a hydroxyl group.

5. The antibacterial polypeptide of claim 1, wherein the antibacterial polypeptide is chemically modified at the C-terminus, N-terminus, or at the amino acid side chain.

6. A gene encoding the antibacterial polypeptide of any one of claims 1 to 5.

7. A recombinant vector carrying the coding gene of claim 6.

8. A recombinant microbial cell carrying the coding gene of claim 6 or expressing the antibacterial polypeptide of any one of claims 1 to 5.

9. The recombinant microbial cell of claim 8, wherein said microbial cell is a prokaryote or a eukaryote.

10. Use of an antibacterial polypeptide according to any one of claims 1 to 5 in the manufacture of a product for the control of microbial infections.