CN102115496A - Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof - Google Patents

Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof Download PDF

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CN102115496A
CN102115496A CN 201010530926 CN201010530926A CN102115496A CN 102115496 A CN102115496 A CN 102115496A CN 201010530926 CN201010530926 CN 201010530926 CN 201010530926 A CN201010530926 A CN 201010530926A CN 102115496 A CN102115496 A CN 102115496A
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cath1
antimicrobial peptide
gene
peptide
arginine
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于海宁
王义鹏
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Dalian University of Technology
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Dalian University of Technology
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Abstract

The invention relates to an antimicrobial peptide Pc-CATH1 from Phasianus colchicus and a gene thereof, a chemical synthesis method and application in the field of biopharmaceuticals and belongs to the technical field of biomedicine. The antimicrobial peptide Pc-CATH1 is a straight-chain polypeptide encoded by (Phasianus colchicus) Cathelicidin genes, and the complete sequence of the antimicrobial peptide Pc-CATH1 is as follows: arginine-isoleucine-lysine-arginine-phenylalanine-tryptophan-proline-valine-valine-isoleucine-arginine-threonine-valine-valine-alanine-glycine-tyrosine-asparaginate-leucine-tyrosine-arginine-alanine-isoleucine-lysine-lysine-lysine; and a gene for encoding a precursor of the antimicrobial peptide Pc-CATH1 consists of 607 nucleotides, wherein the 367th to 445th nucleotides are used for coding mature peptide part. The antimicrobial peptide Pc-CATH1 is smaller in molecular weight, stronger in bactericidal action and broader in spectrum and has strong effect of killing various clinical drug-resistant bacteria; the antimicrobial peptide Pc-CATH1 has a simple structure without a disulfide bond or a ring structure and is convenient for chemical synthesis and the preparation of gene engineering; and in addition, the antimicrobial peptide Pc-CATH1 has the beneficial characteristics of no hemolysis, no cytotoxicity, super-strong serum stability and the like.

Description

A kind of antimicrobial peptide Pc-CATH1 and gene, chemical synthesis process and application
Technical field
The invention provides a kind of wide spectrum antimicrobial peptide Pa-CATH1 of cathelicidin family and gene thereof that derives from Chinese ring-necked pheasant (Phasianus colchicus), chemical synthesis process and application belong to field of biomedicine technology.
Background technology
Cathelicidin is a class by the multi-functional antibacterial peptide of having of constituting of the mature peptide zone of N end signal peptide zone, middle conservative cathelin structural domain and C end high special family, and (mammals, birds, batrachians and fish) all have discovery in the animal body of almost all kinds.(defensin) is the same with alexin, and Cathelicidin comprises the human peculiar host defense peptide of most vertebrate, has constituted the key ingredient of vertebrates natural immunity reaction, is the bridge that connects natural immunity and specific immunity.Cathelicidin has broad-spectrum antibacterial activity, not only gram-positive microorganism, Gram-negative bacteria, some fungi and virus is had very strong fungicidal activity, and many clinical drug-resistant bacteriums are had effect equally.In addition, Cathelicidin also has many other biologicals and learns activity, as panimmunity cell (neutrophil leucocyte, monocyte, mastocyte and T cell) being had chemotaxis, inducing mastocyte threshing and histamine release, adjusting scavenger cell to transcribe, promote wound healing, induction of vascular generation, induce variation cell line cell apoptosis and lymphocyte activation etc.The disappearance of Cathelicidin gene or unconventionality expression all will cause the generation of human serious disease.Current research finds that systemic lupus erythematous is relevant with the overexpression of Cathelicidin LL-37 in the human body with psoriasic morbidity.Because have so numerous activity, Cathelicidin is the focus of studying in the world always.Then containing huge clinical treatment medication preparation at the medicinal exploitation of Cathelicidin is worth.
Because antibiotic abuse causes the chemical sproof problem of invasive organism serious day by day in recent years, has occurred can tolerating fully in a large number traditional antibiotic microorganisms such as penicillin clinically.Recently, a kind of appearance of carrying " superbug " of writing NDM-1 enzyme gene, antibiotic just abuse of arch-criminal and misuse.This kind of enzyme can decompose the beta-lactam ring structure, therefore can make clinical so far the most frequently used microbiotic, comprise penicillin and cynnematin, and other atypia β-Nei Xiananleikangshengsus such as the cephamycin-type of new development, sulfomycin class, monobactams lost efficacy.And the bactericidal mechanism of Cathelicidin family antimicrobial peptide is the integrity mediation by destroying bacterial cell membrane: attract and be attached to electronegative bacterial cell membrane surface by electrostatic interaction, further on bacterial cell membrane, form the hole of striding film, cause leaking of bacterial cell content, thereby cause the death of bacterial cell.In addition, increasing bibliographical information shows that antibacterial peptide also has other bactericidal mechanism, as suppress bacteria cell wall synthetic, change the bacterial cell plasma membrane and suppress barrier film and form, activate autolysin, suppress the desmo enzyme activity, suppress DNA, RNA and proteinic synthetic etc.Just because of the difference of the mode of action, the germicidal action of Cathelicidin mediation is far away faster than traditional microbiotic, and can not produce resistance.In the external antimicrobial test, most of Cathelicidin mature peptides can kill microorganism widely fast under the concentration of micromole and sub-micro mole.The characteristic feature of Cathelicidin mature peptide performance antimicrobial peptide: most of Cathelicidin mature peptide molecular weight are with clean positive charge less than 5000 under the condition of neutral pH, contain a plurality of alkaline amino acid residues because of them; Be rich in hydrophobic base, integral body has amphipathic topological framework.
At present, have many companies carrying out the research and development of antimicrobial peptide abroad, existing multiple antibacterial peptide enters clinical experimental stage.As the hLF-1-1 that derives from people lactoferrin is used for the treatment of the relevant infection of Bone Marrow Stem Cells Transplantation, entered the clinical II phase.The MSI-78 that derives from Africa xenopus magainin has significant curative effect and side effect little to diabetic subject's foot ulcers, has entered clinical III phase experimental stage.The IB-367 that derives from pig protegrin is used for the treatment of the tumour patient stomatocace, has entered the clinical I phase to test.In addition, some are used for wound healing, and intracellular toxin infects, tumour, and the antibacterial peptide of virus infection has also entered clinical experimental stage.
Because not long to the research history of Cathelicidin in the world wide, China also rarely has report in the research of this type of active polypeptide family.Chinese ring-necked pheasant is the widest bird that distributes in China's Phasianidae, have concurrently view and admire, eat, pharmaceutical use, be a kind of important economic bird.The Compendium of Material Medica of Ming Dynasty's LI Shi-Zhen thinks that Chinese ring-necked pheasant meat nature and flavor are sweet, sour, warm, has the merit of invigorating the spleen and replenishing QI; To diarrhea, quench one's thirst, frequency of micturition has certain therapeutic action." Elementary Medicine " claims it " to control mental disorder Zhichuan "." medical center bun will " thinks that its also has the effect of " beneficial liver and blood ".Yet, the Shang Weiyou report is gone back on its pharmaceutical use molecular biology and molecular pharmacology basis behind.
Chinese ring-necked pheasant antimicrobial peptide Pc-CATH1 molecular weight of the present invention is littler, than the most of cathelicidins that identified so far all little (as a Gold-banded Krait cathelicidin-BF:30 amino acid, a chicken fowlicidin2:32 amino acid etc.).Germicidal action stronger (be compared to traditional microbiotic penbritin and kantlex, and people cathelicidin LL-37), wide spectrum more have very strong killing action to the various clinical resistant organism.Simple in structure, do not contain disulfide linkage and ring texture.The sterilization dynamics data shows that there is very strong killing action its germicidal action time rapidly and to the various clinical resistant organism.Have no hemolytic in addition, reach superpower beneficial features such as serum stability.
Summary of the invention
The invention provides a kind of a kind of antimicrobial peptide that derives from Chinese ring-necked pheasant that under the sub-micro molar dose, promptly has very strong antimicrobial acivity, Pc-CATH1 and gene thereof, chemical synthesis process and application.
In order to realize purpose of the present invention, the invention provides following technical scheme:
Pc-CATH1 is a kind of straight-chain polypeptide of Chinese ring-necked pheasant Cathelicidin genes encoding, contain 26 amino-acid residues, theoretical iso-electric point (pI) is 11.60, theoretical molecular is 3175.9, contain 8 alkaline amino acid residues (4 arginine and 4 Methionins), no acidic amino-acid residue, static charge is+8, illustrates that it is a kind of basic polypeptide.Pc-CATH1 does not contain halfcystine, does not therefore have intramolecularly and intermolecular disulfide bond, and is simple in structure.The Pc-CATH1 total order is classified as: arginine-Isoleucine-Methionin-arginine-phenylalanine-tryptophane-proline(Pro)-Xie Ansuan-Xie Ansuan-Isoleucine-arginine-Threonine-Xie Ansuan-Xie Ansuan-L-Ala-glycine-tyrosine-l-asparagine-leucine-tyrosine-arginine-L-Ala-Isoleucine-Methionin-Methionin-Methionin.
Gene sequencing result shows that the gene of coding Chinese ring-necked pheasant Pc-CATH1 precursor cathelicidin is made up of 607 Nucleotide, holds to 3 ' terminal sequence from 5 ' to be:
1 ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
61?ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
121CCCCTGGGCT?ACTCCCAGGC?TCTGGCCCAG?GCTGTGGACT?CCTACAACCA?ACGGCCTGAG
181GTGCAGAATG?CCTTCCGGCT?GCTCAGCGCC?GACCCCGAGC?CCGGCCCGAA?CGTCCAGCTG
241GGCTCCCTGC?ACAACCTCAA?CTTCACCATC?ATAGAGACGC?GGTGCCAGGC?GCGCTCGGGC
301GCCCAGCTCG?ACAGCTGCGA?GTTCAAGGAG?GACGGGCTCG?TCAAGGACTG?CGCTGCGCCC
361GTGGTGCTGC?AAGGCGGCCG?CGCCACGTTC?GATGTCACCT?GCGTGGAGTC?CGTGGCTGAC
421CCTGTCCGCA?TCAAGCGCTT?CTGGCCAGTG?GTCATCAGGA?CTGTGGTTGC?AGGATACAAC
481CTCTACCGGG?CAATCAAGAA?GAAATGAGCC?ATCCCCAGAG?CTGCTGTCAC?CAATGTCCCC
541TTGCTGCTTT?CCATCCAATA?AAGGTGTTTC?CCAGCCTAAA?AAAAAAAAAA?AAAAAAAAAA
601AAAAAAA
Coding Chinese ring-necked pheasant cathelicidin mature peptide Pc-CATH1 is a 367-445 position Nucleotide, and its aminoacid sequence is: Arg 1Ile 2Lys 3Arg 4Phe 5Trp 6Pro 7Val 8Val 9Ile 10Arg 11Thr 12Val 13Val 14Ala 15Gly 16Tyr 17Asn 18Leu 19Tyr 20Arg 21Ala 22Ile 23Lys 24Lys 25Lys 26
The chemical synthesis process of Pc-CATH1:
Mature peptide Pc-CATH1 aminoacid sequence according to coding Chinese ring-necked pheasant cathelicidin antimicrobial peptide gene is inferred synthesizes its complete sequence with automatic Peptide synthesizer (433A, Applied Biosystems).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).
Synthetic Pc-CATH1 antibacterial peptide can be dissolved in the sterilization ultrapure water, is used for pharmacologically active and detects.
Beneficial effect of the present invention is gene clone obtain the encoding gene of Chinese ring-necked pheasant cathelicidin antimicrobial peptide, obtains mature peptide Pc-CATH1 by chemical synthesis process.This antimicrobial peptide molecular weight is little, germicidal action is stronger (is compared to traditional microbiotic penbritin and kantlex, and people cathelicidin LL-37), wide spectrum more, simple in structure, do not contain disulfide linkage and ring texture, make things convenient for the preparation of chemosynthesis and genetically engineered.The sterilization dynamics data shows that there is very strong killing action its germicidal action time rapidly and to the various clinical resistant organism.Have no hemolytic in addition, reach superpower beneficial features such as serum stability.
Description of drawings
Fig. 1 is the sterilization kinetics synoptic diagram of the Pc-CATH1 of 1 times of MIC for concentration.
Fig. 2 is the sterilization kinetics synoptic diagram of the Pc-CATH1 of 2 times of MIC for concentration.
Among the figure: CFU: colony-forming unit;
Figure BDA0000030688860000031
No Peptide: lifeless matter peptide
Figure BDA0000030688860000032
Pc-CATH1
Figure BDA0000030688860000041
Amp:Ampicillin, penbritin
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 thereto.
Embodiment 1
The clone and the gene sequencing of antimicrobial peptide Pc-CATH1 precursor-gene comprise:
Adopt RNeasy Mini Kit to extract the total RNA of Chinese ring-necked pheasant marrow, utilize Creator TMSMART TMCDNA library builds the storehouse test kit and makes up Chinese ring-necked pheasant marrow cDNA library.
Figure BDA0000030688860000042
CDNA first chain is synthesized in Reverse Transcriptase reverse transcription, and primer is:
Forward Oligo dT primer: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 '
Reverse CDSIII primer: 5 '-ATTCTAGAGGCCGAGGCGGCCGACATGT (30) N -1N-3 ' (N=A, G, C, or T; N -1=A, G, or C).
Utilize Advantage DNA Polymerase to synthesize second chain, primer is: forward 5 '-AAGCAGTGGTATCAACGCAGAGT-3 ', reverse primer is all CDSIII.
Designing two specificity forward primers (P1, P2) and a reverse non-specific universal primer (CDSIII), is template with Chinese ring-necked pheasant cDNA, adopts the cDNA of the method amplification cathelicidin of heminested PCR.
Forward P1:5 '-AGGATGCTGAGCTGCTGGGT-3 ';
Forward P2:5 '-ATGCTGAGCTGCTGGGTGCT-3 ';
Reverse non-specific universal primer is CDSIII, and its sequence is: 5 '-ATTCTAGAGGCCGAGGCGGCCGACATGT (30) N -1N-3 ' (N=A, G, C, or T; N -1=A, G, or C).
The positive monoclonal that obtains carries out gene nucleotide series to be measured, the pGEM-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, the total RNA of Chinese ring-necked pheasant marrow extract (used utensil of following experiment and reagent are all through handling no RNase):
A. cut the fritter about 1g from the various flesh tissues of the fresh Chinese ring-necked pheasant of slaughtering respectively, put into the cell cryopreservation pipe of liquid nitrogen precooling respectively, put into liquid nitrogen then rapidly and preserve;
The organization material that B. will be kept in the liquid nitrogen takes out, and puts into the mortar of precooling, fully grind rapidly, during constantly in mortar, add a little liquid nitrogen; The powder of organizing of about 30mg is transferred in the 1.5ml centrifuge tube of precooling,, shakes mixing rapidly to wherein adding 600 μ l Buffer RLT (lysate, RNeasy Mini Kit provides) respectively, after room temperature is placed 20min, 4 ℃, the centrifugal 5min of 12000rpm;
C. the supernatant after centrifugal is transferred in the new 1.5ml centrifuge tube, adds isopyknic 70% ethanol respectively, inhale rapidly and beat mixing; Mixed solution is transferred to centrifugal adsorption column respectively, room temperature, the centrifugal 15s of 12000rpm; Abandon waste liquid, add 700 μ lBuffer RW1 (rinsing liquid) then, 4 ℃, the centrifugal 15s of 12000rpm; Abandon waste liquid, add 500 μ l Buffer RPE (protein liquid removal), 4 ℃, the centrifugal 15s of 12000rpm; Abandon waste liquid, add 500 μ l Buffer RPE (protein liquid removal), 4 ℃, the centrifugal 2min of 12000rpm; Centrifugal adsorption column is transferred in the new 1.5ml centrifuge tube direct Dropwise 35 μ l RNasefree water on adsorption film then, room temperature, the centrifugal 1min of 12000rpm;
The structure in second step, cDNA library
Synthetic (the mRNA reverse transcription) of first chain:
A. in new 0.2ml PCR pipe (no 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, and is behind the mixing, of short duration centrifugal; In the PCR instrument behind 65 ℃ of insulation 5min, rapidly at chilling 2min on ice; Of short duration centrifugal after, in aforementioned tube, be formulated as follows inverse transcription reaction liquid:
Above-mentioned mixed solution 10 μ l
5×PrimeScript?Buffer 4μl
RNase?Inhibitor(40U/μl) 0.5μl
Figure BDA0000030688860000051
Reverse?Transcriptase(200U/μl) 0.5μl
RNase free dH2O is supplemented to 20 μ l systems, and is behind the mixing, of short duration centrifugal; In the PCR instrument, finish following program: 42 ℃, 60min; 70 ℃, 15min; 4 ℃, preserve.CDNA is stored in-80 ℃.
Synthesizing of second chain:
The first chain cDNA, 2 μ l
Deionized water 80 μ l
10×Advantage?2?PCR?buffer 10μl
50× dNTP?Mix 2μl
5’PCR?primer 2μl
CDS?III/3’PCR?primer 2μl
50×Advantage?2?Polymerase?Mix 2μl
The 3rd step, the gene clone of adopting heminested PCR to carry out Chinese ring-necked pheasant cathelicidin are screened
Primer uses the preceding centrifugal 5min of first 12000rpm, adds the ddH of respective volume then according to the mole number of indicating 2O is dissolved to the concentration of 20 μ M.With synthetic marrow cDNA is template, is primer with P1 and CDSIII, carries out the pcr amplification first time.In 0.2ml PCR pipe, add following reagent (cumulative volume 20 μ l):
ddH2O 8μl
CDNA template 1 μ l
Forward primer P1 (20 μ M) 0.5 μ l
Reverse primer CDSIII (20 μ M) 0.5 μ l
2×Pfu?PCR?MasterMix 10μl
Behind the mixing, of short duration centrifugal.The PCR condition is: 94 ℃ of sex change 1min; 20 circulations: 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1min; 72 ℃ are extended 10min; 4 ℃ of preservations.Reaction is got 5 μ l products in 1% agarose gel electrophoresis testing goal band after finishing.
Get step PCR product 1 μ l and add 99 μ l ddH 2O dilutes 100 times as template, is primer with P2 and CDSIII, carries out the pcr amplification second time.In 0.2ml PCR pipe, successively add following reagent (cumulative volume 20 μ l):
ddH2O 7μl
PCR product diluent template 1 a μ l
Forward primer P2 (20 μ M) 1 μ l
Reverse primer CDSIII (20 μ M) 1 μ l
2×Pfu?PCR?Master?Mix 10μl
The PCR condition is: 94 ℃ of sex change 5min; 25 circulations: 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 1min; 72 ℃ are extended 10min; 4 ℃ of preservations.Reaction is got 5 μ l, 1% agarose gel electrophoresis testing goal band after finishing.
Reclaim test kit (day root biology) with glue after amplification is finished and carry out the recovery of purpose fragment.The target DNA fragment that reclaims is connected with sequencing vector pGEM-T vector, 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 lg/ml penbritins (Amp); After dry on the surface, be placed on to be inverted in 37 ℃ of constant incubators and cultivate 12-16h.Picking list bacterium colony detects with the M13 primer PCR and inserts clip size.The positive bacterium colony of picking shakes bacterium and extracts plasmid, uses Applied Biosystems DNA sequencer, and model ABIPRISM 377 carries out nucleotide sequencing.
Gene sequencing and the result of the 4th step, Chinese ring-necked pheasant cathelicidin:
Gene sequencing result shows that the gene of coding Pc-CATH1 precursor cathelicidin is made up of 607 Nucleotide, holds to 3 ' terminal sequence from 5 ' to be:
1 ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
61?ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
121CCCCTGGGCT?ACTCCCAGGC?TCTGGCCCAG?GCTGTGGACT?CCTACAACCA?ACGGCCTGAG
181GTGCAGAATG?CCTTCCGGCT?GCTCAGCGCC?GACCCCGAGC?CCGGCCCGAA?CGTCCAGCTG
241GGCTCCCTGC?ACAACCTCAA?CTTCACCATC?ATAGAGACGC?GGTGCCAGGC?GCGCTCGGGC
301GCCCAGCTCG?ACAGCTGCGA?GTTCAAGGAG?GACGGGCTCG?TCAAGGACTG?CGCTGCGCCC
361GTGGTGCTGC?AAGGCGGCCG?CGCCACGTTC?GATGTCACCT?GCGTGGAGTC?CGTGGCTGAC
421CCTGTCCGCA?TCAAGCGCTT?CTGGCCAGTG?GTCATCAGGA?CTGTGGTTGC?AGGATACAAC
481CTCTACCGGG?CAATCAAGAA?GAAATGAGCC?ATCCCCAGAG?CTGCTGTCAC?CAATGTCCCC
541TTGCTGCTTT?CCATCCAATA?AAGGTGTTTC?CCAGCCTAAA?AAAAAAAAAA?AAAAAAAAAA
601AAAAAAA
The sequence table of the cDNA Nucleotide of Chinese ring-necked pheasant cathelicidin coding region is: sequence length is 607 bases, sequence type: nucleic acid, chain number: strand, topology: straight chain shape, sequence kind: cDNA, source: Chinese ring-necked pheasant marrow.
Coding Chinese ring-necked pheasant cathelicidin mature peptide Pc-CATH1 is a 367-445 position Nucleotide, and its aminoacid sequence is: Arg 1Ile 2Lys 3Arg 4Phe 5Trp 6Pro 7Val 8Val 9Ile 10Arg 11Thr 12Val 13Val 14Ala 15Gly 16Tyr 17Asn 18Leu 19Tyr 20Arg 21Ala 22Ile 23Lys 24Lys 25Lys 26
Embodiment 2
The chemical synthesis process of Pc-CATH1:
I, according to the mature peptide Pc-CATHl aminoacid sequence that coding Chinese ring-necked pheasant cathelicidin gene is inferred, synthesize its complete sequence with automatic Peptide synthesizer (Applied Biosystems), by HPLC reversed phase column chromatography desalting and purifying.
II, molecular weight determination adopt ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF).
Embodiment 3
The pharmacological evaluation of antimicrobial peptide Pc-CATH1:
1.Pc-CATH1 anti-microbial activity detects:
The Pc-CATH1 of chemosynthesis is dissolved in the aseptic ultrapure water with the concentration of 2mg/ml; With the new activatory microorganism of transfering loop picking, be uniformly coated on then on the new LB agar plate; The circular aseptic filter paper sheet of diameter 0.5cm is placed on the above-mentioned agar plate, on the scraps of paper, drips 10 μ l Pc-CATH1 sample solutions then; Put into 37 ℃ of constant incubators and cultivate 12-24h; Observe inhibition zone and whether form, will note the bacterial strain of Pc-CATH1 sensitivity.
2.Pc-CATH1 to sensitive strain minimal inhibitory concentration (Minimum Inhibitory Concentration, mensuration MIC).This experiment is made positive control with people cathelicidin LL-37, traditional microbiotic penbritin and kantlex, and sterile liquid LB makes negative control; Minimal inhibitory concentration is defined as the minimum peptide concentration that naked eyes can observedly suppress microorganism growth fully, or absorbance value is not higher than the minimum concentration of negative control 5%.
The new activatory microorganism of picking is seeded to sterile liquid LB substratum, and 200rpm cultivates 10-16h to logarithmic phase in 37 ℃ of constant temperature oscillators; With the light absorption value at ultraviolet spectrophotometer survey bacterium liquid 600nm light wave place, light absorption value is 1 o'clock, and concentration is approximately 10 9Cfu/ml is diluted to 10 with bacterium liquid with sterile liquid LB substratum 6Cfu/ml, stand-by on ice; The compound concentration gradient is the Pc-CATH1 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 above-mentioned dilution bacterium of 50 μ l liquid; In constant temperature oscillator 37 ℃, 100rpm shaking culture 10-16h; Use microplate reader to survey 600nm light absorption value or visual inspection; Above-mentioned experiment repeats 3 times, averages.Other gets one 96 orifice plate, and same operation only is that 100mMNaCl is added in every hole, still repeats 3 times, averages.
By table 1 as seen, Pc-CATH1 has broad-spectrum antibacterial activity to the microorganism of test, particularly to clinical isolating endurance strain.Compare with the minimal inhibitory concentration value of penbritin, kantlex and LL-37, Pc-CATH1 has stronger antibacterial efficacy.In whole 32 strain bacterial strains, gram-positive microorganism (G+) is compared with Gram-negative bacteria (G-) and fungi, and G+ is extremely responsive to Pc-CATH1, and minimal inhibitory concentration is mostly in 0.09-2.95 μ M scope.Pc-CATH1 even the antibiotic bacterial strain of anti-routine also had powerful sterilization effect, as to the staphylococcus haemolyticus clinical separation strain (IS 2401, minimal inhibitory concentration concentration DRa) in addition only be 0.09 μ M.Pc-CATH1 is to streptococcus aureus ATCC2592 (G+) and a kind of important ward infection pathogenic bacterium, and germ oligotrophy unit cell (G-), anti-microbial activity are also very strong, and minimal inhibitory concentration is respectively 0.18 μ M and 0.74 μ M.In addition, Pc-CATH1 also has very strong anti-mycotic activity, and the minimal inhibitory concentration of Candida albicans ATCC2002 and slime-fungi is reached 0.18 μ M and 0.37 μ M respectively.Moreover, Pc-CATH1 can cause the propionibacterium acnes of facial serious acne to a large amount of populations in a kind of puzzlement whole world, has also demonstrated extremely strong anti-microbial activity, and minimal inhibitory concentration only is 0.74 μ M.
The minimal inhibitory concentration of table 1 Pc-CATH1 (MIC)
Figure BDA0000030688860000081
*MIC: minimal inhibitory concentration, concentration value are the mean value of 3 repeated experiments; LL-37: people cathelicidin, ND:2mg/ml dosage scraps of paper bacteriostatic test does not have obvious activity;＞100 μ g/ml:2mg/ml dosage scraps of paper bacteriostatic tests have inhibition zone, and 100 μ g/ml dosage do not have bacteriostatic activity; IS: clinical isolates strain, Dra: anti-ceftazime, cefoperazone and aztreonam, DRb: anti-trimethoprim-sulfamethoxazole, erythromycin, Ciprofloxacin and penicillin.Above result is three independent repeated experiments mean values.
3.Pc-CATH1 sterilization kinetic determination:
This experiment selects for use intestinal bacteria ATCC25922 (G-) to test respectively; Negative control is an aseptic deionized water, and positive control is a penbritin.The new activatory bacterium of picking mono-clonal is seeded to fresh sterile liquid LB substratum, and 37 ℃, 200rpm shaking culture 10-16h is to logarithmic phase; Measure bacterial concentration, be diluted to 10 6Cfu/ml; Get three parts of above-mentioned bacterium liquid, each 1ml, a copy of it add Pc-CATH1, and making its final concentration is 1 times of (or 2 times) MIC, and second part as negative control, add positive control in the 3rd part, and sampling respectively rapidly, dilute 1000 times after, carry out plate count; Three parts of bacterium liquid are positioned in the constant temperature oscillator 37 ℃, and 100rpm shakes cultivation; Respectively at 0min, 5min, 10min, 15min, 20min, 30min and 60min sampling, carry out plate count after diluting 1000 times; The result as shown in Figure 1 and Figure 2.
As shown in Fig. 1, Fig. 2, the sterilization speed of Pc-CATH1 basically 2 times to penbritin.Pc-CATH1 is a lethality to the anti-microbial activity of streptococcus aureus ATCC2592.Through the streptococcus aureus of above-mentioned concentration Pc-CATH1 processing after 2 hours, can not on agar plate, recover growth.
4.Pc-CATH1 serum stability
This experiment is made negative control with human serum.The preparation human serum: get people 5ml fresh blood, 37 ℃ leave standstill 1h; Place 8-16h for 4 ℃; 4 ℃, the centrifugal 20min of 4000rpm; The serum on careful sucking-off upper strata is transferred in the aseptic 1.5ml centrifuge tube.
Get 900 μ l serum, the Pc-CATH1 sample that adds 100 μ l 10mg/ml, 37 ℃ of incubations behind the mixing take out a little biased sample respectively when 0h, 3h, 6h, 12h, 24h, 36h, 48h, 60h and 72h, measure its MIC to streptococcus aureus (IS).The result shows that Pc-CATH1 has still kept very strong antibacterial ability (MIC 0.37 μ M) to streptococcus aureus ATCC2592 after hatching 72h altogether with human serum.
5.Pc-CATH1 the hemolytic activity analysis
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, place the 15ml centrifuge tube, add 10ml physiological saline, the centrifugal 5min of 2000rpm behind the 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 the small beaker, is diluted to physiological saline to be blush; Every kind of test sample preparation 1000 μ l systems:
10 μ l polypeptide samples+990 μ l erythrocyte diluting fluid Pc-CATH1 (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 Pc-CATH1 and positive and negative contrast.37 ℃ of incubation 30min, the centrifugal 5min of 3000rpm; Collect supernatant 200 μ l, survey 540nm place absorbance value respectively, calculate 3 multiple mean values; Hemolysis rate=(sample 540nm absorbance value-negative control) * 100%/(positive control-negative control).
The experiment structure shows that the Pc-CATH1 under 10 μ g/ml (3.15 μ M) dosage only is 3.6% to the hemolysis rate of human red cell; Shown the strong selectivity of Pc-CATH1 to microorganism cells.
6.Pc-CATH1 cytotoxicity experiment
Human umbilical vein endothelial cells is that HUVEC and rat macrophage are RAW264.7 DMEM+10%FBS culture medium culturing.The cultured cells dilution is inoculated into (2 * 104per well) in 96 orifice plates, and overnight incubation makes cell attachment in the cell culture incubator.The Pc-CATH1 sample that adds different concns then continues to cultivate 48 hours.Mtt assay detects the cytotoxicity of Pc-CATH1 to HUVEC and RAW264.7 cell.
Experimental result shows that Pc-CATH1 is 150 μ g/ml (47.25 μ M) to median lethal concentration (IC50) value of two strain cells, is higher than its MIC value to most bacteriums far away.Illustrate that Pc-CATH1 does not have toxic action to the human normal cell under average MIC value concentration, make the Application and Development of Pc-CATH1 become possibility.

Claims (4)

1. antimicrobial peptide Pc-CATH1, be a kind of straight-chain polypeptide of Chinese ring-necked pheasant Cathelicidin genes encoding, it is characterized in that its total order classifies as: arginine-Isoleucine-Methionin-arginine-phenylalanine-tryptophane-proline(Pro)-Xie Ansuan-Xie Ansuan-Isoleucine-arginine-Threonine-Xie Ansuan-Xie Ansuan-L-Ala-glycine-tyrosine-l-asparagine-leucine-tyrosine-arginine-L-Ala-Isoleucine-Methionin-Methionin-Methionin.
2. the gene of the described antimicrobial peptide Pc-CATH1 of claim 1, the gene of the Chinese ring-necked pheasant antimicrobial peptide Pc-CATH1 precursor cathelicidin that it is characterized in that encoding is made up of 607 Nucleotide, from 5 ' end to 3 ' terminal sequence is:
1 ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
61?ATGCTGAGCT?GCTGGGTGCT?GGTGCTGGCG?CTGCTGGGGG?GGGCCTGCGC?CCTCCCGGCC
121CCCCTGGGCT?ACTCCCAGGC?TCTGGCCCAG?GCTGTGGACT?CCTACAACCA?ACGGCCTGAG
181GTGCAGAATG?CCTTCCGGCT?GCTCAGCGCC?GACCCCGAGC?CCGGCCCGAA?CGTCCAGCTG
241GGCTCCCTGC?ACAACCTCAA?CTTCACCATC?ATAGAGACGC?GGTGCCAGGC?GCGCTCGGGC
301GCCCAGCTCG?ACAGCTGCGA?GTTCAAGGAG?GACGGGCTCG?TCAAGGACTG?CGCTGCGCCC
361GTGGTGCTGC?AAGGCGGCCG?CGCCACGTTC?GATGTCACCT?GCGTGGAGTC?CGTGGCTGAC
421CCTGTCCGCA?TCAAGCGCTT?CTGGCCAGTG?GTCATCAGGA?CTGTGGTTGC?AGGATACAAC
481CTCTACCGGG?CAATCAAGAA?GAAATGAGCC?ATCCCCAGAG?CTGCTGTCAC?CAATGTCCCC
541TTGCTGCTTT?CCATCCAATA?AAGGTGTTTC?CCAGCCTAAA?AAAAAAAAAA?AAAAAAAAAA
601AAAAAAA
Coding Chinese ring-necked pheasant cathelicidin mature peptide Pc-CATH1 is a 367-445 position Nucleotide, and its aminoacid sequence is: Arg 1Ile 2Lys 3Arg 4Phe 5Trp 6Pro 7Val 8Val 9Ile 10Arg 11Thr 12Val 13Val 14Ala 15Gly 16Tyr 17Asn 18Leu 19Tyr 20Arg 21Ala 22Ile 23Lys 24Lys 25Lys 26
3. the chemical synthesis process of the described Pc-CATH1 of claim 1 is characterized in that the mature peptide Pc-CATH1 aminoacid sequence according to the deduction of coding Chinese ring-necked pheasant cathelicidin antimicrobial peptide gene, 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.
4. the application of the described antimicrobial peptide Pc-CATH1 of claim 2 gene is characterized in that, synthetic Pc-CATH1 antibacterial peptide has germicidal action, is dissolved in the sterilization ultrapure water, is used for pharmacologically active and detects.
CN 201010530926 2010-11-03 2010-11-03 Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof Pending CN102115496A (en)

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* Cited by examiner, † Cited by third party
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CN103421100A (en) * 2012-05-22 2013-12-04 中国科学院动物研究所 Antibacterial peptide, preparation method therefor and applications
CN104761629A (en) * 2015-03-05 2015-07-08 大连理工大学 A broadspectrum efficient antimicrobial peptide Pb-CATH-OH1, a gene thereof, a preparing method of the peptide and applications of the peptide
CN105254736A (en) * 2015-09-02 2016-01-20 大连理工大学 Cathelicidin family broad spectrum antimicrobial peptide Pb-CATH4 from python molurus bivittatus and gene, preparation and application thereof

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Publication number Priority date Publication date Assignee Title
CN103421100A (en) * 2012-05-22 2013-12-04 中国科学院动物研究所 Antibacterial peptide, preparation method therefor and applications
CN103421100B (en) * 2012-05-22 2015-11-18 中国科学院动物研究所 A kind of antibacterial peptide and its preparation method and application
CN104761629A (en) * 2015-03-05 2015-07-08 大连理工大学 A broadspectrum efficient antimicrobial peptide Pb-CATH-OH1, a gene thereof, a preparing method of the peptide and applications of the peptide
CN104761629B (en) * 2015-03-05 2017-12-26 大连理工大学 A kind of broad-spectrum high efficacy antimicrobial peptide Pb CATH OH1 and its gene, preparation method and application
CN105254736A (en) * 2015-09-02 2016-01-20 大连理工大学 Cathelicidin family broad spectrum antimicrobial peptide Pb-CATH4 from python molurus bivittatus and gene, preparation and application thereof
CN105254736B (en) * 2015-09-02 2018-06-08 大连理工大学 Derived from the cathelicidin families broad spectrum antimicrobial peptide Pb-CATH4 of Burma boa and its gene, preparation and application

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