CN115974997A - Shibataea kumasiana antibacterial peptide Nv-CATH and gene and application thereof - Google Patents

Abstract

The invention relates to an antibacterial peptide Nv-CATH of rana obovata, a gene and an application thereof, wherein the antibacterial peptide Nv-CATH is a cyclic polypeptide encoded by the specific species rana obovata defense peptide gene of the rana obovata, the molecular weight is 3356.98 dalton, the isoelectric point is 11.56, and the amino acid sequence is shown as SEQ ID NO. 1. The gene (GenBank access No. OP264074) for coding the C.abdominalis antibacterial peptide Nv-CATH precursor consists of 714 nucleotide sequences, the nucleotide sequences are shown in SEQ ID NO:2, wherein, the 373-522 th nucleotides are coding genes of the mature C.abdominalis antibacterial peptide Nv-CATH. Application of the rana obovata antibacterial peptide Nv-CATH in preparing medicaments for treating infectious diseases caused by staphylococcus haemolyticus, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter junii and/or paratyphoid bacillus. The present invention provides a new antibacterial peptide Nv-CATH with strong broad-spectrum antibacterial activity.

Description

Shibataea kumasiana antibacterial peptide Nv-CATH and gene and application thereof

Technical Field

The invention relates to an antibacterial peptide Nv-CATH of rana obovata and a gene and application thereof, belonging to the field of biomedicine.

Background

Recent studies have shown that antimicrobial peptides are an important component of all animal, plant and microbial defense systems. They are small molecular active polypeptides produced by the organism for resisting the invasion of pathogenic microorganisms, and have the characteristics of small molecular weight, good stability, quick sterilization, difficult generation of drug resistance and the like. The characteristics enable the antibacterial peptide to have very wide application prospect, and the antibacterial peptide is expected to replace the traditional antibiotic medicine which is easy to generate drug resistance clinically at present. The reason why the antibacterial peptide is not easy to generate drug resistance is that the sterilization mechanism of the antibacterial peptide is different from that of the traditional antibiotics. They act mainly on the cell membrane of bacteria to destroy its stability, and then the permeability of the cell membrane is changed, so that the contents leak out to cause cell death. The structural form and biological activity of the antibacterial peptide from different species or in the same species are greatly different. Researchers at home and abroad are dedicated to exploring antibacterial peptides with novel structures from different species and directly utilizing the antibacterial peptides or using the antibacterial peptides as templates for molecular modification. Currently, antimicrobial peptides have been used in marine product preservation and animal feed additives. The discovery of antibacterial peptides with potential medicinal value from various natural resources by combining with ecological environment is a hot spot of the research of polypeptide new drugs at present.

Amphibians are the most important resource in the discovery of antimicrobial peptide resources, have exposed skins, and can secrete various antimicrobial peptides with novel molecular structures and complex and diverse functions in order to resist the attack of various microorganisms in the environment. The active polypeptides are widely involved in various physiological activities of organisms, and have various pharmacological activities, such as antimicrobial, anti-tumor, antioxidant, immunoregulation, wound repair, analgesia and the like. The bonobo blossoms often inhabit plateau areas with high altitude and large day-night temperature difference, which means that with the development of adaptability, the bonobo blossoms have necessarily formed an effective set of immune defense systems to resist the attack of microorganisms in the harsh living environment. The frog (Nanora ventricosa) is an amphibian of the genus Bombay of the order Argasanidae, which is a species unique to China, and has not been reported as a potent antimicrobial peptide of the cathelicidin family in the skin.

Disclosure of Invention

The invention aims to provide a novel rana obovata antibacterial peptide with strong broad-spectrum antimicrobial activity (including gram-positive bacteria and gram-negative bacteria), and a gene and application thereof.

The antibacterial peptide Nv-CATH is a cyclic polypeptide encoded by a defense peptide gene of a specific species of the amphibian Shibataea kumasasa, the molecular weight is 3356.98 dalton, the isoelectric point is 11.56, and the full-sequence primary structure (amino acid sequence SEQ ID NO: 1) of the polypeptide is as follows:

(NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG) which cysteine at position 2 and cysteine at position 6 form a pair of intramolecular disulfide bonds to form a cyclic polypeptide.

The gene (GenBank access No. OP264074) for coding the rana obovata antibacterial peptide Nv-CATH precursor consists of 714 nucleotide sequences, the nucleotide sequence is shown as SEQ ID NO:2, and the sequence from 5 'end to 3' end is as follows:

wherein, the 373-522 th nucleotide is a coding gene of mature rana obovata antibacterial peptide Nv-CATH.

The invention relates to application of a bonobo obo frog antibacterial peptide Nv-CATH in preparing a medicament for treating infectious diseases caused by staphylococcus haemolyticus, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter junii and/or typhoid bacillus paratyphi A.

The invention has the beneficial effects that:

the invention provides a novel ranobo obovata antibacterial peptide Nv-CATH with broad-spectrum antimicrobial activity (including gram-positive bacteria and gram-negative bacteria), which has a remarkable effect of inhibiting the growth of bacteria on infectious diseases caused by staphylococcus hemolyticus, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter junii and paratyphoid bacillus.

Drawings

FIG. 1 is a graph of the kinetics of bacterial killing for Nv-CATH, in which: the killing of Nv-CATH pairs (a) s. Aureus ATCC25923, (B) a. Junii 13a15578, (C) k. Pneumoniae 0813343, (D) s. Paratyphi a, (E) e.faecalis ATCC29212, (F) s. Haemolyticus 13a13770 at 0, 1, 2, 4, 6, 8 and 12h at 37 ℃ at a concentration of 4 × MIC was determined; data are the average of three independent experiments.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

1. Cloning and amino acid sequence determination of rana obovata antibacterial peptide Nv-CATH gene

1. Total RNA extraction from skin of Shibataea kumasiana

The living rana obovata was washed with water, put into liquid nitrogen to be quickly frozen for 10 hours, and 300mg of skin tissue was taken, and 3ml of Trizol solution was added to the skin tissue, and homogenized in a 20ml glass homogenizer for 30 minutes. Adding equal volume of phenol/chloroform solution, mixing vigorously, standing at room temperature for 10min, centrifuging at 12000rpm at 4 deg.C for 10min, and removing precipitate. Adding equal volume of isopropanol into the supernatant, standing at room temperature for 10 minutes, centrifuging at 12000rpm for 10 minutes at 4 ℃, washing the precipitate with 75% ethanol once, and drying in the air to obtain the precipitate at the bottom of the tube, namely the total RNA of the skin of the rana obovata.

2. Purification of Japanese buttercup skin mRNA: mRNA isolation and purification Using Promega, USA

mRNA Isolation Systems kit

The extracted total RNA from the skin of Shibataea kumasasa was dissolved in 500. Mu.l of DEPC water, placed in a 65 ℃ water bath for 10 minutes, added with 3. Mu.l of Oligo (dT) probe and 13. Mu.l of 20 XSSC solution, mixed well, and left to cool at room temperature to obtain solution A. And (3) flicking and uniformly mixing magnetic beads (SA-PMP) until the mixture is adsorbed by a magnetic frame for 30 seconds, discarding the supernatant, adding 0.5 XSSC 0.3m1 until the mixture is adsorbed by the magnetic frame for 30 seconds, and finally adding 0.1ml of 0.5 XSSC for suspension to obtain solution B. Adding the solution A into the solution B, standing at room temperature for 10 minutes until the solution is adsorbed by a magnetic rack for 30 seconds, discarding the supernatant, washing with 0.1 XSSC for 4 times, finally discarding the supernatant, adding 0.1ml of DEPC water for suspension, adsorbing by the magnetic rack for 30 seconds, transferring the supernatant to a new test tube, adding 0.15m1 DEPC water for resuspension, adsorbing by the magnetic rack for 30 seconds, and transferring the supernatant to the test tube, wherein the supernatant is purified mRNA of the skin of the Japanese spongia frog. 1/10 volume of 3M sodium acetate, pH5.2, equal volume of isopropanol was added, left at-70 ℃ for 30 minutes, centrifuged at 12000rpm for 10 minutes at 4 ℃, the supernatant discarded, and the precipitate was dissolved in 10. Mu.l of DEPC water.

3. Construction of a Japanese butterhood skin cDNA library

The Creator of CLONTECH is adopted ^TM SMART ^TM cDNA Library Construction Kit of cDNA Library restriction Kit.

(a) First Strand cDNA Synthesis (reverse transcription of mRNA)

Mu.l of Japanese butterflybush skin mRNA, 1. Mu.l of SMART IV oligonucleotide, 1. Mu.l of CDS III/3' PCR primer and 2. Mu.l of deionized water were added to a 0.5ml sterile centrifuge tube to make the total volume 5. Mu.l. Mix the reagents in the centrifuge tubes and centrifuge at 12000rpm for 15 seconds and incubate at 72 ℃ for 2 minutes. The centrifuge tubes were incubated on ice for 2 minutes. The following reagents 2.0. Mu.l of 5 Xprimary strand buffer, 1.0. Mu.l of 20mM dithiothreitol, 1.0. Mu.l of 10mM dNTP mix, and 1.0. Mu.l of PowerScript reverse transcriptase were added to the centrifuge tube. The reagents were mixed in the centrifuge tubes and centrifuged at 12000rpm for 15 seconds and incubated at 42 ℃ for 1 hour. The first strand synthesis was stopped by placing the centrifuge tube on ice. Mu.l of the first strand of the synthesized cDNA was taken from the centrifuge tube and used.

(b) Amplification of the second Strand Using Long-terminal polymerase chain reaction (LD-PCR)

Mu.l of first strand cDNA (reverse transcription of mRNA), 80. Mu.l of deionized water, 10. Mu.l of 10 XDavantage 2PCR buffer, 2. Mu.l of 50 XDNTP mix, 2. Mu.l of 5'PCR primer, 2. Mu.l of CDS III/3' PCR primer and 2. Mu.l of E.coli polymerase centrifuge tube were pre-heated at 95 ℃ for reaction. Amplification was performed in a PCR instrument according to the following procedure: at 95 ℃ for 20 seconds; 22 cycles (95 ℃ C., 5 seconds; 68 ℃ C., 6 minutes). After completion of the circulation, the double strand cDNA synthesized in the centrifuge tube was recovered.

(c) PCR product recovery

By PROMEGA

The SV Gel and PCR Clean-Up System kit is extracted and recovered, and the steps are as follows:

adding cDNA double chains obtained by PCR into equal-volume membrane binding buffer, reversing and uniformly mixing, transferring the mixed solution into a centrifugal purification column, and standing at room temperature for 5 minutes to ensure that the DNA is fully bound with a silica gel membrane. Centrifuge at 12000rpm for 30 seconds and discard the waste liquid from the collection tube. Add 700. Mu.l of eluent (containing ethanol) to the centrifugation and purification column, centrifuge at 12000rpm for 30 seconds, and discard the waste liquid from the collection tube. And (5) repeating the step (2). Centrifuge at 12000rpm for 5 minutes, and place the centrifugal purification column in a new centrifuge tube. 30. Mu.l of ultrapure water was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes. Centrifuging at 12000rpm for 30 seconds, and obtaining the purified cDNA double strand at the bottom of the tube.

(d) Preparation of E.coli DH5 alpha competent cells

A single DH 5. Alpha. Colony was selected, inoculated into 3ml Luria-Bertani (LB) medium containing no ampicillin, cultured overnight at 37 ℃, and the following day, the above-mentioned bacterial solution was re-inoculated into 50ml of LB medium at a ratio of 1. When OD is measured ₆₀₀ When the value reached 0.35, the bacterial culture was harvested. The bacteria were transferred to a sterile, single-use, ice-pre-cooled 50m1 polypropylene tube and placed on ice for 10 minutes and the culture cooled to 0 ℃. The cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The broth was decanted and the tube inverted for l min to allow the last traces of broth to drain. 30ml of precooled 0.1mol/LCaCl per 50ml of initial broth ₂ -MgCl ₂ Solution (80 mmol/L MgCl) ₂ ,20mmol/L CaCl ₂ ) Resuspend each cell pellet. The cells were recovered by centrifugation at 4100rpm for 10 minutes at 4 ℃. The medium was decanted and the tube inverted for l minutes to allow the last traces of medium to drain. Every 50m1 of the initial culture was 2m1 of 0.1mol/L CaCl precooled with ice ₂ Resuspend each cell pellet and dispense for use.

(e) Enzyme cleavage, ligation and transformation of ligation products

Mu.l of Takara pMD18-T vector and 4. Mu.l of a Shibataea kumasasa cDNA double-stranded solution were added to a microcentrifuge tube, and the total amount was 5. Mu.l. Mu.l (equal amount) of ligase buffer mixture was added. The reaction was carried out at 16 ℃ for 2 hours. A total of 10. Mu.l was added to 100. Mu.l of DH 5. Alpha. Competent cells and left on ice for 30 minutes. After heating at 42 ℃ for 90 seconds, the mixture was left on ice for 1 minute. Then, 890. Mu.l of LB medium incubated at 37 ℃ was added thereto, and the mixture was incubated at 37 ℃ for 60 minutes with slow shaking. 200. Mu.l of the suspension was spread on LB medium containing X-Gal, IPTG and Amp and cultured at 37 ℃ for 16 hours to form a single colony. Colonies were washed with 5ml LB liquid medium per LB plate and frozen with 30% glycerol. The constructed cDNA contained approximately 1X 10 ⁶ Individual clones.

4. Cloning and screening of antibacterial peptide gene of squash frog and determination of gene sequence

According to 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)) is designed and combined with a 5' PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3') provided by a kit to amplify a 5' fragment of cDNA encoded by cathelicidin. PCR conditions were pre-denaturation at 95 ℃ for 2min, denaturation at 92 ℃ for 10s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 40s, 30 cycles of denaturation-annealing-extension, and re-extension at 72 ℃ for 10min. The PCR product was purified by gel electrophoresis and cloned into pMD19-T vector (Takara, japan) for DNA sequencing.

Based on the 5' end sequence obtained by sequencing, a forward primer Nv-CATH-F1 (5'-ATGAAGGTCTGGCAGTGTGCG-3') at the 5' end is designed and combined with a PCR primer (5'-ATTCTAGAGGCCGAGGCGGCCG-3') at 3' provided by the kit, and a cDNA full-length sequence coding for cathelicidin is amplified. The PCR reaction conditions were as described above. Finally, the PCR product was cloned into pMD19-T vector and sequenced. The specific sequencing operation steps are as follows: DNA sequencing was performed using an ABI PRISM model 377 applied biosystems DNA sequencer. The final amplified PCR product was recovered using DNA gel recovery kit (DP 209-02, tiangen, china). The recovered fragment was ligated with pMD19-T vector, the ligated product was transformed into E.coli DH 5. Alpha. Competent cells, and after the transformation was completed, the transformed product was spread on a petri dish of LB (Luria-Bertani) solid medium containing AMP, and the petri dish was placed upside down in an incubator and cultured at 37 ℃ for 16 hours. Selecting a single colony as a template to perform colony PCR, determining a PCR product by using agarose gel electrophoresis, selecting a positive clone containing a target fragment, marking the colony, placing the colony in an LB liquid culture medium for culture and amplification, and sending a bacterial liquid to a biological company for DNA sequencing.

The gene sequencing result shows that the gene for encoding the precursor of the rana obovata antibacterial peptide consists of 714 nucleotides (SEQ ID NO: 2) (GenBank Accession Number: OP 264074), and the sequence from the 5 'end to the 3' end is as follows:

wherein, the 373-522 th nucleotide is a coding gene of mature rana obovata antibacterial peptide Nv-CATH.

5. Determination and synthesis of amino acid sequence of antibacterial peptide Nv-CATH of rana obovata

Sequencing results show that the full sequence primary structure of the antibacterial peptide Nv-CATH of the rana obovata is as follows:

NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG, the antibacterial peptide Nv-CATH of the Shibataea kuobovata is synthesized by Shanghai Jie peptide biotechnology limited, and the purity of the polypeptide is more than 95%.

2. Function of antibacterial peptide Nv-CATH of rana obovata for inhibiting bacterial growth

1. Determination of Minimum Inhibitory Concentration (MIC) value of Nv-CATH

Luria-Bertani (LB) liquid medium was used as a culture medium. After the test strain is recovered by LB solid culture medium, the strain is diluted by sterile water to 2X 10 per ml ⁵ Bacterial liquid of each bacterium is reserved. When the minimum inhibitory concentration is determined, the antibacterial detection is carried out by adopting a two-fold dilution method. The specific method comprises the following steps: adding 0.01ml sample into 0.19ml culture medium as the first well, mixing, adding 0.1ml into the 2 nd well (adding 0.1ml fresh culture medium), diluting in multiple times, sucking out 0.1ml from the 6 th well, discarding, comparing the wells at four sides, adding corrected bacteria liquid (2 × 10) ⁵ cfu/ml) 0.1ml, mixed well, incubated at 37 ℃ for 18 hours, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible. The bacterial strains were obtained from the first subsidiary hospital of Kunming medical university, and the test was repeated three times, and the results were averaged as shown in Table 1.

Table 1 effect of the anobotic frog antimicrobial peptide Nv-CATH on inhibiting bacterial growth:

as can be seen from table 1, the rana obovata antimicrobial peptide Nv-CATH has a remarkably strong broad-spectrum antimicrobial activity (including gram-positive bacteria and gram-negative bacteria).

2. Determination of bacterial killing kinetics curves for Nv-CATH

To further determine the bactericidal effect of Nv-CATH, the bacterial killing kinetics of Nv-CATH were examined for s.aureus ATCC25923, a.junii 13a15578, k.pneumoniae 0813343, s.paratyphi a, s.haemolyticus 13a13770, and e.faecalis ATCC 29212.

As shown in fig. 1, the time required for Nv-CATH to induce 100% death of six bacteria, s.aureus ATCC25923, a.junii 13a15578, k.pneumoconiae 0813343, s.paratyphi a, e.faecalis ATCC29212 and s.haemolyticus 13a13770, was 1h, 4h, 6h, 4h and 4h, respectively, at a concentration of 4 × MIC. Among them, nv-CATH showed a rapid bactericidal activity against s.aureus ATCC25923, which requires only 1 hour to kill 100% of bacteria.

Claims (3)

1. The antibacterial peptide Nv-CATH of the bonobo frog is a cyclic polypeptide which is coded by a bonobo frog defense peptide gene of a specific species of the Chinese amphibian and is formed by a pair of intramolecular disulfide bonds consisting of cysteine at the 2 nd position and cysteine at the 6 th position, the molecular weight is 3356.98 dalton, the isoelectric point is 11.56, and the amino acid sequence is shown in SEQ ID NO 1.

2. The gene for coding the rana obovata antibacterial peptide Nv-CATH precursor is characterized in that the precursor gene GenBank access No. OP264074 consists of 714 nucleotides, the nucleotide sequence of the precursor gene is shown as SEQ ID NO. 2, and the 373 th to 522 th nucleotides are coding genes of mature rana obovata antibacterial peptide Nv-CATH.

3. Use of the obobova obovata antibacterial peptide Nv-CATH of claim 1 in the preparation of a medicament for treating infectious diseases caused by staphylococcus haemolyticus, staphylococcus aureus, klebsiella pneumoniae, acinetobacter johnii and/or typhoid bacillus typhi a.

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