Antibacterial peptide atmp7 mutant with improved antibacterial activity and application thereof

Abstract

The application discloses an antibacterial peptide ATMP7 mutant with improved bacteriostatic activity and application thereof. The application, on the basis of an amino acid sequence of a natural antibacterial peptide ATMP7, obtains antibacterial peptide mutants A7-M1, A7-M2, A7-M3, A7-M4, A7-M5, A7-M6 and A7-M7 with significantly improved bacteriostatic activity by combining an amino acid site-directed saturation mutation with a random mutation. The antibacterial peptide ATMP7 mutant provided by the application is convenient to artificially synthesize and low in cost, is significantly superior to the antibacterial peptide ATMP7 in bactericidal effect on gram-positive bacteria, has better stress resistance, has good heat resistance, can significantly reduce a feed-meat ratio of livestock and aquatic animals and improve growth performance indexes of the livestock and the aquatic animals in livestock and aquatic animal breeding, and provides a broader prospect for application of the antibacterial peptide product in the feed, food, medicine and other industries.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry, and particularly relates to an antibacterial peptide ATMP7 mutant with improved antibacterial activity and application thereof. BACKGROUND

[0002] In recent years, the problems of drug resistance, drug residue and environmental pollution caused by long-term use of antibiotic growth promoters in feed have been increasingly concerned by people, and the voice of prohibiting the addition of antibiotics in feed is getting higher and higher. Natural antibacterial peptides have the same broad-spectrum antibacterial effect as antibiotics, and can achieve the purpose of inhibiting bacteria and promoting growth when used as feed additives. Moreover, the antibacterial peptides have the characteristics of no residue and no pathogenic bacteria resistance, and belong to environmentally friendly feed additives. Therefore, it is of great significance to research and develop antibacterial peptides as feed additives for improving the quality of livestock and poultry products and promoting the development of green animal husbandry. However, the natural content of antibacterial peptides in animals is extremely small, and the natural resources are limited. Moreover, the antibacterial peptides have small molecular weight, and are difficult to separate and purify, and the extraction steps are tedious and the yield is low. The artificial design and modification of antibacterial peptides is a fast and effective way to obtain antibacterial peptides, and has become an important content of antibacterial peptide development. Therefore, it is an urgent need in the development and research of antibacterial peptides to obtain antibacterial peptides with simple structure, high antibacterial activity and easy preparation. SUMMARY

[0003] The application provides an antibacterial peptide ATMP7 mutant with improved antibacterial activity and application thereof. The antibacterial peptide ATMP7 mutant provided by the application is obtained by combining the methods of site-directed saturation mutation and random mutation, and has stronger antibacterial activity and better stress resistance, thereby improving the application effect of the antibacterial peptide as a feed additive in livestock and poultry breeding and aquaculture.

[0004] To achieve the above-mentioned purpose, the following technical solutions are adopted:

[0005] The application provides an antibacterial peptide ATMP7 mutant A7-M1 with improved antibacterial activity, and the amino acid sequence of the mutant A7-M1 is shown as SEQ ID NO:4, which is obtained by changing glutamic acid at the 31st position of the antibacterial peptide ATMP7 with the amino acid sequence shown as SEQ ID NO:1 into asparagine.

[0006] The application provides an antibacterial peptide ATMP7 mutant A7-M2 with improved antibacterial activity, and the amino acid sequence of the mutant A7-M2 is shown as SEQ ID NO:21, which is obtained by changing serine at the 35th position of the mutant A7-M1 into alanine.

[0007] This invention provides an antimicrobial peptide ATMP7 mutant A7-M3 with enhanced antibacterial activity. The amino acid sequence of mutant A7-M3 is shown in SEQ ID NO:22, and it is obtained by changing the serine at position 35 of mutant A7-M1 to phenylalanine.

[0008] This invention provides an antimicrobial peptide ATMP7 mutant A7-M4 with enhanced antibacterial activity. The amino acid sequence of mutant A7-M4 is shown in SEQ ID NO:23. It is obtained by changing the serine at position 35 of mutant A7-M1 to leucine.

[0009] This invention provides an antimicrobial peptide ATMP7 mutant A7-M5 with enhanced antibacterial activity. The amino acid sequence of mutant A7-M5 is shown in SEQ ID NO:24. It is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with phenylalanine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

[0010] This invention provides an antimicrobial peptide ATMP7 mutant A7-M6 with enhanced antibacterial activity. The amino acid sequence of mutant A7-M6 is shown in SEQ ID NO:25. It is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with glycine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

[0011] This invention provides an antimicrobial peptide ATMP7 mutant A7-M7 with enhanced antibacterial activity. The amino acid sequence of mutant A7-M7 is shown in SEQ ID NO:26. It is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with leucine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

[0012] The present invention also provides the application of the aforementioned antimicrobial peptide ATMP7 mutant in the preparation of antimicrobial agents.

[0013] Furthermore, the antibacterial agent is a preparation that inhibits both Gram-positive and Gram-negative bacteria.

[0014] Furthermore: the Gram-positive bacteria include Staphylococcus aureus, Clostridium perfringens, and Streptococcus; the Gram-negative bacteria include Escherichia coli, Vibrio parahaemolyticus, Shewanella fulva, Aeromonas hydrophila, and Vibrio alginolyticus.

[0015] The present invention also provides the application of the aforementioned antimicrobial peptide ATMP7 mutant in the preparation of animal feed additives.

[0016] Furthermore: the animals include cattle, sheep, pigs, chickens, ducks, fish, shrimp, and crabs.

[0017] Furthermore: the fish species mentioned are crucian carp, common carp, tilapia, turbot, grass carp, silver carp, perch, bighead carp, black carp, mud carp, Atlantic salmon, grouper, rainbow trout, blunt snout bream, bream, eel, large yellow croaker, yellow catfish, and mandarin fish.

[0018] Furthermore, the antimicrobial peptide ATMP7 mutant can increase the daily weight gain of animals and reduce the feed conversion ratio.

[0019] Furthermore, the antimicrobial peptide ATMP7 mutant can enhance the immunity of animals.

[0020] Furthermore, the antimicrobial peptide ATMP7 mutant can improve the weight gain rate and specific growth rate of animals.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] The antimicrobial peptide ATMP7 mutants A7-M1, A7-M2, A7-M3, A7-M4, A7-M5, A7-M6, and A7-M7 of this invention have small molecular weights, are easy to synthesize artificially, and are inexpensive. They exhibit significantly better bactericidal activity against Gram-positive bacteria than the standard antimicrobial peptide ATMP7, and also demonstrate better stress resistance and heat resistance. In livestock and aquaculture farming, they can significantly reduce the feed conversion ratio and improve the growth performance of farmed animals. Therefore, the antimicrobial peptide ATMP7 mutants produced by this invention can be widely used in animal husbandry, feed, food, and pharmaceutical industries, and have excellent application prospects. Attached Figure Description

[0023] Fig. 1 The effect of the antimicrobial peptide ATMP7 and its mutants on the daily feed intake of weaned piglets is described.

[0024] Fig. 2 The effect of the antimicrobial peptide ATMP7 and its mutants on the daily weight gain of weaned piglets is described.

[0025] Fig. 3 The effect of the antimicrobial peptide ATMP7 and its mutants on the feed conversion ratio of weaned piglets is described. Detailed Implementation

[0026] The following embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims. The protection and scope of the claims of the present invention are not limited to the examples provided.

[0027] Unless otherwise specified, the reagents and biological materials used in the following specific examples are commercially available.

[0028] 1. Strains and vectors

[0029] Pichia pastoris GS115, plasmid pPIC9K, Escherichia coli DH5α, Escherichia coli BL21, and plasmid pET21a(+) were purchased from Invitrogen. The optimized gene was synthesized by Shanghai Jierui Biotechnology Co., Ltd. Sequencing analysis and primer ordering were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0030] 2. Reagents and Culture Media

[0031] Plasmid extraction kit, fragment purification and recovery kit, restriction endonucleases, etc. were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd.; protein marker: Blue Plus II Protein Marker (14-120kDa) was purchased from Beijing TransGen Biotech Co., Ltd.

[0032] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0033] Example 1: Obtaining the site-directed saturation mutant of the antimicrobial peptide ATMP7

[0034] This embodiment refers to the amino acid sequence of the antimicrobial peptide ATMP7 (as shown in SEQ ID NO:1). ATMP7 is an artificially designed and synthesized active polypeptide containing 41 amino acid residues, with a theoretical molecular weight of 4474.38 Da and an isoelectric point of 10.38 Da. Through rational protein engineering analysis, it is believed that the type of amino acid at position 31 of the antimicrobial peptide ATMP7 has a significant impact on its antibacterial activity and stress resistance. Therefore, a saturation mutation was performed on the 31st amino acid, and the amino acid sequences of the designed ATMP7 mutant are shown in SEQ ID NO:2 to SEQ ID NO:20.

[0035] The antimicrobial peptide ATMP7 mutant was synthesized by Shanghai Jierui Biotechnology Co., Ltd. using a chemical synthesis method.

[0036] The antimicrobial activity of the antimicrobial peptide ATMP7 mutant was determined by the agar plate diffusion method. The test microbial strain was Staphylococcus aureus ACCC01337.

[0037] The test microorganisms (OD600 = 0.2-0.3) were mixed with 25 mL of LB solid medium at 55°C and spread on a plate (bacterial suspension:LB medium = 1:100). After solidification, a sterile punch (7 mm diameter) was used to make holes. 70 μL of antimicrobial peptide ATMP7 mutant solution (1 mg / mL) was added to each hole. The plate was incubated at 30°C for 8-12 h. The diameter of the clear zone around the hole was then recorded. Each bacterial species was measured three times, and the average value was calculated.

[0038] Antimicrobial potency assay: While measuring the size of the inhibition zone of the antimicrobial peptide product using the agar diffusion method, samples of different concentrations of chlortetracycline were added as controls. A linear fit was performed with the inhibition zone diameter (mm) as the X-axis and the logarithm of the chlortetracycline potency (ppm) as the Y-axis. The corresponding chlortetracycline potency was then calculated based on the inhibition zone diameter of the antimicrobial peptide product.

[0039] The results of the antimicrobial activity test are shown in Table 1. Among the antimicrobial peptide ATMP7 mutants, the mutant with the 31st amino acid mutated to N (asparagine) showed significantly improved antimicrobial activity compared to the original antimicrobial peptide, and was numbered A7-M1.

[0040] Table 1. Antimicrobial effect of antimicrobial peptides (inhibition zone diameter in mm)

[0041]

[0042]

[0043] Based on the mutant A7-M1, 35 sites were selected for saturation mutation, using the same design method as above.

[0044] The results of the antibacterial activity test are shown in Table 2. Mutants whose antibacterial activity was not improved are not listed in Table 2. After the amino acid at position 35 was changed from serine to alanine, phenylalanine or leucine, the antibacterial activity was further improved, resulting in mutants A7-M2, A7-M3 and A7-M4, whose amino acid sequences are shown in SEQ ID NO:21 to SEQ ID NO:23, respectively.

[0045] Table 2. Antimicrobial peptide antibacterial effect (inhibition zone diameter mm)

[0046]

[0047] Example 2: Random mutation of the antimicrobial peptide A7-M1 mutant

[0048] Using the optimized A7-M1 sequence as a template, mutations were randomly introduced using the GeneMorph II random mutation PCR kit (Stratagene) to construct a mutant library.

[0049] The antibacterial activity of each mutant was determined by the agar plate diffusion method. The tested microbial strain was Staphylococcus aureus ACCC01337.

[0050] The test microorganisms (OD600 = 0.2-0.3) were mixed with 25 mL of LB solid medium at 55°C and spread on a plate (bacterial suspension:LB medium = 1:100). After solidification, holes were punched with a sterile punch (7 mm in diameter), and 70 μL of mutant solution (1 mg / mL) was added to each hole. The plates were incubated at 30°C for 8-12 h. The diameter of the clear zone around the hole was then recorded. Each bacterial species was measured three times, and the average value was calculated.

[0051] Antimicrobial potency assay: While measuring the size of the inhibition zone of the antimicrobial peptide product using the agar diffusion method, samples of different concentrations of chlortetracycline were added as controls. A linear fit was performed with the inhibition zone diameter (mm) as the X-axis and the logarithm of the chlortetracycline potency (ppm) as the Y-axis. The corresponding chlortetracycline potency was then calculated based on the inhibition zone diameter of the antimicrobial peptide product.

[0052] After antibacterial activity assays and sequencing analysis, the antibacterial activities of mutants A7-M5, A7-M6, and A7-M7 were further enhanced, reaching 4556 ppm, 6324 ppm, and 5250 ppm, respectively, equivalent to the potency of chlortetracycline. The amino acid sequences of the A7-M5, A7-M6, and A7-M7 mutants are shown in SEQ ID NO:24 to SEQ ID NO:26, respectively.

[0053] Example 3: Heat resistance analysis of the antimicrobial peptide ATMP7 mutant

[0054] Thermoresistance analysis was performed on mutants A7-M1 to A7-M7, which exhibited higher antibacterial activity than the antimicrobial peptide ATMP7. Solutions of the ATMP7 mutant at a concentration of 1 mg / mL were incubated at 0°C, 80°C, 85°C, and 100°C for 5 min, respectively. The antibacterial activity of the ATMP7 mutant solutions treated at the four different temperatures was then tested using the method described in Example 2 (the test microbial strain was Staphylococcus aureus ACCC01337).

[0055] The results are shown in Table 3. With increasing temperature, the antimicrobial activity of the antimicrobial peptide ATMP7 and its mutants against Staphylococcus aureus ACCC01337 gradually decreased. However, after treatment at high temperatures (80℃, 85℃, and 100℃), the ATMP7 mutant still exhibited good antimicrobial activity, and its antibacterial activity was significantly higher than that of ATMP7. This indicates that the antimicrobial peptide mutants A7-M1 to A7-M7 all possess good heat resistance.

[0056] Table 2. Antimicrobial peptide antibacterial effect (inhibition zone diameter mm)

[0057]

[0058]

[0059] Example 4: Effects of A7-M1, A7-M2, and A7-M3 on the production performance of weaned piglets

[0060] A total of 125 healthy weaned piglets of similar age, litter size, and parity were selected and randomly divided into 5 groups of 25 piglets each. The control group was fed the same diet as nursery pigs. Experimental group 1 was supplemented with antimicrobial peptide ATMP7 150 mg / kg, experimental group 2 with antimicrobial peptide A7-M1 150 mg / kg, experimental group 3 with antimicrobial peptide A7-M2 150 mg / kg, and experimental group 4 with antimicrobial peptide A7-M3 150 mg / kg. The pigs were fed separately for 28 days. On the first day of the experiment and the 29th day, at 8:00 AM, each pen of piglets was weighed on an empty stomach. Feed consumption was recorded, and daily weight gain, daily feed intake, and feed conversion ratio were calculated.

[0061] Depend on Figs. 1-3 It is known that the antimicrobial peptide ATMP7 and its mutants can significantly increase the feed intake and daily weight gain of weaned piglets while reducing the feed conversion ratio, thereby reducing breeding costs. Furthermore, the antimicrobial peptide mutants A7-M1, A7-M2, and A7-M3 have even lower feed conversion ratios than ATMP7, further improving breeding outcomes.

[0062] Example 5: Effects of A7-M1, A7-M2, and A7-M3 on the Immunity of Broiler Chickens under Heat Stress Conditions

[0063] Six hundred high-quality white-feathered broilers aged one day were selected and pre-fed with powdered feed for seven days. After the pre-feeding period, chickens with significant weight differences were screened out, and the remaining 540 chickens were randomly divided into six treatment groups. Each treatment group had three replicates, and each replicate group was one pen (30 chickens per pen). The average weight of chickens in each pen was required to be approximately the same. The experimental treatment methods are shown in Table 3.

[0064] Table 3 Grouping and treatment of broiler chickens

[0065]

[0066] From day 21, broiler chickens were subjected to 8 hours of high-temperature stress daily at 35°C and humidity above 70%. The experiment continued until day 35. Feed intake, average weight, and feed conversion ratio were recorded throughout the experiment. After the rearing period, small intestinal mucosa, jejunal tissue, and liver tissue were collected from broiler chickens. Total RNA was extracted, reverse transcribed, and then amplified by PCR to detect the expression levels of immune factors. The expression levels were expressed as the ratio of the target gene to the internal control β-actin.

[0067] Table 4. Effects of antimicrobial peptides on growth performance of broiler chickens under heat stress.

[0068]

[0069] As shown in Table 4, compared with the control group, the addition of antimicrobial peptides and their mutants significantly increased the average weight of broiler chickens and reduced the feed conversion ratio, achieving similar effects to the antibiotic group. The average weight of the antimicrobial peptide mutant groups A7-M1, A7-M2, and A7-M3 was higher than that of the antimicrobial peptide ATMP7 group, indicating that the mutants have better application effects.

[0070] Table 5. Effects of antimicrobial peptides on the transcriptional level of inflammatory factors in the jejunal mucosa of broiler chickens under heat stress.

[0071]

[0072] Table 6. Effects of antimicrobial peptides on the transcriptional level of anti-inflammatory factors in the jejunal mucosa of broiler chickens under heat stress.

[0073]

[0074] Table 5 shows that the levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, and iNOS in the experimental group were significantly lower than those in the control group and the antibiotic group. Furthermore, the levels of TNF-α and IFN-γ in the mutant group were significantly lower than those in the control group, the antibiotic group, and the antimicrobial peptide ATMP7 group. Table 6 shows that the levels of anti-inflammatory cytokines IL-10, IL-4, and IL-13 in the experimental group were also significantly lower than those in the control group and the antibiotic group, with the mutant group showing a more significant effect. This indicates that the addition of antimicrobial peptides significantly reduced the inflammation level in broiler chickens under heat stress, and the mutant group exhibited a better anti-inflammatory effect.

[0075] Table 7. Effects of antimicrobial peptides on the transcriptional levels of other inflammatory factors in the jejunal mucosa of broiler chickens under heat stress.

[0076]

[0077] As shown in Table 7, the expression levels of cellular oxidative stress response factors NF-κB, interleukins IL-17 and IL-12, and Toll-like receptors TLR4 and TLR5, which are used to recognize bacterial lipopolysaccharides and flagellin, in the experimental group were significantly lower than those in the control group and the antibiotic group. This indicates that the addition of antimicrobial peptides effectively maintained the integrity of the intestinal mucosa under heat stress conditions, preventing the invasion of harmful substances such as bacteria. Moreover, the mutant group showed a slightly better effect than the antimicrobial peptide ATMP7 group.

[0078] The above results indicate that the mutants A7-M1, A7-M2, and A7-M3 in this invention have better antibacterial effects than the antimicrobial peptide ATMP7, and can maintain the integrity of the intestinal mucosa of farmed animals and reduce the occurrence of inflammation, thereby promoting animal growth. Moreover, the antimicrobial peptide ATMP7 mutant, as an alternative to antibiotics in feed additives, can achieve better breeding results under normal and heat stress conditions.

[0079] Example 6: Effects of A7-M1, A7-M2, and A7-M3 on the growth performance of crucian carp

[0080] Forty-fivety crucian carp, temporarily raised to a weight of 8-9 g / fish, were selected and randomly assigned to five treatment groups based on body weight using a single-factor experimental design. The control group received no antimicrobial peptides. Group 1 received antimicrobial peptide ATMP 750 mg / kg, Group 2 received antimicrobial peptide A7-M1 50 mg / kg, Group 3 received antimicrobial peptide A7-M2 50 mg / kg, and Group 4 received antimicrobial peptide A7-M3 50 mg / kg. Each group had six replicates, with 15 fish per replicate. The fish were fed a basal diet (12% fishmeal, 25% soybean meal, 20% rapeseed meal, 7% cottonseed meal, 20% corn germ meal, and 16% wheat bran) for 45 days.

[0081] After 45 days of breeding experiment, the crucian carp were starved for 24 hours, then removed and the surface moisture of the fish was gently absorbed with filter paper before being weighed. The weight gain rate, specific growth rate and feed conversion ratio were calculated.

[0082] The formula for calculating weight gain rate is: WGR(%) = 100 × [(Wt - W0) / W0];

[0083] The formula for calculating the specific growth rate is: SGR(%·day) -1 ) = 100 × [(lnWt - lnW0) / t];

[0084] The formula for calculating the feed conversion ratio is: FCR=F / (Wt-W0);

[0085] In the formula, W0 is the initial average body weight (g), Wt is the final average body weight (g), t is the number of experimental days (d), and F is the amount of food consumed (g, amount fed - amount of uneaten food).

[0086] Table 8. Effects of different doses of antimicrobial peptides on weight gain rate, specific growth rate, and feed conversion ratio of crucian carp.

[0087]

[0088] The effects of antimicrobial peptide ATMP7 and its mutant on the growth performance of crucian carp are shown in Table 8. After 45 days of feeding, antimicrobial peptide ATMP7 and its mutant can significantly improve the weight gain rate and specific growth rate of crucian carp, and also reduce its feed conversion ratio. Moreover, the mutant is significantly better than antimicrobial peptide ATMP7.

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. An antimicrobial peptide ATMP7 mutant A7-M1 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M1 is shown in SEQ ID NO:4, which is obtained by changing the glutamic acid at position 31 of the antimicrobial peptide ATMP7, whose amino acid sequence is shown in SEQ ID NO:1, to asparagine.

2. An antimicrobial peptide ATMP7 mutant A7-M2 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M2 is shown in SEQ ID NO:21, which is obtained by changing the serine at position 35 of the mutant A7-M1 according to claim 1 to alanine.

3. An antimicrobial peptide ATMP7 mutant A7-M3 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M3 is shown in SEQ ID NO:22, which is obtained by changing the serine at position 35 of the mutant A7-M1 according to claim 1 to phenylalanine.

4. An antimicrobial peptide ATMP7 mutant A7-M4 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M4 is shown in SEQ ID NO:23, which is obtained by changing the serine at position 35 of the mutant A7-M1 according to claim 1 to leucine.

5. An antimicrobial peptide ATMP7 mutant A7-M5 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M5 is shown in SEQ ID NO:24, which is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with phenylalanine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

6. An antimicrobial peptide ATMP7 mutant A7-M6 with enhanced antibacterial activity, characterized in that: The amino acid sequence of mutant A7-M6 is shown in SEQ ID NO:25, which is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with glycine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

7. An antimicrobial peptide ATMP7 mutant A7-M7 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A7-M7 is shown in SEQ ID NO:26, which is obtained by replacing proline at position 2 with lysine, proline at position 4 with leucine, tryptophan at position 6 with isoleucine, methionine at position 15 with isoleucine, alanine at position 26 with leucine, proline at position 28 with glutamine, and valine at position 32 with isoleucine.

8. The use of the antimicrobial peptide ATMP7 mutant according to any one of claims 1-7 in the preparation of an antimicrobial agent that inhibits Staphylococcus aureus.

9. The use of the antimicrobial peptide ATMP7 mutant according to any one of claims 1-7 in the preparation of animal feed additives.

10. The application according to claim 9, characterized in that, The animals include cattle, sheep, pigs, chickens, ducks, fish, shrimp, and crabs.

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