Antibacterial-like peptide and application thereof

Abstract

The invention belongs to the technical field of anti-infection of antibacterial peptide-like drugs, and provides an antibacterial peptide-like and application thereof. According to the antibacterial peptide, olefin functionalized amino acid is used as a monomer, and the antibacterial peptide is obtained through free radical polymerization reaction. The antibacterial peptide does not inhibit the growth of bacteria for gram-positive bacteria and gram-negative bacteria, but has the effect of removing bacterial biofilms, and meanwhile, the antibacterial peptide has excellent biocompatibility and is non-toxic. The raw materials and reagents are easy to obtain, the preparation process is simple, and the method has wide prospects in the fields of clinical infection treatment, environmental protection and food safety.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide-like drugs for anti-infection, and in particular to an antimicrobial peptide-like drug and application thereof. Background Art

[0002] Bacterial biofilm, also known as biofilm, is widely present on various wet surfaces containing water, such as food, food processing equipment, water pipes, industrial pipes, ventilation equipment, medical equipment and even the surface of human tissues and organs under pathological conditions. It is a structural bacterial community composed of bacterial cells attached to the surface of inert or active entities and the hydrated matrix that wraps the bacteria. Almost all bacteria can form biofilm under certain conditions. Salmonella, Escherichia coli, Listeria, Staphylococcus aureus, etc. are common pathogens that can easily cause foodborne diseases. They exist in the air, water, dust or human and animal excrement. Food is more likely to be contaminated by them. For example, food processing personnel, cooks or sales personnel carry bacteria, causing food contamination; food carries bacteria before processing, or is contaminated during processing, producing toxins and causing food poisoning; cooked food products are not tightly packaged and are contaminated during transportation; poultry and livestock carry bacteria before slaughter, which can also contaminate food. Bacteria are easy to form biofilms on food, various food processing contact surfaces and non-food processing contact surfaces (such as walls, sewers, dead ends, etc.), and then contaminate food through hands or air, causing food poisoning. In addition to corroding pipes and metal surfaces, the metabolic activities of bacteria in biofilms are more likely to cause animal, plant and human diseases.

[0003] Antimicrobial peptides have broad-spectrum antimicrobial activity and have a strong killing effect on bacteria, especially their killing effect on certain drug-resistant pathogens has attracted people's attention. As a new type of antimicrobial preparation, the use of antimicrobial peptides provides a new idea and means for the treatment of biofilm infection. Antimicrobial peptides have played a unique advantage in inhibiting biofilm formation, killing bacteria in biofilms, and eliminating mature biofilms. However, due to the strong survival ability of bacterial biofilms, the ineffectiveness of conventional antibiotics against biofilms, the drug resistance caused by long-term use, and the harm caused to patients by high doses, the existing antimicrobial preparations are not ideal for removing bacterial biofilms. Therefore, how to develop an anti-biofilm agent with excellent removal effect has become a problem that those skilled in the art need to solve urgently. Summary of the invention

[0004] In view of this, the present invention provides an antimicrobial peptide and its application, the purpose of which is to solve the technical problem that the existing antimicrobial preparations have poor removal effect on bacterial biofilm.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides an antimicrobial peptide having the function of removing bacterial biofilm, which has a general structural formula as shown in formula (1):

[0007]

[0008] Wherein, n is 5 to 100; the structural formula of R is shown in formula (II) to (V):

[0009]

[0010] The present invention provides the use of the antimicrobial peptides in clearing bacterial biofilms, wherein the bacteria in the bacterial biofilms include Gram-negative bacteria and / or Gram-positive bacteria.

[0011] Furthermore, the method for preparing the antimicrobial peptide-like substance comprises the following steps:

[0012] S1, mixing an amino acid, an olefin compound and water and reacting them to obtain a reaction product, and sequentially extracting, drying, rotary evaporating and vacuum drying the reaction product to obtain an olefin-functionalized amino acid monomer;

[0013] S2. The olefin-functionalized amino acid monomer, chain transfer agent, catalyst and solvent are mixed and then subjected to polymerization reaction to obtain the antimicrobial peptide-like peptide.

[0014] Further, in step S1, the amino acid includes N α -tert-Butyloxycarbonyl-D-lysine, N ε -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-tyrosine, N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid and N β -tert-butyloxycarbonyl-D-2,3-diaminopropionic acid;

[0015] The olefin compound comprises acryloyl chloride and / or methacryloyl chloride.

[0016] Furthermore, in step S1, the usage ratio of the amino acid, the olefin compound and water is 800-1200 mg: 300-600 mg: 3-10 mL.

[0017] Furthermore, in step S1, the reaction temperature is 0 to 30° C., and the reaction time is 3 to 8 hours.

[0018] Further, in step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothiothio)propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester and 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid];

[0019] The catalyst includes one or more of 2′,4′,5′,7′-tetrabromofluorescein, tetraphenylporphyrin zinc, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate, and azobisisobutyronitrile;

[0020] The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid and hydrogen peroxide.

[0021] Furthermore, in step S2, the molar ratio of the olefin functionalized amino acid monomer to the chain transfer agent is 20 to 150:1;

[0022] The molar ratio of the olefin functionalized amino acid monomer to the catalyst is 10 3 ~10 5 : 1;

[0023] The mass volume ratio of the olefin functionalized amino acid monomer and the solvent is 0.5-1.5 g:7 mL.

[0024] Furthermore, in step S2, the polymerization reaction time is 4 to 12 hours.

[0025] Furthermore, in step S2, the polymerization reaction includes RAFT thermal polymerization or RAFT photopolymerization;

[0026] The reaction temperature of the RAFT thermal polymerization is 60-90°C; the reaction temperature of the RAFT photopolymerization is 20-30°C.

[0027] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The antimicrobial peptide prepared by the present invention can show the effect of not inhibiting bacterial growth but clearing bacterial biofilm on both Gram-positive bacteria (such as Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), and also has excellent biocompatibility and non-toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Flow chart of preparing antimicrobial peptide-like peptides according to Example 1;

[0030] Figure 2 N prepared in Example 1 β-Acryloyl N α -Hydrogen spectrum of tert-butyloxycarbonyl-D-2,3-diaminopropionic acid monomer;

[0031] Figure 3 This is the hydrogen spectrum of the antimicrobial peptide prepared in Example 1;

[0032] Figure 4 The biofilm removal effects of different types of antimicrobial peptides are shown in Figure 1. (A) Optical photo of biofilm; (B) Statistical results of biofilm removal efficiency;

[0033] Figure 5 The effect of different types of antimicrobial peptides on biofilm components: (A) bacterial number; (B) biofilm protein composition; (C) biofilm polysaccharide composition;

[0034] Figure 6 The biofilm removal effects of antimicrobial peptides of different molecular weights, (A) optical photograph of biofilm; (B) statistical results of biofilm removal efficiency. DETAILED DESCRIPTION

[0035] The present invention provides an antimicrobial peptide having the function of removing bacterial biofilm, which has a general structural formula as shown in formula (1):

[0036]

[0037] wherein n is 5 to 100, preferably 10 to 80, and more preferably 20 to 60;

[0038] The structural formula of R is shown in formula (II) to (V):

[0039]

[0040] In the present invention, the antimicrobial peptide-like peptide is used in clearing bacterial biofilms, and the bacteria in the bacterial biofilms include Gram-negative bacteria and / or Gram-positive bacteria.

[0041] In the present invention, the Gram-negative bacteria are preferably Escherichia coli or Pseudomonas aeruginosa; the Gram-positive bacteria include Staphylococcus aureus or Staphylococcus epidermidis.

[0042] In the present invention, a mature biofilm model cultured on a titanium sheet is used for anti-biofilm detection. The specific model is as follows: a single bacterial colony is dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet is placed in a well plate, and sterile TSB is diluted ten times and then added to the well plate, and static culture is performed at 37°C for 24 hours to obtain a mature biofilm; the culture medium is removed, and sterile TSB with dissolved antimicrobial peptides is added, and the control group is only sterile TSB, and cultured for another 24 hours; the culture medium is removed, crystal violet is added for staining for 30 minutes, the unfixed components are washed off, 30% acetic acid is added to dissolve, and OD595 is measured to characterize the biomass of the biofilm.

[0043] The calculation method of biofilm removal rate is shown in formula (VI):

[0044]

[0045] The present invention also provides a method for preparing the above-mentioned antimicrobial peptide, comprising the following steps:

[0046] S1, mixing an amino acid, an olefin compound and water and reacting them to obtain a reaction product, and sequentially extracting, drying, rotary evaporating and vacuum drying the reaction product to obtain an olefin-functionalized amino acid monomer;

[0047] S2. The olefin-functionalized amino acid monomer, chain transfer agent, catalyst and solvent are mixed and then subjected to polymerization reaction to obtain the antimicrobial peptide-like peptide.

[0048] In the present invention, in step S1, the amino acid comprises N α -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-tyrosine, N ε -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid and N β -tert-butyloxycarbonyl-D-2,3-diaminopropionic acid;

[0049] The olefin compound comprises acryloyl chloride and / or methacryloyl chloride, preferably acryloyl chloride.

[0050] In the present invention, in step S1, the usage ratio of amino acid, olefin compound and water is 800-1200 mg: 300-600 mg: 3-10 mL, preferably 900-1100 mg: 400-500 mg: 5-9 mL, and more preferably 950-1000 mg: 440-480 mg: 6-8 mL.

[0051] In the present invention, in step S1, the reaction temperature is 0 to 30°C, preferably 5 to 25°C, and more preferably 10 to 20°C; the reaction time is 3 to 8h, preferably 4 to 7h, and more preferably 5 to 6h.

[0052] In the present invention, in step S1, the pH of the reaction system is preferably 10.5.

[0053] In the present invention, in step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothiothio)propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester and 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid];

[0054] The catalyst includes one or more of 2′,4′,5′,7′-tetrabromofluorescein, tetraphenylporphyrin zinc, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate, and azobisisobutyronitrile;

[0055] The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid and hydrogen peroxide.

[0056] In the present invention, in step S2, the molar ratio of the olefin functionalized amino acid monomer to the chain transfer agent is 20 to 150:1, preferably 40 to 120:1, and more preferably 60 to 100:1;

[0057] The molar ratio of the olefin functionalized amino acid monomer to the catalyst is 10 3 ~10 5 : 1, preferably 10 4 : 1;

[0058] The mass volume ratio of the olefin functionalized amino acid monomer to the solvent is 0.5-1.5 g:7 mL, preferably 0.8-1.2 g:7 mL, and more preferably 1.0 g:7 mL.

[0059] In the present invention, in step S2, the polymerization reaction time is 4 to 12 hours, preferably 5 to 10 hours, and more preferably 6 to 8 hours.

[0060] In the present invention, in step S2, deoxygenation is also performed before the polymerization reaction, and the deoxygenation time is 40 to 60 minutes, preferably 50 minutes.

[0061] In the present invention, in step S2, the polymerization reaction includes RAFT thermal polymerization or RAFT photopolymerization;

[0062] The reaction temperature of the RAFT thermal polymerization is 60-90°C, preferably 65-85°C, and more preferably 70-80°C; the reaction temperature of the RAFT photopolymerization is 20-30°C, preferably 22-28°C, and more preferably 24-26°C.

[0063] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0064] Example 1

[0065] The flow chart of preparing antimicrobial peptides in this example is as follows Figure 1 shown.

[0066] Weigh 1000 mg N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid, add 5mL of deionized water, adjust the pH to 10.5 with sodium hydroxide solution, stir and dissolve under ice bath, slowly add 532mg of acryloyl chloride to the system under stirring, and adjust the pH to 10.5 in real time. Remove the ice bath after 30min and react at 25℃ for 4h. Then adjust the pH to 2.9, extract with ethyl acetate 3 times, and dry with anhydrous sodium sulfate for 8h. Finally, remove the solvent by rotary evaporation and vacuum dry for 24h to obtain N β -Acryloyl-N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid monomer.

[0067] 10 g N β -Acryloyl-N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid monomer was added to a quartz bottle, and 70 mL of dimethyl sulfoxide was added to dissolve it. The molar ratio of monomer to chain transfer agent was 60:1, and the molar ratio of monomer to catalyst was 10:1. 5 :1, 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid and azobisisobutyronitrile were added to a quartz bottle and sealed with a rubber stopper, then nitrogen was passed through a long and short needle to deoxygenate for 50 minutes, and the needle port was sealed. RAFT thermal polymerization was carried out at 60°C for 10 hours to obtain an antimicrobial peptide (1).

[0068] The prepared N β -Acryloyl-N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid monomer ( Figure 2 ) and antimicrobial peptides ( Figure 3 ) is characterized by Figure 2 and Figure 3 It can be seen that the monomer and polymer were successfully prepared, and the degree of polymerization of the polymer was 33.

[0069] Example 2

[0070] Weigh 800mgN β-tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid, add 5mL of deionized water, adjust the pH to 10.5 with sodium hydroxide solution, stir and dissolve under ice bath, slowly add 532mg of acryloyl chloride to the system under stirring, and adjust the pH to 10.5 in real time. Remove the ice bath after 30min and react at 25℃ for 4h. Then adjust the pH to 2.9, extract with ethyl acetate 3 times, and dry with anhydrous sodium sulfate for 8h. Finally, remove the solvent by rotary evaporation and vacuum dry for 24h to obtain N α -Acryloyl-N β -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid monomer.

[0071] 12g N α -Acryloyl-N β -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid monomer was added to a quartz bottle, and 70 mL of dimethyl sulfoxide was added to dissolve it. The molar ratio of monomer to chain transfer agent was 60:1, and the molar ratio of monomer to catalyst was 10:1. 5 : 1. Add 2-(dodecylthiocarbonylthiothiothio) propionic acid and sodium bicarbonate into a quartz bottle and seal the bottle with a rubber stopper. Then, pass nitrogen through long and short needles to deoxygenate for 50 minutes, and seal the needle opening. Perform RAFT thermal polymerization at 70°C for 8 hours to obtain an antimicrobial peptide (2).

[0072] Example 3

[0073] Weigh 1200mgN α -tert-Butyloxycarbonyl-D-lysine, add 5mL of deionized water, adjust the pH to 10.5 with sodium hydroxide solution, stir and dissolve under ice bath, slowly add 393.2mg of acryloyl chloride to the system under stirring, and adjust the pH to 10.5 in real time. Remove the ice bath after 30min and react at 25℃ for 4h. Then adjust the pH to 2.9, extract with ethyl acetate 3 times, and dry with anhydrous sodium sulfate for 8h. Finally, remove the solvent by rotary evaporation and vacuum dry for 24h to obtain N ε -Acryloyl-N α -tert-Butyloxycarbonyl-D-lysine monomer.

[0074] 9g N ε -Acryloyl-N α -tert-Butyloxycarbonyl-D-lysine monomer was added to a quartz bottle, and 70 mL of dimethyl sulfoxide was added to dissolve it. The molar ratio of monomer to chain transfer agent was 60:1, and the molar ratio of monomer to catalyst was 10:1. 5 : 1. Add 2-(dodecylthiocarbonylthiothiothio) propionic acid and tetraphenylporphyrin zinc into a quartz bottle and seal the quartz bottle with a rubber stopper. Then, pass nitrogen through long and short needles to deoxygenate for 50 minutes, and seal the needle opening. Perform RAFT thermal polymerization at 80°C for 6 hours to obtain an antimicrobial peptide (3).

[0075] Example 4

[0076] Weigh 1000mgN ε -tert-Butyloxycarbonyl-D-lysine, add 5mL of deionized water, adjust the pH to 10.5 with sodium hydroxide solution, stir and dissolve under ice bath, slowly add 393.2mg of acryloyl chloride to the system under stirring, and adjust the pH to 10.5 in real time. Remove the ice bath after 30min and react at 25℃ for 4h. Then adjust the pH to 2.9, extract with ethyl acetate 3 times, and dry with anhydrous sodium sulfate for 8h. Finally, remove the solvent by rotary evaporation and vacuum dry for 24h to obtain N α -Acryloyl-N ε -tert-Butyloxycarbonyl-D-lysine monomer.

[0077] 15 g N α -Acryloyl-N ε -tert-Butyloxycarbonyl-D-lysine monomer was added to a quartz bottle, and 70 mL of dimethyl sulfoxide was added to dissolve it. The molar ratio of monomer to chain transfer agent was 60:1, and the molar ratio of monomer to catalyst was 10:1. 4 : 1, 2-(dodecylthiocarbonylthiothiothio) propionic acid and tetraphenylporphyrin zinc were added to a quartz bottle and sealed with a rubber stopper, then nitrogen was passed through a long and short needle to deoxygenate for 50 minutes, and the needle mouth was sealed. RAFT thermal polymerization was carried out at 80°C for 6 hours to obtain an antimicrobial peptide (4).

[0078] Example 5

[0079] Weigh 1000mgN α -tert-Butyloxycarbonyl-D-tyrosine, add 5mL of deionized water, adjust the pH to 10.5 with sodium hydroxide solution, stir and dissolve under ice bath, slowly add 593mg of methacryloyl chloride to the system under stirring, and adjust the pH to 10.5 in real time. Remove the ice bath after 30min and react at 25℃ for 4h. Then, adjust the pH to 3.8, extract with ethyl acetate 3 times, and dry with anhydrous sodium sulfate for 8h. Finally, remove the solvent by rotary evaporation and vacuum dry for 24h to obtain O-methacryloyl-N α -tert-Butyloxycarbonyl-D-tyrosine monomer.

[0080] 8 g O-methacryloyl-N α -tert-butyloxycarbonyl-D-tyrosine monomer was added to a quartz bottle, and 70 mL of dimethyl sulfoxide was added to dissolve it. The molar ratio of monomer to chain transfer agent was 60:1, and the molar ratio of monomer to catalyst was 10:1. 3: 1. S-(2-cyano-2-propyl)-S-dodecyl trithiocarbonyl ester and sodium bicarbonate were added to a quartz bottle and sealed with a rubber stopper. Subsequently, nitrogen was passed through the long and short needles for deoxygenation for 50 minutes, and the needle opening was sealed. RAFT thermal polymerization was carried out at 70°C for 9 hours to obtain an antimicrobial peptide.

[0081] Example 6

[0082] The same as Example 1, except that the molar ratio of the monomer to the chain transfer agent is 20:1, and an antimicrobial peptide (5) is prepared.

[0083] Example 7

[0084] The same as Example 1, except that the molar ratio of the monomer to the chain transfer agent is 40:1, and an antimicrobial peptide (6) is prepared.

[0085] Example 8

[0086] The same as Example 1, except that the molar ratio of the monomer to the chain transfer agent is 80:1, and an antimicrobial peptide (7) is prepared.

[0087] Example 9

[0088] The same as Example 1, except that the molar ratio of the monomer to the chain transfer agent is 120:1, and an antimicrobial peptide (8) is prepared.

[0089] Example 10

[0090] The same as Example 1, except that the molar ratio of the monomer to the chain transfer agent is 150:1, and an antimicrobial peptide (9) is prepared.

[0091] Application Example 1

[0092] Construction of a mature biofilm clearance model: A single MRSA1857 colony was dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet was placed in a well plate, and sterile TSB was diluted tenfold and added to the well plate. The mature biofilm was statically cultured at 37°C for 24 h. The culture medium was removed, and sterile TSB containing soluble antimicrobial peptides (512 μg / mL) was added. The control group only used sterile TSB and cultured for another 24 h. The culture medium was removed, crystal violet was added for staining for 30 min, the unfixed components were washed away, 30% acetic acid was added to dissolve, and OD595 was measured to characterize the biomass of the biofilm.

[0093] The calculation method of biofilm removal rate is shown in formula (VI):

[0094]

[0095] The biofilm clearance rate of antimicrobial peptides was calculated by formula (VI), and the specific data are as follows: Figure 4As shown, the biofilm removal efficiencies of antimicrobial peptides 1-4 were 57.7%, 47.5%, 37.0% and 39.2%, respectively. Figure 5 The results showed that antimicrobial peptides 1 and 3 mainly achieved biofilm disintegration by removing polysaccharides in the biofilm, and had little effect on the protein components in the biofilm.

[0096] Application Example 2

[0097] Construction of a mature biofilm clearance model: A single S. aureus colony was dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet was placed in a well plate, diluted tenfold with sterile TSB and added to the well plate, and static culture was carried out at 37°C for 24 hours to obtain a mature biofilm; the culture medium was removed, and sterile TSB containing dissolved antimicrobial peptides (512 μg / mL) was added. The control group was treated with sterile TSB only and cultured for another 24 hours; the culture medium was removed, crystal violet was added for staining for 30 minutes, the unfixed components were washed away, 30% acetic acid was added to dissolve, and OD595 was measured to characterize the biomass of the biofilm.

[0098] The calculation method of biofilm removal rate is shown in formula (VI):

[0099]

[0100] The biofilm clearance rate of antimicrobial peptides was calculated by formula (VI), and the specific data are as follows: Figure 6 As shown, the biofilm removal efficiencies of antimicrobial peptides 5, 6, 1, 7, 8, and 9 were 68.6%, 65.4%, 68.8%, 62.6%, 61.2%, and 63.3%, respectively.

[0101] Application Example 3

[0102] Construction of a mature biofilm clearance model: A single colony of P. aeruginosa was dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet was placed in a well plate, and the sheet was added to the well plate after being diluted tenfold with sterile TSB. The sheet was statically cultured at 37°C for 24 h to obtain a mature biofilm; the culture medium was removed, and sterile TSB containing dissolved antimicrobial peptides (512 μg / mL) was added. The control group was treated with sterile TSB only and cultured for another 24 h; the culture medium was removed, crystal violet was added for staining for 30 min, the unfixed components were washed away, 30% acetic acid was added to dissolve, and OD595 was measured to characterize the biomass of the biofilm.

[0103] The calculation method of biofilm removal rate is shown in formula (VI):

[0104]

[0105] The biofilm removal rate of the antimicrobial peptides was calculated by formula (VI). The biofilm removal efficiencies of antimicrobial peptides 5 and 6 were 41.4% and 40.9%, respectively.

[0106] Application Example 4

[0107] Construction of a mature biofilm clearance model: A single E. coli colony was dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet was placed in a well plate, and the sheet was added to the well plate after being diluted tenfold with sterile TSB. The sheet was statically cultured at 37°C for 24 hours to obtain a mature biofilm; the culture medium was removed, and sterile TSB containing dissolved antimicrobial peptides (512 μg / mL) was added. The control group was treated with sterile TSB only and cultured for another 24 hours; the culture medium was removed, crystal violet was added for staining for 30 minutes, the unfixed components were washed away, 30% acetic acid was added to dissolve, and OD595 was measured to characterize the biomass of the biofilm.

[0108] The calculation method of biofilm removal rate is shown in formula (VI):

[0109]

[0110] The biofilm removal rate of the antimicrobial peptide was calculated by formula (VI), and the biofilm removal efficiency of antimicrobial peptide-like 7 was 45.5%.

[0111] Application Example 5

[0112] Construction of a mature biofilm clearance model: A single colony of S. epidermidis was dynamically cultured in TSB to the late logarithmic growth stage, a sterile dry titanium sheet was placed in a well plate, diluted tenfold with sterile TSB and added to the well plate, and statically cultured at 37°C for 24 hours to obtain a mature biofilm; the culture medium was removed, and sterile TSB containing dissolved antimicrobial peptides (512 μg / mL) was added. The control group only used sterile TSB and cultured for another 24 hours; the culture medium was removed, crystal violet was added for staining for 30 minutes, the unfixed components were washed away, 30% acetic acid was added to dissolve, and OD595 was measured to characterize the biomass of the biofilm.

[0113] The calculation method of biofilm removal rate is shown in formula (VI):

[0114]

[0115] The biofilm removal rate of the antimicrobial peptide was calculated by formula (VI), and the biofilm removal efficiency of the antimicrobial peptide-1 was 63.0%.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antimicrobial peptide with the function of removing bacterial biofilm, It is characterized in that It has the general structural formula shown in formula (1): Wherein, n is 5 to 100; the structural formula of R is shown in formula (II) to (V):

2. Use of the antimicrobial peptide according to claim 1 in removing bacterial biofilm, It is characterized in that The bacteria in the bacterial biofilm include Gram-negative bacteria and / or Gram-positive bacteria.

3. Use of the antimicrobial peptide according to claim 2 in removing bacterial biofilm, It is characterized in that The preparation method of the antimicrobial peptide-like peptide comprises the following steps: S1, mixing an amino acid, an olefin compound and water and reacting them to obtain a reaction product, and sequentially extracting, drying, rotary evaporating and vacuum drying the reaction product to obtain an olefin-functionalized amino acid monomer; S2. The olefin-functionalized amino acid monomer, chain transfer agent, catalyst and solvent are mixed and then subjected to polymerization reaction to obtain the antimicrobial peptide-like peptide.

4. Use of the antimicrobial peptide according to claim 3 in removing bacterial biofilm, It is characterized in that In step S1, the amino acids include N α -tert-Butyloxycarbonyl-D-lysine, N ε -tert-Butyloxycarbonyl-D-lysine, N α -tert-Butyloxycarbonyl-D-tyrosine, N α -tert-Butyloxycarbonyl-D-2,3-diaminopropionic acid and N β -tert-butyloxycarbonyl-D-2,3-diaminopropionic acid; The olefin compound includes acryloyl chloride and / or methacryloyl chloride.

5. Use of the antimicrobial peptide according to claim 4 in removing bacterial biofilm, It is characterized in that In the step S1, the usage ratio of the amino acid, the olefin compound and water is 800-1200 mg: 300-600 mg: 3-10 mL.

6. Use of the antimicrobial peptide according to any one of claims 3 to 5 in removing bacterial biofilms, It is characterized in that In the step S1, the reaction temperature is 0 to 30° C., and the reaction time is 3 to 8 hours.

7. Use of the antimicrobial peptide according to claim 6 in removing bacterial biofilm, It is characterized in that In step S2, the chain transfer agent includes one or more of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 2-(dodecylthiocarbonylthiothiothio)propionic acid, S-(2-cyano-2-propyl)-S-dodecyltrithiocarbonyl ester and 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid]; The catalyst includes one or more of 2′,4′,5′,7′-tetrabromofluorescein, tetraphenylporphyrin zinc, tris(2-phenylpyridine)iridium, sodium bicarbonate, ammonium persulfate, and azobisisobutyronitrile; The solvent includes one or more of ethyl acetate, methanol, ethanol, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, concentrated hydrochloric acid and hydrogen peroxide.

8. Use of the antimicrobial peptide according to claim 4, 5 or 7 in removing bacterial biofilms, It is characterized in that In the step S2, the molar ratio of the olefin functionalized amino acid monomer to the chain transfer agent is 20 to 150:1; The molar ratio of the olefin functionalized amino acid monomer to the catalyst is 10 3 ~10 5 : 1; The mass volume ratio of the olefin functionalized amino acid monomer and the solvent is 0.5-1.5 g:7 mL.

9. Use of the antimicrobial peptide according to claim 8 in removing bacterial biofilm, It is characterized in that In the step S2, the polymerization reaction time is 4 to 12 hours.

10. Use of the antimicrobial peptide according to claim 9 in removing bacterial biofilm, It is characterized in that In the step S2, the polymerization reaction includes RAFT thermal polymerization or RAFT photopolymerization; The reaction temperature of the RAFT thermal polymerization is 60-90°C; the reaction temperature of the RAFT photopolymerization is 20-30°C.

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