CN113045629B - Antibacterial peptide BIMix and its application - Google Patents

Abstract

The invention discloses an antibacterial peptide BIMix and an application thereof, belonging to the field of biotechnology. The antibacterial peptide comprises 29 amino acid residues and belongs to an α-helical polypeptide. The amino acid sequence of the antibacterial peptide BIMix is: GLKVIRVKIRQFKRQLKR IKFVRIKNRKP, which is obtained by Antibacterial peptide databases (DRAMP, D BAASP, DAPD) were searched and no sequence completely repeated with the antibacterial peptide BIMix was found, and the antibacterial peptide BIMix belonged to a new type of antibacterial peptide. The antibacterial peptide BIMix of the invention has the advantages of good thermal stability, convenient synthesis and the like, and can directly kill bacteria by acting alone, including strong killing effect on Staphylococcus aureus, drug-resistant Staphylococcus aureus and Escherichia coli , and the antibacterial peptide BIMix can significantly inhibit the formation of bacterial biofilm, and can effectively alleviate the occurrence and development of bacterial drug resistance. prospect.

Description

Antimicrobial Peptide BIMix and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide BIMix and an application thereof.

Background technique

Since the large-scale use of antibiotics, bacterial resistance has been developing rapidly to multiple and complex, including highly virulent pathogenic bacteria such as Staphylococcus aureus (S. aureus), which is a The pathogens that can cause a variety of purulent infections have attracted widespread attention due to their high clinical isolation rate and outstanding drug resistance, among which Methicillin-resistant Staphylococcus aureus (MRSA) is the most common. Typically, in the treatment of MRSA infection, vancomycin is often used as the main drug in clinical practice. However, the sensitivity of bacteria to vancomycin gradually decreases, and due to the obstruction of the biofilm system, it is difficult for such drugs to penetrate into Plays a role in cells that target organs and tissues.

Bacterial biofilm is the main way for bacteria to induce high drug resistance, and it is generally accompanied by the whole process of clinical infection. The formation of bacterial biofilm is a physiological behavior of bacteria to form a defense barrier through self-secreting proteins, free nucleic acids and polysaccharides against environmental stress, which can assist bacteria in resisting most antibacterial drugs and is an important cause of bacterial resistance. One of the reasons is the intractable infection and high drug resistance caused by the formation of bacterial biofilm (BF), which has become increasingly difficult to ignore. According to reports, the minimum inhibitory concentration (MIC) concentration that mature encapsulated bacteria can tolerate is 10 to 1000 times that of planktonic bacteria. If it exists, the infection caused by it will not heal.

Because biofilms can protect bacteria to survive in harsh environments, conventional antibiotics and fungicides cannot penetrate the extracellular matrix, resulting in decreased sensitivity of bacteria to antibiotics and fungicides. Antimicrobial peptides are widely distributed antimicrobial active substances in organisms. It is widely used in the prevention and treatment of bacterial infections due to the advantages of inducing bacteria to develop tolerance and good biocompatibility. In the past few decades, researchers have tried different strategies to improve the effectiveness of antimicrobial peptides as therapeutic drugs. For example, the Chinese invention patent No. CN202010507155.X discloses a natural antimicrobial peptide, which is combined with lemon The acid can inhibit Escherichia coli in vitro after being mixed in a certain proportion. The above antimicrobial peptides need to be used in combination with EDTA or sodium citrate to achieve a better bactericidal effect, and it is difficult to kill bacteria directly. The patent number is CN201910510193.8 Chinese invention , the antibacterial peptide S2, which is designed based on the self-inducing antibacterial peptide that is regulated and secreted by the agr system of Staphylococcus aureus, exhibits a killing effect on Staphylococcus aureus, and the antibacterial peptides in the above-mentioned prior art do not disclose that antibacterial peptides inhibit bacteria Therefore, it is necessary to provide a new type of antimicrobial peptide to alleviate the occurrence and development of bacterial resistance.

SUMMARY OF THE INVENTION

In view of this, the present invention provides an antibacterial peptide BIMix and an application thereof, aiming at solving the deficiencies of conventional antibiotic drugs and effectively alleviating the problem of bacterial drug resistance.

The technical scheme adopted by the present invention to solve its technical problems is as follows:

An antimicrobial peptide BIMix, the amino acid sequence of the antimicrobial peptide BIMix is: GLKVIRVKIRQFKRQLKRIKFVRIKNRKP, as shown in SEQ ID NO.1.

Preferably, the antimicrobial peptide BIMix comprises 29 amino acid residues and belongs to an α-helical antimicrobial peptide.

Preferably, the N-terminus of the antimicrobial peptide BIMix is enriched with strongly hydrophobic amino acids such as L(Leu), V(Val), and I(Ile), and the C-terminus is alternately positively charged with K(Lys) and R(Arg) binding area.

The application of the above antimicrobial peptide BIMix is used for killing bacteria and inhibiting the formation of bacterial biofilm.

Preferably, the bacteria are methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and Escherichia coli (E.coli).

An antibacterial drug containing the above-mentioned antibacterial peptide BIMix.

The beneficial effects of the present invention are as follows: the antibacterial peptide BIMix designed by the present invention belongs to the α-helical antibacterial peptide, has good stability and is convenient to synthesize, and after searching through the antibacterial peptide databases (DRAMP, DBAASP, DAPD), no antibacterial peptides related to the antibacterial peptides (DRAMP, DBAASP, DAPD) are found. The same peptide as the peptide BIMix, the antimicrobial peptide BIMix belongs to a new type of antimicrobial peptide. The antibacterial peptide BIMix can directly kill bacteria by acting alone, including strong killing effect on Staphylococcus aureus, drug-resistant Staphylococcus aureus and Escherichia coli, and the antibacterial peptide BIMix can significantly inhibit bacterial organisms. Therefore, the antibacterial peptide BIMix of the present invention can be used as a new antibacterial drug, and has broad application prospects.

Description of drawings

Fig. 1 is a graph showing the effect of antimicrobial peptide BImix in vitro on bactericidal (Staphylococcus aureus).

Fig. 2 is a graph showing the effect of antimicrobial peptide BImix in vitro on bactericidal (Escherichia coli).

Figure 3 is a schematic diagram of the changes in cell morphology under the transmission electron microscope after the antimicrobial peptide BIMix interacted with the standard methicillin-resistant Staphylococcus aureus strain ATCC33591.

Figure 4 is a schematic diagram of cell morphological changes after the antimicrobial peptide BIMix interacted with the methicillin-sensitive Staphylococcus aureus standard strain ATCC29213 under a transmission electron microscope.

Figure 5 is a schematic diagram of cell morphological changes after the antimicrobial peptide BIMix interacted with Escherichia coli BL21(DE3) under transmission electron microscope.

Figure 6 is a schematic diagram of the changes in cell morphology under the transmission electron microscope after the antimicrobial peptide BIMix interacted with Escherichia coli DH5α.

Figure 7 is a time-kill curve diagram of antimicrobial peptide BIMix against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus standard strains.

Figure 8 is a time-kill curve diagram of antimicrobial peptide BIMix against clinical isolates of Staphylococcus aureus.

Figure 9 is a graph showing the in vitro bactericidal activity of antimicrobial peptide BIMix after different temperature treatments, wherein: A is the standard methicillin-resistant Staphylococcus aureus strain ATCC33591, B is the methicillin-sensitive Staphylococcus aureus standard strain ATCC29213, and C is the golden yellow Staphylococcus clinically isolated strain WLD11, D is Staphylococcus aureus clinically isolated strain WLD10, E is Staphylococcus aureus clinically isolated strain JY21, and F is Staphylococcus aureus clinically isolated strain JY45.

Fig. 10 is the thermal stability colony counting test of antimicrobial peptide BImix.

Figure 11 is the effect of antimicrobial peptide BIMix on the biofilm formation of methicillin-resistant Staphylococcus aureus standard strain ATCC33591.

Figure 12 shows the inhibition of the antimicrobial peptide BIMix on the biofilm of methicillin-resistant Staphylococcus aureus standard strain ATCC33591.

Figure 13 is the effect of antimicrobial peptide BIMix on biofilm formation of methicillin-sensitive Staphylococcus aureus standard strain ATCC29213.

Figure 14 shows the inhibition of the biofilm of the methicillin-sensitive Staphylococcus aureus standard strain ATCC29213 by the antimicrobial peptide BIMix.

Figure 15 is the effect of antimicrobial peptide BIMix on the biofilm formation of clinical isolates of Staphylococcus aureus (MRSA WLD10, MRSA WLD11, MSSA JY21, MSSA JY45).

Detailed ways

The technical solutions and technical effects in the embodiments of the present invention will be further elaborated below with reference to the accompanying drawings of the present invention.

The experimental materials used in the following examples of the present invention are: methicillin-resistant Staphylococcus aureus (MRSA) standard strain ATCC33591, methicillin-susceptible Staphylococcus aureus (MSSA) standard Strain ATCC29213, Escherichia coli BL21(DE3) and DH5α were purchased from ATCC China strain resource bank; clinical isolates MRSA WLD10, MRSA WLD11, MSSA JY21, MSSA JY45 were isolated from Yinchuan City, Ningxia, Wuzhong The milk samples of mastitis from dairy farms in Guyuan City and Guyuan City were determined by the drug susceptibility test. The minimum inhibitory concentration of the above strains to penicillin G was ≥128 μg/mL and the number of multi-drug resistant species was 10 or more, which were highly resistant strains; MH broth (MHB, beef meal, soluble starch, acid hydrolyzed casein; pH 7.4 ± 0.2), MH agar (MHA, the ingredients are added with 1% agar powder on the basis of MH broth) and tryptone soybean broth medium (TSB, tryptone, soybean papain digest, sodium chloride, potassium dihydrogen phosphate, glucose; pH 7.3±0.2) were purchased from Beijing Land Bridge Technology Co., Ltd.; Phosphate buffer solution (PBS, pH=7.0) was purchased from Beijing Soleibo Technology Co., Ltd.

Example 1: Synthesis of antimicrobial peptide BIMix

The amino acid sequence of the antimicrobial peptide BIMix designed in the present invention is: 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), the N-terminal of the antimicrobial peptide BIMix is enriched in L(Leu), V(Val), I(Ile), etc. Hydrophobic amino acids make this region highly hydrophobic, which can significantly increase the hydrophobic moment of the polypeptide BIMix. The C-terminus of the antimicrobial peptide BIMix is alternately formed with K(Lys) and R(Arg) to form a positively charged binding region , the hydrophilic and hydrophobic amino acids are symmetrically distributed at both ends of the antimicrobial peptide, thereby forming an amphiphilic structure; the antimicrobial peptide BIMix can be soluble in water (ddH ₂ O) and various media (150mM NaCl, 50mM SDS and 50% TFE) The formation of a certain number of α-helix structures, especially the core region of Ile9-Arg18, can stably form α-helix structures, which is crucial for the antibacterial and anti-biofilm activities of antimicrobial peptides.

The secondary structure of the antimicrobial peptide BIMix was graded by circular dichroism and homology modeling. The antimicrobial peptide BIMix contains 29 amino acid residues. The antimicrobial peptide BIMix was synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis method. The peptides were synthesized one by one from the C-terminus to the N-terminus on the instrument (synthesized by Gill Biochemical (Shanghai) Co., Ltd.), and the synthesized polypeptides were cut with high concentration of TFA (trifluoroacetic acid) and purified by high performance liquid chromatography (HPLC). , collect the product peaks and finally obtain the polypeptide BIMix with a purity of ≥95%. After vacuum drying, dissolve the polypeptide BIMix with ddH ₂ O (Tiangen Biochemical Technology (Beijing) Co., Ltd.), prepare a stock solution with a concentration of 50 μM, and store it in a -20 ℃ refrigerator medium spare.

Example 2: Detection of bactericidal activity of antimicrobial peptide BIMix

(1) Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) was determined by the micro-broth dilution method, and the results were interpreted according to the CLSI standard (2019 edition). Methicillin-resistant Staphylococcus aureus standard strains (MRSA ATCC33591), methicillin-sensitive Staphylococcus aureus standard strains (MSSAATCC29213), and clinical isolates of Staphylococcus aureus (MRSA WLD10 and MSSA) were cultured to the logarithmic growth phase. JY21), Escherichia coli (E.coliBL21(DE3) and E.coli DH5α) were diluted to 1×10 ⁵ cfu/mL, 100 μL of the above bacterial solution was added to each well of a 96-well plate, and antibacterial was added to Staphylococcus aureus. The final concentration of the peptide BIMix solution was 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 μM, respectively, while the final concentration of the antimicrobial peptide BIMix added in E. coli was increased to 7.5 μM for the test, and the 96-well plate was placed at 37 °C. Incubate in the box, observe the turbidity of the liquid in the well, and take the concentration that can completely inhibit the growth of bacteria as the MIC value.

(2) Determination of minimum bactericidal concentration (MBC)

Methicillin-resistant Staphylococcus aureus standard strains (MRSA ATCC33591), methicillin-sensitive Staphylococcus aureus standard strains (MSSA ATCC29213), Staphylococcus aureus clinical isolates (MRSA WLD10 and MSSA JY21), Escherichia coli (E.coli BL21(DE3) and E.coli DH5α) were diluted to 1×10 ⁵ cfu/mL, and the antimicrobial peptide BImix was added to 400 μL of bacterial solution to make the final concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5 μM, and the final concentration of the antimicrobial peptide BIMix added in E. coli was increased to 7.5 μM, and then placed in a 37 °C incubator for incubation, and 100 μL of bacterial solution was sampled every 15 minutes and coated on MHA plates, in triplicate After the bacterial liquid was completely absorbed, it was placed in a 37°C incubator (Shanghai Boxun Industrial Co., Ltd.) for 10 hours and the colonies were counted. The minimum killing concentration is the MBC value; another 10 μL of the above bacterial solution was dripped on the MHA plate for a plate drop test, and after the bacterial solution was completely absorbed, it was placed in a 37°C incubator for more intuitive expression of its bactericidal activity.

Table 1 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptide BIMix

It can be seen from Table 1, Figure 1 and Figure 2 that when the final concentration of antimicrobial peptide BIMix reaches 2 μM, the standard methicillin-resistant Staphylococcus aureus strain (MRSA ATCC33591) and the methicillin-sensitive Staphylococcus aureus standard strain (MSSAATCC29213) The wells of clinical isolates of Staphylococcus aureus (MRSA WLD10 and MSSA JY21) were obviously clear, indicating that the minimum inhibitory concentration (MIC) of antimicrobial peptide BIMix against Staphylococcus aureus was 2 μM; when the final concentration of antimicrobial peptide BIMix reached 4.5 At μM, the wells of E.coli BL21(DE3) and E.coli DH5α were obviously clarified, indicating that the minimum inhibitory concentration (MIC) of antimicrobial peptide BIMix against Escherichia coli was 4.5 μM. Through colony counting and quantitative analysis by spot test, when the final concentration of BIMix reached 4μM, it could effectively kill more than 98% of Staphylococcus aureus (P<0.001), and when the final concentration of BImix reached 7.5μM, it could effectively kill more than 98% of Staphylococcus aureus. Staphylococcus aureus (P<0.001). The above results show that the antimicrobial peptide BIMix has significant bactericidal activity against highly resistant S. aureus strains (including MRSA strains).

The formula for calculating the sterilization rate is: sterilization rate=1-(the number of colonies in the experimental group/the number of colonies in the blank group×100%)

(3) The effect of antimicrobial peptide BIMix on bacterial cell morphology

The cell morphological changes of bacteria (S. aureus) before and after the antimicrobial peptide BIMix were observed by transmission electron microscopy (TEM). The specific method is: 12000rpm centrifugation to collect the bacteria cultured to log phase (methicillin-resistant Staphylococcus aureus standard strain ATCC33591, methicillin-sensitive Staphylococcus aureus standard strain ATCC29213), E.coli BL21 (DE3) and E.coli The .coli DH5α strain was rinsed three times with PBS buffer (PH=7.0), and the supernatant was discarded; the samples were soaked in 400 μL of 2.5% glutaraldehyde, fixed at 4°C for 12 hours, and stained with 1% osmic acid in a low-speed shaker. For 1 h, use 50%-90% ethanol for gradient dehydration, and then prepare a mixture of (1:1) and (1:2) acetone and epoxy resin. After the above steps, 400 μL of 100% epoxy resin was added and placed in a 60° C. oven to polymerize for 48 hours, and then frozen and ultrathin sectioned after embedding. Finally, the prepared samples were observed with a biological transmission electron microscope (Hitachi HITACHI HT7700 120kv), and 6 fields of view were randomly selected for each sample and photographed (magnification of 98000×).

It can be seen from Figure 3 to Figure 6 that the antimicrobial peptide BIMix mainly acts on the bacterial cell wall, and it is electrostatically attracted to the negatively charged peptidoglycan layer on the surface, thereby changing the membrane potential and destroying the cell wall. The cell wall is damaged and falls off, resulting in incomplete or no cell wall-encapsulated cells; cells without cell wall protection are ruptured due to osmotic pressure, and part of the bacteria can be clearly seen disintegrating in the field of vision, and the contents flow out, leaving shrunken vacuoles. "Shadow".

Example 3: Bactericidal rate of antimicrobial peptide BIMix against Staphylococcus aureus (S. aureus) in vitro

Dilute the strains of Staphylococcus aureus (S. aureus) resistant strains MRSA ATCC33591, MRSA WLD10, MRSA WLD11, and MSSA ATCC29213, MSSA JY21 and MSSA JY45 to a concentration of 1×10 ⁵ cfu/mL that have been cultured to the logarithmic growth phase. Antibacterial peptide BIMix was added to 400 μL of bacterial solution to a final concentration of 4 μM (minimum bactericidal concentration), then placed in a 37°C incubator for incubation, and 100 μL of bacterial solution was sampled every 15 minutes and applied to MHA plates in triplicate; After being completely absorbed, it was placed in an incubator at 37°C for 10 hours and colony counts were performed to quantitatively detect the bactericidal rate of antimicrobial peptide BIMix in vitro; the experiment was repeated 3 times, and IBM SPSS 13.0 software was used for data analysis, with P<0.05 as the presence of statistics. difference.

It can be seen from Figure 7 to Figure 8 that the antimicrobial peptide BIMix with the minimum bactericidal concentration (MBC) can effectively kill more than 90% of the drug-resistant Staphylococcus aureus (S. aureus) (P<0.001); At 15min, 60-70% of bacteria can be effectively killed, and at 45min, the bacteria decreased significantly (P<0.05). Most bacteria were killed after 60min, and the change tended to be gentle. It can be seen that the antibacterial peptide BIMix has the ability to quickly and directly kill bacteria in vitro, and can significantly reduce the number of bacteria per unit volume within 15-45 minutes (P<0.05). In addition, against Staphylococcus aureus standard strains (ATCC33591, ATCC29213) and clinical isolates (MRSA WLD10, MRSA WLD11, MSSA JY21, MSSAJY45) showed good in vitro bactericidal activity and relatively similar bactericidal rate, between MRSA and MSSA strains There is also no difference in action activity, which reflects to a certain extent that the antimicrobial peptide BIMix of the present invention has good, exact and stable bactericidal activity, which can also produce effective bactericidal activity against inherently resistant strains. The calculation formula of the bactericidal rate is: bactericidal rate=1-(the number of colonies in the experimental group/the number of colonies in the blank group×100%).

Example 4: Thermal stability detection of antimicrobial peptide BIMix

The ability to maintain a stable structure under different environmental conditions is the key to the antibacterial and anti-biofilm antimicrobial peptides. The thermal stability of the antimicrobial peptide BIMix was tested, and the temperature of the antimicrobial peptide BIMix was quantitatively analyzed by plate spotting combined with colony counting experiments. effect of activity.

The specific method is as follows: put 30 μL of 50 μM antimicrobial peptide BIMix stock solution in a PCR tube, and place them in a water bath (Yiheng BWS-5) at 20, 40, 60, 80 and 100 °C for 45 minutes, and then take out , in 100 μL of bacterial solution with a concentration of 1×10 ⁵ cfu/mL, add 5 μL of antimicrobial peptide BIMix stock solution to make the final concentration reach 2.5 μM, for Staphylococcus aureus S. aureus standard strain MRSA ATCC33591, MSSA ATCC29213 and clinical isolates MRSA WLD10, MRSA WLD11, MSSA JY21 and MSSA JY45 were subjected to in vitro bactericidal tests, and the effect of temperature on the bactericidal activity of antimicrobial peptide BImix was quantitatively analyzed by plate spot test combined with colony count test. The specific steps of the plate spotting test are as follows: take 20 μL of the bacterial solution incubated with the antimicrobial peptide BIMix at different temperatures (20, 40, 60, 80, 100 °C) and vertically drop it on a 90 mm MHA plate. After the bacterial solution is completely absorbed, place it at 37 Observation after culturing for 10 hours in an incubator at ℃; at the same time, 100 μL of bacterial solution was taken and spread on MHA plates, and the experiment was repeated three times. IBM SPSS13.0 software was used for data analysis.

It can be seen from Figure 9 that in the temperature range of 20-100 °C, compared with the control group, the antimicrobial peptide BIMix treated at each experimental temperature can significantly reduce the number of bacteria per unit volume (P<0.05), and the bactericidal activity increases with temperature. The changes did not change significantly, the activity of antimicrobial peptide BIMix was not affected (P>0.05), and the bactericidal effects of antimicrobial peptide BIMix on MRSA strains and MSSA strains were also significantly different after each experimental temperature treatment (P>0.05). It can be seen from Figure 10 that the colony counting test also showed that the bactericidal activity of the antimicrobial peptide BIMix would not be affected in the above temperature range, that is, under the conditions of 20-100 °C, it could significantly kill planktonic bacteria (P>0.05), indicating that the antibacterial Peptide BIMix has strong thermal stability.

Example 5: Inhibition of antimicrobial peptide BIMix on the formation of Staphylococcus aureus (S. aureus) biofilm

control effect

Crystal violet semi-quantitative staining (Crystal violet assay, CV) and the method of measuring the absorbance value of the formed biofilm at a wavelength of 490 nm were used to detect the inhibitory effect of antimicrobial peptide BIMix on the formation of S. aureus biofilm. The specific method is as follows: add 100 μL of tryptone soybean broth medium (TSB, containing 0.5% NaCl and 1% glucose) to each well of a 96-well plate, and then add bacterial broth (resistant to 1×10 5 cfu/mL) at a concentration of 1×10 ⁵ cfu/mL Methicillin-sensitive Staphylococcus aureus standard strain (MRSA ATCC33591), methicillin-sensitive Staphylococcus aureus standard strain (MSSAATCC29213) Staphylococcus aureus clinical isolates (MRSA WLD10, MRSA WLD11, MSSA JY21, MSSAJY45) and added and Antibacterial peptide BIMix with different final concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 μM) was used as a negative control without antibacterial peptide BIMix, and the 96-well plate containing the samples was placed in a 37°C incubator for incubation 36h, after discarding the supernatant, wash gently 3 times with phosphate buffered saline (PBS, PH=7.0) to remove the planktonic bacteria, add 100 μL of 0.1% crystal violet to stain for 30min in a 37°C incubator, and then discard The dye solution was washed with 95% ethanol to remove the floating color. This step was repeated 3 times, and the gel imaging system (BIO-RADGelDoc XR+) and ImageLab software were used to observe and photograph; The optical absorption value of the formed biofilm at the wavelength of 490nm was used to quantitatively analyze the amount of biofilm formation; the experiment was repeated three times, and IBM SPSS 13.0 software was used for data analysis, with P<0.05 as a statistical difference.

It can be seen from Figure 11 to Figure 15 that the inhibition of biofilm formation was observed by adding the antimicrobial peptide BIMix at a final concentration of 0.5-3.5 μM. Statistical analysis showed that the antimicrobial peptide BIMix at a concentration of 3.5 μM could significantly inhibit the formation of S. aureus biofilm (P<0.05). Morphology turned noticeably thin and dispersed. At the same time, the antimicrobial peptide BIMix at this concentration showed an obvious inhibitory effect on the biofilm of the highly resistant MRSAATCC33591 strain; in addition, it also had the same degree of inhibitory effect on the clinical isolates MRSA WLD10, MRSA WLD11, MSSA JY21, and MSSA JY45 in this region.

The antibacterial peptide BIMix of the invention can directly kill bacteria by acting alone, including strong killing effect on Staphylococcus aureus, drug-resistant Staphylococcus aureus and Escherichia coli. The antibacterial peptide BIMix can significantly inhibit the formation of bacterial biofilm, so the antibacterial peptide BIMix of the present invention can be used as a drug against methicillin-resistant Staphylococcus aureus. After the antibacterial peptide BIMix acts on the bacterial biofilm, Due to the high hydrophobicity and electronegativity of the biofilm surface, this medium induces the α-helical antimicrobial peptide BIMix to form a high-content and stable α-helical structure, which binds to the bacterial biofilm through electrostatic force and destroys its potential. And then destroy the normal structure of the biofilm, this drug can penetrate the biofilm system to play a role in the cells targeting organs and tissues, which can effectively alleviate the drug resistance problem of bacteria.

What is disclosed above is only the preferred embodiment of the present invention, of course, it cannot limit the scope of the right of the present invention. Those of ordinary skill in the art can understand that all or part of the process of realizing the above-mentioned embodiment can be made according to the claims of the present invention. The equivalent changes of the invention still belong to the scope covered by the invention.

sequence listing

<110> Ningxia University

<120> Antibacterial peptide BIMix and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29

<212> PRT

<213> 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 in that: the amino acid sequence of the antimicrobial peptide BIMix is: GLKVIRVKIRQFKRQLKRIKFVRIKNRKP, as shown in SEQ ID NO.1. 2 . The antimicrobial peptide BIMix according to claim 1 , wherein the antimicrobial peptide BIMix comprises 29 amino acid residues and belongs to an α-helical antimicrobial peptide. 3 . 3. The antimicrobial peptide BIMix of claim 1, wherein the N-terminal of the antimicrobial peptide BIMix is enriched with strongly hydrophobic amino acids such as L (Leu), V (Val), I (Ile), and the C-terminal is K(Lys) and R(Arg) alternately form positively charged binding regions. 4. The application 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 biofilm. 5. the application of antimicrobial peptide BIMix as claimed in claim 4 is characterized in that: described bacterium is methicillin-resistant staphylococcus aureus (MRSA), methicillin-sensitive staphylococcus aureus (MSSA), Escherichia coli (E. coli). 6. An antibacterial drug, characterized in that: it contains the antibacterial peptide BIMix described in any one of claims 1 to 3.

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