Antimicrobial peptide mutants and uses thereof

Abstract

The application discloses an antibacterial peptide mutant and application thereof, the amino acid sequence of the mutant is shown in any one of SEQ ID NO:1 or SEQ ID NO:2.The antibacterial peptide has good antibacterial and bacteriostatic performance, and the application thereof in preparing microbial inhibitors and in feed additives is also provided.The feed additive comprises the antibacterial peptide, wherein the antibacterial peptide can replace the growth promoting effect of antibiotics, and the effect is rapid and stable without residue; meanwhile, the feed additive also comprises gamma-aminobutyric acid and vitamin C, which can eliminate the reduction of feed intake caused by high temperature and high humidity, relieve heatstroke symptoms such as no sweating and rapid breathing, resist stress, have a good regulating effect on various physiological and pathological changes, reduce and neutralize toxins, and enhance the detoxification capacity of liver.

Description

[0001] This application is a divisional application. The original application has the application number 2021108924402, the application date is August 4, 2021, and the invention title is "Antibacterial Peptide Mutants and Their Applications". Technical Field

[0002] This invention belongs to the field of functional gene modification technology, specifically relating to an antimicrobial peptide mutant and its application. Background Technology

[0003] Adding antibiotics to feed can alleviate weaning stress in piglets and promote their growth, but antibiotic residues and pathogen resistance have negatively impacted livestock and human health. In 2020, China implemented a complete ban on antibiotics in livestock feed. This ban increases morbidity and mortality rates in piglets during the early nursery period, and slows their growth. Given this ban, finding effective antibiotic alternatives, enhancing intestinal immune regulation in pigs, and developing new, highly effective, and safe products to improve pig gut health are particularly important.

[0004] Antimicrobial peptides are a class of polypeptides with broad-spectrum antibacterial activity. Some antimicrobial peptides can also activate the immune system and play important roles in cell signaling, cell proliferation regulation, and antitumor, antiviral, and antiparasitic activities. Due to their unique bactericidal mechanisms and excellent physicochemical properties, antimicrobial peptides have become a hot topic in the research and development of antibiotic alternatives. They also have broad application prospects in medical anti-infective therapies and livestock production. In the research and development of antimicrobial peptides, modifying existing antimicrobial peptides and designing new molecules are effective ways to improve their activity. Molecular design, structural modification and alteration of antimicrobial peptides, reducing their hemolytic activity, and improving their antibacterial activity and acid-base stability have become important aspects of antimicrobial peptide research and development.

[0005] Gamma-aminobutyric acid (GABA), also known as 4-aminobutyric acid, often exists in solution as a zwitterion (a negatively charged carboxyl group and a positively charged amino group). Due to the electrostatic interaction between the positive and negative charged groups, GABA can exhibit both gaseous (folded) and solid (extended) molecular conformations in solution. This coexistence of multiple molecular conformations allows GABA to bind to various receptor proteins and perform a variety of important physiological functions. GABA promotes animal feed intake, regulates endocrine function, improves immune performance, and enhances stress resistance in livestock and poultry. It has been approved by the Ministry of Health as a new resource food. In animal husbandry, GABA can promote the secretion of gastric juice and growth hormone, regulate animal appetite, increase feed intake, promote livestock and poultry growth, and improve feed efficiency; it also has a detoxifying effect on ammonia toxicity, reducing the incidence of respiratory diseases in livestock and poultry, alleviating heat-induced panting in animals during heat stress, and enhancing animal stress resistance. Summary of the Invention

[0006] The first objective of this invention is to provide a polypeptide.

[0007] The second objective of this invention is to provide a nucleic acid molecule.

[0008] A third objective of this invention is to provide a carrier.

[0009] The fourth object of the present invention is to provide a cell.

[0010] A fifth object of the present invention is to provide applications of the aforementioned polypeptide or nucleic acid molecules, carriers, or cells.

[0011] The sixth object of the present invention is to provide a feed additive.

[0012] The technical solution adopted in this invention is:

[0013] In a first aspect, the present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] A second aspect of the present invention provides a nucleic acid molecule encoding the polypeptide described in the first aspect of the present invention.

[0015] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect of the present invention.

[0016] In a fourth aspect, the present invention provides a cell comprising the carrier described in the third aspect of the present invention, wherein the cell is neither a plant cell nor an animal cell.

[0017] A fifth aspect of the invention provides the use of the polypeptide described in the first aspect of the invention, the nucleic acid molecule described in the second aspect of the invention, the carrier described in the third aspect of the invention, or the cell described in the fourth aspect of the invention in the following (I) or (II):

[0018] (I) Preparation of antibacterial agents;

[0019] (II) Preparation of additives.

[0020] In some embodiments of the present invention, the bacteria are bacteria.

[0021] In some preferred embodiments of the present invention, the bacteria are Salmonella, Escherichia coli, or Staphylococcus aureus.

[0022] In some embodiments of the present invention, the additive is a food additive, feed additive, cosmetic additive, or hygiene product additive.

[0023] In some preferred embodiments of the present invention, the additive is a feed additive.

[0024] A sixth aspect of the present invention provides an additive comprising the polypeptide described in the first aspect of the present invention.

[0025] In some embodiments of the present invention, the additive is a food additive, feed additive, cosmetic additive, or hygiene product additive.

[0026] In some preferred embodiments of the present invention, the additive is a feed additive.

[0027] In some embodiments of the present invention, the feed additive contains, by weight percentage, the following components: 5% to 20% of the polypeptide shown in SEQ ID NO: 1, 5% to 20% of the polypeptide shown in SEQ ID NO: 2, 1% to 30% of γ-aminobutyric acid and 1% to 30% of vitamin C.

[0028] In some embodiments of the present invention, the feed additive contains, by weight percentage, the following components: 5% to 10% of the polypeptide shown in SEQ ID NO: 1, 10% to 20% of the polypeptide shown in SEQ ID NO: 2, 10% to 20% of γ-aminobutyric acid and 5% to 10% of vitamin C.

[0029] In some preferred embodiments of the present invention, the mass ratio of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and γ-aminobutyric acid is 1:2:2.

[0030] In some embodiments of the present invention, the feed additive further comprises excipients.

[0031] In some embodiments of the present invention, the feed additive contains the following components by weight percentage: 5% polypeptide shown in SEQ ID NO: 1, 10% polypeptide shown in SEQ ID NO: 2, 10% γ-aminobutyric acid, 10% vitamin C, and 65% excipients.

[0032] In some embodiments of the present invention, the excipients contain at least one of antioxidants, multivitamins, mineral additives, fermented traditional Chinese medicine, probiotics, yeast culture and zeolite powder.

[0033] The beneficial effects of this invention are:

[0034] This invention provides an antimicrobial peptide, the sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2, a nucleic acid molecule encoding the antimicrobial peptide, and a carrier and cell containing the nucleic acid molecule. The antimicrobial peptide exhibits excellent antimicrobial and bacteriostatic properties. This invention also provides its application in the preparation of antimicrobial, bactericidal, and bacteriostatic products, as well as in product additives. Furthermore, this invention provides a feed additive containing the aforementioned antimicrobial peptide, wherein the antimicrobial peptide can replace the growth-promoting effect of antibiotics, with rapid and stable effects and no residue; it also contains γ-aminobutyric acid and vitamin C, which can relieve reduced feed intake caused by high temperature and humidity, alleviate heatstroke symptoms such as anhidrosis and rapid breathing; it has anti-stress effects, and has a good regulatory effect on various physiological and pathological changes caused by various stresses (relocation, transportation, immunization, fright, weather changes, feed changes, weaning, tail docking); it reduces and neutralizes toxins and enhances liver detoxification capacity. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Example 1: Preparation of antimicrobial peptide mutants

[0037] Antimicrobial peptides with a purity of 95% were synthesized using a 96-well peptide synthesizer by Hangzhou Dangang Biotechnology Co., Ltd. Antimicrobial peptide OdM1 and its mutant OdM1-T, and antimicrobial peptide Myxinidin and its mutant Myxinidin-T were synthesized. The amino acid sequence of antimicrobial peptide OdM1 is shown in SEQ ID NO: 3: ATAWDFGPHGLLPIRPIRIRPLCGKDKS; the amino acid sequence of mutant OdM1-T is shown in SEQ ID NO: 1: ATAWAFGPHGLLPIRPIRIRPACGKDKS. The amino acid sequence of antimicrobial peptide Myxinidin is shown in SEQ ID NO: 4: GIHDILKYGKPS; the amino acid sequence of mutant Myxinidin-T is shown in SEQ ID NO: 2: GIHDILKYGKAR.

[0038] Example 2 Antibacterial performance test

[0039] Using Salmonella CMCC50071, Escherichia coli CICC10899 and Staphylococcus aureus ATCC22023 as indicator bacteria, the minimum inhibitory concentration (MIC) of the antimicrobial peptide OdM1 before and after mutation in Example 1 against the above indicator bacteria was detected.

[0040] The test results are shown in Table 1.

[0041] Table 1 Comparison of Antimicrobial Properties of Antimicrobial Peptides

[0042]

[0043] As can be seen from Table 1, the antimicrobial properties of the antimicrobial peptide mutants OdM1-T and Myxinidin-T were significantly improved compared to the original antimicrobial peptides OdM1 and Myxinidin.

[0044] Example 3

[0045] This embodiment provides a feed additive containing the following components by weight percentage: 5% antimicrobial peptide OdM1-T, 10% antimicrobial peptide Myxinidin-T, 10% γ-aminobutyric acid, 10% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0046] Table 2 Formulations of Examples 3-8 and Comparative Examples 1-5

[0047]

[0048]

[0049] The effective content of γ-aminobutyric acid is 98%.

[0050] The formula for the excipients is: 50% yeast culture, 30% fermented traditional Chinese medicine, 10% compound vitamins, 5% mineral element additives, and 5% zeolite powder. All percentages are by mass.

[0051] The preparation method of the premixed feed additive (hereinafter referred to as premix) in the table above is to thoroughly mix all raw materials in a mixer.

[0052] Example 4

[0053] This embodiment provides a feed additive containing the following components by weight percentage: 10% antimicrobial peptide OdM1-T, 5% antimicrobial peptide Myxinidin-T, 10% γ-aminobutyric acid, 10% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0054] Everything else is the same as in Example 3.

[0055] Example 5

[0056] This embodiment provides a feed additive containing the following components by weight percentage: 10% antimicrobial peptide OdM1-T, 10% antimicrobial peptide Myxinidin-T, 5% γ-aminobutyric acid, 10% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0057] Everything else is the same as in Example 3.

[0058] Example 6

[0059] This embodiment provides a feed additive containing the following components by weight percentage: 20% antimicrobial peptide OdM1-T, 5% antimicrobial peptide Myxinidin-T, 5% γ-aminobutyric acid, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0060] Everything else is the same as in Example 3.

[0061] Example 7

[0062] This embodiment provides a feed additive containing the following components by weight percentage: 5% antimicrobial peptide OdM1-T, 20% antimicrobial peptide Myxinidin-T, 5% γ-aminobutyric acid, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0063] Everything else is the same as in Example 3.

[0064] Comparative Example 1

[0065] This comparative example provides a feed additive containing the following components by weight percentage: 25% antimicrobial peptide OdM1-T, 5% γ-aminobutyric acid, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0066] Everything else is the same as in Example 3.

[0067] Comparative Example 2

[0068] This comparative example provides a feed additive containing the following components by weight percentage: 25% antimicrobial peptide Myxinidin-T, 5% γ-aminobutyric acid, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0069] Everything else is the same as in Example 3.

[0070] Comparative Example 3

[0071] This comparative example provides a feed additive containing the following components by weight percentage: 30% γ-aminobutyric acid, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0072] Everything else is the same as in Example 3.

[0073] Comparative Example 4

[0074] This comparative example provides a feed additive containing the following components by weight percentage: 30% antimicrobial peptide OdM1, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0075] Everything else is the same as in Example 3.

[0076] Comparative Example 5

[0077] This comparative example provides a feed additive containing the following components by weight percentage: 30% antimicrobial peptide Myxinidin, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0078] Everything else is the same as in Example 3.

[0079] Comparative Example 6

[0080] This comparative example provides a feed additive containing the following components by weight percentage: 1% antimicrobial peptide OdM1-T, 1% γ-antimicrobial peptide Myxinidin, 5% vitamin C, and 65% excipients. Details are shown in Table 2. All percentages in the table represent mass percentages.

[0081] Everything else is the same as in Example 3.

[0082] Effect Example: Application Experiment in Livestock Farm

[0083] Experimental Methods: A single-factor experimental design was used. 210 healthy Duroc × Landrace × Large White crossbred weaned piglets at 28 days of age with an average weight of (7.28±1.01) kg were randomly divided into 7 groups based on similar weight and an equal number of males and females, with 5 replicates per group and 6 piglets per replicate. The control group was fed a basal diet. The experimental groups had the same basal diet composition and nutrient levels except for the premix. The experimental groups were named according to the premix group to which they received supplemental feed. The dietary structure of the control and experimental groups is shown in Table 3. The basal diet was formulated according to the NRC (2012) feeding standards, and its composition and nutrient levels are shown in Table 4.

[0084] Table 3 Dietary Structure

[0085]

[0086]

[0087] Table 4. Composition and Nutritional Levels of Basal Diets (Air-Dried Basis)

[0088] raw material content(%) Nutritional components content(%) corn 70 Digestible energy (MJ / kg) 13.95 Puffed full-fat soybeans 5 crude protein 17.9 soybean meal 12 Crude fat 4.33 Fermented soybean meal 5.5 calcium 0.74 fish meal 4 Total phosphorus 0.66 50% choline chloride 0.11 Available phosphorus 0.43 salt 0.4 Lysine 1.29 stone powder 0.84 threonine 0.73 Calcium bicarbonate 0.64 Tryptophan 0.2 Lysine 0.45 threonine 0.05 Tryptophan 0.01 <![CDATA[1% Premix ① > 1 total 100

[0089] Note: The premix provides the following per kilogram of diet: Vitamin A 10000 IU; Vitamin D3 3500 IU; Vitamin E 40 mg; Vitamin K3 3.0 mg; Vitamin B1 1.5 mg; Vitamin B2 8.0 mg; Vitamin B6 4.0 mg; Vitamin B1 4.0 mg; Vitamin B2 ... 12 0.025mg; Folic acid 1.2mg; Biotin 0.15mg; Niacin 50mg; Pantothenic acid 20mg; Copper 50mg; Iron 135mg; Zinc 140mg; Manganese 50mg; Selenium 0.36mg. Nutritional values ​​are calculated.

[0090] During the trial, deworming was carried out uniformly according to the pig farm's regulations. Pigs were fed three times a day, ensuring a small amount of feed remained in the troughs after each feeding. All piglets had free access to food and water. The pigsties were kept clean and well-ventilated, and were regularly cleaned and disinfected. The piglets were observed daily, and their feed intake, mental state, and presence of diarrhea were recorded. The trial period was 30 days.

[0091] Growth performance and diarrhea rate statistics: At the beginning and end of the experiment, piglets were weighed on an empty stomach before morning feeding. Daily feed consumption and diarrhea status were recorded. Feed consumption was calculated on a replicate basis. The average daily weight gain, average daily feed intake, feed conversion ratio, and diarrhea rate were calculated for each group of piglets. Diarrhea rate (%) = Number of diarrhea episodes during the entire experiment / (Number of pigs × Number of days) × 100%.

[0092] Experimental results: The results are shown in Table 5.

[0093] Table 5. Effects of different treatment groups on growth performance and diarrhea rate in weaned piglets.

[0094] Group Average daily weight gain (g / d) Average daily feed intake (g / d) Material weight ratio Diarrhea rate / % control group 263.89±18.52 453.21±25.2 1.72±0.05 15.82±2.36 Example 3 299.41±22.28 481.05±32.96 1.60±0.04 6.29±2.85 Example 4 295.93±12.2 472.56±31.02 1.60±0.08 6.52±1.68 Example 5 289.12±14.34 471.22±15.38 1.63±0.04 6.51±3.18 Example 6 293.25±13.5 468.55±30.25 1.60±0.05 7.58±1.52 Example 7 284.29±14.2 466.9±12.34 1.64±0.07 6.49±1.8 Comparative Example 1 278.93±13.2 463.3±22.65 1.62±0.05 9.68±2.8 Comparative Example 2 277.12±14.32 469.36±21.84 1.63±0.04 8.69±1.19 Comparative Example 3 272.93±30.2 461.84±30.28 1.69±0.05 9.63±1.52 Comparative Example 4 261.21±17.63 456.38±26.3 1.75±0.05 15.88±0.84 Comparative Example 5 265.43±19.67 458.92±29.63 1.73±0.05 13.82±3.58 Comparative Example 6 271.85±13.25 461.21±24.9 1.69±0.05 10.22±3.12

[0095] Table 5 shows that, compared with the control group, the average daily weight gain in Example 3 group increased by 13.46%, which was superior to the other groups. Furthermore, the experimental results of Examples 3 to 7 were similar, with an average daily weight gain of 292.4 g / d, significantly higher than the control group, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6. The experimental results indicate that the combination of the antimicrobial peptide mutants OdM1-T and Myxinidin-T can significantly improve the average daily weight gain, and the effect is superior to that of a single antimicrobial peptide mutant (OdM1-T or Myxinidin-T). The average daily weight gain of the original antimicrobial peptide groups (Comparative Example 4 and Comparative Example 5) was not significantly different from that of the control group, indicating that the antimicrobial peptide mutants have a good effect on improving average daily weight gain.

[0096] Table 5 shows that compared with the control group, the feed conversion ratio (FCR) of Example 3 group was reduced by 7%, which was better than the other groups. Furthermore, the experimental results of Examples 3 to 7 were similar; the average FCR of Examples 3 to 7 was 1.62, significantly lower than the 1.72 of the control group, indicating that the compounded antimicrobial peptide mutant had a good effect on reducing the FCR. The FCR of Examples 3 to 7 was similar to that of Comparative Examples 1 and 2, indicating that the single-component antimicrobial peptide mutant also had a good effect on reducing the FCR. The FCR of Comparative Examples 3, 4, 5, and 6 was close to that of the control group, indicating that low-dose or original antimicrobial peptides could not reduce the FCR. The experimental results show that the compound of antimicrobial peptide mutants OdM1-T and Myxinidin-T can significantly improve the FCR index, and the effect is similar to that of single antimicrobial peptide mutants (OdM1-T or Myxinidin-T). The material weight ratio of the original antimicrobial peptide groups (Comparative Examples 4 and 5) was not significantly different from that of the control group, indicating that the antimicrobial peptide mutant has a good effect on reducing the material weight ratio.

[0097] Table 5 shows that the experimental results of Examples 3 to 7 were similar, with an average diarrhea rate of 6.7%, significantly lower than the control group's diarrhea rate of 15.82%, indicating that the compounded antimicrobial peptide mutant had a better effect on reducing the diarrhea rate. The diarrhea rate of Examples 3 to 7 was better than that of Comparative Examples 1 and 2, indicating that the single-component antimicrobial peptide mutant also had a good effect on reducing the feed conversion ratio, but not as good as the compounded effect. The diarrhea rates of Comparative Examples 3, 4, 5, and 6 were similar to those of the control group, indicating that low-dose or the original antimicrobial peptide could not reduce the diarrhea rate. The experimental results show that the compound of antimicrobial peptide mutants OdM1-T and Myxinidin-T can significantly improve the diarrhea rate, and the effect is better than that of a single antimicrobial peptide mutant (OdM1-T or Myxinidin-T). The diarrhea rate of the original antimicrobial peptide group (Comparative Examples 4 and 5) was not significantly different from that of the control group, indicating that the antimicrobial peptide mutant had a good effect on reducing the diarrhea rate.

[0098] In summary, the premixed feed additives of Examples 3-7 significantly improved the growth indicators of piglets, such as average daily weight gain, average daily feed intake, feed conversion ratio, and diarrhea rate. The mixed feed additives of the present invention under different formulations all significantly improved the growth indicators of piglets, and their effects were significantly higher than those of the formulations in the comparative examples. In particular, when using the premixed feed additive of Example 3, the growth performance of piglets was the best, with an average daily weight gain of 299.41 g / d, an average daily feed intake of 481.05 g / d, a feed conversion ratio of 1.60, and a diarrhea rate of 6.29%.

[0099] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A polypeptide having the amino acid sequence shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A cell comprising the vector of claim 3.

5. The use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 3, or the cell of claim 4 in the following (I) or (II): (I) Preparation of antibacterial agents; (II) Preparation of additives; The bacteria mentioned are Salmonella, Escherichia coli, and Staphylococcus aureus; The additive is a feed additive.

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