CN115043924B

CN115043924B - Modified antibacterial peptide and application thereof

Info

Publication number: CN115043924B
Application number: CN202210465719.7A
Authority: CN
Inventors: 王义鹏; 欧阳建红; 郝伟静; 汪旭
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-10-27
Anticipated expiration: 2042-04-29
Also published as: CN115043924A

Abstract

The invention relates to a modified antibacterial peptide and application thereof, wherein the modified antibacterial peptide is obtained by modifying an antibacterial peptide with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The modified antibacterial peptide has broad-spectrum efficient antibacterial activity, extremely strong anti-inflammatory activity and anti-biofilm activity, has synergistic antibacterial effect with the traditional antibiotics, has the beneficial characteristics of small molecular weight, simple structure, low hemolytic activity, high stability, simple preparation method and the like, and has great potential application prospects in the downstream fields of anti-inflammatory agents, antibacterial agents, preservatives, animal feed additives, cosmetic additives and the like.

Description

Modified antibacterial peptide and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a modified antibacterial peptide and application thereof.

Background

The antibacterial peptide is polypeptide synthesized in ribosome by gene coding, and different types of antibacterial peptide generally have the common characteristics of short peptide, strong cation, good thermal stability, small molecular mass, no drug shielding and small toxicity to eukaryotic cells. Today, antimicrobial peptides have been successfully isolated and classified from most organic organisms ranging from prokaryotes to humans. Antibacterial peptides generally act on bacteria and play an important role in the natural immunity of eukaryotes, and are considered to be immune molecules that are effectively retained in mammals in ancient evolutions. Therefore, the antibacterial peptide can be used as a polypeptide substance widely existing in the natural organisms, can be used as a first defense line of organisms, and can resist the invasion of pathogens. The antibacterial peptide has various biological activities of resisting bacteria, fungi, viruses, inhibiting and killing cancer cells, and the like, and is not easy to generate drug resistance.

At present, the application of the antibacterial peptide in medicine achieves some satisfactory results, and a plurality of novel medicines are gradually put into the medicine market. For example, the antibacterial peptide drug MAI278 developed by using the amphibian antibacterial peptide magainin has nearly completed phase III clinical trials, and shows better killing effect on viruses and tumor cells. daptomycin is an anionic antimicrobial peptide developed by the company Cubitpharmaceuticals and approved by the U.S. food and drug administration for sale at 9, 2003, and is useful in the treatment of skin infections and sepsis caused by gram positive bacteria such as Staphylococcus aureus. Li and other research on the functions of the murine Binlb antibacterial peptide gene has made breakthrough progress. This is the first functional gene found at present and related to inflammation of male reproductive system, and the epididymis has been proved to have the immune system for the first time. MX-226 (CPI-226) is an antimicrobial peptide isolated from bovine endolicidins, has been subjected to clinical trials in IIIb phase, can be used for locally preventing or reducing blood infections associated with central venous catheters, and shows good application prospects. plectasin (mycelial mycin) is an antibacterial peptide separated from an ascomycete, has good bactericidal activity on streptococcus pneumoniae, can inhibit the growth of fungi and viruses, and has been studied in the preclinical stage in 2007. In addition, antibiotics, which are used as feed additives in animal production, play an important role in the development of animal husbandry, but remain in animals and animal products, and cause drug resistance problems due to pathogenic bacteria, which negatively affect human health and the environment. Therefore, the antibacterial peptide is taken as a substance which is most hopeful to replace the traditional antibiotics, and has good application prospect in the fields of medicine industry, food additives and the like. The present invention is therefore aimed at engineering the green sea turtle (chelonian mydas) antimicrobial peptide Cm-CATH2 in the hope of providing a new antimicrobial peptide as a powerful alternative to the new generation of antimicrobial drugs.

Chinese patent CN201610585747.7 discloses an antimicrobial peptide Cm-CATH2, which is modified from Cm-CATH2, and which has good antimicrobial activity; chinese patent CN202011319757.9 discloses the use of the antimicrobial peptide Cm-CATH2 to control fusarium in grains; chinese patent CN202011319760.0 discloses an application of antibacterial peptide Cm-CATH2 to inhibit vibrio parahaemolyticus in seafood. The above patents all show that the antibacterial peptide Cm-CATH2 has better inhibition effect on bacteria, but no research on structural modification and anti-inflammatory effect is involved. Accordingly, it is desirable to provide an engineered body of the antimicrobial peptide Cm-CATH2 having both antimicrobial and anti-inflammatory effects as a potential antimicrobial and anti-inflammatory drug.

Disclosure of Invention

In order to solve the technical problems, the invention firstly shortens the peptide chain of the antibacterial peptide shown in SEQ ID NO.1 to obtain a series of shortened peptides, then researches on antibacterial, anti-inflammatory and hemolytic activities of the shortened peptides, screens out antibacterial peptides with better antibacterial activity, anti-inflammatory activity and lower hemolytic activity, and then replaces Arg and Lys in the antibacterial peptides with unnatural D-type amino acids (D-Arg and D-Lys) to obtain the modified antibacterial peptide.

The first object of the present invention is to provide a modified antibacterial peptide obtained by modifying an antibacterial peptide having an amino acid sequence shown in SEQ ID NO.1, wherein the modification comprises cleavage and removal of 8 amino acids at the N-terminal of the antibacterial peptide having an amino acid sequence shown in SEQ ID NO. 1.

Wherein, the amino acid sequence shown in SEQ ID NO.1 is:

Arg ¹ Arg ² Ser ³ Arg ⁴ Phe ⁵ Gly ⁶ Arg ⁷ Phe ⁸ Phe ⁹ Lys ¹⁰ Lys ¹¹ Val ¹² Arg ¹³ Lys ¹⁴ Gln ¹⁵ Leu ¹⁶ Gly ¹⁷ Arg ¹⁸ Val ¹⁹ Leu ²⁰ Arg ²¹ His ²² Ser ²³ Arg ²⁴ Ile ²⁵ Thr ²⁶ Val ²⁷ Gly ²⁸ Gly ²⁹ Arg ³⁰ Met ³¹ Arg ³² Phe ³³ 。

further, the amino acid sequence of the modified antibacterial peptide is shown as SEQ ID NO.2, and the specific sequence is as follows:

Phe ¹ Lys ² Lys ³ Val ⁴ Arg ⁵ Lys ⁶ Gln ⁷ Leu ⁸ Gly ⁹ Arg ¹⁰ Val ¹¹ Leu ¹² Arg ¹³ His ¹⁴ Ser ¹⁵ Arg ¹⁶ Ile ¹⁷ Thr ¹⁸ Val ¹⁹ Gly ²⁰ Gly ²¹ Arg ²² Met ²³ Arg ²⁴ Phe ²⁵ 。

further, the modification also comprises the steps of replacing arginine Arg in the amino acid sequence shown in SEQ ID NO.2 with unnatural D-type amino acid D-Arg, replacing lysine Lys with unnatural D-type amino acid D-Lys, and remaining natural L-type amino acids.

Further, the molecular weight of the engineered antibacterial peptide is 3026.67Da.

Further, the preparation method of the modified antibacterial peptide comprises the following steps: according to the amino acid sequence of the modified antibacterial peptide, a polypeptide solid-phase synthesis method is adopted to carry out chemical synthesis to obtain a complete sequence, and HPLC reversed phase column chromatography is used to carry out desalination to obtain the modified antibacterial peptide.

A second object of the present invention is to provide the use of the modified antimicrobial peptide described above for the preparation of an antimicrobial agent.

Further, the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.

Further, the gram positive bacteria include, but are not limited to, staphylococcus aureus (e.g., staphylococcus aureus CMCC26003, staphylococcus aureus ATCC43300, staphylococcus aureus 31, staphylococcus aureus 08032706), enterococcus faecalis (e.g., ATCC 29212), enterococcus faecium, clostridium perfringens (e.g., ATCC 13124), staphylococcus epidermidis, etc.

Further, the gram-negative bacteria include, but are not limited to, E.coli (e.g., E.coli ATCC25922, E.coli CMCC 44102), P.aeruginosa (e.g., P.aeruginosa CMCC 10104), A.baumannii (e.g., A.baumannii ATCC19606, A.baumannii 2, A.baumannii 6, A.baumannii 16), shigella flexneri (e.g., shigella flexneri ATCC 12022), salmonella typhimurium (e.g., salmonella typhimurium ATCC 14028), vibrio alginolyticus, vibrio harveyi, etc.

A third object of the present invention is to provide the use of the above-described engineered antibacterial peptide for the preparation of an anti-inflammatory agent.

Further, the anti-inflammatory agent is useful for inhibiting tumor necrosis factor (TNF- α) or interleukin 6 (IL-6).

The fourth object of the invention is to provide the application of the modified antibacterial peptide in preparing an antibacterial film medicament.

Further, the anti-biofilm agent is used for removing or inhibiting the formation of a biofilm.

A fifth object of the present invention is to provide an antibacterial pharmaceutical composition comprising the above-described modified antibacterial peptide and an antibiotic.

Further, the antibiotics may be meropenem, polymyxin B, ampicillin, vancomycin, and the like.

By means of the scheme, the invention has at least the following advantages:

(1) According to the invention, through two-step transformation of the mossback antibacterial peptide Cm-CATH2, the mossback antibacterial peptide Cm-CATH2 is firstly subjected to peptide chain shortening, and then Arg and Lys in the mossback antibacterial peptide are replaced by unnatural D-type amino acids (D-Arg and D-Lys) on the basis of the obtained optimal antibacterial peptide, so that the further transformed antibacterial peptide containing 25 amino acid residues and having a molecular weight of 3026.67Da has broad-spectrum efficient antibacterial activity and extremely strong anti-inflammatory activity.

(2) The modified antibacterial peptide has the advantages of simple structure, low hemolytic activity, high stability, simple preparation method and the like, can be applied to the fields of medicines, cosmetics, food fresh keeping, breeding and the like, and greatly expands downstream application.

The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.

FIG. 1 shows the results of an assay for anti-inflammatory activity of the engineered antimicrobial peptides dNCM2 and NCM 4;

FIG. 2 shows the stability assay results for the engineered antimicrobial peptides dNCM2 and NCM 4;

FIG. 3 is a graph showing the peak area change of stability test of the engineered antimicrobial peptides dNCM2 and NCM4 protease;

FIG. 4 shows the removal activity of the engineered antimicrobial peptides dNCM2 and NCM4 on bacterial biofilms; wherein A is A.baumannii ATCC19606 and B is S.aureus CMCC26003; * Represents P <0.05, < P <0.01, < P <0.001;

FIG. 5 is an inhibition of bacterial biofilm formation by the engineered antimicrobial peptides dNCM2 and NCM 4; wherein A is A.baumannii ATCC19606 and B is S.aureus CMCC26003; * Represents P <0.05, < P <0.01, < P <0.001.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1

Chemical synthesis of modified antibacterial peptide dNCM2

The mossback antibacterial peptide Cm-CATH2 is a polypeptide coded by a gene, contains 33 amino acid residues, has a molecular weight of 4089.9Da and an isoelectric point of 12.96. The complete sequence of the mossback antibacterial peptide Cm-CATH2 is as follows: arg (Arg) ¹ Arg ² Ser ³ Arg ⁴ Phe ⁵ Gly ⁶ Arg ⁷ Phe ⁸ Phe ⁹ Lys ¹⁰ Lys ¹¹ Val ¹² Arg ¹³ Lys ¹⁴ Gln ¹⁵ Leu ¹⁶ Gly ¹⁷ Arg ¹⁸ Val ¹⁹ Leu ²⁰ Arg ²¹ His ²² Ser ²³ Arg ²⁴ Ile ²⁵ Thr ²⁶ Val ²⁷ Gly ²⁸ Gly ²⁹ Arg ³⁰ Met ³¹ Arg ³² Phe ³³ . According to the amino acid sequence of the mossback antimicrobial peptide Cm-CATH2, the modified body NCM4 (the whole sequence is Phe) is designed and obtained by utilizing a molecular modification method ¹ Lys ² Lys ³ Val ⁴ Arg ⁵ Lys ⁶ Gln ⁷ Leu ⁸ Gly ⁹ Arg ¹⁰ Val ¹¹ Leu ¹² Arg ¹³ His ¹⁴ Ser ¹⁵ Arg ¹⁶ Ile ¹⁷ Thr ¹⁸ Val ¹⁹ Gly ² ⁰ Gly ²¹ Arg ²² Met ²³ Arg ²⁴ Phe ²⁵ ) Then, the NCM4 is further subjected to structural transformation of amino acid substitution, arg and Lys in the NCM are replaced by unnatural D-type amino acids (D-Arg and D-Lys), so that the antibacterial peptide dNCM2 is obtained, and the antibacterial peptide dNCM2 is chemically synthesized by utilizing a polypeptide solid-phase synthesis method, wherein the specific preparation method is as follows:

(1) The preparation method of dNCM2 comprises the following steps: based on the amino acid sequence of dNCM2, the complete sequence was synthesized by an automatic polypeptide synthesizer (433A,Applied Biosystems) and desalted by HPLC reversed-phase column chromatography.

(2) Molecular weight determination uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

(3) The purity of the purified dNCM2 was identified by high performance liquid chromatography HPLC.

The measurement results are as follows:

dNCM2 is mossback antibacterial peptideA modification of Cm-CATH2. dNCM2 is a linear polypeptide containing 25 amino acid residues and having a molecular weight of 3026.67Da. dNCM2 total sequence is: phe (Phe) ¹ Lys ² Lys ³ Val ⁴ Arg ⁵ Lys ⁶ Gln ⁷ Leu ⁸ Gly ⁹ Arg ¹⁰ Val ¹¹ Leu ¹² Arg ¹³ His ¹⁴ Ser ¹⁵ Arg ¹⁶ Ile ¹⁷ Thr ¹⁸ Val ¹⁹ Gly ²⁰ Gly ²¹ Arg ²² Met ²³ Arg ²⁴ Phe ²⁵ Wherein all lysine and arginine are unnatural D-amino acids, and the rest are natural L-amino acids.

Example 2

Pharmacological experiments of the engineered antibacterial peptides dNCM2 and NCM 4:

1. determination of antibacterial Activity of the engineered antibacterial peptides dNCM2 and NCM 4:

(1) Test strains stored on the inclined planes are respectively picked up and evenly coated on a flat plate of MH solid culture medium (Beijing Soy Bao technology Co., ltd.), a sterilized filter paper sheet with the diameter of 0.5Cm is placed on the surface of the culture medium, 10 mu l of a 2mg/ml antibacterial peptide dNCM2, NCM4 or Cm-CATH2 sample solution dissolved in sterilized deionized water is dripped, and the culture is inverted at 37 ℃ for 18-20 hours, so that whether a bacteriostasis ring is formed or not is observed. If the sample has antibacterial activity, clear and transparent antibacterial circle can be formed around the filter paper sheet, and the larger the antibacterial circle is, the stronger the antibacterial activity of the sample is.

(2) Determination of minimum inhibitory concentration (Minimum Inhibitory Concentration) of the remodeled antimicrobial peptides dNCM2 and NCM4 (2-fold dilution method):

and selecting the strain with the inhibition zone in the previous experiment for MIC determination experiment. The test strain was inoculated into MH liquid medium (Beijing Soy Bao technology Co., ltd.) and shaking-cultured at 37℃to logarithmic phase, and then the culture broth cultured to logarithmic phase was diluted to 2X 10 with fresh MH liquid medium ⁵ cfu/ml for use.

Mu.l of MH liquid culture medium is added in advance to each well of a sterile 96-well plate, then 100 mu.l of sample solution of the antibacterial peptide dNCM2, NCM4 or Cm-CATH2 which is diluted to a certain concentration by the MH liquid culture medium and filtered by a filter membrane of 0.22 mu m is added to the first well, 100 mu.l of sample solution is added to the 2 nd well after uniform mixing, 100 mu.l of sample solution is sequentially diluted by multiple ratio (see Table 1), 100 mu.l of sample solution is sucked out from the 9 th well and discarded, and the 10 th well is a control tube.

TABLE 1 dilution method

After the above tubes were mixed, the mixture was left to stand at 37℃for 18 hours with slow shaking, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible.

TABLE 2 antibacterial Activity of engineered antibacterial peptides dNCM2 and NCM4

MIC: minimum inhibitory concentration, the above results are the average of three independent replicates.

As can be seen from Table 2, the unmodified antimicrobial peptide Cm-CATH2 and the modified antimicrobial peptides NCM4 and dNCM2 exhibited extremely strong antimicrobial activity against both gram-positive bacteria and gram-negative bacteria, including a portion of clinically isolated pathogenic bacteria, the MIC value of the test bacteria of NCM4 was 18.75. Mu.g/ml or less, and the MIC value of the test bacteria of dNCM2 was in the range of 9.38-75. Mu.g/ml.

2. Determination of the haemolytic activity of the engineered antibacterial peptides dNCM2 and NCM 4:

mixing the collected rabbit blood with Alzhi solution for anticoagulation, washing with physiological saline for 2 times, and re-suspending to 10 times ⁷ -10 ⁸ cell/ml suspension. The diluted erythrocyte suspension is mixed with dNCM2, NCM4 or Cm-CATH2 sample dissolved in physiological saline, heat-preserved for 30min at 37 ℃, centrifuged at 1000rpm for 5min, and the supernatant is measured for absorption at 540 nm. The negative control used physiological saline and the positive control used Triton X-100, percent hemolysis calculated according to the following formula: percent hemolysis H% = a _{Sample of} -A _{Negative control} /A _{Positive control} ×100％。

The results showed that the sample concentration was 100. Mu.g/ml, the percent hemolysis of dNCM2 was 1.29%, the percent hemolysis of NCM4 was 1.51%, and the percent hemolysis of Cm-CATH2 was 4.1%. Indicating that dNCM2 and NCM4 have lower hemolytic activity and are not easy to cause rupture and dissolution of mammalian red blood cells. In particular, the safety is high in the antibacterial activity range.

3. Determination of anti-inflammatory Activity of engineered antibacterial peptides dNCM2 and NCM 4:

abdominal macrophages from 6-8 week old C57 mice were extracted, cultured overnight in 1640 medium with 10% serum, changed to 1640 medium with 2% serum the next day, cells were stimulated with E.coli LPS (Sigma, USA) at a final concentration of 100ng/mL, and the polypeptide dNCM2 or NCM4 at a final concentration of 20. Mu.g/mL, a blank control group without polypeptide and LPS was incubated with a positive control group with LPS only for 16h, the supernatant was removed, and the content of pro-inflammatory factors IL-6 and TNF-. Alpha.in the supernatant was detected with ELISA kit (R & D, USA). Each three in parallel.

The results are shown in FIG. 1, dNCM2 can obviously inhibit the expression of LPS-induced pro-inflammatory factors IL-6 and TNF-alpha in mouse peritoneal macrophages, which indicates that dNCM2 has extremely strong anti-inflammatory activity and the anti-inflammatory activity is equivalent to NCM 4.

4. Stability experimental study of engineered antibacterial peptides dNCM2 and NCM 4:

(1) Enzyme stability test: 0.25% pancreatin for cell digestion was used with the polypeptide samples in a molar ratio of 1:200, incubating at 37 ℃, sampling 50 mu L at 0, 6, 12 and 24 hours respectively, diluting the sampled product by 1 time with polypeptide solvent, filtering with a 0.22 mu m filter membrane, taking 20 mu L, and measuring the residual quantity of the polypeptide sample by reverse high performance liquid chromatography. Wherein phase A was eluted with pure water containing 0.1% trifluoroacetic acid (TFA) and phase B was eluted with acetonitrile containing 0.1% TFA in a gradient to obtain elution peaks and integration areas at various time points after mixing of polypeptide samples dNCM2 or NCM4 with pancreatin, and then plotted with software Origin 2018.

As shown in fig. 2 and 3, the antibacterial peptide dNCM2 has very strong enzyme stability, the peak height and peak area of dNCM2 are still not changed greatly after the antibacterial peptide dNCM2 acts for 24 hours, and the antibacterial peptides NCM4 and Cm-CATH26 hours are obviously degraded, which indicates that the enzyme stability of dNCM2 is obviously better than that of NCM4 and Cm-CATH2.

(2) Salt tolerance, heat tolerance and heat stability test

Salt tolerance: coli ATCC25922 was cultured with MH liquid Medium (Qingdao sea Bo Biotechnology Co., ltd.) at 37℃for 12 hours, and then diluted to 10 with fresh MH liquid Medium containing 0, 50, 100, 150, 200 and 400mM sodium chloride, respectively ⁶ CFU/ml. dNCM2, NCM4 and Cm-CATH2 samples of different concentration gradients were prepared using MH broth with corresponding sodium chloride concentrations. The effect of sodium chloride on the antibacterial activity of dNCM2, NCM4 and Cm-CATH2 was determined by measuring the MIC values of dNCM2, NCM4 and Cm-CATH2 for E.coli ATCC25922 using a 2-fold dilution method.

Heat resistance: dNCM2, NCM4 or Cm-CATH2 was dissolved in sterilized deionized water (2 mg/ml), incubated at 4, 20, 37, 50, 70 and 90℃for 1 hour, and then the MIC value of the sample for E.coli ATCC25922 was determined by a 2-fold dilution method, thereby determining the effect of heating at different temperatures on the antimicrobial activity of the sample.

Thermal stability: dNCM2, NCM4 or Cm-CATH2 was dissolved in sterilized deionized water (2 mg/ml) and incubated at 37℃for 0-96 hours. The samples were assayed for MIC values for E.coli ATCC25922 at 0, 6, 12, 24, 48, 72 and 96 hours, respectively, to determine the thermal stability of the samples.

As shown in Table 3, dNCM2, NCM4 and Cm-CATH2 were very salt tolerant. The antibacterial activity of dNCM2, NCM4 and Cm-CATH2 is kept unchanged at a concentration lower than or equal to the physiological salt concentration of human body (less than or equal to 150mM NaCl). After the salt concentration is higher than the physiological salt concentration of a human body, the antibacterial activity of dNCM2 and NCM4 is only slightly reduced along with the increase of the salt concentration, and particularly the antibacterial activity of dNCM2 is kept unchanged under the condition of 200mM NaCl.

TABLE 3 modified antibacterial peptides dNCM2 and NCM4 salt tolerance

As shown in table 4, dNCM2 has very high heat resistance. The antibacterial activity of the dNCM2 solution was unchanged after 1 hour at 90 ℃. In contrast, NCM4 and Cm-CATH2 were slightly inferior in heat resistance, and after heating at high temperature for 1 hour, MIC values were elevated.

TABLE 4 heat tolerance of engineered antibacterial peptides dNCM2 and NCM4

Many conventional antibiotics, such as solutions of cephalosporins, are extremely unstable and lose activity within a few hours, which greatly limits their use. In contrast, dNCM2 solutions have good thermal stability. The antibacterial activity of the dNCM2 solution was unchanged after 96 hours at 37 ℃ (see Table 5). In contrast, NCM4 and Cm-CATH2 were slightly less thermally stable and had elevated MIC values after 96 hours at 37 ℃.

TABLE 5 engineering antibacterial peptide dNCM2 thermal stability

Example 3

Biofilm removal and inhibition activity assay for engineered antibacterial peptides dNCM2 and NCM4

1. Biofilm removal Activity assay

Taking out the preserved strain from the refrigerator at-80 ℃, quickly melting in a water bath at 37 ℃, dipping a little liquid by using an inoculating loop, marking lines in four Z-shaped areas on an LB solid culture medium, and culturing at constant temperature at 37 ℃ from the last end until single bacterial colony grows; picking single colony in sterile liquid MHB culture medium, shake culturing at 37deg.C and 180rpm to logarithmic phase; detecting bacterial liquid concentration, diluting to 2×10 ⁷ CFU/mL. 200. Mu.L of the above-mentioned bacterial solution was added to a sterile 96-well plate, and the mixture was cultured at 37℃for 48 hours to form a biofilm. The bacterial solution from each well was aspirated and washed three times with PBS. Each hole is added with200. Mu.L of the diluted polypeptide sample was incubated at 37℃for 24 hours at a final concentration of 0.5 XMIC, 1 XMIC, 2 XMIC, 4 XMIC and 8 XMIC. Crystal violet dye solution (0.1%) is added to each hole, the dye solution is sucked out after dyeing for 30min, and the solution is washed three times by sterile PBS and air-dried in an ultra clean bench. Adding 100 mu L of absolute ethyl alcohol into each hole, standing for 20min, and dissolving crystal violet. OD values were measured at ultraviolet wavelength 560nm and the experiment was set up in three replicates. The percentage of biofilm formation (Biofilm Retention%, BR%) was calculated using the following formula: BR (%) =100% - [100% × (F) ₀ -F _peptide )/F ₀ ]PBS was selected as a negative control in this experiment and its measurement was taken as the maximum biofilm residual amount. Biofilm Retention% BR% percent biofilm survival, F ₀ Absorbance for PBS treated group, F _peptide Absorbance values for the polypeptide treatment groups.

2. Biofilm inhibition activity assay

Taking out the preserved strain from the refrigerator at-80 ℃, rapidly melting in a water bath at 37 ℃, dipping a little liquid, marking the strain in a Z shape on an LB solid culture medium, and culturing at the constant temperature of 37 ℃ until single colony grows; picking single colony in sterile liquid MHB culture medium, shaking culture at 37deg.C and 180rpm to logarithmic phase; detecting bacterial liquid concentration, diluting to 2×10 ⁷ CFU/mL. 190. Mu.L of the bacterial liquid is added into a sterile 96-well plate, diluted polypeptide samples are added into each well, and 3 parallel concentrations are set. The final concentration of the polypeptide samples was 0.5×MIC, 1×MIC, 2×MIC, 4×MIC and 8×MIC, and incubated at 37℃for 48 hours. Sucking out the bacterial liquid in each hole, washing the bacterial liquid three times by PBS, and placing the plate on an ultra clean bench for ventilation and blow-drying. Crystal violet dye solution (0.1%) is added to each hole, the dye solution is sucked out after dyeing for 30min, and the solution is washed three times by sterile PBS and air-dried in an ultra clean bench. Adding 100 mu L of absolute ethyl alcohol into each hole, standing for 20min, and dissolving crystal violet. OD was measured at uv wavelength 560 nm. The percentage of biofilm formation (Biofilm Formation%, BF%) was calculated using the following formula: BF (%) =100% - [100% × (F) ₀ -F _peptide )/F ₀ ]Wherein PBS (F) ₀ ) As a negative control, the measured value was regarded as the maximum biofilm formation amount. Biofilm Formation% and BF% are percentages of biofilm formation, F _peptide Absorbance values for the polypeptide treatment groups.

As shown in fig. 4 and 5, both the modified polypeptides dNCM2 and NCM4 were effective in clearing biofilm produced by acinetobacter baumannii (fig. 4A) and staphylococcus aureus (fig. 4B), and concentration dependence was present, and dNCM2 performed better than NCM4 in clearing biofilm produced by acinetobacter baumannii. Both the engineered polypeptides dNCM2 and NCM4 were able to inhibit biofilm production by Acinetobacter baumannii (FIG. 5A) and Staphylococcus aureus (FIG. 5B) at high concentrations. Compared with NCM4, dNCM2 can better inhibit the generation of biological membrane by staphylococcus aureus, and dNCM2 can better inhibit the generation of biological membrane by Acinetobacter baumannii.

Example 4

Determination of synergistic antibacterial effect of modified antibacterial peptides dNCM2 and NCM4 and antibiotics

The polypeptide is prepared into 20X 8MIC concentration by using sterile water, and sequentially subjected to double dilution until 20X 1/64MIC and 11 total concentrations of solvent are added for standby. Weighing antibiotic (meropenem, polymyxin B, ampicillin and vancomycin) medicines, dissolving the medicines into 2mg/mL solution by using sterile water, sequentially diluting the medicines by times to obtain the concentration of 4MIC-1/16MIC, and adding 8 total concentrations of solvent for standby. Diluting the bacterial liquid to 5X 10 ⁵ CFU/mL, ready for use.

Adding 90 mu L diluted bacterial liquid into a 96-well plate for measurement by a chessboard method; the polypeptides were added to the bacteria at 5 μl per well, one concentration per column, 11 columns total: antibiotics were added to the bacteria at 5 μl per well, one concentration per row, for a total of 8 rows; finding MICA, MICB, a, B calculates FIC, fic=fmica+fmicb=a/mica+b/MICB (a, B represents the concentration at the optimal aggregation point for the combination of two drugs, MICA, MICB represents the MIC for the single two drugs), FIC < 0.5 has synergy, FIC > 4 has antagonism, and 0.5 < FIC < 4 has additive effects. The dosing mode is shown in the following table.

Acinetobacter baumannii (A.baumannii ATCC 19606) and methicillin-resistant staphylococcus aureus (S.aureus ATCC 43300) are selected as test bacteria, and the synergistic antibacterial effect of several antibiotics and the modified antibacterial peptide dNCM2 is detected. Wherein, both meropenem and polymyxin B can cooperatively play the role of resisting Acinetobacter baumannii, and FICI indexes are respectively 0.25 and 0.375. The ampicillin and vancomycin can cooperate with the anti-body-modifying antibacterial peptide dNCM2 to resist methicillin-resistant staphylococcus aureus, and FICI indexes are respectively 0.375 and 0.1875.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Sequence listing

<110> university of Suzhou

<120> an engineered antibacterial peptide and use thereof

<140> 2022104657197

<141> 2022-04-29

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<170> SIPOSequenceListing 1.0

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Arg Arg Ser Arg Phe Gly Arg Phe Phe Lys Lys Val Arg Lys Gln Leu

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Gly Arg Val Leu Arg His Ser Arg Ile Thr Val Gly Gly Arg Met Arg

20 25 30

Phe

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Phe Lys Lys Val Arg Lys Gln Leu Gly Arg Val Leu Arg His Ser Arg

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20 25

Claims

1. An application of a modified antibacterial peptide in preparing an antibacterial agent is characterized in that: the amino acid sequence of the modified antibacterial peptide is as follows: phe (Phe) ¹ Lys ² Lys ³ Val ⁴ Arg ⁵ Lys ⁶ Gln ⁷ Leu ⁸ Gly ⁹ Arg ¹⁰ Val ¹¹ Leu ¹² Arg ¹³ His ¹⁴ Ser ¹⁵ Arg ¹⁶ Ile ¹⁷ Thr ¹⁸ Val ¹⁹ Gly ²⁰ Gly ²¹ Arg ²² Met ²³ Arg ²⁴ Phe ²⁵ Wherein, the arginine is D-type arginine and the lysine is D-type lysine.

2. The use according to claim 1, characterized in that: the antibacterial agent is used for inhibiting gram positive bacteria or gram negative bacteria.

3. The use according to claim 2, characterized in that: the gram positive bacteria include staphylococcus aureus, enterococcus faecalis, enterococcus faecium, clostridium perfringens or staphylococcus epidermidis.

4. The use according to claim 2, characterized in that: the gram negative bacteria comprise escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii, shigella flexneri, salmonella typhimurium, vibrio alginolyticus or vibrio harveyi.

5. An antibacterial pharmaceutical composition characterized in that: the antibacterial pharmaceutical composition comprises a modified antibacterial peptide and an antibiotic;

the modified body is antibacterialThe amino acid sequence of the peptide is: phe (Phe) ¹ Lys ² Lys ³ Val ⁴ Arg ⁵ Lys ⁶ Gln ⁷ Leu ⁸ Gly ⁹ Arg ¹⁰ Val ¹¹ Leu ¹² Arg ¹³ His ¹⁴ Ser ¹⁵ Arg ¹⁶ Ile ¹⁷ Thr ¹⁸ Val ¹⁹ Gly ²⁰ Gly ²¹ Arg ²² Met ²³ Arg ²⁴ Phe ²⁵ Wherein, the arginine is D-type arginine and the lysine is D-type lysine;

when the antibiotic is selected from meropenem or polymyxin B, the antibacterial pharmaceutical composition is for use against acinetobacter baumannii; when the antibiotic is selected from ampicillin or vancomycin, the antibacterial pharmaceutical composition is for use against methicillin-resistant staphylococcus aureus.