Antibacterial Peptide of Myotis chinensis and Its Application

Abstract

The present invention belongs to the field of biomedical technologies, and specifically relates to antibacterial peptides derived from Myotis chinensis and their applications. The antibacterial peptides derived from Myotis chinensis are MC-CATH1, MC-CATH2 or MC-CATH3; the antibacterial peptides MC-CATH1, MC-CATH2, and MC-CATH3 derived from Myotis chinensis in the present invention have strong antibacterial activities against Gram-positive bacteria and Gram-negative bacteria, with a broad-spectrum and highly efficient antibacterial effect, and are characterized by low hemolytic activity, low cytotoxicity, high salt tolerance, and strong anti-bacterial biofilm activity. In addition, MC-CATH3 can kill bacteria by inducing an iron death-like mechanism in bacteria. MC-CATH1, MC-CATH2, and MC-CATH3 can be applied to the fields of medicine, cosmetics, food preservation, and aquaculture.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to antimicrobial peptides (MC-CATH1, MC-CATH2, and 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, the overuse of traditional antibiotics in fields such as medicine and animal husbandry has led to an increasing tolerance of microorganisms to these antibiotics. Microbial resistance has become a serious threat to human health. Traditionally, the response to microbial resistance has been to use new or alternative antimicrobial agents that have not been used by resistant microorganisms. However, after a period of use, microorganisms can develop further resistance to new antibiotics, necessitating the continued development of new antimicrobial agents.

[0003] Antimicrobial peptides are small-molecule polypeptides that have antibacterial effects on bacteria, fungi, viruses, and protozoa. They are characterized by their small molecular weight, simple structure, and strong bactericidal activity. Most antimicrobial peptides act by acting on the phospholipid bilayer of bacterial cell membranes, disrupting the integrity of the cell membrane and forming transmembrane channels, leading to the dissolution of cellular contents and cell death. This unique bactericidal mechanism is generally not likely to induce microbial resistance. Furthermore, antimicrobial peptides are generally non-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 antimicrobial 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:

[0006] An antimicrobial peptide derived from myotis sinensis, wherein the antimicrobial peptide derived from myotis sinensis is MC-CATH1, MC-CATH2 or MC-CATH3;

[0007] Among them, MC-CATH1 consists 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 -Leu9 -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)

[0008] MC-CATH2 consists 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 -Pro27 -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)

[0009] 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 -Glu41 -Pro 42 ; (Sequence 3)

[0010] 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;

[0011] ; (Sequence 4)

[0012] 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;

[0013] atggagacccagaggaacagcctgtgctgggggcgctggccgctgttgctgctgctgctgggcctggccctgcccctgcctccggccgccgcccgggccctgagttaccaggaggcggtgcgcctggctgtgcagggcttcaaccagcgctcccgggaggccagcctctaccgcctcctgcagcaggacccgcagccccagggcgacctgaacccagacaccccgaagccggtgagcttcaccctgaaggagaccgtgtgccccaggaccacgcggcagccccctgagcagtgtgacttcaaggagaacgggctggtgaaggcgtgcgcggggaccgtcaccctggaccaggacaccggctactatgacgtccactgcgaggagatcgagggcgttggattgaaggccagagtgctgagggtgaacaacgtcttcaggaggttatgggatacattaaggaatcgtggctggagacccaggctctttatcaggaatctctcgcccgaggaagagccctgagttctgtttggccctggccccggcctctgggctctgaccaataaaatctgggaaagcctcaaaaaaaaaaaaaaaaaaaaaaaaaaa; (Sequence 5)

[0014] 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;

[0015] ; (Sequence 6)

[0016] The antimicrobial peptide MC-CATH1, MC-CATH2, and MC-CATH3 encoding genes were synthesized into first-strand cDNA by reverse transcription of total RNA extracted from the lung tissue of Chinese myotis bat, and the second-strand cDNA was amplified by long-term polymerase chain reaction (LD-PCR).

[0017] Based on the known start codon region of the cathelicidin family of antimicrobial peptides in Chiroptera, a forward primer (5'-ATGGAGACCCAGRGSRRCAGCC-3') was designed and synthesized. Combined with the reverse primer (5'-ATTCTAGAGGCCGAGGCGGCCGAC-3') provided by the kit, PCR amplification was performed. After completion, the target fragment was recovered and ligated into a plasmid vector. Competent cells were transformed, and positive colonies were screened and selected for nucleotide sequencing.

[0018] The coding genes of the active antimicrobial peptides MC-CATH1, MC-CATH2 and MC-CATH3 of Myotis sinensis can be used for polypeptide recombinant expression, transgenic animals, plants, animal cells or plant cells.

[0019] 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.

[0020] 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.

[0021] An application of the antimicrobial peptide derived from the myotis dione, and an application of the antimicrobial peptide derived from the myotis dione in antibacterial biofilm.

[0022] An application of the antimicrobial peptide derived from the myotis divaricata bat, and the application of the antimicrobial peptide derived from the myotis divaricata bat in the preparation of synergistic antibacterial and bacterial growth-inhibiting drugs, fungicides, antimicrobial preparations, animal feed, cosmetics, and preservatives.

[0023] The advantages of the present invention are:

[0024] The Myotis sinensis antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 of the present invention exhibit strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, with broad-spectrum and high efficacy. They also exhibit low hemolytic activity, minimal cytotoxicity, salt tolerance, and strong antibacterial biofilm activity. Furthermore, MC-CATH3 can kill bacteria by inducing a ferroptosis-like mechanism, making it the first antimicrobial peptide discovered in China and abroad to exert its bactericidal effect through a ferroptosis-like mechanism. MC-CATH1, MC-CATH2, and MC-CATH3 are applicable in the fields of medicine, cosmetics, food preservation, and aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 2 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 Staphylococcus aureus CMCC26003.

[0027] Figure 3This is a diagram showing the hemolytic activity effects of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the examples of the present invention.

[0028] Figure 4 The cytotoxic effects of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 1 .

[0029] Figure 5 The cytotoxic effects of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 2 .

[0030] Figure 6 The cytotoxic effects of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in the embodiments of the present invention Figure 3 .

[0031] Figure 7 This is a diagram showing the effectiveness of the antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 provided in an embodiment of the present invention in clearing the biofilm of Escherichia coli ATCC25922.

[0032] 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.

[0033] Figure 9 This is a diagram showing the effects of MC-CATH1, MC-CATH2, and MC-CATH3 solutions containing different salt ions on the MIC of Escherichia coli ATCC25922 provided in an embodiment of the present invention.

[0034] Figure 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.

[0035] Figure 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.

[0036] Figure 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 ROS accumulation in Escherichia coli ATCC25922 cells.

[0037] Figure 13 This is a diagram showing the effect of the antimicrobial peptides MC-CATH2 and MC-CATH3 provided in an embodiment of the present invention on promoting lipid peroxidation in Escherichia coli ATCC25922.

[0038] Figure 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

[0039] 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.

[0040] The Chinese myotis bats mentioned in the following examples were collected from Libo, Guizhou. The sample collection was approved by the animal ethics committee of the unit and did not involve endangered species. Example 1

[0041] Cloning of genes encoding the active peptides MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis:

[0042] (1) Total RNA extraction from lung tissue of Myotis sinensis:

[0043] ① Take 100 mg of lung tissue from Myotis sinensis, put it into a mortar and add liquid nitrogen to 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 thoroughly, and then centrifuge at 4°C, 12000 rpm for 10 minutes.

[0044] ② Centrifuge and take the supernatant, add 0.2 mL of chloroform solution, mix vigorously, let it stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000 rpm for 10 minutes and discard the precipitate.

[0045] ③ Add an equal volume of isopropanol to the supernatant, let it stand at room temperature for 10 minutes, centrifuge it at 4°C, 12000 rpm for 10 minutes, collect the precipitate, wash it once with 75% (v / v) ethanol, and dry it. The precipitate at the bottom of the tube is the total RNA of the lung tissue of Chinese mouse-eared bat.

[0046] (2) Synthesis of the second-strand cDNA from the lung tissue of the Chinese mouse-eared bat: The cDNA was synthesized using the CreatorTM SMARTTM cDNA Library Construction Kit from CLONTECH.

[0047] 1) First-strand cDNA synthesis (mRNA reverse transcription):

[0048] ① Add 1µL of total RNA from the lung tissue of Myotis sinensis, 1µL of 3' SMARTer CDS Primer, and 2.5µL of RNase-free water to an RNase-free PCR tube to a total volume of 4.5µL. Mix well and centrifuge briefly (2000rpm, 30s). After centrifugation, incubate at 72℃ for 3 minutes. After incubation, incubate the tube at 42℃ for 2 minutes.

[0049] ② Add the following reagents (all included in the CLONTECH Creator™ SMART™ cDNA Library Construction Kit) to the above microcentrifuge 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 microcentrifuge tube and briefly centrifuge (2000 rpm, 30 seconds). Incubate at 42°C for 90 minutes, then at 68°C for 10 minutes. After incubation, place the microcentrifuge tube on ice to terminate first-strand synthesis. Remove 2µL of the synthesized first-strand cDNA from the microcentrifuge tube for later use.

[0050] 2) Amplify the second strand using the long-terminal polymerase chain reaction (LD-PCR) method (all reagents used are provided in the Creator™ SMART™ cDNA Library Construction Kit from CLONTECH)

[0051] ① Mix 2µL first-strand cDNA, 80µL deionized water, 10µL 10× Advantage 2 PCR buffer, 2µL 50× dNTP mix, 2µL 5' PCR primer, 2µL CDS III / 3' PCR primer, and 2µL 50× Advantage 2 Polymerase Mix in a PCR tube preheated at 95°C.

[0052] ② Amplify in a PCR instrument according to the following procedure:

[0053] 95°C, 1 minute; 18 cycles: 95°C, 15 seconds, 65°C, 30 seconds, 68°C, 6 minutes. After the cycle is completed, the synthesized cDNA double-stranded in the centrifuge tube is stored at -80°C.

[0054] (3) Cloning of genes encoding the active peptides MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis:

[0055] A forward primer (5'-ATGGAGATCTGGCAGTGTGTGATAT-3') was designed based on the known start codon region of Chiroptera cathelicidin. The reverse primer was the 3'-PCR primer in the CLONTECH Creator™ SMART™ cDNA Library Construction Kit (5'-ATTCTAGAGGCCGAGGCGGCCGAC-3'). PCR reactions were performed under the following conditions: 95°C for 5 minutes, 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, for 30 cycles. After amplification, the target fragment was recovered using a gel extraction kit (Tiangen Biotechnology). The recovered target fragment was ligated into the pMD19-T vector (Takara, Dalian) and transformed into TOP10 competent cells. Plates were plated and selected with ampicillin. Single colonies were picked and the insert size was determined by PCR using the M13 primer. Positive colonies were selected, plasmids were extracted, and samples were sent for nucleotide sequencing.

[0056] Measurement results:

[0057] The sequence of the gene encoding the precursor of the MC-CATH1 active polypeptide of Myotis sinensis from the 5' end to the 3' end is:

[0058] ;

[0059] The nucleotide sequence of the gene encoding the precursor of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH1 is as follows: sequence length: 606 bases, sequence type: nucleic acid, number of strands: single-stranded, topology: linear, sequence type: cDNA, source: lung of the myotis diversifolia (Myotis diversifolia). Nucleotides 396-504 encode the mature peptide of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH1.

[0060] The sequence of the gene encoding the precursor of the MC-CATH2 active polypeptide of Myotis sinensis from the 5' end to the 3' end is:

[0061] ;

[0062] The nucleotide sequence of the gene encoding the precursor of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH2 is as follows: sequence length: 606 bases, sequence type: nucleic acid, number of strands: single-stranded, topology: linear, sequence type: cDNA, source: lung of the myotis diversifolia (Myotis diversifolia). Nucleotides 396-516 encode the mature peptide of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH2.

[0063] The sequence of the gene encoding the precursor of the MC-CATH3 active polypeptide of Myotis sinensis from the 5' end to the 3' end is:

[0064] ;

[0065] The nucleotide sequence of the gene encoding the precursor of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH3 is as follows: sequence length: 603 bases, sequence type: nucleic acid, number of strands: single-stranded, topology: linear, sequence type: cDNA, source: lung of the myotis diversifolia (Myotis diversifolia). Nucleotides 396-522 encode the mature peptide of the myotis diversifolia (Myotis diversifolia) active polypeptide MC-CATH3. Example 2

[0066] Preparation of MC-CATH1, MC-CATH2, and MC-CATH3:

[0067] (1) Chemical synthesis of MC-CATH1, MC-CATH2, and MC-CATH3: Based on the amino acid sequence deduced from the coding gene, the full sequence was synthesized using an automatic peptide synthesizer (433A, Applied Biosystems) and purified by desalting on a reverse-phase HPLC column.

[0068] (2) Molecular weight was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

[0069] (3) The purity of the purified polypeptide 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.

[0070] MC-CATH1 is a small molecule polypeptide encoded by a gene, consisting of 36 amino acid residues, with a molecular weight of 4134.76 Da and an isoelectric point of 12.15. Its amino acid sequence is:

[0071] 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 ;

[0072] MC-CATH2 is a small molecule polypeptide encoded by a gene, consisting of 40 amino acid residues, with a molecular weight of 4931.67 Da and an isoelectric point of 12.06. Its amino acid sequence is:

[0073] 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 ;

[0074] MC-CATH3 is a small molecule polypeptide encoded by a gene, consisting of 42 amino acid residues, with a molecular weight of 4984.76 Da and an isoelectric point of 10.63. Its amino acid sequence is:

[0075] 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

[0076] Antibacterial activity detection of MC-CATH1, MC-CATH2, and MC-CATH3:

[0077] 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, 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.

[0078] Add 90µL of MH liquid medium to the first well of a sterile 96-well plate, and pre-add 50µL of MH liquid medium to the remaining wells. Then, add 10µL of the Chinese myotis diversicolor 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. After mixing, take 50µL and add it to the second well. Dilute it in series, aspirate 50µL from the 8th well and discard it. The 9th well is the control tube.

[0079] Add 50 µL of diluted bacterial strain culture to each well. The 96-well plate was then incubated in a 37°C incubator with gentle shaking for 18 hours. The absorbance was measured at 600 nm. The minimum inhibitory concentration (MIC) was the lowest sample concentration at which bacterial growth was not observed. The results are shown in Table 1.

[0080] As shown in Table 1, MC-CATH1 and MC-CATH2 exhibited 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 exhibited 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.

[0081] Table 1. Antibacterial activity of MC-CATH1, MC-CATH2, and MC-CATH3

[0082] Example 4

[0083] Determination of the bactericidal rate of MC-CATH1, MC-CATH2, and MC-CATH3 antimicrobial peptides from Myotis sinensis:

[0084] The antibacterial peptides from Myotis sinensis were used to test the bactericidal rates of Gram-negative bacteria Escherichia coli ATCC25922 and Gram-positive bacteria Staphylococcus aureus CMCC26003, which have good antibacterial activity. The samples were cultured in MH liquid medium (Thermo Fisher Scientific, USA) at 37°C for 12 hours 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× the MIC (46.9µg / mL). The bacterial suspension containing the active peptide sample from Myotis diversicolor was incubated in a 37°C incubator with shaking. At 0, 15, 30, 60, 120, and 180 minutes, 50µL of the bacterial suspension was diluted 1000-fold. Then, 50µL of the diluted bacterial suspension was spread onto LB solid medium and incubated overnight at 37°C. Colonies were counted. Ampicillin was used as a positive control, and sterile deionized water was used as a negative control.

[0085] The results are as follows Figure 1 、 Figure 2As shown in Tables 2 and 3, MC-CATH1 and MC-CATH2 showed faster killing rates against E. coli ATCC25922 than the positive control, ampicillin. MC-CATH3 showed a comparable killing rate against E. coli ATCC25922, effectively killing all bacteria within 60 minutes. MC-CATH1 and MC-CATH2 also showed faster killing rates against S. aureus CMCC26003 than the positive control, ampicillin, effectively killing all bacteria within 180 and 120 minutes, respectively. MC-CATH3 showed a comparable killing rate against S. aureus CMCC26003 as the positive control, although not all bacteria were completely killed within 180 minutes. However, the killing rate against S. aureus CMCC26003 reached 99.8% compared to 0 minutes.

[0086] Table 2. Bactericidal rates of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 against Escherichia coli ATCC25922

[0087]

[0088] Table 3. Bactericidal rates of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 against Staphylococcus aureus CMCC26003

[0089] Example 5

[0090] Determination of hemolytic activity and cytotoxicity of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis:

[0091] (1) Hemolytic activity: Fresh human blood was collected and mixed with Aldrich's solution for anticoagulation, washed twice with normal saline and resuspended into 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, incubated at 37°C for 30 minutes, and then centrifuged at 1000 rpm for 5 minutes. The supernatant was measured for absorbance 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 MC-CATH1, MC-CATH2, and MC-CATH3 against bacteria, indicating that MC-CATH1, MC-CATH2, and MC-CATH3 have great application potential.

[0092] Table 4. Hemolytic activity of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3

[0093]

[0094] (2) Cytotoxicity: The in vitro cytotoxicity of MC-CATH1, MC-CATH2, and MC-CATH3 was determined using HepG2 (human hepatocellular carcinoma), HEK293T (human embryonic kidney) cells, and Raw 264.7 (mouse macrophage cell line) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 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 Cells were cultured overnight. Different concentrations of MC-CATH1, MC-CATH2, and MC-CATH3 were then added and incubated for 24 hours at 37°C in a 5% CO2 cell culture incubator. After incubation, 20 µL of 5 mg / mL MTT was added to each well of the 96-well plate. After an additional 4 hours of culture, the supernatant was discarded and DMSO was added. The absorbance was measured at 490 nm. The zero-adjustment group received only DMEM medium, while the negative control group received only cells without drug administration. The cytotoxicity percentage (C%) was equal to 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 2. Figure 4-Figure 6As shown in Tables 5-7, when the cell concentration was 2 µM, the lowest MC-CATH1 cell viability in HepG2 cells was 92.59%, while the other two cell lines were all above 95%. The lowest MC-CATH2 cell viability in HEK293T cells was 77.76%, while the other two cell lines were all above 84%. The lowest MC-CATH3 cell viability in HEK293T cells was 77.76%, while the other two cell lines were all above 90%. This indicates that MC-CATH1, MC-CATH2, and MC-CATH3 have weak cytotoxicity 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.

[0095] Table 5. Cytotoxicity of antimicrobial peptide MC-CATH1

[0096]

[0097] Table 6. Cytotoxicity of antimicrobial peptide MC-CATH2

[0098]

[0099] Table 7. Cytotoxicity of antimicrobial peptide MC-CATH3

[0100] Example 6

[0101] Anti-biofilm activity of MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis:

[0102] (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, USA) at 37°C for 12 hours and then diluted to 10% with new medium. 7 cfu / mL bacterial suspension, add 100µL bacterial suspension to each well of a 96-well plate and culture at 37°C for 48 hours. After removing the supernatant, wash three times with PBS, and add 100µL of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 with different concentration gradients in sequence and incubate for another 24 hours. After incubation, remove the supernatant, wash with PBS, fix with 50µL methanol, and add 0.1% crystal violet staining after drying. Finally, discard the staining solution, add anhydrous ethanol to dissolve the crystal violet, and measure the absorbance value at 600nm absorbance. 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 E. 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.

[0103] Table 8. Biofilm clearance ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3

[0104]

[0105] (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 Escherichia coli ATCC25922 biofilm. The strains were cultured in MH liquid medium (Thermo Fisher Scientific, USA) at 37°C for 12 hours and then diluted to 2×10 7 cfu / mL bacterial suspension was added to each well of a 96-well plate, and 50µL of different concentration gradients of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 were added at the same time and cultured at 37°C for 48 hours. The remaining steps were the same as those for biofilm removal. The zero adjustment 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 as follows 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 could reach about 69%. When MC-CATH2 was co-incubated with Escherichia coli, the inhibitory ability against biofilm at a concentration of 8×MIC could reach about 65%. When MC-CATH3 was co-incubated with Escherichia coli, the inhibitory ability against biofilm at a concentration of 8×MIC could reach about 57%. These results indicate that MC-CATH1, MC-CATH2, and MC-CATH3 have great potential for the treatment of biofilm-related infections.

[0106] Table 9. Biofilm inhibition ability of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3

[0107] Example 7

[0108] Salt tolerance of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 from Myotis sinensis:

[0109] (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, USA) at 37°C in an incubator for 12 hours, and then diluted to 2×10 5 cfu / mL. Then, MC-CATH1, MC-CATH2, and MC-CATH3 sample solutions were prepared using MH liquid 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. Figure 9 and shown in Table 10.

[0110] from Figure 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.

[0111] Table 10. MICs of MC-CATH1, MC-CATH2, and MC-CATH3 solutions containing different salt ions against Escherichia coli ATCC25922

[0112] Example 8

[0113] The antimicrobial peptides MC-CATH1 and MC-CATH2 from the Chinese mouse-eared bat exert their bactericidal effects by destroying bacterial cell membranes, while MC-CATH3 exerts its antibacterial effects by inducing bacterial ferroptosis:

[0114] (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, USA) in an incubator at 37°C for 12 hours, and then diluted to 1×10 8 cfu / mL bacterial suspension, take a 96-well plate, add 80µL to each well, and at the same time 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, the bacterial uptake of propidium iodide was detected at an excitation wavelength of 535nm and an emission wavelength of 617nm using a microplate reader, and the bacterial uptake of N-phenyl-1-naphthylamine was detected at an excitation wavelength of 350nm and an emission wavelength of 420nm, which respectively represent the degree of damage of the antimicrobial peptides to the inner and outer membranes of bacteria. The negative control group was a group without antimicrobial peptides. The results are shown in the table. Figure 10 、 Figure 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 them, the uptake of propidium iodide and N-phenyl-1-naphthylamine by MC-CATH1 and MC-CATH2 was significantly stronger than that of MC-CATH3.

[0115] Table 11. Disruptive effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 on the inner membrane of Escherichia coli ATCC25922

[0116]

[0117] Table 12. Disruptive effects of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 on the outer membrane of Escherichia coli ATCC25922

[0118]

[0119] (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 with new medium to 1×10 8cfu / mL bacterial suspension, and add the active oxygen detection probe DCFH-DA (Sigma-Aldrich Co., Ltd., USA) to make the final concentration reach 2µM. Take a 96-well plate, add 90µL to each well, and at the same time add 10µL of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 with different concentration gradients. After incubation 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 active oxygen. The negative control group is the group without adding antimicrobial peptides. The experimental results are as follows: Figure 12 As shown in Table 13, MC-CATH1, MC-CATH2, and MC-CATH3 can all 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 amounts of reactive oxygen species at a concentration of 1×MIC, which is significantly higher than that of MC-CATH1 and MC-CATH2.

[0120] Table 13. Antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 induce ROS accumulation in Escherichia coli ATCC25922 cells

[0121]

[0122] (3) Both MC-CATH2 and MC-CATH3 can promote bacterial lipid peroxidation more effectively: 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 (Germany Cayman Chemical Co., Ltd.) to make the final concentration reach 5µM. Take a 96-well plate, add 90µL to each well, and at the same time add 10µL of antimicrobial peptides MC-CATH1, MC-CATH2, and MC-CATH3 with different concentration gradients. After incubation at 37°C for 60 minutes, centrifuge and wash the bacterial solution twice with PBS. Use a microplate reader to detect the fluorescence intensity of C11-bodipy581 / 591 probe in bacterial cells at an excitation wavelength of 488nm and an emission wavelength of 510nm to represent the level of bacterial lipid peroxidation. The negative control group is a group without antimicrobial peptides. The experimental results are as follows: Figure 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.

[0123] Table 14. Antimicrobial peptides MC-CATH2 and MC-CATH3 promote lipid peroxidation in Escherichia coli ATCC25922

[0124]

[0125] (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 with new medium 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 200W power, repeated 30 times, and centrifuged at 8000 rpm for 10 minutes at 4°C. The intracellular iron content of the bacteria was then detected according to the steps of the Cell Iron Content Detection Kit (Beijing Solebold Biotechnology Co., Ltd.). No antimicrobial peptide was added as a negative control. The results are as follows Figure 14 As shown in Table 15, MC-CATH3 can promote bacteria to accumulate more iron ions, and the iron content of MC-CATH1 and MC-CATH2 is comparable to that of the control group.

[0126] Table 15. Antimicrobial peptide MC-CATH3 induces intracellular iron accumulation in Escherichia coli ATCC25922

[0127]

[0128] 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. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the protection scope of the present invention.

Claims

1. An antibacterial peptide derived from Myotis chinensis, characterized in that: The antibacterial peptides derived from Myotis chinensis 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 -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 。 2. Use of the antibacterial peptide derived from Myotis chinensis described in claim 1, characterized in that: The application of the antibacterial peptides derived from Myotis chinensis in the preparation of drugs for inhibiting Gram-positive bacteria and Gram-negative bacteria.

3. Use of the antibacterial peptide derived from Myotis chinensis according to claim 2, characterized in that: The Gram-positive bacteria include at least one of Enterococcus faecalis, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Staphylococcus epidermidis; the Gram-negative bacteria include at least one of Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae.

4. Use of the antibacterial peptide derived from Myotis chinensis described in claim 1, characterized in that: The application of the antibacterial peptides derived from Myotis chinensis in the preparation of animal feed, cosmetics and anti-corrosion and preservation.

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