Antibacterial peptide capable of promoting anti-gram-negative bacterium activity in serum environment as well as preparation method and application of antibacterial peptide

Abstract

The invention belongs to the technical field of biological medicine, and discloses an antibacterial peptide capable of promoting anti-gram-negative bacterium activity in a serum environment and a preparation method and application thereof, the antibacterial peptide comprises any one of antibacterial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2 and IR3, and the amino acid sequences are respectively shown as SEQ ID NO: 1-SEQ ID NO: 17. The toad antibiosis peptide Cache-DM is obtained by structural modification of the toad antibiosis peptide Cache-DM. The method comprises the following steps: intercepting a core sequence of the Cache-DM, and replacing the truncated sequence with amino acid Try / Ile-Arg / Lys. The antibacterial peptide disclosed by the invention is short in sequence, small in molecular weight and low in hemolysis. After being acted by serum, the group of antibacterial peptides has an efficient inhibiting effect on gram-negative bacteria, but does not damage gram-positive bacteria, and the group of polypeptides has the potential of becoming a narrow-spectrum antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an antimicrobial peptide having the activity of promoting anti-Gram-negative bacteria in a serum environment, and a preparation method and application thereof. Background Art

[0002] The emergence of antibiotics has improved the prognosis of patients with bacterial infections and reduced the mortality rate caused by bacterial infections. However, with the overuse of antibiotics, accompanied by the evolution and selection of bacteria, bacteria gradually develop resistance to antibiotics. In order to face the increasingly serious problem of bacterial resistance, it is necessary to develop new antibiotics or alternative drugs.

[0003] Antimicrobial peptides (AMPs) are a class of polypeptides that are widely present in nature and are mainly involved in immune regulation in organisms. As research has found, antimicrobial peptides also have biological activities such as antibacterial, antiviral, and anticancer. In addition, the advantages of small toxic side effects and non-immunogenicity have also made antimicrobial peptides receive much attention in the field of biomedicine. Antimicrobial peptides can be used to destroy cell membranes or enter cells to target intracellular substances such as nucleic acids and mitochondria to exert their biological activities. Antimicrobial peptides can directly destroy cell membranes and can act simultaneously through a single or multiple mechanisms to achieve antibacterial effects, which also makes antimicrobial peptides less likely to develop drug resistance. Therefore, antimicrobial peptides are expected to become a substitute for traditional antibiotics.

[0004] Since serum contains abundant enzymes, natural antimicrobial peptides are easily inactivated by enzymatic hydrolysis in serum. Improving the stability of antimicrobial peptide molecules in serum is also a problem that needs to be solved in the development of antimicrobial peptides. In summary, the development of antimicrobial peptides with high safety, high biological activity, and stability in serum is of great significance for promoting the application of antimicrobial peptides. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an antimicrobial peptide having the activity of promoting anti-Gram-negative bacteria in a serum environment, and a preparation method and application thereof.

[0006] The present invention is achieved by providing an antimicrobial peptide having an activity of promoting anti-Gram-negative bacteria under a serum environment, wherein the amino acid sequences of the antimicrobial peptides are as shown in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, and SEQ ID No: 17.

[0007] Another object of the present invention is to provide a method for preparing an antimicrobial peptide having an activity promoting anti-Gram-negative bacteria in a serum environment, the method for preparing the antimicrobial peptide comprising the following steps:

[0008] Cath-DM-NCT' disclosed in CN117986342A was further modified, and the R at positions 1 and 15 and the K at position 11 of Cath-DM-NCT' were replaced with tryptophan (Trp, W) or isoleucine (Ile, I), and the L at positions 9 and 14 were replaced with lysine (Lys, K) or arginine (Arg, R). In addition, only SEQ ID No: 14 was amidated at the C-terminus.

[0009] The specific names and sequences of antimicrobial peptides are as follows:

[0010] W1: amino acid sequence as shown in SEQ ID NO.1;

[0011] W2: amino acid sequence as shown in SEQ ID NO.2;

[0012] WK1: amino acid sequence as shown in SEQ ID NO.3;

[0013] WK2: amino acid sequence as shown in SEQ ID NO.4;

[0014] WK3: amino acid sequence as shown in SEQ ID NO.5;

[0015] WR1: amino acid sequence as shown in SEQ ID NO.6;

[0016] WR2: amino acid sequence as shown in SEQ ID NO.7;

[0017] WR3: amino acid sequence as shown in SEQ ID NO.8;

[0018] I1: amino acid sequence as shown in SEQ ID NO.9;

[0019] I2: amino acid sequence as shown in SEQ ID NO.10;

[0020] IK1: amino acid sequence as shown in SEQ ID NO.11;

[0021] IK2: amino acid sequence as shown in SEQ ID NO.12;

[0022] IK3: amino acid sequence as shown in SEQ ID NO.13;

[0023] IK3-NH2: amino acid sequence as shown in SEQ ID NO.14;

[0024] IR1: amino acid sequence as shown in SEQ ID NO.15;

[0025] IR2: amino acid sequence as shown in SEQ ID NO.16;

[0026] IR3: The amino acid sequence is shown in SEQ ID NO.17.

[0027] Furthermore, the preparation method of sequence-derived antimicrobial peptides is as follows: according to the amino acid sequence of the derived antimicrobial peptides W1 to IR3, the full sequence is obtained by solid phase synthesis, and then the group of antimicrobial peptides is obtained by HPLC reverse phase column chromatography desalting, and the peptides are identified by ultra high performance liquid chromatography-mass spectrometry analyzer.

[0028] Another object of the present invention is to provide the use of any one or more of the antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 in the preparation of a medicament for treating Gram-negative bacterial infections in the blood.

[0029] Furthermore, the Gram-negative bacteria include Escherichia coli.

[0030] Another object of the present invention is to provide an antimicrobial peptide preparation for use against Gram-negative bacteria, characterized in that the antimicrobial peptide includes any one or a combination of antimicrobial peptide W1, antimicrobial peptide W2, antimicrobial peptide WK1, antimicrobial peptide WK2, antimicrobial peptide WK3, antimicrobial peptide WR1, antimicrobial peptide WR2, antimicrobial peptide WR3, antimicrobial peptide I1, antimicrobial peptide I2, antimicrobial peptide IK1, antimicrobial peptide IK2, antimicrobial peptide IK3, antimicrobial peptide IK3-NH2, antimicrobial peptide IR1, antimicrobial peptide IR2, and antimicrobial peptide IR3.

[0031] Furthermore, the preparation further comprises an excipient for enhancing the stability and biological activity of the antimicrobial peptide in a serum environment.

[0032] Furthermore, the preparation is in the form of an injection, a spray or a paste, and is used to treat or prevent Gram-negative bacterial infection.

[0033] Another object of the present invention is to provide an antimicrobial peptide composition having the ability to promote anti-Gram-negative bacteria activity in a serum environment, characterized in that the composition comprises a mixture of antimicrobial peptide IK3-NH2 and antimicrobial peptide IR3, and the antimicrobial peptide is prepared by solid phase synthesis and identified by HPLC purification and mass spectrometry analysis.

[0034] Furthermore, the antimicrobial peptide IK3-NH2 is amidated at the C-terminus to improve its stability and activity in a serum environment.

[0035] Furthermore, the composition is used to treat systemic or local infections caused by Gram-negative bacteria, and can be used in combination with other antibiotics.

[0036] Another object of the present invention is to provide an antimicrobial peptide delivery system for resisting Gram-negative bacterial infection, characterized in that the system comprises nanoparticles encapsulated with antimicrobial peptides W1, W2, WK1, WK2, and WK3, and the antimicrobial peptides are prepared by solid phase synthesis and purified by HPLC.

[0037] Furthermore, the nanoparticles are made of chitosan or PLGA (polylactic acid-glycolic acid copolymer) materials, have a sustained release function, and can continuously release antimicrobial peptides in vivo.

[0038] Furthermore, the system is used for treating infections caused by Gram-negative bacteria through intravenous injection or local application, and can reduce the occurrence of traditional antibiotic resistance problems.

[0039] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0040] First, the present invention is based on shortening the peptide chain of the black-orbiting toad antimicrobial peptide Cath-DM disclosed in CN104119434A, further obtaining Cath-DM-NCT' disclosed in CN117986342A, and replacing it with amino acids Try / Ile-Arg / Lys, and performing C-terminal amidation treatment on some antimicrobial peptides to obtain derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, IR3. Based on the above, the seventeen derivative antimicrobial peptides can significantly improve their antibacterial activity against Escherichia coli under the action of serum, so that this group of polypeptides has the potential to become a narrow-spectrum antibiotic substitute.

[0041] Second, the method for preparing the antimicrobial peptide with enhanced anti-Gram-negative bacterial activity in serum environment provided by the present invention demonstrates significant technological progress. The following are some specific aspects:

[0042] 1. Innovative antimicrobial peptide design: Traditional antimicrobial peptides have a wide range of sources, but using antimicrobial peptides from Bufo bufo gargarizans for derivative design, especially by precisely modifying and replacing their sequences to obtain peptides with stronger antimicrobial activity, is an innovative attempt.

[0043] 2. Precise amino acid replacement and modification: The specific amino acids of the derivative peptide Cath-DM-NCT' derived from the toad antimicrobial peptide Cath-DM were precisely replaced and modified, such as tryptophan (Trp, W), lysine (Lys, K) and arginine (Arg, R). This regulation not only enhances the antibacterial activity of the antimicrobial peptide, but also improves its stability and bioavailability.

[0044] 3. C-terminal amidation treatment: C-terminal amidation treatment of the peptide not only increases the stability of the peptide, but also improves its solubility in aqueous solution, making it easier for subsequent drug development and application.

[0045] 4. Efficient preparation method: The entire preparation process is simple and efficient, and it is easy to achieve large-scale production, which facilitates subsequent drug development and clinical application.

[0046] 5. Broad application prospects: Since these antimicrobial peptides have strong antimicrobial activity, they have broad application prospects in the fields of medicine, agriculture, food industry, etc. In particular, these antimicrobial peptides play an important role in combating antibiotic-resistant bacteria.

[0047] The method for preparing an antimicrobial peptide with anti-Gram-negative bacteria activity in a serum environment provided by the present invention achieves a significant improvement in antimicrobial activity by precisely regulating the amino acid sequence of the antimicrobial peptide, while simplifying the preparation process and improving production efficiency, demonstrating significant technological progress and innovation.

[0048] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0049] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0050] Antibacterial drugs are in urgent need of development, and the market demand for antibacterial drugs is huge. As a natural product, antimicrobial peptides have unique advantages, are safer for organisms, and have great potential for the development of antibacterial drugs. The seventeen polypeptides displayed in the present invention have great potential for the development of in vivo anti-Gram-negative bacteria drugs. Since this group of derivative peptides has improved antibacterial activity against Escherichia coli in a serum environment, this group of polypeptides has the potential to become a narrow-spectrum antibiotic substitute.

[0051] (2) The technical solution of the present invention overcomes technical prejudice:

[0052] At present, the means of improving the stability and activity of polypeptides in serum are to add metal ions and physically encapsulate the polypeptides. However, the group of antimicrobial peptides shown in the present invention, without other modifications, still maintain good biological activity against Escherichia coli in Gram-negative bacteria in serum, and even improve its biological activity. The invention provides a polypeptide without additional modifications to improve its biological activity in serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0054] Figure 1 It is a flow chart of a method for preparing an antimicrobial peptide having activity promoting anti-Gram-negative bacteria in a serum environment provided by an embodiment of the present invention;

[0055] Figure 2It is a high performance liquid chromatography (HPLC) analysis chart of the derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 provided in Example 1 of the present invention. A is W1, B is W2, C is WK1, D is WK2, E is WK3, F is WR1, G is WR2, H is WR3, I is I1, J is I2, K is IK1, L is IK2, M is IK3, N is IK3-NH2, O is IR1, P is IR2, and Q is IR3;

[0056] Figure 3 : is a mass spectrometry (MS) analysis chart of the derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 provided in Example 1 of the present invention. A is W1, B is W2, C is WK1, D is WK2, E is WK3, F is WR1, G is WR2, H is WR3, I is I1, J is I2, K is IK1, L is IK2, M is IK3, N is IK3-NH2, O is IR1, P is IR2, and Q is IR3. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] Based on the above method for preparing an antimicrobial peptide having anti-Gram-negative bacterial activity in a serum environment, the following are two specific examples:

[0059] Example 1: Preparation of antimicrobial peptide IK3 for the treatment of bacterial infections in the blood

[0060] 1. Peptide design and synthesis:

[0061] According to the above steps, the middle 15 amino acids were cut from the antimicrobial peptide Cath-DM derived from Bufo bufo molitor and subjected to specific amino acid replacement treatment to synthesize the antimicrobial peptide IK3.

[0062] 2. In vitro antibacterial activity test:

[0063] In vitro antibacterial activity tests were performed using a variety of Gram-negative bacterial strains. The synthesized IK3 was co-cultured with bacteria and its antibacterial effect was evaluated by determining the minimum inhibitory concentration (MIC).

[0064] 3. In vivo effect verification:

[0065] Select a suitable animal model (such as mice) to establish an infection model, inject IK3 through the tail vein, observe the survival rate, pathological changes and bacterial clearance of the animals after treatment, and evaluate its in vivo antibacterial effect and safety.

[0066] 4.Drug preparation development:

[0067] Based on the results of in vivo and in vitro tests, IK3 is developed into pharmaceutical preparations, such as solutions, injectable gels, etc., to meet different clinical application needs.

[0068] Example 2: Development of new antibacterial materials using antimicrobial peptide WK3

[0069] 1. Peptide synthesis:

[0070] According to the above steps, WK3 was extracted and modified from the antimicrobial peptide Cath-DM from Bufo nigropunctatus.

[0071] 2. Preparation of antibacterial materials:

[0072] The synthesized WK3 is mixed with appropriate carrier materials (such as polymers, nanoparticles) to prepare antibacterial materials. The preparation of the material needs to consider the stability, release rate and antibacterial activity of the peptide segment.

[0073] 3. Evaluation of antibacterial properties of materials:

[0074] Antibacterial performance tests were carried out using a variety of bacteria to evaluate the antibacterial effect and biocompatibility of the materials.

[0075] 4. In vivo effect test:

[0076] Construct animal models of specific diseases (such as mice), such as a colorectal cancer model associated with Escherichia coli infection, to evaluate the antibacterial properties of the antimicrobial peptides in the material against pathogenic bacteria in the tumor and the overall anti-cancer ability of the material.

[0077] These two examples demonstrate the broad application potential of antimicrobial peptides with enhanced anti-Gram-negative bacterial activity in a serum environment in the fields of medicine and materials science, which not only provide new strategies for antimicrobial therapy, but also provide valuable bioactive molecules for the development of new antimicrobial materials.

[0078] The antimicrobial peptides provided in the embodiments of the present invention have anti-Gram-negative bacteria activity in a serum environment, and the antimicrobial peptides include any one of antimicrobial peptide W1, antimicrobial peptide W2, antimicrobial peptide WK1, antimicrobial peptide WK2, antimicrobial peptide WK3, antimicrobial peptide WR1, antimicrobial peptide WR2, antimicrobial peptide WR3, antimicrobial peptide I1, antimicrobial peptide I2, antimicrobial peptide IK1, antimicrobial peptide IK2, antimicrobial peptide IK3, antimicrobial peptide IK3-NH2, antimicrobial peptide IR1, antimicrobial peptide IR2, and antimicrobial peptide IR3.

[0079] like Figure 1 As shown, an embodiment of the present invention provides a method for preparing an antimicrobial peptide having activity of promoting anti-Gram-negative bacteria in a serum environment, and the method for preparing the antimicrobial peptide comprises the following steps:

[0080] The 15 amino acids in the middle of the antimicrobial peptide Cath-DM from Bufo nigrostachianus were intercepted to obtain a peptide Cath-DM-NCT', and the R at positions 1 and 15 or the K at position 11 of Cath-DM-NCT' were replaced with tryptophan (Trp, W) or isoleucine (Ile, I), and the L at positions 9 and 14 were replaced with lysine (Lys, K) or arginine (Arg, R). In addition, some antimicrobial peptides were amidated at the C-terminus.

[0081] Furthermore, the preparation method of antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 comprises the following steps: according to the amino acid sequence of the antimicrobial peptide, the full sequence is obtained by solid phase synthesis, and then HPLC reverse phase column chromatography desalting is used to obtain the derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3, and the peptides are identified by ultra high performance liquid chromatography-mass spectrometry analyzer.

[0082] The invention relates to the use of any one or more of the antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 provided in the embodiments of the invention in the preparation of a medicament for treating Gram-negative bacterial infections in the blood.

[0083] The Gram-negative bacteria include Escherichia coli.

[0084] The antibacterial activity against Gram-negative bacteria is enhanced in the presence of human serum.

[0085] Case study 1: Design of antimicrobial peptides and synthesis of peptides

[0086] The amino acid sequence of the antimicrobial peptide Cath-DM from Bufo nigropunctatus is:

[0087] SSRRKPCKGWLCKLKLRGGYTLIGSATNLNRPTYVRA (the two cysteines at positions 7 and 12 form a disulfide bond).

[0088] The peptide Cath-DM-NCT' was obtained by cutting the middle 15 amino acids of the antimicrobial peptide Cath-DM from Bufo bufo molitor, and its amino acid sequence is: RRKPCKGWLCKLKLR (the two cysteines at the 5th and 10th positions form a disulfide bond).

[0089] The R at position 1 of Cath-DM-NCT' was replaced with W to obtain W1, whose amino acid sequence is: WRKPCKGWLCKLKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0090] The R at positions 1 and 15 of Cath-DM-NCT' were replaced with W to obtain W2, whose amino acid sequence is: WRKPCKGWLCWLKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0091] The 1st R and 11th K of Cath-DM-NCT' were replaced with W, and the 9th L was replaced with K to obtain WK1, whose amino acid sequence is: WRKPCKGWKCWLKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0092] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with W, and the L at positions 9 and 14 were replaced with K to obtain WK2, whose amino acid sequence is: WRKPCKGWKCWLKKR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0093] The R at positions 1 and 15 and the K at position 11 of Cath-DM-NCT' were replaced with W, and the L at positions 9 and 14 were replaced with K to obtain WK3, whose amino acid sequence is: WRKPCKGWKCWLKKW (the two cysteines at positions 5 and 10 form a disulfide bond).

[0094] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with W, and L at position 9 was replaced with R to obtain WR1, whose amino acid sequence is: WRKPCKGWRCWLKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0095] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with W, and the L at positions 9 and 14 were replaced with R to obtain WR2, whose amino acid sequence is: WRKPCKGWRCWLKRR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0096] The R at positions 1 and 15 and R at position 11 of Cath-DM-NCT' were replaced with W, and L at positions 9 and 14 were replaced with R to obtain WR3, whose amino acid sequence is: WRKPCKGWRCWLKRW (the two cysteines at positions 5 and 10 form a disulfide bond).

[0097] The R at position 1 of Cath-DM-NCT' was replaced with I to obtain I1, whose amino acid sequence is: IRKPCKGWLCKLKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0098] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with I to obtain I2, whose amino acid sequence is: IRKPCKGWLCILKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0099] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with I, and L at position 9 was replaced with K to obtain IK1, whose amino acid sequence is: IRKPCKGWKCILKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0100] The 1st R and the 11th K of Cath-DM-NCT' were replaced with I, and the 9th and 14th L were replaced with K to obtain IK2, whose amino acid sequence is: IRKPCKGWKCILKKR (the two cysteines at the 5th and 10th positions form a disulfide bond).

[0101] The R at positions 1 and 15 and the K at position 11 of Cath-DM-NCT' were replaced with I, and the L at positions 9 and 14 were replaced with K to obtain IK3, whose amino acid sequence is: IRKPCKGWKCILKKI (the two cysteines at positions 5 and 10 form a disulfide bond).

[0102] The R at positions 1 and 15 and the K at position 11 of Cath-DM-NCT' were replaced with I, the L at positions 9 and 14 were replaced with K, and the C-terminus was amidated to obtain IK3-NH2, whose amino acid sequence is: IRKPCKGWKCILKKI-NH2 (the two cysteines at positions 5 and 10 form a disulfide bond).

[0103] The R at position 1 and K at position 11 of Cath-DM-NCT' were replaced with I, and L at position 9 was replaced with R to obtain IR1, whose amino acid sequence is: IRKPCKGWRCILKLR (the two cysteines at positions 5 and 10 form a disulfide bond).

[0104] The 1st R and the 11th K of Cath-DM-NCT' were replaced with I, and the 9th and 14th L were replaced with R to obtain IR2, whose amino acid sequence is: IRKPCKGWRCILKRR (the two cysteines at the 5th and 10th positions form a disulfide bond).

[0105] The R at positions 1 and 15 and K at position 11 were replaced with I, and L at positions 9 and 14 were replaced with R in Cath-DM-NCT' to obtain IR3, whose amino acid sequence is: IRKPCKGWRCILKRI (the two cysteines at positions 5 and 10 form a disulfide bond).

[0106] The sequences of the seventeen antimicrobial peptides are shown in Table 1 . The seventeen derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 were synthesized by solid phase chemical synthesis. The results of peptide synthesis HPLC analysis are shown in Figure 2 The mass spectrometry results are shown in Figure 3 In addition, the ProtParam function of ExPASy was used to calculate the charge number, and HeliQuest was used to evaluate the hydrophobic moment index of amphiphilicity.

[0107] Table 1 Amino acid sequence of polypeptide

[0108] Peptides Amino acid sequence Molecular weight (Da) Charge Hydrophobic moment <μH> W1 WRKPCKGWLCKLKLR 1913.42 +5.9 0.425 W2 WRKPCKGWLCWLKLR 1971.46 +4.9 0.502 WK1 WRKPCKGWKCWLKLR 1986.49 +5.9 0.570 WK2 WRKPCKGWKCWLKKR 2001.50 +6.9 0.624 WK3 WRKPCKGWKCWLKKW 2031.53 +5.9 0.836 WR1 WRKPCKGWRCWLKLR 2014.49 +5.9 0.571 WR2 WRKPCKGWRCWLKRR 2057.52 +6.9 0.625 WR3 WRKPCKGWRCWLKRW 2087.56 +5.9 0.837 I1 IRKPCKGWLCKLKLR 1840.37 +5.9 0.395 I2 IRKPCKGWLCILKLR 1825.36 +4.9 0.459 IK1 IRKPCKGWKCILKLR 1840.38 +5.9 0.524 IK2 IRKPCKGWKCILKKR 1855.40 +6.9 0.580 IK3 IRKPCKGWKCILKKI 1812.38 +5.9 0.761 <![CDATA[IK3-NH2]]> <![CDATA[IRKPCKGWKCILKKI-NH2]]> 1811.39 +6.9 0.761 IR1 IRKPCKGWRCILKLR 1868.39 +5.9 0.525 IR2 IRKPCKGWRCILKRR 1911.42 +6.9 0.581 IR3 IRKPCKGWRCILKRI 1868.40 +5.9 0.762

[0109] Note: In the amino acid sequences in this table, a disulfide bond is formed between the two cysteines.

[0110] Case study 2: Hemolytic activity of antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, IR3

[0111] The hemolytic activity of the peptides was detected using 4% Mianyang erythrocytes. The peptides were diluted with PBS to a final concentration of 512μg / mL and a volume of 50μL. An equal volume of sterile saline was used as a negative control, and an equal volume of 1% TritonX-100 was used as a positive control. An equal volume of 4% Mianyang erythrocytes was added and incubated at 37°C for 1h. After incubation, centrifuge at 1000g for 5min, aspirate 80μL of the supernatant into a clean 96-well plate, and use a full-wavelength microplate reader to detect the absorbance of the supernatant at a wavelength of 490nm. The experiment was repeated three times. The results are shown in Table 3. At a concentration of 512μg / mL, the hemolytic activity of the seventeen peptides was low, all ≤10%, indicating that this group of antimicrobial peptides has a high safety.

[0112] Table 2 Hemolytic activity of peptides

[0113] Peptides Hemolysis rate (%) Peptides Hemolysis rate (%) W1 6 I1 0 W2 5 I2 1 WK1 1 IK1 1 WK2 6 IK2 0 WK3 2 IK3 5 WR1 4 <![CDATA[IK3-NH2]]> 0 WR2 5 IR1 2 WR3 3 IR2 0 IR3 2

[0114] Case study 3: Activity of antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 against Escherichia coli in human serum

[0115] According to McFarland turbidimetric method, the bacterial solution in logarithmic growth phase was diluted to 2×10 5 of bacterial suspension. The peptide was diluted twice with blank MHB to a final concentration of 128-2μg / mL in a volume of 50μL. The peptide solution was mixed with the diluted bacterial suspension in a ratio of 1:1 (the final concentration of human serum was 20%). Incubate at 37°C, 100rpm for 18h and determine the MIC value. The results are shown in Table 3. After the action of 20% human serum, the killing activity against Staphylococcus aureus was inhibited, while the killing activity against Escherichia coli was significantly increased. This phenomenon makes this group of peptides have the potential to become a substitute for narrow-spectrum antibiotics against Gram-negative bacteria.

[0116] Table 3 Antibacterial activity of peptides in 20% serum solution

[0117]

[0118] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. An antimicrobial peptide having an activity to promote anti-Gram-negative bacteria in a serum environment, characterized in that: Antimicrobial peptides with enhanced anti-Gram-negative bacteria activity are obtained by replacing the middle 15 amino acids of the antimicrobial peptide Cath-DM derived from Bufo bufo gargarizans at specific sites; these antimicrobial peptides are prepared by introducing tryptophan (Trp, W) or isoleucine (Ile, I) at the 1st, 15th or 11th position, and introducing lysine (Lys, K) or arginine (Arg, R) at the 9th and 14th positions; the stability of SEQ ID No: 14 is further improved by C-terminal amidation treatment; the antimicrobial peptides are synthesized by solid phase synthesis, purified by HPLC reverse phase chromatography, and identified by ultra-high performance liquid chromatography-mass spectrometry, thereby obtaining antimicrobial peptides with significant anti-Gram-negative bacteria activity in a serum environment.

2. The method for preparing an antimicrobial peptide having an activity promoting anti-Gram-negative bacteria in a serum environment according to claim 1, characterized in that: The amino acid sequence of the antimicrobial peptide includes any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.

17.

3. The method for preparing an antimicrobial peptide having an activity promoting anti-Gram-negative bacteria in a serum environment as claimed in claim 1, characterized in that: The method for preparing an antimicrobial peptide that promotes anti-Gram-negative bacteria activity under a serum environment comprises the following steps: The 15 amino acids in the middle of the antimicrobial peptide Cath-DM from Bufo nigromaculata were intercepted to obtain a polypeptide Cath-DM-NCT', and the R at positions 1 and 15 or the K at position 11 of Cath-DM-NCT' were replaced with tryptophan (Trp, W) or isoleucine (Ile, I), and the L at positions 9 and 14 were replaced with lysine (Lys, K) or arginine (Arg, R). In addition, only SEQ ID No: 14 was amidated at the C-terminus.

4. The method for preparing an antimicrobial peptide having an activity promoting anti-Gram-negative bacteria in a serum environment as claimed in claim 2, characterized in that: The preparation method of antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3 comprises the following steps: according to the amino acid sequence of the derivative antimicrobial peptide, the full sequence is obtained by solid phase synthesis, and then HPLC reverse phase column chromatography desalting is used to obtain the derivative antimicrobial peptides W1, W2, WK1, WK2, WK3, WR1, WR2, WR3, I1, I2, IK1, IK2, IK3, IK3-NH2, IR1, IR2, and IR3, and the peptides are identified by ultra high performance liquid chromatography-mass spectrometry analyzer.

5. Use of the antimicrobial peptide according to claim 1, wherein the amino acid sequence comprises any one or more of the sequences shown in SEQ ID NO.1 to SEQ ID NO.17, in the preparation of a drug for treating blood infections caused by Gram-negative bacteria.

6. The use of the antimicrobial peptide amino acid sequence according to claim 4, comprising any one or more of the sequences shown in SEQ ID NO.1 to SEQ ID NO.17, in the preparation of a drug for treating blood gram-negative bacterial infections, characterized in that: The drug is used to inhibit Gram-negative bacteria.

7. The use of the antimicrobial peptide amino acid sequence according to claim 5, comprising any one or more of the sequences shown in SEQ ID NO.1 to SEQ ID NO.17, in the preparation of a drug for treating blood-borne Gram-negative bacterial infections, characterized in that: The Gram-negative bacteria include Escherichia coli.

8. An antimicrobial peptide preparation for use against Gram-negative bacteria, characterized in that: The antimicrobial peptides include any one or a combination of antimicrobial peptide W1, antimicrobial peptide W2, antimicrobial peptide WK1, antimicrobial peptide WK2, antimicrobial peptide WK3, antimicrobial peptide WR1, antimicrobial peptide WR2, antimicrobial peptide WR3, antimicrobial peptide I1, antimicrobial peptide I2, antimicrobial peptide IK1, antimicrobial peptide IK2, antimicrobial peptide IK3, antimicrobial peptide IK3-NH2, antimicrobial peptide IR1, antimicrobial peptide IR2, and antimicrobial peptide IR3.

9. The antimicrobial peptide preparation according to claim 8, characterized in that The preparation further comprises an excipient for enhancing the stability and biological activity of the antimicrobial peptide in a serum environment.

10. The antimicrobial peptide preparation according to claim 8, characterized in that The preparation is in the form of injection, spray or ointment, and is used for treating or preventing Gram-negative bacteria infection.

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