Antibacterial peptide mutant and application thereof

Abstract

The invention provides an antibacterial peptide mutant and application thereof. The amino acid sequence of the antibacterial peptide mutant is as shown in SEQ ID NO: 1. The nucleotide sequence codes the antibacterial peptide mutant, and the nucleotide sequence is as shown in SEQ ID NO: 2. The antibacterial peptide mutant provided by the invention can resist heat up to 120 DEG C, and can resist extreme strong acid and strong alkali environments with pH of 1-13 at normal temperature. The protein has stable physiological and biochemical properties, can tolerate hydrolysis of pepsin and trypsin with certain concentration, and has no hemolytic activity. The mutant of the Mccj25 antibacterial peptide provided by the embodiment of the invention has good bactericidal activity, can reduce the use cost of the antibacterial peptide to a certain extent, reduces the addition amount of the antibacterial peptide, and has a good application prospect.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to an antimicrobial peptide mutant and its application. Background Art

[0002] Antimicrobial peptides, as a bioactive substance with a unique antimicrobial mechanism, are gradually emerging. They have broad-spectrum antimicrobial activity, can effectively inhibit the growth of a variety of pathogens, and are not prone to causing bacterial resistance. Antimicrobial peptides have diverse mechanisms of action. They can not only directly act on bacterial cell membranes to destroy their structure and function, but also enhance the body's antimicrobial ability by regulating the host's immune response. Compared with antibiotics, antimicrobial peptides have higher safety and biocompatibility, lower toxicity to normal human cells, and are more suitable for long-term use.

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

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

[0005] Based on the above purpose, the present invention provides an antimicrobial peptide mutant, the amino acid sequence of the antimicrobial peptide mutant is as described in SEQ ID NO:1.

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

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

[0008] The present application also provides a recombinant bacterium, wherein the recombinant bacterium comprises the vector as described above.

[0009] The embodiments of the present application also provide a use 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 bacteria as described above in inhibiting bacteria, preparing an antibacterial agent, or preparing an additive.

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

[0011] In some embodiments, the bacteria is Escherichia coli.

[0012] In some embodiments, the additive is a food additive, a feed additive, a cosmetic additive or a hygiene product additive.

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

[0014] The present application also provides an antibacterial agent, comprising the antimicrobial peptide mutant as described above.

[0015] The present application also provides an additive comprising the antimicrobial peptide mutant as described above.

[0016] The MIC of the Mccj25 antimicrobial peptide provided in 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 antimicrobial peptide. At the same time, the antimicrobial peptide mutant provided in the embodiment of the present application can be heat-resistant up to 120°C, and can withstand extreme strong acid and strong alkaline environments of pH 1 to 13 at room temperature. It has stable physiological and biochemical properties, can withstand hydrolysis by a certain concentration of pepsin and trypsin, and is non-hemolytic. The mutant of the Mccj25 antimicrobial peptide provided in the embodiment of the present application has good bactericidal activity, can reduce the cost of using the antimicrobial peptide mutant to a certain extent, and reduce the amount of antimicrobial peptide mutant added, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

[0019] Figure 2 This is a plate map of the mutant strain colony screening of the antimicrobial peptide Mccj25 in Example 1.

[0020] Figure 3 This is the peak spectrum of the pure mutant of the antimicrobial peptide Mccj25 in Example 2 detected by high performance liquid chromatography at a wavelength of 280 nm.

[0021] Figure 4 This is the peak spectrum of the mutant strain fermentation broth of the antimicrobial peptide Mccj25 in Example 2 detected by high performance liquid chromatography at a wavelength of 280 nm.

[0022] Figure 5 This is a graph for verifying the minimum inhibitory concentration of the mutant of the antimicrobial peptide Mccj25 in Example 3.

[0023] Figure 6 This is a verification diagram of the inhibition zone of Mccj25 and the mutant of the antimicrobial peptide Mccj25 against Escherichia coli 25922 strain in Example 4.

[0024] Figure 7 This is a graph verifying the hemolytic activity of Mccj25 and the mutants of the antimicrobial peptide Mccj25 in Example 6. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to 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 common meanings understood by persons with ordinary skills in the field to which the present application belongs.

[0027] In today's medical and biological fields, finding safer and more effective antibacterial methods has become an urgent task. Antibiotics, once a powerful antibacterial weapon, have made great contributions to human health, but have also gradually exposed many serious problems. With the widespread use and even abuse of antibiotics, bacterial resistance has continued to increase, resulting in a sharp decrease in the effectiveness of many originally effective antibiotics against pathogens, which has brought huge challenges to disease treatment and seriously threatened human health and life safety.

[0028] Mccj25 is a bacteriocin class I antibacterial 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 sequence is shown in SEQ ID NO:5. Among them, the amino acid sequence is GGAGHVPEYFVGIGTPISFYG, the nucleotide sequence is ATGATTAAACATTTTCACTTTAACAAACTGAGCAGCGGCAAAAAAAACAATGTGCCGAGCCCGGCCAAAGGCGTGATTCAGATTAAAAAATCAGCCAGCCAGCTGACCAAAGGCGGCGCGGGCCATGTGCCGGAATATTTTGTGGGCATTGGCACCCCGATTAGCTTTTATGGCTAA. The precursor peptide sequence is MIKHFHFNKLSSGKKNNVPSPAKGVIQIKKSASQLTKGGAGHVPEYFVGIG TPISFYG. The N-terminal Gly1~Glu8 of Mccj25 antimicrobial peptide are connected by covalent bonds to form a ring, forming a lasso ring structure. This structure is very stable and can withstand strong denaturation conditions, so it has good tolerance to temperature, protease, acid and alkali, etc. It has good killing and inhibitory effects on Escherichia coli, Salmonella and Shigella.

[0029] In E. coli, the production of Mccj25 mainly involves four genes, namely mcjA, mcjB, mcjC and mcjD, of which mcjA is responsible for encoding the precursor of Mccj25. Fully mutating the mcjA gene and screening antimicrobial peptide mutant sequences with higher antimicrobial activity will help reduce the cost of using antimicrobial peptide mutants and reduce the amount of antimicrobial peptide mutants added.

[0030] Based on this, the embodiment of the present application provides a mutant of the Mccj25 antimicrobial peptide and its related applications. The mcjA gene is saturated mutated by error-prone PCR to change the amino acid sequence of its peptide chain, and the mutated gene is introduced into a plasmid mediated by a constitutive promoter. The recombinant plasmid is transformed into the chassis cells of the Escherichia coli engineered strain containing the mcjD gene and the mcjB and mcjC genes to obtain a series of mutant strains. Then, the obtained mutant strain colonies are inoculated on a plate containing the indicator bacteria of Escherichia coli ATCC25922, and the mutant with the strongest antibacterial activity is selected according to the size of the inhibition zone around the colony, and the Mccj25 antimicrobial peptide mutant having the amino acid sequence described in SEQ ID NO: 1 is obtained. The MIC of the mutant of the Mccj25 antimicrobial peptide against ATCC25922 is 0.025-0.05 μg / mL, and the bactericidal ability is three to four times that of the wild-type Mccj25 antimicrobial peptide. Therefore, the mutant of Mccj25 antimicrobial peptide provided in the embodiment of the present application has good bactericidal activity, can reduce the cost of using the antimicrobial peptide mutant to a certain extent, and reduce the amount of antimicrobial peptide mutant added. At the same time, it also has good application prospects.

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

[0032] The antimicrobial peptide mutant provided in the examples of the present application is heat-resistant up to 120° C. and can withstand extremely strong acid and alkaline environments with a pH of 1 to 13 at room temperature.

[0033] The antimicrobial peptide mutant provided in the examples of the present application has stable and safe biological properties, can withstand hydrolysis by a certain concentration of pepsin and trypsin, and is insoluble in blood, thus laying a foundation for subsequent applications as a feed additive, etc.

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

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

[0036] In some embodiments, the vector may be an expression vector containing a constitutive promoter, such as pET-9a. The constitutive promoter is a promoter that allows the gene to be continuously expressed without special conditions, such as lpp, Trc and other promoters.

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

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

[0039] Based on the same inventive concept, the embodiments of the present application also provide 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 bacteria as described above in inhibiting bacteria, preparing an antibacterial agent, or preparing an additive.

[0040] In some embodiments, the application in antibacterial can be an application of antibacterial, bactericidal or antibacterial.

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

[0042] In some embodiments, the bacteria may be Escherichia coli.

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

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

[0045] Based on the same inventive concept, an embodiment of the present application also provides an antibacterial agent, comprising the antimicrobial 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 comprising the antimicrobial 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 is further described below in conjunction with specific implementation methods.

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

[0052] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

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

[0054] Experimental method: According to the gene sequence of mcjA gene in NCBI database, the gene sequence fragment was synthesized, and then the gene fragment was connected to plasmid pMD19 by seamless cloning and introduced into Escherichia coli DH5α to obtain a plasmid template containing mcjA gene.

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

[0056] According to the gene sequence of mcjA, the pMD 19 plasmid containing the mcjA gene was used as a template, and different concentrations of Mn were selected, including 0.05mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, and 0.5mM. 2+ , the mcjA gene fragment was amplified by error-prone PCR. The amplification reaction system is shown in Table 1. 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 amplification reaction program was set as follows: pre-denaturation: 95℃5min; denaturation: 95℃15s; annealing: 56℃15s; extension: 72℃20s; reaction 30 cycles; extension: 72℃, 10min. The PCR products were purified and recovered by agarose gel electrophoresis to obtain multiple mcjA gene mutation fragments.

[0057] Table 1 Error-prone PCR amplification system

[0058]

[0059] Through seamless cloning, multiple fragments of the mcjA gene after mutation were connected one by one to the plasmid pET-9a containing the constitutive promoter expression element to form Figure 1 The plasmid expression schematic diagram of the mcjA mutant gene is shown, and it is introduced into the chemical transformation competent cells of the Escherichia coli engineering strain chassis cells containing the mcjD gene, mcjB gene and mcjC gene to obtain the recombinant strain, which is revived in LB liquid culture medium for 1 hour after heat shock, and then spread on kanamycin resistance plates and cultured at 37°C for 12 hours.

[0060] The positive transformants obtained by resistance plate screening were subjected to an inhibition zone test. A mutant strain of the antimicrobial peptide Mccj25 with the most obvious inhibition zone was screened out and named as strain GA-5. Then, the identification primers JD-1 and JD-2 were used for colony PCR identification. 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 shown in SEQ ID NO:8. The nucleotide sequence of primer JD-2 is shown in SEQ ID NO:9. The amplification reaction program was set as follows: pre-denaturation: 95℃5min; denaturation: 95℃15s; annealing: 56℃15s; extension: 72℃20s; reaction 30 cycles; extension: 72℃, 10min. 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: Figure 2 As shown. Among them, the strain corresponding to the inhibition zone in sequence number 3 is GA-5, which is a mutant of the Mccj25 antimicrobial peptide. The amino acid sequence of the antimicrobial peptide mutant is as described in SEQ ID NO:1, and the nucleic acid sequence encoding the antimicrobial peptide mutant is as described in SEQ ID NO:2. The amino acid sequence is GGAGHVPEYFVHAGGPISFYG. The nucleic acid sequence is GGCGGCGCGGGCCATGTGCCGGAATATTTTGTGCATGCGGGCGGCCCG ATTAGCTTTTATGGCTAA.

[0066] Example 2 Detection of mutants of antimicrobial 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. The products were separated by gradient elution at a flow rate of 1 mL / min using a Shim-pack Scepter C18 column at 33°C and 280 nm UV light. The operation steps were as follows:

[0068] (1) Take an appropriate amount of fermentation liquid using an EP tube and centrifuge at 13000 rpm for 3 minutes to obtain the supernatant. The culture medium used for fermentation may be LB liquid culture medium. The culture conditions may be constant temperature culture at 37°C for 12 hours.

[0069] (2) Organic mobile phase B: 90% acetonitrile solution preparation: Use a dedicated 100mL measuring cylinder to measure 100mL of deionized water and pour it into a reagent bottle. Then measure 900mL of pure acetonitrile and pour it into the reagent bottle to mix. Pass through a 0.2μm organic membrane and ultrasonically degas 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 use a volumetric flask to make up to 1000 mL. Then pass it through a 0.2 μm water system membrane and ultrasonically degas for 15 min.

[0071] (4) Preparation of standard solution: Weigh 0.1 g of pure mutant of antimicrobial peptide Mccj25 into a 100 mL volumetric flask, add purified water to the mark, shake and mix, and filter through a 0.22 μm filter membrane.

[0072] After the chromatographic column is balanced, set the following gradient elution program and draw 1 mL of the standard into the EP tube, add pure water and dilute it to 0.8 g / L, 0.4 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, 0.025 g / L in sequence, filter it through a 0.22 μm filter membrane and place it in the sample rack. The injection volume is 10 μL and the gradient elution program shown in Table 4 is started for gradient elution. The peak area at each concentration is obtained to draw the surface curve.

[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 area at the corresponding time, and then compare it with the surface curve.

[0074] Table 4 Binary gradient elution program

[0075]

[0076]

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

[0078] Example 3 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptide Mccj25 Mutants

[0079] Experimental method: 1) Sample preparation:

[0080] The fermentation liquid of the mutant of antimicrobial peptide Mccj25 was centrifuged and the peptide content was detected by liquid phase. The pure antimicrobial peptide Mccj25 was used as the control group.

[0081] 2) MIC experiment:

[0082] Prepare LB liquid culture medium, take 5 ml of it and add it to the corresponding test tubes, then add the diluted mutant solution of antimicrobial peptide Mccj25 / antimicrobial peptide Mccj25 pure solution, gradient dilution, the concentration gradient is 32ug / ml, 16ug / ml, 8ug / ml, 4ug / ml, 2ug / ml, 1ug / ml, 0.5ug / ml, 0.25ug / ml, 0.125ug / ml and 0.0625ug / ml. Cover the test tube stopper and sterilize it in a high pressure steam sterilizer at 121℃ for 20 minutes for use. After diluting the activated Escherichia coli 25922 bacterial solution in the test tube, inoculate it in the test tube in turn according to the inoculation amount of 1%. Cultivate it in a constant temperature shaker at 37℃, 220rmp / min for 7 hours, observe the sample, the bacterial growth is turbid when the naked eye can see it, and the clear test tube with the lowest concentration is judged as the minimum inhibitory concentration (MIC value).

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

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

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

[0086] Experimental method: Escherichia coli ATCC 25922 and Salmonella enterica were inoculated on nutrient plates and cultured at 35-37°C for 16-24 hours. Then, a single colony on the cultured nutrient plate was inoculated into 50 ml of sterile LB liquid culture medium and cultured in an incubator at 37°C and 220 rpm for 18 hours. Dilute to OD 600 = around 0.1 for standby use.

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

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

[0089] Result analysis: Figure 6 It can be seen that the mutant of the antimicrobial peptide Mccj25 has obvious inhibition zones in Escherichia coli ATCC 25922 and Salmonella enterica plates, indicating that the mutant has a strong inhibitory effect on Escherichia coli, Salmonella and other bacteria.

[0090] Example 5 Physical and chemical properties test of mutants of antimicrobial peptide Mccj25

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

[0092] Experimental method: The mutant of antimicrobial peptide Mccj25 and the pure product of Mccj25 control group were prepared into a working solution with a concentration of 100 mg / L, and EP tubes with a temperature gradient of 40, 60, 80, and 100°C were set up, and 1 mL was taken from each EP tube, and then placed in a constant temperature water bath for 30 minutes. In addition, 1 mL of the working solution of the mutant of antimicrobial peptide Mccj25 and the Mccj25 control group was taken from each test tube, and placed in a high temperature sterilizer at 121°C for 30 minutes. The above samples were set up in three parallel groups. After the above experimental groups were treated, they were placed in cold water to return to room temperature, and the changes in the content of each tube were determined by high performance liquid chromatography.

[0093] Experimental results: The test 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 mutant of the antimicrobial peptide Mccj25 remained basically unchanged under different temperature gradient treatment conditions of 40°C, 60°C, 80°C, 100°C and 120°C, indicating that the mutant of the antimicrobial peptide Mccj25 has strong thermal stability.

[0097] 2) Acid and alkali resistance experiment of antimicrobial peptide Mccj25 mutant

[0098] Experimental method: The mutant of antimicrobial peptide Mccj25 and the pure product of Mccj25 control group were prepared into working solution with concentration of 100 mg / L and pH=7.0. 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 were set up. 10 mL of the above working solution was taken in each corresponding test tube, and the pH was adjusted to the corresponding value. Three groups of parallel samples were set up, and then treated at 37°C for 6 hours. The changes in the content of each tube were determined by high performance liquid chromatography.

[0099] Experimental results: The test 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 mutant of the antimicrobial peptide Mccj25 remained basically unchanged under different acid-base conditions of pH=1.0-13.0. Therefore, the mutant of the antimicrobial peptide Mccj25 has good ability to resist strong acid and strong base environments.

[0104] Example 6 Biological property stability test of antimicrobial peptide Mccj25 mutants

[0105] 1) Protease resistance experiment of antimicrobial peptide Mccj25 mutant

[0106] After entering the body, the active peptide needs to ensure that it reaches the intestine with a complete structure in order to retain its biological activity. Therefore, after oral administration, the active peptide needs to resist digestion and hydrolysis by enzymes such as pepsin and trypsin in the gastrointestinal tract. If the active peptide is degraded in this process, its biological activity will be reduced or even inactivated. Therefore, the protease resistance of the active peptide is of great significance. Therefore, two key enzymes in the gastrointestinal tract (pepsin and trypsin) were selected to simulate in vitro digestion of the mutant of the antimicrobial peptide Mccj25.

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

[0108] Experimental results: The test 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 results of protease treatment analysis in Table 7, it can be seen that the content of the mutant of antimicrobial peptide Mccj25 can still be maintained above 90% after 30-60 minutes of pepsin and trypsin digestion. Therefore, the mutant of antimicrobial peptide Mccj25 has good digestion stability in the gastrointestinal tract, can maintain its sequence integrity in the stomach, and has good application prospects.

[0113] 2) Hemolytic activity experiment of antimicrobial peptide Mccj25 mutant

[0114] Experimental method: First, prepare Columbia agar blood agar medium. The preparation process is as follows. Weigh the Columbia agar medium powder of 4.1 into a conical flask, add purified water to make up to 100 mL, heat and continue to stir until the medium is completely dissolved, and sterilize at 121℃ for 20 minutes. When the medium cools to about 50℃, add 5% sterile defibrinated sheep blood and mix thoroughly. Pour the mixture into a sterile plate, wait for it to cool and solidify, and place it in a refrigerator at 4℃.

[0115] Use a sterilized hole puncher to punch holes on the Columbia blood agar culture dish, with the center of each hole at least 25 mm apart. Use a micropipette to pipette the hemolytic phospholipid control group, saline, and 1 g / L antimicrobial peptide Mccj25 mutant fermentation liquid into the agar wells. Cover the culture dish, place it in a 37°C incubator for 12 hours, and then observe the size of the hemolytic zone.

[0116] Experimental results: Hemolysis in the culture dish is as follows Figure 7 shown.

[0117] Result analysis; Figure 7 It can be seen that the mutant of the antimicrobial peptide Mccj25 has no hemolytic activity, while the positive control lysophospholipid has a hemolytic zone. Therefore, the mutant of the antimicrobial peptide Mccj25 has stable biological properties.

[0118] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0119] While the disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of 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 should be included in the scope of protection 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 nucleic acid sequence, characterized in that The nucleic acid sequence encodes the antimicrobial peptide mutant according to claim 1, and the nucleic acid sequence is as described in SEQ ID NO:

2.

3. A carrier, characterized in that The vector comprises the nucleic acid sequence 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 nucleic acid sequence according to claim 2, the vector according to claim 3, or the recombinant bacteria according to claim 4 in inhibiting bacteria or preparing 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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