CN102516382B - Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis - Google Patents

Abstract

The invention discloses novel antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a separation method and a chemical synthesis method for the antimicrobial peptide Hainanenin-5 of Amolops hainanensis and application of the gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and belongs to the technical field of biomedicine. The Hainanenin-5 is small peptide which is obtained by the steps of electrically stimulating the Amolops hainanensis so as to collect a skin exudate, centrifuging, freeze-drying, performing gel filtration column chromatography and reverse phase-high performance liquid chromatography (RP-HPLC) and purifying, and has a C terminal containing a seven-membered ring; the molecular weight is 2,337.91; and the isoelectric point is 10.11. A primary structure of the Hainanenin-5 is Phe<1>a<2>Leu<3>Gly<4>Ala<5>Val<6>Thr<7>Lys<8>Arg<9>Leu<10>Pro<11>Ser<12>Leu<13>Phe<14>Cys<15>Leu<16>Ile<17>Thr<18>Arg<19>Lys<20>Cys<21>. A gene for encoding a Hainanenin-5 precursor consists of 321 nucleotides, wherein a mature peptide part is encoded by 139th to 201st nucleotides. The Hainanenin-5 has low molecular weight and a simple structure, only contains one disulfide bond, and can be conveniently prepared through chemical synthesis and genetic engineering. The Hainanenin-5 is used for preparing a therapeutic medicine for pathogenic microorganism infectious diseases, has broad-spectrum antimicrobial activity, has the activities on gram-positive bacteria, gram-negative bacteria and fungi (including clinically separated medicine-resistant strains), and also has the advantages of low hemolysis.

Description

The rapid frog antimicrobial peptide Hainanenin-5 in a kind of Hainan and gene thereof, application

Technical field

The invention provides a kind of wide spectrum antimicrobial peptide Hainanenin-5 and gene, separation and chemical synthesis process and application that derives from the rapid frog in Hainan (Amolops hainanensis), belong to field of biomedicine technology.

Background technology

Along with antibiotic, use extensively, in a large number, the resistance that pathogenic micro-organism produces microbiotic also improves gradually, become the difficult point of infectious diseases treatment, occurred especially in the recent period the threat of " superbug ", all forced people to remove to find novel anti-microbial agents.Recent research finds, peptide antibiotics has the anti-microbial activity of wide spectrum, has " traditional microbiotic " incomparable superiority simultaneously: as when the least action concentration, fast and wide spectrum ground killing microorganisms (comprising clinical anti-medicine bacterium at present); Fungi is also had to restraining effect; Can not induce the generation of drug resistance strain; All effective for local infection and systemic infection, promise to be antiseptic-germicide of new generation, its development is in widespread attention at present.Simultaneously also to have molecular weight little for antibacterial peptide, and stability is high, be difficult for the features such as the power of developing immunity to drugs.Antibacterial peptide is that the class producing through induction in organism has bioactive micromolecule polypeptide, and antibacterial peptide is extensively present in bacterium, plant and animal body, is an important component part of host immune system of defense.

Different from traditional microbiotic, batrachians antibacterial skin peptide is by disturbing prokaryotic cell prokaryocyte membrane structure to cause plasma membrane permeability to increase, thereby causes antibacterial or germicidal action, and pathogenic agent is difficult for developing immunity to drugs to it.Batrachians antibacterial peptide is efficient except having, wide spectrum and be difficult for producing the anti-microbial activity of resistance, also have antitumor, virus, protozoon isoreactivity, and to human normal cell almost without acting on.Therefore, in antibiotics resistant pathogenic strains, constantly occur, the diseases such as virus and tumour cause the today that has threat to class health, and antibacterial peptide will be expected to become resisting pathogenic microbes of new generation and the medicine of tumour.According to domestic and foreign literature, from various biogenetic derivation separation, obtain different active polypeptide, and some has entered clinical treatment.Active polypeptide Magainin tool wide spectrum anti-microbial effect as obtained from Xenopus laevis (Xenopus laevis) Skin exudate has anti-tumor activity simultaneously, in the U.S., has got permission as extensive pedigree antibiotic, and it is clinical that its gene engineering product entered for three phases; The active polypeptide IB-367 that Intrabiotics company produces has got permission to be used for the treatment of stomatocace one clinical trial phase that cancer patient causes because of various bacteria infection; Applied Microbiology company and the cooperation of Astra company also obtain good curative effect with the clinical trial of active polypeptide nisin treatment stomach ulcer.

China has abundant amphibian animal resource, and it is the important component part of Chinese biological resource, and wherein a lot of species have the value such as edible or medicinal, have very large exploitation potentiality.Amphibians is owing to living in for a long time under the environment that humidity, darkness, microorganism grow in a large number, three cover defense mechanisms in long-term natural evolution process, have been formed: (1) physical barriers, mucous gland is secreted a large amount of glycoprotein and proteoglycan, be wrapped in skin appearance face, hinder the intrusion of pathogenic bacteria; (2) acquired immune system, contains IgG antibody in Amphibians skin; (3) innate immune system, mainly consists of a large amount of antibacterial peptides.At present, the clearest to the research of batrachia antibacterial peptide in batrachians especially skin and enteron aisle antibacterial peptide.From Zasloff, from water frog skin, be separated to after Magainins, in succession from different batrachias, be separated to many antibacterial peptide families (as Dermaseptins, Temporins, Ranatuerins, Brevinins, Tigerinins) with the bacteriostatic activity of wide spectrum, some also have unique anti-mycotic activity.There is long history in China to the application of batrachians medicine, but the research of its activeconstituents and pharmacological properties is mainly concentrated on to the organic molecules such as alkaloid, and the research of its skin activity peptide matters is also rarely had to report.The rapid frog in Hainan (Amolops hainanensis) is the Amphibians of the rapid Rana of Ranidae, is Chinese endemic species, is distributed in the ground such as Hainan.

Hainan of the present invention rapid frog (Amolops hainanensis) ring-type antibacterial peptide Hainanenin-5 molecular weight is little, simple in structure, only, containing a disulfide linkage, facilitates chemosynthesis and preparation.Other family's antibacterial peptides of having reported in the Amphibians of comparing source, Hainanenin-5 antimicrobial acivity is stronger, and the antimicrobial acivity with wide spectrum, gram positive bacterium, gram negative bacterium and fungi are all had to activity, comprising a large amount of clinical separated Resistant strain, there is in addition the low beneficial features of hemolytic, all make Hainanenin-5 become the good template of externally used antimicrobial drug development.

Summary of the invention

The invention provides a kind of a kind of antimicrobial peptide Hainanenin-5 that derives from the rapid frog in Hainan (Amolops hainanensis) with very strong antimicrobial acivity and gene thereof, separation and purification, chemosynthesis analysis and application.

In order to realize object of the present invention, the invention provides following technical scheme:

Hainan rapid frog antimicrobial peptide Hainanenin-5 is first through Sephadex G-50 gel-filtration from the rapid frog Skin exudate in batrachians Hainan, then the ring type polypeptide that a kind of C end that reverse phase HPLC (RP-HPLC) separation obtains contains seven yuan of Rana Box, molecular weight is 2337.91 dalton, and iso-electric point is 10.11.Polypeptide complete sequence primary structure is: phenylalanine L-Ala leucine glycine L-Ala α-amino-isovaleric acid Threonine Methionin arginine leucine proline(Pro) Serine leucine phenylalanine halfcystine leucine Isoleucine Threonine Arginine Lysine halfcystine.Contain 4 alkaline amino acid residues (2 arginine and 2 Methionins), no acidic amino-acid residue, static charge is+4, illustrates that it is a kind of basic polypeptide.The 15th and the 21st halfcystine form intramolecular disulfide bond.

Built the rapid frog (Amolops hainanensis) the skin cDNA library in Hainan, therefrom screening obtains the precursor sequence of Hainanenin-5, gene sequencing result show the to encode gene of the rapid frog (Amolops hainanensis) the Hainanenin-5 precursor in Hainan is comprised of 321 Nucleotide, from 5 ' end to 3 ' terminal sequence, is:

1 ATGTTCACCA TGAAGAAATC CATGTTACTC CTTTTCTTCC TTGGGACCAT CAACTTATCT

61 CTCTGTGAGC AACAGAGAGA TGCCGAAGAA GAAAGAAGAG ACGATGAAGA TAAAAGGGAT

121 GTTGAAGTGG AAAAACGATT TGCATTAGGT GCGGTTACTA AGCGTTTGCC ATCACTGTTT

181 TGTTTGATTA CCAGAAAATG TTGAAGCTTG AGCTGGAAAT CATCTGATGA GAAATATAAT

241 TTAGCTAAAT GCACATCAGA TATCTTATAA AAAATAAAAA TTCTCACATG CAAAAAAAAA

301 AAAAAAAAAA AAAAAAAAAA A

The rapid frog (Amolops hainanensis) the mature peptide Hainanenin-5 in coding Hainan is 139-201 position Nucleotide, and its aminoacid sequence is: Phe ¹ala ²leu ³gly ⁴ala ⁵val ⁶thr ⁷lys ⁸arg ⁹leu ¹⁰pro ¹¹ser ¹²leu ¹³phe ¹⁴cys ¹⁵leu ¹⁶ile ¹⁷thr ¹⁸arg ¹⁹lys ²⁰cys ²¹(FALGAVTKRLPSLFCLITRKC).

The chemical synthesis process of Hainanenin-5: according to the aminoacid sequence of the coding Hainan rapid frog (Amolops hainanensis) antibacterial peptide hainanenin-5 mature peptide, with synthetic its complete sequence of automatic Peptide synthesizer; By HPLC reversed phase column chromatography desalting and purifying, and determine that its purity is greater than 95%; With ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS), measure its molecular weight.

The high purity Hainanenin-5 antimicrobial peptide that chemosynthesis obtains can be dissolved in sterilizing ultrapure water, for pharmacologically active, detects.

Beneficial effect of the present invention is that separation and purification obtains antimicrobial peptide Hainanenin-5 from the rapid frog skin secretion lyophilized powder of Hainan, from build the rapid frog (Amolops hainanensis) cDNA library in Hainan, clone's gene of antimicrobial peptide Hainanenin-5 precursor that obtains encoding, obtains mature peptide Hainanenin-5 by chemical synthesis process.This ring-type antibacterial peptide hainanenin-5 molecular weight is little, simple in structure, only, containing a disulfide linkage, facilitates chemosynthesis and preparation.Other family's antibacterial peptides of having reported in the Amphibians of comparing source, Hainanenin-5 antimicrobial acivity is stronger, and the antimicrobial acivity with wide spectrum, gram positive bacterium, gram negative bacterium and fungi are all had to activity, comprising a large amount of clinical separated Resistant strain, there is in addition the low beneficial features of hemolytic, all make Hainanenin-5 become the good template of externally used antimicrobial drug development.

Accompanying drawing explanation:

Figure 1A is the SephadexG-50 gel filtration chromatography figure of the rapid frog (Amolops hainanensis) the antimicrobial peptide Hainanenin-5 in Hainan of the present invention.Arrow is depicted as Peak Activity.

Figure 1B is the anti-phase high pressure liquid chromatography figure of the rapid frog (Amolops hainanensis) the antibacterial peptide Hainanenin-5 in Hainan of the present invention.Arrow a is depicted as Hainanenin-5.

Embodiment

Below in conjunction with technical scheme, describe specific embodiments of the invention in detail, but content of the present invention is not limited to this.

Embodiment 1

The separation and purification of Hainanenin-5: choose the rapid frog in adult Hainan (A.hainanensis) that is in a good state of health (n=5), with ultrapure water drip washing back.Adopt electrostimulation to collect skin secretion, with electric shock device, frog skin of back is carried out to electricity irritation, voltage I0V, electric shock time 3ms, after stimulating, by skin secretion with 0.9% normal saline flushing extremely in clean container, the centrifugal 20min of 12000rpm, preserves after abandoning supernatant, lyophilize.-20 ℃ save backup.

The first step Sephadex G-50 gel permeation chromatography: 10ml0.1M phosphoric acid salt (Na for 0.9g A.hainanensis skin of back secretory product lyophilized powder ₂hPO ₄-NaH ₂pO ₄pH 6.0) damping fluid dissolving, the centrifugal 10min of 12000rpm, get supernatant liquor and be splined on the Sephadex G-50 gel exclusion chromatography post (1.6cm x 90cm, Amersham Bioscience) that balance is good, use same buffer solution elution, flow velocity 3mL/10min, with automatic Fraction Collector, collect 3mL/ pipe, 220nm detects and collects each peak and detects anti-microbial activity, and freeze-drying is standby.

Second step reverse phase HPLC (RP-HPLC) is: the peak of the activeconstituents that the separation of Sephadex G-50 gel exclusion chromatography obtains is dissolved in pure water again, 4 ℃, the centrifugal 15min of 12000rpm, get supernatant liquor, with 0.45 μ m membrane filtration, collect filtrate and be splined on C18 reversed-phase column (the Hypersil BDS C18 through the abundant balance of ultrapure water containing 1 ‰ trifluoroacetic acids, 30cm x 0.46cm) by the elution system that acetonitrile (containing 1 ‰ trifluoroacetic acids) forms, carry out gradient elution, 215nm detects peptide concentration.Each peak that collection obtains, freeze-drying is concentrated, with the deionized water of sterilizing, again dissolves and carries out anti-microbial activity detection.

The Hainanenin-5 molecular weight determination of the 3rd step Primary Structure Analysis purifying adopts electron spray(ES) level Four bar flight time tandem mass spectrometry (ESI-Q-TOF-MS, Biosystems/MDS Sciex Toronto, Canada).Isoelectric focusing electrophoresis is measured iso-electric point, with automatic Protein Sequencer, measures aminoacid sequence structure.

Embodiment 2

Clone and the gene sequencing of antimicrobial peptide Hainanenin-5 precursor-gene, comprising:

Adopt AxyPrepTM Multisource Total RNA Miniprep Kit to extract the total RNA of the rapid frog (Amolops hainanensis) skin in Hainan, utilize Creator ^tMsMART ^tMcDNA library builds storehouse test kit and builds the rapid frog (Amolops hainanensis) the skin cDNA library in Hainan.

cDNA the first chain is synthesized in Reverse Transcriptase reverse transcription, and primer is:

Forward SMARTer V Oligonucleotide:5 '-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3 ' (X=undisclosed base in the proprietary SMARTer oligo sequence)

Reverse 3 ' In-Fusion SMARTer CDS Primer:

5’-CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTVN-3’(N＝A，C，G，or T；V＝A，G，or C)。

Utilize Advantage DNA Polymerase to synthesize the second chain, primer is: forward 5 '-primer:5 '-AAGCAGTGGTATCAACGCAGAGT-3 '.Reverse primer is all 3 ' In-Fusion SMARTer CDS Primer.

Design a specificity forward primer (P1) and a reverse non-specific universal primer (3 '-primer), the rapid frog (Amolops hainanensis) the skin cDNA library in Hainan of take is template, the cDNA of pcr amplification Hainanenin-5 precursor.

Forward P1:5 '-CCAAAGATGTTSMCCWYGAAG-3 ' (M=A or C; W=A or T; Y=C or T)

Reverse non-specific universal primer is 3 '-primer, its sequence by: 5 '-CGGGGTACGATGAGACACCAT-3 ' is obtained positive monoclonal and is carried out gene nucleotide series mensuration, pMD ^tMthe 19-T vector universal primer that checks order:

Forward M13F:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ';

Reverse M13R:5 '-GAGCGGATAACAATTTCACACAGG-3 ';

Concrete steps are as follows:

The first step, AxyPrepTM Multisource Total RNA Miniprep Kit extract the total RNA of the rapid frog (Amolops hainanensis) skin in Hainan (testing below utensil used and reagent all through processing, without RNase):

A. from the rapid frog (Amolops hainanensis) back, fresh Hainan of slaughtering, cut a fritter skin histology, put into the cell cryopreservation tube of Liquid nitrogen precooler, then put into rapidly liquid nitrogen and preserve;

B. the organization material being kept in liquid nitrogen is taken out, put into the mortar of precooling, fully grind rapidly, constantly in mortar, add a little liquid nitrogen during this time, grind into powder, add 400 μ l Buffer R-I, with syringe, repeatedly aspirate 8-10 time, proceed in 1.5ml centrifuge tube (test kit provides).Add 150 μ l Buffer R-II, vortex oscillation 15-30s, the centrifugal 5min of 12,000 * g.

C. the supernatant after centrifugal is transferred in new 1.5ml centrifuge tube, adds 250 μ l Virahols, mix.By preparing pipe, be placed in 2ml centrifuge tube (test kit provides), the mixed solution in transfer step 4 is to preparing in pipe, the centrifugal 1min of 6,000 * g.Abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, prepare and in pipe, add 500 μ l Buffer W1A, the centrifugal 1min of 12,000 * g.Abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, prepare and in pipe, add 700 μ l Buffer W2, the centrifugal 1min of 12,000 * g; With same method again with 700 μ l Buffer W2 washings once.Abandon filtrate, by preparing pipe, put and get back in 2ml centrifuge tube, the centrifugal 1min of 12,000 * g.By preparing pipe, put into a clean 1.5ml centrifuge tube (test kit provides), add 70-100 μ l Buffer TE preparing periosteum central authorities.The standing 1min of room temperature, the centrifugal 1min of 12,000 * g, wash-out obtains RNA.

Second step, Creator ^tMsMART ^tMcDNA library builds storehouse test kit and builds the rapid frog (Amolops hainanensis) the skin cDNA library in Hainan:

Synthetic (the mRNA reverse transcription) of the first chain:

A. in new 0.2ml PCR pipe (without DNase and RNase), prepare following mixed solution:

Template ribonucleic acid 1 μ g

Oligo dT Primer(50μM) 1μl

dNTP Mixture(10mM each) 1μl

RNase free ddH2O is supplemented to 10 μ l, after mixing, of short duration centrifugal; In PCR instrument after 65 ℃ of insulation 5min, rapidly at chilling 2min on ice; Of short duration centrifugal after, in above-mentioned pipe, be formulated as follows inverse transcription reaction liquid:

RNase free dH2O is supplemented to 20 μ l systems, after mixing, of short duration centrifugal; In PCR instrument, complete following program: 42 ℃, 60min; 70 ℃, 15min; 4 ℃, preserve.CDNA is stored in-80 ℃.

Synthesizing of the second chain:

The gene clone screening of the 3rd step, the rapid frog (Amolos hainanensis) the antibacterial peptide hainanenin-5 in Hainan

1, design of primers

According to signal peptide conserved sequence design forward degenerated primer 5 '-CCAAAGATG TTSMCCWYGAAG-3 ' (M=A or C of the batrachia antibacterial peptide precursor of having reported; W=A or T; Y=C or T).The centrifugal 5min of first 12000rpm before primer is used, the mole number that then basis is indicated adds the ddH of respective volume ₂o is dissolved to the concentration of 20 μ M.

2, pcr amplification

The skin cDNA synthesizing of take is template, with above-mentioned degenerated primer, in conjunction with building the joint primer that storehouse test kit provides: 3 '-primer:5 '-ATTCTAGAGGCCGAGGCGGCCGAC-3 ', carries out pcr amplification.In 0.2ml PCR pipe, add following reagent (cumulative volume 20 μ l):

After mixing, of short duration centrifugal.PCR condition is: 94 ℃ of sex change 5min; 30 circulations: 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 40s; 72 ℃ are extended 10min; 4 ℃ of preservations.After reaction finishes, get 5 μ l products in 1% agarose gel electrophoresis testing goal band.

3, object fragment reclaims

Increased and with glue, reclaimed test kit (day root biological) and carry out the recovery of object fragment afterwards.By the target DNA fragment and the sequencing vector pMD that reclaim ^tM19-T vector connects, and is transformed into CaCl ₂-MgCl ₂the DH5 α competent cell that method prepares.Getting 100 μ l transformed bacteria liquid is uniformly coated on the LB nutrient agar flat board that contains 100 μ g/ml penbritins (Amp); After dry on surface, be placed on 37 ℃ of constant temperature culture carton upside downs and cultivate 12-16h, the bacterium colony growing carries out next step bacterium colony PCR and measures.

4, object fragment sequence is measured

Picking list bacterium colony is done template, take M13-F and M13-R as primer, and bacterium colony PCR detects Insert Fragment size in single bacterium colony.Primer sequence is as follows:

M13-F：5’-GTAAAACGACGGCCAGTG-3’

M13-R：5’-CAGGAAACAGCTATGACC-3’

Select the positive colony that contains object fragment and deliver to the order-checking of DNA sequencing company.In sequencing result and GeneBank database, sequence is compared.

Gene sequencing and the result of the 4th step, Hainan rapid frog (Amolops hainanensis) antibacterial peptide Hainanenin-5 precursor: the gene of the coding Hainan rapid frog (Amolops hainanensis) antimicrobial peptide Hainanenin-5 precursor is comprised of 321 Nucleotide, from 5 ' end to 3 ' terminal sequence, is:

1 ATGTTCACCA TGAAGAAATC CATGTTACTC CTTTTCTTCC TTGGGACCAT CAACTTATCT

61 CTCTGTGAGC AACAGAGAGA TGCCGAAGAA GAAAGAAGAG ACGATGAAGA TAAAAGGGAT

121 GTTGAAGTGG AAAAACGATT TGCATTAGGT GCGGTTACTA AGCGTTTGCC ATCACTGTTT

181 TGTTTGATTA CCAGAAAATG TTGAAGCTTG AGCTGGAAAT CATCTGATGA GAAATATAAT

241 TTAGCTAAAT GCACATCAGA TATCTTATAA AAAATAAAAA TTCTCACATG CAAAAAAAAA

301 AAAAAAAAAA AAAAAAAAAA A

The sequence table of the cDNA Nucleotide of the rapid frog (Amolops hainanensis) antimicrobial peptide Hainanenin-5 coding region, Hainan is: sequence length is 321 bases, sequence type: nucleic acid, chain number: strand, topology: ring texture, sequence kind: cDNA, source: the rapid frog (Amolops hainanensis) skin histology in Hainan.

The rapid frog (Amolops hainanensis) the mature peptide Hainanenin-5 in coding Hainan is 139-201 position Nucleotide, and its aminoacid sequence is: Phe ¹ala ²lue ³gly ⁴ala ⁵val ⁶thr ⁷lys ⁸arg ⁹lue ¹⁰pro ¹¹ser ¹²lue ¹³phe ¹⁴cys ¹⁵lue ¹⁶ile ¹⁷thr ¹⁸arg ¹⁹lys ²⁰cys ²¹(FALGAVTKRLPSLFCLITRKC).

Embodiment 3

The chemical synthesis process of antimicrobial peptide Hainanenin-5:

1, according to the mature peptide Hainanenin-5 aminoacid sequence of inferring, with synthetic its complete sequence of automatic Peptide synthesizer (Applied Biosystems), by HPLC reversed phase column chromatography desalting and purifying.

2, molecular weight determination adopts ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS).

Embodiment 4

The pharmacological evaluation of antimicrobial peptide Hainanenin-5:

1.Hainanenin-5 antimicrobial acivity detects:

1.1 inhibition zone analytical methods: the Hainanenin-5 of chemosynthesis is dissolved in aseptic ultrapure water with the concentration of 2mg/ml.Microbionation, on Luria-Bertani (LB) solid medium, is inverted and is cultivated in 37 ℃ of incubators.After bacterium colony grows, with transfering loop picking list bacterium colony, evenly coat LB solid culture primary surface, the little filter paper that is 0.5cm by the diameter through sterilizing is pasted on media surface, gets 10 μ l testing samples and drips on filter paper.In 37 ℃ of incubators, be inverted and cultivate 18-20h, observe inhibition zone and whether form, have inhibition zone to form and represent that sample has bacteriostatic activity.The size of inhibition zone can be weighed the power of anti-microbial activity.To the bacterial strain of hainanenin-5 sensitivity be recorded.

1.2Hainanenin-5 measures sensitive strain minimal inhibitory concentration (Minimum Inhibitory Concentration, MIC).

This experiment is made positive control with traditional microbiotic penbritin, and sterile liquid MH makes negative control; Minimal inhibitory concentration is defined as the minimum peptide concentration that suppresses microorganism growth completely that naked eyes can be observed, or absorbance value is not higher than the minimum concentration of negative control 5%.

The microorganism of the new activation of picking, is seeded to sterile liquid MH substratum, and in 37 ℃ of constant temperature oscillators, 200rpm cultivates 10-16h to logarithmic phase; The light absorption value of surveying bacterium liquid 600nm light wave place with ultraviolet spectrophotometer, light absorption value is 1 o'clock, concentration is approximately 10 ⁹cFU/ml, is diluted to 10 by bacterium liquid with sterile liquid MH substratum ⁶cFU/ml, stand-by on ice; On 96 microwell plates, compound concentration gradient is the hainanenin-5 sample solution through 0.22 μ m aperture membrane filtration of 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 625 μ g/ml, 3.13 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml, 0.39 μ g/ml, 0.20 μ g/ml, every hole 50 μ l; Every hole adds the above-mentioned dilution bacterium of 50 μ l liquid; In constant temperature oscillator 37 ℃, 100rpm shaking culture 18h; Use microplate reader to survey 600nm light absorption value or visual inspection; Above-mentioned experiment repeats 3 times, averages.

From table 1, Hainanenin-5 shows very strong anti-microbial activity to most test strain, particularly the endurance strain to clinical separation.In the bacterial strain detecting 17 kinds of experiments, Hainanenin-5 shows extremely strong anti-microbial activity to most gram negative bacteriums (comprising reference culture and clinical separated Resistant strain).In 5 strain gram-positive microorganism (G+) bacterial strains of all tests, Enterococcus faecium 1299 (IS), extremely responsive to Hainanenin-5, minimal inhibitory concentration is 4.69 μ g/ml, and positive control penbritin is greater than 100 μ g/ml to the MIC of this bacterium.In addition, Hainanenin-5 also has stronger anti-mycotic activity.The minimal inhibitory concentration of Candida albicans ATCC 2002 and Slime mould 090413 (IS) is respectively to 2.34 μ g/ml and 9.38 μ g/ml.

The anti-microbial activity of table 1 Hainanenin-5 antimicrobial peptide

* MIC: minimal inhibitory concentration, concentration value is the mean value that repeats experiment for 3 times; > 100 μ g/ml:2mg/ml dosage scraps of paper bacteriostatic tests have inhibition zone, and 100 μ g/ml dosage are without bacteriostatic activity; IS: clinical isolates strain.Above result is three independent empirical average values that repeat.

2, the hemolytic activity analysis of Hainanenin-5

This experiment is made positive control with 1%Triton X-100, with physiological saline, makes negative control.Prepare red corpuscle: get 1ml human blood, be placed in 15ml centrifuge tube, add 10ml physiological saline, mix the centrifugal 5min of rear 2000rpm, resuspended with physiological saline, till repeated centrifugation to supernatant liquor does not take on a red color; With the resuspended erythroprecipitin of 1ml physiological saline, the resuspended liquid that takes a morsel adds in small beaker, with normal saline dilution to being blush; Every kind of test sample preparation 1000 μ l systems:

10 μ l polypeptide sample+990 μ l erythrocyte diluting fluid Hainanenin-5 (final concentration is 20 μ g/ml)

10 μ l 1 ‰ Triton X-100+990 μ l erythrocyte diluting fluid positive controls

10 μ l physiological saline+990 μ l erythrocyte diluting fluid negative controls

3 repetition systems are respectively done in Hainanenin-5 and positive and negative contrast.37 ℃ of incubation 30min, the centrifugal 5min of 3000rpm; Collect supernatant 200 μ l, survey respectively 540nm place absorbance value, calculate the mean value of 3 repetitions; Hemolysis rate=(sample 540nm absorbance value-negative control) * 100%/(positive control-negative control).

Experimental result shows, when Hainanenin-5 concentration is 25 μ g/ml and 50 μ g/ml (for 5～10 times of minimal inhibitory concentration), it is only 13.31% and 19.14% respectively to the hemolysis rate of human red cell, shows its strong selectivity to microorganism cells.

Claims (4)

1. the rapid frog antimicrobial peptide in Hainan Hainanenin-5 is the ring type polypeptide that the separated a kind of C end obtaining contains seven yuan of Rana Box from the rapid frog Skin exudate in batrachians Hainan, molecular weight is 2337.91 dalton, iso-electric point is 10.11, polypeptide complete sequence primary structure is: phenylalanine L-Ala leucine glycine L-Ala α-amino-isovaleric acid Threonine Methionin arginine leucine proline(Pro) Serine leucine phenylalanine halfcystine leucine Isoleucine Threonine Arginine Lysine halfcystine, the 15th and the 21st halfcystine form intramolecular disulfide bond.

2. the gene of the antimicrobial peptide Hainanenin-5 that screening obtains from the rapid frog (Amolops hainanensis) the skin cDNA library in Hainan having built, the gene of Hainan rapid frog (Amolops hainanensis) antimicrobial peptide Hainanenin-5 precursor of it is characterized in that encoding forms (Genbank accession:JF329956) by 321 Nucleotide, from 5 ' end to 3 ' terminal sequence, is:

1 ATGTTCACCA TGAAGAAATC CATGTTACTC CTTTTCTTCC TTGGGACCAT CAACTTATCT

61 CTCTGTGAGC AACAGAGAGA TGCCGAAGAA GAAAGAAGAG ACGATGAAGA TAAAAGGGAT

121 GTTGAAGTGG AAAAACGATT TGCATTAGGT GCGGTTACTA AGCGTTTGCC ATCACTGTTT

181 TGTTTGATTA CCAGAAAATG TTGAAGCTTG AGCTGGAAAT CATCTGATGA GAAATATAAT

241 TTAGCTAAAT GCACATCAGA TATCTTATAA AAAATAAAAA TTCTCACATG CAAAAAAAAA

301 AAAAAAAAAA AAAAAAAAAA A

The rapid frog (Amolops hainanensis) the mature peptide Hainanenin-5 in coding Hainan is 139-201 position Nucleotide, and its aminoacid sequence is: Phe ¹ala ²leu ³gly ⁴ala ⁵val ⁶thr ⁷lys ⁸arg ⁹leu ¹⁰pro ¹¹ser ¹²leu ¹³phe ¹⁴cys ¹⁵leu ¹⁶ile ¹⁷thr ¹⁸arg ¹⁹lys ²⁰cys ²¹(FALGAVTKRLPSLFCLITRKC).

3. the purposes of the rapid frog antimicrobial peptide in Hainan according to claim 1 Hainanenin-5 in the medicine of preparing cause pathogeny imcrobe infection disease.

4. purposes according to claim 3, is characterized in that, described antimicrobial peptide Hainanenin-5 is dissolved in sterilizing ultrapure water, for pharmacologically active, detects.

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