Marine antibacterial healing-promoting peptide and application thereof in preparation of wound infection prevention and treatment product

Abstract

The application discloses a marine antibacterial and healing-promoting peptide and application thereof to preparation of a wound infection prevention and treatment product. Through site-directed mutagenesis and other methods, 273 peptide segments with more remarkable antibacterial and healing-promoting activity are preferably obtained. The antibacterial and healing-promoting peptide has broad-spectrum antibacterial activity, can rapidly increase membrane permeability, cause bacterial breakdown, and effectively inhibit growth of gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Vibrio vulnificus and gram-positive bacteria such as Staphylococcus aureus. The antibacterial and healing-promoting peptide has good biocompatibility, no cytotoxicity, does not cause hemolysis, and can significantly promote cell proliferation and migration. In terms of prevention and treatment of wound infection, the antibacterial and healing-promoting peptide can effectively inhibit bacterial growth, reduce damage of toxins to the body, significantly reduce the level of pro-inflammatory factors, accelerate wound contraction, and improve the wound healing speed and repair quality. The antibacterial and healing-promoting peptide has a wide range of applications and can be applied to drugs and medical biomaterials, and has a very remarkable effect on prevention and treatment of wound infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a marine antibacterial and healing-promoting peptide with antibacterial and healing-promoting effects and application thereof in preparation of a wound infection prevention and treatment product. BACKGROUND

[0002] Trauma often occurs in young adults, and has a great impact on social labor loss and family burden. Depending on whether the body surface structure is damaged, trauma can be divided into two categories: open and closed. Common open injuries mainly include abrasions (only the epidermis is peeled off, there are a few bleeding points and oozing, and it can self-heal within 1-2 days), lacerations (wounds are often contaminated and more serious), cuts or slashes (wounds are deep, and the inflammatory response is obvious), and puncture wounds (wounds are small but deep, are easily blocked by blood clots, and even complicated with infection, especially anaerobic bacterial infection). If classified according to the injury factors, trauma can also be divided into cold weapon injury, firearm injury, burn injury, frostbite, impact injury, chemical injury, radiation injury, and combined injury, etc.

[0003] Generally speaking, open trauma is easy to diagnose, but is more likely to be contaminated or even infected. Skin and mucous membranes are the "first line of defense" against infection in the host. If the skin is damaged or burned, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and other bacteria can invade and cause pyogenic infection. Under the stimulation of injury factors, inflammatory response will occur within a few hours after injury, and if there is bacterial contamination, foreign matter retention or more necrotic tissue, the inflammatory response will be more severe, and even a large number of pathogenic bacteria will continuously or frequently enter the blood circulation, or a large amount of inflammatory mediators produced by local infection will enter the blood, thereby triggering systemic inflammatory response, and finally causing sepsis.

[0004] There are many types of common pathogenic bacteria that cause sepsis. Gram-positive bacteria include Staphylococcus aureus, hemolytic streptococcus, enterococcus, etc.; Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Bacteroides, Klebsiella pneumoniae, etc.; anaerobic bacteria include fragile bacillus, anaerobic streptococcus, etc.; fungi include Candida, etc. These bacteria can produce toxic substances. Gram-negative bacteria mainly produce endotoxin-lipopolysaccharide (LPS), which is released from the cell wall after bacterial death, and acts on phagocytes to cause pro-inflammatory cytokine release, thereby triggering a series of chain reactions. Gram-positive bacteria mainly produce exotoxins, such as enterotoxins, toxic shock syndrome toxin-1, etc. Other cell wall products and antigens of Gram-positive bacteria and fungi can also trigger systemic inflammatory response.

[0005] Therefore, it is essential to prevent and treat trauma infection in time and effectively. Generally, it is started from the aspects of treating primary infection focus, applying antibiotics and enhancing body resistance, etc. The wound is the most important source of infection, so the eschar should be removed as soon as possible and fully covered; abscess should be incised and drained in time; foreign matter and necrotic tissue should be removed to open the dead space and fully drain. According to the nature of the primary focus, the antibacterial drugs are selected empirically, usually broad-spectrum antibiotics or two kinds of antibacterial drugs are combined, and the drug use time is reasonably selected. Systemic antibiotics are generally not used for small area and shallow trauma, and even if used, the application time is generally not more than 3-5 days; high-efficiency broad-spectrum antibiotics can be used in the early stage of deep trauma, but long-term continuous use should be avoided. Subsequently, the antibacterial drugs are adjusted according to the therapeutic effect, disease evolution, bacterial culture and drug sensitivity test.

[0006] In recent decades, antibacterial drugs have played a significant role in the prevention, control and treatment of trauma infection. There are hundreds of commonly used antibacterial drugs in clinical practice, but due to wide application, the phenomenon of misuse of antibacterial drugs occurs from time to time. The misuse of antibacterial drugs increases the drug resistance of pathogenic bacteria, leading to dysbiosis and secondary infection, and also causes allergic or toxic reactions. Bacterial drug resistance has become a difficult and focal point of current research. In addition to studying the drug resistance mechanism of antibacterial drugs, the development of natural anti-infection drugs should also be emphasized.

[0007] Antibacterial peptides, also known as host defense peptides, are small molecule polypeptides with broad-spectrum antibacterial activity and immunomodulatory activity, which are induced by the body and are an inherent part of the body's non-specific defense system. They widely exist in organisms (plants, animals, microorganisms) in nature and have good killing or inhibiting effect on bacteria, fungi, viruses and parasites. They are also an important part of the innate immune system of marine invertebrates. In seawater with a maximum of 10 6 C FU / mL of microorganisms, antibacterial peptides can help aquatic animals quickly identify and kill invading pathogenic microorganisms using their broad-spectrum antibacterial activity.

[0008] Compared with traditional antibiotics, antibacterial peptides mainly act on the bacterial cell membrane, killing bacteria by changing the permeability of the cell membrane, and almost no residue is left in the human body after use, and it is not easy to develop drug resistance. As part of the body's non-specific immune activity, long-term use of antibacterial peptides can stimulate the body's immune system and enhance the body's resistance to disease. Due to the high biological safety, high stability, broad-spectrum bactericidal, no residue, and not easy to develop drug resistance of antibacterial peptides, they are widely used in the fields of medicine, etc. In addition, with the progress of science and technology, bioinformatics technology has been widely used in scientific research. A series of online software can be used to predict and screen antibacterial peptides, greatly improving the efficiency of research. SUMMARY

[0009] One of the purposes of the present application is to provide a marine antibacterial healing-promoting peptide which can effectively inhibit the growth of gram-negative bacteria and gram-positive bacteria, has good biocompatibility, has no cytotoxicity, can significantly improve cell viability, promote cell proliferation and migration, reduce inflammatory response, effectively promote high-quality repair of wound infection, and reduce scar proliferation.

[0010] Another purpose of the present application is to provide an application of the marine antibacterial healing-promoting peptide in the preparation of a wound infection prevention and / or healing-promoting product.

[0011] The present application achieves the above-mentioned purposes through the following technical solutions:

[0012] On one hand, the present application provides a marine antibacterial healing-promoting peptide which can effectively inhibit bacterial growth and has good biocompatibility. The amino acid sequence of the antibacterial peptide is one or several of the following 273 peptide segments:

[0013] The second amino acid in the original peptide segment 9 (RSARAGLQFPVGRVHRFLRR) reported by the inventors in the literature is replaced by I or W;

[0014] The 12th amino acid in the original peptide segment 10 (RGKGGKAWAKAKSRSARAGLQFPVGRVHRFLRR) is replaced by I or V, or the 13th amino acid is replaced by A, F, I, L, V or G, or the 15th amino acid is replaced by A, F, I, K, L, R, V, W or G, or the 19th amino acid is replaced by K, or the 21st amino acid is replaced by F, I, K, L, R, V or W, or the 23rd amino acid is replaced by A, F, I, K, L, R, V or W, or the 28th amino acid is replaced by F, I, L, V or W;

[0015] The 2nd amino acid in the original peptide segment 12 (FLGIKQTLKSLRQGKAKLII) is replaced by I, or the 3rd, 9th or 14th amino acid is replaced by F, or the 5th amino acid is replaced by F or I, or the 6th amino acid is replaced by A, F, I, L or V, or the 7th amino acid is replaced by R, K, A, F, I, L, V, W or G, or the 10th amino acid is replaced by A, F, I, K, L, V, W or G, or the 12th amino acid is replaced by A, F, I, K, L, Q, V, W or G, or the 13th amino acid is replaced by F or L, or the 15th or 17th amino acid is replaced by F, I, L or V, or the 16th amino acid is replaced by L;

[0016] substituting the 7th amino acid in the original peptide segment 13 (KAWAKAKSRSARAGLQFPVGRVHRFLRR) with A, I or V, or substituting the 8th amino acid in the original peptide segment 13 with A, F, I, K, L, R, V or G, or substituting the 10th amino acid in the original peptide segment 13 with A, I, K, L, R, V or G, or substituting the 16th amino acid in the original peptide segment 13 with I, L or V, or substituting the 18th amino acid in the original peptide segment 13 with A, I, L or V, or substituting the 23rd amino acid in the original peptide segment 13 with A, F, I, L or V;

[0017] substituting the 2nd amino acid in the original peptide segment 15 (ILRLAVGLKGLPSVNPAWLV) with F, N or G, or substituting the 3rd or 11th amino acid in the original peptide segment 15 with K, or substituting the 4th, 8th, 10th, 15th, 16th, 17th or 19th amino acid in the original peptide segment 15 with K or R, or substituting the 6th amino acid in the original peptide segment 15 with K, L, N, R or G, or substituting the 12th amino acid in the original peptide segment 15 with A, K, N, Q, R or G, or substituting the 13th amino acid in the original peptide segment 15 with K, R or G, or substituting the 14th or 20th amino acid in the original peptide segment 15 with K, N, R or G, or substituting the 18th amino acid in the original peptide segment 15 with A, F, I, K, L, N, R, V or G;

[0018] removing 2 amino acids from the N-terminal end of the original peptide segment 16 (ANFITHPYKRILRLAVGLKG), or substituting the 1st amino acid in the original peptide segment 16 with I or V, or substituting the 2nd amino acid in the original peptide segment 16 with A, I, K, L, R or V, or substituting the 5th amino acid in the original peptide segment 16 with A, F, I, K, L, N, Q, R or V, or substituting the 6th or 7th amino acid in the original peptide segment 16 with A, F, I, L or V, or substituting the 8th amino acid in the original peptide segment 16 with A, F, I, K, L, N, Q, R, S, V or W, or substituting the 10th or 13th amino acid in the original peptide segment 16 with K, or substituting the 12th, 14th, 15th, 16th, 17th or 18th amino acid in the original peptide segment 16 with I, or substituting the 20th amino acid in the original peptide segment 16 with I, K or R;

[0019] substituting the 4th amino acid in the original peptide segment 17 (IKVELKRLAANNVLVHTKGT) with A, C, F, I, K, L, N, Q, R, V or W, or substituting the 6th amino acid in the original peptide segment 17 with I, or substituting the 7th or 16th amino acid in the original peptide segment 17 with I, L or V, or substituting the 11th or 12th amino acid in the original peptide segment 17 with I or K, or substituting the 17th or 20th amino acid in the original peptide segment 17 with I, K, L or V;

[0020] removing 2, 3, 4 or 5 amino acids from the N-terminal end of the original peptide segment 19 (KYFLGIKQTLKSLRQGKAKLIIL), or substituting the 1st amino acid in the original peptide segment 19 with A, F, I, L, V, W or G, or substituting the 2nd amino acid in the original peptide segment 19 with A, C, D, E, F, I, K, L, N, Q, R, V, W or G.

[0021] The above-mentioned antibacterial healing-promoting peptide can not only effectively inhibit the normal growth of Gram-negative bacteria including Escherichia coli, multi-drug resistant Escherichia coli, Acinetobacter baumannii, drug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio alginolyticus, but also significantly inhibit the growth of Gram-positive bacteria including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus luteus, and Enterococcus faecalis.

[0022] On the other hand, the present application provides the use of the marine antibacterial healing-promoting peptide in the preparation of a wound infection prevention and / or healing-promoting product.

[0023] Preferably, the wound infection prevention product is a product against Escherichia coli, multi-drug resistant Escherichia coli, Acinetobacter baumannii, drug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus luteus, and / or Enterococcus faecalis.

[0024] As an optional embodiment, the wound infection prevention and / or healing-promoting product of the present application is a pharmaceutical product that can be used for the prevention of infection of open wounds including burns, cuts, traffic injuries, surgical injuries, war injuries, firearm injuries combined with seawater immersion injuries, and chronic refractory wounds.

[0025] In a preferred embodiment of the present application, the marine antibacterial healing-promoting peptide can be used in a pharmaceutical product; preferably, the marine antibacterial healing-promoting peptide is particularly suitable for use in a pharmaceutical product that can effectively prevent wound infection, and the pharmaceutical product dosage forms include lotions, gels, tinctures, spirit agents, powders, oils, pastes, plasters, film-coating agents, aerosols, etc. When used in a pharmaceutical product, the effective dose of the marine antibacterial healing-promoting peptide in the pharmaceutical product is 62.5-250 μg / mL.

[0026] In a preferred embodiment of the present application, the marine antibacterial healing-promoting peptide can also be used in medical biomaterials; preferably, the marine antibacterial healing-promoting peptide is particularly suitable for use in medical biomaterials that can effectively prevent wound infection, and the medical biomaterial dosage forms include lotions, solutions, dressings, gels, injections, etc., and the dressing types include films, hydrocolloids, hydrogels, sponges, sprays, etc. When used in medical biomaterials, the effective dose of the marine antibacterial healing-promoting peptide is 62.5-250 μg / mL.

[0027] It is worth mentioning that the marine antibacterial healing-promoting peptide provided by the present application has significant bacteriostatic, anti-inflammatory and healing-promoting effects, good biocompatibility, does not cause body allergy, and can effectively promote the rapid and high-quality repair of wound infection. Therefore, when the marine antibacterial healing-promoting peptide of the present application is applied in a drug or a medical biological material, the selection of the type, dosage and preparation process of the medical raw material and the medical biological material raw material is relatively wide. All components used in the medicine and the medical biological material should be human acceptable and should not affect the performance of the original marine antibacterial healing-promoting peptide of the present application, that is, when in contact with the human body or in compounding with other components, it should not cause improper toxicity, incompatibility, instability and allergic reactions, etc.

[0028] In another aspect, the present application also provides a wound infection prevention and / or healing-promoting product containing the above marine antibacterial healing-promoting peptide as an active ingredient.

[0029] The solution of the present application is based on the understanding of the inventors on the mechanism of wound infection occurrence, development and prevention, the structure-activity relationship of the marine antibacterial healing-promoting peptide, and the research results of modern pharmacology and medical biological materials, and through a large amount of creative labor, a marine biological peptide with good biocompatibility, no allergenicity, and significant bacteriostatic, anti-inflammatory and healing-promoting effects is found, excavated and optimized.

[0030] The present application has the following outstanding beneficial effects:

[0031] (1) On the basis of the 27 peptide segments with antibacterial potential reported by the inventors in the literature, the original peptide segments are optimized in structure by means of point mutation and deletion of amino acids, etc., and finally 273 peptide segments with more significant antibacterial activity are obtained. It is found through research that compared with the original peptide segments, the antibacterial and healing-promoting effects of the antibacterial healing-promoting peptides of the present application optimized in structure are obviously improved.

[0032] (2) The marine antibacterial healing-promoting peptides of the present application can rapidly increase the permeability of cell membranes, cause cell rupture, and thus effectively inhibit the normal growth of gram-negative bacteria and gram-positive bacteria. The antibacterial healing-promoting peptides have no cytotoxicity, do not cause hemolysis, have good biocompatibility, can significantly improve cell activity, and promote rapid proliferation and migration of cells. In the prevention and treatment of wound infection, these antibacterial healing-promoting peptides can effectively inhibit the growth of bacteria in infected wounds, reduce the further damage of toxins to the body, significantly reduce the level of pro-inflammatory factors, accelerate the contraction of the wound surface, and improve the healing speed and repair quality of the infected wound surface.

[0033] (3) The marine antibacterial healing-promoting peptides of the present application have good water solubility, good biocompatibility, do not cause skin irritation and allergy, and have significant antibacterial, anti-inflammatory and healing-promoting effects, and can be widely used in the treatment of wound infection in biological medicine and medical biological materials. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 7 is the determination of the antibacterial activity of the marine antibacterial healing-promoting peptide against S. aureus in Example 2 (compared to the blank control, **p<0.01).

[0035] Figure 2 Figure 8 is the determination of the antibacterial activity of the marine antibacterial healing-promoting peptide against P. aeruginosa in Example 2 (compared to the blank control, **p<0.01).

[0036] Figure 3 Figure 9 is the determination of the antibacterial activity of the marine antibacterial healing-promoting peptide against V. vulnificus in Example 2 (compared to the blank control, **p<0.01).

[0037] Figure 4 Figure 10 is the antibacterial curve of the marine antibacterial healing-promoting peptide against S. aureus in Example 2.

[0038] Figure 5 Figure 11 is the antibacterial curve of the marine antibacterial healing-promoting peptide against P. aeruginosa in Example 2.

[0039] Figure 6 Figure 12 is the antibacterial curve of the marine antibacterial healing-promoting peptide against V. vulnificus in Example 2.

[0040] Figure 7 Figure 13 is the effect of the marine antibacterial healing-promoting peptide on the cell membrane permeability of S. aureus in Example 2.

[0041] Figure 8 Figure 14 is the effect of the marine antibacterial healing-promoting peptide on the cell membrane permeability of P. aeruginosa in Example 2.

[0042] Figure 9 Figure 15 is the effect of the marine antibacterial healing-promoting peptide on the cell membrane permeability of V. vulnificus in Example 2.

[0043] Figure 10 Figure 16 is the hemolytic evaluation of the marine antibacterial healing-promoting peptide in Example 2.

[0044] Figure 11 Figure 17 is the cell viability assay after co-incubation of the marine antibacterial healing-promoting peptide with L929 cells for 24 h in Example 2 (compared to the blank control, **p<0.01).

[0045] Figure 12 Figure 18 is the migration area assay after co-incubation of the marine antibacterial healing-promoting peptide with L929 cells for 24 h in Example 2 (compared to the blank control, **p<0.01).

[0046] Figure 13 Figure 2 is a graph showing the change in the area of the infected wound in rats in Example 2.

[0047] Figure 14 Figure 3 is a graph showing the expression level of inflammatory factors in the skin tissue on day 7 in Example 2 (compared with the infection control, **p<0.01). DETAILED DESCRIPTION

[0048] The application will be further described below in conjunction with the accompanying drawings, but the embodiments of the application are not limited to the following examples. Any equivalent changes or variations made in accordance with the method of the application should be considered within the scope of the application. The raw materials used below are commercially available, unless otherwise specified.

[0049] Example 1

[0050] The 27 peptide segments obtained by the inventors were used as templates, and methods such as shortening the length and site-directed mutagenesis were used to modify the structures. The newly generated peptide segments were subjected to antibacterial activity analysis using the CAMPR3 tools: random forest (RF), support vector machine (SVM), discriminant analysis (DA), and artificial neural network (ANN).

[0051] Through antibacterial activity evaluation, a total of 273 antibacterial peptides were finally selected, including 2 original peptide segment 9 structure optimized antibacterial peptides, 38 original peptide segment 10 structure optimized antibacterial peptides, 48 original peptide segment 12 structure optimized antibacterial peptides, 30 original peptide segment 13 structure optimized antibacterial peptides, 50 original peptide segment 15 structure optimized antibacterial peptides, 50 original peptide segment 16 structure optimized antibacterial peptides, 30 original peptide segment 17 structure optimized antibacterial peptides, and 25 original peptide segment 19 structure optimized antibacterial peptides.

[0052] It was found that the antibacterial activity of the structure-optimized peptide segments of the application was significantly improved compared with the original peptide segments. Specifically, as follows:

[0053] For example, as shown in Table 1, the 2nd amino acid S in the original peptide segment 9 was replaced by a hydrophobic amino acid I or W, and the antibacterial activity of the newly generated peptide segments was significantly improved (the RF value was increased from 0.811 to 0.949 and 0.942, respectively).

[0054] Table 1 Structure optimization and antibacterial activity of original peptide segment 9

[0055]

[0056] For example, as shown in Table 2, the 12th cationic amino acid K in the original peptide segment 10 is replaced by hydrophobic amino acids I or V, respectively, and the antibacterial activity of the newly generated peptide segment is significantly improved (RF value is increased from 0.9195 to 0.9615, 0.9545, respectively); the 13th amino acid S is replaced by hydrophobic amino acids A, F, I, L, V or G, and the antibacterial activity of the newly generated peptide segment is significantly improved (RF value is increased from 0.9195 to 0.935, 0.952, 0.966, 0.956, 0.957, 0.922, respectively).

[0057] Table 2 Structure optimization and antibacterial activity of original peptide segment 10

[0058]

[0059]

[0060] For example, as shown in Table 3, the 7th amino acid T in the original peptide segment 12 is replaced by cationic amino acids R, K, hydrophobic amino acids A, F, I, L, V, W or G, respectively, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.981 to 0.985, 0.989, 0.991, 0.993, 0.992, 0.994, 0.992, 0.995, 0.99, respectively); the 17th amino acid K is replaced by hydrophobic amino acids F, I, L or V, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.981 to 0.987, 0.989, 0.99, 0.987, respectively).

[0061] Table 3 Structure optimization and antibacterial activity of original peptide segment 12

[0062]

[0063]

[0064] For example, as shown in Table 4, the 10th amino acid S in the original peptide segment 13 is replaced by A, I, K, L, R, V or G, respectively, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.963 to 0.98, 0.985, 0.974, 0.987, 0.98, 0.984, 0.975, respectively, and RF value is increased from 0.9735 to 0.9975, 0.999, 0.997, 0.9975, 0.995, 0.998, 0.996, respectively); the 23rd cationic amino acid H is replaced by A, F, I, L or V, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.963 to 0.973, 0.982, 0.98, 0.982, 0.979, respectively, and RF value is increased from 0.9735 to 0.9955, 0.9985, 0.999, 0.999, 0.9985, respectively).

[0065] Table 4 Structure optimization and antibacterial activity of original peptide segment 13

[0066]

[0067] For example, as shown in Table 5, the 6th amino acid V in the original peptide segment 15 is replaced by K, L, N, R or G, respectively, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.933 to 0.973, 0.936, 0.938, 0.957, 0.939, respectively, and RF value is increased from 0.9725 to 0.995, 0.9735, 0.9845, 0.9925, 0.9855, respectively); the C-terminal amino acid V is replaced by K, N, R or G, respectively, and the antibacterial activity of the newly generated peptide segment is significantly improved (SVM value is increased from 0.933 to 0.966, 0.938, 0.941, 0.939, respectively, and RF value is increased from 0.9725 to 0.995, 0.9845, 0.992, 0.9855, respectively).

[0068] Table 5 Structure optimization and antibacterial activity of original peptide segment 15

[0069]

[0070]

[0071] For example, as shown in Table 6, replacing the first amino acid A in the original peptide 16 with I or V significantly improved the antibacterial activity of the newly generated peptide (SVM value increased from 0.924 to 0.946 and 0.929, respectively; RF value increased from 0.98 to 0.991 and 0.987, respectively; and DA value increased from 0.987 to 0.995 and 0.993, respectively). Similarly, replacing the fifth amino acid T with A, F, I, K, L, N, Q, R, or V significantly improved the antibacterial activity of the newly generated peptide (SVM value increased from 0.924 to 0.946 and 0.929, respectively). The values ​​were 56, 0.946, 0.959, 0.967, 0.956, 0.945, 0.942, 0.967, and 0.953, respectively. The RF value increased from 0.98 to 0.9935, 0.9895, 0.9925, 0.995, 0.9915, 0.992, 0.9925, 0.9935, and 0.9905, respectively. The DA value increased from 0.987 to 0.992, 0.991, 0.994, 0.994, 0.995, 0.992, 0.99, 0.994, and 0.994, respectively.

[0072] Furthermore, surprisingly, removing two amino acids (AN) from the N-terminus of the original peptide 16 not only reduced the cost of solid-phase synthesis but also significantly improved the antibacterial activity (SVM value increased from 0.924 to 0.926, RF value increased from 0.98 to 0.985, and DA value increased from 0.987 to 0.995).

[0073] Table 6. Structural optimization and antibacterial activity of the original peptide 16

[0074]

[0075]

[0076] For example, as shown in Table 7, the fourth amino acid E in the original peptide segment 17 is replaced by A, C, F, I, K, L, N, Q, R, V or W, respectively, and the newly generated peptide segment has significantly improved antibacterial activity (SVM value is increased from 0.904 to 0.97, 0.946, 0.971, 0.979, 0.965, 0.977, 0.963, 0.961, 0.94, 0.974, 0.966, respectively; RF value is increased from 0.8545 to 0.976, 0.982, 0.989, 0.997, 0.945, 0.991, 0.96, 0.962, 0.925, 0.989, 0.996, respectively; DA value is increased from 0.841 to 0.983, 0.939, 0.989, 0.989, 0.957, 0.987, 0.976, 0.971, 0.95, 0.984, 0.982, respectively).

[0077] Table 7 Structure optimization and antibacterial activity of original peptide segment 17

[0078]

[0079] For example, as shown in Table 8, the N-terminal amino acid K in the original peptide segment 19 is replaced by A, F, I, L, V, W or G, respectively, and the newly generated peptide segment has significantly improved antibacterial activity (SVM value is increased from 0.811 to 0.948, 0.963, 0.969, 0.932, 0.964, 0.973, 0.974, respectively; RF value is increased from 0.9455 to 0.9945, 0.9935, 0.985, 0.99, 0.9905, 0.992, 0.9935, respectively).

[0080] In addition, surprisingly, after removing 2-5 amino acids from the N-terminal of the original peptide segment 19, the newly generated peptide segment not only has reduced solid-phase synthesis cost, but also has significantly improved antibacterial activity (SVM value is increased from 0.811 to 0.987, 0.971, 0.975, 0.9, respectively; RF value is increased from 0.9455 to 0.998, 0.9955, 0.9615, 0.9945, respectively).

[0081] Table 8 Structure optimization and antibacterial activity of original peptide segment 19

[0082]

[0083] In summary, the 273 peptides after structural optimization have significantly improved antibacterial activity compared to the original peptides.

[0084] To further evaluate the antibacterial activity of the above peptides, considering factors such as saving manpower cost and improving work efficiency, the inventors selected 14 peptides with the smallest improvement in antibacterial prediction value (control is the original peptide) from the above, and evaluated their antibacterial activity through in vitro experiments. We can reasonably speculate that if these structural optimization peptides with the smallest improvement in antibacterial prediction value are found to have significant antibacterial efficacy, then structural optimization peptides with greater improvement in antibacterial prediction value should also have significant antibacterial efficacy.

[0085] The amino acid sequences of the structural optimization peptides (14) and their corresponding original peptides (8) are as follows:

[0086] The structural optimization peptide RWARAGLQFPVGRVHRFLRR (2→W, referred to as P9a) and the control RSARAGLQFPVGRVHRFLRR (original peptide 9, referred to as P9);

[0087] The structural optimization peptides RGKGGKAWAKAKSRSARAKLQFPVGRVHRFLRR (19→K, referred to as P10a), RGKGGKAWAKAKGRSARAGLQFPVGRVHRFLRR (13→G, referred to as P10b), and the control RGKGGKAWAKAKSRSARAGLQFPVGRVHRFLRR (original peptide 10, referred to as P10);

[0088] The structural optimization peptides FLGIKVTLKSLRQGKAKLII (6→V, referred to as P12a), FLGIKQTLKSLRQGK LKLII (16→L, referred to as P12b), and the control FLGIKQTLKSLRQGKAKLII (original peptide 12, referred to as P12);

[0089] The structural optimization peptides KAWAKAASRSARAGLQFPVGRVHRFLRR (7→A, referred to as P13a), KAWAKAKSRSARAGLLFPVGRVHRFLRR (16→L, referred to as P13b), and the control KAWAKAKSRSARAGLQFPVGRVHRFLRR (original peptide 13, referred to as P13);

[0090] The structural optimization peptide ILRLALGLKGLPSVNPAWLV (6→L, referred to as P15a) and the control ILRLAVGLKGLPSVNPAWLV (original peptide 15, referred to as P15);

[0091] ​Structure-optimized peptide segment AVFITHPYKRILRLAVGLKG (2→V, abbreviated as P16a), FITHPYKRILRLAVGLKG (shortened length, abbreviated as P16s), and control ANFITHPYKRILRLAVGLKG (original peptide segment 16, abbreviated as P16);

[0092] Structure-optimized peptide segment IKVELKRLAAKNVLVHTKGT (11→K, abbreviated as P17a), IKVELKRLAANNVLVHTKGK (20→K, abbreviated as P17b), and control IKVELKRLAANNVLVHTKGT (original peptide segment 17, abbreviated as P17);

[0093] Structure-optimized peptide segment LYFLGIKQTLKSLRQGKAKLIIL (1→L, abbreviated as P19a), IKQTLKSLRQGKAKLIIL (shortened length, abbreviated as P19s), and control KYFLGIKQTLKSLRQGKAKLIIL (original peptide segment 19, abbreviated as P19).

[0094] According to the above-mentioned amino acid sequences, solid-phase polypeptide synthesis method was used to synthesize the peptides by Shanghai Qiangyao Biotechnology Co., Ltd. (purity > 96%), and the final products were stored in the form of dry powder at -80℃ in an ultra-low temperature refrigerator.

[0095] The structure-optimized peptide segments and original peptide segments synthesized above were subjected to the following antibacterial activity detection. The gram-negative bacteria used were Escherichia coli, multi-drug resistant Escherichia coli, Acinetobacter baumannii, drug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio alginolyticus; the gram-positive bacteria used were Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus luteus, and Enterococcus faecalis. The bacteria (non-Vibrio) were cultured in NB nutrient broth and MH broth medium, and the Vibrio was cultured in 2216E medium. The bacteria were cultured at the optimal temperature to the logarithmic phase, centrifuged at 4000×g for 5 min, washed 3 times with TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4), and resuspended to OD 600 nm 0.6.

[0096] The antibacterial peptides (final concentration 250 μg / mL in the detection system, solvent TBS buffer) were added to the bacterial suspension, and 10 mM CaCl2 was added to make OD 600 nm 0.3, and the bacterial agglutination was observed under an optical microscope. The blank control was bovine serum albumin (2 mg / mL), and each group had 3 biological replicates.

[0097] Table 9 Peptide segment antibacterial activity detection

[0098]

[0099] Wherein, "++" represents > 50% bacteria agglutination, precipitation; "+" represents > 30% bacteria agglutination, precipitation; "-" represents no agglutination, precipitation.

[0100] Continued table

[0101]

[0102]

[0103] Wherein, "++" represents > 50% bacteria agglutination, precipitation; "+" represents > 30% bacteria agglutination, precipitation; "-" represents no agglutination, precipitation.

[0104] It is found that, as shown in Table 9, the original peptide segments 9, 17 and 19 have no agglutination and precipitation effect on bacteria, and the original peptide segments 10, 12, 13, 15 and 16 have relatively weak agglutination and precipitation effect on bacteria (> 30% bacteria agglutination, precipitation). However, the structure-optimized peptide segments have significant precipitation and agglutination effect on gram-negative bacteria and gram-positive bacteria (> 50% bacteria agglutination, precipitation), and have very obvious agglutination and precipitation effect on drug-resistant strains (multidrug-resistant Escherichia coli, drug-resistant Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis). Therefore, compared with the original peptide segments, the antibacterial activity of the structure-optimized peptide segments is significantly improved.

[0105] Since the 14 structure-optimized peptide segments selected have the smallest improvement range of antibacterial activity prediction value, and experiments prove that the actual antibacterial activity (compared with the corresponding original peptide segment) is obviously improved, we can boldly speculate that the actual antibacterial activity of other structure-optimized peptide segments with greater improvement range of antibacterial prediction value will be more significantly improved. In short, the antibacterial healing-promoting peptide has broad-spectrum and significant antibacterial activity, and the antibacterial activity is significantly better than that of the previously reported original peptide segments.

[0106] It is also found that the antibacterial healing-promoting peptide can rapidly increase the cell membrane permeability, cause cell rupture, and effectively inhibit the growth of gram-negative bacteria (Escherichia coli, multidrug-resistant Escherichia coli, Acinetobacter baumannii, drug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus) and gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus luteus and Enterococcus faecalis).

[0107] In addition, the experiment found that the antibacterial healing-promoting peptides have no cytotoxicity, do not cause red blood cell rupture, have good biocompatibility, can significantly improve cell activity, and promote rapid proliferation and migration of cells. In the prevention and treatment of wound infection, the antibacterial healing-promoting peptides can effectively inhibit the growth of bacteria in the infected wound, reduce the secondary damage of toxins to the body, significantly reduce the level of pro-inflammatory factors, accelerate the contraction of the wound, and improve the healing speed and repair quality of the infected wound.

[0108] Example 2

[0109] Since the patent application relates to a large number of marine antibacterial healing-promoting peptides, considering the experimental cost, work efficiency and other factors, only 14 structure-optimized peptide segments with the smallest increase in antibacterial prediction value of the original peptide segment are selected to carry out antibacterial, healing-promoting, wound infection prevention and treatment experiments. We can reasonably speculate that if the experiment finds that the structure-optimized peptide segments with the smallest increase in antibacterial prediction value all have significant antibacterial, healing-promoting and other effects, then the remaining structure-optimized peptide segments with a greater increase in antibacterial prediction value also have significant antibacterial, healing-promoting and other effects.

[0110] Next, several clinically common and highly representative bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio vulnificus) will be selected to describe the antibacterial activity and mechanism, biocompatibility evaluation, cell activity, wound infection prevention and treatment efficacy of the antibacterial healing-promoting peptides in more detail.

[0111] (1) Antibacterial activity determination of antibacterial peptides

[0112] Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Vibrio vulnificus (V. vulnificus) were incubated at 35°C for 20 h, and a bacterial suspension (OD 600 nm The bacterial suspension was diluted 50 times, and 50 μL per well was added to a 96-well plate, followed by the addition of 50 μL of broth medium (blank control), ciprofloxacin (S. aureus, P. aeruginosa: 0.59 μg / mL; V. vulnificus: 9.37 μg / mL), or the antibacterial peptides of the present application (solvent: broth medium) (final concentration 31.25 μg / mL), and incubation at 35°C for 20 h (S. aureus, P. aeruginosa) or 37°C for 24 h (V. vulnificus). The OD 600 nm .

[0113] The experiment found that, for example, Figures 1-3As shown, after adding the structurally optimized antimicrobial peptide, the number of bacteria in each treatment group was significantly reduced (Staphylococcus aureus ≤64.19%, Pseudomonas aeruginosa ≤83.82%, Vibrio vulnificus ≤53.09%), showing a significant difference compared with the blank control (p<0.01). This result indicates that the structurally optimized antimicrobial peptide of this invention has significant activity against both Gram-negative and Gram-positive bacteria.

[0114] (2) Plotting the antimicrobial curve of antimicrobial peptides

[0115] Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio vulnificus were incubated at 35°C for 20 hours, and then a suspension was prepared in broth medium (S. aureus: OD). 600nm 0.30-0.35; P. aer uginosa: OD 600nm 0.20-0.30; V. vulnificus: OD 600nm For a concentration of 0.10-0.20, take 50 μL of each well and add it to a 96-well plate, then add 50 μL of broth medium (blank control), ciprofloxacin (299.90 μg / mL) or the antimicrobial peptide of this invention (final concentration 1000 μg / mL, with the original peptide at the same concentration as a control), and detect with an ELISA reader at 600 nm, recording once every 10 min.

[0116] like Figure 4 As shown, compared with the blank control, the addition of the structure-optimized antimicrobial peptide P9a significantly reduced the number of Staphylococcus aureus at 180 min (p<0.01); moreover, the reduction in bacterial count became more pronounced over time (p<0.01 compared with the blank control and the original peptide P9a). The study also found that the antimicrobial peptide of this invention exhibits Staphylococcus aureus growth-inhibiting activity similar to that of the positive control drug ciprofloxacin. These results indicate that, compared with the original peptide, the structure-optimized antimicrobial peptide of this invention significantly enhances the inhibitory activity against Staphylococcus aureus.

[0117] like Figure 5 As shown, compared with the blank control, the positive control drug ciprofloxacin, and the original peptide P10, the addition of the structure-optimized antimicrobial peptides P10a and P10b, respectively, resulted in a rapid decrease in the number of *Pseudomonas aeruginosa* (p<0.01). With further time, after co-culturing the antimicrobial peptides with *P. aeruginosa* for 250 min, the bacterial count in the P10b treatment group gradually began to exceed that of the positive control drug ciprofloxacin. These results indicate that, compared with the original peptide, the structure-optimized antimicrobial peptides of this invention significantly enhance the inhibitory activity against *P. aeruginosa*.

[0118] likeFigure 6 As shown, compared with the blank control, the positive control drug ciprofloxacin, and the original peptide P13, the bacterial count began to decrease significantly after co-culturing the structure-optimized antimicrobial peptides P13a and P13b with Vibrio vulnificus for 150 min (p<0.05); the decrease in bacterial count became more pronounced with further time. These results indicate that, compared with the original peptide, the structure-optimized antimicrobial peptides of this invention exhibit significantly enhanced inhibitory activity against Vibrio vulnificus.

[0119] In summary, the antibacterial and healing-promoting peptide of this invention can significantly inhibit the growth of Gram-positive and Gram-negative bacteria. Compared with the original peptide, the antibacterial activity of the structurally optimized antibacterial and healing-promoting peptide of this invention is significantly improved.

[0120] (3) Effects of antimicrobial peptides on bacterial cell membrane permeability

[0121] Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio vulnificus were cultured at 35°C for 20 h. A suspension was prepared in PBS buffer (0.01 M, pH 7.20-7.40), centrifuged at 3500 rpm for 5 min, and the supernatant was discarded. The suspension was then resuspended in PBS and centrifuged again, repeated three times. The OD of the PBS buffer suspension was then calculated. 600 nm The concentration was 0.50-0.64. In a black 96-well plate, 10 μL of propargyl iodide (final concentration 20 μg / mL), 40 μL of bacterial suspension, and 50 μL of the antimicrobial peptide of this invention (final concentration 1000 μg / mL, with the original peptide at the same concentration as a control) were added sequentially. The plate was then analyzed using a microplate reader (excitation wavelength 535 nm, emission wavelength 614 nm), with data recorded every minute. The blank control was PBS buffer, and the positive control was 0.50% Triton X-100.

[0122] like Figure 7 As shown, compared with the blank control and the original peptide P15, the addition of the structure-optimized antimicrobial peptide P15a to the Staphylococcus aureus suspension significantly enhanced the intracellular propidium iodide fluorescence signal (p<0.01). This result indicates that the antimicrobial peptide of this invention can rapidly increase cell membrane permeability, causing cell rupture and thus inhibiting the normal growth of Staphylococcus aureus; compared with the original peptide, the structure-optimized antimicrobial peptide of this invention has a more significant effect in increasing Staphylococcus aureus membrane permeability.

[0123] like Figure 8As shown, compared with the blank control, the positive control Triton X-100, and the original peptide P16, the addition of the structure-optimized antimicrobial peptides P16a and P16s to the *Pseudomonas aeruginosa* suspension rapidly and significantly enhanced the intracellular propidium iodide fluorescence signal (p<0.01). These results indicate that the antimicrobial peptides of this invention can significantly increase cell membrane permeability, causing cell rupture and thus inhibiting the normal growth of *Pseudomonas aeruginosa*; compared with the original peptide, the structure-optimized antimicrobial peptides of this invention have a more significant effect in increasing *Pseudomonas aeruginosa* membrane permeability.

[0124] like Figure 9 As shown, compared with the blank control and the original peptide P17, the addition of the structure-optimized antimicrobial peptides P17a and P17b to Vibrio vulnificus suspension rapidly and significantly enhanced the intracellular propidium iodide fluorescence signal. After co-culturing the structure-optimized antimicrobial peptides P17a and P17b with the bacterial suspension for 12 min, the intracellular propidium iodide fluorescence signal intensity was significantly higher than that of the positive control Triton X-100 (p<0.01), and the difference in fluorescence signal intensity further increased with time. These results indicate that the antimicrobial peptides of the present invention can rapidly increase cell membrane permeability, induce cell rupture, and thus inhibit the normal growth of Vibrio vulnificus; compared with the original peptide, the structure-optimized antimicrobial peptides of the present invention have a more significant effect on increasing Vibrio vulnificus membrane permeability.

[0125] In summary, the antimicrobial healing peptide of this invention can rapidly increase cell membrane permeability, causing cell rupture, and thereby inhibiting the normal growth of Gram-negative and Gram-positive bacteria. Compared with the original peptide, the structure-optimized antimicrobial peptide of this invention significantly enhances its efficacy in increasing bacterial membrane permeability.

[0126] (4) Evaluation of the hemolytic properties of antimicrobial peptides

[0127] Add one volume of physiological saline to anticoagulated rabbit blood, centrifuge at 1500 rpm for 15 min, discard the supernatant, resuspend in physiological saline, and centrifuge again. Repeat this process three times to prepare a 2% erythrocyte suspension. Add 100 μL of the erythrocyte suspension to a 96-well plate, add 100 μL of the antimicrobial peptide of this invention (final concentration 2000 μg / mL), incubate at 37°C for 1 h, centrifuge at 1000 rpm, and add 150 μL of the supernatant to a 96-well plate. Detect the OD using a microplate reader. 540 nm The blank control was physiological saline, and the positive control was 0.50% Triton X-100.

[0128] like Figure 10 As shown, none of the antimicrobial peptide treatment groups of the present invention caused erythrocyte rupture, and there was no significant difference compared with the blank control. This result indicates that the antimicrobial healing peptides of the present invention have good biocompatibility at concentrations ≤2000 μg / mL and do not cause hemolysis.

[0129] (5) Evaluation of antibacterial peptide cell proliferation activity

[0130] After L929 cell recovery and stable passage, 5.00x10 3 cells / well were inoculated in a 96-well plate, incubated at 37℃, 5% CO2 for 24h, 100μL of the antibacterial peptide of the application (final concentration 62.50, 125, 250μg / mL) was added, and cultured for 24h. The liquid in the hole was sucked off, 100μL of CCK8 working solution was added, and incubated at 37℃, 5% CO2 for 1h, and the OD 450 nm .

[0131] It was found that under the condition of each drug concentration, the cell activity of the antibacterial peptide treatment group was significantly higher than that of the blank control (p<0.01). And under the condition of final concentration 125μg / mL, the cell activity of the antibacterial peptide treatment group was most obviously improved Figure 11 . The results show that the antibacterial healing-promoting peptide has the effect of significantly improving cell activity, and the best drug concentration is 62.5-250μg / mL.

[0132] (6) Evaluation of antibacterial peptide cell migration activity

[0133] After L929 cell recovery and stable passage, 3.00x10 4 cells / well were inoculated in a 48-well plate, incubated at 37℃, 5% CO2 for 24h, and starved with blank medium for 24h. A 20μL pipette tip was used to draw a cell scratch at the bottom of the hole plate, and PBS buffer was used to wash the floating cells and debris, 400μL of the antibacterial peptide of the application (final concentration 62.50, 250μg / mL) was added, and at 0, 6, 24, 30, 48h, optical microscope was used to take pictures, and the cell migration area was counted.

[0134] It was found that at 24h, the antibacterial peptide promoted the cell migration area significantly higher than the blank control group (p<0.01); under the condition of final concentration 62.50μg / mL, the cell migration area of the antibacterial peptide treatment group was most obviously improved Figure 12 . And at 30h, 48h, the antibacterial peptide (final concentration 62.50μg / mL) promoted the cell migration area significantly higher than the blank control group (p<0.01).

[0135] The results show that the antibacterial healing-promoting peptide has the effect of significantly promoting cell migration, and the best drug concentration is 62.5-250μg / mL.

[0136] (7) Evaluation of antibacterial peptide promoting seawater immersion infected wound repair efficiency

[0137] Due to the large workload, numerous testing indicators, and high personnel consumption involved in conducting physiological efficacy evaluation experiments, to save costs and improve efficiency, only P17a, with the lowest mean antibacterial prediction value, was selected from the 273 antibacterial and healing-promoting peptides in this invention for experimental purposes. We can reasonably infer that if the experiments find that the structurally optimized peptides with the lowest antibacterial prediction values ​​have significant effects in promoting wound infection repair, then other structurally optimized peptides with higher antibacterial prediction values ​​will also have significant effects in promoting wound infection repair.

[0138] Forty-five SD rats were weighed and anesthetized by intraperitoneal injection of 3% sodium pentobarbital (60 mg / kg). Hair was removed from the back of the rats, and the exposed skin was disinfected with iodine and 75% alcohol. The back of the rats was then excised using steam-sterilized tissue scissors to create three 1.2 cm diameter wounds, corresponding to the blank control (Control), seawater-infected control (Infected), and seawater-infected antimicrobial peptide (Infected+P17a), respectively. Each wound was treated with 0.1 g of gel per rat, covered with sterile gauze, and then the rats were housed individually. The treatment was administered once daily for 21 consecutive days. Healing was observed and the wound area was calculated on days 3, 7, 10, 14, and 21. Subsequently, the rats were euthanized, and skin tissue was harvested. E-LISA was used to detect serum endotoxin levels and the expression of inflammatory factors in the skin tissue.

[0139] In the seawater infection control group and the seawater infection antimicrobial peptide treatment group, sterile gauze soaked in seawater was applied to the wound for 15 minutes to establish a wound infection model. The blank control group received sterile gauze soaked in physiological saline for 15 minutes. The blank control group and the seawater infection control group received the drug gel matrix, while the seawater infection antimicrobial peptide treatment group received the antimicrobial peptide gel. The gel matrix consisted of 2.0% sodium hyaluronate, with the remainder being physiological saline; the antimicrobial peptide gel consisted of 125 μg / mL antimicrobial peptide, with the remainder being the gel matrix.

[0140] like Figure 13 As shown, on days 7 and 10, the wound area in the seawater-infected antimicrobial peptide treatment group was significantly smaller than that in the seawater-infected control group (p<0.01). By day 14, all wounds in this group had healed, while the wounds in the blank control group and the seawater-infected control group had not completely healed. These results indicate that the antimicrobial healing-promoting peptide of this invention can effectively promote the rapid healing of seawater-infected wounds.

[0141] On day 3, skin tissue from the damaged wound was taken, weighed, and immersed in 5 mL of physiological saline. The tissue was then aseptically homogenized and continuously diluted with physiological saline to a final concentration of 10. -1 10 -2 10 -3 10 -4The dilution liquid is 0.9% sodium chloride injection. According to the surface coating method, 0.1 mL of each dilution concentration is coated on a 9 cm blood plate with an L-shaped glass rod, and incubated at 35 DEG C for 24 hours, and the bacterial colony number is detected. It is found that the bacterial number of the seawater infection antibacterial peptide treatment group is 4.13 x 10 8 CFU / g, which is significantly lower than that of the seawater infection control group (bacterial number 1.75 x 10 10 CFU / g, p<0.01). The results show that the antibacterial and healing promoting peptide has good antibacterial performance, and can effectively reduce the number of bacteria in the seawater infected wound.

[0142] The serum endotoxin is a polymer of protein and lipopolysaccharide organization, which is mainly released by gram-negative bacteria, gram-positive bacteria, anaerobic bacteria, etc. It is found that at the 7th day, the serum endotoxin concentration of the seawater infection antibacterial peptide treatment group is 0.79 EU / mL, which is significantly lower than that of the seawater infection control group (1.82 EU / mL, p<0.01) and the blank control group (1.33 EU / mL, p<0.01). The results show that the antibacterial and healing promoting peptide can significantly reduce the serum endotoxin level, and further relieve the inflammatory response.

[0143] It is also found that, as shown in Figure 14 At the 7th day, compared with the seawater infection control group, the protein level of the anti-inflammatory factor (TGF-β1) in the skin tissue of the seawater infection antibacterial peptide treatment group is significantly increased (p<0.01), and the protein level of the pro-inflammatory factor (TNF-α, IL-1β, IL-6) is significantly decreased (p<0.01). The results show that the antibacterial and healing promoting peptide has good anti-inflammatory efficacy.

[0144] In summary, the antibacterial and healing promoting peptide can not only significantly inhibit bacterial growth and reduce secondary damage of toxins to the body, but also greatly reduce the generation of pro-inflammatory factors, accelerate wound contraction, and effectively improve the healing speed and repair quality of the infected wound.

Claims

1. An antibacterial healing-promoting peptide, characterized in that, The amino acid sequence is one or several of the following peptide segments: RWARAGLQFPVGRVHRFLRR; FLGIKVTLKSLRQGKAKLII; KAWAKAASRSARAGLQFPVGRVHRFLRR; LYFLGIKQTLKSLRQGKAKLIIL.

2. Use of the antibacterial healing-promoting peptide of claim 1 in the preparation of a wound infection healing-promoting product.

3. Use according to claim 2, characterized in that, The wound infection healing-promoting product is an Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and / or Enterococcus faecalis healing-promoting product.

4. Use according to claim 3, characterized in that, The wound infection healing-promoting product is a drug product that can be used for the prevention and treatment of open wound infections, including burn wounds, cutting wounds, traffic wounds, surgical wounds, war wounds, firearm wounds combined with seawater immersion wounds, and chronic refractory wounds.

5. Use according to claim 4, characterized in that, The chronic refractory wound includes diabetic ulcers, inflammatory ulcers and / or pressure ulcers.

6. Use according to claim 2, characterized in that, The wound infection healing-promoting product is a drug product that can effectively prevent and treat wound infections, and the drug dosage forms include lotions, gels, tinctures, spirit agents, powders, oils, pastes, plasters, film-coating agents and aerosols.

7. Use according to claim 6, characterized in that, The effective dose of the antibacterial healing-promoting peptide in the drug product is 62.5-250 μg / mL. 5-250 μg / mL.

8. The use according to claim 2, characterized in that, The wound infection healing-promoting product is a medical biomaterial, and the medical biomaterial dosage forms include lotions, solutions, dressings, gels and injections.

9. Use according to claim 8, characterized in that, When used in medical biomaterials, the effective dose of the antibacterial healing-promoting peptide is 62.5-250 μg / mL.

10. Use according to claim 8, characterized in that, The dressing types include films, hydrocolloids, hydrogels, sponges and / or sprays.

11. A wound infection prohealing product, characterized in that, It contains the antibacterial healing-promoting peptide of claim 1 as an active ingredient.

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