CN101168564B - Human antibiotic peptide and use for derivative thereof - Google Patents
Human antibiotic peptide and use for derivative thereof Download PDFInfo
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- CN101168564B CN101168564B CN2007100663029A CN200710066302A CN101168564B CN 101168564 B CN101168564 B CN 101168564B CN 2007100663029 A CN2007100663029 A CN 2007100663029A CN 200710066302 A CN200710066302 A CN 200710066302A CN 101168564 B CN101168564 B CN 101168564B
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Abstract
The invention relates to human antimicrobial peptide and the purpose of the derivant thereof. The human antimicrobial peptide has amino acid sequences chosen from a SEQ ID NO: 1 listing, and also comprises function equivalents obtained by substitution, cyclization, change from L-amino acid into D-amino acid, lacunae or addition of respective amino acid in amino acid sequence polypeptide. Compared with other source base antimicrobial polypeptide, the human antimicrobial peptide has the advantages that the structure is simple, the artificial synthesis is convenient, and the human antimicrobial peptide is applied to human body non-antigenicity. The human antimicrobial peptide and the derivant of the human antimicrobial peptide of the invention has the remarkable functions of inhibiting bacteria and funguses from growing, thus the human antimicrobial peptide can be used to prepare anti-microorganism infective preparation.
Description
Technical field
The present invention relates to the purposes of human antibiotic peptide and verivate thereof, be specifically related to people's semenogelin degrade aminoacid sequence, nucleotide coding sequence and their antibacterial application of little peptide SgI-29 and verivate thereof.
Background technology
Infection is the one of the main reasons that threatens the human life always, and the eighties of last century microbiotic thirties comes to light and is widely used in clinical treatment, has saved countless patients' life.Because the antibiotic a large amount of uses of tradition have caused increasing rapidly of multidrug resistant disease pathogenic microorganism.Therefore demanding developing new antibiotic now urgently tackles pathogenic micro-organism resistance serious day by day in the world wide.
Antibacterial peptide (having another name called peptide antibiotics) is extensive in distributed in nature, is the endogenous material of resisting exogenous infection that organism produces, and goes through millions of years and seldom produces tolerance, has characteristics such as effect is rapid, powerful, wide spectrum.Thereby in it is found; Just caused (the Koczulla AR that attractes attention of pharmacy circle and high-tech biological industry circle; Bals R, 2003.Antimicrobialpeptides:current status and therapeutic potential.Drugs 63,389-406).
Antibacterial peptide is distributed in batrachians, insect, plant and the Mammals widely.Discover that antibacterial peptide content in amphibian animal skin is especially abundant; People have found nearly thousand antibacterial peptides (can retrieve at http://www.bbcm.univ.trieste.it/～tossi/pag1.htm) from nature over more than 30 year; Wherein there be more than 800 from batrachians skin (mainly being batrachia) (Apponyi MA; Et al.; 2004.Host-defence peptides of Australian anurans:structure, mechanism of action and evolutionary significance.Peptides 25,1035-1054).
Since the microbiotic invention; The mankind have obtained bigger achievement in control and treatment infected by microbes; But along with antibiotic lasting use at present; The mikrobe resistance has become the significant problem in the infected by microbes control at present, to such an extent as to the certain micro-organisms bacterium has not been controlled a line material of killing.As in clinical drug therapy, it all is worldwide clinical difficult problems at present that the staphylococcus of vancomyein resistance, faecalis and other Gram-negatives catch.Three types cause that mainly strong resistance has also appearred in meningitic bacterium clinically, anti-penicillium mould, paraxin meningococcus, and streptococcus pneumoniae, antibiotic streptococcus pneumoniae also extensively occurs the antagonism cephalosporin.Therefore the discovery of new class antimicrobial material and development have become the task of top priority and international focus.Compare with present widely used microbiotic; Peptide antibiotics has lot of advantages: as when the least action concentration, fast and the killing microorganisms of wide spectrum (comprising clinical anti-medicine bacterium at present) also has restraining effect to fungi; The resistant strain generative nature is little; All effective to local infection and systemic infection, just becoming the new class microbiotic, its development receives extensive attention (Brogden KA at present; 2005.Antimicrobial peptides:pore formers or metabolic inhibitors in bacteria? Nat.Rev.Microbiol.3,238-250).
Human antibiotic peptide is one of focus of research always; It does not have antigenicity; Therefore more be prone to use (DeSmet K, Contreras R, 2005.Human antimicrobial peptides:defensins as medicine; Cathelicidins and histatins.Biotechnol.Lett.27,1337-1347).
Summary of the invention
An object of the present invention is to provide human antibiotic peptide and verivate thereof; Has the beneficial features that simple in structure, synthetic is convenient, be applied to the human body no antigen; Another object of the present invention provides the purposes of human antibiotic peptide and verivate thereof, and it has the effect of significant inhibition bacterium and fungal growth.
The objective of the invention is to realize like this:
The present invention relates to a kind of human antibiotic peptide, separate obtaining from the healthy male semen liquefaction, its aminoacid sequence is (SEQ ID NO:1) as follows:
Histidine - Asn - Lys - Gln - Glu - Gly - Arg - Asp - histidine - Asp - Lys - Ser - Lys - glycine - histidine - phenylalanine - histidine - Arg - Val - Val - isoleucine - histidine - histidine - Lys - Gly - Gly - lysine acid - alanine - histidine (His? Asn? Lys? Gln? Glu? Gly? Arg? Asp? His? Asp? Lys? Ser? Lys? Gly? His? Phe? His? Arg? Val? Val? IleHis? His? Lys? Gly? Gly? Lys? Ala? His).
Human antibiotic peptide of the present invention comprises that indivedual amino acid whose replacements in the aforementioned polypeptides, cyclisation, L-type amino acid become D-type amino acid, disappearance or adding and the polypeptide of the function equivalent that obtains.
Promptly substitute and have the antibacterial peptide of anti-microbial activity derived from the isolating human antibiotic peptide amino-acid residue of above-mentioned human antibiotic peptide, said antibacterial peptide has and is selected from the aminoacid sequence shown in the sequence table SEQ ID NO:2: His Asn Lys Gln Glu GlyLys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His.
Can also be derived from a plurality of aminoacid deletion of above-mentioned human antibiotic peptide and have the antibacterial peptide of anti-microbial activity that said antibacterial peptide has and is selected from the aminoacid sequence shown in the sequence table SEQ ID NO:3: Lys Gln Glu Gly Arg Asp His Asp LysSer Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys.
Human antibiotic peptide provided by the invention, its preparation method can be mechanochemical methods, also can the encoding sox of antibacterial peptide be cloned on the carrier, in host cell, express the back then and obtain.Wherein expression vector can be a kind of in plasmid or the virus.Host cell can be a prokaryotic cell prokaryocyte, comprises intestinal bacteria or subtilis etc., and host cell also can be an eukaryotic cell, comprises yeast cell, vegetable cell, insect cell or mammalian cell etc.The antibacterial peptide of preparation can be identified through mass spectrum.
The separation method of human antibiotic peptide of the present invention separates antibacterial peptide and has the aminoacid sequence shown in the SEQ ID NO:1.
The preparation method of human antibiotic peptide of the present invention and verivate thereof adopts mechanochemical method synthetic.
A kind of isolating nucleotide sequence or its complementary sequence, said nucleotide sequence coded antibacterial peptide recited above.
A kind of construction, described construction contain and have the nucleotide sequence that is selected from the aminoacid sequence shown in the sequence table SEQ ID NO:3.
A kind of cell, described cell contain peptide recited above, above said nucleotide sequence or construction recited above.
The method of said antibacterial peptide above the preparation, this method comprises:
(1) under the condition that is fit to peptide expression recited above, cultivates cell recited above;
(2) isolate antibacterial peptide recited above.
The purposes of human antibiotic peptide of the present invention and verivate thereof, the application in the medicine of preparation anti-microbial infection of described antibacterial peptide and verivate thereof.
In order to further investigate the structure-function relationship of this human antibiotic peptide of the present invention and verivate thereof, utilize full-automatic polypeptide synthetic instrument to synthesize one group of polypeptide, to study.
With the SgI-29 with following amino acid sequences of preparation, SgI-29R7K and SgI-25 antibacterial peptide are that example is narrated below:
SgI-29(SEQ?ID?NO：1)：
His?Asn?Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?HisHis?Lys?Gly?Gly?Lys?Ala?His
SgI-29R7K(SEQ?ID?NO：2)：
His?Asn?Lys?Gln?Glu?Gly?Lys?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?HisHis?Lys?Gly?Gly?Lys?Ala?His
SgI-25(SEQ?ID?NO：3)：
Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?His?His?LysGly?Gly?Lys
Utilize the agar diffusion experiment to detect the bacteriostatic activity of polypeptide.The result shows that synthetic antibacterial peptide of the present invention has strong fungicidal activity.
Antibacterial peptide function equivalent to aminoacid deletion verivate in the polypeptide has also carried out preparation and biological activity determination equally, and the result shows that these polypeptide also have fungicidal activity preferably.
Beneficial effect of the present invention is: human antibiotic peptide and verivate thereof have the effect of significant inhibition bacterium and fungal growth.Compare with other alkaline antimicrobial polypeptide of originating, that this human antibiotic peptide has is simple in structure, synthetic convenient, be applied to the beneficial features of human body no antigen.
Description of drawings
Fig. 1 is the SP-Sepharose ion exchange chromatography figure of human antibiotic peptide of the present invention.
Fig. 2 is the G-25 sieve chromatography figure of human antibiotic peptide of the present invention.
Fig. 3 is the Mono-S ion exchange chromatography figure of human antibiotic peptide of the present invention.
Fig. 4 is the HPLC anti-phase C18 column chromatography figure of human antibiotic peptide of the present invention.
Fig. 5 is the mass spectrum at human antibiotic peptide HPLC anti-phase C18 column chromatography III of the present invention peak.
Fig. 6 is the nucleotide sequence of human antibiotic peptide SgI-29 gene of the present invention.
Fig. 7 is the aminoacid sequence of human antibiotic peptide SgI-29 of the present invention.
Fig. 8 is the mass spectrum of synthetic human antibiotic peptide SgI-29 of the present invention.
Embodiment
Embodiment 1
1, the separation and purification of human antibiotic peptide:
The normal people after ascetic 3-7 days the masturbation extracting extract in the dry disinfection measuring cup; After the room temperature liquefaction; Through sperm mikrobe sound attitude area of computer aided automatic analysis system (model: CASAS-QH-III; Beijing Tsing Hua Tong Fang product) confirm the normal sample of sperm each item index in 10000 rev/mins of centrifugal 10 minutes separated sperms and seminal fluid, carefully collect supernatant put-20 ℃ subsequent use.
The first step: SP-Sepharose IX: the normal people's seminal fluid (n=40) that obtains as stated above is splined on uses 50mM Na2HPO4-NaH2PO4 in advance; PH=7.4 damping fluid equilibrated SP-Sepharose (Pharmacia product) cationic exchange coloum (130mm is long, diameter 20mm).After finishing, last appearance carries out linear elution with 0-0.5M NaCl.Every pipe is collected 3ml.Bacteriostatic activity detects and shows that the 58-131 pipe has very strong fungicidal activity.Merge 58-131 pipe frost drying and concentrate, see accompanying drawing 1.
Second step: molecular sieve Sephadex G-25 chromatography: after the first step freeze-drying concentrating sample dissolves with the Milli-Q ultrapure water; Be splined on and use 50mM Tris-HCl in advance; PH8.8 damping fluid equilibrated AKTA
HiPrepTM G-25 desalting column (26/10; The Pharmacia product), used instrument is AKTA
Explore 100 (Amersham Biosciences product).Collect the II peak and carry out frost drying and concentrate, referring to accompanying drawing 2.
The 3rd step: Mono-S IX: after the second step freeze-drying concentrating sample dissolves with the Milli-Q ultrapure water; Be splined on and use 50mM Tris-HCl in advance; PH8.8 damping fluid equilibrated Mono-S cationic exchange coloum (1ml; HR5/5, the Pharmacia product), used instrument is AKTAExplore 100 (an Amersham Biosciences product).Collect the VII peak, see accompanying drawing 3.
The 4th step: reverse phase HPLC: through Mono-S cation exchange column chromatography gained VII peak Yi Shui (containing 0.1% trifluoroacetic acid): the elution system that second fine (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient elution; Collect the III peak and be the people's semenogelin I derived peptide that contains anti-microbial activity, see accompanying drawing 4.
Contain the evaluation of people's semenogelin I derived peptide of anti-microbial activity:
The first step: the determined amino acid sequence at reverse phase HPLC III peak: adopt full-automatic Protein Sequencer (model: 491, ABI Company products) to measure the aminoacid sequence structure.The result shows that the aminoacid sequence at reverse phase HPLC III peak is: His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser.
Second step: fast atom bombardment mass spectroscopy(FABMS) method (Fast atom bombardment mass spectrometry; FAB-MS) determining molecular weight: with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1: 1: 1; V: V: V, volume ratio) be substrate, Cs+ is as projectile; Electric current is 1mA, and emission voltage is 25Kv.The result shows that reverse phase HPLC III peak contains 4 molecules, and their molecular weight is respectively 3377.17 dalton, 3850.67 dalton, 5117.64 dalton and 5174.18 dalton, sees accompanying drawing 5.
The 3rd step: the evaluation of people's semenogelin I derived peptide: the result that the reverse phase HPLC III peak aminoacid sequence His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser of mensuration carries out the BLAST analysis discloses the degraded that it derives from people's semenogelin I, is the derived products of people's semenogelin I.It is that 3377.17 daltonian polypeptide derive from mature human semenogelin I that the mass spectral accurate molecular weight of FAB-MS is measured the proof molecular weight, and its shearing site is the 85th and the 113rd of mature human semenogelin I.Resultant derived fragment is called after SgI-29 in the present invention, and its total order is classified as: His AsnLys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly GlyLys Ala His.The theoretical molecular-weight average [M+H] of SgI-29+be 3377.74, determining molecular weight [M+H]+be 3377.17.Experimental result has proved that fully SgI-29 is hydrolyzed the back by people's semenogelin I and produces.
Embodiment 2
1, the preparation of human antibiotic peptide and separation and purification:
Present embodiment adopts the solid state chemistry synthesis method to carry out, and utilizes full-automatic polypeptide synthetic instrument to synthesize their complete sequence respectively, and the title of each synthetic antibacterial peptide and complete sequence amino acid primary structure thereof are following:
SgI-29(SEQ?ID?NO：1)：
His?Asn?Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?HisHis?Lys?Gly?Gly?Lys?Ala?His
SgI-29R7K(SEQ?ID?NO：2)：
His?Asn?Lys?Gln?Glu?Gly?Lys?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?HisHis?Lys?Gly?Gly?Lys?Ala?His
SgI-25(SEQ?ID?NO：3)：
Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg?Val?Val?Ile?His?His?LysGly?Gly?Lys。
Through the desalination of HPLC anti-phase C18 column chromatography, purifying.
2, the evaluation of human antibiotic peptide:
The human antibiotic peptide of purifying is identified its purity with the high-efficient liquid phase chromatogram HPLC method, and molecular-weight determination adopts the fast atom bombardment mass spectroscopy(FABMS) method, measures the aminoacid sequence structure with automatic Protein Sequencer.
Embodiment 3
1, the expression of human antibiotic peptide SgI-29 gene in intestinal bacteria
According to known the 86th to the 112nd the gene order that is encoded into acquaintance's semenogelin I; It is cloned on the expression vector pMAL-p2X plasmid; Transformed into escherichia coli DH5 α then, in colibacillus periplasm, bacterium pericentral siphon Partial Protein is crossed the MBP affinity column after hypotonic processing through IPTG abduction delivering MBP-SgI-29 fusion rotein; Obtain purifying MBP-SgI-29 fusion rotein, after the FXa cutting, obtain antibacterial peptide SgI-29 again.
The high-fidelity pfu enzyme that uses among the embodiment; The T4DNA ligase enzyme is available from U.S. Promega company; Restriction enzyme Xmn I, Hind III, affine resin (Amylose resin) and expression vector pMAL-p2X are available from Britain New England BioLabs company, and Tryptones, yeast extract are available from Oxoid company, and acrylic amide, methylene diacrylamide, Ammonium Persulfate 98.5, isopropyl-(IPTG) etc. are available from Sigma company.FXa is available from Novagen company.
People's seminal vesicle take from one 56 years old because of bladder urothelium cancer row bladder, prostate gland, seminal vesicle total resection male patient, pathologic finding confirms that seminal vesicle is not by tumor invading.The seminal vesicle that operation is downcut places liquid nitrogen to preserve immediately, up to extracting total RNA.
2, the extraction of total RNA and cDNA are synthetic
Total RNA adopts Qiangen company total RNA extraction reagent box to extract.The synthetic TAKALA company test kit that adopts of cDNA, the concrete operations by specification carries out.
3, design of primers and synthetic
According to the sequences Design primer of SgI gene, SgI-1 5 ' GGT TTT CCA AGC AAG ATG AAG 3 '; SgI-1R:5 ' AGG TGG TGT CAT CCA TGG ACC AAG 3 '; SgI-EX1 5 ' CAT AAT AAA CAAGAA GGC AGA GAC C 3 '; SgI52-EXR 5 ' CCT AAC AAG CTT TTA ATA TTG ACT GGATAT TCC 3 ' is synthesized by Beijing hundred victory companies that match.Wherein AAG CTT is the restriction enzyme site of HindIII, and the base CCTAAC of its front is the protection sequence.
4, SgI52 gene amplification and clone
Use the cDNA that has obtained to be masterplate, adopting SgI-1 and SgI-1R is that primer carries out pcr amplification, condition: cDNA 1 μ l; 10 X buffer, 5 μ l, dNTP (10mM) 1 μ l, SgI-1 (20p/ μ l) 1 μ l; SgI-1R (20p/ μ l) 1 μ l, pfu 0.25 μ l, ddH2O 40.75 μ l.Pcr amplification condition: 94 ℃ of 2min; 92 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.PCR product row agarose gel electrophoresis reclaims test kit with the obvious band in 1.3Kb place with Tiangen DNA glue and carries out the DNA recovery.DNA with reclaiming does substrate, does primer with SgI-EX1 and SgI52-EXR and carries out pcr amplification SgI-52 gene fragment.Condition: the SgI DNA 0.5 μ l of recovery, 10 X buffer, 5 μ l, dNTP (2.5mM) 4 μ l, SgI-EX1 (20p/ μ l) 1 μ l, SgI52-EXR (20p/ μ l) 1 μ l, pfu 0.25 μ l, H2O 38.25 μ l.PCR expands condition once: 94 ℃ of 2min; 92 ℃ of 15s, 50 ℃ of 30s, 72 ℃ of 20s, 35 circulations; 72 ℃ of 2min.The PCR product finds near the 150bp obvious band is arranged through electrophoresis.The PCR product is cut through the HindIII enzyme, cuts the glue purification gene fragment.Adopt the pMAL-p2X plasmid in the test kit extracting e. coli jm109, cut plasmid with restriction endonuclease HindIII and XmnI, electrophoresis confirms.With the T4 ligase enzyme plasmid is connected with the SgI-52 gene that cuts with restriction endonuclease HindIII.4 ℃ are spent the night.The transformed competence colibacillus bacillus coli DH 5 alpha.Picking colony is identified, and is extracted the plasmid order-checking.
5, the abduction delivering of SgI-52 polypeptide
Incubated overnight has the pMAL-p2X plasmid bacillus coli DH 5 alpha of SgI-52 gene.The bacillus coli DH 5 α that adds 1/100 incubated overnight gets into RB substratum 1000ml, 37 ℃ of 200rpm shake the about 2-3 of bacterium hour to O.D.600 be 0.5.Adding IPTG (final concentration is 0.3mM) and continue to cultivate and induce 3 hours, is contrast not add each recombinant plasmid transformed bacterial cultures of IPTG inductive.
6, recombinant protein detects and identifies
1 liter of bacterial cultures is collected bacterium in the centrifugal 10min of 4000g, with the resuspended bacterium of 30mM Tris-HCl+20%Sucrose pH8.0 (every g bacterium adds 80ml).It is 1mM that the EDTA that adds 0.5M makes final concentration, and room temperature was shaken 5-10 minute.Centrifugal 10 minutes of 4 ℃ of 8000g.MgSO4 with the ice-cold 5mM of 2.5ml is resuspended.Shook under the ice bath 10 minutes.Centrifugal 10 minutes of 4 ℃ of 8000g.Supernatant is the protein of periplasmic expression.Add suitable albumen sample-loading buffer, carry out the SDS-PAGE electrophoresis after boiling 5min, and, confirm that recombinant protein has or not expression through coomassie brilliant blue staining.With the X factor recombinant protein is excised from fusion rotein, carry out mass spectroscopy and sequencing.
Embodiment 4
Anti-microbial activity detects; Reference literature method (Lehrer et al.; 1991.Ultrasensitive assays for endogenousantimicrobial polypeptides.J.Immunol.Methods 137:167-173), adopt the double-deck punch method of agarose to measure MEC MEC (Minimal Effective Concentration).Samples using solid phase method chemosynthesis in the present embodiment, the title of each synthetic antibacterial peptide and complete sequence amino acid primary structure thereof are seen embodiment 2.
Wherein, the nucleotide sequence of SgI-29 sees that accompanying drawing 6, aminoacid sequence see that the mass spectrum of accompanying drawing 7, synthetic human antibiotic peptide SgI-29 sees accompanying drawing 8.
The bottom culture medium prescription is: 1% low melting-point agarose (SigmaA6013), 0.3mg/ml Trypsin freeze (Oxoid product) and are dissolved in the 10mM pH7.4 Na2HPO4-NaH2PO4 damping fluid.Bottom substratum 20ml adds incubated overnight logarithmic phase bacterium (about 105-106CFU) respectively in the time of 42 ℃, make it in diameter 76mm petridish, evenly spread out cloth as bottom after shaking up.After waiting to solidify, beat the circular hole of 3mm on the bottom substratum, every hole adds the antibacterial peptide aqueous solution of 5 μ l different concns.37 ℃ hatch 3 hours after; On bottom, cover one deck nutrient agar medium (top layer substratum: 1%Sigma A6013 low melting-point agarose; Be dissolved in the 10mM pH7.4 Na2HPO4-NaH2PO4 damping fluid 0.6mg/ml the Oxoid Trypsin freezes), 37 ℃ were continued to hatch 12-16 hour.Measure the transparent ring diameter of asepsis growth.The calculating of anti-microbial activity: the U=of anti-microbial activity unit (the diameter mm-3 of antibiotic ring) X10.Logarithmic value with antibacterial peptide concentration is an X-coordinate, and anti-microbial activity is that ordinate zou is made regression equation, and calculates the Cmin MEC of the antibacterial peptide of ability bacteria growing inhibiting.Bacterial isolates derives from No.1 Hospital Attached to Kunming Medical College, and result such as table 1 are averaged in this test repetition four times.
The bacteriostatic activity of table 1. human antibiotic peptide SgI-29
| Microorganisms | MEC MEC (μ g/ml) | ||
| SgI-29 | ?SgI-29R7K | SgI-25 | |
| Escherichia coli ATCC 25922 Escherichia coli ML-3 5P Escherichia coli clinic strain 1 Escherichia coli clinic strain 2 Bacillus pyocyaneus ATCC 27853 Bacillus pyocyaneus CMCCB 10104 Bacillus pyocyaneus PA 01 Bacillus pyocyaneus clinic strain Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 43300 | ?2.831?7.433?3.556?3.937?5.292?7.334?12.864?10.405?＞100?＞100 | 3.062 6.882 4.079 3.638 4.996 6.378 10.638 11.909 undetermined undetermineds | 5.361 10.231 6.823 7.231 11.734 15.634 18.885 25.362 undetermined undetermineds |
| ?Candida?albicans ATCC?10231?Pichia?pastoris?GS?115 | ?20.523?18.244 | 25.768 21.224 | ?30.665?24.315 |
Test the explanation of used bacterium and fungal bacterial strain:
Streptococcus aureus (Staphylococcus aureus) ATCC 43300 is methicillin resistant staphylococcus aureus ATCC type strains, to PCs, and the beta-lactam resistance, responsive to vancomyein.
Intestinal bacteria (Escherichia coli) ML-35P is an ammonia benzyl resistance type strain.
Intestinal bacteria (Escherichia coli) clinical drug-resistant strain 1 and 2 is the clinical separation strain that produces extended spectrum, to penicillium mould, 1 generation of cephalo, 2 generations, 3 generations and 4 generation resistance, to safe can, aminoglycosides is responsive.
Pseudomonas aeruginosa (Bacillus pyocyaneus) clinical drug-resistant strain is the multidrug resistance clinical separation strain, to the equal resistance of all kinds of microbiotic of present clinical use, comprises safe ability, MEM.
The Candida albicans of Mycophyta (Candida albicans) ATCC 10231 and pichia spp (Pichia pastoris) GS115 are type strain.
Visible by table 1, synthetic human antibiotic peptide SgI-29 and amino acid replacement thereof and aminoacid deletion verivate have the effect of significant inhibition bacterium and fungal growth, can be used as antimicrobial material and are used to prepare the anti-microbial infection preparation.
Sequence table .SEQ
< 110>Kunming Institute of Zoology, Chinese Academy of Sciences
< 120>human antibiotic peptide and antimicrobial application thereof
<160>6
<210>1
<211>29
<212>PRT
< 213>homo sapiens (Homo sapiens)
<400>1
His?Asn?Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe
1 5 10 15
His?Arg?Val?Val?Ile?His?His?Lys?Gly?Gly?Lys?Ala?His
20 25
<210>2
<211>29
<212>PRT
< 213>artificial sequence
<400>2
His?Asn?Lys?Gln?Glu?Gly?Lys?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe
1 5 10 15
His?Arg?Val?Val?Ile?His?His?Lys?Gly?Gly?Lys?Ala?His
20 25
<210>3
<211>25
<212>PRT
< 213>artificial sequence
<400>3
Lys?Gln?Glu?Gly?Arg?Asp?His?Asp?Lys?Ser?Lys?Gly?His?Phe?His?Arg
1 5 10 15
Val?Val?Ile?His?His?Lys?Gly?Gly?Lys
20 25
<210>4
<211>87
<212>DNA
< 213>homo sapiens (Homo sapiens)
<400>4
cataataaac?aagaaggcag?agaccatgat?aaatcaaaag?gtcattttca?cagggtagtt 60
atacaccata?aaggaggcaa?agctcat 87
<210>5
<211>87
<212>DNA
< 213>artificial sequence
<400>5
cataataaac?aagaaggcaa?agaccatgat?aaatcaaaag?gtcattttca?cagggtagtt 60
atacaccata?aaggaggcaa?agctcat 87
<210>6
<211>75
<212>DNA
< 213>artificial sequence
<400>6
aaacaagaag?gcaaagacca?tgataaatca?aaaggtcatt?ttcacagggt?agttatacac 60
cataaaggag?gcaaa 75
Claims (9)
1. isolating human antibiotic peptide, it is characterized in that said human antibiotic peptide is by the aminoacid sequence shown in the sequence table SEQ ID NO:1: His Asn Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His forms.
2. substitute and have the antibacterial peptide of anti-microbial activity derived from the isolating human antibiotic peptide amino-acid residue of claim 1, it is characterized in that said antibacterial peptide is by the aminoacid sequence shown in the sequence table SEQ ID NO:2: His Asn Lys Gln Glu Gly Lys Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys Ala His forms.
3. derived from a plurality of aminoacid deletion of claim 1 and have the antibacterial peptide of anti-microbial activity, it is characterized in that said antibacterial peptide is by being selected from the aminoacid sequence shown in the sequence table SEQ ID NO:3: Lys Gln Glu Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val Val Ile His His Lys Gly Gly Lys forms.
4. the preparation method of claim 1,2 or 3 described antibacterial peptides is characterized in that adopting mechanochemical method synthetic.
5. an isolating nucleic acid molecule is characterized in that any described antibacterial peptide of said nucleotide sequence coded claim 1 to 3.
6. a construction is characterized in that described construction contains the nucleotide sequence of any described antibacterial peptide of coding claim 1 to 3.
7. a cell is characterized in that described cell contains any described antibacterial peptide of claim 1 to 3, the said nucleotide sequence of claim 5 or the described construction of claim 6.
8. method for preparing any said antibacterial peptide of claim 1 to 3 is characterized in that this method comprises:
(1) under the condition that is fit to any described peptide expression of claim 1 to 3, cultivates the described cell of claim 7;
(2) isolate any described antibacterial peptide of claim 1 to 3.
9. the purposes of any described antibacterial peptide of claim 1 to 3 is characterized in that: the application of described antibacterial peptide in the medicine of preparation anti-microbial infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100663029A CN101168564B (en) | 2007-10-22 | 2007-10-22 | Human antibiotic peptide and use for derivative thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100663029A CN101168564B (en) | 2007-10-22 | 2007-10-22 | Human antibiotic peptide and use for derivative thereof |
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| Publication Number | Publication Date |
|---|---|
| CN101168564A CN101168564A (en) | 2008-04-30 |
| CN101168564B true CN101168564B (en) | 2012-03-21 |
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| CN110438139A (en) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | A method of antibacterial peptide T9W is prepared using Pichia yeast |
| CN112263712B (en) * | 2020-10-14 | 2022-04-29 | 中山大学附属第六医院 | Peritoneal repair material with antibacterial property and preparation method thereof |
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