CN103172721A - Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof - Google Patents
Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof Download PDFInfo
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Abstract
一种海南湍蛙的抗微生物肽Hainanenin-1及其基因，分离纯化、化学合成和应用，属于生物医学技术领域。Hainanenin-1是电刺激海南湍蛙收集皮肤分泌液离心，冻干后经凝胶过滤柱层析和RP-HPLC后纯化后得到的一种C端含有七元环的小肽，分子量为2278.90，等电点9.50。其一级结构为苯丙氨酸-丙氨酸-亮氨酸-甘氨酸-丙氨酸-缬氨酸-苏氨酸-赖氨酸-亮氨酸-亮氨酸-脯氨酸-丝氨酸-亮氨酸-亮氨酸-半胱氨酸-蛋氨酸-异亮氨酸-苏氨酸-精氨酸-赖氨酸-半胱氨酸。编码Hainanenin-1前体基因由320个核苷酸组成，其中编码成熟肽部分的为第139-201位核苷酸。Hainanenin-1作为制备病原微生物感染疾病的治疗药物被应用，具有广谱的抗微生物活性，同时还具有溶血性低的特点。
The antimicrobial peptide Hainanenin-1 and its gene of Hainan turbulent frog are separated and purified, chemically synthesized and applied, and belong to the field of biomedical technology. Hainanenin-1 is a small peptide containing a seven-membered ring at the C-terminal obtained after being electrically stimulated to collect skin secretions from the Hainan frog, centrifuged, lyophilized, and purified by gel filtration column chromatography and RP-HPLC. The molecular weight is 2278.90. The isoelectric point is 9.50. Its primary structure is phenylalanine-alanine-leucine-glycine-alanine-valine-threonine-lysine-leucine-leucine-proline-serine- Leucine-Leucine-Cysteine-Methionine-Isoleucine-Threonine-Arginine-Lysine-Cysteine. The precursor gene encoding Hainanenin-1 consists of 320 nucleotides, of which the nucleotides 139-201 encode the mature peptide. Hainanenin-1 is used as a therapeutic drug for the preparation of pathogenic microorganism infection diseases, has broad-spectrum antimicrobial activity, and also has the characteristics of low hemolysis.
Description
Technical field
The invention provides a kind of wide spectrum antimicrobial peptide Hainanenin-1 that derives from the rapid frog in Hainan (Amolops hainanensis) and gene, separation and chemical synthesis process and in the application of field of biological pharmacy, belong to field of biomedicine technology.
Background technology
Along with antibiotic extensive, a large amount of use, pathogenic micro-organism also improves gradually to the resistance that microbiotic produces, become the difficult point of infectious diseases treatment, the threat of " superbug " occurred especially in the recent period, all forced people to remove to seek novel anti-microbial agents.Recent research finds, peptide antibiotics has the anti-microbial activity of wide spectrum, has simultaneously " traditional microbiotic " incomparable superiority: as when the least action concentration, fast and wide spectrum ground killing microorganisms (comprising present clinical anti-medicine bacterium); Fungi also there is 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 a molecular weight little for antibacterial peptide, and stability is high, be difficult for the characteristics such as the power of developing immunity to drugs.Antibacterial peptide is that in organism, the class through inducing generation has bioactive micromolecule polypeptide, and antibacterial peptide extensively is 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 to cause the plasma membrane permeability to increase by interference prokaryotic cell prokaryocyte membrane structure, thereby causes antibacterial or germicidal action, and pathogenic agent is difficult for developing immunity to drugs to it.The batrachians antibacterial peptide is efficient except having, wide spectrum and be difficult for producing the anti-microbial activity of resistance, also have antitumor, virus, the protozoon isoreactivity, and to the human normal cell almost without acting on.Therefore, constantly occur in the antibiotics resistant pathogenic strains, the diseases such as virus and tumour cause the today that has threat to class health, and antibacterial peptide will be expected to become the resisting pathogenic microbes of a new generation and the medicine of tumour.According to domestic and foreign literature, separated obtaining different active polypeptide from various biogenetic derivations, and some has entered clinical treatment.Active polypeptide Magainin tool wide spectrum anti-microbial effect as obtaining from Xenopus laevis (Xenopus laevis) Skin exudate has anti-tumor activity simultaneously, has got permission as extensive pedigree antibiotic in the U.S., 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 cancer patient and has infected stomatocace one clinical trial phase that causes because of various bacteria; 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 have been formed in long-term natural evolution process: (1) physical barriers, the mucous gland a large amount of glycoprotein of secretion and proteoglycan, be wrapped in the skin appearance face, hinder the intrusion of pathogenic bacteria; (2) acquired immune system contains IgG antibody in Amphibians skin; (3) innate immune system mainly is made 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 is separated to Magainins from water frog skin after, in succession be separated to many antibacterial peptide families (as Dermaseptins, Temporins, Ranatuerins, Brevinins, Tigerinins) with bacteriostatic activity of wide spectrum from different batrachias, 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 the organic molecules such as alkaloid, and the research of its skin activity peptide matters is also rarely had report.The rapid frog in Hainan (Amolops hainanebsis) is the Amphibians of the rapid Rana of Ranidae, is the endemic species of China, is distributed in the ground such as Hainan.
Hainan of the present invention rapid frog (Amolops hainanensis) ring-type antibacterial peptide Hainanenin-1 molecular weight is little, simple in structure, only contains a disulfide linkage, facilitates chemosynthesis and preparation.Other family's antibacterial peptides of having reported in the Amphibians of comparing source, the Hainanenin-1 antimicrobial acivity is stronger, and the antimicrobial acivity with wide spectrum, gram positive bacterium, gram negative bacterium and fungi all there is activity, comprising a large amount of clinical separation Resistant strain, have in addition the low beneficial features of hemolytic, all make Hainanenin-1 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-1 and gene, separation and purification, chemosynthesis and application of deriving from the rapid frog in Hainan (Amolops hainanensis) with very strong antimicrobial acivity.
In order to realize purpose of the present invention, the invention provides following technical scheme:
Hainan rapid frog antimicrobial peptide Hainanenin-1 is first through Sephadex G-50 gel-filtration from the rapid frog Skin exudate in batrachians Hainan, then reverse phase HPLC (RP-HPLC) separates the ring type polypeptide that a kind of C end that obtains contains seven yuan of Rana Box, molecular weight is 2278.90 dalton, and iso-electric point is 9.50.Polypeptide complete sequence primary structure is: phenylalanine-Ala-Leu-Gly-Ala-α-amino-isovaleric acid-Threonine-Methionin-Leu-Leu-proline(Pro)-Serine-Leu-Leu-halfcystine-methionine(Met)-Isoleucine-Threonine-arginine-Methionin-halfcystine.Contain 3 alkaline amino acid residues (1 arginine and 2 Methionins), no acidic amino-acid residue, static charge is+3, illustrates that it is a kind of basic polypeptide.The 15 and the 21 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-1, the gene sequencing result shows that the gene of the rapid frog (Amolops hainanensis) the Hainanenin-1 precursor in coding Hainan is comprised of 321 Nucleotide, from 5 ' end to 3 ' terminal sequence is:
1?ATGTTGCCCT?TGAAGAAATC?CATGTTACTC?CTTTTCTTCC?TTGGGACCAT?CAACTTATCT?61?CTCTGTGAGCAAGAGAGAGA?TGCCGAAGAA?GAAAGAAGAG?ACGATGAAGA?TAAAAGGGAT
121?GTTGAAGTGG?AAAAACGATT?TGCATTAGGT?GCGGTTACTA?AGCTTTTGCC?ATCACTGTTA
181?TGTATGATTA?CCAGAAAATG?TTGAAGCTTG?AGCTGGAAAT?CATCTGATGA?GAAATATAAT?241TTAGCTAAAT?GCACATCAGA?TATCTTATAA?AAAATAAAAA?TTCTCACATA?TAAAAAAAAA?301?AAAAAAAAAAAAAAAAAAAA
The rapid frog (Amolops hainanensis) the mature peptide Hainanenin-1 in coding Hainan is 139-201 position Nucleotide, and its aminoacid sequence is: Phe
1Ala
2Leu
3Gly
4Ala
5Val
6Thr
7Lys
8Leu
9Leu
10Pro
11Ser
12Leu
13Leu
14Cys
15Met
16Ile
17Thr
18Arg
19Lys
20Cys
21(FALGAVT KLLPSLLCMITRKC).
The chemical synthesis process of Hainanenin-1: according to the aminoacid sequence of the coding Hainan rapid frog (Amolops hainanensis) antibacterial peptide Hainanenin-1 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%; Measure its molecular weight with ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS).
The high purity Hainanenin-1 antimicrobial peptide that chemosynthesis obtains can be dissolved in the sterilization ultrapure water, is used for pharmacologically active and detects.
Beneficial effect of the present invention is that separation and purification obtains antimicrobial peptide Hainanenin-1 from the rapid frog skin secretion lyophilized powder of Hainan, clone's gene of antimicrobial peptide Hainanenin-1 precursor that obtains encoding, obtain mature peptide Hainanenin-1 by chemical synthesis process from build the rapid frog (Amolops hainanensis) cDNA library in Hainan.This ring-type antibacterial peptide Hainanenin-1 molecular weight is little, simple in structure, only contains a disulfide linkage, facilitates chemosynthesis and preparation.Other family's antibacterial peptides of having reported in the Amphibians of comparing source, the Hainanenin-1 antimicrobial acivity is stronger, and the antimicrobial acivity with wide spectrum, gram positive bacterium, gram negative bacterium and fungi all there is activity, comprising a large amount of clinical separation Resistant strain, have in addition the low beneficial features of hemolytic, all make Hainanenin-1 become the good template of externally used antimicrobial drug development.
Description of drawings
Figure 1A is the SephadexG-50 gel filtration chromatography figure of the rapid frog (Amolops hainanensis) the antimicrobial peptide Hainanenin-1 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-1 in Hainan of the present invention.Arrow h is depicted as Hainanenin-1.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme, but content of the present invention is not limited to this.
Embodiment 1
The separation and purification of Hainanenin-1: 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 electricity irritation, voltage I0V, electric shock time 3ms, after stimulating, with 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: 0.9g A.hainanensis skin of back secretory product lyophilized powder 10ml0.1M phosphoric acid salt (Na
2HPO
4-NaH
2PO
4PH 6.0) the 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, collect the 3mL/ pipe with automatic Fraction Collector, 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 that Sephadex G-50 gel exclusion chromatography is separated the activeconstituents that obtains is dissolved in pure water again, 4 ℃, the centrifugal 15min of 12000rpm, get supernatant liquor, with 0.45 μ m membrane filtration, collect C18 reversed-phase column (the Hypersil BDS C18 that filtrate is splined on the abundant balance of ultrapure water through containing 1 ‰ trifluoroacetic acids, 30cm x 0.46cm) elution system that consists of with acetonitrile (containing 1 ‰ trifluoroacetic acids) is carried out gradient elution, and 215nm detects peptide concentration.Each peak that collection obtains, freeze-drying is concentrated, again dissolves and carries out anti-microbial activity and detect with the deionized water of sterilizing.
The Hainanenin-1 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, measures the aminoacid sequence structure with automatic Protein Sequencer.
Embodiment 2
Clone and the gene sequencing of antimicrobial peptide Hainanenin-1 precursor-gene comprise:
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 the 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), take Hainan rapids frog (Amolops hainanensis) skin cDNA library as template, the cDNA of pcr amplification Hainanenin-1 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 to carry out gene nucleotide series and measures, 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 (following experiment utensil used and reagent are all through processing, without RNase):
A. cut a fritter skin histology from the rapid frog (Amolops hainanensis) back, fresh Hainan of slaughtering, put into the cell cryopreservation tube of Liquid nitrogen precooler, then put into rapidly liquid nitrogen and preserve;
The organization material that B. will be kept in liquid nitrogen takes out, put into the mortar of precooling, fully grind rapidly, constantly add a little liquid nitrogen in mortar during this time, grind into powder, add 400 μ l Buffer R-I, repeatedly aspirate 8-10 time with syringe, change 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.To prepare pipe and be placed in 2ml centrifuge tube (test kit provides), the mixed solution in transfer step 4 to the preparation pipe in, the centrifugal 1min of 6,000 * g.Abandon filtrate, will prepare pipe and put and get back in the 2ml centrifuge tube, add 500 μ l Buffer W1A, the centrifugal 1min of 12,000 * g in the preparation pipe.Abandon filtrate, will prepare pipe and put and get back in the 2ml centrifuge tube, add 700 μ l Buffer W2, the centrifugal 1min of 12,000 * g in the preparation pipe; With same method again with 700 μ l Buffer W2 washings once.Abandon filtrate, will prepare pipe and put and get back in the 2ml centrifuge tube, the centrifugal 1min of 12,000 * g.The preparation pipe is put into a clean 1.5ml centrifuge tube (test kit provides), and central authorities add 70-100 μ l Buffer TE at the preparation periosteum.The standing 1min of room temperature, the centrifugal 1min of 12,000 * g, wash-out gets RNA.
Second step, Creator
TMSMART
TMCDNA library builds the 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. prepare following mixed solution in new 0.2ml PCR pipe (without DNase and RNase):
RNase free ddH2O is supplemented to 10 μ l, and is after mixing, of short duration centrifugal; In the PCR instrument after 65 ℃ of insulation 5min, rapidly at chilling 2min on ice; Of short duration centrifugal after, be formulated as follows inverse transcription reaction liquid in above-mentioned pipe:
RNase free dH2O is supplemented to 20 μ l systems, and is after mixing, of short duration centrifugal; Complete following program in the PCR instrument: 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 (Amolops hainanensis) the antibacterial peptide Hainanenin-1 in Hainan
1, design of primers
Signal peptide conserved sequence design forward degenerated primer according to the batrachia antibacterial peptide precursor of having reported
5’-CCAAAGATGTTSMCCWYGAAG-3’(M＝A?or?C；W＝A?or?T；Y＝C?or?T)。Primer uses the front centrifugal 5min of first 12000rpm, then adds the ddH of respective volume according to the mole number of indicating
2O is dissolved to 20 μ M concentration.
2, pcr amplification
Take synthetic skin cDNA as template, build with above-mentioned degenerated primer combination the joint primer that the storehouse test kit provides: 3 '-primer:5 '-ATTCTAGAGGCCGAGGCGGCCGAC-3 ', carry out pcr amplification.Add following reagent (cumulative volume 20 μ l) in 0.2ml PCR pipe:
After mixing, of short duration centrifugal.The 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.Reaction is got 5 μ l products in 1% agarose gel electrophoresis testing goal band after finishing.
3, the purpose fragment reclaims
Reclaim test kit (it root is biological) with glue after amplification is completed and carry out the recovery of purpose fragment.With target DNA fragment and the sequencing vector pMD that reclaims
TM19-T vector connects, and transforms 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 the surface, be placed on to be inverted in 37 ℃ of constant incubators and cultivate 12-16h, the bacterium colony that grows carries out bacterium colony PCR of lower step and measures.
4, the purpose fragment sequence is measured
Picking list bacterium colony is done template, and take M13-F and M13-R as primer, bacterium colony PCR detects Insert Fragment size in single bacterium colony.
Primer sequence is as follows:
M?13-F：5’-GTAAAACGACGGCCAGTG-3’
M?13-R：5’-CAGGAAACAGCTATGACC-3’
Select the positive colony that contains the purpose 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-1 precursor: the gene of the coding Hainan rapid frog (Amolops hainanensis) antimicrobial peptide Hainanenin-1 precursor is comprised of 320 Nucleotide, from 5 ' end to 3 ' terminal sequence is:
1?ATGTTGCCCT?TGAAGAAATC?CATGTTACTC?CTTTTCTTCC?TTGGGACCAT?CAACTTATCT
61?CTCTGTGAGC?AAGAGAGAGA?TGCCGAAGAA?GAAAGAAGAG?ACGATGAAGA?TAAAAGGGAT
121?GTTGAAGTGG?AAAAACGATT?TGCATTAGGT?GCGGTTACTA?AGCTTTTGCC?ATCACTGTTA
181?TGTATGATTA?CCAGAAAATG?TTGAAGCTTG?AGCTGGAAAT?CATCTGATGA?GAAATATAAT
241?TTAGCTAAAT?GCACATCAGA?TATCTTATAA?AAAATAAAAA?TTCTCACATA?TAAAAAAAAA
301?AAAAAAAAAA?AAAAAAAAAA
The sequence table of the cDNA Nucleotide of the rapid frog (Amolops hainanensis) antimicrobial peptide hainanenin-5 coding region, Hainan is: sequence length is 320 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-1 in coding Hainan is 139-201 position Nucleotide, and its aminoacid sequence is: Phe
1Ala
2Leu
3Gly
4Ala
5Val
6Thr
7Lys
8Leu
9Leu
10Pro
11Ser
12Leu
13Leu
14Cys
15Met
16Ile
17Thr
18Arg
19Lys
20Cys
21(FALGAVT KLLPSLLCMITRKC).
Embodiment 3
The chemical synthesis process of antimicrobial peptide Hainanenin-1:
1, according to the mature peptide Hainanenin-1 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-1:
1.Hainanenin-1 antimicrobial acivity detects:
1.1 inhibition zone analytical method: the Hainanenin-1 of chemosynthesis is dissolved in aseptic ultrapure water with the concentration of 2mg/ml.Microbionation is inverted in 37 ℃ of incubators and is cultivated on Luria-Bertani (LB) solid medium.After bacterium colony grows, evenly coat LB solid culture primary surface with transfering loop picking list bacterium colony, will be that the little filter paper of 0.5cm is pasted on media surface through the diameter of sterilization, get 10 μ l testing samples and drip on filter paper.Be inverted in 37 ℃ of incubators 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 record the bacterial strain of Hainanenin-1 sensitivity.
1.2Hainanenin-1 sensitive strain minimal inhibitory concentration (Minimum Inhibitory Concentration, MIC) is measured.
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 fully 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; Survey the light absorption value at bacterium liquid 600nm light wave place with ultraviolet spectrophotometer, light absorption value is 1 o'clock, and concentration is approximately 10
9CFU/ml is diluted to 10 with bacterium liquid with sterile liquid MH substratum
6CFU/ml, stand-by on ice; The compound concentration gradient is the Hainanenin-1 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, 6.25 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 on 96 microwell plates, every hole 50 μ l; Every hole adds the 50 above-mentioned dilution bacterium of 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.
We can find out that Hainanenin-1 shows the strong anti-microbial activity of wide spectrum from table 1, particularly to the endurance strain of clinical separation.Test result show Hainanenin-1 to the MIC of many bacterium than little many of penbritin, illustrate that Hainanenin-1 is stronger than penbritin to the anti-microbial activity of these bacterium.For example in the gram-positive microorganism of 5 kinds of tests, Hainanenin-1 is the strongest to the anti-microbial activity of faecium 1299, and MIC is only 4.69 μ g/ml, and the positive control penbritin is to the MIC of faecium＞100 μ g/ml.Hainanenin-1 is except having anti-microbial effect gram-positive microorganism and Gram-negative bacteria, also have stronger anti-mycotic activity for example the Hainanenin-1 dialogue MIC that reads ATCC2002 be only 2.34 μ g/ml.
The anti-microbial activity of table 1 Hainanenin-1 antimicrobial peptide
*MIC: minimal inhibitory concentration, concentration value are the mean value of 3 repeated experiments;＞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 repeated experiments mean values.
2, the hemolytic activity analysis of Hainanenin-1
This experiment is made positive control with 1%Triton X-100, makes negative control with physiological saline.The preparation red corpuscle: get the 1ml human blood, be placed in the 15ml centrifuge tube, add 10ml physiological saline, the centrifugal 5min of 2000rpm after mixing, 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 specimen prepares 1000 μ l systems:
10 μ l polypeptide samples+990 μ l erythrocyte diluting fluid Hainanenin-1 (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-1 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, the mean value of 3 repetitions of calculating; Hemolysis rate=(sample 540nm absorbance value-negative control) * 100%/(positive control-negative control).
Experimental result shows, when Hainanenin-1 concentration is 25 μ g/ml and 50 μ g/ml (for 5～10 times of minimal inhibitory concentration), its hemolysis rate to human red cell is only 10.72% and 12.34% respectively, shows that it is to the strong selectivity of microorganism cells.
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| CN103755796A (en) * | 2014-01-22 | 2014-04-30 | 大连理工大学 | Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof |
| CN106046122A (en) * | 2016-06-01 | 2016-10-26 | 潍坊科技学院 | Amphibian-animal-source bradykinin and application thereof |
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| CN1803835A (en) * | 2006-01-12 | 2006-07-19 | 南京农业大学 | Active polypeptide of brown-spotted torrential frog, its gene and application in drug preparation |
| CN1827641A (en) * | 2006-04-18 | 2006-09-06 | 河北师范大学 | Antimicrobial Polypeptide of Brown-spotted Frog and Its Application in Pharmaceuticals |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103755796A (en) * | 2014-01-22 | 2014-04-30 | 大连理工大学 | Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof |
| CN103755796B (en) * | 2014-01-22 | 2016-05-11 | 大连理工大学 | The rapid frog anticancer peptide Hainanenin-1 in Hainan and gene and application |
| CN106046122A (en) * | 2016-06-01 | 2016-10-26 | 潍坊科技学院 | Amphibian-animal-source bradykinin and application thereof |
| CN106046122B (en) * | 2016-06-01 | 2019-06-04 | 潍坊科技学院 | An amphibian-derived bradykinin and its application |
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