CN110386972B

CN110386972B - Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof

Info

Publication number: CN110386972B
Application number: CN201910787413.1A
Authority: CN
Inventors: 周长林; 吴皓旻; 黄亚; 马菱蔓; 窦洁
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2021-08-13
Anticipated expiration: 2039-08-23
Also published as: CN110386972A

Abstract

The invention discloses an antibacterial polypeptide Pb2-1 or PCL-1 and a preparation method and application thereof, wherein the amino acid sequences of the antibacterial polypeptide Pb2-1 and the PCL-1 are respectively shown as SEQ ID NO 1 and 2. The invention starts from polypeptide CATHPB2 derived from Python bivittatu of Mycoplana ambda, obtains polypeptide Pb2-1 with high alpha-helix degree through structure-activity relationship research, and is covalently connected with a section of undecapeptide with trypsin inhibition activity at the N-end thereof to obtain polypeptide PCL-1 with a new structure consisting of 24 amino acids. The alpha-helix degree and the hydrophobicity of the pharmacodynamic modules of the polypeptides Pb2-1 and PCL-1 prepared by the invention are improved; in addition, the PCL-1 is connected with an undecapcyclic peptide sequence, so that the stability of the polypeptide to trypsin is obviously improved while the polypeptide has good in-vitro antibacterial activity, and the polypeptide can be applied to treatment of bacterial and fungal infectious diseases.

Description

Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of polypeptide medicines in biochemistry, and particularly relates to an antibacterial polypeptide Pb2-1 or PCL-1, and a preparation method and application thereof.

Background

1928 since the discovery of adonimycin, people are gradually entering the age of controlling and treating bacterial infectious diseases. However, the abuse of antibiotics has accelerated the evolution of pathogenic bacteria, resulting in the emergence of a large number of drug-resistant bacteria and multi-drug resistant bacteria in recent years. Because the research and development of novel antibiotics are not accelerated along with the rapid increase of drug-resistant bacteria, and because the antibiotics have low economic benefit and high approval difficulty, many pharmaceutical companies quit the research and development of the antibiotic drugs, and the discovery of the antibiotic drugs is slowed down year by year. The emergence of multidrug resistant bacteria and the lack of antibiotic drugs has made the search for new antibacterial drugs urgent.

Antibacterial peptides are an important component of the natural immune defense mechanism of most organisms, are ubiquitous in nature, and the number of antibacterial peptides currently found in animals, plants and microorganisms is more than 2900. Although the sources and sequences of the antibacterial peptides are different, the antibacterial peptides have some commonalities in structure, the antibacterial peptides generally consist of 10-60 amino acid residues, the natural antibacterial peptides usually contain lysine and arginine, and can be combined with hydrogen ions in water to endow polypeptide molecules with electropositivity, so the common antibacterial peptides are cationic polypeptides, and the secondary structures of the common antibacterial peptides can be generally divided into an alpha-helical structure, a beta-folded structure and an extended structure. Most of the antibacterial peptides have broad-spectrum antibacterial activity and have good killing effect on gram-negative bacteria, gram-positive bacteria, protozoa, fungi and even viruses. Besides good antibacterial activity, the antibacterial peptide also has the effects of resisting tumors, regulating immunity, promoting wound healing and the like. The unique membrane disruption sterilization mechanism of the cationic antibacterial peptide is not easy to induce pathogens to generate drug resistance, which has great advantages compared with the traditional antibiotics and is expected to become a novel substitute for clinical treatment of infectious diseases; however, its poor stability, especially the instability to trypsin, limits its clinical use. In recent years, the stability problem of polypeptide drugs has become a hot spot of domestic and foreign research.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the antibacterial polypeptide Pb2-1 or PCL-1, the polypeptide has good in vivo and in vitro antibacterial activity and better stability to trypsin, can be applied to various common infections and stubborn infectious diseases, and can be used as an excellent substitute drug or an auxiliary drug of the existing antibiotics.

The invention also provides a preparation method and application of the antibacterial Pb2-1 or PCL-1.

The technical scheme is as follows: in order to achieve the aim, the antibacterial polypeptide Pb2-1 has an amino acid sequence shown as SEQ ID NO. 1: GFRKFMRRLKKFF are provided.

The amino acid sequence of the antibacterial polypeptide PCL-1 is SEQ ID NO. 2: CWTKSIPPKPCGFRKFMRRLKKFF are provided.

The preparation method of the antibacterial polypeptide Pb2-1 comprises the following steps:

the polypeptide CATHPB2 is taken as a template, the region with conserved sequence and high concentration of alpha-helix is intercepted, the interception is carried out from the 3 rd position of the N-end, the number of the amino acids of the polypeptide is reduced from 29 to 13, and the polypeptide Pb2-1 is obtained.

The preparation method of the antibacterial polypeptide PCL-1 comprises the following steps:

(1) taking the polypeptide CATHPB2 as a template, intercepting a region with conserved sequence and high concentration of alpha-helix, and reducing the number of polypeptide amino acids from 29 to 13 from the 3 rd position of the N-end to obtain polypeptide Pb 2-1;

(2) an undecapcyclic peptide with the sequence of CWTKSIPPKPC (SEQ ID NO:3) is covalently linked with the N-terminal of Pb2-1 to obtain a polypeptide PCL-1 with intramolecular disulfide bonds.

Wherein the sequence CWTKSIPPKPC is an undecapeptide which is disulfide-bonded and cyclized and has trypsin inhibitory activity. The undecapeptide only has trypsin inhibitory activity and does not have any antibacterial activity.

The invention relates to application of the antibacterial polypeptide Pb2-1 or the antibacterial polypeptide PCL-1 in preparing a medicament for resisting pathogenic bacteria infection.

Wherein the anti-pathogenic infection drug comprises an anti-bacterial and anti-fungal infection drug.

Further, the bacteria are staphylococcus aureus, methicillin-resistant staphylococcus aureus (MRSA), staphylococcus epidermidis, streptococcus pyogenes, streptococcus pneumoniae, pseudomonas aeruginosa, escherichia coli, shigella dysenteriae, and klebsiella pneumoniae; the fungi are Candida albicans and Cryptococcus neoformans.

Wherein the antibacterial polypeptides Pb2-1 and PCL-1 are synthesized by a conventional polypeptide solid phase synthesis method.

The present invention relates to pathogenic bacteria and pathogenic fungi comprising: standard strains of ATCC origin and conventional strains isolated clinically.

The antibacterial polypeptide Pb2-1 or PCL-1 of the invention both takes polypeptide CATHPB2(SEQ ID NO: 4: KRNGFRKFMRRLKKFFAGGGSSIAHIKLH) as a template, the CATHPBs are cathelicidins family cationic polypeptides extracted from the lung tissue of Python bivittatu of Mycoplana mylonto, and the polypeptide CATHPB2 is one of the polypeptides, consists of 29 amino acids, and has antibacterial effect. According to the invention, through the structure-activity relationship research, 13 amino acid residues with better alpha-helix degree at the N-terminal are intercepted, the positive charge number of the obtained polypeptide Pb2-1 is reduced, but the alpha-helix degree, hydrophobicity and hydrophobic moment are improved, the amino acid number is greatly reduced, the cost of polypeptide synthesis is reduced, and meanwhile, the antibacterial peptide has a stronger antibacterial effect compared with the CATHPB 2; the antibacterial polypeptide PCL-1 with greatly improved trypsin stability is also obtained by covalently connecting the undecapeptide with the N-end of Pb 2-1. The polypeptide PCL-1 of the invention not only has good in vitro antibacterial activity, but also has good stability to trypsin, can be applied to various common infections and stubborn infectious diseases, and can be used as a good substitute drug or an auxiliary drug of the existing antibiotics.

The antibacterial polypeptide Pb2-1 is obtained by taking the polypeptide CATHPB2 as a template and intercepting the N-terminal amino acid of the polypeptide, meanwhile, the N-terminal of Pb2-1 is covalently connected with a section of undecapeptide with trypsin inhibition activity to obtain PCL-1, and the polypeptides Pb2-1 and PCL-1 are obtained by adopting a solid phase synthesis method. The amino acid sequences of the antibacterial polypeptides Pb2-1 and PCL-1 are shown in SEQ ID NO.1 and 2.

The modification of the antibacterial polypeptide PCL-1 is designed by taking CATHPB2 as a template, 13 amino acids are intercepted from the 3 rd site of the N-end of CATHPB2 to obtain Pb2-1, and an undecapeptide with trypsin inhibition activity is covalently connected with the N-end of the intercepted sequence, so that the aim of maintaining the antibacterial activity and improving the stability of the trypsin is fulfilled. The full sequence is cysteine-tryptophan-threonine-lysine-serine-isoleucine-proline-lysine-proline-cysteine-glycine-phenylalanine-arginine-lysine-phenylalanine-methionine-arginine-leucine-lysine-phenylalanine.

In vitro antibacterial test results show that the antibacterial polypeptide Pb2-1 is obtained by intercepting 13 amino acids at the tail end of CATHPB2-N, and has better broad-spectrum antibacterial activity and greatly reduced cost compared with the parent peptide CATHPB 2. The antibacterial polypeptide PCL-1 can still maintain better broad-spectrum antibacterial activity under the condition that 13 amino acids at the N-terminal of CATHPB2 are intercepted and are covalently connected with undecapicin. The polypeptide Pb2-1TI (GFRKFMRRLKKKFFCWTKIPPKPC) obtained by covalently linking an undecapcycline at the C-terminus of Pb2-1 had weaker antibacterial activity against each strain of bacteria or fungi than the antibacterial polypeptide PCL-1 obtained by covalently linking an undecapycline at the N-terminus of Pb 2-1. Meanwhile, the sterilization curve result shows that the polypeptide PCL-1 has good sterilization activity on staphylococcus aureus, escherichia coli and cryptococcus neoformans clinical isolates, can kill the escherichia coli and the cryptococcus neoformans within 8 hours, has more obvious sterilization effect on the staphylococcus aureus, and can completely kill the staphylococcus aureus within 1 hour.

In vitro toxicity studies show that the antibacterial polypeptides Pb2-1 and PCL-1 of the invention have low hemolytic activity on sheep erythrocytes, and the hemolytic activity can be ignored within the effective action range of Pb2-1 and PCL-1.

The in vitro stability of the antibacterial polypeptides Pb2-1 and PCL-1 prepared by the invention is researched by HPLC and antibacterial tests. The results show that the residual amounts of Pb2-1 and PCL-1 do not change obviously under different pH conditions and different temperatures, and the structural integrity can be maintained, which indicates that the compound has pH stability and thermal stability; when the concentration of NaCl is 200mM or less, the antibacterial activity of Pb2-1 and PCL-1 on Escherichia coli is kept unchanged, which shows that Pb2-1 and PCL-1 can better retain the antibacterial activity under the condition of high-concentration salt ions; PCL-1 covalently linked with undecapricopeptide still has higher integrity after being incubated for 1h with trypsin, and the MIC value of Escherichia coli is improved by 4 times.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. compared with the polypeptide CATHPB2, the antibacterial polypeptide Pb2-1 prepared by the invention has better broad-spectrum antibacterial activity and greatly reduced cost.

2. The prepared antibacterial polypeptide PCL-1 has broad-spectrum antibacterial activity, and compared with the mutant peptide Pb2-1, the stability of trypsin is obviously improved.

3. The design and preparation method of the antibacterial polypeptides Pb2-1 and PCL-1 is simple and convenient, the design is novel, the raw material sources are easy to obtain, and the industrial production and application can be realized.

4. The antibacterial polypeptides Pb2-1 and PCL-1 prepared by the invention can be applied to the preparation of antibacterial and antifungal infection medicines, and have good in vivo and in vitro antibacterial activity; wherein, after connecting a section of undecapeptide which has trypsin inhibitory activity but no antibacterial activity, PCL-1 still has excellent antibacterial activity, and the stability of the trypsin is greatly improved, so that the undecapeptide can be applied to various common infections and stubborn infectious diseases and can be used as an excellent substitute drug or an auxiliary drug of the existing antibiotics.

Drawings

FIG. 1 is an HPLC chromatogram of the antibacterial polypeptide PCL-1 of the present invention;

FIG. 2 is a mass spectrum of PCL-1;

FIG. 3 is an HPLC chromatogram of the antibacterial polypeptide Pb2-1 of the present invention;

FIG. 4 is a mass spectrum of Pb 2-1;

FIG. 5 is a graph of the bactericidal effect of PCL-1, Pb2-1 on Staphylococcus aureus;

FIG. 6 is a graph showing the sterilization of Escherichia coli by PCL-1 and Pb 2-1;

FIG. 7 is a graph showing the bactericidal activity of PCL-1, Pb2-1 against Cryptococcus neoformans;

FIG. 8 is a graph showing hemolytic activity of PCL-1 and Pb2-1 on sheep red blood cells;

FIG. 9 is a graph quantifying the stability of PCL-1, Pb2-1 against temperature;

FIG. 10 is a graph quantifying the stability of PCL-1, Pb2-1 against pH;

FIG. 11 is a graph quantifying the stability of PCL-1, Pb2-1 against trypsin;

FIG. 12 is a graph showing the effect of PCL-1 on the number of bacteria in blood of mice infected with Escherichia coli.

Detailed Description

The present invention will be further described with reference to the following examples and the accompanying drawings.

Example 1

Preparation of antibacterial polypeptides Pb2-1 and PCL-1:

(2) an undecapeptide which has the sequence of CWTKSIPPKPC, is subjected to disulfide bond cyclization and has trypsin inhibition activity is covalently connected with the N-end of Pb2-1 to obtain the polypeptide PCL-1 with intramolecular disulfide bond.

(3) The polypeptides Pb2-1 and PCL-1 are synthesized by a solid phase synthesis method.

The amino acid sequence of the prepared Pb2-1 is as follows: glycine-phenylalanine-arginine-lysine-phenylalanine-methionine-arginine-leucine-lysine-phenylalanine.

The amino acid sequence of the prepared PCL-1 is as follows: cysteine-tryptophan-threonine-lysine-serine-isoleucine-proline-lysine-proline-cysteine-glycine-phenylalanine-arginine-lysine-phenylalanine-methionine-arginine-leucine-lysine-phenylalanine.

Synthesis of the Linear polypeptide Pb2-1 or PCL-1: and the C end and the N end are carried out one by one. Soaking Rink amide AM Resin in dichloromethane for 15min, and after the Resin expands, extracting dichloromethane; piperidine/DMF solution (10 ml per gram of resin) was added at a volume ratio of 1:4, the reaction was carried out 2 times for 5min and 15min with nitrogen bubbling, and the resin was washed 9 times with DMF after the reaction was completed. And (3) adding 2-3 drops of each of 20-40 resins into an assay reagent ABC (liquid A is ninhydrin/absolute ethyl alcohol solution, liquid B is pyridine, liquid C is phenol/absolute ethyl alcohol solution, and liquid A, B, C is added with 2-3 drops) and heating for 3min at 100 ℃, wherein the color of the solution and the resin is changed into blue to remove amino protection. An excess of Fmoc-Phe-OH and HOBT, which reacted twice in moles, was added, dissolved in 10ml of DMF per gram of resin, DIC and Collidine, which reacted twice in moles, with nitrogen sparge for 1 h. After the reaction is finished, washing the resin with DMF for 6 times, repeatedly carrying out condensation reaction, sequentially connecting each Fmoc protected amino acid to complete the synthesis of a straight chain sequence, soaking the resin with dichloromethane and ether, and then pumping to dry. Adding TFA, reacting for 2h in a constant temperature shaker at a shaker rotation speed of 110r/min and a temperature of 25 ℃. Filtering to remove resin, adding anhydrous diethyl ether into the filtrate, centrifuging to obtain solid, adding anhydrous diethyl ether, washing, centrifuging, repeating for several times, and oven drying to obtain crude polypeptide.

Cyclization of polypeptide PCL-1 disulfide bond: the crude polypeptide was dissolved in purified water to a concentration of 1 mg/ml. Adjusting the pH value to 8-9 with dilute ammonia water, and reacting for 24h at normal temperature. The end of the reaction was detected by HPLC and MASS.

And (3) polypeptide purification: taking a certain amount of polypeptide Pb2-1 or PCL-1 aqueous solution, and removing large-particle impurities by using a filter. Meanwhile, a preparative liquid chromatograph is adopted to pass through the column, and samples are collected in sections. And (4) performing gradient analysis by using an analytical chromatograph, and reserving a sample reaching the required purity. Then the polypeptide Pb2-1 or PCL-1 is obtained after freeze drying treatment.

Example 2

Purity determination (HPLC method) and mass spectrometry analysis results of the polypeptides Pb2-1 and PCL-1:

example 1 the polypeptides Pb2-1 and PCL-1 were synthesized and purified to obtain the final product, which was identified by high performance liquid chromatography and mass spectrometry.

Liquid chromatography conditions: c18 column (4.6X 250mm,5 μm); mobile phase a was acetonitrile containing 0.1% trifluoroacetic acid and mobile phase B was purified water containing 0.1% trifluoroacetic acid. The detection wavelength is 220 nm; the flow rate is 1.0 ml/min; the sample was taken in an amount of 10. mu.l and gradient elution was carried out. Elution conditions were linear gradient concentration increasing: the initial concentration of the organic phase was 27%, which increased to 52% at 25min, and the column was washed with 100% organic phase from 25min to 30 min.

As can be seen from FIGS. 1 and 3, the purity of the polypeptides PCL-1 and Pb2-1 prepared in example 1 of the present invention is greater than 98%.

As can be confirmed from FIGS. 2 and 4, the molecular weight of PCL-1 is 2999.78, and the molecular weight of Pb2-1 is 1761.23, which is consistent with the theoretical values.

Example 3

Determination of in vitro antibacterial activity of polypeptides Pb2-1 and PCL-1 of the invention

(1) Recovery and activation of bacterial strain

The experimental strain frozen at-20 ℃ is transferred from the glycerol tube to the corresponding agar slant medium (bacteria to nutrient agar slant, fungi to Sabouraud dextrose agar slant). Culturing the bacteria at 37 deg.C for 24 hr, culturing the fungi at 28 deg.C for 48 hr, and storing in refrigerator at 4 deg.C.

(2) Preparation of bacterial liquid

Taking a small amount of thallus on the inclined plane, transferring into 2ml of corresponding liquid culture medium, culturing the bacteria at 37 deg.C and the fungi at 28 deg.C for 8h, and diluting to 10% with corresponding liquid culture medium⁵CFU/ml bacterial suspension for use.

(3) Preparation of drugs

The polypeptide Pb2-1 or PCL-1 prepared in example 1 was weighed and dissolved in physiological saline, respectively, and prepared into mother solution of 1024 μ g/ml, filtered and sterilized by a 0.22 μm aqueous phase filter, subpackaged by an EP tube, and stored at-20 ℃ for later use.

(4) The polypeptides Pb2-1 and PCL-1 were used for determination of the Minimal Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) of the test strains.

The MIC and MBC detection methods for both polypeptides were identical. That is, after polypeptide drugs are diluted into different concentration gradients by multiple times with liquid culture medium, 50 ul of each drug is put into a 96-well plate and inoculated with 50 ul of 10 with equal volume⁵CFU/ml bacterial suspension, so that the final concentration of the medicine is 256, 128, 64, 32, 16, 8, 4, 2 and 1 mu g/ml respectively, and meanwhile, 100 mu l of liquid culture medium is used as a blank control group, and 50 mu l of bacterial suspension is added into the liquid culture medium with the same volume as the negative control group. Culturing the bacteria at 37 ℃ for 16-18 h, culturing the fungi at 25 ℃ for 48h, and detecting the absorbance value of each hole at 595nm by using an enzyme-labeling instrument. To be able to completely inhibit the growth of bacteria or fungiThe lowest drug concentration is the Minimum Inhibitory Concentration (MIC). Meanwhile, Pb2-1TI and CATHPB2 are used as comparison, and the synthesis of Pb2-1TI and CATHPB2 polypeptides, the preparation of medicaments and the determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the medicaments are consistent with the polypeptides Pb2-1 and PCL-1.

Taking 100 ul of culture without bacteria or fungi growth in a 96-well plate determined by the MIC, adding 900 ul of sterile culture medium for dilution, transferring to a sterile culture dish, adding about 10.0ml of culture medium with the temperature of 55 ℃, culturing bacteria at 37 ℃ for 24h, culturing fungi at 28 ℃ for 48h, observing and recording the colony number on the plate, and recording the minimum drug concentration with the colony number less than 5 as the MBC value. The results are shown in Table 1:

TABLE 1 MIC and MBC values of the antimicrobial polypeptides PCL-1, Pb2-1TI and Pb2-1 against various bacteria and fungi

Continuing with Table 1:

^aan amoxicillin resistant strain;^bgentamicin resistant strains.

As can be seen from Table 1, Pb2-1 has better broad-spectrum antibacterial activity relative to the parent peptide CATHPB2, and has better antibacterial activity on both standard and clinical bacteria and fungi, the MIC value is in the range of 4-32 mu g/ml, and the MBC value is in the range of 4-32 mu g/ml; the polypeptide PCL-1 obtained by connecting a section of undecapeptide with trypsin inhibitory activity and no antibacterial activity to the N-terminal of Pb2-1 has slightly reduced antibacterial activity compared with Pb2-1, but still has better antibacterial activity to bacteria and fungi, the MIC value to standard and clinical bacteria and fungi is in the range of 8-64 mu g/ml, and the MBC value is in the range of 8-128 mu g/ml. And Pb2-1TI has weak antibacterial activity to bacteria and fungi, and the MIC value to bacteria is 4 times of that of PCL-1.

(5) Sterilization curve of polypeptides Pb2-1 and PCL-1

The bacterial suspension diluted in nutrient broth or Sabouraud's dextrose liquid medium is adjusted to a bacterial concentration of about 10⁵CFU/ml, adding polypeptide PCL-1, Pb2-1 (final concentration: 4 × MIC) or amoxicillin (final concentration: 1024 μ g/ml), and culturing at 37 deg.C. At 0, 1, 2, 4, 8, 12, 24h, 200. mu.l of the culture broth was taken out for gradient dilution and plate counting. The normal saline group is set as a negative control group, the polymyxin group is set as a positive control group, three dilution gradients are arranged in parallel, the colony logarithm value is used as a ordinate, the action time of the polypeptide is used as an abscissa, and a sterilization curve is drawn. The results are shown in FIGS. 5, 6 and 7.

Figures 5, 6 and 7 show the bactericidal effect of polypeptides PCL-1 and Pb2-1 on each experimental strain respectively, the PCL-1 has a remarkable bactericidal effect, can kill Escherichia coli and cryptococcus neoformans within 8 hours, has a more remarkable bactericidal effect on staphylococcus aureus, and can kill all staphylococcus aureus within 1 hour. The result shows that the antibacterial polypeptide PCL-1 has good bactericidal action.

Example 4

Effect of antibacterial polypeptides Pb2-1 and PCL-1 on hemolytic activity of sheep red blood cells

Defibered sheep blood was added to a sterile EP tube, centrifuged at 3000rpm for 10min, washed 3 times with PBS (pH 7.4) and resuspended to make a 3% (V/V) suspension of erythrocytes. 50 mul of sheep red blood cell suspension is respectively sucked and added into a 96-well plate, and polypeptide solution diluted by equal volume is added, so that the final concentration of the polypeptide is respectively 512, 256, 128, 64, 32, 16 and 8 mug/ml. The PBS-treated group was set as a negative control, the 1% Triton X-100-treated group was set as a positive control, and 3 duplicate wells were used for each group. After the drug treatment, the 96-well plate is placed at 37 ℃ for constant temperature culture for 1h and then taken out, and centrifuged at 3000rpm for 10 min. The supernatant was aspirated, the OD of each well at 450nm was read with a microplate reader, and the hemolysis rate was calculated as follows:

hemolysis rate (Jia medicine OD)₄₅₀Negative control group OD₄₅₀) /(Positive control OD₄₅₀Negative control group OD₄₅₀)×100％

As can be seen from FIG. 8, even when the polypeptide concentration is as high as 512. mu.g/ml, the hemolytic rate of Pb2-1 and PCL-1 to sheep red blood cells is low, not exceeding 5%. The range of the combined MIC values can be seen as: PCL-1 has a lower hemolytic activity than Pb2-1 in the effective action range, and has a hemolytic rate of less than 1% for erythrocytes below a concentration of 256. mu.g/ml.

Example 5

In vitro stability research of the antibacterial polypeptides Pb2-1 and PCL-1 of the invention

(1) Thermal stability

The polypeptide with the concentration of 1024 mu g/ml is mixed with PBS (pH 7.4) with the same volume, and the mixture is respectively placed in thermostatic water baths at 0, 20, 37, 50, 70 and 90 ℃ for 1 h. Detecting the incubated sample by using RT-HPLC (reverse transcription-high performance liquid chromatography), wherein the analysis conditions of the liquid chromatogram are as follows: c18 column (4.6X 250mm,5 μm); mobile phase a was acetonitrile containing 0.1% trifluoroacetic acid and mobile phase B was purified water containing 0.1% trifluoroacetic acid. The detection wavelength is 220 nm; the flow rate is 1.0 ml/min; the sample was taken in an amount of 20. mu.l and gradient elution was carried out. The elution condition is that the concentration of the linear gradient is increased, wherein the elution condition of Pb2-1 is specifically as follows: the initial concentration of the organic phase is 22%, the concentration is increased to 42% in 20min, and the chromatographic column is washed by 100% organic phase in 20min to 25 min; the elution conditions of PCL-1 are specifically as follows: the initial concentration of the organic phase was 25%, increasing to 45% at 20min, and the column was washed with 100% organic phase from 20min to 25 min. The results are shown in FIG. 9.

As can be seen from FIG. 9, after PCL-1 and Pb2-1 were incubated at different temperatures for 1h, the residual amounts of the polypeptides were not decreased compared to the 0 ℃ group, and even at incubation temperatures as high as 90 ℃, both polypeptides could maintain high integrity, indicating that both polypeptides have high thermal stability.

(2) Stability of pH

Solutions of

pH

1, 3, and 5 were prepared with 1M HCl, and solutions of pH 9 and 11 were prepared with 1M NaOH. The polypeptide solution was prepared with solutions of different pH to a final concentration of 512. mu.g/ml and incubated at 37 ℃ for 24 h. The incubated sample was subjected to RT-HPLC analysis under the same conditions as in example 5 (1). The results are shown in FIG. 10.

As can be seen from FIG. 10, after 24h of incubation in an extremely acidic environment with pH 1, the residual amount of PCL-1 did not decrease significantly, while Pb2-1 remained intact by 90%, indicating that PCL-1 can be stably present in human gastric acid (pH about 1.5), while Pb2-1 is slightly unstable in gastric acid. When the pH is up to 11, the residual quantity of the two polypeptides is not obviously reduced, which indicates that the two polypeptides can maintain high integrity in different pH environments.

(3) Salt stability

Culturing Escherichia coli to logarithmic phase, and diluting the culture solution to 10% with MHB culture medium containing NaCl of different concentrations⁵CFU/ml. Diluting the polypeptide with MH culture medium to different concentration gradients, respectively taking 50 μ l in 96-well plate, and inoculating 50 μ l of 10 containing NaCl with different concentrations in equal volume⁵CFU/ml bacterial suspension, so that the final concentration of the medicine is 256, 128, 64, 32, 16, 8, 4, 2 and 1 mu g/ml, and the final concentration of NaCl is 0, 50, 100, 150, 200 and 400 mM. Meanwhile, 100 mul of MHB culture medium containing NaCl with different concentrations is used as a blank control group, 50 mul of bacterial suspension is added into the same volume of MHB culture medium containing NaCl with different concentrations to be used as a negative control group, the MHB culture medium is cultured at 37 ℃ for 16-18 h, and the influence of different salt ion concentrations on the MIC value of the polypeptide against Escherichia coli is detected. The results are shown in Table 2:

table 2 effect of different concentrations of NaCl on anti-e.coli activity of polypeptides

As can be seen from Table 2, when the concentration of NaCl was as high as 200mM, the MIC values of PCL-1 and Pb2-1 for Escherichia coli were not changed, whereas normal NaCl in human body was about 150mM, indicating that both polypeptides maintain good antibacterial activity even when the concentration of NaCl was higher than normal physiological concentration, and both polypeptides have high salt tolerance.

(4) Trypsin stability

The stability of the polypeptides in trypsin solution was first checked by liquid chromatography. Mixing polypeptide with concentration of 2048 μ g/ml and trypsin (not less than 250 N.F.u/mg) with concentration of 0.02mg/ml in equal volume, incubating at 37 deg.C for 0.5, 1, 2h, and using enzyme-free group as blank control. Acetonitrile with the same volume as the incubation system is added, the mixture is stored at 4 ℃ for 2h, trypsin is denatured and precipitated, then the sample is centrifuged at 10000rpm for 10min, the supernatant is filtered and detected by RT-HPLC, and the conditions of liquid chromatography are the same as those of example 5 (1). The results are shown in FIG. 11.

In addition to the effect of trypsin on the polypeptides, changes in the antimicrobial activity of the polypeptides treated with trypsin were also examined by liquid chromatography. Culturing Escherichia coli to logarithmic growth phase, and diluting to 10% with MH culture medium⁵CFU/ml. Mixing the polypeptide and trypsin (more than or equal to 250 N.F.u/mg) according to a molar ratio of 300:1, and incubating at 37 ℃ for 0.5, 1, 2, 4 and 8 hours while using a blank group without enzyme as a blank control. Then, inactivating trypsin in boiling water bath for 5min, diluting each group of polypeptides with MH culture medium to different concentration gradients, placing 50 μ l each in 96-well plate, and inoculating 50 μ l 10⁵And (3) CFU/ml bacterial suspension, wherein the final concentrations of the medicines are respectively 256, 128, 64, 32, 16, 8, 4, 2 and 1 mu g/ml, meanwhile, 100 mu l of liquid culture medium is used as a blank control group, 50 mu l of bacterial suspension is added into the liquid culture medium with the same volume as the blank culture medium to be used as a positive control group, the positive control group is cultured for 16-18 h at 37 ℃, and the influence of trypsin on the MIC value of the polypeptide against Escherichia coli is detected. The results are shown in Table 3.

TABLE 3 Effect of Trypsin on the Activity of polypeptides against Escherichia coli

As can be seen from FIG. 11, after 0.5h of incubation with trypsin, 90% of the polypeptide PCL-1 remains intact, and when the incubation time is prolonged to 1h, the PCL-1 is decomposed by about 40%, and when the incubation time is as long as 2h, 25% of the polypeptide PCL-1 remains intact, indicating that part of the polypeptide remains undegraded.

Meanwhile, as can be seen from table 3, the MIC of the polypeptide PCL-1 against escherichia coli is increased by 2 times after 0.5h of trypsin treatment, and the stability of trypsin is better.

Therefore, the stability of the PCL-1 in the trypsin is greatly improved, and the influence of the trypsin on the activity of the polypeptide against Escherichia coli can be effectively avoided.

Example 6

The invention also discloses the protective effect of the antibacterial polypeptide PCL-1 on bacteremia model mice

Female ICR mice with the healthy weight of 20-22 g are taken and randomly divided into 6 groups, and each group comprises 13 mice. And (3) taking the liquid of the Escherichia coli with MLD (bacterial density distribution) bacteria to inject into mice with each 0.5ml, and establishing a septicemia model. The administration components are 5 groups, a polypeptide PCL-1 high dose group (10mg/kg), a polypeptide PCL-1 medium dose group (5.0mg/kg), a polypeptide PCL-1 low dose group (2.5mg/kg), a polymyxin group (2.5mg/kg) and a physiological saline group, wherein the PCL-1 is prepared in example 1. The polypeptide administration group and the normal saline group are respectively administrated in the abdominal cavity twice 0.5h and 2h after infection, and the polymyxin group is administrated in the tail vein once 2h after infection. Meanwhile, the group without bacteria is set as a blank control group.

After 8h of infection, the mice were bled about 400 μ l from the orbit and anticoagulated with heparin sodium. Diluting to different gradients, adding 1.0ml of each diluent into a sterile culture dish, adding a Macconyya agar culture medium, placing at 37 ℃ for constant-temperature culture for 18-24 h, observing and recording the number of bacterial colonies on a plate, and calculating the survival number of bacteria in the blood of the mouse. The results are shown in FIG. 12.

As can be seen from FIG. 12, the number of bacteria in the blood of the mice was about 10 after 8h infection⁸CFU/ml, polymyxin can obviously reduce the number of bacteria in the blood of mice, compared with a model group, the number of bacteria in a dose group in the polypeptide PCL-1 is reduced by 1.6 log values, and the number of bacteria in a high dose group is reduced by nearly 2.5 log values, and compared with the model group, the significant difference is shown, and the polypeptide PCL-1 can obviously eliminate the bacteria in the blood of infected mice.

Sequence listing

<110> university of Chinese pharmacy

<120> antibacterial polypeptide Pb2-1 or PCL-1, and preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Phe Arg Lys Phe Met Arg Arg Leu Lys Lys Phe Phe

1 5 10

<210> 2

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Cys Trp Thr Lys Ser Ile Pro Pro Lys Pro Cys Gly Phe Arg Lys Phe

1 5 10 15

Met Arg Arg Leu Lys Lys Phe Phe

20

<210> 3

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Cys Trp Thr Lys Ser Ile Pro Pro Lys Pro Cys

1 5 10

<210> 4

<211> 29

<212> PRT

<213> Mycoplana Python (Python bivittatu)

<400> 4

Lys Arg Asn Gly Phe Arg Lys Phe Met Arg Arg Leu Lys Lys Phe Phe

1 5 10 15

Ala Gly Gly Gly Ser Ser Ile Ala His Ile Lys Leu His

20 25

Claims

1. An antibacterial polypeptide Pb2-1, which is characterized in that the amino acid sequence is SEQ ID NO: 1:

GFRKFMRRLKKFF。

2. an antibacterial polypeptide PCL-1 is characterized in that the amino acid sequence is SEQ ID NO:

CWTKSIPPKPCGFRKFMRRLKKFF。

3. a method for preparing the antibacterial polypeptide Pb2-1 of claim 1, which comprises the following steps:

the polypeptide CATHPB2 is used as a template, and the amino acid sequence is SEQ ID NO: KRNGFRKFMRRLKKFFAGGGSSIAHIKLH, cutting the conserved sequence region with high concentration of alpha-helix, and reducing the number of polypeptide amino acids from 29 to 13 from the 3 rd position of the N-end to obtain the polypeptide Pb 2-1.

4. A method for preparing the antibacterial polypeptide PCL-1 of claim 2, which comprises the following steps:

(1) the polypeptide CATHPB2 is used as a template, and the amino acid sequence is SEQ ID NO: KRNGFRKFMRRLKKFFAGGGSSIAHIKLH, intercepting the region with conserved sequence and highly concentrated alpha-helix, and reducing the number of polypeptide amino acids from 29 to 13 from the 3 rd position of the N-end to obtain the polypeptide Pb 2-1;

(2) an undecapcyclic peptide with the sequence of CWTKSIPPKPC is covalently linked with the N-end of Pb2-1 to obtain a polypeptide PCL-1 with intramolecular disulfide bonds.

5. The method according to claim 4, wherein the sequence CWTKSIPPKPC is an undecapeptide having a disulfide bond to form a ring and having trypsin inhibitory activity.

6. The use of the antibacterial polypeptide Pb2-1 of claim 1 or the antibacterial polypeptide PCL-1 of claim 2 in the preparation of a medicament for resisting bacterial and fungal infections.

7. The use according to claim 6, wherein the bacteria are Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Shigella dysenteriae and Klebsiella pneumoniae; the fungi are Candida albicans and Cryptococcus neoformans.