Antibacterial peptide ATMP23 mutant with improved antibacterial activity and application of antibacterial peptide ATMP23 mutant in preparation of antibacterial agent
By mutating the antimicrobial peptide ATMP23 with amino acids, ATMP23 mutants A23-M4, A23-M5, and A23-M6 were designed, solving the problems of low natural content and difficulty in isolation and purification of antimicrobial peptides in animal breeding. This achieved highly efficient antibacterial effects against Gram-positive and Gram-negative bacteria, improving the growth performance of livestock, poultry, and aquaculture.
Patent Information
- Application Number
- CN202511388200.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-26
- Publication Date
- 2026-02-06
AI Technical Summary
Existing antimicrobial peptides have problems such as low natural content, difficulty in separation and purification, high cost, and insufficient stress resistance in animal breeding, making them difficult to be effectively used for antibacterial and growth-promoting applications.
By mutating the antimicrobial peptide ATMP23 with amino acids, mutants A23-M4, A23-M5, and A23-M6 were designed to improve its antibacterial activity and stress resistance, and were prepared into antimicrobial agents for use in livestock, poultry, and aquaculture.
The mutant significantly enhances the bactericidal effect against Gram-positive and Gram-negative bacteria, reduces the feed conversion ratio, and improves the growth performance of farmed animals, making it suitable for the feed, food, and pharmaceutical industries.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of bioengineering, and particularly relates to an antibacterial peptide ATMP23 mutant with improved antibacterial activity and application thereof in preparation of an antibacterial agent. 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 call for prohibiting the addition of antibiotics in feed is getting louder. 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, research and development of antibacterial peptides as feed additives have important significance 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, with complicated extraction steps and low yield. 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 ATMP23 mutant with improved antibacterial activity and application thereof in preparation of an antibacterial agent. The antibacterial peptide ATMP23 mutant provided by the application is subjected to amino acid mutation and screening based on the original antibacterial peptide ATMP23, and the screened antibacterial peptide ATMP23 mutant has stronger antibacterial activity and better stress resistance, and improves the application effect of the antibacterial peptide ATMP23 mutant as a feed additive in livestock and poultry breeding and aquaculture and in food preservation.
[0004] To achieve the above-mentioned purpose, the following technical solutions are adopted:
[0005] The application provides an antibacterial peptide ATMP23 mutant A23-M4 with improved antibacterial activity, the amino acid sequence of the mutant A23-M4 is shown as SEQ ID NO: 2, the nucleotide sequence of the coding gene is shown as SEQ ID NO: 6, and the mutant A23-M4 is obtained by changing the 19th amino acid of the amino acid sequence shown as SEQ ID NO: 1 from proline to alanine.
[0006] The application further provides the antibacterial peptide ATMP23 mutant A23-M5 with improved antibacterial activity, wherein the amino acid sequence of the mutant A23-M5 is shown as SEQ ID NO: 3, the nucleotide sequence of the encoding gene is shown as SEQ ID NO: 7, and the mutant A23-M5 is obtained by changing the 14th amino acid in the amino acid sequence shown as SEQ ID NO: 1 from isoleucine to valine, the 19th amino acid from proline to alanine, and the 20th amino acid from lysine to arginine.
[0007] The application further provides the antibacterial peptide ATMP23 mutant A23-M6 with improved antibacterial activity, wherein the amino acid sequence of the mutant A23-M6 is shown as SEQ ID NO: 4, the nucleotide sequence of the encoding gene is shown as SEQ ID NO: 8, and the mutant A23-M6 is obtained by changing the 14th amino acid in the amino acid sequence shown as SEQ ID NO: 1 from isoleucine to valine, the 19th amino acid from proline to alanine, and the 31st amino acid from phenylalanine to tryptophan.
[0008] The application further provides the antibacterial peptide ATMP23 mutant in the preparation of an antibacterial agent.
[0009] Further, the antibacterial agent is a preparation for inhibiting gram-positive bacteria and gram-negative bacteria.
[0010] Further, the gram-positive bacteria include Staphylococcus aureus, Clostridium welchii, Streptococcus agalactiae and Streptococcus iniae, and the gram-negative bacteria include Vibrio splendidus, Escherichia coli, Salmonella, Vibrio parahaemolyticus and Vibrio alginolyticus.
[0011] The application further provides the antibacterial peptide ATMP23 mutant in the preparation of a feed additive for livestock and aquatic animals.
[0012] Further, the feed additive can effectively improve the growth index of livestock and aquatic animals, and the growth index includes body weight.
[0013] Further, the livestock animals include chickens, ducks, pigs, geese, cows and sheep.
[0014] Further, the aquatic animals include crucian carp, common carp, tilapia, turbot, grass carp, silver carp, sea bass, bighead carp, blue carp, catfish, Atlantic salmon, grouper, rainbow trout, megalobrama terminalis, bream, eel, large yellow croaker, yellow catfish, mandarin fish, prawn, crayfish, freshwater shrimp, sea cucumber and crab.
[0015] Compared with the prior art, the application has the advantages and technical effects that the antibacterial peptide ATMP23 mutant A23-M4, A23-M5 and A23-M6 in the application have small molecular weight, are convenient to artificially synthesize and low in cost, have a bactericidal effect on gram-positive bacteria which is significantly better than that of ATMP23, and have better stress resistance, and application in livestock and poultry and aquatic feed can significantly reduce the feed-meat ratio of farmed animals and improve the growth performance index of the farmed animals. The antibacterial peptide mutant produced by the application is a chicken immunopeptide, is safe and environmentally friendly, and can be widely applied to the feed, food, medicine and other industries. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 Heat resistance effect of the antibacterial peptide ATMP23 and its mutants.
[0017] Figure 2 Endogenous enzyme tolerance effect of the antibacterial peptide ATMP23 and its mutants.
[0018] Figure 3 MIC antibacterial effect of the mutant A23-M6 on Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio splendidus.
[0019] Figure 4 Plate antibacterial result of the mutant A23-M6 on Staphylococcus aureus.
[0020] Figure 5 Standard curve for determining the diameter of the antibacterial circle and the titer of aureomycin hydrochloride with Staphylococcus aureus as the indicator bacteria. DETAILED DESCRIPTION
[0021] The following examples are only a preferred scheme of the application, and do not limit the application in any form, and other variants and modifications can be made without exceeding the technical scheme recorded in the claims, and the protection and claim scope of the application are not limited to the provided cases.
[0022] The reagents and biological materials used in the following specific examples can be obtained from commercial channels unless otherwise specified.
[0023] 1. Strains and vectors
[0024] Pichia pastoris GS115, plasmid pPIC9K, Escherichia coli DH5α, Escherichia coli BL21, and plasmid pET 21a(+) were purchased from Invitrogen Company; the optimized gene was synthesized by Shanghai Jeery Bioengineering Co., Ltd.; sequencing analysis and primer ordering were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.
[0025] 2. Reagents and media
[0026] Plasmid extraction kit, fragment purification and recovery kit, restriction endonuclease were purchased from Baosheng Bioengineering (Dalian) Co., Ltd.; GeneMorph II random mutation PCR kit was purchased from Stratagene Company; Ampicillin, IPTG, etc. were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; Protein Marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Zhenxi Gold Biotechnology Co., Ltd.
[0027] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.
[0028] MD medium: 1.34% YNB, 0.4 mg / L biotin, 2% glucose;
[0029] YPD medium: 1% yeast extract, 2% peptone, 2% glucose;
[0030] BMGY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% glycerol;
[0031] BMMY medium: 1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 0.4 mg / L biotin, 1% methanol;
[0032] When the above medium is solid, 2% agar powder is added.
[0033] Example 1: Preparation of antibacterial peptide ATMP23
[0034] Chemical synthesis method of antibacterial peptide ATMP23: according to the sequence of SEQ ID NO: 1, the full sequence of ATMP23 was synthesized by using an automatic polypeptide synthesizer (ABI433), and was purified by HPLC reverse phase column chromatography desalting. The molecular weight of the purified antibacterial peptide ATMP23 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 by automatic amino acid sequence analyzer.
[0035] The antibacterial peptide ATMP23 is an active polypeptide artificially designed and synthesized, which contains 31 amino acid residues, and the theoretical molecular weight and isoelectric point are 3.76 KDa and 13.35, respectively. The amino acid sequence is shown in SEQ ID NO: 1: LVQRGRFGRFLRKIRRFRPKVTITIQGSARF.
[0036] According to the amino acid sequence optimization and synthesis of nucleotide sequence as shown in SEQ ID NO: 5, the synthesized nucleotide base sequence is cloned into pPIC9K plasmid, and GS115 is used as the host for expression.
[0037] Example 2: Random mutation and mutant screening of antibacterial peptide ATMP23
[0038] The optimized ATMP23 sequence is used as a template, and mutations are randomly introduced using the GeneMorph II random mutation PCR kit (Stratagene) to construct a mutant library.
[0039] The agar plate diffusion method is used to determine the antibacterial activity of the antibacterial peptide ATMP23 mutant, and the test microbial strain is Staphylococcus aureus ACCC01337 (purchased commercially).
[0040] The above test microorganism (OD600=0.2~0.3) is mixed with 25mL of 55℃ LB solid medium and plated (bacterial suspension:LB medium=1:100), and after it is solidified, a sterile puncher (diameter 7mm) is used to punch holes, 70μL of antibacterial peptide ATMP23 mutant fermentation broth is added to the holes, and it is cultured at 30℃ for 8~12h, then the diameter of the inhibition zone (clear zone) around the hole is recorded, each strain is measured three times, and the average value is calculated.
[0041] After repeated fermentation and antibacterial activity determination, and sequencing analysis, the mutants with larger inhibition zones are A23-M4 (SEQ ID NO: 2), A23-M5 (SEQ ID NO: 3), and A23-M6 (SEQ ID NO: 4), and their mutant amino acid sequences are as follows, respectively:
[0042] SEQ ID NO: 2: LVQRGRFGRFLRKIRRFR A KVTITIQGSARF;
[0043] SEQ ID NO: 3: LVQRGRFGRFLRK V RRFR AR VTITIQGSARF;
[0044] SEQ ID NO: 4: LVQRGRFGRFLRK V RRFR A KVTITIQGSAR W .
[0045] The nucleotide sequence of the gene encoding mutant A23-M4 is shown in SEQ ID NO: 6, the nucleotide sequence of the gene encoding mutant A23-M5 is shown in SEQ ID NO: 7, and the nucleotide sequence of the gene encoding mutant A23-M6 is shown in SEQ ID NO: 8.
[0046] Example 3: Antibacterial activity profile analysis of antimicrobial peptide mutants A23-M4, A23-M5, and A23-M6
[0047] The antimicrobial activity of antimicrobial peptide mutants A23-M4, A23-M5, and A23-M6 was determined using the agar plate diffusion method. The tested microbial strains were: Escherichia coli, Salmonella, Staphylococcus aureus O1337, Clostridium perfringens, Vibrio splenicum (preserved in our laboratory), Vibrio parahaemolyticus 4# (sourced from Guangzhou, Ocean University of China No. 4#), Vibrio parahaemolyticus 3# (sourced from the Yellow Sea Fisheries Research Institute, Yellow Sea Fisheries Research Institute No. 3#), Vibrio alginolyticus (Yellow Sea Fisheries Research Institute No. 20140824001-7, Ocean University of China No. HR), Streptococcus agalactiae (Wuxi, commercially available), and Streptococcus dolphinus (Wuxi, commercially available).
[0048] The tested microorganisms (OD600 = 0.2~0.3) were mixed with 25 mL of LB solid medium at 55℃ and spread on a plate (bacterial suspension:LB medium = 1:100). After solidification, holes were punched with a sterile punch (7 mm in diameter). 70 μL of antimicrobial peptide ATMP23 (0.2 g / mL fermentation spray powder or centrifuged fermentation broth) was added to the holes. The plates were incubated at 30℃ for 8~12 h. The diameter of the transparent zone around the hole was then recorded. Each bacterial species was measured three times, and the average value was calculated.
[0049] Table 1. Results of antimicrobial peptide mutants A23-M4, A23-M5, and A23-M6.
[0050]
[0051] As shown in Table 1, the antibacterial activity test results of the present invention, the antimicrobial peptides A23-M4, A23-M5, and A23-M6 have a very broad antibacterial spectrum and are effective against Gram-positive bacteria (Staphylococcus aureus, Clostridium perfringens, Streptococcus, etc.) and Gram-negative bacteria (Escherichia coli, Salmonella, Vibrio parahaemolyticus, Vibrio splenium, and Vibrio alginolyticus, etc.) and pathogenic bacteria in livestock, poultry, and aquatic animals. They have good application prospects in the prevention of diseases in livestock, poultry, and aquatic animals.
[0052] Example 4: Stress resistance analysis of antimicrobial peptide ATMP23 and its mutants
[0053] Heat resistance: Antimicrobial peptide ATMP23 and its mutant solutions with a concentration of 0.2 g / mL were placed at room temperature, 80 °C and 100 °C for 5 min respectively; then the antimicrobial activity of the antimicrobial peptide ATMP23 and its mutant solutions treated at four different temperatures was analyzed according to the method of antimicrobial activity analysis in Example 3 (antimicrobial activity was analyzed by the agar plate diffusion method described above). The test microbial strain was Staphylococcus aureus O1337.
[0054] Endogenous enzyme tolerance: Under different conditions, the simulated product needs to be resistant to the effects of acid, pepsin and trypsin in the gastrointestinal tract. After testing, it was found that the antimicrobial peptide ATMP23 and its mutants can tolerate the degradation of gastric acid and proteases, and the antibacterial effect is not affected.
[0055] The results are as follows Figure 1 and Figure 2 As shown, the antimicrobial activity of antimicrobial peptide ATMP23 and its mutants against Staphylococcus aureus ACCC01337 gradually decreased with increasing temperature. However, after high-temperature treatment (80℃ and 100℃), the mutant of antimicrobial peptide ATMP23 still exhibited good antimicrobial activity, and its antibacterial activity was much higher than that of ATMP23; moreover, it was resistant to degradation by gastric acid and proteases. This indicates that the antimicrobial peptide mutants A23-M4, A23-M5, and A23-M6 all possess good resistance to adverse conditions.
[0056] Example 5: MIC inhibitory effect of antimicrobial peptide mutant A23-M6 against Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio splenium.
[0057] A fermentation solution of the antimicrobial peptide mutant A23-M6 at a concentration of 0.2 g / mL was used, and its minimum inhibitory concentration (MIC) was determined using *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, and *Vibrio splenicum* as indicator bacteria. MIC detection of the antimicrobial peptide mutant A23-M6: (The antimicrobial peptides mentioned in this example are all antimicrobial peptide mutants A23-M6.)
[0058] (1) Preparation of 200,000 ppm antimicrobial peptide (A23-M6)
[0059] Weigh 2.00 g of antimicrobial peptide spray-dried powder, add water to make up to 10 ml, stir magnetically for 30 min, centrifuge at 10000 rpm for 10 min, and take the supernatant and filter it through a 0.22 μm filter membrane for sterilization.
[0060] (2) Preparation of indicator bacteria
[0061] After inoculating the indicator bacteria at a 1% inoculum, culture at 30°C and 200 rpm on a shaker until the late stationary phase (approximately 16-20 hours). Dilute the bacterial culture with blank medium to below 500 CFU / ml (the concentration of indicator bacteria used in actual operation is shown in the table).
[0062] (3) Antimicrobial peptides were added using a 2-fold dilution method.
[0063] Add 100 μl of LB medium to each well of a 96-well plate. Add 100 μl of sterile 200,000 ppm antimicrobial peptide mutant A23-M6 to column 1 of the medium, mix well, and then add 100 μl to column 2. Mix again, and then add 100 μl to column 3, and so on, until column 11, at which point 100 μl is discarded. Column 12 contains no antimicrobial peptide. The antimicrobial peptide concentration (ppm) in each well is as follows:
[0064] Table 2. Concentration of antimicrobial peptides in each well (ppm)
[0065]
[0066] (4) Add indicator bacteria
[0067] The indicator bacteria cultured to the late stationary phase were diluted 10% with LB medium. 5 10 6 10 7 10 8 Add 100 μl of blank LB medium to rows AB (only one negative control group without indicator bacteria is needed for the same antimicrobial peptide), and add 100 μl of diluted 10 μl to rows CD. 8 Dilute the bacterial culture to 100 μl in column EF with 10 times the volume of the culture. 7 Dilute the bacterial culture (10 times the volume) with GH by adding 100 μl to a 10-fold dilution. 6 10 times the bacterial solution (or 10 times) 5 (At appropriate dilution). The dilution factors of the indicator bacteria in each well are as follows:
[0068] Table 3. Dilution factor of indicator bacteria in each well
[0069]
[0070] Table 4 shows the concentrations (ppm) of antimicrobial peptides in each well at this time:
[0071]
[0072] (5) Cultivation and detection
[0073] After mixing the liquid in the 96-well plate, let it stand at room temperature for 4 hours, then incubate at 30°C with shaking at 100 rpm for 8-12 hours. Use an ELISA reader to detect the absorbance value at 620 nm (incubate until the negative control group containing only indicator bacteria shows significant growth), or directly observe whether there is turbidity such as bacterial growth.
[0074] (6) Results are as follows Figure 3As shown, the minimum inhibitory concentration (MIC) of antimicrobial peptide A23-M6 against these three pathogens is 195 ppm, which is less than 200 ppm.
[0075] Example 6: Fermentation of antimicrobial peptide mutant A23-M6 and determination of its antimicrobial activity
[0076] The A23-M6 recombinant strain with the best antimicrobial peptide mutant was selected for activation and seed culture preparation. It was then fermented in a 30L tank, and its antimicrobial activity was measured, as well as in aquaculture experiments.
[0077] The fermentation process is divided into three stages: (1) Cell culture stage: seed liquid is introduced at a ratio of 8%, and cultured at 30℃ for 20~24h to deplete the glycerol in the fermentation liquid; (2) Starvation stage: when the carbon source glycerol is depleted, no carbon source is added temporarily, and the starvation stage ends when the dissolved oxygen rises to 80%; (3) Induction expression stage: pH is adjusted to the required value with ammonia or phosphoric acid, methanol is added for induction, and dissolved oxygen is kept above 20%, and the induction time is 160~200h; After the fermentation is completed, the fermentation liquid is processed by plate and frame filter press to obtain metabolite supernatant, and then spray-dried through post-processing to obtain antimicrobial peptide powder for subsequent property and application testing.
[0078] Antimicrobial potency assay: Using Staphylococcus aureus as the indicator bacterium, and following the method described in Example 3, different concentrations of chlortetracycline samples were added as controls while measuring the size of the inhibition zone of the antimicrobial peptide product using the agar diffusion method. 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.
[0079] The data obtained from the measurements are shown in Table 5. The standard curve for the diameter of the inhibition zone and the potency of chlortetracycline are as follows: Figure 4 As shown, the calculated antibacterial potency of the antimicrobial peptide spray powder is 226255.95 ppm.
[0080] Table 5 Fermentation potency of antimicrobial peptide A23-M6
[0081]
[0082] Example 7: Effects of antimicrobial peptide mutant A23-M6 on the growth performance of Litopenaeus vannamei
[0083] (1) Experimental design
[0084] Two hundred and seventy Litopenaeus vannamei shrimp weighing 4-5 g each were selected and randomly assigned to three treatment groups, one control group and two experimental groups according to their body weight. The control group was fed a basal diet without antimicrobial peptides. Experimental group 1 was fed a basal diet supplemented with 100 mg / kg of antimicrobial peptide mutant A23-M6. Experimental group 2 was fed a basal diet supplemented with 200 mg / kg of antimicrobial peptide mutant A23-M6. Each group had three replicates, with 30 shrimp in each replicate.
[0085] Three meals a day were provided at 8:30, 12:30, and 16:30, with feeding amounts based on a 1-hour satiety period, ensuring a small amount of feed remained in the tank after 1 hour. Uneaten feed was then collected by tank using a siphon, dried at 105 ℃ to constant weight, weighed, and recorded. The breeding experiment lasted for 30 days.
[0086] (2) Determination of growth performance indicators
[0087] After 30 days of culture trials, Litopenaeus vannamei was cultured in an empty stomach for 24 hours. After being removed and the surface moisture of the shrimp was gently dried with filter paper, the shrimp were weighed and the weight gain rate, specific growth rate and feed conversion ratio were calculated.
[0088] The formula for calculating weight gain rate is: WGR (%) = 100 × [(Wt - W0) / W0];
[0089] The formula for calculating the specific growth rate is: SGR (%·day) -1 ) = 100 × [(lnWt - lnW0) / t];
[0090] The formula for calculating the feed conversion ratio is: FCR = F / (Wt - W0);
[0091] 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).
[0092] (3) Test results
[0093] The effects of the antimicrobial peptide mutant A23-M6 on the growth performance of Litopenaeus vannamei are shown in Table 6. After 30 days of feeding, the results showed that the antimicrobial peptide mutant A23-M6 significantly improved the weight gain rate and specific growth rate of Litopenaeus vannamei, and also reduced its feed conversion ratio to some extent.
[0094] Table 6. Effects of different doses of the antimicrobial peptide mutant A23-M6 on weight gain, specific growth rate, and feed conversion ratio of Litopenaeus vannamei.
[0095]
[0096] The results of Examples 2-7 above demonstrate that the antimicrobial peptide ATMP23 and its mutants A23-M4, A23-M5, and A23-M6 of this invention have a broad antibacterial spectrum. Among them, A23-M6 exhibits the best overall antibacterial effect, showing significant antibacterial effects against Gram-positive bacteria including Staphylococcus aureus, Clostridium perfringens, Streptococcus agalactiae, and Streptococcus dolphinii; and Gram-negative bacteria including Vibrio splenium, Escherichia coli, Salmonella, Vibrio parahaemolyticus, and Vibrio alginolyticus. Furthermore, it exhibits good thermal stability. It can be widely used in the feed, food, and pharmaceutical industries, especially as a feed additive in livestock and poultry farming and aquaculture of fish, shrimp, and sea cucumbers.
[0097] 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. A mutant A23-M4 of the antimicrobial peptide ATMP23 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M4 is shown in SEQ ID NO: 2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:
6. A23-M4 is obtained by changing the 19th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from proline to alanine.
2. An antimicrobial peptide ATMP23 mutant A23-M5 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M5 is shown in SEQ ID NO: 3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:
7. A23-M5 is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, the 19th amino acid from proline to alanine, and the 20th amino acid from lysine to arginine.
3. A mutant ATMP23 mutant A23-M6 with enhanced antibacterial activity, characterized in that: The amino acid sequence of the mutant A23-M6 is shown in SEQ ID NO: 4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:
8. A23-M6 is obtained by changing the 14th amino acid of the amino acid sequence shown in SEQ ID NO: 1 from isoleucine to valine, the 19th amino acid from proline to alanine, and the 31st amino acid from phenylalanine to tryptophan.
4. The use of the antimicrobial peptide ATMP23 mutant according to any one of claims 1-3 in the preparation of antimicrobial agents.
5. The application according to claim 4, characterized in that: The antibacterial agent is a preparation that inhibits Gram-positive and Gram-negative bacteria.
6. The application according to claim 5, characterized in that: The Gram-positive bacteria include Staphylococcus aureus, Clostridium perfringens, Streptococcus agalactiae, and Streptococcus dolphinus; the Gram-negative bacteria include Vibrio brilliance, Escherichia coli, Salmonella, Vibrio parahaemolyticus, and Vibrio alginolyticus.
7. The use of the antimicrobial peptide ATMP23 mutant according to any one of claims 1-3 in the preparation of feed additives for livestock and aquatic animals.
8. The application according to claim 7, characterized in that: The dosage of the antimicrobial peptide ATMP23 mutant is 90-220 mg / kg, applied 2-4 times / day, for 15-35 days.
9. The application according to claim 7, characterized in that: The livestock and poultry include chickens, ducks, pigs, geese, cattle, and sheep.
10. The application according to claim 7, characterized in that: The aquatic animals include crucian carp, 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, mandarin fish, prawn, red swamp crayfish, freshwater shrimp, sea cucumber, and crab.