An antibacterial peptide against the causative agent of ranaviruses, ranavirus, and a method for producing the same

Abstract

The application discloses an antibacterial peptide against the Mirabilis pyxidis pathogen of blue eye disease of Rana spinosa, relates to the technical field of antibacterial peptides, and the antibacterial peptide is AMP-New2, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No. 5. The antibacterial peptide against the Mirabilis pyxidis pathogen of blue eye disease of Rana spinosa is designed and the antibacterial effect of the antibacterial peptide AMP-New2 on the Mirabilis pyxidis is discovered, and the antibacterial peptide is expected to be applied to the treatment of blue eye disease of Rana spinosa, and new Mirabilis pyxidis resistant drugs are prepared by replacing antibiotics.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to an antimicrobial peptide against Elizabethan mil, the pathogen causing blue-eye disease in the spiny-breasted frog. Background Technology

[0002] Elizabethan bacillus is a Gram-negative, obligate aerobic, non-spore-forming, non-fermenting, non-motile, slender, slightly curved bacillus. It can decompose fructose, glucose, lactose, maltose, mannitol, and trehalose, but cannot decompose arabinose, raffinose, salicin, sucrose, or xylose to produce acid. It produces H2S, is malonic acid utilization negative, and nitrate reduction negative. It grows well at 37°C, but does not grow at 5°C and 42°C. It can hydrolyze aescin.

[0003] Elizabethkingia miricola is an important zoonotic pathogenic microorganism. In aquaculture, this bacterium can infect various farmed and wild frogs, exhibiting high infectivity and mortality, leading to a variety of symptoms in frogs, including meningitis (head tilt) and cataracts, protruding eyes, blue eyes, spinning in the water, lethargy, loss of appetite, and abdominal distension.

[0004] Existing research indicates that Elizabethanella miltiorrhiza may exhibit natural resistance to antibiotics commonly used to treat Gram-negative bacterial infections (such as aminoglycosides, β-lactam antibiotics, tetracyclines, and chloramphenicol). Therefore, empirical treatment is not only ineffective in controlling the disease but may also contribute to its spread. Furthermore, all Elizabethanella miltiorrhiza strains isolated from wry neck disease currently exhibit high levels of drug resistance, making the disease difficult to treat.

[0005] Current treatment primarily involves using antibiotics to suppress Elizabethan mil, but the extensive use of antibiotics poses significant safety risks to frog products. Antibiotic abuse negatively impacts environmental microbial resistance, animal immunity, and gut microbiota, and can even be transmitted from animals to humans through the food chain. Therefore, reducing or even avoiding antibiotic use in commercial frog (such as the spiny-breasted frog) farming has become an urgent issue.

[0006] To overcome the shortcomings of traditional antibacterial methods, antimicrobial peptides have long been a focus of attention. Antimicrobial peptides are a class of small polypeptide molecules with antibacterial activity, exhibiting excellent antibacterial properties. Due to their small molecular weight, high thermal stability, good water solubility, lack of immunogenicity, low likelihood of inducing drug resistance, and broad antibacterial spectrum, antimicrobial peptides are considered the optimal alternative molecules to antibiotics. Summary of the Invention

[0007] The purpose of this invention is to provide an antimicrobial peptide against Elizabethan mil, the pathogen causing blue-eye disease in the spiny-breasted frog, to replace traditional antibiotics in the breeding of commercial frogs and overcome the shortcomings of traditional antimicrobial methods.

[0008] To achieve the above objectives, on the one hand, the present invention provides an antimicrobial peptide against Elizabethan mil, the pathogen causing blue-eye disease in the spiny-breasted frog, wherein the antimicrobial peptide is AMP-New2, and its amino acid sequence is shown in SEQ ID No. 5.

[0009] On the other hand, the present invention provides the use of the above-mentioned antimicrobial peptide in the preparation of a drug for inhibiting Elizabethan mil.

[0010] On the other hand, the present invention provides an application of the above-mentioned antimicrobial peptide in the breeding of spiny-breasted frogs.

[0011] Therefore, the antimicrobial peptide of the present invention, which is effective against Elizabethanella miltiorrhiza, the pathogen causing blue-eye disease in the spiny-breasted frog, has the following beneficial effects:

[0012] (1) In this invention, Elizabethanella miltiorrhiza was obtained by extracting and amplifying bacteria from the eyes of blue-eyed frogs;

[0013] (2) AMP-New1, AMP-New2 and three other broad-spectrum antimicrobial peptides were subjected to drug sensitivity tests to demonstrate that AMP-New2 has the ability to inhibit the growth of Elizabethan mil;

[0014] (3) This invention is the first to discover the antimicrobial effect of the antimicrobial peptide AMP-New2 on Elizabethan mil, which is expected to be applied to the treatment of blue-eye disease in the spiny-breasted frog and to replace antibiotics in the preparation of new anti-Elizabethan mil drugs.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 A spiny-breasted frog suffering from blue-eye disease;

[0017] Figure 2 To compare the 16S rDNA sequencing results of bacteria isolated from diseased frogs with the BLAST results in the NCBI database;

[0018] Figure 3 The inhibition zones of different antimicrobial peptides (5 mg / mL) are shown. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0021] Example 1

[0022] S1. Strain activation and identification

[0023] S11. Streaking: Take typical spiny-breasted frogs with blue-eye disease, scrape the eyes to collect bacteria, and then inoculate them onto antibiotic-free LB plates using the conventional streaking method. Incubate at 28°C for 20 hours.

[0024] Images of sick frogs Figure 1 As shown, the spiny-breasted frog suffering from blue-eye disease has cloudy corneas and pale blue pupils.

[0025] S12. Expanding culture: Pick a single colony from a solid LB plate and transfer it to a liquid LB plate. Incubate overnight at 28°C.

[0026] S13. Bacterial Strain Identification: Using bacterial culture as a PCR template, PCR amplification was performed using the universal primers 27F / 1522R for bacterial 16S rDNA provided by Zhejiang Shangya Biotechnology Co., Ltd. The amplification products were sent to Zhejiang Shangya Biotechnology Co., Ltd. for gene sequencing. The sequencing results were then analyzed using BLAST in the NCBI database. Figure 2 As shown in the figure. The results showed 100% identity with Elizabethkingia miricola, indicating that the pathogen of blue-eye disease in the spiny-breasted frog was Elizabethkingia miricola.

[0027] S2, Drug sensitivity test

[0028] S21. Bacterial propagation: Elizabethan bacillus mirabilis was transferred at a dilution of 1:1000 to 50 mL of antibiotic-free liquid LB, cultured on a shaker at 28°C for 14 h, and then the bacteria were harvested. OD was measured and adjusted. 600 Make it equal to 1.

[0029] S22. Plate preparation: Pour the sterilized LB solid medium into sterile petri dishes, about 20 mL per dish.

[0030] S23, Bacterial Count: Take OD 600 The bacterial culture with a concentration equal to 1 was serially diluted 10-fold to 10. -7 100 μL of the diluted bacterial solution was plated and incubated at 28°C for about 24 hours.

[0031] Count the specific number of colonies with a colony count between 30 and 300, then multiply by the corresponding dilution factor to calculate the OD. 600 The total number of viable bacteria was roughly calculated using a bacterial solution with a value approximately equal to 1.

[0032] The results showed that when diluted to 10 -6 At that time, the colony count was 87, which was then multiplied by the dilution factor of 10. 6With a bacterial culture volume of 100 μL coated on the plate, the OD can be obtained. 600 The total number of viable bacteria in a bacterial solution with a concentration of 1 is approximately 8.7 × 10⁻⁶. 8 CFU / mL.

[0033] According to the WS / T 650—2019 standard, the bacterial concentration used in the inhibition zone test is approximately 5.0 × 10⁻⁶. 5 -5.0×10 6 CFU / mL, in subsequent experiments, OD 600 Dilute the bacterial solution equal to 1 by 200 times before use.

[0034] S24. Bacterial plating: Take an antibiotic-free plate, take 100 μL of diluted bacterial solution and spread it onto a sterile plate with glass beads. Incubate the plate upright in a 28℃ incubator for 15 min. Set up a negative control at the same time.

[0035] S25. Flat plate punching: Use a puncher to punch holes in the coated flat plate, one hole in the center and 4-5 holes around it.

[0036] S26, Drug sensitivity test

[0037] (1) Synthesis and dilution of antimicrobial peptides:

[0038] Two antimicrobial peptides, AMP-New1 and AMP-New2, were designed and subjected to drug sensitivity testing along with previously reported broad-spectrum antimicrobial peptides AMP-1, AMP-2, and AMP-3 to determine whether these five antimicrobial peptides exhibit inhibitory effects against Elizabethan mil. All five antimicrobial peptides were synthesized by Nanjing Jietai Biotechnology Co., Ltd.

[0039] The amino acid sequence of AMP-1 is shown in SEQ ID No. 1: FLPLIGRVLS GIL.

[0040] The amino acid sequence of AMP-2 is shown in SEQ ID No. 2: LLPIVGNLL KSLL.

[0041] The amino acid sequence of AMP-3 is shown in SEQ ID No.3: FMPILSCSRF KRC.

[0042] The amino acid sequence of AMP-New1 is shown in SEQ ID No.4: GWKKG LKLGLKLL.

[0043] The amino acid sequence of AMP-New2 is shown in SEQ ID No. 5: GWKKL LKLGLKLL.

[0044] (2) The total mass of each tube of antimicrobial peptide is 2 mg. It is diluted with DMSO or sterile water to a solution of 5 mg / mL depending on the solubility.

[0045] (3) Antimicrobial peptide susceptibility test: 100 μL of diluted bacterial solution was spread onto a sterile agar plate using glass beads, and wells were punched. 18 μL of antimicrobial peptide solution was added to each well, and sterile water or DMSO was added to the center well as a negative control. The plate was incubated at 28℃ for approximately 24 hours. The presence and diameter of inhibition zones were observed. The test results are shown in Table 1 below. Figure 3 As shown:

[0046] Table 1

[0047]

[0048] The results showed that AMP-1, AMP-2, AMP-3, and AMP-New1 had no inhibitory effect on Elizabethan mil, while AMP-New2 was an antimicrobial peptide with a significant inhibitory effect on Elizabethan mil.

[0049] Therefore, this invention provides an antimicrobial peptide against Elizabethan mil, the pathogen causing blue-eye disease in the spiny-breasted frog. The antimicrobial peptide AMP-New2 was designed and its antibacterial effect against Elizabethan mil was discovered, and it is expected to be applied to the treatment of blue-eye disease in the spiny-breasted frog, replacing antibiotics to prepare new anti-Elizabethan mil drugs.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antimicrobial peptide against Elizabethanella mil, the pathogen causing blue-eye disease in the spiny-breasted frog, characterized in that: The antimicrobial peptide is AMP-New2, and its amino acid sequence is shown in SEQ ID No.

5.

2. The use of the antimicrobial peptide as described in claim 1 in the preparation of a drug for inhibiting Elizabethan mil.

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