Antibacterial peptide for resisting blue eye disease pathogen Elizabeth mileli of giant frog

Abstract

The invention discloses an antibacterial peptide for resisting a blue eye disease pathogen Elizabeth mileli of a giant frog, and relates to the technical field of antibacterial peptides, the antibacterial peptide is AMP-New2, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.5. The invention further discloses a preparation method of the antibacterial peptide for resisting the blue eye disease pathogen Elizabeth mileli of the giant frog. According to the antibacterial peptide for resisting the ilizabeth mileli pathogen of the blue eye disease of the giant frog, the antibacterial effect of the antibacterial peptide AMP-New2 on the ilizabeth mileli is designed and found, and the antibacterial peptide AMP-New2 is expected to be applied to treatment of the blue eye disease of the giant frog to replace antibiotics to prepare a new medicine for resisting the ilizabeth mileli.

Description

Technical Field

[0001] The invention relates to the technical field of antimicrobial peptides, in particular to an antimicrobial peptide resistant to Elizabeth Milleissima, a pathogen of blue eye disease of Rana spinosa. Background Art

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

[0003] Elizabethkingia miricola is an important pathogenic microorganism that affects both humans and frogs. In the aquaculture industry, the bacteria can infect a variety of farmed and wild frogs. It is highly contagious and has a high mortality rate, causing frogs to develop symptoms such as meningitis (head tilt) and cataracts, bulging eyes, blue eyes, swimming in the water, lack of energy, loss of appetite, and abdominal distension.

[0004] Existing studies have shown that Elizabeth Millipore may be naturally resistant to antibiotics generally used to treat Gram-negative bacterial infections (such as aminoglycosides, β-lactam antibiotics, tetracycline and chloramphenicol, etc.), so empirical medication is not only not conducive to disease control, but may also cause its spread. In addition, Elizabeth Millipore strains isolated from wry head disease have shown high drug resistance, making the disease difficult to treat.

[0005] The existing treatment options mainly use antibiotics to inhibit Elizabeth MilliporeSigma, but the extensive use of antibiotics will cause huge safety risks to frog products. The abuse of antibiotics has a negative impact on environmental microbial resistance, animal immunity, intestinal microorganisms, and even passes from animals to humans through the food chain. Therefore, reducing or even avoiding the use of antibiotics in the breeding process of commercial frogs (such as Rana spinosa) has become an urgent problem to be solved.

[0006] In order to overcome the shortcomings of traditional antibacterial methods, antimicrobial peptides have always attracted much attention. Antimicrobial peptides are a class of small polypeptide molecules with antimicrobial activity, showing good antimicrobial activity. Antimicrobial peptides are regarded as the best alternative molecules to antibiotics due to their small molecular weight, strong thermal stability, good water solubility, non-immunogenicity, low resistance to drug resistance, and broad antimicrobial spectrum. Summary of the invention

[0007] The purpose of the present invention is to provide an antimicrobial peptide against Elizabeth Milleissima, the pathogen of blue eye disease in Rana spinosa, so as to replace traditional antibiotics and be applied to the breeding of commercial frogs, thereby overcoming the defects of traditional antimicrobial methods.

[0008] To achieve the above objectives, on the one hand, the present invention provides an antimicrobial peptide against Elizabeth Milleissima, the pathogen of blue eye disease in Rana spinosa. 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 a use of the above-mentioned antimicrobial peptide in the preparation of a drug for inhibiting Elizabeth II bacteria.

[0010] On the other hand, the present invention provides an application of the antimicrobial peptide in breeding of Rana spinosa.

[0011] Therefore, the antimicrobial peptide of the present invention against Elizabeth Milleissima, the pathogen of blue eye disease of Rana spinosa, has the following beneficial effects:

[0012] (1) The present invention obtains Elizabethella miltiorrhiza by extracting and isolating bacteria from the eyes of frogs with blue eye disease of Rana spinosa;

[0013] (2) AMP-New1 and AMP-New2 were subjected to drug susceptibility testing together with three other broad-spectrum antimicrobial peptides, demonstrating that AMP-New2 has the ability to inhibit the growth of Elizabeth Millipore;

[0014] (3) The present invention discovered for the first time the antibacterial effect of the antimicrobial peptide AMP-New2 on Mielizabethiae, and it is expected to be applied to the treatment of blue eye disease in Rana spinosa, and to prepare new anti-Mielizabethiae drugs instead of antibiotics.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for the sick frog Rana spinosa suffering from blue eye disease;

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

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

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0021] Embodiment 1

[0022] S1. Bacterial activation and identification

[0023] S11. Streak: Take a typical frog with blue eye disease of Rana spinosa, scrape the eyes to obtain bacteria, and then inoculate onto an antibiotic-free LB plate using the conventional streak method and culture at 28°C for 20 hours.

[0024] Sick frog pictures Figure 1 As shown, the cornea of ​​the spinous-chested frog suffering from blue eye disease is cloudy and the pupil is light blue.

[0025] S12. Cultivation: Pick a single clone from the solid LB plate and transfer it to liquid LB, and culture it at 28°C overnight.

[0026] S13, strain identification: The bacterial solution was used as a PCR template, and the bacterial 16S rDNA universal primer 27F / 1522R provided by Zhejiang Shangya Biotechnology Co., Ltd. was used for PCR amplification. The amplified product was sent to Zhejiang Shangya Biotechnology Co., Ltd. for gene sequencing. The sequencing results were subjected to BLAST analysis in the NCBI database, such as Figure 2 The results showed that it had 100% consistency with Elizabethkingia miricola, indicating that the pathogen of blue eye disease of Rana spinosa isolated was Elizabethkingia miricola.

[0027] S2. Drug sensitivity test

[0028] S21. Bacterial propagation: transfer Elizabeth Millipore into 50 mL of antibiotic-free liquid LB at a ratio of 1:1000, incubate at 28°C in a shaking incubator for 14 h, and then harvest the bacteria. 600 Make it equal to 1.

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

[0030] S23. Bacterial count: Take OD 600 The bacterial solution was diluted 10 times to 10 -7 , take 100 μL of the diluted bacterial solution to plate and culture at 28℃ for about 24h.

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

[0032] The results showed that when diluted to 10 -6 When the colony count is 87, multiply it by the dilution factor of 10 6The amount of bacterial solution coated on the plate is 100 μL, and the OD 600 The total number of viable bacteria in a bacterial solution equal to 1 is approximately 8.7×10 8 CFU / mL.

[0033] According to WS / T 650-2019 standard, the bacterial concentration used in the inhibition ring test is about 5.0×10 5 -5.0×10 6 CFU / mL, in subsequent experiments, OD 600 The bacterial solution equal to 1 was diluted 200 times before use.

[0034] S24. Bacterial coating: Take a non-antibiotic plate, take 100 μL of diluted bacterial solution and coat the plate with glass beads on a sterilized plate, place it upright in a 28°C incubator for 15 minutes, and set up a negative control at the same time.

[0035] S25. Punching holes on the plate: Use a puncher to punch holes on the coated plate, with one hole in the middle 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 simultaneously tested for drug sensitivity with the reported broad-spectrum antimicrobial peptides AMP-1, AMP-2, and AMP-3 to test whether the five antimicrobial peptides have inhibitory effects on Elizabeth Millipore. The above five antimicrobial peptides were commissioned to be synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

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

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

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

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

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

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

[0045] (3) Antimicrobial peptide susceptibility test: Take 100 μL of diluted bacterial solution and use a sterilized plate coated with glass beads to coat the plate and punch holes. Add 18 μL of antimicrobial peptide solution to each well around the plate, and add sterile water or DMSO solvent to the middle well as a negative control. Incubate at 28°C for about 24 hours. Observe the presence of an inhibition zone and measure the diameter of the inhibition zone. The test results are shown in Table 1 and 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 Elizabethia miltiorrhiza, and AMP-New2 was an antimicrobial peptide with a significant inhibitory effect on Elizabethia miltiorrhiza.

[0049] Therefore, the present invention discloses an antimicrobial peptide against Mielizabethia miltiorrhiza, the pathogen of blue eye disease in Rana spinosa. The antibacterial effect of the antimicrobial peptide AMP-New2 against Mielizabethia miltiorrhiza is designed and discovered. It is expected to be applied to the treatment of blue eye disease in Rana spinosa, and to prepare new anti-Mielizabethia miltiorrhiza drugs instead of antibiotics.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An antimicrobial peptide against Elizabeth Millipore, the pathogen of blue eye disease in Rana spinosa, characterized in that: The antimicrobial peptide is AMP-New2, and its amino acid sequence is shown in SEQ ID No.

5.

2. Use of the antimicrobial peptide as claimed in claim 1 in the preparation of drugs for inhibiting Elizabeth II bacteria.

3. Use of the antimicrobial peptide as claimed in claim 1 in breeding of Rana spinosa.

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