Abdominal spotted frog antibacterial peptide Nv-CATH, gene and application thereof

Abstract

The present application relates to a kind of abdominal spot frog antibacterial peptide Nv-CATH and its gene and application, the antibacterial peptide Nv-CATH is the circular polypeptide coded by the defense peptide gene of amphibian species unique species abdominal spot frog in China, molecular weight 3356.98 Dalton, isoelectric point 11.56, its amino acid sequence is as shown in SEQ ID NO:1.The gene (GenBank accession No.OP264074) of the precursor of the abdominal spot frog antibacterial peptide Nv-CATH is composed of 714 nucleotide sequences, its nucleotide sequence is as shown in SEQ ID NO:2, wherein the 373-522th nucleotide is the encoding gene of mature abdominal spot frog antibacterial peptide Nv-CATH.The application of abdominal spot frog antibacterial peptide Nv-CATH in preparing the therapeutic drug of the infection disease caused by hemolytic staphylococcus, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter junni and / or paratyphi A.The present application provides a new antibacterial peptide Nv-CATH with strong broad-spectrum antibacterial activity.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide Nv-CATH from the spotted dwarf frog (Nanorana ventripunctata), its gene, and its applications, belonging to the field of biomedicine. Background Technology

[0002] Recent studies have shown that antimicrobial peptides are an important component of the defense systems of all animals, plants, and microorganisms. They are a class of small-molecule bioactive polypeptides produced by the body to resist pathogenic microorganisms, characterized by small molecular weight, good stability, rapid bactericidal action, and low likelihood of inducing drug resistance. These characteristics give antimicrobial peptides a very broad application prospect and the potential to replace traditional antibiotics that are prone to inducing drug resistance in clinical practice. The reason why antimicrobial peptides are less likely to induce drug resistance is that their bactericidal mechanism differs from that of traditional antibiotics. They mainly act on the bacterial cell membrane, disrupting its stability, thereby altering cell membrane permeability, causing leakage of contents, and leading to cell death. The structural forms and biological activities of antimicrobial peptides vary considerably between different species or even within the same species. Researchers at home and abroad have been committed to discovering novel antimicrobial peptides from different species, using them directly or as templates for molecular modification. Currently, antimicrobial peptides are used in seafood preservation and animal feed additives. Discovering antimicrobial peptides with potential medicinal value from various natural resources in conjunction with the ecological environment is currently a hot topic in peptide drug research.

[0003] Amphibians are among the most important resources for the discovery of antimicrobial peptides. Their exposed skin allows them to secrete a variety of novel and functionally complex antimicrobial peptides to defend against various microorganisms in the environment. These bioactive peptides participate extensively in various physiological activities and possess diverse pharmacological activities, such as antimicrobial, antitumor, antioxidant, immunomodulatory, wound repair, and analgesic effects. The spotted pygmy frog (Nanorana ventripunctata) typically inhabits high-altitude, diurnal temperature variations in plateau marshes, indicating that with adaptive development, it must have developed an effective immune defense system to resist microbial invasion in harsh environments. The spotted pygmy frog (Nanorana ventripunctata) is an amphibian belonging to the genus Nanorana in the family Ranidae of the order Anura. It is endemic to China, and potent antimicrobial peptides of the cathelicidin family in its skin have not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a novel antimicrobial peptide from the spotted frog (Rana ventricosa) with potent and broad-spectrum antimicrobial activity (including Gram-positive and Gram-negative bacteria), its gene, and its applications.

[0005] The antimicrobial peptide Nv-CATH of this invention is a cyclic polypeptide encoded by the defense peptide gene of the Chinese amphibian endemic species, the spotted dwarf frog. It has a molecular weight of 3356.98 Daltons and an isoelectric point of 11.56. The primary structure of the polypeptide (amino acid sequence SEQ ID NO: 1) is as follows:

[0006]

[0007] (NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG), where the cysteine ​​residues at position 2 and 6 form an intramolecular disulfide bond to create a cyclic polypeptide.

[0008] The gene encoding the Nv-CATH precursor, an antimicrobial peptide from the spotted dwarf frog (GenBank accession No. OP264074), consists of 714 nucleotide sequences, as shown in SEQ ID NO:2. Its sequence from the 5' end to the 3' end is as follows:

[0009]

[0010] Nucleotides 373–522 are the encoding gene for the mature dwarf frog antimicrobial peptide Nv-CATH.

[0011] The present invention relates to the use of the antimicrobial peptide Nv-CATH from the dwarf frog in the preparation of a medicament for treating infectious diseases caused by hemolytic Staphylococcus, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter jumbo and / or Salmonella paratyphi A.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention provides a novel antimicrobial peptide, Nv-CATH, from the dwarf frog with broad-spectrum antimicrobial activity (including Gram-positive and Gram-negative bacteria). This invention has a significant inhibitory effect on bacterial growth in infectious diseases caused by hemolytic Staphylococcus, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter jumbo, and Salmonella paratyphi A. Attached Figure Description

[0014] Figure 1The figure shows the bacterial killing kinetics curves of Nv-CATH. In the figure, the killing effect of Nv-CATH at a concentration of 4×MIC on (A) S. aureus ATCC25923, (B) A. junii 13A15578, (C) K. pneumoniae 0813343, (D) S. paratyphi A, (E) E. faecalis ATCC29212, and (F) S. haemolyticus 13A13770 was measured at 0, 1, 2, 4, 6, 8, and 12 h at 37 °C. The data are the average values ​​of three independent experiments. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings.

[0016] I. Cloning and amino acid sequence determination of the Nv-CATH gene, an antimicrobial peptide from the spotted dwarf frog.

[0017] 1. Total RNA extraction from the skin of the spotted frog

[0018] Live *Phoebe zhennan* frogs were cleaned with water and flash-frozen in liquid nitrogen for 10 hours. 300 mg of skin tissue was taken and 3 ml of Trizol solution was added, then homogenized in a 20 ml glass homogenizer for 30 minutes. An equal volume of phenol / chloroform solution was added, and the mixture was vigorously mixed. The mixture was incubated at room temperature for 10 minutes, then centrifuged at 12000 rpm for 10 minutes at 4°C, and the precipitate was discarded. An equal volume of isopropanol was added to the supernatant, and the mixture was incubated at room temperature for 10 minutes, then centrifuged at 12000 rpm for 10 minutes at 4°C. The precipitate was washed once with 75% ethanol, air-dried, and the precipitate at the bottom of the tube was the total RNA from the *Phoebe zhennan* skin.

[0019] 2. Purification of skin mRNA from the spotted frog: mRNA isolation and purification were performed using equipment from PROMEGA (USA). mRNA Isolation Systems Kit

[0020] Total RNA extracted from the skin of the spotted frog was dissolved in 500 μl of DEPC water and incubated at 65°C for 10 minutes. Then, 3 μl of Oligo(dT) probe and 13 μl of 20×SSC solution were added, mixed thoroughly, and allowed to cool to room temperature; this was called solution A. Magnetic beads (SA-PMP) were gently tapped to mix, and the mixture was allowed to adhere to a magnetic rack for 30 seconds. The supernatant was discarded, and 0.3 ml of 0.5×SSC was added, allowing the mixture to adhere to the magnetic rack for 30 seconds. Finally, 0.1 ml of 0.5×SSC was added to suspend the mixture; this was called solution B. Add solution A to solution B, incubate at room temperature for 10 minutes, then allow to adhere to a magnetic rack for 30 seconds. Discard the supernatant. Wash four times with 0.1×SSC solution, discarding the supernatant again. Resuspend the sample in 0.1 ml of DEPC-treated water, allowing it to adhere to a magnetic rack for 30 seconds. Transfer the supernatant to a new tube, add 0.15 ml of DEPC-treated water to resuspend the sample, and allow it to adhere to a magnetic rack for 30 seconds. Transfer the supernatant to the same tube. The supernatant will contain purified *Pleurotus ostreatus* skin mRNA. Add 1 / 10 volume of 3M sodium acetate (pH 5.2) and an equal volume of isopropanol. Incubate at -70°C for 30 minutes, then centrifuge at 12000 rpm for 10 minutes at 4°C. Discard the supernatant and dissolve the precipitate in 10 μl of DEPC-treated water.

[0021] 3. Construction of skin cDNA library of the spotted frog

[0022] Using CLONTECH's Creator TM SMART TM cDNA Library Construction Kit (cDNA Library Construction Kit)

[0023] (a) cDNA first-strand synthesis (mRNA reverse transcription)

[0024] Add 1 μl of *Pleurotus ostreatus* skin mRNA, 1 μl of SMART IV oligonucleotides, and 1 μl of CDS III / 3' PCR primers to a 0.5 ml sterile centrifuge tube, and add 2 μl of deionized water to bring the total volume to 5 μl. Mix the reagents in the centrifuge tube and centrifuge at 12000 rpm for 15 seconds, then incubate at 72°C for 2 minutes. Incubate the centrifuge tube on ice for 2 minutes. Add the following reagents to the centrifuge tube: 2.0 μl of 5× first-strand buffer, 1.0 μl of 20 mM dithiothreitol, 1.0 μl of 10 mM dNTP mixture, and 1.0 μl of PowerScript reverse transcriptase. Mix the reagents in the centrifuge tube and centrifuge at 12000 rpm for 15 seconds, then incubate at 42°C for 1 hour. Place the centrifuge tube on ice to stop the first-strand synthesis. Take 2 μl of the synthesized cDNA first strand from the centrifuge tube for later use.

[0025] (b) Amplification of the second strand using long-terminated polymerase chain reaction (LD-PCR)

[0026] Preheat the PCR instrument to 95℃. In a centrifuge tube, combine 2 μl of cDNA first strand (mRNA reverse transcription), 80 μl of deionized water, 10 μl of 10×Advantage 2 PCR buffer, 2 μl of 50×dNTP mixture, 2 μl of 5' PCR primers, 2 μl of CDS III / 3' PCR primers, and 2 μl of E. coli polymerase. Amplify the PCR using the following program: 95℃ for 20 seconds; 22 cycles (95℃ for 5 seconds; 68℃ for 6 minutes). After cycling, recover the synthesized double-stranded cDNA from the centrifuge tube.

[0027] (c) PCR product recovery

[0028] Using PROMEGA The SV Gel and PCR Clean-Up System kit was used for extraction and recovery, and the steps are as follows:

[0029] Add an equal volume of membrane binding buffer to the cDNA double strands obtained by PCR, invert and mix well. Transfer the mixture to a centrifuge purification column and incubate at room temperature for 5 minutes to allow the DNA to fully bind to the silica membrane. Centrifuge at 12000 rpm for 30 seconds and discard the waste liquid in the collection tube. Add 700 μl of elution buffer (containing ethanol) to the centrifuge purification column and centrifuge at 12000 rpm for 30 seconds, discarding the waste liquid in the collection tube. Repeat step 2. Centrifuge at 12000 rpm for 5 minutes, then transfer the centrifuge purification column to a new centrifuge tube. Add 30 μl of ultrapure water and incubate at room temperature for 5 minutes. Centrifuge at 12000 rpm for 30 seconds; the solution at the bottom of the tube is the purified cDNA double strands.

[0030] (d) Preparation of Escherichia coli DH5α competent cells

[0031] Pick a single DH5α colony and inoculate it into 3 ml of Luria-Bertani (LB) medium without ampicillin. Incubate overnight at 37°C. The next day, take the above bacterial culture and inoculate it into 50 ml of LB medium at a 1:100 ratio. Shake at 37°C for 2 hours. When OD 600When the pH reaches 0.35, harvest the bacterial culture. Transfer the bacteria to a sterile, single-use, ice-cold 50ml polypropylene tube and place it on ice for 10 minutes to cool the culture to 0°C. Centrifuge at 4100 rpm for 10 minutes at 4°C to recover the cells. Discard the culture medium and invert the tube for 1 minute to allow the last trace culture medium to drain completely. Resuspend each cell pellet in 50ml of initial culture medium and 30ml of ice-cold 0.1mol / L CaCl2-MgCl2 solution (80mmol / L MgCl2, 20mmol / L CaCl2). Centrifuge at 4100 rpm for 10 minutes at 4°C to recover the cells. Discard the culture medium and invert the tube for 1 minute to allow the last trace culture medium to drain completely. Resuspend each cell pellet in 2ml of ice-cold 0.1mol / L CaCl2 for 50ml of initial culture and aliquot for later use.

[0032] (e) Enzyme digestion, ligation, and transformation of ligation products

[0033] Add 1 μl of Takara pMD18-T vector and 4 μl of *Rana ventricosa* cDNA double-stranded solution to a microcentrifuge tube, bringing the total volume to 5 μl. Add 5 μl (equal volume) of ligase buffer mixture. Incubate at 16°C for 2 hours. Add 10 μl of the total volume to 100 μl of DH5α competent cells and incubate on ice for 30 minutes. Heat at 42°C for 90 seconds, then incubate on ice for 1 minute. Add 890 μl of LB medium that has been warmed to 37°C and incubate at 37°C with gentle shaking for 60 minutes. Spread 200 μl of the mixture onto LB medium containing X-Gal, IPTG, and Amp and incubate at 37°C for 16 hours to form single colonies. Wash each LB plate with 5 ml of LB liquid medium and freeze with 30% glycerol. The constructed cDNA contains approximately 1 × 10⁻⁶ cells / mL. 6 A single clone.

[0034] 4. Cloning and sequencing of antimicrobial peptide genes in the spotted dwarf frog

[0035] Based on the reported highly conserved cathelin domain sequence of amphibian cathelicidins, a 3' reverse primer Nv-CATH-R1 (5'-WSCRCAGRYCTTCACCTCC-3' (W=A / T; S=G / C; R=A / G; Y=C / T)) was designed and combined with the 5' PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') provided in the kit to amplify the 5' fragment of the cDNA encoded by cathelicidin. PCR conditions were: 95℃ pre-denaturation for 2 min, 92℃ denaturation for 10 s, 50℃ annealing for 30 s, 72℃ extension for 40 s, repeated 30 times, followed by a final extension at 72℃ for 10 min. The PCR product was purified by gel electrophoresis and cloned into the pMD19-T vector (Takara, Japan) for DNA sequencing.

[0036] Based on the 5' end sequence obtained from sequencing, a 5' forward primer Nv-CATH-F1 (5'-ATGAAGGTCTGGCAGTGTGCG-3') was designed and combined with the 3' PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCG-3') provided in the kit to amplify the full-length cDNA sequence encoding cathelicidin. The PCR reaction conditions were as described above. Finally, the PCR product was cloned into the pMD19-T vector and sequenced. The specific sequencing steps are as follows: DNA sequencing was performed using an ABI PRISM 377 Applied Biosystems DNA sequencer. The final amplified PCR product was recovered using a DNA gel recovery kit (DP209-02, Tiangen, China). The recovered fragment was ligated into the pMD19-T vector, and the ligation product was transformed into *E. coli* DH5α competent cells. After transformation, the transformation product was plated onto LB (Luria-Bertani) solid medium containing AMP, and the plates were inverted and incubated at 37°C for 16 hours. Single colonies were selected as templates for colony PCR. The PCR products were determined by agarose gel electrophoresis, and positive clones containing the target fragment were selected, labeled, and cultured in LB liquid medium for amplification. The bacterial culture was then sent to a biotechnology company for DNA sequencing.

[0037] Gene sequencing results indicate that the gene encoding the antimicrobial peptide precursor of the dwarf frog (Rana ventricosa) consists of 714 nucleotides (SEQ ID NO:2) (GenBank Accession Number: OP264074), and the sequence from the 5' end to the 3' end is as follows:

[0038]

[0039] Nucleotides 373–522 are the encoding gene for the mature dwarf frog antimicrobial peptide Nv-CATH.

[0040] 5. Determination and synthesis of amino acid sequence of Nv-CATH, an antimicrobial peptide from the spotted dwarf frog.

[0041] Sequencing results show that the full-length primary structure of the antimicrobial peptide Nv-CATH from the dwarf frog is as follows:

[0042] NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG, the antimicrobial peptide Nv-CATH from the spotted dwarf frog, was synthesized by Shanghai Jietai Biotechnology Co., Ltd., with a peptide purity greater than 95%.

[0043] II. The inhibitory effect of the antimicrobial peptide Nv-CATH from the spotted frog on bacterial growth.

[0044] 1. Determination of the minimum inhibitory concentration (MIC) of Nv-CATH

[0045] Luria-Bertani (LB) liquid medium was used as the culture medium. After the test strains were revived on LB solid medium, the colonies were diluted with sterile water to a concentration of 2 × 10⁻⁶ cells / mL. 5 Prepare bacterial suspensions for later use. When determining the minimum inhibitory concentration (MIC), use the two-fold dilution method for antibacterial testing. The specific method is as follows: Add 0.01 ml of sample to 0.19 ml of culture medium as the first well, mix well, and then add 0.1 ml to the second well (which already contains 0.1 ml of fresh culture medium). Continue with serial dilutions, discarding 0.1 ml from the sixth well. The surrounding wells serve as controls. Add the calibrated bacterial suspension (2 × 10⁻⁶) to each well. 5 Mix 0.1 ml of cfu / ml solution, incubate at 37°C for 18 hours, and measure the absorbance at 600 nm. The minimum inhibitory concentration (MIC) is the lowest sample concentration at which no bacterial growth is observed. The bacterial strain was obtained from the First Affiliated Hospital of Kunming Medical University. This experiment was repeated three times, and the average value was taken. The results are shown in Table 1.

[0046] Table 1. The inhibitory effect of the antimicrobial peptide Nv-CATH from the spotted frog on bacterial growth:

[0047]

[0048] As shown in Table 1, the antimicrobial peptide Nv-CATH from the dwarf frog exhibits significant and potent broad-spectrum antimicrobial activity (including Gram-positive and Gram-negative bacteria).

[0049] 2. Determination of the bacterial killing kinetics curve of Nv-CATH

[0050] To further determine the bactericidal effect of Nv-CATH, the bactericidal kinetics of Nv-CATH against S. aureus ATCC25923, A. junii 13A15578, K. pneumoniae 0813343, S. paratyphi A, S. haemolyticus 13A13770, and E. faecalis ATCC29212 were investigated.

[0051] like Figure 1 As shown, at a concentration of 4×MIC, the times required for Nv-CATH to induce 100% mortality of six bacterial strains—S. aureus ATCC25923, A. junii 13A15578, K. pneumoniae 0813343, S. paratyphi A, E. faecalis ATCC29212, and S. haemolyticus 13A13770—were 1 h, 4 h, 6 h, 4 h, 6 h, and 4 h, respectively. Among these, Nv-CATH exhibited rapid bactericidal activity against S. aureus ATCC25923, achieving 100% bacterial death in just 1 h.

Claims

1. A dwarf frog antimicrobial peptide Nv-CATH, characterized in that, The antimicrobial peptide is a cyclic polypeptide encoded by the defense peptide gene of the spotted dwarf frog, an amphibian species endemic to China. It consists of a pair of intramolecular disulfide bonds formed by cysteine ​​residues at positions 2 and 6. The polypeptide has a molecular weight of 3356.98 Daltons and an isoelectric point of 11.

56. Its amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the antimicrobial peptide Nv-CATH from the spotted frog as described in claim 1 in the preparation of a medicament for treating infectious diseases caused by hemolytic Staphylococcus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter jumbo and / or Salmonella paratyphi A.

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