Chinese mouse hepialus antibacterial peptide and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a Chinese mouse ear-derived antibacterial peptide and application thereof. The antibacterial peptide derived from the Chinese mouse ear is MC-CATH1, MC-CATH2 or MC-CATH3, and the antibacterial peptide derived from the Chinese mouse ear is MC-CATH3; the antibacterial peptides MC-CATH1, MC-CATH2 and MC-CATH3 derived from the hepialus sinensis have relatively strong antibacterial activity on gram positive bacteria and gram negative bacteria, have broad-spectrum and efficient antibacterial action, and have the characteristics of low hemolytic activity, small cytotoxicity, salt tolerance and strong antibacterial biofilm activity, so that the antibacterial peptides MC-CATH1, MC-CATH2 and MC-CATH3 can be used for preparing the antibacterial peptides of the hepialus sinensis, and the antibacterial peptides MC-CATH1, MC-CATH2 and MC-CATH3 can be used for preparing the antibacterial peptides of the hepialus sinensis. In addition, the MC-CATH3 can kill bacteria by inducing the bacteria to generate a ferroptosis-like mechanism. The MC-CATH1, the MC-CATH2 and the MC-CATH3 can be applied to the fields of medicines, cosmetics, food preservation and the breeding industry.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to antimicrobial peptides (MC-CATH1, MC-CATH2, MC-CATH3) derived from myotis sinensis and applications thereof. Background Art

[0002] Traditional antibiotics have achieved remarkable results in treating bacterial infections. However, in recent years, with the abuse of traditional antibiotics in medicine and aquaculture, microorganisms have developed increasingly strong tolerance to traditional antibiotics, and microbial resistance has become a serious threat to human health. The means of dealing with microbial resistance has always been to use new or alternative antimicrobial agents that have not been used by resistant microorganisms. However, after a period of use, microorganisms will further develop resistance to new antibiotics, so there is a need to continuously develop new antimicrobial agents.

[0003] Antimicrobial peptides are small molecule polypeptides that have killing effects on bacteria, fungi, viruses and protozoa. Antimicrobial peptides have the characteristics of small molecular weight, simple structure and strong bactericidal activity. The bactericidal mechanism of most antimicrobial peptides is to act on the phospholipid bilayer on the bacterial cell membrane, destroy the integrity of the cell membrane and form transmembrane channels on the cell membrane, causing the dissolution of cell contents and leading to cell death. This unique bactericidal mechanism is generally not easy to cause microbial resistance, and antimicrobial peptides are generally not toxic to normal mammalian cells and tissues, and there is no residue problem. Therefore, antimicrobial peptides are expected to become a new type of highly effective antibacterial drug with broad development and application prospects. Summary of the invention

[0004] The object of the present invention is to provide antimicrobial peptides (MC-CATH1, MC-CATH2, MC-CATH3) derived from Myotis sinensis and applications thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is: An antimicrobial peptide derived from Myotis diffusa, wherein the antimicrobial peptide derived from Myotis diffusa is MC-CATH1, MC-CATH2 or MC-CATH3; Among them, MC-CATH1 is composed of 36 amino acid residues, with a molecular weight of 4134.76Da, an isoelectric point of 12.15, and an amino acid sequence of: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg 5 -Ala 6 -Gly 7 -Arg 8 -Leu 9 -Pro 10-Gly 11 -Leu 12 -Phe 13 -Gly 14 -Leu 15 -Leu 16 -Trp 17 -Asp 18 -Arg 19 -Ile 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Arg 25 -Arg 26 -Pro 27 -Arg 28 -Asp 29 -Val 30 -Phe 31 -Glu 32 -Asn 33 -Leu 34 -Ser 35 -Ala 36 ; (Sequence 1) MC-CATH2 is composed of 40 amino acid residues, with a molecular weight of 4931.67Da, an isoelectric point of 12.06, and an amino acid sequence of: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg 5 -Val 6 -Leu 7 -Arg 8 -Val 9 -Asn 10 -Asn 11 -Val 12 -Phe 13 -Arg 14 -Arg 15 -Leu 16 -Trp 17 -Asp 18 -Thr 19 -Leu 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Trp 25 -Arg 26 -Pro 27 -Arg 28 -Leu 29-Phe 30 -Ile 31 -Arg 32 -Asn 33 -Leu 34 -Ser 35 -Pro 36 -Glu 37 -Glu 38 -Glu 39 -Pro 40 ; (Sequence 2) MC-CATH3 consists of 42 amino acid residues, with a molecular weight of 4984.76Da, an isoelectric point of 10.63, and an amino acid sequence of: Lys 1 -Leu 2 -Asn 3 -Ala 4 -Glu 5 -Asn 6 -Leu 7 -Gly 8 -Glu 9 -Arg 10 -Ile 11 -Lys 12 -Asn 13 -Ala 14 -Lys 15 -Lys 16 -Lys 17 -Val 18 -Trp 19 -Glu 20 -Lys 21 -Ile 22 -Lys 23 -Ser 24 -Phe 25 -Gly 26 -Arg 27 -Arg 28 -Ile 29 -Lys 30 -Glu 31 -Phe 32 -Phe 33 -Arg 34 -Lys 35 -Pro 36 -Ser 37 -Pro 38 -Glu 39 -Gly 40 -Glu 41 -Pro 42 ; (Sequence 3) The coding gene of the MC-CATH1 precursor is shown in the following sequence, wherein nucleotides 396-504 in the coding gene correspond to the MC-CATH1 polypeptide; ; (Sequence 4) The coding gene of the MC-CATH2 precursor is shown in the following sequence, wherein nucleotides 396-516 in the coding gene correspond to the MC-CATH2 polypeptide; atggagacccagaggaacagcctgtgctgggggcgctggccgctgttgctgctgctgctgggcctggccctgcccctgcctccggccgccgcccgggccctgagttaccaggaggcggtgcgcctggctgtgcagggcttcaaccagcgctcccgggaggccagcctctaccgcctcctgcagcaggacccgcagccccagggcgacctgaacccagacaccccgaagccggtgagcttcaccctgaaggagaccgtgtgccccaggaccacgcggcagccccctgagcagtgtgacttcaaggagaacgggctggtgaaggcgtgcgcggggaccgtcaccctggaccaggacaccggctactatgacgtccactgcgaggagatcgagggcgttggattgaaggccagagtgctgagggtgaacaacgtcttcaggaggttatgggatacattaaggaatcgtggctggagacccaggctctttatcaggaatctctcgcccgaggaagagccctgagttctgtttggccctggccccggcctctgggctctgaccaataaaatctgggaaagcctcaaaaaaaaaaaaaaaaaaaaaaaaaaa; (Sequence 5) The coding gene of the precursor of MC-CATH3 is shown in the following sequence, wherein the nucleotides at positions 396-522 in the coding gene correspond to the MC-CATH3 polypeptide; ; (Sequence 6) The antimicrobial peptide MC-CATH1, MC-CATH2, and MC-CATH3 encoding genes were synthesized by reverse transcription of total RNA extracted from the lung tissue of Chinese mouse-eared bats, and the second-chain cDNA was amplified by long-terminal polymerase chain reaction (LD-PCR) method.

[0006] According to the known start codon region of the cathelicidin family antimicrobial peptides of Chiroptera, the forward primer 5'-ATGGAGACCCAGRGSRRCAGCC-3' was artificially designed and synthesized, combined with the reverse primer 5'-ATTCTAGAGGCCGAGGCGGCCGAC-3' provided by the kit. PCR reaction amplification, after amplification, the target fragment was recovered, the recovered target fragment was connected to the plasmid vector, transformed into competent cells, and the positive colonies were screened and picked for nucleotide sequencing.

[0007] The coding genes of the active antimicrobial peptides MC-CATH1, MC-CATH2 and MC-CATH3 of Myotis sinensis can be applied to polypeptide recombinant expression, transgenic animals, plants, animal cells or plant cells.

[0008] An application of the antimicrobial peptide derived from Myotis diffusa, and an application of the antimicrobial peptide derived from Myotis diffusa in inhibiting Gram-positive bacteria and Gram-negative bacteria.

[0009] The Gram-positive bacteria include at least one of Enterococcus faecalis, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, and Staphylococcus epidermidis; the Gram-negative bacteria include at least one of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Shigella dysenteriae.

[0010] An application of the antimicrobial peptide derived from myotis diffusa, and an application of the antimicrobial peptide derived from myotis diffusa in antibacterial biofilm.

[0011] An application of the antimicrobial peptide derived from myotis diffusa, the application of the antimicrobial peptide derived from myotis diffusa in the preparation of synergistic antibacterial and bacterial growth inhibiting drugs, bactericides, antimicrobial preparations, animal feed, cosmetics and preservation.

[0012] The advantages of the present invention are: The Chinese mouse-eared bat antimicrobial peptides MC-CATH1, MC-CATH2 and MC-CATH3 of the present invention have strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, broad-spectrum and efficient antimicrobial action, and have the characteristics of low hemolytic activity, low cytotoxicity, salt tolerance and strong antibacterial biofilm activity. In addition, MC-CATH3 can kill bacteria by inducing an iron-like mechanism of death in bacteria, and is the first antimicrobial peptide discovered at home and abroad that exerts a bactericidal effect through an iron-like mechanism of death. MC-CATH1, MC-CATH2 and MC-CATH3 can be applied to the fields of medicine, cosmetics, food preservation and aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram showing the sterilization speed of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention against Escherichia coli ATCC25922.

[0014] Figure 2 This is a diagram showing the sterilization speed effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on Staphylococcus aureus CMCC26003.

[0015] Figure 3This is a diagram showing the hemolytic activity effects of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention.

[0016] Figure 4 Cytotoxic effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 1 .

[0017] Figure 5 Cytotoxic effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 2 .

[0018] Figure 6 Cytotoxic effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 3 .

[0019] Figure 7 This is a diagram showing the effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on the removal of Escherichia coli ATCC25922 biofilm.

[0020] Figure 8 This is a graph showing the inhibitory effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on the biofilm of Escherichia coli ATCC25922.

[0021] Fig. 9 This is a diagram showing the effect of MC-CATH1, MC-CATH2, and MC-CATH3 solutions containing different salt ions provided in an embodiment of the present invention on the MIC of Escherichia coli ATCC25922.

[0022] Fig.10 This is a diagram showing the destructive effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on the inner membrane of Escherichia coli ATCC25922.

[0023] Fig.11 This is a diagram showing the destructive effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on the outer membrane of Escherichia coli ATCC25922.

[0024] Fig.12 This is a diagram showing the effect of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention on inducing intracellular ROS accumulation in Escherichia coli ATCC25922.

[0025] Fig.13 This is a diagram showing the effect of the antimicrobial peptides MC-CATH2 and MC-CATH3 provided in the embodiments of the present invention on promoting lipid peroxidation in Escherichia coli ATCC25922.

[0026] Fig.14 This is a diagram showing the effect of the antimicrobial peptide MC-CATH3 provided in an embodiment of the present invention inducing intracellular iron ion accumulation in Escherichia coli ATCC25922. DETAILED DESCRIPTION

[0027] The following examples are used to further describe the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0028] The Chinese myotis bats mentioned in the following examples were collected from Libo, Guizhou. The sample collection was approved by the unit's animal ethics committee and did not involve endangered species. Example 1

[0029] Cloning of genes encoding the active peptides MC-CATH1, MC-CATH2 and MC-CATH3 of Myotis sinensis: (1) Total RNA extraction from lung tissue of Myotis diversicolor: ① Take 100 mg of lung tissue from Myotis diversicolor, put it into a mortar, add liquid nitrogen and grind it into powder, transfer it to an EP tube, add 1 mL of total RNA extraction buffer (Trizol, a product of Life Science, USA), mix it thoroughly, and then centrifuge it at 4°C, 12000 rpm for 10 minutes.

[0030] ② Centrifuge and take the supernatant, add 0.2 mL of chloroform solution, mix vigorously, leave at room temperature for 10 minutes, then centrifuge at 4°C, 12000 rpm for 10 minutes and discard the precipitate.

[0031] ③ Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 minutes, centrifuge at 4°C, 12000rpm for 10 minutes, collect the precipitate, wash once with 75% (V / V) ethanol, and dry. The precipitate at the bottom of the tube is the total RNA of the lung tissue of Chinese mouse-eared bat.

[0032] (2) Synthesis of second-strand cDNA from lung tissue of Myotis diffusa: using the CreatorTM SMARTTM cDNA Library Construction Kit from CLONTECH.

[0033] 1) First-strand cDNA synthesis (mRNA reverse transcription): ① Add 1µL total RNA from lung tissue of Myotis diversicolor, 1µL 3' SMARTer CDS Primer and 2.5µL RNase-free water to an RNase-free PCR tube to make the total volume reach 4.5µL. Mix well and centrifuge briefly (2000rpm, 30s). After centrifugation, keep it at 72℃ for 3 minutes. After keeping it warm, incubate the centrifuge tube at 42℃ for 2 minutes.

[0034] ② Add the following reagents (all included in the CreatorTM SMARTTM cDNA Library Construction Kit from CLONTECH) to the above centrifuge tube: 2.0µL 5× first-strand buffer, 0.25µL 100mM DTT, 1.0µL 10mM dNTP Mix, 1.0µL SMARTer V Oligonucleotide, 0.25µL RNase Inhibitor, and 1.0µL SMARTScribe Reverse Transcriptase. Mix the reagents in the centrifuge tube and centrifuge briefly (2000rpm, 30s), incubate at 42℃ for 90 minutes, and then incubate at 68℃ for 10 minutes. After incubation, place the centrifuge tube on ice to terminate the synthesis of the first strand. Take 2µL of the synthesized cDNA first strand from the centrifuge tube for later use.

[0035] 2) Amplify the second strand using the long-terminal polymerase chain reaction (LD-PCR) method (the reagents used are all provided in the CreatorTM SMARTTM cDNA Library Construction Kit of CLONTECH) ① Mix 2µL cDNA first strand, 80µL deionized water, 10µL 10×Advantage 2 PCR buffer, 2µL 50×dNTP mixture, 2µL 5'PCR primer, 2µL CDS III / 3'PCR primer and 2µL 50×Advantage 2Polymerase Mix in a PCR tube preheated at 95℃.

[0036] ② Amplify in the PCR instrument according to the following procedure: 95℃, 1 minute; 18 cycles: 95℃, 15sec, 65℃, 30sec, 68℃, 6 minutes. After the cycle, the synthesized cDNA double strand in the centrifuge tube was stored at -80℃.

[0037] (3) Cloning of genes encoding the active peptides MC-CATH1, MC-CATH2 and MC-CATH3 of Myotis sinensis: The forward specific primer 5'-ATGGAGATCTGGCAGTGTGTGATAT-3' was designed based on the known start codon region of cathelicidin in Chiroptera, and the reverse primer was the 3'-PCR primer in the CreatorTM SMARTTM cDNA Library Construction Kit of CLONTECH, whose sequence was 5'-ATTCTAGAGGCCGAGGCGGCCGAC-3'. The PCR reaction was carried out under the following conditions: 95℃ for 5 minutes, 95℃ for 30 sec, 50℃ for 30 sec and 72℃ for 1 minute, 30 cycles. After amplification, the target fragment was recovered using a gel recovery kit (Tiangen Bio). The recovered target fragment was connected to the pMD19-T vector (Takara, Dalian) and transformed into TOP10 competent cells. The plate was coated and ampicillin was selected, and a single colony was picked to detect the size of the inserted fragment using M13 primer PCR. Positive colonies were picked, plasmids were extracted by shaking, and samples were sent for nucleotide sequencing.

[0038] Measurement results: The sequence of the coding gene encoding the precursor of the active polypeptide MC-CATH1 of Myotis sinensis from the 5' end to the 3' end is: ; The nucleotide sequence of the gene encoding the precursor of the active polypeptide MC-CATH1 of the Chinese mouse-eared bat is as follows: sequence length is 606 bases, sequence type: nucleic acid, number of chains: single chain, topology: linear, sequence type: cDNA, source: lung of the Chinese mouse-eared bat. The nucleotides encoding the mature peptide of the active polypeptide MC-CATH1 of the Chinese mouse-eared bat are nucleotides 396-504.

[0039] The sequence of the coding gene encoding the precursor of the active polypeptide MC-CATH2 of Myotis sinensis from the 5' end to the 3' end is: ; The nucleotide sequence table of the coding gene of the precursor of the active polypeptide MC-CATH2 of the Chinese mouse-eared bat is as follows: sequence length is 606 bases, sequence type: nucleic acid, number of chains: single chain, topology: linear, sequence type: cDNA, source: lung of the Chinese mouse-eared bat. The nucleotides encoding the mature peptide of the active polypeptide MC-CATH2 of the Chinese mouse-eared bat are nucleotides 396-516.

[0040] The sequence of the coding gene encoding the precursor of the active polypeptide MC-CATH3 of Myotis sinensis from the 5' end to the 3' end is: ; The nucleotide sequence of the gene encoding the precursor of the active polypeptide MC-CATH3 of the Chinese mouse-eared bat is as follows: sequence length is 603 bases, sequence type: nucleic acid, number of chains: single chain, topology: linear, sequence type: cDNA, source: lung of the Chinese mouse-eared bat. The nucleotides encoding the mature peptide of the active polypeptide MC-CATH3 of the Chinese mouse-eared bat are nucleotides 396-522. Example 2

[0041] Preparation of MC-CATH1, MC-CATH2, and MC-CATH3: (1) Chemical synthesis of MC-CATH1, MC-CATH2, and MC-CATH3: Based on the amino acid sequence deduced from the coding gene, the complete sequence was synthesized using an automatic peptide synthesizer (433A, Applied Biosystems) and purified by desalting on a reverse-phase HPLC column.

[0042] (2) Molecular weight was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

[0043] (3) The purity of the purified peptide was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

[0044] MC-CATH1 is a small molecule polypeptide encoded by a gene, consisting of 36 amino acid residues, with a molecular weight of 4134.76Da and an isoelectric point of 12.15. Its amino acid sequence is: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg 5 -Ala 6 -Gly 7 -Arg 8 -Leu 9 -Pro 10 -Gly 11 -Leu 12 -Phe 13 -Gly 14 -Leu 15 -Leu 16 -Trp 17 -Asp 18 -Arg 19 -Ile 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Arg 25 -Arg 26 -Pro 27 -Arg 28 -Asp 29 -Val 30 -Phe 31 -Glu 32 -Asn 33 -Leu 34 -Ser 35 -Ala 36 ; MC-CATH2 is a small molecule polypeptide encoded by a gene, consisting of 40 amino acid residues, with a molecular weight of 4931.67Da and an isoelectric point of 12.06. Its amino acid sequence is: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg5 -Val 6 -Leu 7 -Arg 8 -Val 9 -Asn 10 -Asn 11 -Val 12 -Phe 13 -Arg 14 -Arg 15 -Leu 16 -Trp 17 -Asp 18 -Thr 19 -Leu 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Trp 25 -Arg 26 -Pro 27 -Arg 28 -Leu 29 -Phe 30 -Ile 31 -Arg 32 -Asn 33 -Leu 34 -Ser 35 -Pro 36 -Glu 37 -Glu 38 -Glu 39 -Pro 40 ; MC-CATH3 is a small molecule polypeptide encoded by a gene, consisting of 42 amino acid residues, with a molecular weight of 4984.76Da and an isoelectric point of 10.63. Its amino acid sequence is: Lys 1 -Leu 2 -Asn 3 -Ala 4 -Glu 5 -Asn 6 -Leu 7 -Gly 8 -Glu 9 -Arg 10 -Ile 11 -Lys 12 -Asn 13 -Ala 14 -Lys 15 -Lys 16 -Lys 17 -Val 18-Trp 19 -Glu 20 -Lys 21 -Ile 22 -Lys 23 -Ser 24 -Phe 25 -Gly 26 -Arg 27 -Arg 28 -Ile 29 -Lys 30 -Glu 31 -Phe 32 -Phe 33 -Arg 34 -Lys 35 -Pro 36 -Ser 37 -Pro 38 -Glu 39 -Gly 40 -Glu 41 -Pro 42 . Example 3

[0045] Antibacterial activity detection of MC-CATH1, MC-CATH2 and MC-CATH3: The minimum inhibitory concentration (MIC) was determined by the two-fold dilution method: the test strains listed in Table 1 were inoculated into MH liquid medium (Thermo Fisher Scientific, Inc., USA), and then cultured in an incubator at 37°C with shaking until the logarithmic growth phase. The culture medium of the strains cultured to the logarithmic growth phase was then diluted to 2×10 5 cfu / mL for later use.

[0046] Add 90µL MH liquid medium to the first well of a sterile 96-well plate, add 50µL MH liquid medium to the remaining wells in advance, then add 10µL of the Chinese mouse-eared bat active polypeptide sample solution diluted to a certain concentration with MH liquid medium and filtered through a 0.22mm pore filter membrane to the first well, mix well, take 50µL and add it to the second well, dilute it in multiples, aspirate 50µL from the 8th well and discard it, and the 9th well is the control tube.

[0047] Add 50 µL of the diluted strain culture solution to each well, then place the 96-well plate in an incubator at 37°C with slow shaking for 18 hours, and measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is observed. The results are shown in Table 1.

[0048] As shown in Table 1, MC-CATH1 and MC-CATH2 showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria, with MIC values ​​ranging from 0.78 to 75 µg / mL. MC-CATH3 also had strong antibacterial activity against strains other than Bacillus cereus CMCC63301, Staphylococcus epidermidis, and Escherichia coli CMCC44102, with MIC values ​​ranging from 2.34 to 75 µg / mL.

[0049] Table 1. Antibacterial activity of MC-CATH1, MC-CATH2, and MC-CATH3 Example 4

[0050] Determination of the bactericidal rate of antimicrobial peptides MC-CATH1, MC-CATH2 and MC-CATH3 from Myotis sinensis: The antibacterial activity of the antimicrobial peptides of Myotis sinensis against the Gram-negative bacteria Escherichia coli ATCC25922 and the Gram-positive bacteria Staphylococcus aureus CMCC26003 was determined respectively. The samples were cultured in MH liquid medium (Thermo Fisher Scientific, Inc., USA) at 37°C for 12 h and then diluted to 10% with fresh MH liquid medium. 6 cfu / mL bacterial suspension. MC-CATH1, MC-CATH2, and MC-CATH3 samples dissolved in sterile deionized water were added to the bacterial suspension to a final concentration of 5×MIC (46.9µg / mL). The bacterial solution with the active peptide sample of Myotis diffusa was placed in an incubator for shaking culture at 37°C. 50µL of the bacterial solution was diluted 1000 times at 0, 15, 30, 60, 120, and 180 minutes, and then 50µL of the diluted bacterial solution was spread on LB solid culture medium. After overnight culture in a 37°C incubator, the colonies were counted. This experiment used ampicillin as a positive control and sterile deionized water as a negative control.

[0051] The results are as follows Figure 1 , Figure 2As shown in Tables 2 and 3, the sterilization speed of MC-CATH1 and MC-CATH2 against Escherichia coli ATCC25922 is faster than that of the positive control ampicillin, and the sterilization speed of MC-CATH3 against Escherichia coli ATCC25922 is comparable to that of the positive control ampicillin, and all bacteria can be effectively killed within 60 minutes. The sterilization speed of MC-CATH1 and MC-CATH2 against Staphylococcus aureus CMCC26003 is faster than that of the positive control ampicillin, and all bacteria are effectively killed within 180 minutes and 120 minutes, respectively. The sterilization speed of MC-CATH3 against Staphylococcus aureus CMCC26003 is comparable to that of the positive control ampicillin. Although all bacteria were not completely killed within 180 minutes, the killing effect on Staphylococcus aureus CMCC26003 reached 99.8% compared with 0 minutes.

[0052] Table 2. Bactericidal rate of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 against Escherichia coli ATCC25922

[0053] Table 3. Bactericidal rate of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 against Staphylococcus aureus CMCC26003 Example 5

[0054] Determination of hemolytic activity and cytotoxicity of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis: (1) Hemolytic activity: Fresh human blood was collected and mixed with Aldrich's solution for anticoagulation, washed twice with saline and resuspended in 10 7 -10 8 The diluted red blood cell suspension was mixed with gradient concentrations of MC-CATH1, MC-CATH2, and MC-CATH3 sample solutions dissolved in physiological saline, respectively, incubated at 37°C for 30 minutes, and then centrifuged at 1000 rpm for 5 minutes. The supernatant was measured for light absorption at a wavelength of 540 nm. The negative control used physiological saline, and the positive control used TritonX-100. The hemolysis percentage was calculated according to the following formula: Hemolysis percentage H%=A 样品 -A 阴性对照 / A 阳性对照 ×100%. The experimental results are as follows Figure 3As shown in Table 4, MC-CATH1, MC-CATH2, and MC-CATH3 have low hemolytic activity. The hemolytic activity of MC-CATH1 at a concentration of 64 µM is only 5.68%, the hemolytic activity of MC-CATH2 at a concentration of 64 µM is only 10.50%, and the hemolytic activity of MC-CATH3 at a concentration of 64 µM is only 1.12%. Moreover, the dosage concentration is much greater than the MICs value of the bacteria, indicating that MC-CATH1, MC-CATH2, and MC-CATH3 have great application potential.

[0055] Table 4. Hemolytic activity of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3

[0056] (2) Cytotoxicity: HepG2 (human hepatocellular carcinoma), HEK293T (human embryonic kidney cells), Raw 264.7 (mouse macrophage cell line), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method were used to determine the in vitro cytotoxicity of MC-CATH1, MC-CATH2, and MC-CATH3. The cells were cultured in DMEM complete medium containing 10% fetal bovine serum (Biological Industries, Israel) and 100 U / mL streptomycin penicillin (Shanghai Yuanye Biotechnology Co., Ltd.). Before the formal experiment, the above cells were seeded in 96-well plates (2×10 4 / well) and cultured overnight, then added different concentrations of MC-CATH1, MC-CATH2, and MC-CATH3 and incubated in a 37°C, 5% carbon dioxide cell culture incubator for 24 hours. After the incubation, 20µL, 5mg / mL MTT was added to each well of the 96-plate, and the supernatant was discarded after another 4 hours of culture. DMSO was added to measure the light absorption value at a wavelength of 490nm. The zero adjustment group only added DMEM culture medium, and the negative control group only added cells without drug administration. The cytotoxicity percentage C%=A 样品 -A 调零组 / A 阴性对照 -A 调零组 ×100%. The results of MC-CATH1, MC-CATH2, and MC-CATH3 cytotoxicity experiments are shown in Figure 4-Figure 6As shown in Tables 5-7, when the cell concentration was 2 µM, the lowest MC-CATH1 cell survival rate in HepG2 cells was 92.59%, and the other two cell lines were both higher than 95%. The lowest MC-CATH2 cell survival rate in HEK293T cells was 77.76%, and the other two cell lines were both higher than 84%. The lowest MC-CATH3 cell survival rate in HEK293T cells was 77.76%, and the other two cell lines were both higher than 90%. This indicates that MC-CATH1, MC-CATH2, and MC-CATH3 have weak toxicity to mammalian cells at a dosage concentration equivalent to the bacterial MICs value, and also shows that MC-CATH1, MC-CATH2, and MC-CATH3 have great application potential.

[0057] Table 5. Cytotoxicity of antimicrobial peptide MC-CATH1

[0058] Table 6. Cytotoxicity of antimicrobial peptide MC-CATH2

[0059] Table 7. Cytotoxicity of antimicrobial peptide MC-CATH3 Example 6

[0060] Anti-biofilm activity of MC-CATH1, MC-CATH2 and MC-CATH3 from Myotis sinensis: (1) Determination of the biofilm removal ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3: The crystal violet staining method was used to evaluate the biofilm removal ability of MC-CATH1, MC-CATH2, and MC-CATH3 against Escherichia coli ATCC25922. The strains were cultured in MH liquid medium (Thermo Fisher Scientific, Inc., USA) at 37°C for 12 hours and then diluted to 10 with new medium. 7 cfu / mL bacterial suspension, add 100µL of bacterial suspension to each well of a 96-well plate and culture at 37℃ for 48 hours. After removing the supernatant, wash three times with PBS, and add 100µL of different concentration gradients of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 in turn and incubate for another 24 hours. After incubation, remove the supernatant, wash with PBS, fix with 50µL methanol, and add 0.1% crystal violet to stain after drying. Finally, discard the stain, add anhydrous ethanol to dissolve the crystal violet, and measure the absorbance at 600nm. The zero adjustment group only added culture medium, and the negative control group only added bacterial suspension without drug administration. The biofilm clearance rate B%=A 样品 -A 调零组 / A 阴性对照 -A 调零组×100%. The result is Figure 7 As shown in Table 8, the clearance rate of Escherichia coli biofilm using MC-CATH1 at 8×MIC can reach about 77%, the clearance rate of MC-CATH2 at 8×MIC can reach about 75%, and the clearance rate of MC-CATH3 at 8×MIC can reach about 60%, indicating that the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 have great potential for the treatment of biofilm-related infections.

[0061] Table 8. Biofilm removal ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3

[0062] (2) Determination of the biofilm inhibition ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3: The crystal violet staining method was used to evaluate the inhibitory ability of MC-CATH1, MC-CATH2, and MC-CATH3 on the biofilm of Escherichia coli ATCC25922. The strains were cultured in MH liquid medium (Thermo Fisher Scientific, Inc., USA) at 37°C for 12 h and then diluted to 2×10 7 cfu / mL bacterial suspension, 50µL was added to each well of a 96-well plate, and 50µL of different concentrations of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 were added at 37°C for 48 hours. The remaining steps were the same as those for biofilm removal. The zeroing group was treated with only culture medium, and the negative control group was treated with only bacterial suspension without drug administration. The biofilm removal rate B%=A 样品 -A 调零组 / A 阴性对照 -A 调零组 ×100%, the result is Figure 8 As shown in Table 9, when MC-CATH1 was co-incubated with Escherichia coli, the inhibitory ability against biofilm at a concentration of 8×MIC was about 69%, when MC-CATH2 was co-incubated with Escherichia coli, the inhibitory ability against biofilm at a concentration of 8×MIC was about 65%, and when MC-CATH3 was co-incubated with Escherichia coli, the inhibitory ability against biofilm at a concentration of 8×MIC was about 57%, indicating that MC-CATH1, MC-CATH2, and MC-CATH3 have great potential for the treatment of biofilm-related infections.

[0063] Table 9. Biofilm inhibition ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 Example 7

[0064] Salt tolerance of antimicrobial peptides MC-CATH1, MC-CATH2 and MC-CATH3 from Myotis diffusa: (1) Determination of salt tolerance of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3: Escherichia coli ATCC25922 was cultured in MH liquid medium (Thermo Fisher Scientific, Inc., USA) at 37°C in an incubator for 12 h, and then diluted to 2×10 with fresh MH liquid medium containing 150 mM sodium chloride, 4.5 mM potassium chloride, 6 µM ammonium chloride, 1 mM magnesium chloride, 2.5 mM calcium chloride, and 4 µM ferric chloride. 5 cfu / mL. Then, MC-CATH1, MC-CATH2, and MC-CATH3 sample solutions were prepared using MH liquid culture medium containing different salt concentrations. The MIC values ​​of MC-CATH1, MC-CATH2, and MC-CATH3 were determined using the 2-fold dilution method described in Example 2. Finally, the degree of inhibition was observed after incubation for 16-18 hours. The results are shown in Figure 2. Fig. 9 As shown in Table 10.

[0065] from Fig. 9 As can be seen from Table 10, the concentrations of different salt ions in the human body are simulated, except for Na + and Ca 2+ Except for the significant effect on the antibacterial activity of MC-CATH1, MC-CATH2, and MC-CATH3, the MICs values ​​of MC-CATH1, MC-CATH2, and MC-CATH3 at other salt ion concentrations were between 9.38 and 18.75 µg / mL.

[0066] Table 10. MIC of MC-CATH1, MC-CATH2, and MC-CATH3 solutions containing different salt ions against Escherichia coli ATCC25922 Example 8

[0067] The antimicrobial peptides MC-CATH1 and MC-CATH2 of Chinese mouse-eared bat exert their bactericidal effects by destroying bacterial cell membranes, and MC-CATH3 exerts its antibacterial effects by inducing bacterial ferroptosis: (1) MC-CATH1 and MC-CATH2 exert their antibacterial effects by destroying bacterial cell membranes: Escherichia coli ATCC25922 was cultured in MH liquid medium (Thermo Fisher Scientific, Inc., USA) at 37°C in an incubator for 12 hours, and then diluted to 1×10 8cfu / mL of bacterial suspension, take a 96-well plate, add 80µL to each well, and add 10µL of different concentration gradients of antimicrobial peptides MC-CATH1, MC-CATH2, MC-CATH3 and 10µL of 500µL / mL propidium iodide bacterial inner membrane activity detection probe (PI) (Sigma-Aldrich Co., Ltd., USA) or 10µL of 100µM N-phenyl-1-naphthylamine bacterial outer membrane activity detection probe (NPN) (Sigma-Aldrich Co., Ltd., USA). After incubation at 37°C for 60 minutes, use an enzyme reader to detect the bacterial uptake of propidium iodide at an excitation wavelength of 535nm and an emission wavelength of 617nm, and the bacterial uptake of N-phenyl-1-naphthylamine at an excitation wavelength of 350nm and an emission wavelength of 420nm, which represent the degree of damage of antimicrobial peptides to the inner and outer membranes of bacteria, respectively. The negative control group is a group without antimicrobial peptides. Results are shown in Fig.10 , Fig.11 As shown in Tables 11 and 12, the three antimicrobial peptides from Myotis sinensis enhanced the destruction of bacterial cell membranes in a concentration-dependent manner, among which MC-CATH1 and MC-CATH2 had significantly stronger uptake of propidium iodide and N-phenyl-1-naphthylamine than MC-CATH3.

[0068] Table 11. Destructive effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 on the inner membrane of Escherichia coli ATCC25922

[0069] Table 12. Destructive effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 on the outer membrane of Escherichia coli ATCC25922

[0070] (2) MC-CATH3 has a stronger ability to promote bacterial accumulation of reactive oxygen species (ROS): Escherichia coli ATCC25922 was cultured in MH liquid medium (Thermo Fisher Scientific, USA) in an incubator at 37°C for 12 hours, and then diluted to 1×10 8cfu / mL bacterial suspension, and add reactive oxygen detection probe DCFH-DA (Sigma-Aldrich, USA) to make the final concentration reach 2µM, take a 96-well plate, add 90µL to each well, and add 10µL of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 of different concentration gradients at the same time, culture at 37°C for 60 minutes, centrifuge and wash the bacterial solution twice with PBS, use a microplate reader at an excitation wavelength of 488nm and an emission wavelength of 525nm to detect the fluorescence intensity of the DCFH-DA probe in the bacterial cells to represent the level of reactive oxygen, and the negative control group is the group without adding antimicrobial peptides. The experimental results are as follows Fig.12 As shown in Table 13, MC-CATH1, MC-CATH2, and MC-CATH3 can induce bacteria to accumulate a large amount of reactive oxygen species at a concentration of 1×MIC. MC-CATH3 can induce bacteria to accumulate very high reactive oxygen species at a concentration of 1×MIC, which is significantly higher than MC-CATH1 and MC-CATH2.

[0071] Table 13. Antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 induce intracellular ROS accumulation in Escherichia coli ATCC25922

[0072] (3) Both MC-CATH2 and MC-CATH3 can promote bacterial lipid peroxidation more strongly: Escherichia coli ATCC25922 was cultured in MH liquid medium (Thermo Fisher Scientific, USA) in an incubator at 37°C for 12 hours, and then diluted to 1×10 7 cfu / mL bacterial suspension, and add lipid peroxidation probe C11-bodipy581 / 591 (German Cayman Chemical Co., Ltd.) to make the final concentration reach 5µM, take a 96-well plate, add 90µL to each well, and add 10µL of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 of different concentration gradients at the same time, incubate at 37°C for 60 minutes, centrifuge and wash the bacterial solution twice with PBS, use a microplate reader at an excitation wavelength of 488nm and an emission wavelength of 510nm to detect the fluorescence intensity of C11-bodipy581 / 591 probe in bacterial cells to represent the level of bacterial lipid peroxidation, and the negative control group is a group without antimicrobial peptides. The experimental results are as follows Fig.13 As shown in Table 14, MC-CATH1, MC-CATH2, and MC-CATH3 can all increase the level of bacterial lipid peroxidation in a concentration-dependent manner. MC-CATH3 can significantly promote a higher level of bacterial lipid peroxidation at a concentration of 0.25×MIC. At a concentration of 4×MIC, MC-CATH2 and MC-CATH3 promote bacterial lipid peroxidation more strongly than MC-CATH1.

[0073] Table 14. Antimicrobial peptides MC-CATH2 and MC-CATH3 promote lipid peroxidation in Escherichia coli ATCC25922

[0074] (4) MC-CATH3 forces bacteria to accumulate more iron ions: Escherichia coli ATCC25922 was cultured in MH liquid medium (Thermo Fisher Scientific, USA) in an incubator at 37°C for 12 hours, and then diluted to 5×10 7 cfu / mL bacterial suspension, MC-CATH1, MC-CATH2, and MC-CATH3 were added to the final concentration of 5×MIC, cultured for 60 minutes, and ultrasonicated for 3 seconds with an interval of 10 seconds using an ultrasonic disruptor at a power of 200 W. After repeating 30 times, centrifuged at 8000 rpm for 10 minutes at 4°C, and then the intracellular iron content of the bacteria was detected according to the steps of the Cell Iron Content Detection Kit (Beijing Solebold Biotechnology Co., Ltd.), without adding antimicrobial peptides as a negative control. The results are as follows Fig.14 As shown in Table 15, MC-CATH3 can induce bacteria to accumulate more iron ions, and the iron content of MC-CATH1 and MC-CATH2 is comparable to that of the control group.

[0075] Table 15. Antimicrobial peptide MC-CATH3 induces intracellular iron accumulation in Escherichia coli ATCC25922

[0076] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by those skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. An antimicrobial peptide derived from Myotis sinensis, characterized in that: The antimicrobial peptides from Myotis sinensis are MC-CATH1, MC-CATH2 or MC-CATH3; Among them, the amino acid sequence of MC-CATH1 is: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg 5 -Ala 6 -Gly 7 -Arg 8 -Leu 9 -Pro 10 -Gly 11 -Leu 12 -Phe 13 -Gly 14 -Leu 15 -Leu 16 -Trp 17 -Asp 18 -Arg 19 -Ile 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Arg 25 -Arg 26 -Pro 27 -Arg 28 -Asp 29 -Val 30 -Phe 31 -Glu 32 -Asn 33 -Leu 34 -Ser 35 -Ala 36 ; The amino acid sequence of MC-CATH2 is: Gly 1 -Leu 2 -Lys 3 -Ala 4 -Arg 5 -Val 6 -Leu 7 -Arg 8 -Val 9 -Asn 10 -Asn 11 -Val 12 -Phe 13 -Arg 14 -Arg 15 -Leu 16 -Trp 17 -Asp 18 -Thr 19 -Leu 20 -Arg 21 -Asn 22 -Arg 23 -Gly 24 -Trp 25 -Arg 26 -Pro 27 -Arg 28 -Leu 29 -Phe 30 -Ile 31 -Arg 32 -Asn 33 -Leu 34 -Ser 35 -Pro 36 -Glu 37 -Glu 38 -Glu 39 -Pro 40 ; The amino acid sequence of MC-CATH3 is: Lys 1 -Leu 2 -Asn 3 -Ala 4 -Glu 5 -Asn 6 -Leu 7 -Gly 8 -Glu 9 -Arg 10 -Yes 11 -Lys 12 -Asn 13 -Ala 14 -Lys 15 -Lys 16 -Lys 17 -Val 18 -Trp 19 -Glu 20 -Lys 21 -Yes 22 -Lys 23 -Sir 24 -Phe 25 -Gly 26 -Arg 27 -Arg 28 -Yes 29 -Lys 30 -Glu 31 -Phe 32 -Phe 33 -Arg 34 -Lys 35 -Pro 36 -Sir 37 -Pro 38 -Glu 39 -Gly 40 -Glu 41 -Pro 42 。 2. The antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The coding gene of the MC-CATH1 precursor is shown in the following sequence, wherein nucleotides 396-504 in the coding gene correspond to the MC-CATH1 polypeptide; .

3. The antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The coding gene of the MC-CATH2 precursor is shown in the following sequence, wherein nucleotides 396-516 in the coding gene correspond to the MC-CATH2 polypeptide; atggagacccagaggaacagcctgtgctgggggcgctggccgctgttgctgctgctgctgggcctggccctgcccctgcctccggccgccgcccgggccctgagttaccaggaggcggtgcgcctggctgtgcagggcttcaaccagcgctcccgggaggccagcctctaccgcctcctgcagcaggacccgcagccccagggcgacctgaacccagacaccccgaagccggtgagcttcaccctgaaggagaccgtgtgccccaggaccacgcggcagccccctgagcagtgtgacttcaaggagaacgggctggtgaaggcgtgcgcggggaccgtcaccctggaccaggacaccggctactatgacgtccactgcgaggagatcgagggcgttggattgaaggccagagtgctgagggtgaacaacgtcttcaggaggttatgggatacattaaggaatcgtggctggagacccaggctctttatcaggaatctctcgcccgaggaagagccctgagttctgtttggccctggccccggcctctgggctctgaccaataaaatctgggaaagcctcaaaaaaaaaaaaaaaaaaaaaaaaaaa。 4. The antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The coding gene of the precursor of MC-CATH3 is shown in the following sequence. Among them, the nucleotides at positions 396-522 in the coding gene correspond to the MC-CATH3 polypeptide; .

5. A use of the antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The application of the antimicrobial peptide derived from Myotis diffusa in inhibiting Gram-positive bacteria and Gram-negative bacteria.

6. The use of the antimicrobial peptide derived from Myotis sinensis according to claim 5, characterized in that: The Gram-positive bacteria include at least one of Enterococcus faecalis, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, and Staphylococcus epidermidis; the Gram-negative bacteria include at least one of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Shigella dysenteriae.

7. A use of the antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The application of the antimicrobial peptide derived from Myotis sinensis in antibacterial biofilm.

8. A use of the antimicrobial peptide derived from Myotis sinensis according to claim 1, characterized in that: The antimicrobial peptide derived from Myotis sinensis is used in the preparation of synergistic antibacterial and bacterial growth inhibiting drugs, bactericides, antimicrobial preparations, animal feed, cosmetics and preservation.

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