An antimicrobial peptide GK18 with broad-spectrum antimicrobial activity and its application

Abstract

The present invention relates to the field of antimicrobial peptide application technology, and specifically to an antimicrobial peptide GK18 with broad-spectrum antimicrobial activity and its application. The amino acid sequence of the antimicrobial peptide GK18 is shown in SEQ ID NO. 1. The antimicrobial peptide GK18 of the present invention has antimicrobial activity against a variety of bacteria (Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and their drug-resistant strains, and has excellent biosafety and physiological stability. It has broad development prospects and can be used in alternative treatments for bacterial infections or in bacteriostatic or bactericidal applications.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide applications, and in particular to an antimicrobial peptide GK18 with broad-spectrum antimicrobial activity and applications thereof. Background Art

[0002] Antimicrobial peptides (AMPs), as a crucial component of the innate immune system, provide the body's first line of defense against microbial infection. As a class of active small-molecule polypeptides, AMPs are not only active against bacteria, fungi, and viruses, but are also less likely to induce bacterial resistance than traditional small-molecule antibiotics. Furthermore, AMPs can be combined with commonly used clinical antibiotics to restore antibacterial or bactericidal activity, effectively reducing the dosage of more toxic drugs and minimizing adverse drug reactions. These unique activity advantages make AMPs an ideal alternative to next-generation antibiotics, potentially alleviating the AMR crisis caused by drug-resistant bacterial infections.

[0003] However, most of the existing antimicrobial peptides are narrow-spectrum antimicrobial peptides and do not have antimicrobial effects on a variety of bacteria. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an antimicrobial peptide GK18 with broad-spectrum antimicrobial activity and its application.

[0005] The present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an antimicrobial peptide GK18 having broad-spectrum antimicrobial activity. The amino acid sequence of the antimicrobial peptide GK18 is shown in SEQ ID NO.1.

[0007] The present invention screened out a highly effective and low-toxic cationic active AMP molecule. Experiments confirmed that the antimicrobial peptide GK18 exhibited broad-spectrum antimicrobial activity against the standard strains tested (Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and their resistant strains. In addition, the antimicrobial peptide GK18 also has good biocompatibility (hemolytic / cytotoxicity) and physiological stability. These biological properties indicate that the antimicrobial peptide GK18 has good development prospects and can be used for alternative treatments for bacterial infections or in antibacterial or bactericidal applications.

[0008] In a second aspect, the present invention provides the use of the antimicrobial peptide GK18 in the preparation of an antimicrobial product, wherein the antimicrobial product is used to inhibit or kill Staphylococcus aureus and its resistant strains, Escherichia coli and its resistant strains, Acinetobacter baumannii and its resistant strains, Pseudomonas aeruginosa and its resistant strains, and Klebsiella pneumoniae and its resistant strains.

[0009] Furthermore, the antibacterial product contains the antibacterial peptide GK18 as the only active ingredient.

[0010] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0011] Specifically, the antimicrobial peptide can be used as a broad-spectrum antimicrobial drug for the treatment of various diseases caused by drug-resistant bacterial infections, and is particularly suitable for combating Staphylococcus aureus and its drug-resistant strains, Escherichia coli and its drug-resistant strains, Acinetobacter baumannii and its drug-resistant strains, Pseudomonas aeruginosa and its drug-resistant strains, and Klebsiella pneumoniae and its drug-resistant strains; the antibacterial agent can be used in the fields of medical care, food preservation, personal care products, and environmental disinfection.

[0012] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the auxiliary material is an auxiliary material required for preparing one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

[0014] In a third aspect, the present invention provides the use of the antimicrobial peptide GK18 in the preparation of a drug for treating cancer.

[0015] Furthermore, the cancer includes lung cancer and cervical cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a functionally diverse and high-performance polypeptide in the field of antimicrobial peptides, offering important technical support and alternative resources for the development of antimicrobial drugs and the treatment of multidrug-resistant bacterial infections. It also supports the development of anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The protein structure of the antimicrobial peptide GK18, A is the helical wheel analysis diagram of the antimicrobial peptide GK18, and B is the protein structure modeling diagram.

[0019] Figure 2 The antibacterial activity of the antimicrobial peptide GK18 against the tested bacteria, A: the tested bacteria is S. aureus ATCC25923, B: the tested bacteria is MRSA_WM, C: the tested bacteria is E. coli ATCC25922, D: the tested bacteria is A. baumannii ATCC19606, E: the tested bacteria is P. aeruginosa CMCC10104, F: the tested bacteria is K. pneumoniae ATCC700603.

[0020] Figure 3These are the biocompatibility test results of the antimicrobial peptide GK18, A: hemolysis test results, B: toxicity test results of the antimicrobial peptide GK18 on LO2 cells, B: toxicity test results of the antimicrobial peptide GK18 on HK-2 cells, C: toxicity test results of the antimicrobial peptide GK18 on A549 cells, D: toxicity test results of the antimicrobial peptide GK18 on HeLa cells.

[0021] Figure 4 These are the results of physiological stability test of antimicrobial peptide GK18. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0023] Example 1: Preparation and characterization of antimicrobial peptide GK18

[0024] The amino acid sequence of the antimicrobial peptide GK18 of the present invention is shown in SEQ ID NO.1. It is prepared by Gill Biochemical (Shanghai) Co., Ltd. using a solid-phase chemical synthesis method. The synthesis steps include: ① connecting the first amino acid to an insoluble polymer resin support; ② protecting the active functional groups that do not participate in peptide chain extension; ③ activating the functional groups that participate in peptide chain extension and then connecting the next amino acid; ④ after the polypeptide sequence is extended to a specified length, it is cut from the resin support, the protected functional groups are deprotected, and the resulting crude peptide is purified; ⑤ subsequently, using HPLC-MS mass spectrometry to detect the purity of the polypeptide and identify the actual molecular weight of the polypeptide.

[0025] To investigate whether the activity of the antimicrobial peptide GK18 is correlated with structural factors, prediction analysis was performed using helical wheel diagrams, three-dimensional structure projection, and ProtParam software. Figure 1 A shows the helical wheel map of the antimicrobial peptide GK18; Figure 1 Table B shows the crystal structure of the antimicrobial peptide GK18, which exhibits an α-helical structure. Table 1 details the molecular weight, isoelectric point, net charge, half-life, hydrophobicity, and hydrophobic moment of the antimicrobial peptide GK18. These results indicate that the antimicrobial peptide GK18 is a typical cationic α-helical peptide.

[0026] SEQ ID NO. 1: GKKIKKFVWVVMRKLIMH.

[0027] Table 1 Analysis results of the physicochemical properties of antimicrobial peptide GK18

[0028] Physical and chemical indicators feature Relative molecular mass 2240.91 Structural formula <![CDATA[C 187 H 181 N 29 O 19 S2]]> Isoelectric point 11.39 Net charge +6 half life In vitro half-life 30h / in vivo half-life >20h hydrophobicity 0.535 hydrophobic moment 0.342

[0029] Example 2: Verification of the antibacterial activity of antimicrobial peptide GK18

[0030] To clarify the drug sensitivity of the antimicrobial peptide GK18 against clinical Gram-positive and Gram-negative bacteria, the minimum inhibitory concentration (MIC) was determined using the microdilution method. The MIC is expressed as molar concentration / mass concentration, with the molar concentration unit being μM and the mass concentration unit being μg / mL. The specific method is as follows: the concentration of the test bacterial solution in the logarithmic phase was adjusted to 2.0×10 6 CFU / mL; then, equal volumes of bacterial solution were added to 96-well plates with different concentrations of antimicrobial peptide GK18, and equal volumes of 100 μL MHB culture medium and 100 μL of the test bacterial solution were set as blank control and growth control, respectively; the above 96-well plates were incubated at 37°C for 24 hours and the results were observed. The drug concentration corresponding to the clear or no growth wells observed by naked eye compared with the negative control group was the MIC value of the test drug.

[0031] The results are shown in Table 2. The antimicrobial peptide GK18 showed good in vitro antibacterial effects on the tested bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae), with MIC values ​​ranging from 1.30 / 4 to 2.60 / 8. In order to further clarify the activity of the antimicrobial peptide GK18 against clinical drug-resistant bacteria, we measured the MIC values ​​of the antimicrobial peptide GK18 against 3 strains of MRSA, 3 strains of CRAB, 3 strains of ESBL-EC, 3 strains of CRPA and 3 strains of CRKP. The results are still encouraging: the antimicrobial peptide GK18 also showed good antibacterial activity against the above-mentioned drug-resistant bacteria (Table 3). To further verify the activity of the antimicrobial peptide GK18, we used the changes in the MIC value after 24 hours of action of the antimicrobial peptide GK18. The results are as follows Figure 2 As shown in the figure, antimicrobial peptide GK18 can effectively kill the tested bacteria at the MIC concentration. The above results show that antimicrobial peptide GK18 is a new type of antimicrobial peptide with high efficiency and broad spectrum, and has good research value.

[0032] Table 2 In vitro antimicrobial spectrum of antimicrobial peptide GK18

[0033]

[0034] Note: Polymyxin B: PMB; Vancomycin: Van; “NA” means not detected, the same below.

[0035] Table 3 In vitro activity of antimicrobial peptide GK18 against drug-resistant bacteria

[0036]

[0037] Example 3: Biocompatibility of antimicrobial peptide GK18

[0038] To investigate the safety of the antimicrobial peptide GK18, we tested the hemolytic and cytotoxic properties of the antimicrobial peptide GK18. The specific methods are as follows: ① Hemolytic assay: 8% hRBCs were incubated with different concentrations of GK18 for 24 hours, and the supernatant was taken and the A level in each dose group was detected using a multifunctional microplate reader. 540 nm absorbance value. 10mM PBS and 1% Triton-X 100 were used as negative and positive controls respectively. ② Cytotoxicity assay: 2×10 4 Cells were transferred to 96-well plates and cultured overnight. The next day, different concentrations of GK18 were added to the 96-well plates and cultured for 24 hours. Cells alone and culture medium alone served as negative and blank controls, respectively, and assayed using the CCK8 detection kit.

[0039] The hemolytic results showed that the antimicrobial peptide GK18 did not show obvious hemolysis on human red blood cells at a concentration of 32 μM ( Figure 3 A); Cytotoxicity results also showed that at a concentration of 32 μM, the antimicrobial peptide GK18 had a low cytotoxic effect on human normal liver cells LO2 and human renal proximal tubular epithelial cells HK-2 ( Figure 3 BC); and showed anticancer activity against cancer cells (A549 and HeLa) ( Figure 3 DE). The above results indicate that the antimicrobial peptide GK18 has good biocompatibility and has the potential to be prepared into an anticancer drug.

[0040] Example 4: Physiological stability of antimicrobial peptide GK18

[0041] To evaluate the physiological stability of the antimicrobial peptide GK18, this study used a microdilution method to examine the changes in the MIC values ​​of the novel peptide under different environmental conditions. The specific method is as follows: Different concentrations of the antimicrobial peptide GK18 were added to test bacterial solutions containing varying concentrations of NaCl, KCl, CaCl2, serum, and proteases (trypsin, pepsin, and proteinase K). Enzyme stability testing required eliminating enzyme activity in a 60°C water bath. MHB culture medium served as a control group. After incubation at 37°C for 24 hours, the MIC values ​​of GK18 were measured.

[0042] The results are as follows Figure 4 As shown in the results, 5mM NaCl, 5mM KCl, 5mM CaCl2, and 10% serum had no significant effect on the activity of the antimicrobial peptide GK18. Surprisingly, the activity of the antimicrobial peptide GK18 increased only 2-4 times in the presence of pepsin and proteinase K. Like other peptides, the antimicrobial peptide GK18 is sensitive to trypsin and is easily inactivated in the presence of trypsin.

[0043] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. An application of an antimicrobial peptide GK18 with broad-spectrum antimicrobial activity in the preparation of an antimicrobial product, characterized in that: The amino acid sequence of the antimicrobial peptide GK18 is shown in SEQ ID NO.

1. The antimicrobial product is used to inhibit or kill Staphylococcus aureus and its drug-resistant strains, Escherichia coli and its drug-resistant strains, Acinetobacter baumannii and its drug-resistant strains, Pseudomonas aeruginosa and its drug-resistant strains, and Klebsiella pneumoniae and its drug-resistant strains.

2. The use of the antimicrobial peptide GK18 in the preparation of antimicrobial products according to claim 1, characterized in that: The antibacterial product uses the antibacterial peptide GK18 as the only active ingredient.

3. The use of the antimicrobial peptide GK18 in the preparation of antimicrobial products according to claim 1, characterized in that: The antibacterial product is a medicine or a bacteriostatic agent.

4. The use of the antimicrobial peptide GK18 in the preparation of antimicrobial products according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use of the antimicrobial peptide GK18 in the preparation of antimicrobial products according to claim 4, characterized in that: The auxiliary material is an auxiliary material required for preparing one of water extracts, powders, lotions, tinctures, oils, emulsions, ointments, plasters or aerosols.

6. Use of the antimicrobial peptide GK18 according to claim 1 in the preparation of a drug for treating cancer, characterized in that: The cancers are lung cancer and cervical cancer.

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