CN110950947B

CN110950947B - Rana temporaria chensinensis host defense peptide DMS-PS2, and gene and application thereof

Info

Publication number: CN110950947B
Application number: CN201911314267.7A
Authority: CN
Inventors: 高艺恬; 吴迪; 吴明江; 宋昕宇; 佟海滨; 张旭
Original assignee: Wenzhou University
Current assignee: Wenzhou University
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2022-04-29
Anticipated expiration: 2039-12-19
Also published as: CN110950947A

Abstract

The invention discloses a rana naemarginata host defense peptide DMS-PS2, a gene and application thereof, and belongs to the field of biomedicine. The host defense peptide DMS-PS2 is a polypeptide consisting of 28 amino acids, and the molecular weight is 2953.5 daltons. The gene coding DMS-PS2 precursor peptide (GenBank: LT718217.1) consists of 346 nucleotide sequences, wherein the 136-th 228 nucleotide sequence is the coding gene of DMS-PS2 mature peptide, and the mature peptide DMS-PS2 is obtained by further post-translational modification. The DMS-PS2 has broad-spectrum antibacterial activity, has strong antibacterial effect on free bacteria and bacterial biofilms, and has the action mode of directly acting on the bacterial biofilms to break the bacterial plasma membranes, so that bacterial drug resistance is not easy to generate, and theoretical basis is provided for developing anti-infectious disease treatment drugs and medical equipment coating drugs.

Description

Rana temporaria chensinensis host defense peptide DMS-PS2, and gene and application thereof

Technical Field

The invention relates to a rana naemarginata host defense peptide DMS-PS2 and application thereof, belonging to the technical field of biomedicine.

Background

Antibiotics are widely used to treat infections caused by susceptible microorganisms, often bacteria or fungi. However, many bacteria develop resistance to these "traditional antibiotics" due to their inappropriate use, or even abuse. New antimicrobial alternatives are then sought. In recent years, researches show that the polypeptide antibiotics not only have broad-spectrum antimicrobial activity, but also have incomparable advantages compared with the traditional antibiotics: at very low concentrations, it is possible to rapidly kill various bacteria (including clinically resistant strains); it also has inhibitory effect on fungi and viruses; the drug resistance to microorganisms is not easy to generate; the polypeptide drug is effective for local infection and systemic infection, so the polypeptide drug is expected to become a new generation antimicrobial drug. At present, the development of polypeptide antimicrobial drugs is receiving increasingly extensive attention.

Many amphibians are widely used in medicine, and recent researches show that skin secretions of the amphibians have wide pharmacological activities, such as antimicrobial, anti-tumor, antioxidant, immunoregulation, wound repair, analgesic effect and the like. Therefore, the screening of some pharmacologically active monomer compounds from the skin of amphibians is a hotspot of the new drug invention. According to the literature reports at home and abroad, different active polypeptides have been separated from various biological sources, and some active polypeptides enter the clinical stage. Although China has a long history of application to amphibians, the amphibians are used as medicines as a whole, and the research on active ingredients and pharmacological properties of the amphibians is weak.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a novel rana nandiniana host defense peptide DMS-PS2 with broad-spectrum antimicrobial property, and a gene and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme: a Rana temporaria chensinensis David host defense peptide DMS-PS2 amino acid sequence and application thereof, wherein the molecular weight of a polypeptide consisting of 28 amino acids encoded by Rana temporaria chensinensis David defense peptide gene is 2953.5 daltons, and the amino acid sequence is shown as SEQ ID NO. 1:

Ala Leu Trp Lys Thr Leu Leu Lys Asn Val Gly Lys Ala Ala Gly Lys Ala Val

1 5 10 15

Leu Asn Ala Val Thr Asp Met Val Asn Gln-amide

20 25

further, the gene GenBank: LT718217.1 is composed of 346 nuclear amino acid sequences, the nuclear amino acid sequence is shown in SEQ ID NO.2, the gene sequencing result shows that the gene for coding the precursor of the defending peptide of the rana naemarginata is composed of 346 nucleotides (SEQ ID NO:2) (GenBank Accession Number: LT718217.1), and the sequence from 5 'end to 3' end is:

1 atggcgttcc taaagaaatc tcttttcctt gtactattcc ttggacttgt ctctctttct

61 atctgtgaag aagagaaaag agaaaatgaa gatgaggaga aacaagaaga tgatgagcaa

121 agtgaagaga agagagctct gtggaaaact ttattaaaaa atgtagggaa agctgcagga

181 aaagcggttt taaatgcagt tactgatatg gtaaatcaag gagagcaata aagttaagaa

241 aatgtaaatc aaattgctct aaggagtgca attatcaata attgtaggca aacttatatt

301 aaagcatatt gaacataaaa aaaaaaagaa aaaaaaaaaa aaaaaa

wherein, the 136-position 228 nuclear amino acid is the coding gene of mature Rana arborescens host defense peptide DMS-PS2, and the mature peptide DMS-PS2 is obtained through further post-translational modification.

The defensive peptide has strong inhibiting and killing effects on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, candida albicans and methicillin-resistant staphylococcus aureus through experimental investigation, and does not show hemolytic activity under the active concentration, so that the defensive peptide can be used as a potential antibacterial drug. In addition, experiments investigate the strong membrane-penetrating killing effect of the host defense peptide on bacterial biofilms of staphylococcus aureus, escherichia coli and pseudomonas aeruginosa, so that the host defense peptide can be used as a medical instrument coating antibacterial agent.

In conclusion, the invention has the following beneficial effects: the invention provides a novel broad-spectrum antimicrobial rana nanmeiensis host defense peptide DMS-PS2, which has obvious effect of inhibiting the growth of bacteria and fungi on infectious diseases caused by escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, candida albicans and methicillin-resistant staphylococcus aureus, has the effect mode of directly acting on a bacterial biomembrane to break the bacterial plasma membrane, and can be used as an antibacterial agent and a medical device coating medicine.

Drawings

FIG. 1 shows the skin exudate of the purified Rana catesbeiana of this example, with arrows indicating the components having antibacterial activity;

FIG. 2 shows the mechanism of action of the dendrobe defense peptide DMS-PS2 of this example in inhibiting bacterial growth.

Detailed Description

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

Firstly, preparation and amino acid sequence determination of rana chensinensis host defense peptide DMS-PS2

1. Collecting skin secretion of Rana temporaria chensinensis David

The living body rana naemarginata is cleaned by water and placed in a cylindrical sample bottle with the diameter of 10 cm and the height of 10 cm. The frog back surface was rubbed and the frog back foam liquid was rinsed with 100ml of deionized water. The secretion is frozen in liquid nitrogen, freeze dried in freeze drier and freeze dried at-80 deg.c.

2. Separation and purification of rana naemarginata host defense peptide DMS-PS2

The collected secretion freeze-dried powder is used as a raw material, the south American frog host defense peptide DMS-PS2 is purified according to the following procedures, and each separated and purified component is subjected to molecular weight detection, and the specific method is as follows:

(a) 5mg of the above secretion lyophilized powder was dissolved in 1ml of trifluoroacetic acid/deionized water (0.05/99.95; v/v). Centrifugation (3000 Xg, 30 min) was carried out and the supernatant was collected and stored at-20 ℃.

(b) The supernatant adopts reversed phase high pressure liquid phase (RP-HPLC) C₁₈The column was purified in a 2795 quaternary gradient HPLC, U.S.A., by eluting with a linear gradient of trifluoroacetic acid/deionized water (0.05/99.95; v/v) to trifluoroacetic acid/deionized water/acetonitrile (0.05/20/79.95; v/v/v) at a flow rate of 1ml/min, collecting the eluted fractions per minute, and detecting the molecular weight of the fractions per minute by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS), which was performed in a Voyager DE Mass spectrometer, U.S.A. Lyophilizing the fraction having a molecular weight of 2953.5 daltons of the mature peptide deduced from the sequence obtained by molecular cloning, and continuing to use RP-HPLC C₁₈Further purifying with chromatographic column to obtain purified Nanmeia frog host defense peptide DMS-PS 2.

3. Determination of amino acid sequence of Rana dybowskii host defense peptide DMS-PS2

The purified rana naxatilis host defense peptide DMS-PS2 was sequenced using MS/MS fragment sequencing in an LCQ flash ion trap mass spectrometer from Thermo Fisher Scientific, usa. Sequencing results confirm that the full-sequence primary structure of the rana chensinensis defense peptide DMS-PS2 is as follows: ALWKTLLKNVGKAAGKAVLNAVTDMVNQ-amide.

Cloning of DMS-PS2 gene as defense peptide of rana naemarginata host

1. Purification of mRNA from Rana temporaria chensinensis skin secretion

mRNA isolation and purification A Dynabead mRNA DIRECT kit was used. Adding liquid nitrogen into the above lyophilized powder 5mg, grinding for three times, dissolving in 1ml of lysate, cracking on ice for 5min, intermittently shaking for several times, mixing, centrifuging (12000 Xg for 20 min), and collecting supernatant. Mu.l of magnetic beads (Dynabeads Oligo (dT)₂₅) Flicking and mixing evenly, adsorbing for 30 seconds by a magnetic frame, abandoning the supernatant, adding 250 mu l of lysine/Binding Buffer for resuspending magnetic beads, then adsorbing for 30 seconds by the magnetic frame, abandoning the supernatant. Adding the frog skin secretion lysate to resuspend the magnetic beads, incubating for 15 minutes on ice until the magnetic frame adsorbs for 2 minutes, discarding the supernatant, and Washing three times with 500. mu.L of Washing Buffer A and Washing Buffer B respectivelyAnd adsorbing the supernatant on a magnetic frame for 30 seconds, and discarding the supernatant. Add 18. mu.L of Elution Buffer, incubate at 80 ℃ for 2 min, adsorb on magnetic rack for 30 sec, transfer supernatant to new tube.

3. Construction of Rana temporaria chensinensis skin cDNA library

Using SMART from CLONTECH^TMAnd constructing a Kit of the RACE Kit plasmid cDNA library.

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

Adding 1 μ l 3 'PCR primer and 5' PCR primer into 2 centrifuge tubes, sequentially adding 4 μ l Rana Nigromaculata skin mRNA and 1 μ l Smart II An oligo, mixing, incubating at 70 deg.C for 2 min, standing on ice for 2 min, sequentially adding 2 μ l 5 XFirst-Strand Buffer, 1 μ l DTT, 1 μ l dNTP Mix, and 1 μ l BD PowerScript into each tube^TMThe Reverse Transcriptase was mixed well, incubated at 42 ℃ for 1.5 hours, then 50. mu.l of PCR water was added to each tube and mixed well, incubated at 72 ℃ for 7 minutes and stored at-20 ℃.

(b) And amplifying the second strand by using a 3' cDNA end rapid amplification polymerase chain reaction (RACE-PCR) method

Mu.l of first strand cDNA (reverse transcription of mRNA), 2.6. mu.l of PCR-Grade water, 1. mu.l of 10 × BD Advantage 2 PCR buffer, 0.2. mu.l of dNTP Mix, 0.5. mu.l of NUP universal PCR primers, 0.5. mu.l of Gene-specific sense primer and 0.2. mu.l of 50 × BD Advantage 2 Polymerase Mix were placed in a centrifuge tube for reaction. Amplification was performed in a PCR instrument according to the following procedure: 1 minute at 94 ℃; 35 cycles (94 ℃, 30 seconds; 50-68 ℃, 30 seconds; 72 ℃, 3 minutes); 72 ℃ for 3 minutes. After the circulation, the mixture was stored at 4 ℃. Gene amplification specific primer sequence 5 '-TCTGAATTRYAAGMSCARACATG-3', nested primer (NUP) primer sequence 5'-AAGCAGTGGTATCAACGCAGAGT-3'.

(c) And recovering the PCR product

Extraction recovery was performed using the E.Z.N.A. Cycle-pure kit from Omega Bio-Tek, according to the following steps:

the cDNA double strand obtained by PCR is mixed with 5 XTE buffer, then the mixture is transferred to a Hibind DNA centrifugal purification column, centrifuged at 12800rpm for 1 minute, and the waste liquid in the collection tube is poured off. Add 700. mu.l of washing buffer to the centrifugation and purification column, centrifuge at 12800rpm for 1 minute, and discard the waste from the collection tube. Then 500. mu.l of washing buffer was added to the centrifugation and purification column and centrifuged at 12800rpm for 1 minute, and the waste liquid in the collection tube was discarded. The column was then centrifuged at 12800rpm for 2 minutes and the column was placed in a new centrifuge tube. Add 30. mu.l of PCR Water and let stand at room temperature for 2 minutes. Centrifuging at 12800rpm for 30 seconds, and obtaining the purified cDNA double strand at the bottom of the tube.

(d) Ligation and conversion of ligation products

The purified sample was dissolved with 5. mu.L of PCR-grade water, and 1.5. mu.L of the sample solution was added with 0.5. mu.L of pGEM-T Easy vector, 0.5. mu. L T4 DNA ligand, 2.5. mu.L of 2 × Rapid Ligation Buffer, left at room temperature for 1 hour, and then left overnight at 4 ℃. mu.L of the mixture was mixed with 50. mu.L of E.coli competent cells, and the mixture was allowed to stand on ice for 20 minutes, at 42 ℃ for 47 seconds, and on ice for 2 minutes. 950. mu.L of SOC medium was added and incubated at 37 ℃ for 2.5 hours with slow shaking. 100. mu.l of the suspension was spread on LB medium containing X-Gal, IPTG and Amp overnight at 37 ℃ to form a single colony. The white colonies successfully recombined were inoculated onto IPTG/X-Gal/Ampicillin LB agar plates using sterile inoculating loops and cultured at 37 ℃ for 24 h. White colonies were picked and re-inoculated onto IPTG/X-Gal/Ampicillin LB agar plates, secondary blue-white screening was performed, after incubation at 37 ℃ for 24h, the white colonies were lysed with 20. mu.L of PCR-grade water, the solution was incubated at 100 ℃ for 5 minutes, then left on ice for 5 minutes, and centrifuged at 12000 Xg for 10 minutes.

4. Clone screening of south American tree frog host defense peptide gene

The amplification primer sequence was 5'-GTAACGCCAGGGTTTTCCCAG-3', and the other primer sequence was 5'-TGTGAGCGGATAACAATTTCAC-3'. The PCR reaction was performed under the following conditions: 30 seconds at 94 ℃; 45 seconds at 52 ℃; 30 cycles at 72 ℃ for 2 min 30 sec. Purification was performed using the E.Z.N.A. Cycle-pure kit from Omega Bio-Tek and the above experimental method.

5. Determination of gene sequence of defensive peptide of Rana temporaria chensinensis David

(a) Sequencing reaction

A BigDye Terminator v3.1 cycle sequencing kit is adopted, an equivalent reagent and a purified sample are mixed and then are amplified by a PCR method, and the PCR reaction is carried out under the following conditions: at 96 ℃ for 1 minute; at 96 deg.C for 20 seconds; at 55 deg.C for 10 seconds; 60 ℃ for 4 minutes, for 26 cycles.

(b) Product purification and sequencing reactions

The reaction product is purified by ethanol precipitation. And uniformly mixing 72 mu L of 95% ethanol with an equivalent sample, further transferring 10 mu L of pure water to all the mixtures, placing the samples at room temperature for 20 minutes, centrifuging at 12000 x g for 20 minutes, removing supernatant, adding 260 mu L of 70% ethanol for 10 minutes, washing precipitates, collecting DNA samples by using a centrifuge, and performing vacuum concentration and drying. After the sample is redissolved by 10 mu L HiDiDNA, the sample is heated at 95 ℃ for 4.5 minutes and then placed on ice for 5 minutes. The prepared samples were loaded onto a sequencing plate and analyzed using an ABI 3100 DNA sequencer, USA.

The gene sequencing result shows that the gene for coding the precursor of the defending peptide of the rana naemarginata consists of 346 nucleotides (SEQ ID NO:2) (GenBank Accession Number: LT718217.1), and the sequence from the 5 'end to the 3' end is as follows:

1 atggcgttcc taaagaaatc tcttttcctt gtactattcc ttggacttgt ctctctttct

61 atctgtgaag aagagaaaag agaaaatgaa gatgaggaga aacaagaaga tgatgagcaa

121 agtgaagaga agagagctct gtggaaaact ttattaaaaa atgtagggaa agctgcagga

181 aaagcggttt taaatgcagt tactgatatg gtaaatcaag gagagcaata aagttaagaa

241 aatgtaaatc aaattgctct aaggagtgca attatcaata attgtaggca aacttatatt

301 aaagcatatt gaacataaaa aaaaaaagaa aaaaaaaaaa aaaaaa

wherein, the 136-228 nuclear amino acid is a coding gene of mature rana arborescens host defense peptide DMS-PS2, and is further subjected to posttranslational modification to obtain DMS-PS 2.

Thirdly, the antibacterial effect of the tree frog defense peptide DMS-PS2 on bacteria

(a) The effect of the defensive peptide DMS-PS2 in inhibiting the growth of free bacteria:

Mueller-Hinton Broth (MHB) liquid medium was used as the culture medium. When the Minimum Inhibitory Concentration (MIC) is determined, twice dilution is adoptedThe method is used for antibacterial detection. The specific method comprises the following steps: adding 10 μ l sample into 190 μ l culture medium as the first well, mixing 100 μ l with 100 μ l fresh culture medium, adding into the 2 nd well, diluting in multiple times, sucking out 0.1ml from the 6 th well, discarding, comparing the wells at four sides, adding corrected bacteria solution (2 × 10)⁵cfu/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 Guangdong province culture Collection, and the test was repeated three times, and the results were averaged as shown in Table 1.

(b) The elimination effect of the defense peptide DMS-PS2 on bacterial biofilms:

Mueller-Hinton Broth (MHB) liquid medium was used as the culture medium. The bacteria were cultured under the above-mentioned three (a) conditions for 24 hours, washed with a sterile phosphate buffer solution, and then cultured at 37 ℃ for 1 hour with the addition of 200. mu.l of a polypeptide (1-512. mu.g/ml) of a series of concentrations dissolved in MHB medium. Free bacteria were washed 2 times with sterile PBS. After 30 minutes of fixation by addition of 200. mu.l of methanol, the sample was air-dried. The dye was applied with 0.1% crystal violet for 30 minutes and then washed 2 times with pure water. The content was completely dissolved in 33% glacial acetic acid, and then the absorbance was measured at a wavelength of 550nm, and the results are shown in Table 2.

Fourthly, hemolysis of the tree frog defense peptide DMS-PS 2:

DMS-PS2 was dissolved in PBS, serial concentrations were obtained by a two-fold dilution method, and the prepared 4% horse blood red cell suspensions were incubated with serial concentrations of the polypeptide for 2 hours at 37 ℃. The final test concentration ranged from 512. mu.g/ml to 1. mu.g/ml. After incubation for 10 minutes at 5000 xg, the positive control was a 1% Triton100 and horse blood mixture, and the results were measured for absorbance at λ =550nm using a microplate reader, and the results were treated with PRIZM7, as shown in table 1.

TABLE 1 Rana arborina defence peptide DMS-PS2 inhibition of free bacterial growth and haemolysis

Bacterial strains	Minimum inhibitory concentration (μ M)
		Gram-positive bacteria:
staphylococcus aureus (NCTC 10788)	2.71
		Methicillin-resistant staphylococcus aureus (ATCC 12493)	5.42
Methicillin-resistant staphylococcus aureus (ATCC 43300)	2.71
		Gram-negative bacteria:
escherichia coli (NCTC 10418)	2.71
		Pseudomonas aeruginosa (ATCC 27853)	5.42
Fungi:
		candida albicans (NCPF 1467)	5.42
50% hemolytic concentration	46.95

As can be seen from table 1, the dendrocalamus defensive peptide DMS-PS2 has a significant effect of inhibiting the growth of bacteria and fungi, and particularly has a potent antibacterial effect against drug-resistant bacteria.

TABLE 2 Elimination of bacterial biofilms by Rana catesbiana defense peptide DMS-PS2

Bacterial strains	Minimum transmembrane antibacterial concentration (μ M)
		Gram-positive bacteria:
staphylococcus aureus (NCTC 10788)	5.42
		Gram-negative bacteria:
escherichia coli (NCTC 10418)	5.42
		Pseudomonas aeruginosa (ATCC 27853)	86.67

As can be seen from Table 2, the dendrocalamus defensive peptide DMS-PS2 has strong membrane-penetrating bacteriostatic action on the biomembrane generated by bacteria, and is suggested to be used as a medical appliance coated with an antibacterial agent.

Fourthly, the action mechanism of the tree frog defense peptide for inhibiting the growth of bacteria;

the results of staining by using a thermo fisher LIVE/DEAD Baclight bacterial viability kit, examining the influence of DMS-PS2 on the permeability of an MRSA plasma membrane under MIC and MBC concentrations, observing the influence of DMS-PS2 on the cell morphology of MRSA bacteria by a scanning electron microscope, and comparing with bacteria which are not treated by drugs under normal conditions are shown in figure 2.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the design concept of the present invention should be included in the scope of the present invention.

SEQUENCE LIST

<110> university of Wenzhou

<120> rana nandiniana host defense peptide DMS-PS2, and gene and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 28

<212> PRT

<213> Phyllomedusa sauvagii

<220>

<221> ACETYLATION

<221> (28) ..(28)

<221> c-terminal Linked amide

<400> 1

Ala Leu Trp Lys Thr Leu Leu Lys Asn Val Gly Lys Ala Ala Gly Lys Ala Val

Leu Asn Ala Val Thr Asp Met Val Asn Gln-amide

<210> 2

<211> 346

<212> DNA

<213> Phyllomedusa sauvagii

<400> 2

atggcgttcc taaagaaatc tcttttcctt gtactattcc ttggacttgt ctctctttct

atctgtgaag aagagaaaag agaaaatgaa gatgaggaga aacaagaaga tgatgagcaa

agtgaagaga agagagctct gtggaaaact ttattaaaaa atgtagggaa agctgcagga

aaagcggttt taaatgcagt tactgatatg gtaaatcaag gagagcaata aagttaagaa

aatgtaaatc aaattgctct aaggagtgca attatcaata attgtaggca aacttatatt

aaagcatatt gaacataaaa aaaaaaagaa aaaaaaaaaa aaaaaa

Claims

1. A Rana dybowskii host defense peptide DMS-PS2, comprising: the amino acid sequence is shown in SEQ ID NO. 1.

2. The use of the rana nigromaculata host defense peptide DMS-PS2 according to claim 1 for the preparation of therapeutic drugs and antibacterial coated drugs for medical devices, wherein the peptide DMS-PS2 is selected from the group consisting of: the therapeutic drug and the medical instrument antibacterial coating drug are used for treating infection caused by escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, candida albicans and methicillin-resistant staphylococcus aureus.