Antibacterial neuropeptide and application thereof

Abstract

The invention belongs to the technical field of biochemistry and biological medicine, and discloses an antibacterial neuropeptide Urocortin III and application thereof, and the antibacterial neuropeptide Urocortin III has an amino acid conserved sequence of xxxTLSLDVPTNxMNxLFxxxKAKNxRAxAxxNAxLx AQI-NH2. The antibacterial neuropeptide Urocortin III synthesized by the invention has obvious inhibition and killing effects on important pathogenic bacteria of fishes and human beings, such as vibrio splendidus, vibrio harveyi, streptococcus agalactiae, bacillus subtilis, escherichia coli and staphylococcus aureus, and has relatively good inhibition and killing effects on gram-positive bacteria and gram-negative bacteria; the compound can be used for research and development of novel bacteriostatic and bactericidal drugs or compositions, and has good application prospects in the fields of aquatic fishery drugs, fish feeds, human food additives, cosmetics, medicines and the like.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biochemistry and biomedicine, and particularly relates to an antibacterial neuropeptide and its application. Background Art

[0002] Bacterial infections cause a wide variety of diseases in the fields of human health and fishery farming, with a wide impact. In the face of bacterial infectious diseases, traditional treatment methods often rely on the application of a large number of antibiotics. However, with the increasingly severe problem of antibiotic resistance, researchers are turning their focus to the development of new antibacterial active substances in order to find effective alternatives to antibiotics. Against this background, endogenous polypeptides are becoming new potential candidates. Antimicrobial peptides have been discovered by scholars due to their unique structure and antibacterial principle. Antimicrobial peptides are an important part of the innate immune system and are small molecules with antibacterial, antifungal, antiparasitic, and antiviral activities. Many antimicrobial peptides have been reported. Recent studies have revealed that certain neuropeptides secreted by nerve cells exhibit broad-spectrum antibacterial activity. These neuropeptides are significantly similar to known antimicrobial peptides in terms of molecular size, structural configuration, amino acid sequence, amphiphilic characteristics, and net cationic charge. Experiments have proven that these neuropeptides do have antibacterial ability in vitro. In addition, neuropeptides play a potential role in regulating immune responses and neuroinflammatory processes. They can not only directly fight infections but also provide protection for nerve tissues against microbial invasion. This discovery provides a new perspective and potential molecular basis for the development of new antibacterial strategies.

[0003] The mechanism of action of antibacterial neuropeptides is similar to that of antimicrobial peptides, and the process includes: ① electrostatic binding of positively charged antimicrobial peptides to the negatively charged surface of microorganisms; ② destabilization of the negatively charged phospholipid bilayer leading to the destruction of membrane integrity; ③ change in membrane permeability; ④ death of microorganisms due to hypotonicity. Research has shown that the topological (rather than linear) amphiphilic structure of polypeptides will directly affect their ability to permeate membranes. Therefore, the regular clustered distribution of charges on the surface of polypeptides is the basis for their antibacterial ability. Compared with traditional antibiotics that have only one target, antibacterial neuropeptides have multiple targets and can destroy pathogens from more directions, greatly reducing the emergence of drug-resistant bacteria. Antibacterial neuropeptides are more difficult to develop drug resistance than antibiotics. The mechanisms for treating drug-resistant bacterial infections can be summarized into the following five categories: biofilm penetration, resensitization, intracellular bacteriostatic function, immune activity regulation, and biofilm inhibition. This unique antibacterial mechanism is not easy to cause bacterial drug resistance, has no toxicity to normal cells and tissues, and there will be no residue problem. It is expected to replace antibiotics as a new type of highly effective antibacterial drug. Therefore, researching more types of antibacterial neuropeptides is of great significance for the treatment of bacterial infections and the development of antibiotic alternatives. Summary of the Invention

[0004] The object of the present invention is to provide an antibacterial neuropeptide and its application, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: an antibacterial neuropeptide, the neuropeptide includes neuropeptide Urocortin III, and the amino acid conserved sequence of the neuropeptide Urocortin III is xxxTLSLDVPTNxMNxLFxxxKAKNxRAxAxxNAxLxAQI-NH2 (SEQ ID NO.1);

[0006] Among them, the first x is S or T; the second x is K, R or Q; the third x is F, L or V; the 13th x is I or L; the 16th x is I, V or L; the 19th x is D or N; the 20th x is I or V; the 21st x is A or D; the 26th x is L or M; the 29th x is K or Q; the 31st x is A or D; the 32nd x is A, E or D; the 35th x is R, Q or H; the 37th x is L or M.

[0007] Preferably, the amino acid sequence of the neuropeptide Urocortin III is SRLTLSLDVPTNIMNVLFDVAKAKNLRAKAAENARLLAQI-NH2 (SEQ ID NO.2).

[0008] Preferably, the amino acid sequence of the neuropeptide Urocortin III is TKFTLSLDVPTNIMNVLFNIAKAKNLRAKAAANAQLMAQI-NH2 (SEQ ID NO.3).

[0009] A gene encoding the neuropeptide Urocortin III of SEQ ID NO.2, with the sequence: ATGCTGATGCCGGTCCACTTCCTGCTGCTCCTGCTGCTGCTCCTGGGGGGCCCCAGGACAGGCCTCCCCCACAAGTTCTACAAAGCCAAGCCCATCTTCAGCTGCCTCAACACCGCCCTGTCTGAGGCTGAGAAGGGCCAGTGGGAGGATGCATCCCTGCTGAGCAAGAGGAGCTTCCACTACCTGCGCAGCAGAGACGCCTCTTCGGGAGAGGAGGAGGAGGGCAAAGAGAAAAAGACTTTCCCCATCTCTGGGGCCAGGGGTGGAGCCAGAGGCACCCGGTACAGATACGTGTCCCAAGCACAGCCCAGGGGAAAGCCACGCCAGGACACGGCCAAGAGTCCCCACCGCACCAAGTTCACCCTGTCCCTCGACGTCCCCACCAACATCATGAACCTCCTCTTCAACATCGCCAAGGCCAAGAACCTGCGTGCCCAGGCGGCCGCCAATGCCCACCTGATGGCGCAAATTGGGAGGAAGAAGTAG(SEQ ID NO.4).

[0010] A gene that encodes the neuropeptide Urocortin III with SEQ ID NO.3, and its sequence is: ATGCTGTCGTCCCTAAAGACCCTGCTGCTGTTCTCGGTCCTGTGCACACCGACCTCCAGCCTGTGCCTCCGTCTCTACCAGACGCGCCCCGACCTCCTCTGCGACGACCAGCTGGCGCTCGGGGTCCGGAATGACCAAACTGAGCCTGGTTACTCCACTGTGGACGGCTGGGGATCCCTCATGCAGTCCGCGGAGTATCTCTCCTCCTCATCCTCCACGTCGTCGTCCTCTTTCTCCTCTGCTGAATCCAGTCGGGAAAAAAGGACTTCAAGTCCCGCAAACTACCGCTTCTTGAGCCGGACCAAGCTCAGGGGCCAGATGCTCCGCAACAGCAACAAAGGGGACCGGAGGAGCAGACTGACCCTGTCCCTGGACGTCCCGACCAACATCATGAACGTGCTCTTTGATGTGGCCAAGGCCAAAAACCTGCGAGCCAAGGCAGCTGAGAACGCACGTCTGCTGGCGCAAATCGGACGGAGGAAATAA(SEQ ID NO.5).

[0011] A pharmaceutical composition comprising the neuropeptide Urocortin III as claimed in claim 1.

[0012] Preferably, the neuropeptide Urocortin III comprises the amino acid sequence as claimed in claim 2 or 3.

[0013] Use of an antibacterial neuropeptide in the preparation of aquatic feeds, fishery drugs, human food additives, cosmetics and medicines.

[0014] Use of a pharmaceutical composition in the preparation of aquatic feeds, fishery drugs, human food additives, cosmetics and medicines.

[0015] Use of an antibacterial neuropeptide or a pharmaceutical composition in the preparation of drugs for the treatment of tropical fish meningoencephalitis, neonatal meningoencephalitis and septicemia.

[0016] An antibacterial or bactericidal composition comprising a neuropeptide or a pharmaceutical composition, which has inhibitory and killing effects on Vibrio splendidus, Vibrio harveyi, Streptococcus agalactiae, Bacillus subtilis, Escherichia coli and Staphylococcus aureus.

[0017] Technical effects and advantages of the present invention: The neuropeptide Urocortin III (hereinafter referred to as UCN3) provided by the present invention has very sensitive antibacterial and bactericidal activities against Vibrio splendidus, Vibrio harveyi, Escherichia coli, Streptococcus agalactiae, Bacillus subtilis and Staphylococcus aureus, and can be used to prepare antibacterial or bactericidal drugs or compositions, and can be used in the fields of aquatic fishery drugs, fish feed, as well as human food additives, cosmetics and medicine. It can also be used to prepare drugs for preventing and treating tropical fish meningoencephalitis, treating neonatal meningoencephalitis and septicemia, promoting wound healing and other diseases, so as to solve the problem of antibiotic resistance. Description of the Drawings

[0018] Figure 1 It is a comparison diagram of the conserved amino acid sequences of the neuropeptide UCN3 precursor proteins from different species in Example 1. Among them, the arrow indicates the UCN3 of tilapia, and the dotted box indicates the amino acid sequence of the mature neuropeptide UCN3 of each species;

[0019] Figure 2 It is a result diagram of the amino acid conserved sequence analysis of the neuropeptide UCN3 from different species in Example 1;

[0020] Figure 3 It is a liquid chromatography-mass spectrometry detection diagram of the neuropeptide UCN3 from tilapia in Example 2;

[0021] Figure 4 It is a detection result diagram of the minimum inhibitory concentration (MIC) of the neuropeptide UCN3 from tilapia against various bacteria in Example 3;

[0022] Figure 5 It is a detection result diagram of the half inhibitory concentration (IC 50 ) of the neuropeptide UCN3 from tilapia against various bacteria;

[0023] Figure 6 It is an ultrastructural observation of the damage effect of the neuropeptide UCN3 from tilapia on various bacteria in Example 4; The scale bar is 1μm;

[0024] Figure 7 It is a plate culture result diagram after co-culturing the human-derived neuropeptide UCN3 with Escherichia coli (A), Streptococcus agalactiae (B) and Staphylococcus aureus (C) in Example 6;

[0025] Figure 8 It is a cytotoxicity analysis of the neuropeptide UCN3 from tilapia against various bacteria in Example 7. Detailed Embodiments

[0026] The following further describes the specific embodiments of the present invention in conjunction with the accompanying drawings. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation on the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Example 1:

[0028] Through bioinformatics analysis of the sequence SRLTLSLDVPTNIMNVLFDVAKAKNLRAKAAENARLLAQI-NH2 (SEQ ID NO.2), we found that the polypeptide encoded by it is very similar to antimicrobial peptides in terms of structure, amphiphilicity, and net positive charge, and may have antimicrobial activity. Subsequently, we retrieved the amino acid sequences of the precursor proteins of neuropeptide UCN3 from different species on the NCBI website. These species include: Homo sapiens, Mus musculus, Xenopus tropicalis, Danio rerio, Oreochromis niloticus, Thunnus maccoyii, Nibea albiflora, and Larimichthys crocea. The alignment results are as Figure 1 shown, and the dotted square indicates the amino acid sequences of the mature neuropeptide UCN3 of each species.

[0029] Further in-depth analysis found that the amino acid conserved sequence of neuropeptide UCN3 is as Figure 2 shown, which is xxxTLSLDVPTNxMNxLFxxxKAKNxRAxAxxNAxLxAQI-NH2 (SEQ ID NO.1), where the first x is S or T; the second x is K or R or Q; the third x is F or L or V; the 13th x is I or L; the 16th x is I or V or L; the 19th x is D or N; the 20th x is I or V; the 21st x is A or D; the 26th x is L or M; the 29th x is K or Q; the 31st x is A or D; the 32nd x is A or E or D; the 35th x is R or Q or H; the 37th x is L or M.

[0030] Subsequent experiments showed that UCN3 from different sources all had excellent antimicrobial activity, and through retrieval, it was found that the amino acid sequence of neuropeptide UCN3 was significantly different from known antimicrobial peptides, making it a new antimicrobial neuropeptide.

[0031] Example 2:

[0032] According to the amino acid conserved sequence of urocortin 3 (UCN3) derived from tilapia, as Figure 1 indicated by the arrow in 193 H 334 N 58 O 55 S1, the UCN3 polypeptide was synthesized by C-terminal amide synthesis method. The amino acid sequence is SRLTLSLDVPTNIMNVLFDVAKAKNLRAKAAENARLLAQI-NH2 (SEQ ID NO.2). The molecular formula is C

[0033] The obtained image is as Figure 3 shown. In the liquid chromatography diagram, UCN3 eluted at a retention time of 8.021 min. According to the peak area normalization method analysis, its purity was higher than 95%. At the same time, the mass spectrometry diagram detected the [M+3H] 3+ , [M+4H] 4+ , [M+5H] 5+ and [M+6H] 6+ mass-to-charge ratios (m / Z) of 1460.50, 1095.70, 876.75 and 730.85 respectively. The relative molecular mass (M) of UCN3 deduced from the mass-to-charge ratio was 4378.50, 4378.80, 4378.10 and 4379.10. The fitting detection value was 4378.80, which was consistent with the theoretical value of 4379.17. Therefore, it was indicated that the synthesized neuropeptide UCN3 sequence was correct and reliable.

[0034] Example 3: Antibacterial activity assay was performed on the synthesized tilapia-derived neuropeptide UCN3 in Example 2:

[0035] Bacteria acquisition: Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 23857), Streptococcus agalactiae (ATCC 51487) and Staphylococcus aureus (ATCC 12600) were purchased commercially; Vibrio splendidus and Vibrio harveyi were provided by the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0036] Escherichia coli, Bacillus subtilis and Staphylococcus aureus were activated using LB medium (formula: 1% tryptone, 0.5% yeast extract, 1% sodium chloride); Vibrio splendidus and Vibrio harveyi were activated using TSB medium containing 1.5% sodium chloride (formula: 1.7% tryptone, 0.3% soy peptone, 4% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.25% glucose); Streptococcus agalactiae was activated using BHI medium (formula: 0.4% bovine brain infusion powder, 0.4% bovine heart infusion powder, 0.5% peptone, 1.6% casein peptone, 0.5% sodium chloride, 0.2% glucose, 0.25% disodium hydrogen phosphate). After inoculation, the cultures were incubated overnight at 150 rpm and 37 °C on a shaker. Subsequently, the overnight cultures were diluted 1 / 100 into fresh corresponding media and cultured at 37 °C until the absorbance at 600 nm reached 0.4 - 0.6 (mid-exponential growth phase). The supernatant was discarded by centrifugation, and the bacterial impurities were washed with PBS; the supernatant was discarded by centrifugation, and the bacterial solution was fixed with 1 mL of PBS, pipetted and mixed evenly to dilute the bacterial solution to 10 6 CFU / mL; A series of working solutions with neuropeptide UCN3 concentrations of 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM were prepared in advance, and a negative control (PBS) was set simultaneously; 50 μL of the series of working solutions was mixed with 50 μL of the working dilution of the bacterial solution, and they were co-incubated at 150 rpm and 37 °C for 3 h. The reaction was stopped by adding cold PBS solution (dilution 1 / 10) on ice for 10 min. For the above bacterial solutions (UCN3 actual concentrations were 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM), 90 μL of fresh medium was added to a 96-well plate and cultured for 2 - 6 h, and the OD 600 value was measured using an enzyme-linked immunosorbent assay reader. The experiment was repeated 3 times for determination.

[0037] The antibacterial effect was expressed by the bacterial survival rate: Bacterial survival rate (%) = [(OD value after treatment with the peptide - background OD of the medium) / (OD value without the peptide - background OD of the medium)] × 100%. With the bacterial survival rate as the ordinate and the actual concentration of neuropeptide UCN3 in the co-incubated bacterial solution as the abscissa, an MIC graph was made. As 600 shown, neuropeptide UCN3 had excellent antibacterial effects against various bacteria. The MIC values for Vibrio splendidus, Escherichia coli, Vibrio harveyi, Bacillus subtilis and Streptococcus agalactiae were 32 μM, and the MIC value for Staphylococcus aureus was 64 μM (see Table 1); In addition, the antibacterial activity could be evaluated by the half-maximal inhibitory concentration (IC 600 50%); With the bacterial inhibition rate as the ordinate and log(actual concentration of neuropeptide UCN3 in the co-incubated bacterial solution) as the abscissa, as Figure 4 shown, 50 ) assessment: Figure 5Shown: Using GraphPad Prism to fit the IC 50 curve, the fitted IC 50 value of Vibrio splendidus was 1.66 ± 1.09 μM, the fitted IC 50 value of Vibrio harveyi was 5.11 ± 1.17 μM, the fitted IC 50 value of Escherichia coli was 4.21 ± 1.11 μM, the fitted IC 50 value of Streptococcus agalactiae was 8.31 ± 1.19 μM, the fitted IC 50 value of Bacillus subtilis was 8.82 ± 1.18 μM, and the fitted IC 50 value of Staphylococcus aureus was 16.6 ± 1.24 μM (see Table 1). From the MIC values and IC 50 values, it can be seen that neuropeptide UCN3 has excellent antibacterial and bactericidal activities against Vibrio splendidus, Vibrio harveyi, Streptococcus agalactiae, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Compared with the MIC and IC 50 values of antibacterial peptides reported by predecessors, UCN3 has a higher ability to inhibit and kill Vibrio splendidus, Vibrio harveyi, Escherichia coli, Streptococcus agalactiae, Bacillus subtilis, and Staphylococcus aureus.

[0038] Table 1 Antibacterial activities of the antibacterial neuropeptide UCN3 derived from tilapia.

[0039]

[0040] Example 4: Ultrastructural observation of the effect of the tilapia-derived neuropeptide UCN3 synthesized in Example 2 on bacterial damage:

[0041] The effect of the antibacterial peptide on bacteria was observed using a transmission electron microscope. Each bacterium was cultured to the logarithmic growth phase, the bacterial liquid was taken, the bacteria were washed twice with PBS buffer, resuspended with PBS buffer, and a bacterial suspension with a bacterial concentration of 10 8 CFU / mL was prepared. The bacterial liquid was divided into two groups: a control group (PBS) and a UCN3 treatment group (UCN3 concentration was 2 × IC50). Incubate at 37 °C for 3 h, centrifuge to discard the supernatant, wash twice with PBS buffer, fix the treated bacteria with glutaraldehyde, wash with PBS, dehydrate with a series of ethanol gradients, infiltrate with different ratios of acetone epoxy resin, embed, section and stain, and observe the morphological changes of the bacteria with a transmission electron microscope to analyze the effect of the antibacterial peptide on bacterial damage.

[0042] The results are as Figure 6As shown, the results of transmission electron microscopy observation revealed significant differences in bacterial cells between the control group and the UCN3-treated group. The bacterial cells in the control group presented a complete appearance, with a smooth and continuous surface and uniform electron density. In contrast, the bacterial cells in each group treated with UCN3 showed obvious morphological changes: the cell morphology was significantly deformed, the cell membrane was abnormal and began to disintegrate, and the cell wall became wrinkled and rough. In addition, vacuoles appeared inside the cells, the cell volume swelled, and there was leakage of cell contents, with uneven distribution of electron density. These observation results together confirmed the severe damaging effect of the antimicrobial peptide UCN3 on the bacterial cell membrane, which destroyed the integrity of the cell membrane, led to the leakage of cell contents, and thus achieved the bactericidal effect.

[0043] Example 5:

[0044] The human-derived neuropeptide UCN3 was used as a representative to detect its antibacterial activity. The human neuropeptide UCN3 was synthesized by C-terminal amidation method, and its amino acid sequence was TKFTLSLDVPTNIMNVLFNIAKAKNLRAKAAANAQLM AQI-NH2 (SEQ ID NO.3).

[0045] Using the method of Example 3, the antibacterial activity of the human neuropeptide UCN3 against Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus was tested, and the MIC values and IC 50 values of different bacteria were calculated.

[0046] The experimental results are shown in Table 2. The MIC values of the human neuropeptide UCN3 against Escherichia coli and Streptococcus agalactiae were 32 μM, and the MIC value against Staphylococcus aureus was 64 μM, achieving a similar antibacterial effect to the neuropeptide UCN3 derived from tilapia. The IC 50 values of the human neuropeptide UCN3 against Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus were 6.92 ± 1.79 μM, 14.46 ± 3.13 μM, and 21.5 ± 2.48 μM, respectively. Compared with the neuropeptide UC

[0047] N3 derived from tilapia, the antibacterial activity was slightly lower, but still showed excellent antibacterial and bactericidal activities.

[0048] Table 2 Antibacterial activity of the human antibacterial neuropeptide UCN3.

[0049]

[0050] Example 6: The co-culture-plate coating method was used to further verify the antibacterial effect of the human neuropeptide UCN3 synthesized in Example 5:

[0051] Activate Escherichia coli (Escherichia coli, ATCC 25922), Streptococcus agalactiae (ATCC 51487), and Staphylococcus aureus (ATCC 12600), and culture them overnight at 150 rpm and 37 °C. The next day, culture them again until the absorbance value (OD value) at 600 nm is between 0.4 and 0.6 (mid-exponential growth phase), centrifuge to discard the supernatant, and wash with PBS; centrifuge to discard the supernatant, and resuspend the bacterial solution with 1 mL of PBS, pipette and mix well to dilute the bacterial solution to 10 6 CFU / ml; Prepare a series of working solutions of neuropeptide UCN3 with concentrations of 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM in advance, and set a negative control (PBS) at the same time; Take 50 μL of the series of working solutions and mix them with 50 μL of the diluted bacterial solution for co-culture, incubate at 150 rpm and 37 °C for 3 h in a microplate oscillator, stop the reaction by adding cold PBS solution (dilution 1 / 10) on ice for 10 min, take 100 μL of the bacterial solution (the actual concentration of UCN3 is 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM), and perform strict aseptic operation in a laminar flow hood, spread it on a solid medium, and culture it overnight in a 37 °C incubator, then take it out and count.

[0052] As Figure 7 shown, when the actual concentration of UCN3 is 32 μM, no colony growth was observed in Escherichia coli and Streptococcus agalactiae; while when the actual concentration of UCN3 is 64 μM, almost no colony growth was observed in Staphylococcus aureus. The above results correspond to the OD 600 measurement results in Example 5.

[0053] Example 7: Cytotoxicity detection of the novel antimicrobial peptide UCN3 (the tilapia-derived neuropeptide UCN3 synthesized in Example 2):

[0054] Resuscitated HEK 293T cells were prepared with complete medium (its formula is: 90% DMEM, 10% fetal bovine serum, 100 U / mL penicillin-streptomycin) to a density of 3 - 5×10 4A cell suspension of [[[number]]] cells / mL was taken, and 100 μL per well was added to a 96-well plate and cultured overnight at 37 °C until the cell confluence was about 50%. Working solutions of the UCN3 series with concentrations of 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM were prepared with complete medium, and a negative control (PBS) was set simultaneously. The original cell culture medium in the 96-well plate was aspirated, and 100 μL per well of the above working solution was added and cultured overnight at 37 °C. Then, 10 μL of CCK-8 solution was added to each well. After culturing in an incubator for 1 hour, the absorbance OD at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. 450 The calculation formula for the CCK-8 cell survival rate is: survival rate (100%) = (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) × 100%. Among them, the OD value of the experimental group is the absorbance value of the cells after treatment with the antimicrobial peptide, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0055] The results are as Figure 8 shown. When the concentration of the antimicrobial peptide neuropeptide UCN3 was 2 μM, compared with the control group, it could significantly improve cell activity and promote cell growth; when the concentration of the antimicrobial peptide was 1 μM, 4 μM, 8 μM, 16 μM, 32 μM, and 64 μM, there was no significant difference in cell activity compared with the control group. Generally speaking, when the concentration of the antimicrobial peptide neuropeptide UCN3 was less than or equal to 64 μM (greater than the MIC values of most bacteria), it showed low cytotoxicity.

[0056] In summary, as can be seen from the above examples, the antimicrobial neuropeptide UCN3 in the present invention can be obtained by chemical synthesis. This antimicrobial peptide has strong antibacterial and bactericidal activities against both Gram-positive and Gram-negative bacteria. This antimicrobial peptide neuropeptide is simple to obtain and has good water solubility. It can be applied to fields such as medicine, cosmetics, food preservation, and aquaculture in the future. Compared with other antimicrobial peptides, UCN3 shows superior antibacterial and bactericidal abilities, and the antibacterial concentration is more sensitive. In particular, the MIC value for Vibrio splendidus is as low as 32 μM, and the IC 50 is as low as 1.66 μM. The MIC values for Escherichia coli, Vibrio splendidus, Vibrio harveyi, and Streptococcus agalactiae are lower than 32 μM, and the IC 50 is lower than 10 μM.

[0057] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibacterial neuropeptide, characterized in that: The neuropeptide includes Urocortin III, and the amino acid sequence of Urocortin III is xxxTLSLDVPTNxMNxLFxxxKAKNxRAxAxxNAxLxAQI-NH 2; wherein, the x at the 1st position is S or T; the x at the 2nd position is K, R or Q; the x at the 3rd position is F, L or V; the x at the 13th position is I or L; the x at the 16th position is I, V or L; the x at the 19th position is D or N; the x at the 20th position is I or V; the x at the 21st position is A or D; the x at the 26th position is L or M; the x at the 29th position is K or Q; the x at the 31st position is A or D; the x at the 32nd position is A, E or D; the x at the 35th position is R, Q or H; the x at the 37th position is L or M.

2. An antibacterial neuropeptide according to claim 1, characterized in that: The amino acid sequence of the neuropeptide Urocortin III is SRLTLSLDVPTNIMNVLFDVAKAKNLRAKAAENARLLAQI-NH2.

3. An antibacterial neuropeptide according to claim 1, characterized in that: The amino acid sequence of the neuropeptide Urocortin III is TKFTLSLDVPTNIMNVLFNIAKAKNLRAKAAANAQLMAQI-NH2.

4. A gene, characterized in that: The gene encodes the neuropeptide Urocortin III described in claim 2.

5. A gene, characterized in that: The gene encodes the neuropeptide Urocortin III described in claim 3.

6. A pharmaceutical composition, characterized in that: The pharmaceutical composition includes the neuropeptide Urocortin III as described in claim 1.

7. Use of a neuropeptide according to any one of claims 1-3 in the preparation of aquatic feed, fishery drugs and human food additives.

8. Use of a pharmaceutical composition according to claim 6 in the preparation of aquatic feed, fishery drugs and human food additives.

9. Use of a neuropeptide according to any one of claims 1-3 or a pharmaceutical composition according to claim 6 in the preparation of drugs for treating tropical fish meningoencephalitis and septicemia.

10. An antibacterial or bactericidal composition, characterized in that: It includes a neuropeptide according to any one of claims 1-3 or a pharmaceutical composition according to claim 6.

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