Antibacterial peptide atmp7 mutant for improving the antioxidant stress capacity of animals after challenge and application thereof

Abstract

The application discloses an antibacterial peptide ATMP7 mutant for improving the oxidation stress resistance of animals after an attack and application thereof. The mutants are A7-S1, A7-S2 and A7-S3 with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:21 and SEQ ID NO:22 respectively. The mutant is convenient to artificially synthesize and low in cost, and has significantly better bacteriostatic activity and application effect than ATMP7, and has better acid resistance. The mutant can improve the oxidation resistance of white-feathered broilers after an attack. The white-feathered broilers are attacked by Clostridium welchii and Escherichia coli, and the oxidation stress reaction of the body is generated, and the mutant can relieve the oxidation stress of the white-feathered broilers after an attack, improve the immunity of the body, restore the morphological structure of the jejunum, regulate the intestinal flora structure, and improve the intestinal barrier function, thereby providing a reference for the application of the antibacterial peptide in breeding.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry, and in particular relates to an antimicrobial peptide ATMP7 mutant that enhances the antioxidant stress resistance of animals after viral challenge and its application. Background Technology

[0002] The gut is not only the largest digestive and absorptive organ in the body, but also an important immune organ. A physiologically intact intestinal mucosa forms a barrier against bacteria and endotoxins. However, under stress, large numbers of bacteria and toxins cross the gastrointestinal tract and enter the host, reducing the resistance of intestinal mucosal epithelial cells, disrupting tight junctions, and damaging the intestinal mucosal barrier. Therefore, enhancing animal immune function and disease resistance through nutritional regulation, and alleviating intestinal damage caused by oxidative stress, has attracted increasing attention from researchers.

[0003] Natural antimicrobial peptides are characterized by being residue-free and not inducing antibiotic resistance in pathogenic bacteria. They can enhance animal immune function and antioxidant capacity, adjust intestinal flora structure, improve animal health, and increase animal production performance, making them a green, safe, and highly efficient novel feed additive. However, the natural content of antimicrobial peptides in animals is extremely low, and natural resources are limited. Furthermore, antimicrobial peptides have small molecular weights, making separation and purification difficult, and extraction procedures cumbersome with low yields. Artificial design and modification of antimicrobial peptides is a rapid and effective way to obtain them and has become an important aspect of antimicrobial peptide development. Therefore, obtaining antimicrobial peptides with simple structures, high antimicrobial activity, and easy preparation is an urgent need in antimicrobial peptide development research. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide ATMP7 mutant that enhances the antioxidant stress resistance of animals after challenge with pathogens and its applications. The ATMP7 mutant of this invention is obtained by site-directed saturation mutation of the 11th amino acid in the ATMP7 amino acid sequence through artificial synthesis, combined with random mutation. The resulting mutant exhibits stronger antibacterial activity and better stress resistance. It can improve the antioxidant stress resistance of animals after challenge with harmful bacteria, helping them to quickly recover normal physiological functions, thereby reducing the harm of stress to animals, improving production efficiency, and promoting the healthy and rapid development of the industry.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention provides an antimicrobial peptide ATMP7 mutant A7-S1 that enhances the antioxidant stress resistance of animals after challenge, the amino acid sequence of which is shown in SEQ ID NO:2.

[0007] Furthermore, the mutant A7-S1 was obtained by changing the lysine at position 11 of the antimicrobial peptide ATMP7, as shown in SEQ ID NO:1, to asparagine.

[0008] The present invention also provides an antimicrobial peptide mutant obtained by mutation of the mutant A7-S1, which is mutant A7-S2 with an amino acid sequence as shown in SEQ ID NO:21, or mutant A7-S3 with an amino acid sequence as shown in SEQ ID NO:22.

[0009] Furthermore, the mutant A7-S2 is obtained by changing the aspartic acid at position 21 of mutant A7-S1, whose amino acid sequence is as shown in SEQ ID NO:2, to isoleucine.

[0010] Furthermore, the mutant A7-S3 was obtained by changing arginine at position 5 to leucine, glutamic acid at position 13 to isoleucine, arginine at position 20 to phenylalanine, and glycine at position 27 to asparagine from the mutant A7-S1 with the amino acid sequence shown in SEQ ID NO:2.

[0011] The present invention also provides the application of the mutant A7-S1 or the antimicrobial peptide mutant in the preparation of antimicrobial agents.

[0012] Furthermore, the antibacterial agent inhibits Clostridium perfringens and Escherichia coli.

[0013] The present invention also provides the application of the mutant A7-S1 or the antimicrobial peptide mutant in the preparation of feed additives that enhance the antioxidant stress resistance of animals after challenge.

[0014] Furthermore, the dosage of the mutant A7-S1 or the antimicrobial peptide mutant is 50 mg / kg feed to 200 mg / kg feed.

[0015] Furthermore: the animals mentioned are chickens, ducks, and pigs.

[0016] Furthermore, the feed additive can improve the production performance of livestock and poultry.

[0017] Furthermore, the feed additive can improve the antioxidant stress resistance of broiler chickens and ducks after viral challenge.

[0018] Furthermore, the feed additive helps restore the intestinal morphology and structure.

[0019] Furthermore, the feed additive can be added directly to feed or added to drinking water.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The antimicrobial peptides ATMP7 mutants A7-S1, A7-S2, and A7-S3 of this invention have small molecular weights, are easy to synthesize artificially, and are low in cost. They exhibit significantly better bactericidal activity against Clostridium perfringens than ATMP7 and also demonstrate better stress resistance. This invention experimentally verified the effects of the antimicrobial peptide mutants on serum antioxidant indices, intestinal morphology, and relative expression levels of intestinal mucosal immune factors mRNA in broiler chickens and ducks under oxidative stress. This demonstrates that when animals are under stress or experiencing gastrointestinal diseases, the addition of the antimicrobial peptides ATMP7 mutants A7-S1, A7-S2, and A7-S3 described in this invention has a beneficial effect on the animals, providing a theoretical basis and reference for the application of antimicrobial peptides in animal husbandry. Attached Figure Description

[0022] Figure 1 The antibacterial effect of antimicrobial peptides treated at pH 2.0;

[0023] Figure 2 The antibacterial effect of antimicrobial peptides treated at pH 3.0;

[0024] Figure 3 The antibacterial effect of antimicrobial peptides treated at pH 4.0. Implementation

[0025] 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.

[0026] Unless otherwise specified, the reagents and biological materials used in the following specific examples are commercially available.

[0027] Example 1: Preparation of the antimicrobial peptide ATMP7 mutant

[0028] This invention references the amino acid sequence of the antimicrobial peptide ATMP7 (as shown in SEQ ID NO:1). The antimicrobial peptide 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.

[0029] Using bioinformatics methods, the secondary structure, conserved regions, and active sites of the antimicrobial peptide ATMP7 were analyzed. It was found that the type of amino acid at position 11 has a significant impact on its antimicrobial activity. Therefore, a saturation mutation was performed on the amino acid at position 11, and the designed amino acid sequences are shown in SEQ ID NO:2-SEQ ID NO:20.

[0030] Chemical synthesis method of the antimicrobial peptide ATMP7 mutant: Based on the amino acid sequence of the above-mentioned antimicrobial peptide ATMP7 mutant (SEQ ID NO:2-SEQ ID NO:20), the full sequence of the ATMP7 mutant was synthesized using a fully automated peptide synthesizer (ABI433), and purified by HPLC reversed-phase column chromatography. The molecular weight of the purified antimicrobial peptide ATMP7 mutant was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was analyzed using an automated amino acid sequencer.

[0031] Example 2: Antibacterial activity analysis of the 11th amino acid saturated mutant of antimicrobial peptide ATMP7

[0032] The antimicrobial activity of the antimicrobial peptide ATMP7 mutant was determined by the agar plate diffusion method. The test microbial strain was Clostridium perfringens ATCC13124.

[0033] The test microorganism Clostridium perfringens ATCC13124 (OD600=0.4~0.5) was mixed with 15mL of RCM medium at 55℃ and poured into agar plates (bacterial suspension: RCM medium=1:100). After solidification, a sterile punch (7mm diameter) was used to make holes. 70μL of antimicrobial peptide ATMP7 mutant solution (1mg / mL) was added to each hole. The plates were then anaerobically incubated at 37℃ for 8~12h. The diameter of the clear zone around the hole was recorded. Each bacterial species was measured three times, and the average value was calculated.

[0034] Antimicrobial potency assay: While measuring the size of the inhibition zone of the antimicrobial peptide product using the agar diffusion method, penicillin samples of different concentrations were added as controls. A linear fit was performed with the inhibition zone diameter (mm) as the X-axis and the logarithm of the penicillin potency (ppm) as the Y-axis. Then, the penicillin potency of each antimicrobial peptide mutant was calculated based on the inhibition zone diameter.

[0035] Table 1. Antibacterial effect of antimicrobial peptides (inhibition zone diameter in mm)

[0036]

[0037] As shown in Table 1, the antibacterial activity test results show that the antimicrobial peptide ATMP7 mutant of the present invention, the mutant with the 11th amino acid mutated to N, namely A7-S1, has significantly improved antibacterial activity compared with the original antimicrobial peptide.

[0038] Example 3: Improving the acid resistance of mutant A7-S1 according to the application scenario

[0039] Random mutations and screening were performed on mutant A7-S1 (SEQ ID NO:2) to obtain two mutants, A7-S2 and A7-S3, with greatly enhanced acid resistance. Their amino acid sequences are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0040] The antimicrobial activity of acid-resistant mutants A7-S2 and A7-S3 of antimicrobial peptide A7-S1 was determined using the agar plate diffusion method. The test microbial strain was Clostridium perfringens ATCC13124. A 20% aqueous solution of the antimicrobial peptide A7-S1 mutant was prepared and adjusted to pH 2.0, pH 3.0, and pH 4.0 with 1M hydrochloric acid. The solutions were then incubated at 37℃ for 60 min, and after removal, adjusted back to neutral (pH 6.5) with 2M sodium hydroxide for later use.

[0041] Mix the test microorganism (OD600 = 0.4~0.5) with 25 mL of RCM solid medium at 55℃ and spread it on a plate (bacterial suspension: RCM medium = 1:100). After solidification, punch holes with a sterile punch (7 mm in diameter). Add 70 μL of antimicrobial peptide A7-S1 mutant acid treatment solution (1 mg / mL) to the holes and anaerobic culture at 37℃ for 8~12 h. Then record the diameter of the clear zone around the hole. Repeat the measurement three times for each bacterial species and calculate the average value.

[0042] Antimicrobial potency assay: While measuring the size of the inhibition zone of the antimicrobial peptide product using the agar diffusion method, penicillin samples of different concentrations were added as controls. A linear fit was performed with the inhibition zone diameter (mm) as the X-axis and the logarithm of the penicillin potency (ppm) as the Y-axis. The penicillin potency corresponding to the inhibition zone diameter of the antimicrobial peptide product was then calculated.

[0043] like Figure 1-3 As shown, the antimicrobial peptides A7-S1 mutants A7-S2 and A7-S3 of the present invention can greatly improve their antimicrobial titer after treatment in acidic environments of pH 2.0, pH 3.0 and pH 4.0, and improve the retention rate after acid treatment.

[0044] Example 4: Effects of low concentrations of ATMP7, A7-S1, A7-S2, and A7-S3 on antioxidant stress in broiler chickens challenged with Clostridium perfringens

[0045] This experiment selected 400 high-quality 1-day-old white-feathered broilers and pre-fed them with powdered feed for 7 days. After the pre-feeding period, chickens with significant weight differences were screened out, and the remaining 360 chickens with similar weights were randomly divided into 6 treatment groups, with 3 replicates for each treatment group. Each replicate group was one pen (20 chickens per pen). The experimental treatments are shown in Table 2.

[0046] Table 2 Experimental Design

[0047]

[0048] Clostridium perfringens is widely distributed in the natural environment and is a common bacterium in the intestines of humans and animals. It produces exotoxins that can cause necrotic enteritis and enterotoxemia in animals, severely impacting intestinal health in broilers, reducing production performance, and increasing mortality. The experiment lasted 21 days. After the experiment, the abdominal skin was carefully cut open to expose the thoracic cavity. The ileum was separated and longitudinally cut, and the mucosa was scraped off with a glass slide for measuring antioxidant indices. The thymus, bursa of Fabricius, and spleen were collected and weighed to determine immune organ indices. The total length and weight (including contents) of the intestines were measured separately, and the relative weight of the intestines was calculated.

[0049] Table 3. Effects of antimicrobial peptides on antioxidant indices of ileal mucosa in broiler chickens after viral challenge.

[0050] Group Glutathione (GSH) μg / mg prot Total antioxidant capacity T-AOC mmol / mg prot Heme oxygenase-1 HO-1pg / mg prot negative control group 71.40b 2.19bc 23.67bc Positive control group 63.04c 1.45c 21.14c Experiment 1 group 73.28b 2.20bc 24.30bc Experimental Group 2 75.28ab 2.62ab 27.10a Experiment 3 groups 80.56a 2.79a 26.72a Experiment 4 groups 81.03a 2.98a 27.23a SEM 2.15 0.43 0.80 value 0.02 0.02 0.03

[0051] Table 3 shows that, compared with the positive control group, the addition of 50 mg / kg of antimicrobial peptide ATMP7 and ATMP7 mutant to the feed effectively improved the antioxidant capacity of broilers. Specifically, the mutant group significantly increased GSH content, total antioxidant capacity, and heme synthase-1 content. P＜ (0.05), A7-S3 exhibits the best antioxidant effect.

[0052] Table 4. Effects of antimicrobial peptides on immune organ indices in broiler chickens after viral challenge.

[0053] Group Thymus index, % Bursa of Fabricius index, % Spleen index, % negative control group 0.73 0.46 0.16 Positive control group 0.56 0.35 0.14 Experiment 1 group 0.62 0.39 0.15 Experimental Group 2 0.70 0.39 0.15 Experiment 3 groups 0.69 0.46 0.17 Experiment 4 groups 0.77 0.45 0.19 SEM 0.024 0.021 0.006 value 0.91 0.42 0.22

[0054] Table 4 shows that adding 50 mg / kg of antimicrobial peptide ATMP7 and ATMP7 mutant to the feed tended to increase the thymus index, bursa of Fabricius index, and spleen index, but the levels were not yet significant. P >0.05). Compared with the control group and the ATMP7 group, A7-S2 and A7-S3 can better improve the body's immunity.

[0055] Table 5. Effects of antimicrobial peptides on intestinal development in broiler chickens after viral challenge.

[0056]

[0057] Table 5 shows that adding 50 mg / kg of the antimicrobial peptide ATMP7 and its mutant to the feed can increase intestinal length, intestinal weight, and relative weight, but the increase is not statistically significant. P >0.05). Compared with the control group and the ATMP7 group, A7-S2 and A7-S3 can better promote intestinal development.

[0058] In conclusion, the ATMP7 mutants A7-S2 and A7-S3 have more significant advantages over other groups in improving the antioxidant capacity, enhancing immunity, and promoting intestinal development in challenged broilers.

[0059] Example 5: Effects of added ATMP7, A7-S2, and A7-S3 on antioxidant stress in broiler chickens challenged with Clostridium perfringens

[0060] This experiment selected 400 high-quality broiler chickens aged 1 day and pre-fed them with powdered feed for 7 days. After the pre-feeding period, chickens with significant weight differences were screened out, and the remaining 360 chickens with similar weights were randomly divided into 5 treatment groups, with 6 replicates for each treatment group. Each replicate group consisted of 1 pen (12 chickens per pen). The experimental treatments are shown in Table 6.

[0061] Table 6 Experimental Design

[0062]

[0063] The experiment lasted 42 days. After the experiment, feed intake, body weight, average daily weight gain, average daily feed intake, and feed conversion ratio of the broilers were recorded. Vein blood was collected from the wings to determine antioxidant indicators, and jejunal mucosa was scraped using sterile glass slides to determine the mRNA expression level of inflammatory factors. Intestinal contents were also collected to determine the gut microbiota.

[0064] Table 7. Effects of antimicrobial peptides on the production performance of broiler chickens after viral challenge.

[0065] Group Average daily weight gain (g / (animal·d)) Average daily feed intake (g / (animal·d)) Meat-to-fat ratio negative control group 53.70 98.23 1.83a Positive control group 50.71 95.62 1.89a Experiment 1 group 57.22 102.45 1.79ab Experimental Group 2 59.90 103.42 1.73b Experiment 3 groups 58.90 101.30 1.72b SEM 4.12 7.11 0.26 value 0.39 0.69 0.02

[0066] Table 7 shows that adding 100 mg / kg of the antimicrobial peptide ATMP7 and its mutants A7-S2 and A7-S3 to the feed can increase the average daily weight gain and average daily feed intake of broilers after challenge, but the effect is not significant. P >0.05). Compared to the control group, mutants A7-S2 and A7-S3 significantly reduced the feed conversion ratio. (P <0.05 ) This indicates that mutants A7-S2 and A7-S3 can improve the production performance of broilers after challenge.

[0067] Table 8. Effects of antimicrobial peptides on the antioxidant properties of serum from broiler chickens after viral challenge.

[0068] Group Total antioxidant capacity T-AOC (U / ml) Superoxide dismutase (SOD) (U / ml) Glutathione peroxidase (GSH-Px) (U / ml) Malondialdehyde (MDA) (μmol / L) negative control group 1.17b 67.87 441.14 4.66ab Positive control group 1.10b 65.13 429.98 4.82a Experiment 1 group 1.28ab 69.05 450.24 4.33ab Experimental Group 2 1.36a 70.21 465.19 4.19bc Experiment 3 groups 1.43a 74.69 478.18 4.02c SEM 0.03 0.95 5.26 0.33 value 0.04 0.11 0.14 0.05

[0069] Table 8 shows that adding 100 mg / kg of the antimicrobial peptide ATMP7 and mutants A7-S2 and A7-S3 to the feed, compared with the control group, can increase the SOD and GSH-Px content of broilers after challenge. P>0.05), mutants A7-S2 and A7-S3 significantly increased T-AOC content and significantly decreased MDA content. (P <0.05 ) This indicates that mutants A7-S2 and A7-S3 can improve the antioxidant properties of broilers after challenge with the virus.

[0070] Table 9. Effects of antimicrobial peptides on the relative expression levels of immune factors mRNA in the jejunal mucosa of broiler chickens after viral challenge.

[0071] Group TNF-α IFN-γ IL-10 negative control group 1.00a 1.00a 1.00b Positive control group 1.84b 1.64b 0.41a Experiment 1 group 1.70bc 1.60b 1.10b Experimental Group 2 1.73b 1.58b 1.16b Experiment 3 groups 1.67c 1.58b 1.27c SEM 0.10 0.17 0.14 value 0.04 0.02 0.01

[0072] As shown in Table 9, the relative mRNA expression levels of inflammatory factors TNF-α and IFN-γ in the positive control group were significantly higher than those in the negative control group after challenge. P <0.05), while the relative expression level of the anti-inflammatory factor IL-10 mRNA was significantly lower than that of the control group ( P <0.05 indicates an imbalance in the expression of pro-inflammatory and anti-inflammatory cytokines mRNA in the jejunal mucosa of broiler chickens after challenge, with abnormally elevated levels of pro-inflammatory cytokines mRNA, leading to an inflammatory state in the animals. Compared to the positive control group, the relative expression level of the anti-inflammatory cytokine IL-10 mRNA in the mucosa of the groups fed with the antimicrobial peptide ATMP7 and mutants A7-S2 and A7-S3 was significantly increased. P <0.05). The mutant A7-S3 significantly reduced the relative mRNA expression level of the pro-inflammatory cytokine TNF-α. This indicates that the antimicrobial peptide mutants A7-S2 and A7-S3 promoted the balance between pro-inflammatory and anti-inflammatory factors.

[0073] Table 10 Effects of antimicrobial peptides on the gut microbiota structure of broiler chickens after viral challenge.

[0074] Group Escherichia coli (1g CFU / g) Lactobacillus (1g CFU / g) negative control group 9.21ab 8.70bc Positive control group 10.38a 7.86c Experiment 1 group 8.90abc 8.80b Experimental Group 2 8.46bc 8.91b Experiment 3 groups 7.49c 9.59a SEM 0.35 0.48 value 0.03 0.02

[0075] Table 10 shows that adding 100 mg / kg of the antimicrobial peptide ATMP7 and its mutants A7-S2 and A7-S3 to the feed can reduce the number of harmful bacteria (Escherichia coli) and increase the number of beneficial bacteria (Lactobacillus) in the intestines of broilers after challenge, with mutant A7-S3 showing a significant increase. P <0.05). This indicates that the mutant A7-S3 has a better effect in regulating the gut microbiota.

[0076] In summary, mutants A7-S2 and A7-S3 can improve the production performance and antioxidant capacity of broilers after viral challenge, promote immunity, regulate intestinal flora structure, and protect intestinal health.

[0077] Example 6: Effects of high concentrations of ATMP7, A7-S2, and A7-S3 on antioxidant stress in broiler chickens challenged with Clostridium perfringens

[0078] This experiment selected 480 high-quality broiler chickens aged 1 day and pre-fed them with powdered feed for 7 days. After the pre-feeding period, chickens with significant weight differences were screened out, and the remaining 450 chickens with similar weights were randomly divided into 5 treatment groups, with 6 replicates for each treatment group. Each replicate group was one pen (15 chickens per pen). The experimental treatments are shown in Table 11.

[0079] Table 11 Experimental Design

[0080]

[0081] The experiment lasted 42 days. After the experiment, the feed intake, body weight, average daily weight gain, average daily feed intake, and feed conversion ratio of the broilers were recorded. Vein blood was collected from the wings to determine antioxidant and immune indicators, and a section of jejunum was taken for intestinal morphology analysis.

[0082] Table 12 Effects of antimicrobial peptides on the production performance of broiler chickens after viral challenge.

[0083] Group Average daily weight gain (g / (animal·d)) Average daily feed intake (g / (animal·d)) Meat-to-fat ratio negative control group 57.46c 94.98 1.65ab Positive control group 55.10c 93.45 1.70a Experiment 1 group 58.49bc 95.27 1.63b Experimental Group 2 59.54ab 95.88 1.61bc Experiment 3 groups 60.72a 96.11 1.58c SEM 0.32 0.33 0.02 value 0.01 0.69 0.02

[0084] Table 12 shows that adding 200 mg / kg of the antimicrobial peptide ATMP7 and mutants A7-S2 and A7-S3 to the feed can increase the average daily weight gain and average daily feed intake of broilers after challenge, and reduce the feed conversion ratio. Among them, mutants A7-S2 and A7-S3 showed significant differences compared to the challenge group. P <0.05). This indicates that mutants A7-S2 and A7-S3 can significantly improve the production performance of broilers.

[0085] Table 13 Effects of antimicrobial peptides on serum antioxidant indices of broiler chickens after viral challenge

[0086] Group SOD (U / ml) GSH-Px (U / ml) CAT (U / ml) negative control group 204.78c 612.53bc 2.01 Positive control group 200.56c 607.41c 1.89 Experiment 1 group 216.23b 620.03ab 2.37 Experimental Group 2 220.45b 629.18a 2.89 Experiment 3 groups 236.51a 630.45a 2.97 SEM 5.32 5.30 0.13 value 0.01 0.04 0.07

[0087] Table 13 shows that, compared with the challenge group, the addition of 200 mg / kg of antimicrobial peptide A7 and mutants A7-S2 and A7-S3 to the feed significantly increased the levels of SOD and GSH-Px in the serum of broilers after challenge. P <0.05), among which, mutant A7-S3 was significantly better than the other two groups with added antimicrobial peptides (experimental group 1 and experimental group 2). This indicates that antimicrobial peptide A7 and mutants A7-S2 and A7-S3 can significantly improve the production performance of broilers, with A7-S3 showing the best application effect.

[0088] Table 14 Effects of antimicrobial peptides on serum immune indicators in broiler chickens after viral challenge.

[0089] Group IL-2 (ng / L) IL-6 (ng / L) IgG (g / L) IgA (g / L) IgM (g / L) negative control group 275.56 153.96 3.45bc 0.39bc 1.66b Positive control group 279.98 160.27 2.67c 0.31c 1.51c Experiment 1 group 275.77 159.43 4.67b 0.45b 1.74b Experimental Group 2 269.12 155.34 4.78b 0.47b 1.97ab Experiment 3 groups 262.07 150.87 5.43a 0.58a 2.29a SEM 18.65 11.45 0.67 0.15 0.31 value 0.17 0.38 0.04 0.03 0.04

[0090] Table 14 shows that, compared with the challenge group, the addition of 200 mg / kg of the antimicrobial peptide ATMP7 and mutants A7-S2 and A7-S3 to the feed significantly increased the levels of immunoglobulins IgA, IgG, and IgM in broilers after challenge. P <0.05). After the addition of antimicrobial peptides, the pro-inflammatory factors IL-2 and IL-6 were reduced to varying degrees, but did not reach significant levels. P >0.05). This indicates that mutants A7-S2 and A7-S3 can effectively enhance the body's immunity.

[0091] Table 15 Effects of antimicrobial peptides on jejunal morphology in broiler chickens after viral challenge.

[0092] Group Pile height / μm crypt depth / μm Fleece-like sleekness negative control group 1180.9 160.78 7.34 Positive control group 1123.61 162.26 6.92 Experiment 1 group 1216.43 153.27 7.94 Experimental Group 2 1230.23 155.48 7.91 Experiment 3 groups 1247.45 156.32 7.98 SEM 50.35 10.51 0.94 value 0.83 0.62 0.33

[0093] Table 15 shows that, compared with the challenge group, the addition of 200 mg / kg of the antimicrobial peptide ATMP7 and mutants A7-S2 and A7-S3 to the feed can increase the villus height, decrease the crypt depth, and increase the villus-crypt ratio in the broiler intestine after challenge. P >0.05). Among them, compared with other antimicrobial peptide groups, the mutant A7-S3 group has a more superior effect on intestinal morphology.

[0094] In conclusion, mutants A7-S2 and A7-S3 can improve the production performance, antioxidant capacity, and immune capacity of broilers after viral challenge, maintain intestinal morphology, and ensure healthy growth.

[0095] Example 7: Effects of high addition levels of A7-S2 and A7-S3 on antioxidant stress in E. coli-infected ducks

[0096] This experiment selected 300 healthy, one-day-old male Cherry Valley ducks of similar weight and randomly assigned them to 5 treatments based on the principle of no difference in weight. Each treatment had 6 replicates, and each replicate had 10 ducks. The experimental treatments are shown in Table 16.

[0097] Table 16 Experimental Design

[0098]

[0099] Escherichia coli is a Gram-negative bacterium that can produce enterotoxins in animals, causing severe intestinal infections, disrupting the intestinal flora balance, and resulting in symptoms such as diarrhea. The experiment lasted for 15 days. After the experiment, the feed intake and body weight of the ducks were recorded from days 7 to 14. Body weight gain, feed intake, feed conversion ratio, and mortality rate were also recorded. Blood was collected from the jugular vein, and serum was separated to determine antioxidant and small intestinal permeability indicators. After euthanasia by exsanguination from the jugular vein, the abdominal cavity of the ducks was opened, and the thymus, spleen, and bursa of Fabricius were completely removed, cleaned with filter paper, and weighed.

[0100] Table 17 Effects of antimicrobial peptides on the production performance of ducks challenged with Escherichia coli

[0101]

[0102] Table 17 shows that, compared with the challenge group, adding antimicrobial peptides to the feed can increase the body weight gain and feed intake of ducks after challenge, and reduce the feed conversion ratio and mortality rate. Among them, the mutant A7-S3 group can significantly increase body weight gain and reduce mortality rate. P <0.05). This indicates that mutants A7-S2 and A7-S3 can effectively improve the production performance of meat ducks.

[0103] Table 18 Effects of antimicrobial peptides on immune organ indices in ducks challenged with Escherichia coli

[0104] Group Bursa of Fabricius index (mg / g) Thymus index (mg / g) Spleen index (mg / g) negative control group 0.47bc 1.21 0.47abc Positive control group 0.31c 1.20 0.38c Experiment 1 group 0.60ab 1.27 0.49ab Experimental Group 2 0.61ab 1.30 0.51ab Experiment 3 groups 0.73a 1.35 0.57a SEM 0.12 0.25 0.03 value 0.02 0.06 0.01

[0105] As shown in Table 18, compared with the challenge group, the addition of antimicrobial peptides to the feed can increase the thymus index of ducks after challenge ( P >0.05), significantly improving the bursa of Fabricius index and spleen index ( P <0.05). Comparison among antimicrobial peptide groups showed that the mutant A7-S3 group had a better effect on improving the body's immunity.

[0106] Table 19 Effects of antimicrobial peptides on the antioxidant properties of serum from ducks challenged with Escherichia coli

[0107]

[0108] Table 19 shows that, compared with the challenge group, the addition of antimicrobial peptides to the feed significantly increased the levels of GSH-Px and T-AOC in the serum of broiler ducks after challenge. P <0.05), the A7-S3 group can significantly increase SOD ( <0.05), P <0.05). Comparison among antimicrobial peptide groups showed that the mutant A7-S3 group had a better effect on improving the body's antioxidant capacity.

[0109] Table 20 Effects of antimicrobial peptides on the intestinal barrier of ducks challenged with Escherichia coli

[0110] Group Diamine oxidase U / mL Nitric oxide μmol / L Endotoxin - 1 μmol / L negative control group 1.60a 37.34 65.18a Positive control group 1.68a 37.98 67.12a Experiment 1 group 1.51c 36.56 64.23a Experimental Group 2 1.57b 36.42 58.04b Experiment 3 groups 1.50c 36.79 59.25b SEM 0.16 0.73 1.64 value 0.04 0.96 0.02

[0111] As shown in Table 20, compared with the challenge group, the addition of antimicrobial peptide mutants A7-S2 and A7-S3 to the feed significantly increased the content of intestinal diamine oxidase and significantly decreased the content of endotoxin-1. P <0.05). This indicates that the antimicrobial peptide mutants A7-S2 and A7-S3 can maintain the function of the intestinal barrier in ducks and reduce changes in intestinal permeability.

[0112] In conclusion, mutants A7-S2 and A7-S3 can improve the production performance, antioxidant capacity, and immune capacity of ducks after viral challenge, maintain intestinal morphology, ensure the integrity of the intestinal barrier function, and promote healthy growth.

[0113] 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-S1 that enhances the antioxidant stress resistance of animals after viral challenge, characterized in that, The amino acid sequence of the mutant A7-S1 is shown in SEQ ID NO:

2.

2. The mutant A7-S1 according to claim 1, characterized in that, The mutant A7-S1 was obtained by changing the lysine at position 11 of the antimicrobial peptide ATMP7, as shown in SEQ ID NO:1, to asparagine.

3. An antimicrobial peptide mutant obtained by the A7-S1 mutant of claim 1, characterized in that, The antimicrobial peptide mutant is mutant A7-S2 with an amino acid sequence as shown in SEQ ID NO:21, or mutant A7-S3 with an amino acid sequence as shown in SEQ ID NO:

22.

4. The antimicrobial peptide mutant according to claim 3, characterized in that, The mutant A7-S2 was obtained by changing the aspartic acid at position 21 of mutant A7-S1, whose amino acid sequence is shown in SEQ ID NO:2, to isoleucine.

5. The antimicrobial peptide mutant according to claim 3, characterized in that, The mutant A7-S3 was obtained by changing arginine at position 5 to leucine, glutamic acid at position 13 to isoleucine, arginine at position 20 to phenylalanine, and glycine at position 27 to asparagine from the mutant A7-S1, whose amino acid sequence is shown in SEQ ID NO:

2.

6. The application of the mutant A7-S1 according to claim 1 or the antimicrobial peptide mutant according to claim 3 in the preparation of antimicrobial agents, characterized in that, The antibacterial agent inhibits Clostridium perfringens and Escherichia coli.

7. The application of the mutant A7-S1 according to claim 1 or the antimicrobial peptide mutant according to claim 3 in the preparation of feed additives that enhance the antioxidant stress resistance of animals after challenge, characterized in that, The animal in question is either a chicken or a duck.

8. The application according to claim 7, characterized in that, The dosage of the mutant A7-S1 or the antimicrobial peptide mutant is 50 mg / kg feed to 200 mg / kg feed.

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