Neuropeptide UTS1 variant and application thereof

Abstract

The invention belongs to the technical field of biochemistry and biological medicine, and discloses a neuropeptide UTS1 variant which is [R39, K40]-UTS1, the [R39, K40]-UTS1 is obtained by optimizing 39 and 40 amino acid sites of the neuropeptide UTS1 into R and K, and the amino acid conserved sequence of the [R39, K40]-UTS1 is SEEPPLSIDLTFHLLRNMIQMAKMESQREQAQLNRKVLRKV-NH2. The [R39, K40]-UTS1 provided by the invention has obvious effects of inhibiting and killing vibrio splendidus, vibrio harveyi, bacillus subtilis, streptococcus agalactiae, escherichia coli and staphylococcus aureus, has a better inhibiting effect on gram-positive bacteria and gram-negative bacteria, can be used for researching and developing novel bacteriostatic and bactericidal drugs or compositions, and has wide application prospects. The method has good application potential in the fields of aquatic fishery medicines or human 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 a neuropeptide UTS1 variant and its application. Background Art

[0002] Bacteria are ubiquitous and pose a great harm to human health and livestock, aquatic product aquaculture. Among them, Streptococcus agalactiae, as a zoonotic bacterium, causes fish septicemia and neonatal meningitis. Some Escherichia coli infections in the human body result in diseases such as urinary tract infections and septicemia. Vibrio splendidus and Vibrio harveyi are both common marine conditional pathogens and important pathogenic bacteria causing bacterial diseases in humans and aquatic animals, and have once caused outbreaks of diseases in marine cultured fish and shrimp worldwide. With the abuse and blind use of traditional antibiotics in fields such as medicine and aquaculture, microorganisms have developed increasing tolerance to traditional antibiotics.

[0003] Therefore, finding new antimicrobial agents is an important breakthrough to solve antibiotic resistance. Antimicrobial peptides have a simple structure and are a type of small molecule polypeptide, which show good inhibitory or killing effects on many harmful microorganisms such as bacteria, fungi, and viruses. Its principle is to destroy the integrity of the microbial cell membrane, resulting in the dissolution of intracellular substances and ultimately causing the death of bacteria. This mechanism does not cause toxicity to normal mammalian cells and there is no residue problem, so it is not easy to cause microbial drug resistance. Therefore, antimicrobial peptides are expected to become a new type of highly effective antibacterial drug, which has important practical significance for the prevention and treatment of fish septicemia, sea cucumber skin ulcer syndrome, and the treatment of neonatal meningitis, and has broad application potential in the development of antibacterial agents. Summary of the Invention

[0004] The purpose of the present invention is to provide a neuropeptide UTS1 variant and its application to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A neuropeptide UTS1 variant, the neuropeptide UTS1 variant is [R 39 ,K 40 -UTS1, which is obtained by optimizing the 39th and 40th amino acid sites of neuropeptide UTS1 to R and K, and its amino acid conserved sequence is SEEPPLSIDLTFHLLRNMIQMAKMESQREQAQLNRKVLRKV-NH2 (SEQ ID NO.1).

[0006] Preferably, the [R 39 ,K40 -The molecular formula of UTS1 is C 211 H 359 N 65 O 61 S3, with a molecular weight of 4878.68, an isoelectric point of 10.82, a net charge number of +3.1, and an average hydrophobicity of the polypeptide of 0.353.

[0007] Preferably, in any of the above solutions, all the amino acids are L-type.

[0008] A preparation comprising a neuropeptide UTS1 variant.

[0009] A pharmaceutical composition comprising a neuropeptide UTS1 variant.

[0010] Preferably, the pharmaceutical composition has the function of inhibiting or killing Gram-positive bacteria and Gram-negative bacteria.

[0011] The application of a neuropeptide, a bacterial agent, and a pharmaceutical composition in the preparation of aquatic feed and fishery drugs.

[0012] The application of a neuropeptide, a bacterial agent, and a pharmaceutical composition in the preparation of drugs for preventing and treating fish septicemia and sea cucumber skin ulcer syndrome.

[0013] The technical effects and advantages of the present invention: The neuropeptide UTS1 variant [R 39 ,K 40 -UTS1 has good water solubility and very sensitive antibacterial and bactericidal activities against Vibrio splendidus, Vibrio harveyi, Escherichia coli, Streptococcus agalactiae, Bacillus subtilis, and Staphylococcus aureus. It can be used to prepare antibacterial / bactericidal drugs or compositions for aquatic fishery drugs or human cosmetics and medicine, etc. It can also be used to prepare drugs for preventing and treating tropical fish septicemia and treating neonatal meningitis and other diseases to solve the problem of antibiotic resistance and provide a new way for the research and development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the three-dimensional structure of [R 39 ,K 40 -UTS1 in Example 1;

[0015] Figure 2 It is the liquid chromatography-mass spectrometry detection chart of [R 39 ,K 40 -UTS1 in Example 2; wherein, A is the chromatogram and B is the mass spectrum;

[0016] Figure 3 It is [R 39 ,K 40-UTS1 minimum inhibitory concentration (MIC) test results against various bacteria;

[0017] Figure 4 For [R 39 ,K 40 -UTS1 in Example 3 50 -UTS1 half maximal inhibitory concentration (IC

[0018] Figure 5 For [R 39 ,K 40 -UTS1 in Example 4

[0019] Figure 6 For [R 39 ,K 40 -UTS1 in Example 5 Detailed implementation manners

[0020] The following further describes the detailed implementation manners of the present invention with reference to the accompanying drawings. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Example 1:

[0022] [R 39 ,K 40 -UTS1 has an amino acid sequence of SEEPPLSIDLTFHLLRNMIQMAKMESQREQAQLNRKVLRKV-NH2 ([R39,K40]-UTS1). Its three-dimensional structure is as shown in Figure 1 . It has more excellent antibacterial and bactericidal activities. And through retrieval, it is found that the amino acid sequence of the antimicrobial peptide [R39,K40]-UTS1 is significantly different from known antimicrobial peptides, and it is a new antimicrobial peptide.

[0023] Example 2: Chemical synthesis and confirmation of the antimicrobial peptide [R 39 ,K 40 -UTS1.

[0024] The antimicrobial peptide [R 39 ,K 40-The amino acid conserved sequence of UTS1 is SEEPPLSIDLTFHLLRNMIQMAKMESQREQAQLNRKVLRKV-NH2 (SEQ ID NO.1), and the antimicrobial peptide [R 39 ,K 40 -UTS1 was chemically synthesized by solid-phase peptide C-terminal amide synthesis method using an automatic amino acid synthesizer (433A, Applied Biosystems). The antimicrobial peptide [R 39 ,K 40 -UTS1 was analyzed and verified by high performance liquid chromatography-mass spectrometry (HPLC-MS) technology (commercial service provided by Shanghai Sangon Biotech Co., Ltd.). The results are as Figure 2 shown.

[0025] HPLC-MS conditions: Mobile phase A: 0.1% aqueous trifluoroacetic acid solution, Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution; Chromatographic column: SHIMADZU shim-pack GIST (4.6×250mm×5μm); Injection volume 30 μL; Flow rate: 1.0 mL / min; Detection wavelength: 214 nm; Mobile phase conditions: At 0.01 min, 36% of Mobile phase B, at 20 min, 56% of Mobile phase B; Mass spectrometry ion source: ESI ionization source; Detection mode: Positive ion detection mode (+); Prerod bias: +4.5 kv; Nebulizing Gas Flow: 1.50 L / min; Detector: -0.2 kv; CDL Temp: 250 °C; T.Flow: 0.2 mL / min; CDL Volt: 0 v; Block Temp: 200 °C.

[0026] The obtained image is as Figure 2 shown. In the liquid chromatogram ( Figure 2 A), at a retention time of 10.553 min, [R 39 ,K 40 -UTS1 eluted, and according to the peak area normalization method analysis, its purity was higher than 95%. In the mass spectrometry diagram ( Figure 2 B) of the same period, the [M+4H] 39 ,K 40 , [M+5H] 4+ , [M+6H] 5+ , [M+7H] 6+ , and [M+8H] 7+ of the antimicrobial peptide [R 8+ -UTS1 could be detected, and the mass-to-charge ratios (m / Z) were 1220.55, 976.60, 813.95, 697.80, and 610.75 respectively. Based on the mass-to-charge ratios, the [R 39 ,K40 - The relative molecular masses (M) of UTS1 are 4878.20, 4878.00, 4877.70, 4877.60 and 4878.00, and the fitted detected value is 4877.90, with a deviation from the theoretical value of 4878.739 less than 1. Therefore, it shows that the synthesized neuropeptide [R 39 ,K 40 -UTS1 has a correct and reliable sequence.

[0027] Example 3: For the newly discovered antibacterial peptide [R 39 ,K 40 -UTS1 synthesized in Example 2, its antibacterial activity was determined (minimum inhibitory concentration MIC determination and half-maximal inhibitory concentration IC 50 determination).

[0028] Bacteria acquisition: Escherichia coli (ATCC 25922), Streptococcus agalactiae (ATCC 51487), Bacillus subtilis 23857 and Staphylococcus aureus (ATCC 12600) were purchased through commercial channels. Vibrio splendidus and Vibrio harveyi were provided by the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0029] Activate each strain: Inoculate Escherichia coli, Bacillus subtilis and Staphylococcus aureus into LB liquid medium (its formula is: 1% tryptone, 0.5% yeast extract, 1% sodium chloride), and inoculate Streptococcus agalactiae into BHI medium (its formula is: 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). Inoculate Vibrio splendidus and Vibrio harveyi into TSB medium containing 1.5% NaCl (its formula is: 1.7% tryptone, 0.3% soy peptone, 4% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.25% glucose). Culture overnight at 150 rpm and 37 °C on a shaker. The next day, dilute the overnight culture 1 / 100 into fresh respective corresponding media and culture at 37 °C until the absorbance value at 600 nm is between 0.4 - 0.6 (mid-exponential growth phase). Centrifuge at 7000 rpm for 5 min, discard the upper bacterial medium, use 1 mL of PBS solution to pipette and resuspend the bottom bacterial precipitate to mix the bacteria and wash the bacterial impurities; centrifuge at 7000 rpm for 5 min, discard the supernatant, and make the bacterial solution volume up to 1 mL with PBS, pipette and mix well to dilute the bacterial solution to 10 6CFU / mL. Prepare the antibacterial peptide [R 39 ,K 40 -UTS1 in advance at a series of working solution concentrations of 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, and 2 μM, and set up 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, incubate at 150 rpm and 37 °C for 3 h, stop the reaction by adding cold PBS solution (dilution 1 / 10) on ice for 10 min, take 10 μL of the above-mentioned bacterial solution (the actual concentration of antibacterial peptide [R 39 ,K 40 -UTS1 is 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM), add 90 μL of fresh medium to a 96-well plate, culture for 2 - 6 h, and measure the OD 600 value with an enzyme-linked immunosorbent assay reader. The experiment is measured in parallel 3 times. The antibacterial effect is expressed by the bacterial survival rate: Bacterial survival rate (%) = [(OD value after adding the peptide treatment - background OD 600 value of the medium) / (OD value without the peptide - background OD 600 value of the medium)] × 100%. Taking the bacterial survival rate as the ordinate and the actual concentration of antibacterial peptide [R 39 ,K 40 -UTS1 in the co-incubated bacterial solution as the abscissa to plot a graph. As shown in Figure 3 , the MIC value of antibacterial peptide [R 39 ,K 40 -UTS1 against Escherichia coli is 32 μM, and the MIC values against Streptococcus agalactiae, Vibrio harveyi, Vibrio splendidus, and Staphylococcus aureus are 64 μM. It has a certain inhibitory effect on Bacillus subtilis, and its MIC value is greater than 128 μM (see Table 1). In addition, the antibacterial activity can be evaluated by the half-maximal inhibitory concentration (IC 50 ): Taking the bacterial inhibition rate as the ordinate and log(antibacterial peptide [R 39 ,K 40 -UTS1 in the co-incubated bacterial solution) as the abscissa to plot a graph. As shown in Figure 4 : Use GraphPad Prism to fit the IC 50 curve, and obtain the fitted IC 50 value of Escherichia coli as 3.22 ± 1.12 μM, the fitted IC 50 value of Vibrio splendidus as 6.14 ± 1.24 μM, the fitted IC 50 value of Streptococcus agalactiae as 7.64 ± 1.22 μM, the fitted IC 50 value of Vibrio harveyi as 14.33 ± 1.18 μM, and the fitted IC 50 value of Staphylococcus aureus as 18.75 ± 1.21 μM (see Table 1). Through the MIC value and IC50 value, it can be seen that the antimicrobial peptide [R 39 ,K 40 -UTS1 has superior inhibitory activity against Escherichia coli, Streptococcus agalactiae, Vibrio splendidus, Staphylococcus aureus and Vibrio harveyi, and shows weak antibacterial effect against Bacillus subtilis.

[0030] Table 1 [R 39 ,K 40 -UTS1 antibacterial activity.

[0031]

[0032] Example 4: Ultrastructural observation of the bactericidal effect of the novel antimicrobial peptide [R 39 ,K 40 -UTS1 on bacteria.

[0033] The bactericidal effect of the antimicrobial peptide on bacteria was observed by transmission electron microscopy. 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 cell concentration of 10 8 CFU / mL was prepared. The bacterial liquid was divided into two groups: the control group (PBS) and the [R 39 ,K 40 -UTS1 treatment group (the concentration of the antimicrobial peptide was 2×IC 50 , and the concentration of the antimicrobial peptide in the Bacillus subtilis treatment group was 64 μM). 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 bactericidal effect of the antimicrobial peptide on bacteria.

[0034] The results are as Figure 5 shown. The bacterial cell morphology in the control group was regular, the surface was smooth and continuous, the bacterial cells were intact, and the electron density was uniform; in each group of bacteria in the [R 39 ,K 40 -UTS1 treatment group, it was found that the cell membrane of the bacteria was disrupted, the cell wall was wrinkled and rough, vacuoles appeared in the cells, the cells swelled and were accompanied by the outflow of contents, and the electron density was uneven. The above results indicate that [R 39 ,K 40 -UTS1 can destroy the integrity of the cell membrane, resulting in the outflow of cell contents, and ultimately play a role in killing bacteria. However, [R 39 ,K 40 -UTS1 has a weak bactericidal effect on the probiotic Bacillus subtilis, and even when the concentration of the antimicrobial peptide is 64 μM, some cells still have a complete morphology.

[0035] Example 5: Cytotoxicity Detection of the Novel Antimicrobial Peptide [R 39 ,K 40 -UTS1.

[0036] Resuscitated HEK 293T cells were used to prepare a cell suspension with a density of 3 - 5×10 4 cells / mL in complete medium (formulation: 90% DMEM, 10% fetal bovine serum, 100 U / mL penicillin - streptomycin). 100 μL / well was added to a 96 - well plate and cultured overnight at 37°C until the cell confluence was about 50%. A series of working solutions of [R 39 ,K 40 -UTS1 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 / well of the above - mentioned 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 the incubator for 1 hour, the absorbance OD450 at 450 nm was measured with an enzyme - linked immunosorbent assay (ELISA) reader. 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.

[0037] The results are as Figure 6 shown. When the concentration of the antimicrobial peptide [R 39 ,K 40 -UTS1 is greater than or equal to 2 μM and less than or equal to 16 μM, it can significantly improve cell activity and promote cell growth; when the concentration of the antimicrobial peptide is 1 μM, 32 μM, and 64 μM, there is no significant difference in cell activity compared with the control group. The above results indicate that the antimicrobial peptide [R 39 ,K 40 -UTS1 shows low cytotoxicity when the concentration is less than or equal to 64 μM (the MIC value of most bacteria).

[0038] In summary, as can be seen from the above examples, the antimicrobial peptide [R 39 ,K 40 -UTS1 of the present invention can be obtained by chemical synthesis. This antimicrobial peptide has strong antibacterial and bactericidal activities against Gram - positive and Gram - negative bacteria. The antimicrobial peptide is simple to obtain, has good water solubility, and can be applied to fields such as medicine, cosmetics, food preservation, and aquaculture in the future. Compared with other antimicrobial peptides, the antibacterial concentration is more sensitive, and the MIC value for Escherichia coli is as low as 32 μM, IC 50It is also as low as 3.22 μM. The MIC values of Staphylococcus aureus, Vibrio splendidus, Vibrio harveyi, and Streptococcus agalactiae are as low as 64 μM, among which the IC of Vibrio splendidus and Streptococcus agalactiae 50 is lower than 10 μM, and the IC of Staphylococcus aureus and Vibrio harveyi 50 is lower than 20 μM. In addition, the antimicrobial peptide [R 39 ,K 40 -UTS1 shows weak lethality to the probiotic Bacillus subtilis (MIC > 128 μM), which shows certain advantages in the application of this antimicrobial peptide, being able to kill pathogenic bacteria while preserving the activity of the probiotic Bacillus subtilis.

[0039] 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. A neuropeptide UTS1 variant, characterized in that: The neuropeptide UTS1 variant is [R 39 ,K 40 -UTS1, which is obtained by optimizing the 39th and 40th amino acid sites of the neuropeptide UTS1 to R and K, and its amino acid conserved sequence is SEEPPLSIDLTFHLLRNMIQMAKMESQREQAQLNRKVLRKV-NH2.

2. A neuropeptide UTS1 variant according to claim 1, characterized in that: The [R 39 ,K 40 -UT S1 has a molecular formula of C 211 H 359 N 65 O 61 S3, a molecular weight of 4878.68, an isoelectric point of 10.82, a net charge number of +3.1, and an average hydrophobicity of 0.353 for the polypeptide.

3. A neuropeptide UTS1 variant according to claim 1, characterized in that: All of the amino acids are of the L-type.

4. A preparation, characterized in that: It includes the neuropeptide UTS1 variant described in any one of claims 1-3.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition includes the neuropeptide UTS1 variant described in any one of claims 1-3.

6. A pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition has the function of inhibiting or killing Gram-positive bacteria and Gram-negative bacteria.

7. Use of the neuropeptide described in any one of claims 1-3, the bacterial agent described in claim 5, and the pharmaceutical composition described in claim 6 in the preparation of aquatic feeds and fishery drugs.

8. Use of the neuropeptide described in any one of claims 1-3, the bacterial agent described in claim 5, and the pharmaceutical composition described in claim 6 in the preparation of drugs for preventing and treating fish septicemia and sea cucumber skin ulcer syndrome.

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