Antimicrobial peptide mutants and their applications

Abstract

The present application provides an antimicrobial peptide mutant and its application. The amino acid sequence of the antimicrobial peptide mutant is as described in SEQ ID NO: 1. The nucleic acid sequence encodes the antimicrobial peptide mutant as described above, and the nucleic acid sequence is as described in SEQ ID NO: 2. The antimicrobial peptide mutant provided by the present application can withstand heat up to 120 °C and can tolerate extreme strong acid and strong base environments with pH ranging from 1 to 13 at room temperature. It has stable physiological and biochemical properties, can tolerate hydrolysis by a certain concentration of pepsin and trypsin, and has no hemolytic property. The mutant of the Mccj25 antimicrobial peptide provided by the examples of the present application has good bactericidal activity, can reduce the usage cost of the antimicrobial peptide to a certain extent, reduce the addition amount of the antimicrobial peptide, and has good application prospects.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and particularly to an antibacterial peptide mutant and its application. Background Art

[0002] As a bioactive substance with a unique antibacterial mechanism, antibacterial peptides have gradually emerged. They have broad-spectrum antibacterial activity, can effectively inhibit the growth of various pathogenic bacteria, and are not easily prone to bacterial drug resistance. The antibacterial peptide has diverse action mechanisms. It can not only directly act on the bacterial cell membrane, destroying its structure and function, but also enhance the antibacterial ability of the body by regulating the host immune response. Compared with antibiotics, antibacterial peptides have higher safety and biocompatibility, lower toxicity to normal human cells, and are more suitable for long-term use.

[0003] Screening antibacterial peptide mutant sequences with higher antibacterial activity is of great significance. Summary of the Invention

[0004] In view of this, the purpose of this application is to provide an antibacterial peptide mutant and its application.

[0005] Based on the above purpose, the embodiment of this application provides an antibacterial peptide mutant, and the amino acid sequence of the antibacterial peptide mutant is as described in SEQ ID NO:1.

[0006] The embodiment of this application also provides a nucleic acid sequence, which encodes the antibacterial peptide mutant as described above, and the nucleic acid sequence is as described in SEQ ID NO:2.

[0007] The embodiment of this application also provides a vector, which contains the nucleic acid sequence as described above.

[0008] The embodiment of this application also provides a recombinant bacterium, which contains the vector as described above.

[0009] The embodiment of this application also provides the application of the antibacterial peptide mutant as described above, or the nucleic acid sequence as described above, or the vector as described above, or the recombinant bacterium as described above in antibacterial or preparing antibacterial agents or preparing additives.

[0010] In some of these embodiments, the bacteria include at least one of Escherichia coli and Salmonella.

[0011] In some of these embodiments, the bacterium is Escherichia coli.

[0012] In some of these embodiments, the additives are food additives, feed additives, cosmetic additives or hygiene product additives.

[0013] In some of these embodiments, the additive is a feed additive.

[0014] The embodiments of the present application also provide an antibacterial agent, which contains the antibacterial peptide mutant as described above.

[0015] The embodiments of the present application also provide an additive, which contains the antibacterial peptide mutant as described above.

[0016] The MIC of the Mccj25 antibacterial peptide provided by the present application against ATCC25922 is 0.025 - 0.05 μg / mL, and the bactericidal ability is three to four times that of the wild-type Mccj25 antibacterial peptide. At the same time, the antibacterial peptide mutant provided by the embodiments of the present application can withstand heat up to 120 °C and can tolerate the extreme strong acid and strong base environment with pH 1 to 13 at room temperature. Its physiological and biochemical properties are stable, it can tolerate hydrolysis by pepsin and trypsin at a certain concentration, and it has no hemolytic property. The mutant of the Mccj25 antibacterial peptide provided by the embodiments of the present application has good bactericidal activity, can reduce the usage cost of the antibacterial peptide mutant to a certain extent, reduce the addition amount of the antibacterial peptide mutant, and has good application prospects. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the present application or related technologies, the following will briefly introduce the drawings required for use in the description of the embodiments or related technologies. Obviously, the drawings in the following description are only the embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Schematic diagram of plasmid expression of the mcjA mutant gene in Example 1.

[0019] Figure 2 Colony screening plate diagram of the mutant strain of the antibacterial peptide Mccj25 in Example 1.

[0020] Figure 3 High-performance liquid chromatography detection peak spectrum diagram of the pure product of the mutant of the antibacterial peptide Mccj25 at a wavelength of 280 nm in Example 2.

[0021] Figure 4 High-performance liquid chromatography detection peak spectrum diagram of the fermentation broth of the mutant strain of the antibacterial peptide Mccj25 at a wavelength of 280 nm in Example 2.

[0022] Figure 5 Minimum inhibitory concentration verification diagram of the mutant of the antibacterial peptide Mccj25 in Example 3.

[0023] Figure 6 Inhibitory zone verification diagram of Mccj25 and the mutant of the antibacterial peptide Mccj25 against Escherichia coli 25922 strain in Example 4.

[0024] Figure 7 Verification diagram of the hemolytic activity of Mccj25 and mutants of the antimicrobial peptide Mccj25 in Example 6. Specific implementation manners

[0025] In order to make the objectives, technical solutions and advantages of the present application more clear and understandable, the present application will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0026] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should have the ordinary meanings understood by those of ordinary skill in the art to which the present application belongs.

[0027] In today's medical and biological fields, finding safer and more effective antibacterial means has become an urgent task. Antibiotics, once the "sharp weapons" against bacteria, while making great contributions to human health, have gradually exposed many serious problems. With the extensive use and even abuse of antibiotics, the bacterial resistance has been continuously enhanced, resulting in a sharp reduction in the efficacy of many originally effective antibiotics against pathogens, which has brought great challenges to disease treatment and seriously threatened human health and life safety.

[0028] Mccj25 is a class I antibacterial small peptide composed of 21 amino acids found in Escherichia coli in infant feces. The amino acid sequence is shown in SEQ ID NO:3, the nucleotide sequence is shown in SEQ ID NO:4, and the precursor peptide chain sequence is shown in SEQ ID NO:5. Among them, the amino acid sequence is GGAGHVPEYFVGIGTPISFYG, the nucleotide sequence is ATGATTAAACATTTTCACTTTAACAAACTGAGCAGCGGCAAAAAAAACAATGTGCCGAGCCCGGCCAAAGGCGTGATTCAGATTAAAAAATCAGCCAGCCAGCTGACCAAAGGCGGCGCGGGCCATGTGCCGGAATATTTTGTGGGCATTGGCACCCCGATTAGCTTTTATGGCTAA. The precursor peptide chain sequence is MIKHFHFNKLSSGKKNNVPSPAKGVIQIKKSASQLTKGGAGHVPEYFVGIGTPISFYG. The N-terminal Gly1 to Glu8 of the Mccj25 antimicrobial peptide are covalently linked to form a ring, forming a lasso-like ring structure. This structural property is very stable and can resist strong denaturing conditions, so it has good tolerance to temperature, protease, acid and alkali. It has good killing and inhibitory effects on Escherichia coli, Salmonella and Shigella.

[0029] In Escherichia coli, the production of Mccj25 mainly involves four genes, namely mcjA, mcjB, mcjC, and mcjD, where mcjA is responsible for encoding the precursor of Mccj25. Sufficiently mutating the mcjA gene and screening for antibacterial peptide mutant sequences with higher antibacterial activity can help reduce the usage cost of antibacterial peptide mutants and decrease the addition amount of antibacterial peptide mutants.

[0030] Based on this, the embodiments of the present application provide mutants of the Mccj25 antibacterial peptide and their related applications. By error-prone PCR, the mcjA gene is subjected to saturation mutagenesis, thereby changing the amino acid sequence of its peptide chain. The mutated gene is introduced into a plasmid mediated by a constitutive promoter, and the recombinant plasmid is transformed into the chassis cells of an Escherichia coli engineering strain containing the mcjD gene and the mcjB and mcjC genes, obtaining a series of mutant strains. Then, the obtained mutant strain colonies are spot-inoculated onto a plate containing Escherichia coli ATCC25922 as the indicator bacterium. According to the size of the inhibition zone around the colonies, the mutant with the strongest antibacterial activity is selected, and an Mccj25 antibacterial peptide mutant with the amino acid sequence as set forth in SEQ ID NO:1 is obtained. The MIC of this Mccj25 antibacterial peptide mutant against ATCC25922 is 0.025 - 0.05 μg / mL, and its bactericidal ability is three to four times that of the wild-type Mccj25 antibacterial peptide. Therefore, the mutants of the Mccj25 antibacterial peptide provided by the embodiments of the present application have good bactericidal activity, can reduce the usage cost of antibacterial peptide mutants to a certain extent, and decrease the addition amount of antibacterial peptide mutants. At the same time, they also have good application prospects.

[0031] The embodiments of the present application provide an antibacterial peptide mutant. The amino acid sequence of the antibacterial peptide mutant is as set forth in SEQ ID NO:1, which is GGAGHVPEYFVHAGGPISFYG. This antibacterial peptide mutant is a mutant of the Mccj25 antibacterial peptide. The antibacterial spectrum of the antibacterial peptide mutant is the same as that of the wild-type antibacterial peptide, and it has good killing and inhibitory effects on Escherichia coli and Salmonella. Its MIC against ATCC25922 is 0.06 - 0.12 μg / mL, and its bactericidal ability is three to four times that of the wild-type antibacterial peptide.

[0032] The antibacterial peptide mutant provided by the embodiments of the present application can withstand heat up to 120°C and can tolerate extreme strong acid and strong base environments with pH ranging from 1 to 13 at room temperature.

[0033] The biological properties of the antibacterial peptide mutant provided by the embodiments of the present application are stable and safe. It can tolerate hydrolysis by pepsin and trypsin at a certain concentration and has non-hemolytic properties, laying a foundation for subsequent applications such as being used as a feed additive.

[0034] Based on the same inventive concept, an embodiment of the present application further provides a nucleic acid sequence encoding the antimicrobial peptide mutant as described above. The nucleic acid sequence is GGCGGCGCGGGCCATGTGCCGGAATATTTTGTGCATGCGGGCGGCCCGATTAGCTTTTATGGCTAA, as described in SEQ ID NO:2.

[0035] Based on the same inventive concept, an embodiment of the present application further provides a vector, which contains the nucleic acid sequence as described in the previous embodiment.

[0036] In some embodiments, the vector can be an expression vector containing a constitutive promoter, such as pET-9a. The constitutive promoter is a promoter that can continuously express genes without special conditions, and can include promoters such as lpp and Trc.

[0037] Based on the same inventive concept, an embodiment of the present application further provides a recombinant bacterium, which contains the vector as described in the previous embodiment.

[0038] In some embodiments, the recombinant bacterium can be an Escherichia coli engineering strain chassis cell containing the mcjD gene, mcjB gene, and mcjC gene. The Escherichia coli engineering strain can be any one of Escherichia coli BL21, Escherichia coli MC4100, and Escherichia coli K-12MG1655.

[0039] Based on the same inventive concept, an embodiment of the present application further provides an application of the antimicrobial peptide mutant as described above, or the nucleic acid sequence as described above, or the vector as described above, or the recombinant bacterium as described above in antibacterial or preparing an antibacterial agent or preparing an additive.

[0040] In some embodiments, the application in antibacterial can be applications such as antibacterial, bactericidal, or antimicrobial.

[0041] In some embodiments, the bacterium can include at least one of Escherichia coli and Salmonella.

[0042] In some embodiments, the bacterium can be Escherichia coli.

[0043] In some embodiments, the additive can be a food additive, a feed additive, a cosmetic additive, or a hygiene product additive.

[0044] In some embodiments, the additive can be a feed additive.

[0045] Based on the same inventive concept, an embodiment of the present application further provides an antibacterial agent, which comprises the antibacterial peptide mutant as described above.

[0046] In some embodiments, the bacteria may include at least one of Escherichia coli and Salmonella.

[0047] Based on the same inventive concept, an embodiment of the present application further provides an additive, which comprises the antibacterial peptide mutant as described above.

[0048] In some embodiments, the additive may be a food additive, a feed additive, a cosmetic additive or a hygiene product additive.

[0049] In some embodiments, the additive may be a feed additive.

[0050] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0051] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0052] The test materials used in the following examples are all obtained by purchasing from a conventional biochemical reagent store unless otherwise specified.

[0053] Example 1 Screening of mutants of Mccj25 antibacterial peptide

[0054] Experimental method: According to the gene sequence of the mcjA gene in the NCBI database, the gene sequence fragment was synthesized, and then the gene fragment was ligated to the plasmid pMD 19 by seamless cloning and introduced into Escherichia coli DH5α to obtain a plasmid template containing the mcjA gene.

[0055] Prepare Escherichia coli engineering strain chassis cell chemical transformation competent cells containing the mcjD gene, the mcjB gene and the mcjC gene. The specific preparation process is as follows: (1) Inoculate the Escherichia coli chassis cell strain on the LB solid medium with an inoculation loop and culture it in a constant temperature incubator at 37°C for 12 h. (2) Inoculate a single colony of the Escherichia coli chassis cell strain into 5 mL of LB liquid medium and culture it in a constant temperature shaking incubator at 37°C and 220 r / min for 12 h. (3) Inoculate an appropriate amount of the bacterial culture into 100 mL of LB liquid medium prepared in advance and culture it in a constant temperature shaking incubator at 37°C and 220 r / min for 2 - 3 h until OD 600= 0.2 - 0.3. After taking it out, place it on ice for pre-cooling for 20 min. (4) Transfer the above-mentioned bacterial culture to a pre-cooled sterile centrifuge cup, centrifuge at 7000 r / min at 4°C for 5 min in a centrifuge, and discard the supernatant. (5) Add a small amount of pre-cooled 0.1 moL / L sterile CaCl2 solution to resuspend the bacteria, then continue to add CaCl2 to 30 mL, place it on ice for 20 min, centrifuge at 6000 r / min at 4°C for 5 min, and discard the supernatant. (6) Add 4 mL of pre-cooled 0.1 moL / L sterile CaCl2 to resuspend, add 1 mL of 80% sterile glycerol, mix well, and aliquot 90 μL into pre-cooled sterile EP tubes, and store at -80°C.

[0056] According to the gene sequence of mcjA, using the pMD 19 plasmid containing the mcjA gene as a template, different concentrations of Mn of 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM were selected 2+ , and the mcjA gene fragment was amplified by error-prone PCR. Among them, the amplification reaction system is shown in Table 1. Among them, the nucleotide sequence of primer mcjA-1 is shown in SEQ ID NO:6. The nucleotide sequence of primer mcjA-2 is shown in SEQ ID NO:7. The settings of the amplification reaction program are: pre-denaturation: 95°C for 5 min; denaturation: 95°C for 15 s; annealing: 56°C for 15 s; extension: 72°C for 20 s; react for 30 cycles; extension: 72°C for 10 min. The PCR products were purified and recovered by agarose gel electrophoresis to obtain multiple fragments after mcjA gene mutation.

[0057] Table 1 Error-prone PCR amplification system

[0058]

[0059] By seamless cloning, multiple fragments after mcjA gene mutation were respectively ligated one by one to the plasmid pET-9a containing the constitutive promoter expression element, forming a plasmid expression schematic diagram of the mcjA mutant gene as shown in Figure 1 , and introduced into the chemically competent cells of the chassis cells of the Escherichia coli engineering strain containing the mcjD gene, mcjB gene, and mcjC gene to obtain recombinant strains. After heat shock, they were resuscitated in LB liquid medium for 1 h, and then spread on a kanamycin-resistant plate and cultured at 37°C for 12 h.

[0060] The positive transformants obtained by screening on resistant plates were subjected to an antibacterial circle test. A mutant strain of the antibacterial peptide Mccj25 with the most obvious antibacterial circle was screened out from them, named strain GA-5. Then, colony PCR identification was performed on it using the identification primers JD-1 and primer JD-2. The colony PCR identification system is shown in Table 2. Among them, as shown in Table 3, the nucleotide sequence of primer JD-1 is as shown in SEQ ID NO:8. The nucleotide sequence of primer JD-2 is as shown in SEQ ID NO:9. The amplification reaction program was set as follows: pre-denaturation: 95°C for 5 min; denaturation: 95°C for 15 s; annealing: 56°C for 15 s; extension: 72°C for 20 s; 30 cycles of reaction; extension: 72°C for 10 min. Sequencing was performed after the colony PCR identification result was positive.

[0061] Table 2 Colony PCR Identification System

[0062]

[0063] Table 3 Primer Sequences

[0064]

[0065] Experimental results: As Figure 2 shown. Among them, the strain corresponding to the antibacterial circle in No. 3 is GA-5, that is, the mutant of the Mccj25 antibacterial peptide. The amino acid sequence of this antibacterial peptide mutant is as shown in SEQ ID NO:1, and the nucleic acid sequence encoding this antibacterial peptide mutant is as shown in SEQ ID NO:2. The amino acid sequence is GGAGHVPEYFVHAGGPISFYG. The nucleic acid sequence is GGCGGCGCGGGCCATGTGCCGGAATATTTTGTGCATGCGGGCGGCCCGATTAGCTTTTATGGCTAA.

[0066] Example 2 Detection of Mutants of Antibacterial Peptide Mccj25

[0067] Experimental method: High performance liquid chromatography was used to quantitatively analyze the mutants of Mccj25 in the fermentation broth of the recombinant strain obtained in Example 1. Under the conditions of 33°C and ultraviolet light with a wavelength of 280 nm, the products were separated by gradient elution through a Shim-pack Scepter C18 chromatographic column at a flow rate of 1 mL / min. The operation steps are as follows:

[0068] (1) Take an appropriate amount of fermentation broth with an EP tube, centrifuge at 13000 rpm for 3 minutes, and take the supernatant. Among them, the medium used for fermentation can be LB liquid medium. The culture conditions can be constant temperature culture at 37°C for 12 h.

[0069] (2) Organic mobile phase B: Preparation of 90% acetonitrile solution: Use a dedicated 100 mL graduated cylinder to measure 100 mL of deionized water and pour it into a reagent bottle. Then measure 900 mL of pure acetonitrile and pour it into the reagent bottle for mixing. Filter through a 0.2 μm organic membrane filter and degas by ultrasonic treatment for 15 minutes.

[0070] (3) Inorganic mobile phase A: Preparation of 0.1% trifluoroacetic acid solution: Take 100 μL of trifluoroacetic acid, dissolve it in deionized water, and finally make up the volume to 1000 mL with a volumetric flask. Then filter through a 0.2 μm aqueous membrane filter and degas by ultrasonic treatment for 15 minutes.

[0071] (4) Preparation of standard solution: Weigh 0.1 g of the mutant pure product of antimicrobial peptide Mccj25 into a 100 mL volumetric flask, add pure water to make up the volume to the scale line, shake well, and filter through a 0.22 μm filter membrane.

[0072] After the chromatographic column is balanced, set the following gradient elution program and pipette 1 mL of the standard into an EP tube. Add pure water to sequentially dilute it to 0.8 g / L, 0.4 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, and 0.025 g / L. After filtering through a 0.22 μm filter membrane, place it on the sample rack. Start gradient elution with an injection volume of 10 μL according to the gradient elution program shown in Table 4. Plot the external standard curve with the peak areas at each concentration obtained.

[0073] Take 1 mL of the fermentation supernatant, filter it through a 0.22 μm filter membrane, and then start gradient elution. Record the chromatogram and the peak areas at the corresponding times, and then compare with the external standard curve.

[0074] Table 4 Binary gradient elution program

[0075]

[0076]

[0077] Experimental results: The results are as Figure 3 and Figure 4 shown. Among them, Figure 3 is the high-performance liquid chromatography detection peak spectrum of the mutant pure product of antimicrobial peptide Mccj25 at a wavelength of 280 nm. Figure 4 is the high-performance liquid chromatography detection peak spectrum of the fermentation broth of the mutant strain of antimicrobial peptide Mccj25 at a wavelength of 280 nm.

[0078] Example 3 Determination of the minimum inhibitory concentration of the mutant of antimicrobial peptide Mccj25

[0079] Experimental method: 1) Sample preparation:

[0080] After centrifuging the fermentation broth of the mutant of antimicrobial peptide Mccj25, detect the peptide content by liquid phase. Take the pure product of antimicrobial peptide Mccj25 as the control group.

[0081] 2) MIC experiment:

[0082] Prepare LB liquid medium, dispense and transfer 5 ml into corresponding test tubes respectively, then add the mutant solution of antibacterial peptide Mccj25 / the pure product solution of antibacterial peptide Mccj25 after dilution, and perform gradient dilution. The concentration gradients are 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml and 0.0625 μg / ml in sequence. Cover the test tube plugs and sterilize at 121 °C in a high-pressure steam sterilizer for 20 minutes for standby. After diluting the activated Escherichia coli 25922 bacterial solution in the test tubes, inoculate it into the test tubes in sequence according to an inoculation amount of 1%. Incubate in a constant temperature shaker at 37 °C and 220 rmp / min for 7 hours, observe the samples. If the bacteria grow visibly turbid to the naked eye, it is turbid; if there is no visible growth to the naked eye, it is clear. The lowest concentration of the clear test tube is judged as the minimum inhibitory concentration (MIC value).

[0083] Experimental results: The antibacterial results of MIC are as Figure 5 shown. Among them, Figure 5 the first row in is the antibacterial result of the pure product solution of antibacterial peptide Mccj25. Figure 5 the second row in is the antibacterial result of the mutant solution of antibacterial peptide Mccj25. Figure 5In test tubes corresponding to different serial numbers, the concentrations of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 are as follows: 1: 32 μg / ml, 2: 16 μg / ml, 3: 8 μg / ml, 4: 4 μg / ml, 5: 2 μg / ml, 6: 1 μg / ml, 7: 0.5 μg / ml, 8: 0.25 μg / ml, 9: 0.125 μg / ml, 10: 0.0625 μg / ml. That is, in the test tube corresponding to serial number 1, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 32 μg / ml. In the test tube corresponding to serial number 2, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 16 μg / ml. In the test tube corresponding to serial number 3, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 8 μg / ml. In the test tube corresponding to serial number 4, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 4 μg / ml. In the test tube corresponding to serial number 5, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 2 μg / ml. In the test tube corresponding to serial number 6, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 1 μg / ml. In the test tube corresponding to serial number 7, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 0.5 μg / ml. In the test tube corresponding to serial number 8, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 0.25 μg / ml. In the test tube corresponding to serial number 9, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 0.125 μg / ml. In the test tube corresponding to serial number 10, the concentration of the mutant of antimicrobial peptide Mccj25 / pure antimicrobial peptide Mccj25 is 0.0625 μg / ml.

[0084] Result analysis: As can be seen from Figure 5 the first line in, the minimum inhibitory concentration MIC of pure antimicrobial peptide Mccj25 is 0.25 - 0.5 μg / ml. As can be seen from Figure 5 the second line in, the minimum inhibitory concentration of the mutant of antimicrobial peptide Mccj25 is 0.0625 - 0.125 μg / ml. Therefore, the bactericidal ability of the mutant of antimicrobial peptide Mccj25 in the embodiments of the present application is three to four times that of antimicrobial peptide Mccj25.

[0085] Example 4 Determination of the antibacterial activity of the mutant of antimicrobial peptide Mccj25

[0086] Experimental method: Escherichia coli ATCC 25922 and Salmonella enterica were respectively inoculated on nutrient plates and cultured at 35 - 37°C for 16 - 24 h. Then, single colonies on the cultured nutrient plates were inoculated into 50 ml of sterile LB liquid medium and cultured in an incubator at 37°C with 220 rmp for 18 h. Dilute to an OD 600 of about 0.1 for standby.

[0087] Dilute the mutants of antimicrobial peptide Mccj25 into working solutions with different concentrations, and use distilled water as a blank control. Use a pipette to aspirate 0.2 ml of the above-activated Escherichia coli ATCC 25922 and Salmonella enterica bacterial solutions, transfer them respectively into the already poured nutrient agar culture dishes, spread them evenly with a spreading rod, and cover the culture dishes. Use a sterilized puncher to punch holes in the culture dishes, with a distance of more than 25 mm between the centers of each hole. Use a micropipette to aspirate the mutant solution of antimicrobial peptide Mccj25 into the agar holes, cover the culture dishes, and incubate upright. After culturing in a 37°C incubator for 16 - 20 h, observe the size of the inhibition zone.

[0088] Experimental results: The inhibition zone results of the mutants of antimicrobial peptide Mccj25 and the blank control are as Figure 6 shown. Among them, Figure 6 part (a) is the inhibition zone result of Escherichia coli ATCC 25922. Figure 6 part (b) is the inhibition zone result of Salmonella enterica.

[0089] Result analysis: As can be seen from Figure 6 , the mutants of antimicrobial peptide Mccj25 have relatively obvious inhibition zones in Escherichia coli ATCC 25922, Salmonella enterica, and the plates, indicating that the mutants have strong inhibitory effects on bacteria such as Escherichia coli and Salmonella.

[0090] Example 5 Physicochemical property test of mutants of antimicrobial peptide Mccj25

[0091] 1) Thermal stability experiment of mutants of antimicrobial peptide Mccj25

[0092] Experimental method: Prepare working solutions with a concentration of 100 mg / L for the mutants of antimicrobial peptide Mccj25 and the pure product of the Mccj25 control group. Set up EP tubes with temperature gradients of 40, 60, 80, and 100 °C. Take 1 mL of each solution and place it in an EP tube, then place it in a constant temperature water bath for 30 min. In addition, take 1 mL of the working solutions of the mutants of antimicrobial peptide Mccj25 and the Mccj25 control group and place them in test tubes, and treat them at 121 °C in a high-temperature sterilizer for 30 min. The above samples are all set up in three parallels. After the above experimental group treatments are completed, place them in cold water to return to room temperature, and use high-performance liquid chromatography to measure the content changes in each tube.

[0093] Experimental results: The detection results are shown in Table 5.

[0094] Table 5 Thermal stability test of mutants of antimicrobial peptide Mccj25

[0095]

[0096] Result analysis: As shown in Table 5, the content of the mutants of antimicrobial peptide Mccj25 is basically unchanged under different temperature gradient treatment conditions of 40 °C, 60 °C, 80 °C, 100 °C, and 120 °C. This indicates that the mutants of antimicrobial peptide Mccj25 have strong thermal stability.

[0097] 2) Acid and alkali tolerance experiment of mutants of antimicrobial peptide Mccj25

[0098] Experimental method: Prepare working solutions with a concentration of 100 mg / L and pH = 7.0 for the mutants of antimicrobial peptide Mccj25 and the pure product of the Mccj25 control group. Set up test tube experimental groups with different acid-base conditions of pH = 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0. Take 10 mL of the above working solutions and place them in the corresponding test tubes, and adjust the pH to the corresponding values respectively. The above samples are all set up in three parallels, and then treat them at 37 °C for 6 h, and use high-performance liquid chromatography to measure the content changes in each tube.

[0099] Experimental results: The detection results are shown in Table 6.

[0100] Table 6 Acid-base stability test of mutants of antimicrobial peptide Mccj25

[0101]

[0102]

[0103] Result analysis: As shown in Table 6, the concentration of the mutants of antimicrobial peptide Mccj25 is basically unchanged under different acid-base range conditions of pH = 1.0 - 13.0. Therefore, the mutants of antimicrobial peptide Mccj25 have good ability to tolerate strong acid and strong alkali environments.

[0104] Biological Property Stability Test of Mutants of Antimicrobial Peptide Mccj25

[0105] 1) Protease Resistance Experiment of Mutants of Antimicrobial Peptide Mccj25

[0106] After the bioactive peptide enters the body, it needs to reach the intestine with an intact structure to retain its biological activity. Therefore, after oral administration, the bioactive peptide needs to resist the digestion and hydrolysis of enzymes such as pepsin and trypsin in the gastrointestinal tract. If the bioactive peptide is degraded during this process, its biological activity will be reduced or even inactivated. Therefore, the protease resistance performance of bioactive peptides is of great significance. Therefore, two key enzymes (pepsin and trypsin) in the gastrointestinal tract were selected to perform in vitro simulated digestion on mutants of antimicrobial peptide Mccj25.

[0107] Experimental method: Mutants of antimicrobial peptide Mccj25 were diluted to 100 mg / L with an aqueous solution of pH = 2, pepsin (1:3000) was added, and hydrolysis was carried out in a water bath at 37 °C for 60 min. Samples were taken every 30 min and stored in a refrigerator at 4 °C for later use. After sample treatment, the pH was adjusted to 7, trypsin (1:250) was added, and hydrolysis was carried out in a water bath at 37 °C for 60 min. Samples were taken every 30 min. The mutant sample solutions of antimicrobial peptide Mccj25 in the blank group and each experimental group were filtered through a filter membrane with a pore size of 0.22 μm, and then analyzed by high-performance liquid chromatography. Then, the content of mutants of antimicrobial peptide Mccj25 after treatment with pepsin and trypsin was detected by high-performance liquid chromatography.

[0108] Experimental results: The detection results are shown in Table 7.

[0109] Table 7 Protease Resistance Test of Mutants of Antimicrobial Peptide Mccj25

[0110]

[0111]

[0112] Result analysis: From the protease treatment analysis results in Table 7, it can be seen that the content of mutants of antimicrobial peptide Mccj25 can still remain above 90% after 30 - 60 min of digestion by pepsin and trypsin. Therefore, mutants of antimicrobial peptide Mccj25 have good digestive stability in the gastrointestinal tract, can maintain the integrity of their sequences in the stomach, and have good application prospects.

[0113] 2) Hemolytic Experiment of Mutants of Antimicrobial Peptide Mccj25

[0114] Experimental method: First, prepare Columbia agar blood agar medium, and the preparation process is as follows. Weigh 4.1 g of dry powder of Columbia agar medium into an Erlenmeyer flask, add pure water to make the volume up to 100 mL, heat and continuously stir until the medium is completely dissolved, sterilize at 121 °C under high pressure for 20 minutes. When the medium cools to about 50 °C, add 5% sterile defibrinated sheep blood and mix well. Pour the mixture into a sterile petri dish, and after it cools and solidifies, place it in a refrigerator at 4 °C.

[0115] Use a sterilized punch to punch holes in the Columbia blood agar culture dish, with a distance of more than 25 mm between the centers of each hole. Use a micropipette to suck the lysophospholipid control group, physiological saline, and 1 g / L fermentation broth of the antimicrobial peptide Mccj25 mutant into the agar holes, cover the culture dish, and after standing in a 37 °C incubator for 12 hours, observe the size of the hemolytic zone.

[0116] Experimental results: The hemolysis situation of the culture dish is as Figure 7 shown.

[0117] Result analysis; As Figure 7 can be seen, no hemolysis was observed in the mutant of the antimicrobial peptide Mccj25, while the positive control lysophospholipid had a hemolytic zone. Therefore, the mutant of the antimicrobial peptide Mccj25 has stable biological properties.

[0118] Those of ordinary skill in the art should understand that: The discussion of any of the above embodiments is only exemplary, and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples; Under the concept of the present disclosure, the technical features between the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the embodiments of the present disclosure as described above, and they are not provided in detail for the sake of brevity.

[0119] Although the present disclosure has been described in connection with specific embodiments of the present disclosure, many substitutions, modifications, and variations of these embodiments will be apparent to those of ordinary skill in the art based on the foregoing description.

[0120] The embodiments of the present disclosure are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. An antimicrobial peptide mutant, characterized in that: The amino acid sequence of the antimicrobial peptide mutant is as shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that The polynucleotide encodes the antimicrobial peptide mutant according to claim 1, and the sequence of the polynucleotide is as shown in SEQ ID NO:

2.

3. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacterium comprises the vector as claimed in claim 3.

5. Use of the antimicrobial peptide mutant according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, or the recombinant bacterium according to claim 4 in the preparation of an antibacterial agent or an additive.

6. The use according to claim 5, characterized in that: The bacteria include at least one of Escherichia coli and Salmonella; the additive is a food additive, a feed additive, a cosmetic additive or a sanitary product additive.

7. The use according to claim 6, characterized in that: The bacteria is Escherichia coli.

8. The use according to claim 6, characterized in that: The additive is a feed additive.

9. An antibacterial agent, characterized in that Comprising the antimicrobial peptide mutant as claimed in claim 1.

10. An additive, characterized in that Comprising the antimicrobial peptide mutant as claimed in claim 1.

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