A vancomycin-antimicrobial peptide conjugate and its application

Abstract

The present invention relates to a vancomycin-antimicrobial peptide conjugate and its application. After a cysteine ​​is added to the N-terminus of the antimicrobial peptide M2 ​​for modification, vancomycin and the antimicrobial peptide modified body are coupled through a two-step reaction through a bifunctional protein cross-linking agent sulfo-SMCC. Compared with vancomycin, antimicrobial peptide M2, and a mixture of vancomycin and antimicrobial peptide M2, the vancomycin-antimicrobial peptide conjugate has significantly improved antibacterial activity, overcomes the drug resistance of Gram-positive bacteria to vancomycin, and also produces antibacterial activity against some Gram-negative bacteria, thereby increasing the antibacterial spectrum of vancomycin. At the same time, it also has a stronger anti-biofilm ability, can kill retained bacteria, inhibit bacterial clustering movement, and can also form a synergistic effect with antibiotics to kill bacteria together.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to a vancomycin-antimicrobial peptide conjugate and application thereof. Background Art

[0002] Since the discovery of antibiotics, they have been playing the most important role in the field of antibacterial and anti-infection. However, in recent years, with the abuse of traditional antibiotics in medicine and animal husbandry, more and more drug-resistant bacteria have emerged. The world has entered the post-antibiotic era. Microbial resistance has become a major challenge that seriously threatens human life and health.

[0003] It is reported that bacterial infection has become the second leading cause of death in the world. Among them, infections caused by drug-resistant bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae are the top threats to human life and health. At present, more than 1 million people die from drug-resistant bacterial infections every year. According to the World Health Organization, this number will rise to 10 million by 2050. Therefore, in the post-antibiotic era, it is urgent to seek new and more effective drugs against drug-resistant bacteria.

[0004] Since its discovery in 1952, vancomycin has been widely used to treat infections caused by serious drug-resistant Staphylococcus aureus and other Gram-positive bacteria. Vancomycin mainly binds to two D-alanine sequences in the cell wall peptidoglycan precursor lipopeptide, inhibiting cell wall synthesis and causing bacterial death. However, continuous abuse has led to the emergence of many vancomycin-resistant bacteria. The main resistance mechanism is that the binding target D-alanine of vancomycin is replaced by D-lactic acid and D-serine, which increases the steric hindrance of vancomycin binding to the target and reduces the binding affinity of vancomycin to the target. The reduced permeability of bacterial cell membrane also leads to bacterial resistance to vancomycin.

[0005] Antimicrobial peptides are natural active peptides mainly derived from animals, plants and microorganisms in nature. They are short peptides formed by 10-50 amino acids connected by amide bonds. They mainly destroy the bacterial cell membrane structure and bind to some intracellular targets to inhibit the synthesis of DNA and protein to play a bactericidal role. In summary, antimicrobial peptides have the following advantages: wide sources, easy to obtain, strong and broad antimicrobial activity, unique mechanism, not easy to cause drug resistance, simple structure, and easy to transform. In addition, antimicrobial peptides also have certain immunomodulatory activity. At present, more than 30 antimicrobial peptides are in the clinical research stage. Among them, M2 is a derivative peptide derived from frog antimicrobial peptides, which is used in the treatment of skin infectious diseases such as psoriasis and diabetic foot. In addition, it also shows good killing effects on viruses and tumor cells. In summary, in the post-antibiotic era, antimicrobial peptides have become the most promising antibacterial and anti-infective drugs to replace traditional antibiotics.

[0006] At present, some researchers have noticed that antimicrobial peptides may become a breakthrough in solving the problem of bacterial resistance, and the most commonly used method is the combination of antibiotics and antimicrobial peptides. This combination method can only achieve a simple superposition of the effects of antibiotics and antimicrobial peptides, but cannot really solve the problem of bacterial resistance to antibiotics. Therefore, it is urgent to find a method that can maximize the joint effect of antimicrobial peptides and antibiotics in the bactericidal process. Summary of the invention

[0007] In order to solve the problem that the combined effect of antibiotics and antimicrobial peptides is not ideal, and to enable antibiotics and antimicrobial peptides to work together to the greatest extent, the present invention provides a vancomycin-antimicrobial peptide conjugate.

[0008] The first object of the present invention is to provide a vancomycin-antimicrobial peptide conjugate, wherein the vancomycin-antimicrobial peptide conjugate is obtained by covalently cross-linking vancomycin and an antimicrobial peptide having an amino acid sequence as shown in SEQ ID NO.1 through a cross-linking agent.

[0009] Furthermore, the terminal of the antimicrobial peptide is modified with cysteine.

[0010] Furthermore, the terminal lysine of the antimicrobial peptide is modified by acylation.

[0011] Furthermore, all amino acids in the modified antimicrobial peptide are L-type amino acids.

[0012] Furthermore, the covalent cross-linking is achieved by a bifunctional protein cross-linking agent Sulfo-SMCC.

[0013] Furthermore, the method for preparing the antimicrobial peptide modification comprises the following steps: chemically synthesizing the antimicrobial peptide modification using solid phase synthesis technology according to the amino acid sequence of the antimicrobial peptide modification, purifying the antimicrobial peptide modification using preparative reversed phase high performance liquid chromatography, and identifying the molecular weight using high resolution mass spectrometry.

[0014] Furthermore, vancomycin was coupled with the bifunctional protein cross-linker sulfo-SMCC via an amide bond to obtain the vancomycin modified body Vm-SMCC with a molecular weight of 1667.55 Da.

[0015] Furthermore, the preparation method of the vancomycin modified body Vm-SMCC comprises the following steps: purification by preparative reversed phase high performance liquid chromatography and molecular weight identification by high resolution mass spectrometry.

[0016] Furthermore, the antimicrobial peptide and the modified vancomycin Vm-SMCC are coupled to obtain the vancomycin-antimicrobial peptide conjugate Vm-M2.

[0017] Furthermore, the preparation method of the vancomycin-antimicrobial peptide conjugate Vm-M2 comprises the following steps: purification using preparative reversed-phase high performance liquid chromatography and molecular weight identification using high-resolution mass spectrometry.

[0018] The second object of the present invention is to provide the use of vancomycin-antimicrobial peptide conjugates in the preparation of antibacterial agents.

[0019] Furthermore, the antibacterial agent is used to inhibit Gram-positive bacteria or Gram-negative bacteria.

[0020] Further, the Gram-positive bacteria include but are not limited to Staphylococcus aureus (such as Staphylococcus aureus CMCC26003, Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC43300, Staphylococcus aureus 15772, Staphylococcus aureus 15192, vancomycin-resistant Staphylococcus aureus 52 and vancomycin-resistant Staphylococcus aureus 11), Enterococcus faecalis (such as vancomycin-resistant Enterococcus faecalis ATCC29212 and Enterococcus faecalis 2), Clostridium perfringens and Bacillus cereus.

[0021] Furthermore, the Gram-negative bacteria include but are not limited to Escherichia coli (such as Escherichia coli ATCC25922 and Escherichia coli CMCC44102), Acinetobacter baumannii (such as Acinetobacter baumannii ATCC19606 and pan-resistant Acinetobacter baumannii 1), Klebsiella pneumoniae (Klebsiella pneumoniae 9883, pan-resistant Klebsiella pneumoniae 2, pan-resistant Klebsiella pneumoniae 3, pan-resistant Klebsiella pneumoniae 4), Pseudomonas aeruginosa (such as Pseudomonas aeruginosa ATCC27 853, Pseudomonas aeruginosa CMCC10104, Pseudomonas aeruginosa 60357, Pseudomonas aeruginosa 52097 and pan-resistant Pseudomonas aeruginosa 5), ​​Shigella flexneri (such as Shigella flexneri ATCC12022 and Shigella flexneri CMCC51571), Salmonella typhimurium (such as Salmonella typhimurium ATCC14028 and Salmonella typhimurium CICC21483) and Vibrio parahaemolyticus (such as Vibrio parahaemolyticus ATCC17802).

[0022] The third object of the present invention is to provide the use of vancomycin-antimicrobial peptide conjugates in antibacterial biofilm.

[0023] The fourth object of the present invention is to provide an antibacterial pharmaceutical composition, comprising the vancomycin-antimicrobial peptide conjugate and an antibiotic.

[0024] Furthermore, the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention couples vancomycin and antimicrobial peptide M2 ​​through the bifunctional protein crosslinker sulfo-SMCC, and the resulting vancomycin-antimicrobial peptide conjugate Vm-M2 greatly improves the antibacterial activity of vancomycin and effectively overcomes the problem of vancomycin resistance of some Gram-positive bacteria. The conjugate Vm-M2 significantly expands the inherent antibacterial spectrum of vancomycin and has strong antibacterial activity against major Gram-negative pathogens (including resistant and pan-resistant clinical isolates). In addition, at the same molar concentration, the conjugate exhibits more efficient bactericidal activity than vancomycin, antimicrobial peptide M2, and a mixture of vancomycin and antimicrobial peptide M2.

[0027] (2) The vancomycin-antimicrobial peptide conjugate Vm-M2 provided by the present invention is obtained through a two-step coupling reaction, and is easy to synthesize, the raw materials are easily available, and the synthesis cost is low. In addition, the conjugate has the advantages of low hemolytic activity, high stability, and strong anti-biofilm activity, and can be applied to the fields of medicine, cosmetics, and aquaculture, greatly expanding the downstream application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the result of mass spectrometry and HPLC analysis of the vancomycin modified form Vm-SMCC;

[0029] Figure 2 It is the mass spectrometry and HPLC analysis results of vancomycin-antimicrobial peptide conjugate Vm-M2;

[0030] Figure 3 This is the synthetic route of vancomycin-antimicrobial peptide conjugate Vm-M2;

[0031] Figure 4 It is the residual bactericidal effect of vancomycin-antimicrobial peptide conjugate Vm-M2;

[0032] Figure 5 It is the salt ion stability of vancomycin-antimicrobial peptide conjugate Vm-M2;

[0033] Figure 6 It is the effect of vancomycin-antimicrobial peptide conjugate Vm-M2 on bacterial swarming movement. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] Experimental strains: Clinical isolates were collected from the Affiliated Hospital of Soochow University.

[0036] Example 1: Chemical Synthesis of Vancomycin-Antibacterial Peptide Conjugate Vm-M2

[0037] Antimicrobial peptide M2 ​​is a modified form of frog antimicrobial peptide, consisting of 22 amino acids with the sequence: Gly 1 Ile 2 Gly 3 Lys 4 Phe 5 Leu 6 Lys 7 Lys 8 Ala 9 Lys 10 Lys 11 Phe 12 Gly 13 Lys 14 Ala 15 Phe 16 Val 17 Lys 18 Ile 19 Leu 20 Lys 21 Lys 22 (SEQ ID NO.1), wherein the C-terminal lysine is amidated.

[0038] In order to facilitate coupling, the modified C-M2 was designed by molecular modification method according to the amino acid sequence of frog antimicrobial peptide M2, and the amino acid sequence was Cys 1 Gly 2 Ile 3 Gly 4 Lys 5 Phe 6 Leu 7 Lys 8 Lys 9 Ala 10 Lys 11 Lys 12 Phe 13 Gly 14 Lys 15 Ala 16 Phe 17 Val 18 Lys 19 Ile 20 Leu 21 Lys 22 Lys 23 (SEQ ID NO.2), wherein the C-terminal lysine is amidated and the specific preparation method is as follows:

[0039] Ⅰ. Based on the amino acid sequence of C-M2, the entire sequence was synthesized using an automatic peptide synthesizer (433A, Applied Biosystems) and desalted using HPLC reverse phase column chromatography;

[0040] Ⅱ. Molecular weight determination was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF);

[0041] III. The purity of purified C-M2 was identified by high performance liquid chromatography (HPLC).

[0042] The secondary amine of vancomycin reacts with the succinimide ester on the bifunctional protein cross-linker sulfo-SMCC in a weakly alkaline environment to form a stable amide bond. Vancomycin and sulfo-SMCC react in equal amounts. Vancomycin is dissolved in PBS buffer (pH = 8.16). After vancomycin is completely dissolved, the pH value of the solution is measured with a pH meter. If the pH value is lower than 8.16, it is necessary to adjust the pH value to 8.16 with NaOH (0.1 mol / L). Sulfo-SMCC is dissolved in an appropriate amount of DMSO, and then the two are mixed and stirred at room temperature for 3 hours. The reaction is monitored by reverse HPLC. After the reaction is complete, the product is purified by preparative HPLC and the molecular weight is identified by high-resolution mass spectrometry. The results of liquid chromatography and mass spectrometry analysis are as follows: Figure 1 As shown, the reaction product obtained in this step is named: Vm-SMCC.

[0043] The maleimide group at the other end of the bifunctional protein cross-linker sulfo-SMCC can react with the sulfhydryl group on the N-terminal cysteine ​​of the antimicrobial peptide in a weakly acidic environment (pH = 5.5) to form a stable thioether bond. Vm-SMCC and the antimicrobial peptide C-M2 react in equal amounts. The two are dissolved in PBS buffer (pH = 5.5). After complete dissolution, the pH value of the solution is measured with a pH meter. If the pH is lower than 5.5, it is necessary to adjust the pH to 5.5 with NaOH (0.1 mol / L) solution. The reaction is stirred at room temperature for 3 hours, and the reaction is monitored by reverse HPLC. After the reaction is complete, the product is purified by preparative HPLC and the molecular weight is identified by high-resolution mass spectrometry. The results of liquid chromatography and mass spectrometry analysis are as follows. Figure 2 As shown, the final product was named: Vm-M2.

[0044] Synthesis route diagram Figure 3 shown.

[0045] Example 2: Pharmacological experiments of vancomycin-antimicrobial peptide conjugate Vm-M2

[0046] 1. Determination of antibacterial activity of Vm-M2:

[0047] (1) Determination of the minimum inhibitory concentration (MIC) of Vm-M2 (2-fold dilution method).

[0048] Eleven strains of Gram-positive bacteria and 18 strains of Gram-negative bacteria were selected for MIC determination. The test strains were inoculated into MH liquid medium (Mei Lun Biotechnology) and cultured at 37°C with shaking until the logarithmic growth phase. Then, the culture medium cultured to the logarithmic growth phase was diluted to 2×10 5 cfu / mL for later use.

[0049] 90 μL MH liquid culture medium was pre-added to each well of a sterile 96-well plate, and then 10 μL of vancomycin, M2 and Vm-M2 sample solutions diluted to a certain concentration with MH liquid culture medium and filtered through a 0.22 μm pore filter membrane were added to the first well. In addition, a physical mixing group of vancomycin and antimicrobial peptide M2 ​​was set up, and the system was 80 μL MH + 10 μL vancomycin + 10 μL M2. After mixing, 50 μL was added to the second well, and then diluted in multiples (Table 1) in sequence, and 50 μL was aspirated from the 8th well and discarded.

[0050] Table 1 Dilution method

[0051]

[0052] After mixing the above tubes, place them at 37°C and shake them slowly at 110 rpm for 16 hours, and measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which bacterial growth is not seen.

[0053] Table 2 Antibacterial activity of vancomycin-antimicrobial peptide conjugate Vm-M2

[0054]

[0055]

[0056] MIC: minimum inhibitory concentration. The above results are the average of three independent repeated experiments.

[0057] As shown in Table 2, the conjugate Vm-M2 has stronger and broader antibacterial activity than vancomycin alone and antimicrobial peptide alone. For Gram-positive bacteria, Vm-M2 overcomes the resistance of Staphylococcus aureus and Enterococcus faecalis to vancomycin. For many Gram-negative bacteria that are not sensitive to vancomycin, especially for some pan-resistant clinical isolates, the conjugate Vm-M2 also exhibits extremely strong antibacterial activity, which is higher than the antimicrobial peptide M2 ​​added alone. At the same time, the physical mixture of vancomycin and antimicrobial peptide was used as a control. The results showed that simple physical mixing did not produce stronger antibacterial activity, which further demonstrated that chemical coupling produced better antibacterial effects. The GM value is the arithmetic mean of the MIC value of the antimicrobial drug against the tested bacteria. The lower the GM value, the stronger the antimicrobial activity. As shown in Table 2, the GM value of vancomycin is 56.48 μM, the GM value of antimicrobial peptide M2 ​​is 8.66 μM, the GM value of the physical mixture group is 4.40 μM, and the GM value of the conjugate Vm-M2 is 2.60 μM. Therefore, it can be considered that the overall antimicrobial activity of the conjugate is 21.72 times higher than that of vancomycin, 3.33 times higher than that of the single antimicrobial peptide M2, and 1.69 times higher than that of the physical mixture group. This shows that the conjugate Vm-M2 has excellent antimicrobial activity and clinical application value.

[0058] (2) Bactericidal kinetics of vancomycin-antimicrobial peptide conjugate Vm-M2

[0059] Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 were cultured in MH liquid medium at 37°C for 6 hours, and then diluted to 10 with fresh MH liquid medium. 6 cfu / mL bacterial suspension. Vancomycin, M2 and Vm-M2 samples dissolved in sterile deionized water were added to the bacterial suspension to a final concentration of 4 μM. The bacterial solution after administration was placed in an incubator at 37°C for shaking culture. 10 μL of the bacterial solution was diluted 1000 times at 0, 15, 30, 60, 120 and 180 minutes, and then 50 μL of the diluted bacterial solution was spread on MH solid culture medium. After overnight culture in a 37°C incubator, the colonies were counted. Because vancomycin is ineffective against Gram-negative bacteria, polymyxin B was used as a positive control for Escherichia coli ATCC25922, and sterilized PBS was used as a negative control.

[0060] Table 3 Bactericidal rate of vancomycin, M2 and Vm-M2 (Escherichia coli ATCC25922)

[0061]

[0062] Table 4 Bactericidal rate of vancomycin, M2 and Vm-M2 (Staphylococcus aureus CMCC26003)

[0063]

[0064] As shown in Tables 3 and 4, for Staphylococcus aureus CMCC26003, the conjugate Vm-M2 exhibited an extremely fast bactericidal rate, killing all live bacteria within 30 minutes. The antimicrobial peptide M2 ​​still had a small amount of live bacteria at 120 minutes. The bactericidal rate of vancomycin was very slow, and it could not effectively kill bacteria within 180 minutes, and could only inhibit bacterial growth. For Escherichia coli ATCC25922, the conjugate Vm-M2 exhibited a bactericidal rate comparable to that of the positive control polymyxin B, killing all bacteria within 30 minutes. Vancomycin, however, had no bactericidal or antibacterial activity.

[0065] (3) Retention bacteria experiment

[0066] After being hit by antimicrobial drugs, most bacteria will die quickly, but there are still a small number of subpopulations that cannot be completely killed. Unlike traditional drug-resistant bacteria, they do not undergo changes in their genetic sequences. This bacterial subpopulation is called retained bacteria, which is a phenotypic variant of bacteria in a dormant state. Although the drug can bind to its target, it cannot kill the bacteria. Therefore, retained bacteria are tolerant to almost all antimicrobial drugs, but their resistance characteristics cannot be inherited to offspring. After collection and re-cultivation, their resistance characteristics disappear and the MIC value does not change significantly. In recent years, more and more clinical cases have shown that retained bacteria are an important cause of chronic infectious diseases that are not cured and symptoms recur. Research on the mechanism of retained bacteria, prevention, and elimination has also received more and more attention. In order to detect the killing effect of the conjugate Vm-M2 on retained bacteria, Staphylococcus aureus CMCC26003 was selected and cultured in a 37°C shaker at 200rpm for 6 hours until the logarithmic growth phase, and then diluted to 10 with MH liquid culture medium. 8 CFU / mL, take 900μL and add it to a centrifuge tube, add ciprofloxacin (CIP) with a final concentration of 20 times MIC to the bacterial solution, and culture it on a shaker at 37℃ for 8 hours. Take 10μL of bacterial solution and dilute it 1000 times at 0h, 2h, 4h, 6h, and 8h, and take 50μL of the diluted bacterial solution to plate. At the 8h time point, take out 300μL of bacterial solution, centrifuge and discard the culture medium, wash 3 times with sterile PBS, remove ciprofloxacin, resuspend with 300μL MH liquid culture medium, divide it into 3 equal parts, 100μL in each tube, add vancomycin, M2 and Vm-M2 samples with a final concentration of 4μM, then take samples at 24h and 48h time points, dilute 1000 times, plate, place in a 37℃ incubator for 16h, and take out the plate for colony counting. This experiment also set up a physical mixing group with vancomycin and antimicrobial peptide M2 ​​co-incubated at 4μM each.

[0067] Depend on Figure 4The experimental results show that after 8 hours of incubation with ciprofloxacin, stable and drug-resistant retained bacteria were formed. When ciprofloxacin was continued to be given for incubation for 48 hours, the number of live bacteria remained almost unchanged. The bactericidal effect of vancomycin on retained bacteria was not significant. The number of retained bacteria colonies only decreased by 1 order of magnitude after incubation for 48 hours. The bactericidal effect of antimicrobial peptide M2 ​​on retained bacteria was stronger than that of vancomycin. The number of retained bacteria colonies after incubation for 48 hours decreased by 3 orders of magnitude. The bactericidal effect of retained bacteria in the physical mixing group was similar to that of the antimicrobial peptide M2 ​​alone. The conjugate Vm-M2 showed a very strong bactericidal effect on retained bacteria. The number of retained bacteria colonies after incubation for 48 hours decreased by more than 4 orders of magnitude. Statistical analysis showed that when the drug was incubated for 48 hours, there were significant differences in the number of retained bacteria colonies in the conjugate group and the vancomycin group alone, the antimicrobial peptide group alone and the physical mixing group. Therefore, the bactericidal effect of the conjugate on retained bacteria was significantly stronger than that of vancomycin and antimicrobial peptide M2 ​​alone, and the bactericidal effect of the physical mixture of vancomycin and antimicrobial peptide on retained bacteria was also inferior to that of the conjugate group.

[0068] 2. Determination of hemolytic activity of vancomycin-antimicrobial peptide conjugate Vm-M2

[0069] The collected human blood was mixed with Aldrich's solution for anticoagulation, washed twice with saline and resuspended into 10 7 -10 8 The diluted red blood cell suspension was mixed with M2 and Vm-M2 samples dissolved in saline, incubated at 37°C for 30 min, and then centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was measured at 540 nm. The negative control used saline, and the positive control used Triton X-100. The hemolysis percentage was calculated according to the following formula: Hemolysis percentage H% = A 样品 -A 阴性对照 / A 阳性对照 × 100%. The results are shown in Table 5.

[0070] Table 5 Hemolytic activity of M2 and Vm-M2

[0071]

[0072] The results showed that when the sample concentration was close to 8 times the GM (arithmetic mean of MIC) value, the hemolysis percentage of Vm-M2 was 8.16%, and at the same concentration, the hemolysis percentage of M2 was 13.45%, indicating that the conjugate Vm-M2 had lower hemolytic activity than the single antimicrobial peptide M2, and was not likely to cause mammalian erythrocyte rupture and lysis. Especially within the range of antimicrobial activity, it was highly safe.

[0073] 3. Experimental study on the salt ion stability of vancomycin-antimicrobial peptide conjugate Vm-M2

[0074] There are many common salt ions in the human body, which are indispensable for maintaining normal life activities of the human body. Most of these salt ions are positively charged. The conjugate Vm-M2 is positively charged due to the conjugation of antimicrobial peptides, while the lipid components of bacterial cell membranes are negatively charged. Studies have shown that positively charged salt ions will compete with antimicrobial peptides to bind to bacterial cell membranes, resulting in the weakening or disappearance of the antimicrobial activity of antimicrobial peptides. Therefore, it is necessary to detect the salt ion stability of the conjugate Vm-M2. Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were selected and cultured at 37°C for 12 hours using MH liquid culture medium (Suzhou Alpha Biotechnology Co., Ltd.). Then, MH liquid culture medium containing physiological salt ion concentrations (NaCl150mM, KCl 4.5mM, CaCl 2 2 mM, MgCl 2 1mM, ZnCl 2 8 μM, FeCl 3 4 μM, NH 4 Cl 6 μM) diluted to 10 5 CFU / ml. Vm-M2 samples with different concentration gradients were prepared using MH liquid culture medium containing corresponding salt ion concentrations. The MIC values ​​of Vm-M2 against Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were determined by the 2-fold dilution method to determine the effect of different salt ions on the antibacterial activity of vancomycin-antimicrobial peptide conjugates.

[0075] like Figure 5 As shown in Figure 2, the conjugate Vm-M2 has strong salt ion tolerance. Under the reference human physiological salt ion concentration (NaCl 150mM, KCl 4.5mM, CaCl 2 2 mM, MgCl 2 1mM, ZnCl 2 8 μM, FeCl 3 4 μM, NH 4 Cl 6μM), the antibacterial activity of the conjugate Vm-M2 remained basically unchanged (the MIC value did not increase by more than one time).

[0076] Example 3: Effect of vancomycin-antimicrobial peptide conjugate Vm-M2 on bacterial colonization effect

[0077] Bacterial swarming is the flagella-dependent migration of bacteria from the inoculation point to the surrounding area on the surface of the culture medium in a group manner. It is a response behavior of bacteria to self-adapt to the environment, which is of great significance to bacterial resistance and biofilm formation. In order to study the effect of the conjugate Vm-M2 on the bacterial clustering effect, we conducted the following experiment: the culture medium was prepared as follows: tryptone (10 g / L), NaCl (10 g / L), yeast extract (5 g / L), agar (0.3%), each component was dissolved in deionized water, and after high-temperature sterilization, vancomycin, M2 and Vm-M2 with a final concentration of 0.5 μM, 1 μM, and 2 μM, as well as a physical mixture of vancomycin and antimicrobial peptides with the same final concentration were added while hot, and an equal volume of PBS buffer was added as a blank control. The plates were placed in six-well plates and cooled for use. Escherichia coli ATCC25922 and Staphylococcus aureus CMCC26003 were taken and cultured at 37°C for 6 hours to the logarithmic growth phase using MH liquid culture medium (Suzhou Alpha Biotechnology Co., Ltd.), respectively. The plates were diluted to 10 6 CFU / mL, aspirate 5 μL and drop it on the cooled solid culture medium, then place it in a 37°C incubator for 24 hours, take it out and take pictures, and use imageJ software to measure the colony area.

[0078] like Figure 6 As shown in the figure, for Escherichia coli ATCC25922, the colony area of ​​the vancomycin group was not significantly different from that of the PBS blank control group, and it had no inhibitory effect on the swarming movement of the tested bacteria. For Staphylococcus aureus CMCC26003, the inhibitory effect of vancomycin on swarming movement was dose-dependent, but its inhibitory effect was weaker than that of the single antimicrobial peptide M2 ​​and the conjugate Vm-M2. In addition, for the above two test bacteria, at the same dosage concentration (2 μM), the colony area of ​​the conjugate Vm-M2 group was significantly smaller than that of the single antimicrobial peptide M 2 drug administration group and physical mixing group. It is worth noting that when the Vm-M2 administration concentration was 2 μM, no Escherichia coli ATCC25922 colonies were observed on the culture medium, so the colony area was 0. This may be because all Escherichia coli were killed at this concentration. In summary, the conjugate Vm-M2 can effectively block signal transduction between bacterial cells and effectively inhibit bacterial clustering movement behavior, which will help inhibit the production of biofilms and drug resistance, further illustrating that the conjugate Vm-M2 has great application value.

[0079] Example 4: Study on the anti-biofilm activity of vancomycin-antimicrobial peptide conjugate Vm-M2

[0080] 1. Biofilm Removal Activity Assay

[0081] Take out the stored strain from the -80℃ refrigerator, thaw quickly in a 37℃ water bath, dip a little liquid with an inoculation loop, and streak in four areas on the LB solid medium in a Z shape, starting from the end of the previous streak, and culture at 37℃ until a single colony grows; pick a single colony in sterile liquid MHB medium, and culture at 37℃, 200rpm with shaking until the logarithmic growth phase; test the bacterial solution concentration and dilute it to 1×10 7 CFU / mL. Add 100 μL of the above bacterial solution to a sterile 96-well plate and culture at 37°C for 48 hours to allow biofilm formation. Aspirate the bacterial solution from each well and wash three times with PBS. Add 100 μL of diluted vancomycin, M2 and conjugate Vm-M2 samples to each well. The physical mixing group is 50 μL vancomycin + 50 μL M2. The final concentrations of the samples are 1 μM, 2 μM, 4 μM and 8 μM. After adding the samples, place them at 37°C for 24 hours. Add crystal violet stain (0.1%) to each well, aspirate the stain after 30 minutes of staining, wash three times with sterile PBS, and air-dry in a clean bench. Add 100 μL of anhydrous ethanol to each well and let it stand for 20 minutes to dissolve the crystal violet. Detect the OD value at an ultraviolet wavelength of 560 nm, and set up three parallel experiments. The percentage of biofilm formation (BiofilmRetention%, BR%) is calculated using the following formula: BR (%) = 100% - [100% × (F 0 -F drug ) / F 0 ], PBS was selected as the negative control in this experiment, and its measured value was regarded as the maximum biofilm residue. Biofilm Retention%, BR% is the biofilm retention percentage, F 0 is the absorbance value of the PBS-treated group, F drug It is the absorbance value of the drug treatment group.

[0082] Table 6 Biofilm removal activity of vancomycin, M2 and Vm-M2 (Escherichia coli ATCC25922)

[0083]

[0084] Table 7 Biofilm removal activity of vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA52)

[0085]

[0086] 2. Biofilm Inhibitory Activity Assay

[0087] Take out the stored strain from the -80℃ refrigerator, thaw quickly in a 37℃ water bath, take a little liquid and make a Z-shaped line on the LB solid medium, and culture at 37℃ until a single colony grows; pick a single colony in sterile liquid MHB medium, and culture at 37℃, 200rpm shaking until the logarithmic growth phase; test the bacterial solution concentration and dilute it to 2×10 7 CFU / mL. Add 50 μL of the above bacterial solution to a sterile 96-well plate, add 50 μL of diluted vancomycin, M2 and conjugate Vm-M2 samples to each well, the physical mixing group is 25 μL vancomycin + 25 μL M2, the final concentration of the sample is 1 μM, 2 μM, 4 μM and 8 μM, and culture at 37°C for 48 hours. Aspirate the bacterial solution from each well, wash three times with PBS, and place the plate on a clean bench for ventilation and air drying. Add crystal violet stain (0.1%) to each well, aspirate the stain after staining for 30 minutes, wash three times with sterile PBS, and air dry in a clean bench. Add 100 μL of anhydrous ethanol to each well and let it stand for 20 minutes to dissolve the crystal violet. Detect the OD value at an ultraviolet wavelength of 560 nm. Calculate the percentage of biofilm formation (BiofilmFormation%, BF%) using the following formula: BF(%) = 100% - [100% × (F 0 -F drug ) / F 0 ], where PBS(F 0 ) is the negative control, and its measured value is regarded as the maximum biofilm formation amount. Biofilm Formation%, BF% is the percentage of biofilm formation, F drug It is the absorbance value of the drug treatment group.

[0088] Table 8 Biofilm inhibition activity of vancomycin, M2, and Vm-M2 (Escherichia coli ATCC25922)

[0089]

[0090] Table 9 Biofilm Inhibitory Activity of Vancomycin, M2 and Vm-M2 (Staphylococcus aureus VRSA52)

[0091]

[0092] As shown in Tables 6, 7, 8, and 9, for Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52, vancomycin had no biofilm removal and inhibition activity, while antimicrobial peptide M2 ​​and conjugate Vm-M2 both showed concentration-dependent antibiofilm activity. Interestingly, at the same administration concentration, conjugate Vm-M2 showed stronger biofilm removal and inhibition activity than antimicrobial peptide M2 ​​alone, and the antibiofilm activity of the physical mixture group was also inferior to that of the conjugate group. Therefore, it can be considered that conjugate Vm-M2 has good application potential in chronic refractory diseases caused by bacterial infections.

[0093] Example 5: Determination of the synergistic antibacterial effect of vancomycin-antimicrobial peptide conjugate Vm-M2 and traditional antibiotics

[0094] Weigh antibiotics (meropenem, polymyxin B, ciprofloxacin and gentamicin) and dissolve them in sterile water to make a 2 mg / mL solution. Dilute them in multiples to get a concentration of 8-1 / 64 MIC. Add solvent to make 11 concentrations for later use. Make the conjugate Vm-M2 in sterile water to make a concentration of 4×MIC. Dilute them in multiples to get a concentration of 4-1 / 16 MIC. Add solvent to make 8 concentrations for later use. Dilute the bacterial solution to 5×10 5 CFU / mL for later use. Add 90 μL of diluted bacterial solution to a 96-well plate; add antibiotics to the bacteria, 5 μL per well, one concentration per column, for a total of 11 columns: add the conjugate Vm-M2 to the bacterial solution, 5 μL per well, one concentration per row, for a total of 8 rows; find MICA, MICB, A,, B to calculate FIC, FIC = FMICA + FMICB = A / MICA + B / MICB (A, B represent the concentrations at the optimal convergence point for the combination of the two drugs, MICA, MICB represent the MICs when the two drugs are used alone), when FIC < 0.5, there is a synergistic effect, when FIC > 4 (including 4), there is an antagonistic effect, when 0.5 < FIC < 2 (including 0.5), the two drugs have an additive effect, when 2 < FIC < 4 (including 2), the two drugs have an irrelevant effect, and the dosing method is shown in the following table.

[0095]

[0096] Table 10 Synergistic antibacterial effect of Vm-M2 and traditional antibiotics

[0097]

[0098] Escherichia coli ATCC25922 and vancomycin-resistant Staphylococcus aureus VRSA52 were selected as test bacteria to detect the synergistic antibacterial effects of the above four first-line clinical antibiotics and the conjugate Vm-M2. According to the experimental results, the conjugate has synergistic effects with other antibiotics except meropenem, which may be related to the inherent action mechanism of different antibiotics. In general, the conjugate has a synergistic antibacterial effect with most first-line clinical antibiotics, showing its great potential in clinical combination therapy.

[0099] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A vancomycin-antimicrobial peptide conjugate, characterized in that: The vancomycin-antimicrobial peptide conjugate is obtained by covalently cross-linking vancomycin and an antimicrobial peptide with an amino acid sequence as shown in SEQ ID NO.2, wherein the N-terminus of the antimicrobial peptide is modified with cysteine, and the covalent cross-linking is achieved by a bifunctional protein cross-linker Sulfo-SMCC.

2. Use of the vancomycin-antimicrobial peptide conjugate according to claim 1 in the preparation of an antibacterial agent, wherein the antibacterial agent is used to inhibit Gram-positive bacteria or Gram-negative bacteria; The Gram-positive bacteria are Staphylococcus aureus ATCC29213, Enterococcus faecalis ATCC29212, Clostridium perfringens ATCC13124, and Bacillus cereus CMCC63301; The Gram-negative bacteria are Escherichia coli ATCC25922, Escherichia coli CMCC44102, Acinetobacter baumannii ATCC19606, and Shigella flexneri ATCC12022.

3. Use of the vancomycin-antimicrobial peptide conjugate according to claim 1 in inhibiting bacterial biofilm formation.

4. An antibacterial drug composition, characterized in that: The antibacterial pharmaceutical composition comprises the vancomycin-antimicrobial peptide conjugate according to claim 1 and an antibiotic, wherein the antibiotic is selected from one of polymyxin B, ciprofloxacin and gentamicin.

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