CN113045629B - Antibacterial peptide BIMix and application thereof - Google Patents
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Abstract
The invention discloses an antibacterial peptide BIMix and application thereof, belonging to the technical field of biology, wherein the antibacterial peptide comprises 29 amino acid residues, belonging to alpha-helix type polypeptide, and the amino acid sequence of the antibacterial peptide BIMix is as follows: GLKVIRVKIRQFKRQLKR IKFVRIKNRKP, no sequence completely repeated with the antibacterial peptide BIMix is found after searching through antibacterial peptide database (DRAMP, D BAASP, DAPD), and the antibacterial peptide BIMix belongs to novel antibacterial peptide. The antibacterial peptide BIMix has the advantages of good thermal stability, convenience in synthesis and the like, can be used for directly killing bacteria under independent action, and has a strong killing effect on staphylococcus aureus, drug-resistant staphylococcus aureus and escherichia coli, the antibacterial peptide BIMix can obviously inhibit the formation of bacterial biofilm, and can effectively relieve the occurrence and development of bacterial drug resistance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide BIMix and application thereof.
Background
Since the large-scale use of antibiotics, bacterial drug resistance is rapidly developing towards multiple and complicated processes, including very virulent pathogenic bacteria such as Staphylococcus aureus (s.aureus), which is a pathogenic bacterium capable of causing various pyogenic infections, and has been widely noticed due to its high clinical isolation rate and outstanding drug resistance, wherein Methicillin-resistant Staphylococcus aureus (MRSA) is the most typical example, and vancomycin is clinically used as a main drug in the treatment of MRSA infection, however, the bacteria have gradually reduced sensitivity to vancomycin, and the drugs are difficult to penetrate into cells of targeted organs and tissues due to the obstruction of biological membrane systems to play a role.
Bacterial biofilms serve as the primary means of bacterial induction of high drug resistance and are generally associated with the entire course of clinical infection. The formation of bacterial Biofilm is a physiological behavior of bacteria against environmental stress, which is shown by forming a defense barrier through self-secreted protein, free nucleic acid and polysaccharide substances, can assist bacteria to resist most antibacterial drugs, is one of important reasons for causing bacterial drug resistance, and the problem of intractable infection and high drug resistance caused by the formation of bacterial Biofilm (Biofilm, BF) is becoming more and more important. The Minimum Inhibitory Concentration (MIC) that the mature tunica adventitia bacteria can tolerate is reported to be 10-1000 times of that of planktonic bacteria, and therefore, as long as the biofilm is not cleared, bacteria highly tolerant to drugs exist for a long time, and the infection caused by the bacteria is delayed and not healed.
Because the biofilm can protect bacteria to live in a severe environment, conventional antibiotics and bactericides cannot penetrate through extracellular matrix, so that the sensitivity of the bacteria to the antibiotics and bactericides is reduced, and the antibacterial peptide serving as an antibacterial active substance widely distributed in organisms is widely applied to prevention and treatment of bacterial infection because the antibacterial peptide is not easy to induce the bacteria to generate the advantages of good tolerance, good biocompatibility and the like. In the past decades, researchers have tried to improve the effectiveness of antibacterial peptides as therapeutic drugs by different strategies, for example, chinese patent No. cn202010507155.x discloses a natural antibacterial peptide, which can inhibit escherichia coli in vitro after being mixed with citric acid in a certain proportion, the antibacterial peptide can achieve a good bactericidal effect only by being matched with EDTA or sodium citrate, and is difficult to directly kill bacteria, chinese patent No. CN201910510193.8 discloses that antibacterial peptide S2 which is modified and designed based on self-induced antibacterial peptide secreted by agr system regulation of staphylococcus aureus shows a bactericidal effect on staphylococcus aureus, and none of the antibacterial peptides in the prior art discloses that antibacterial peptide inhibits the formation of bacterial biofilm, so that it is necessary to provide a novel antibacterial peptide to alleviate the occurrence and development of bacterial drug resistance.
Disclosure of Invention
In view of the above, the invention provides an antibacterial peptide BIMix and an application thereof, and aims to solve the defects of conventional antibiotic drugs and effectively alleviate the problem of bacterial drug resistance.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an antibacterial peptide BIMix, wherein the amino acid sequence of the antibacterial peptide BIMix is as follows: GLKVIRVKIRQFKRQLKRIKFVRIKNRKP, as shown in SEQ ID NO. 1.
Preferably, the antibacterial peptide BIMix comprises 29 amino acid residues and belongs to alpha-helix type antibacterial peptides.
Preferably, the antibacterial peptide BIMix is enriched in strong hydrophobic amino acids such as L (Leu), V (Val), I (Ile) and the like at the N-terminal, and K (Lys) and R (Arg) alternately form an electropositive binding region at the C-terminal.
The application of the antibacterial peptide BIMix is used for killing bacteria and inhibiting the formation of bacterial biofilms.
Preferably, the bacteria are methicillin-resistant staphylococcus aureus (MRSA), methicillin-sensitive staphylococcus aureus (MSSA), escherichia coli (e.
An antibacterial medicine contains the antibacterial peptide BIMix.
The invention has the beneficial effects that: the designed antibacterial peptide BIMix belongs to alpha-helical antibacterial peptide, has good stability and convenient synthesis, does not find the same polypeptide as the antibacterial peptide BIMix after being searched by antibacterial peptide databases (DRAMP, DBAASP and DAPD), and belongs to novel antibacterial peptide. The antibacterial peptide BIMix can be used for directly killing bacteria under the independent action, and has a strong killing effect on staphylococcus aureus, drug-resistant staphylococcus aureus and escherichia coli, and the antibacterial peptide BIMix can obviously inhibit the formation of bacterial biofilms, so that the antibacterial peptide BIMix can be used as a novel antibacterial drug, and has a wide application prospect.
Drawings
FIG. 1 is a graph showing the in vitro bactericidal (Staphylococcus aureus) effect of the antimicrobial peptide BIMix.
FIG. 2 is a graph showing the in vitro bactericidal (E.coli) effect of the antimicrobial peptide BIMix.
FIG. 3 is a schematic diagram of the change of cell morphology after the action of antibacterial peptide BIMix and methicillin-resistant Staphylococcus aureus standard strain ATCC33591 under a transmission electron microscope.
FIG. 4 is a schematic diagram of the change of cell morphology after the action of antibacterial peptide BIMix and methicillin-sensitive Staphylococcus aureus standard strain ATCC29213 under a transmission electron microscope.
FIG. 5 is a schematic diagram of the change of cell morphology after the antibacterial peptide BIMix acts with Escherichia coli BL21(DE3) under a transmission electron microscope.
FIG. 6 is a schematic diagram of the change of cell morphology after the action of antibacterial peptide BIMix and Escherichia coli DH5 alpha under a transmission electron microscope.
FIG. 7 is a time-sterilization graph of the antimicrobial peptide BIMix against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus standard strains.
FIG. 8 is a time-sterilization graph of antimicrobial peptide BIMix against a clinical isolate of Staphylococcus aureus.
Fig. 9 is a graph of the in vitro bactericidal activity of the antimicrobial peptide BIMix after treatment at different temperatures, wherein: a is methicillin-resistant staphylococcus aureus standard strain ATCC33591, B is methicillin-sensitive staphylococcus aureus standard strain ATCC29213, C is staphylococcus aureus clinical separation strain WLD11, D is staphylococcus aureus clinical separation strain WLD10, E is staphylococcus aureus clinical separation strain JY21, and F is staphylococcus aureus clinical separation strain JY 45.
Figure 10 is an antimicrobial peptide BIMix thermostable colony count assay.
FIG. 11 is a graph of the effect of the antimicrobial peptide BIMix on biofilm formation by the methicillin-resistant Staphylococcus aureus standard strain ATCC 33591.
FIG. 12 is a graph showing the inhibition of the biofilm by the antimicrobial peptide BIMix against methicillin-resistant Staphylococcus aureus standard strain ATCC 33591.
FIG. 13 is a graph of the effect of the antimicrobial peptide BIMix on biofilm formation by methicillin-sensitive Staphylococcus aureus Standard strain ATCC 29213.
FIG. 14 is a graph showing the biofilm inhibition of the antimicrobial peptide BIMix against methicillin-sensitive Staphylococcus aureus Standard strain ATCC 29213.
FIG. 15 is a graph of the effect of antimicrobial peptide BIMix on biofilm formation by clinical isolates of Staphylococcus aureus (MRSA WLD10, MRSA WLD11, MSSA JY21, MSSA JY 45).
Detailed Description
The technical solutions and effects in the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings of the present invention.
The experimental materials used in the following examples of the invention were: methicillin-resistant Staphylococcus aureus (MRSA) standard strain ATCC33591, Methicillin-sensitive Staphylococcus aureus (MSSA) standard strain ATCC29213, escherichia coli BL21(DE3) and DH5 alpha are purchased from ATCC Chinese strain resource libraries; clinical isolated strains MRSA WLD10, MRSA WLD11, MSSA JY21 and MSSA JY45 are isolated from cow farm mastitis milk samples in Ningxia Yinchuan city, Wu faith city and Guyuan city, and the minimum inhibitory concentration of the strains on penicillin G is more than or equal to 128 mu G/mL and the number of multiple drug-resistant strains is more than 10, and the strains belong to high-drug-resistant strains; MH broth (MHB, powdered beef, soluble starch, acid hydrolyzed casein; pH 7.4 + -0.2), MH agar (MHA, 1% agar powder added to MH broth) and tryptone soy broth medium (TSB, tryptone, soy papain digest, sodium chloride, potassium dihydrogen phosphate, glucose; pH 7.3 + -0.2) were purchased from Beijing Luqiao technology, Inc.; phosphate buffered saline (PBS, PH 7.0) was purchased from beijing solibao technologies ltd.
Example 1: synthesis of antibacterial peptide BIMix
The invention isThe designed amino acid sequence of the antibacterial peptide BIMix is as follows: GLKVIRVKIRQFKRQLKRIKFVRIKNRKP (Gly-Leu-Lys-Val-Ile-Arg-Val-Lys-Ile-Arg-Gln-Phe-Lys-Arg-Gln-Leu-Lys-Arg-Ile-Lys-Phe-Val-Arg-Ile-Lys-Asn-Arg-Lys-Pro), wherein the N-terminal of the antibacterial peptide BIMix is enriched with strong hydrophobic amino acids such as L (Leu), V (Val), I (Ile) and the like, so that the region presents high hydrophobicity, and the hydrophobic moment of the polypeptide BIMix can be obviously increased, the C-terminal of the antibacterial peptide BIMix alternately forms a positive electricity binding region by (K Lys) and R (Arg), and the hydrophilic amino acids are symmetrically distributed at the two ends of the antibacterial peptide, so as to form an amphiphilic structure; the antimicrobial peptide BIMix in water (ddH)2O) and various mediums (150mM NaCl, 50mM SDS and 50% TFE) can form a certain amount of alpha-helical structures, and especially the core region of Ile9-Arg18 can stably form the alpha-helical structures, which is important for the exertion of the antibacterial and anti-biofilm activities of the antibacterial peptide.
Classifying the secondary structure of the antibacterial peptide BIMix by circular dichroism chromatography and homologous modeling, wherein the antibacterial peptide BIMix comprises 29 amino acid residues, the antibacterial peptide BIMix is synthesized one by one from the C end to the N end on a polypeptide synthesizer (synthesized by Gill Biochemical (Shanghai) Co., Ltd.) by adopting Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis method, the synthesized polypeptide is cut by high-concentration TFA (trifluoroacetic acid), then purified by a High Performance Liquid Chromatograph (HPLC), a product peak is collected to finally obtain the polypeptide BIMix with the purity of more than or equal to 95%, and the polypeptide BIMix is dried in vacuum and then is subjected to ddH2O (Tiangen Biochemical technology, Beijing) Ltd.) dissolved the polypeptide BIMix, prepared a stock solution with a concentration of 50. mu.M, and stored in a refrigerator at-20 ℃ for later use.
Example 2: bactericidal activity detection of antibacterial peptide BIMix
(1) Determination of Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration (MIC) was determined by broth dilution with interpretation according to CLSI standard (2019 version). Culturing methicillin-resistant Staphylococcus aureus standard strain (MRSA ATCC33591), methicillin-sensitive Staphylococcus aureus standard strain (MSSA ATCC29213), Staphylococcus aureus clinical isolates (MRSA WLD10 and MSSA JY21), Escherichia coli (E.coli BL21(DE3) and E.coli DH5 alpha) were diluted to 1X 105cfu/mL, adding 100 μ L of the above bacterial liquid into each well of a 96-well plate, adding an antibacterial peptide BIMix solution into staphylococcus aureus until the final concentration is 0.5, 1, 1.5, 2, 2.5, 3 and 3.5 μ M respectively, increasing the final concentration of the antibacterial peptide BIMix added into escherichia coli to 7.5 μ M for test, placing the 96-well plate in a 37 ℃ incubator for incubation, observing the turbid condition of the liquid in the wells, and taking the concentration which can completely inhibit the growth of bacteria as an MIC value.
(2) Determination of Minimum Bactericidal Concentration (MBC)
The methicillin-resistant staphylococcus aureus standard strain (MRSA ATCC33591), methicillin-sensitive staphylococcus aureus standard strain (MSSA ATCC29213), staphylococcus aureus clinical isolated strains (MRSA WLD10 and MSSA JY21), escherichia coli (E.coli BL21(DE3) and E.coli DH5 alpha) which are cultured to logarithmic growth phase are all diluted to 1 × 105cfu/mL, adding antibacterial peptide BIMix to 400 μ L of bacterial liquid to make the final concentration 0.5, 1, 1.5, 2, 2.5, 3, 3.5 μ M, and increasing the final concentration of antibacterial peptide BIMix added to Escherichia coli to 7.5 μ M, then placing in 37 ℃ incubator, sampling 100 μ L of bacterial liquid every 15min, coating on MHA plate, and making triplicate; after the bacteria liquid is completely absorbed, putting the bacteria liquid in a 37 ℃ incubator (Shanghai Bogomi Co., Ltd.) to culture for 10h, counting bacterial colonies, quantitatively detecting the Minimum Bactericidal Concentration (MBC) of the antibacterial peptide BIMix in vitro, and taking the minimum Concentration at which the bacteria are completely killed as the MBC value; and dripping 10 mu L of the bacterial liquid on an MHA plate to perform a plate dripping test, and culturing in a 37 ℃ incubator after the bacterial liquid is completely absorbed so as to more intuitively express the bactericidal activity.
TABLE 1 determination of the antimicrobial peptides BIMix Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
As can be seen from Table 1, FIG. 1 and FIG. 2, when the final concentration of the antimicrobial peptide BIMix reaches 2 μ M, clarification obviously appears in the wells of a methicillin-resistant Staphylococcus aureus standard strain (MRSA ATCC33591), a methicillin-sensitive Staphylococcus aureus standard strain (MSSA ATCC29213) and a Staphylococcus aureus clinical isolate strain (MRSA WLD10 and MSSA JY21), which indicates that the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide BIMix to Staphylococcus aureus is 2 μ M; when the final concentration of the antimicrobial peptide BIMix reaches 4.5 mu M, clarification appears in the wells of E.coli BL21(DE3) and E.coli DH5 alpha, which indicates that the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide BIMix to Escherichia coli is 4.5 mu M. Through colony counting and spot test quantitative analysis, more than 98% of staphylococcus aureus (P <0.001) can be effectively killed when the final concentration of BIMix reaches 4 MuM, and more than 98% of staphylococcus aureus (P <0.001) can be effectively killed when the final concentration of BIMix reaches 7.5 MuM. The results show that the antibacterial peptide BIMix has relatively remarkable bactericidal activity on highly drug-resistant S.aureus strains (including MRSA strains).
The sterilization rate calculation formula is as follows: the bactericidal rate was 1- (number of colonies in experiment group/number of colonies in blank group. times.100%)
(3) Effect of the antimicrobial peptide BIMix on the morphology of bacterial cells
The change in cell morphology of the bacteria (s. aureus) before and after the action of the antimicrobial peptide BIMix was observed by means of Transmission Electron Microscopy (TEM). The specific method comprises the following steps: bacteria (methicillin-resistant staphylococcus aureus standard strain ATCC33591, methicillin-sensitive staphylococcus aureus standard strain ATCC29213), e.coli BL21(DE3) and e.coli DH5 α strains cultured to the log phase were collected by centrifugation at 12000rpm, rinsed 3 times with PBS buffer (PH 7.0), and the supernatant was discarded; the sample was immersed in 400. mu.L of 2.5% glutaraldehyde, fixed at 4 ℃ for 12 hours at low temperature, stained with 1% osmic acid for 1 hour with shaking on a slow shaker, gradient dehydrated with 50% -90% ethanol, and then acetone and epoxy resin were formulated into a mixture of (1:1) and (1: 2). After the above steps are finished, 400 microlitre of 100% epoxy resin is added and placed in an oven at 60 ℃ for polymerization for 48 hours, and after embedding, frozen ultrathin sections are carried out. Finally, the finished samples were observed and photographed (98000 x magnification) using a transmission electron microscope of biotype (hitachi HITACHI HT 7700120 kv) with 6 fields per specimen randomly selected.
As can be seen from fig. 3 to 6, the antimicrobial peptide BIMix mainly acts on bacterial cell walls, and changes the membrane potential to destroy the cell walls through electrostatic attraction with the peptidoglycan layer with negative electricity on the surface, that is, the bacteria are obviously damaged and shed after incubation with the antimicrobial peptide BIMix, so as to form incomplete or thallus without cell wall wrapping; the thallus without cell wall protection is broken due to osmotic pressure, part of the bacteria are clearly seen in the visual field to disintegrate, the content flows out, and a shrivelled vacuole-like bacterial ghost is left.
Example 3: antibacterial peptide BIMix bactericidal rate against staphylococcus aureus (s. aureus) in vitro
The drug-resistant strains MRSA ATCC33591, MRSA WLD10, MRSA WLD11, MSSA ATCC29213, MSSA JY21 and MSSA JY45 cultured to logarithmic growth phase are diluted to 1 × 105cfu/mL, adding antibacterial peptide BIMix to 400. mu.L of bacterial liquid to a final concentration of 4. mu.M (minimum bactericidal concentration), then placing in an incubator at 37 ℃ for incubation, sampling 100. mu.L of bacterial liquid every 15min, and coating on an MHA plate in triplicate; after the bacteria liquid is completely absorbed, putting the bacteria liquid in a 37 ℃ incubator for culturing for 10h and counting bacterial colonies to quantitatively detect the in-vitro sterilization rate of the antibacterial peptide BIMix; the experiment was repeated 3 times and data analysis was performed using IBM SPSS13.0 software, as P<0.05 as there was a statistical difference.
As can be seen from fig. 7 to 8, the antimicrobial peptide BIMix at the Minimum Bactericidal Concentration (MBC) is finally effective in killing more than 90% of drug-resistant staphylococcus aureus (s.aureus) (P < 0.001); wherein, the antibacterial peptide BIMix can effectively kill 60-70% of bacteria in 15min, the bacteria are remarkably reduced (P is less than 0.05) in 45min, most of bacteria in 60min and later are killed, and the change tends to be gentle. Therefore, the antibacterial peptide BIMix has the capability of quickly killing bacteria directly from the outside of the body, and the number of bacteria in a unit volume can be obviously reduced within 15-45min (P is less than 0.05). In addition, both standard strains (ATCC33591 and ATCC29213) and clinical isolates (MRSA WLD10, MRSA WLD11, MSSA JY21 and MSSA JY45) of staphylococcus aureus show good in-vitro bactericidal activity and relatively similar bactericidal rate, and the difference in action activity between MRSA and MSSA strains is not shown, so that the antibacterial peptide BIMix has good, exact and stable bactericidal activity to a certain extent, namely can generate effective bactericidal activity on inherent drug-resistant strains. The sterilization rate calculation formula is as follows: the bactericidal rate was 1- (number of experimental colonies/number of blank colonies × 100%).
Example 4: thermal stability assay for antimicrobial peptide BIMix
The key to the function of the antibacterial and anti-biofilm antibacterial peptide is that the stable structure can be maintained under different environmental conditions, the heat stability test is carried out on the antibacterial peptide BIMix, and the influence of the temperature on the bactericidal activity of the antibacterial peptide BIMix is quantitatively analyzed by combining a flat plate drip with a colony counting experiment.
The specific method comprises the following steps: placing 30 μ L of 50 μ M antibacterial peptide BIMix stock solution in PCR tube, sequentially placing in 20, 40, 60, 80 and 100 deg.C water bath (constant BWS-5) for 45min, taking out, and adding into 100 μ L of 1 × 105Adding 5 mu L of antibacterial peptide BIMix stock solution into cfu/mL bacterial solution to enable the final concentration to reach 2.5 mu M, carrying out in-vitro sterilization tests on staphylococcus aureus S.aureus standard strains MRSA ATCC33591 and MSSA ATCC29213 and clinical isolates MRSA WLD10, MRSA WLD11, MSSA JY21 and MSSA JY45, and quantitatively analyzing the influence of temperature on the antibacterial activity of the antibacterial peptide BIMix by combining a plate drop test and a colony counting test. The flat plate spot test comprises the following specific steps: dripping 20 μ L of bacterial liquid incubated with antibacterial peptide BIMix at different temperatures (20, 40, 60, 80, 100 deg.C) on 90mm MHA plate, culturing in 37 deg.C incubator for 10 hr after the bacterial liquid is completely absorbed, and observing; at the same time, 100. mu.L of each suspension was applied to MHA plates, the test was repeated 3 times, and data analysis was performed using IBM SPSS13.0 software as P<0.05 as there was a statistical difference.
As can be seen from fig. 9, in the temperature range of 20-100 ℃, compared with the control group, the antimicrobial peptide BIMix treated at each experimental temperature can significantly reduce the number of bacteria in unit volume (P <0.05), the bactericidal activity is not significantly changed with the change of temperature, the antimicrobial peptide BIMix activity is not affected (P >0.05), and the bactericidal effects of the antimicrobial peptide BIMix treated at each experimental temperature on MRSA strains and MSSA strains are also significantly different (P > 0.05). As shown in fig. 10, the colony counting test also shows that the bactericidal activity of the antimicrobial peptide BIMix is not affected in the above temperature range, i.e., the antimicrobial peptide BIMix can significantly kill planktonic bacteria (P >0.05) at 20-100 ℃, which indicates that the antimicrobial peptide BIMix has strong thermal stability.
Example 5: inhibition of biofilm formation by antimicrobial peptide BIMix on staphylococcus aureus (s
Effect of making
The inhibition effect of the antibacterial peptide BIMix on the formation of the biofilm of staphylococcus aureus (S.aureus) is detected by a Crystal violet semi-quantitative staining method (CV) and a method for measuring the light absorption value of the formed biofilm at 490 nm. The specific method comprises the following steps: to each well of a 96-well plate, 100. mu.L of tryptone Soy Broth medium (TSB, containing 0.5% NaCl and 1% glucose) was added, followed by addition of 1X 10 concentration5cfu/mL bacterial solution (methicillin-resistant Staphylococcus aureus Standard Strain (MRSA ATCC33591), methicillin-sensitive Staphylococcus aureus Standard Strain (MSSA ATCC29213) Staphylococcus aureus clinical isolates (MRSA WLD10, MRSA WLD11, MSSA JY21, MSSAJY45) and adding the antibiotic peptides BIMix at different final concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5. mu.M), with no antibiotic peptide BIMix as a negative control, placing the 96-well plate containing the specimen in a 37 ℃ incubator for 36h, discarding the supernatant, gently washing 3 times with phosphate buffer (PBS, pH 7.0) to remove the planktonic bacteria, adding 100. mu.L of 0.1% crystal violet to stain in the incubator at 37 ℃ for 30min, then washing the stain solution with 95% ethanol to remove the loose color, repeating the steps 3 times, using gel imaging system (BIO-Doc software for Lab X + visualization, and observing the Immunity X, measuring the light absorption value of the formed biofilm at 490nm wavelength by a microplate reader (Beckmann, USA) to quantitatively analyze the formation amount of the biofilm; the experiment was repeated 3 times and data analysis was performed using IBM SPSS13.0 software, as P<0.05 as there was a statistical difference.
As can be seen from FIGS. 11 to 15, the inhibition of biofilm formation was observed by the addition of antimicrobial peptide BIMix at a final concentration of 0.5 to 3.5. mu.M. Statistical analysis shows that the antibacterial peptide BIMix with the concentration of 3.5 MuM can obviously inhibit the formation of S.aureus biofilm (P is less than 0.05), and the biofilm is obviously changed into thinning and dispersion from the form of thick connected sheets along with the increase of the antibacterial peptide BIMix concentration. Meanwhile, the antibacterial peptide BIMix with the concentration has an obvious inhibiting effect on a biofilm of a highly drug-resistant MRSA ATCC33591 strain; in addition, the compound also has the same inhibitory effect on local clinical isolates MRSA WLD10, MRSA WLD11, MSSA JY21 and MSSA JY 45.
The antibacterial peptide BIMix can directly kill bacteria under independent action, and has stronger killing action on staphylococcus aureus, drug-resistant staphylococcus aureus and escherichia coli, the bacterial biofilm is a main mode for generating high drug resistance through bacterial induction, and the antibacterial peptide BIMix can obviously inhibit the formation of the bacterial biofilm, so the antibacterial peptide BIMix can be used as a drug for resisting methicillin-resistant staphylococcus aureus, after the antibacterial peptide BIMix acts on the bacterial biofilm, because the surface of the biofilm has high hydrophobicity and electronegativity, the medium induces the alpha-helical antibacterial peptide BIMix to form a high-content and stable alpha-helical structure, the medium is combined with the bacterial biofilm through electrostatic acting force and destroys the potential of the bacterial biofilm, the normal structure of the biofilm is further destroyed, and the drug can penetrate through a biological membrane system to play a role in cells of targeted organs and tissues, can effectively relieve the drug resistance problem of bacteria.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> Ningxia university
<120> antibacterial peptide BIMix and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Leu Lys Val Ile Arg Val Lys Ile Arg Gln Phe Lys Arg Gln Leu
1 5 10 15
Lys Arg Ile Lys Phe Val Arg Ile Lys Asn Arg Lys Pro
20 25
Claims (6)
1. An antimicrobial peptide BIMix, characterized by: the amino acid sequence of the antibacterial peptide BIMix is as follows: GLKVIRVKIRQFKRQLKRIKFVRIKNRKP, as shown in SEQ ID NO. 1.
2. The antimicrobial peptide BIMix of claim 1, wherein: the antibacterial peptide BIMix comprises 29 amino acid residues and belongs to alpha-helical antibacterial peptide.
3. The antimicrobial peptide BIMix of claim 1, wherein: the N end of the antibacterial peptide BIMix is enriched with strong hydrophobic amino acids such as L (Leu), V (Val), I (Ile) and the like, and the C end alternately forms an electropositive binding region by K (Lys) and R (Arg).
4. The use of the antimicrobial peptide BIMix according to any one of claims 1 to 3, wherein: the antimicrobial peptide BIMix is used to kill bacteria and inhibit the formation of bacterial biofilms.
5. The use of the antimicrobial peptide BIMix of claim 4, wherein: the bacteria are methicillin-resistant staphylococcus aureus (MRSA), methicillin-sensitive staphylococcus aureus (MSSA), and Escherichia coli (E.
6. An antibacterial drug, which is characterized in that: comprising the antimicrobial peptide BIMix according to any one of claims 1 to 3.
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| CN118324876B (en) * | 2024-06-13 | 2024-08-20 | 青岛华大基因研究院 | Marine-derived antibacterial peptide FD116 and application thereof |
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