CN101845092A - Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof - Google Patents

Abstract

The invention relates to an antimicrobial peptide Ea-CATH1 of Equus asinus and genes and applications thereof, belonging to the technical field of biological medicine. Ea-CATH1 is one genetic coding straight-chain polypeptide of Cathelicidin of Equus asinus, and the complete sequence of Ea-CATH1 is as follows: lysine-arginine-arginine-glycin-serine-valine-threonine-threonine-arginine-tyrosine-glutamine-phenylalanine-leucine-methionine-isoleucine-histidine-leucine-leucine-arginine-proline-lysine-lysine-leucine-phenylalanine-alanine. The gene coding Ea-CATH1 precursor is composed of 550 nucleotides and the part coding mature peptide is the 391-465th nucleotides. Ea-CATH1 has smaller molecular weight, stronger bactericidal effect and broader spectrum and extremely strong killing effect on various clinical drug-resistance bacteria; Ea-CATH1 has simple structure, does not contain disulfide bond and ring structure and is suitable to be prepared by chemical synthesis and gene engineering; and Ea-CATH1 also has useful characteristics such as no hemolysis and ultrastrong serum stability.

Description

A kind of antimicrobial peptide Ea-CATH1 and gene and the application of domestic animal donkey

Technical field

The invention provides a kind of cathelicidin wide spectrum antimicrobial peptide Ea-CATH1 of family and gene and the application of domestic animal donkey (Equus asinus), 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.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.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 bactericidal mechanism of Cathelicidin antimicrobial peptide is by the integrity mediation that destroys 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.And traditional microbiotic mainly is some enzymes that act in the bacterial cell.Just because of the difference of the mode of action, the germicidal action of Cathelicidin mediation is far away faster than traditional microbiotic, and the difficult resistance that produces.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.

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.The domestic animal donkey is one of draught animal with diversified economy value of China's native country breed, and the nationwide big and small meat donkey plant that distributes reaches thousands of families.Because the feeding cost of donkey is low, being of high nutritive value of donkey meat itself, so the production of donkey meat is the novel meat product of a great exploitation potential for its, in addition, donkey is the animal with higher pharmaceutical use, the solid gums that the skin of meat donkey is made through decocting and concentrating claims donkey-hide gelatin, claims Colla Corii Asini again, is the traditional Chinese medicine material of China.Its meat, skin, bone, hair, hoof, penis, fat and breast can also be used as medicine.Yet, the research of its activeconstituents and pharmacological properties is mainly concentrated on organic molecule, the Shang Weiyou report is gone back in its activated protein peptide matters research.

Domestic animal donkey antimicrobial peptide Ea-CATH1 molecular weight of the present invention is littler, than the most of cathelicidins that identified so far all little (as 30 amino acid of Gold-banded Krait cathelicidin-BF, a chicken fowlicidin232 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 Ea-CATH1 and gene and application that under the sub-micro molar dose, promptly has the domestic animal donkey of very strong antimicrobial acivity.

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

Domestic animal donkey antimicrobial peptide Ea-CATH1 is a kind of straight-chain polypeptide of donkey Cathelicidin genes encoding, contain 25 amino-acid residues, theoretical iso-electric point (pI) is 12.02, theoretical molecular is 3060.75, contain 7 alkaline amino acid residues (4 arginine and 3 Methionins), no acidic amino-acid residue, static charge is+7, illustrates that it is a kind of basic polypeptide.Ea-CATH1 does not contain halfcystine, does not therefore have intramolecularly and intermolecular disulfide bond, and is simple in structure.

The Ea-CATH1 total order is classified as: Methionin-arginine-arginine-glycine-Serine-Xie Ansuan-Threonine-Threonine-arginine-tyrosine-glutamine-Phe-Leu-methionine(Met)-Isoleucine-HIS-LEU-leucine-Arg-Pro-Methionin-Methionin-Ile-Phe-L-Ala.

Gene sequencing result shows that the gene of coding domestic animal donkey antimicrobial peptide Ea-CATH1 precursor cathelicidin is made up of 550 Nucleotide, holds to 3 ' terminal sequence from 5 ' to be:

1 ATGGAGACCC?AGAGGGACAG?TTGTTCCCTG?GGCTGGTGGT?CACTGTTGCT?ACTGCTACTG

61 GGCCTGATGA?TCCCTCTGGC?CACCACTCAG?GCCCTCAGCT?ACAAGGAGGC?CGTACTCCGT

121?GCCGTGGATG?GCCTCAACCA?GTGGTCCTCA?GATGAGAATC?TCTACCGCCT?CCTGGAGCTG

181?GACCCTCTGC?CCAAGGGAGA?CGAGGCCCCA?GACACCCCAA?AGCCTGTGAG?CTTCACGGTC

241?AAGGAGACTG?TGTGCCCCAG?GACAACGCAG?CAGCCACTGG?AGCAGTGTGA?CTTCAAGGAG

301?AATGGGCTGG?TGAAACAGTG?TGTGGGGACA?GTCATCCTGG?ACCCGGTTAA?GGCCTCCGTT

361?GACATCGGTT?GTGATGAGCC?CCAGCGTGTC?AAGAGACGCG?GCTCGGTGAC?TACTCGTTAC

421?CAGTTCCTGA?TGATTCACCT?TCTTCGACCT?AAGAAGCTTT?TCGCCTAGAA?TCGGCTTGCC

481?CTGGCTTGGG?CTTCTGGACT?CTGAAAAATA?AATTATGTGA?AAGCCGCTTA?AAAAAAAAAA

541?AAAAAAAAAA

Coding domestic animal donkey cathelicidin mature peptide Ea-CATH1 is a 391-465 position Nucleotide, and its aminoacid sequence is: Lys ¹Arg ²Arg ³Gl ^Y4Ser ⁵Val ⁶Thr ⁷Thr ⁸Arg ⁹Tyr ¹⁰Gln ¹¹Phe ¹²Leu ¹³Met ¹⁴Ile ¹⁵His ¹⁶Leu ¹⁷Leu ¹⁸Arg ¹⁹Pro ²⁰Lys ²¹Lys ²²Leu ²³Phe ²⁴Ala ²⁵

The chemical synthesis process of Ea-CATH1:

Mature peptide Ea-CATH1 aminoacid sequence according to coding domestic animal donkey 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).

Domestic animal donkey antimicrobial peptide Ea-CATH1 synthetic antibacterial peptide can be dissolved in the sterilization ultrapure water, is used for pharmacologically active and detects.

Beneficial effect of the present invention is the gene clone domestic animal donkey cathelicidin antimicrobial peptide gene that obtains encoding, and obtains mature peptide Ea-CATH1 by chemical synthesis process.This antimicrobial peptide molecular weight is littler, 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.

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 domestic animal donkey antimicrobial peptide Ea-CATH1 precursor-gene comprise:

Adopt RNeasy Mini Kit to extract the total RNA of donkey spleen, utilize

CDNA first chain is synthesized in Reverse Transcriptase reverse transcription.Designing two specificity forward primers (P1, P2) and a reverse non-specific universal primer (CDSIII), is template with strand cDNA, adopts the cDNA of the method amplification donkey cathelicidin gene of heminested PCR.

Forward P1:5 '-GGACCATGGAGACCCAGAGG-3 ';

Forward P2:5 '-ATGGAGACCCAGAGGGACAGTT-3 ';

Reverse non-specific universal primer is the Creator of CLONTECH company ^TMSMART ^TM3 ' PCR Primer primer CDSIII among the cDNA LibraryConstruction Kit, its sequence is: 5 '-ATTCTAGAGGCCGAGGCGGCCGACATGT (30) N _-1N-3 ' (N=A, G, C, or T; N _-1=A, G, orC).

The positive monoclonal that obtains carries out gene nucleotide series to be measured, the pMD19-T vector universal primer that checks order:

Forward M 13F:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ';

Reverse M13R:5 '-GAGCGGATAACAATTTCACACAGG-3 ';

Concrete steps are as follows:

The first step, the total RNA of donkey spleen 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 donkey of slaughtering respectively, put into the cell cryopreservation pipe of liquid nitrogen precooling respectively, put into liquid nitrogen then rapidly and preserve and take back the laboratory;

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 RNase free water on adsorption film then, room temperature, the centrifugal 1min of 12000rpm;

Synthetic (the mRNA reverse transcription) of second step, cDNA 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/μd) 0.5μl

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 ℃.

The 3rd step, the gene clone of adopting heminested PCR to carry out donkey 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 spleen cDNA strand 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 single-stranded 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 the 0.2mlPCR 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 CDS III (20 μ M) 1 μ l

2×Pfu?PCR?MasterMix 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 pMD19-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, donkey cathelicidin:

Gene sequencing result shows that the gene of coding domestic animal donkey antimicrobial peptide Ea-CATH1 precursor cathelicidin is made up of 550 Nucleotide, holds to 3 ' terminal sequence from 5 ' to be:

1 ATGGAGACCC?AGAGGGACAG?TTGTTCCCTG?GGCTGGTGGT?CACTGTTGCT?ACTGCTACTG

61 GGCCTGATGA?TCCCTCTGGC?CACCACTCAG?GCCCTCAGCT?ACAAGGAGGC?CGTACTCCGT

121?GCCGTGGATG?GCCTCAACCA?GTGGTCCTCA?GATGAGAATC?TCTACCGCCT?CCTGGAGCTG

181?GACCCTCTGC?CCAAGGGAGA?CGAGGCCCCA?GACACCCCAA?AGCCTGTGAG?CTTCACGGTC

241?AAGGAGACTG?TGTGCCCCAG?GACAACGCAG?CAGCCACTGG?AGCAGTGTGA?CTTCAAGGAG

301?AATGGGCTGG?TGAAACAGTG?TGTGGGGACA?GTCATCCTGG?ACCCGGTTAA?GGCCTCCGTT

361?GACATCGGTT?GTGATGAGCC?CCAGCGTGTC?AAGAGACGCG?GCTCGGTGAC?TACTCGTTAC

421?CAGTTCCTGA?TGATTCACCT?TCTTCGACCT?AAGAAGCTTT?TCGCCTAGAA?TCGGCTTGCC

481?CTGGCTTGGG?CTTCTGGACT?CTGAAAAATA?AATTATGTGA?AAGCCGCTTA?AAAAAAAAAA

541?AAAAAAAAAA

The sequence table of the gene nucleotide of domestic animal donkey antibacterial peptide cathelicidin is: sequence length is 550 bases, sequence type: nucleic acid, chain number: strand, topology: straight chain shape, sequence kind: cDNA, source: domestic animal donkey spleen.

Coding domestic animal donkey cathelicidin mature peptide Ea-CATH1 is a 391-465 position Nucleotide, and its aminoacid sequence is: Lys ¹Arg ²Arg ³Gly ⁴Ser ⁵Val ⁶Thr ⁷Thr ⁸Arg ⁹Tyr ¹⁰Gln ¹¹Phe ¹²Leu ¹³Met ¹⁴Ile ¹⁵His ¹⁶Leu ¹⁷Leu ¹⁸Arg ¹⁹Pro ²⁰Lys ²¹Lys ²²Leu ²³Phe ²⁴Ala ²⁵

Embodiment 2

The chemical synthesis process of Ea-CATH1:

I, according to the mature peptide Ea-CATH1 aminoacid sequence that coding domestic animal donkey cathelicidin antimicrobial peptide gene is inferred, synthesize its complete sequence with automatic Peptide synthesizer (433A, 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 domestic animal donkey antimicrobial peptide Ea-CATH1:

1.Ea-CATH1 anti-microbial activity detects:

The Ea-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 Ea-CATH 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 Ea-CATH1 sensitivity.

2.Ea-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 spectrophotometric instrumentation 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; The compound concentration gradient is the Ea-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.

By table 1 as seen, Ea-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, Ea-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 responsive more to Ea-CATH1, and minimal inhibitory concentration is mostly in 0.6-4.7 μ g/ml scope.Ea-CATH1 even the antibiotic bacterial strain of anti-whole routines also had powerful sterilization effect is 9.4 μ g/ml as the minimal inhibitory concentration concentration to faecium IS091299.Ea-CATH1 is the strongest to the anti-microbial activity of streptococcus aureus ATCC2592 and staphylococcus haemolyticus IS092401 (DRa), and minimal inhibitory concentration is all 0.6 μ g/ml; Minimal inhibitory concentration to streptococcus aureus (IS) and Nocardia asteroides is all 1.2 μ g/ml.In addition, Ea-CATH1 also has very strong anti-mycotic activity, and the minimal inhibitory concentration of Candida albicans ATCC2002 and slime-fungi is reached 9.4 μ g/ml and 4.7 μ g/ml respectively.Moreover, Ea-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 4.7 μ g/ml.

The minimal inhibitory concentration (MIC) of table 1Ea-CATH1

^*MIC: minimal inhibitory concentration, concentration value are the mean value of 3 repeated experiments; LL-37: people cathelicidin, Amp: penbritin, Kana: kantlex; ND:2mg/ml dosage scraps of paper bacteriostatic test does not have obvious activity;＞100:2mg/ml dosage scraps of paper bacteriostatic test has 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.Ea-CATH1 sterilization kinetic determination:

This experiment select for use respectively streptococcus aureus ATCC2592 (G+) and Bao formula acinetobacter calcoaceticus (IS7178) (G-) two kinds of bacterial strains test; Negative control is an aseptic deionized water, and positive control is penbritin Amp.

The new activatory bacterium of picking mono-clonal is seeded to fresh sterile liquid LB substratum, and 35 ℃, 200rpm shaking culture 10-16h is to logarithmic phase; Measure bacterial concentration, be diluted to 10 ⁶Cfu/ml; In bacterium liquid, add the polypeptide sample, respectively to 1,5,10 times of MIC of final concentration, 35 ℃ then, 100rpm shaking culture; Pick up counting to add polypeptide, respectively get 1 μ l bacterium liquid at 0min, 10min, 30min, 1h, 1.5h, 2h, 3h and 6h time point respectively, dilute 1000 times; Get 50 μ l dilution bacterium liquid coated plate respectively; Be inverted for 37 ℃ and cultivate 10-16h; Enumeration.The result is as shown in table 2.

The sterilization kinetics of table 2Ea-CATH1

Annotate: CFU: colony-forming unit; * 1MIC, * 5MIC and * 10MIC:1,5 and 10 times of corresponding M IC; MIC ¹: 0.6 μ g/ml; MIC ²: 2.4 μ g/ml; MIC ³: 4.7 μ g/ml.

As shown in table 2, Ea-CATH1 than Amp with the performance of kinetics faster anti-microbial activity.The Ea-CATH1 of 10 times of minimal inhibitory concentrations can kill whole streptococcus aureuses (Amp needs 2 hours) rapidly in 0.5 hour; The Ea-CATH1 of 5 times of minimal inhibitory concentrations needs 1 hour (Amp needs 3 hours); The Ea-CATH1 of 1 times of minimal inhibitory concentration needs 2 hours (Amp needs 6 hours).Proof Ea-CATH1 is a lethality to the anti-microbial activity of streptococcus aureus ATCC2592.Through the streptococcus aureus of above-mentioned concentration Ea-CATH1 processing after 2 hours, can not on agar plate, recover growth.By contrast, conventional Amp can not remove bacterium fully in 2 hours.

4.Ea-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 Ea-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 stability of table 3Ea-CATH1 in human serum

The serum stability experimental result shows (table 3), Ea-CATH1 is after hatching 12 hours altogether with human serum, MIC compare hatch before without any reduction, on the contrary may be because ionic concn or proteic influence in the serum, cause its structural change (spirane structure increases), thereby make the antibacterial ability at the initial stage of hatching increase by one times than in water; Between 24-48 hour, the Ea-CATH1 antibacterial ability in the serum has only decline a little; After 72 hours, Ea-CATH1 to still keeping of streptococcus aureus (IS) very strong antibacterial ability (MIC 9.4 μ g/ml).

5.Ea-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 Ea-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 Ea-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 Ea-CATH1 under 20 μ g/ml dosage only is 1.8% to the hemolysis rate of human red cell; This dosage is 10-30 times of most minimal inhibitory concentrations.

Sequence table

<110>National?Center?for?Biotechnology?Information

<120>dut2010082001

<130>dut2010082001

<160>1

<170>PatentIn?version?3.3

<210>1

<211>1956

＜212〉gene order of domestic animal donkey antimicrobial peptide Ea-CATH1 precursor cathelicidin

SEQ?ID?NO：1

1 ATGGAGACCC?AGAGGGACAG?TTGTTCCCTG?GGCTGGTGGT?CACTGTTGCT?ACTGCTACTG

61 GGCCTGATGA?TCCCTCTGGC?CACCACTCAG?GCCCTCAGCT?ACAAGGAGGC?CGTACTCCGT

121?GCCGTGGATG?GCCTCAACCA?GTGGTCCTCA?GATGAGAATC?TCTACCGCCT?CCTGGAGCTG

181?GACCCTCTGC?CCAAGGGAGA?CGAGGCCCCA?GACACCCCAA?AGCCTGTGAG?CTTCACGGTC

241?AAGGAGACTG?TGTGCCCCAG?GACAACGCAG?CAGCCACTGG?AGCAGTGTGA?CTTCAAGGAG

301?AATGGGCTGG?TGAAACAGTG?TGTGGGGACA?GTCATCCTGG?ACCCGGTTAA?GGCCTCCGTT

361?GACATCGGTT?GTGATGAGCC?CCAGCGTGTC?AAGAGACGCG?GCTCGGTGAC?TACTCGTTAC

421?CAGTTCCTGA?TGATTCACCT?TCTTCGACCT?AAGAAGCTTT?TCGCCTAGAA?TCGGCTTGCC

481?CTGGCTTGGG?CTTCTGGACT?CTGAAAAATA?AATTATGTGA?AAGCCGCTTA?AAAAAAAAAA

541?AAAAAAAAAA

Claims (3)

1. a kind of antimicrobial peptide Ea-CATH1 of domestic animal donkey, be a kind of straight-chain polypeptide of donkey Cathelicidin genes encoding, it is characterized in that its total order classifies as: Methionin-arginine-arginine-glycine-Serine-Xie Ansuan-Threonine-Threonine-arginine-tyrosine-glutamine-Phe-Leu-methionine(Met)-Isoleucine-HIS-LEU-leucine-Arg-Pro-Methionin-Methionin-Ile-Phe-L-Ala.

2. the gene of the described domestic animal donkey of claim 1 antimicrobial peptide Ea-CATH1, the gene of the domestic animal donkey antimicrobial peptide Ea-CATH1 precursor cathelicidin that it is characterized in that encoding is made up of 550 Nucleotide, from 5 ' end to 3 ' terminal sequence is:

1?ATGGAGACCC?AGAGGGACAG?TTGTTCCCTG?GGCTGGTGGT?CACTGTTGCT?ACTGCTACTG

61?GGCCTGATGA?TCCCTCTGGC?CACCACTCAG?GCCCTCAGCT?ACAAGGAGGC?CGTACTCCGT

121?GCCGTGGATG?GCCTCAACCA?GTGGTCCTCA?GATGAGAATC?TCTACCGCCT?CCTGGAGCTG

181?GACCCTCTGC?CCAAGGGAGA?CGAGGCCCCA?GACACCCCAA?AGCCTGTGAG?CTTCACGGTC

241?AAGGAGACTG?TGTGCCCCAG?GACAACGCAG?CAGCCACTGG?AGCAGTGTGA?CTTCAAGGAG

301?AATGGGCTGG?TGAAACAGTG?TGTGGGGACA?GTCATCCTGG?ACCCGGTTAA?GGCCTCCGTT

361?GACATCGGTT?GTGATGAGCC?CCAGCGTGTC?AAGAGACGCG?GCTCGGTGAC?TACTCGTTAC

421?CAGTTCCTGA?TGATTCACCT?TCTTCGACCT?AAGAAGCTTT?TCGCCTAGAA?TCGGCTTGCC

481?CTGGCTTGGG?CTTCTGGACT?CTGAAAAATA?AATTATGTGA?AAGCCGCTTA?AAAAAAAAAA

541?AAAAAAAAAA

Coding domestic animal donkey cathelicidin mature peptide Ea-CATH1 is a 391-465 position Nucleotide, and its aminoacid sequence is: Lys ¹Arg ²Arg ³Gly ⁴Ser ⁵Val ⁶Thr ⁷Thr ⁸Arg ⁹Tyr ¹⁰Gln ¹¹Phe ¹²Leu ¹³Met ¹⁴Ile ¹⁵His ¹⁶Leu ¹⁷Leu ¹⁸Arg ¹⁹Pro ²⁰Lys ²¹Lys ²²Leu ²³Phe ²⁴Ala ²⁵

3. the application of the described domestic animal donkey of claim 2 antimicrobial peptide Ea-CATH1 gene is characterized in that, domestic animal donkey antimicrobial peptide Ea-CATH1 synthetic antibacterial peptide has germicidal action, is dissolved in the sterilization ultrapure water, is used for pharmacologically active and detects.