CN101412753A - Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof - Google Patents

Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof Download PDF

Info

Publication number
CN101412753A
CN101412753A CNA2008100589769A CN200810058976A CN101412753A CN 101412753 A CN101412753 A CN 101412753A CN A2008100589769 A CNA2008100589769 A CN A2008100589769A CN 200810058976 A CN200810058976 A CN 200810058976A CN 101412753 A CN101412753 A CN 101412753A
Authority
CN
China
Prior art keywords
cathelicidin
antibacterial peptide
methionin
lys
lysine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CNA2008100589769A
Other languages
Chinese (zh)
Inventor
赖仞
王义鹏
杨海龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kunming Institute of Zoology of CAS
Original Assignee
Kunming Institute of Zoology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kunming Institute of Zoology of CAS filed Critical Kunming Institute of Zoology of CAS
Priority to CNA2008100589769A priority Critical patent/CN101412753A/en
Publication of CN101412753A publication Critical patent/CN101412753A/en
Pending legal-status Critical Current

Links

Abstract

The invention discloses cathelicidin-BF and a gene and application thereof, which belong to the field of biomedicine. The cathelicidin-BF is straight chain polypeptide and contains thirty amino acid residues, the molecular weight is 3,637.54Da, and the isoelectric point is 11.79. The complete sequence of the cathelicidin-BF is lysine-phenyl alanine-phenyl alanine-arginine-lysine-leucine- lysine-lysine-serine-valine-lysine-lysine-arginine-lactamine-lysine-glutamic acid-phenyl alanine- phenyl alanine-lysine-lysine-proline-arginine-valine-isoleucine-glycin-valine-serine-isoleucine- praline-phenyl alanine. The gene for encoding the cathelicidin-BF consists of 750 ribonucleotides, wherein 484th to 573rd ribonucleotides are used for encoding a mature peptide part. The cathelicidin-BF has small molecular weight, strong sterilization effect, and quick action time, and has quite strong killing function to a plurality of kinds of clinical drug-fast bacteria. In addition, the cathelicidin-BF also has the advantages of broad-spectrum antibiotics, salt independence and so on.

Description

Bungarus fasciatus antibacterial peptide cathelicidin-BF and gene thereof and application
Technical field:
The invention provides a kind of Gold-banded Krait (Bungarus fasciatus) antibacterial peptide cathelicidin-BF and gene and application, belong to field of biomedicine technology.
Background technology:
Cathelicidin is one and has multi-functional antibacterial peptide family, only in mammals, birds and fish discovery arranged at present.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 the panimmunity 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.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.
Along with antibiotic extensive and incorrect uses of tradition such as penicillin, microorganism has produced more and more stronger tolerance to traditional microbiotic, occurred can tolerating fully in a large number traditional antibiotic microorganisms such as penicillin clinically, existing microbiotic is powerless to these pathogenic micro-organisms.Antibacterial peptide is a kind of novel antimicrobial polypeptide, and most of antibacterial peptide molecular weight are less than 10000Da, and positively charged is rich in hydrophobic base, can form amphipathic structure.The bactericidal mechanism of antibacterial peptide mainly is by electrostatic interaction attraction and is attached to electronegative bacterial cell membrane surface, further forms the hole of striding film on bacterial cell membrane, causes leaking of bacterial cell content, thereby causes 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 antibacterial peptide mediation is far away faster than traditional microbiotic, and is difficult for making bacterium to produce tolerance.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 antibacterial 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.
In the traditional Chinese medicine and national medicine of China, many snake classes are used as medicinal material and are widely used, as black-snake, and boa, pallas pit viper, Gold-banded Krait, coral snake, Naja, Agkistrodon etc.The snake whole body all can be used as medicine, and meat of snake has strong nerve, the effect of promoting longevity, and can treat weak after being ill, diseases such as wandering arthritis is numb, arthralgia.Snake gall have dispel the wind, heat-clearing, the effect of reducing phlegm, making eye bright, cure mainly infantile malnutrition, swelling and pain of hemorrhoid.The Fel Serpentis ethanolic soln can effectively be treated keratitis, keratohelcosis, corneal macula.With Fel Serpentis, Unibract Fritillary Bulb, almond is " Fel Serpentis et Bulbus Fritillariae Cirrhosae Liquidus " that main raw material is made, be present clinical cough-relieving apophlegmatic good medicine easy to use, evident in efficacy, snake slough has expelling wind for relieving convulsion, desinsection, move back the screen detumescence, clearing heat and detoxicating function, can control diseases such as order screen, sore furuncle, anal fistula, mumps, malaria, pediatric epilepsy scared, mange, scrofula.Snake venom is a very expensive rare medicinal material on the present international medicinal material market, and the injection liquid that utilizes agkistrodon halyx pallas venom to make has significantly anticancer, anticoagulant effect.
Meanwhile, the complicacy of traditional Chinese medicine pharmaceutical cpd and the limitation of concocting method thereof cause active constituents of medicine can better not play a role, thereby the specific reactive monomer compound of searching is one of important content of the modernization of Chinese medicine from these conventional medicaments.Up to now, Chinese scholars has been isolated large number of biological activated protein and polypeptide from snake venom, and many active polypeptide wherein have good drug development prospect.
Bungarus is the poisonous snake with proteroglyphic tooth in the Elapidae Anilius, is distributed widely in southern china each province and Southeast Asian countries.About krait venom albumen, various proteolytic enzyme and inhibitor have a large amount of reports at present, but also do not report about the research of antibacterial peptide in the krait venom.
The contriver searches comparison with Bungarus fasciatus antibacterial peptide cathelicidin-BF complete sequence amino acid structure of the present invention through the NCBI Protein Data Bank, finds no any phase homopolypeptide.The contriver searches comparison with Bungarus fasciatus antibacterial peptide cathelicidin-BF encoding gene of the present invention through the NCBI gene database, finds no any homologous genes.
Summary of the invention:
The objective of the invention is based on above-mentioned theory research and prior art basis, a kind of Bungarus fasciatus antibacterial peptide cathelicidin-BF and gene and application with intensive anti-microbial activity is provided.
In order to realize purpose of the present invention, the invention provides following technical scheme:
Bungarus fasciatus antibacterial peptide cathelicidin-BF is a kind of straight-chain polypeptide of Gold-banded Krait cathelicidin genes encoding, contains 30 amino-acid residues, molecular weight 3637.54Da, iso-electric point 11.79.Bungarus fasciatus antibacterial peptide cathelicidin-BF total order is classified as: Methionin-phenylalanine-phenylalanine-arginine-Methionin-leucine-Methionin-Methionin-Serine-Xie Ansuan-Methionin-Methionin-arginine-L-Ala-Methionin-L-glutamic acid-phenylalanine-phenylalanine-Methionin-Methionin-proline(Pro)-arginine-Xie Ansuan-Isoleucine-glycine-Xie Ansuan-Serine-Isoleucine-proline(Pro)-phenylalanine.
The clone of Bungarus fasciatus antibacterial peptide cathelicidin-BF gene comprises:
The total RNA of Gold-banded Krait poison gland extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library construction, the design primer utilizes PCR method screening Bungarus fasciatus antibacterial peptide cathelicidin-BF gene.Amplimer length is 24 Nucleotide, and its sequence is
5 ' AA (A/G) TT (T/C) TT (T/C) AG (A/G) AA (A/G) is T (A/T/C/G) AA (A/G) AA (A/G) 3 ' (C/T), and another amplimer of PCR is the Creator of CLONTECH company TMSMART TM3 ' PCR Primer primer among the cDNA LibraryConstruction Kit, its sequence is
5’ATTCTACAGGCCGAGGCGGCCGACATG?3′。The positive monoclonal that obtains carries out gene nucleotide series and measures.Gene sequencing result shows that the gene of coding Bungarus fasciatus antibacterial peptide cathelicidin-BF is made up of 750 Nucleotide, holds to 3 ' terminal sequence from 5 ' to be:
atggaagggt?tcttctggaa?gaccttgctg?gtggttggag?ctcttgccat?tgctgggacc 60
tcctcacttc?cacacaaacc?cctgatctat?gaagaggctg?tggaccttgc?agtgagcatc 120
tacaacagca?aatctgggga?agactctctc?taccgtctcc?tggaggctgt?ttctccaccc 180
aagtgggatc?ctctttctga?aagcaaccaa?gagctgaact?tcaccatgaa?ggagacggtg 240
tgcctggtgg?ccgaagaacg?atccttggag?gaatgcgact?tccaggaaga?cggggtcgtc 300
atgggatgca?caggctacta?tttcttcggg?gagtcgcccc?cggtggtcgt?tctcacctgc 360
aagcctgtgg?gtgaagaagg?ggagcagaag?caggaggagg?ggaacgagga?ggagaaggaa 420
gtggaggagg?aggaacagga?ggaagacgag?aaggatcagc?ccaggagggt?caagagattc 480
aagaaatttt?tcaggaagct?gaagaagagc?gtgaagaaac?gtgccaagga?attcttcaag 540
aagccgaggg?tcatcggggt?ctccatcccc?ttctaagaga?ggggctcaga?aggacctgcg 600
gcctccgctc?tccgatccca?ggaaaaccgg?cagagaagac?gatgcgggat?gctccagtcc 660
gtcaaatcat?ttcccaatag?gatgctccgc?cattcaatcc?atgaataaat?aaatatatac 720
ttgaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 750
Coding Bungarus fasciatus antibacterial peptide cathelicidin-BF mature peptide is the 484-573 Nucleotide, and its aminoacid sequence is:
Lys 1?Phe 2?Phe 3?Arg 4?Lys 5?Leu 6?Lys 7?Lys 8?Ser 9?Val 10?Lys 11?Lys 12?Arg 13?Ala 14?Lys 15
Glu 16?Phe 17?Phe 18?Lys 19?Lys 20?Pro 21?Arg 22?Val 23?Ile 24?Gly 25?Val 26?Ser 27?Ile 28?Pro 29?Phe 30
Bungarus fasciatus antibacterial peptide cathelicidin-BF gene prepares the application of Bungarus fasciatus antibacterial peptide cathelicidin-BF as genetically engineered.
The separation purification method of Bungarus fasciatus antibacterial peptide cathelicidin-BF:
The thick malicious 0.4g of freeze dried Gold-banded Krait at first crosses gel permeation chromatography post Sephadex G-50, collects the peak with anti-microbial activity, and freeze-drying is dialysed with phosphate buffered saline buffer; Cross cationic exchange coloum CM-SephadexC-25 then, collect peak, freeze-drying with anti-microbial activity; Cross anti-phase high-pressure liquid phase (RP-HPLC) C4 post at last, purifying obtains Bungarus fasciatus antibacterial peptide cathelicidin-BF.
The chemical synthesis process of Bungarus fasciatus antibacterial peptide cathelicidin-BF:
Record the aminoacid sequence of sequence and the deduction of coding Gold-banded Krait cathelicidin-BF antibacterial peptide gene according to the Edman edman degradation Edman, with synthetic its complete sequence of 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).The synthetic antibacterial peptide is dissolved in aqua sterilisa, is used for active the detection.
Beneficial effect of the present invention is:
Separation and purification obtains Bungarus fasciatus antibacterial peptide cathelicidin-BF, and the clone obtains its cDNA sequence.This antibacterial peptide molecular weight is little, germicidal action is strong, action time is rapid, the various clinical resistant organism is had very strong killing action.Have beneficial features such as broad-spectrum antimicrobial and non-salt dependence in addition.
Embodiment:
Further specify essentiality content of the present invention with embodiment below, but content of the present invention is not limited thereto.
Gold-banded Krait cathelicidin-BF antibacterial peptide gene clone:
I, the total RNA of Gold-banded Krait poison gland extract:
A. live body Gold-banded Krait haircut is put to death, and gets poison gland, weighs, and gets 300mg poison gland tissue, adds the total RNA of 10ml and extracts damping fluid (Trizol solution, U.S. GIBCOBRL company product), homogenate in the 20ml glass homogenizer.
B. add equal-volume phenol/chloroformic solution, violent mixing, room temperature was placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, reject precipitation.
C. supernatant adds isopyknic Virahol, and room temperature was placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, precipitation is washed once with 75% ethanol, dries, and pipe end throw out is the total RNA of Gold-banded Krait poison gland.
The purifying of II, Gold-banded Krait poison gland mRNA:
U.S. PROMEGA company is adopted in Gold-banded Krait poison gland mRNA separation and purification
Figure A200810058976D0007153854QIETU
MRNAIsolation Systems test kit.
A. get the total RNA 500 μ g of Gold-banded Krait poison gland and be dissolved in the 500 μ l DEPC water, put into 65 ℃ of water-baths 10 minutes, add Oligo (dT) probe and 13 μ l, the 20 * SSC solution of people 3 μ l, mixing is placed the room temperature cooling, is called A liquid.
B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked mixing,, abandon supernatant, add 0.5 * SSC0.3ml,, add 0.1ml 0.5 * SSC at last and suspend, be referred to as B liquid to magnetic force frame absorption 30 seconds to magnetic force frame absorption 30 seconds.
C. A liquid is added in the B liquid, room temperature was placed 10 minutes, to magnetic force frame absorption 30 seconds, abandon supernatant, with 0.1 * SSC washing 4 times, abandon supernatant at last, add 0.1ml DEPC aqueous suspension, to the magnetic force frame, adsorbed 30 seconds, supernatant is moved to new test tube, add 0.15ml DEPC water again and suspend again, to magnetic force frame absorption 30 seconds, moving supernatant to above-mentioned test tube, then is the Rana grahami skin mRNA of purifying in the supernatant.
D. add 1/10 volume 3M sodium acetate, pH5.2, the equal-volume Virahol, in-70 ℃ of placements 30 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm abandoned supernatant, and resolution of precipitate is in 10 μ l DEPC water.
III, Gold-banded Krait poison gland cDNA library construction: adopt the CLONTECH Creator of company TMSMART TMCDNALibrary Construction Kit.
A.cDNA first chain synthesizes (mRNA reverse transcription):
1. add 1 μ l Gold-banded Krait poison gland mRNA, 1 μ l SMART IV oligonucleotide, 1 μ l CDS III/3 ' PCR primer in the centrifuge tube that DEPC handles, the water that adds 2 μ l DEPC processing makes cumulative volume reach 5 μ l.
2. the reagent in the mixing centrifuge tube is also of short duration centrifugal, and 72 ℃ are incubated 2 minutes.
3. centrifuge tube was hatched on ice 2 minutes.
4. in centrifuge tube, add following reagent 2.0 μ l 5 * the first chains buffering, 1.0 μ l 20mM dithiothreitol (DTT), 1.0 μ l 10mM dNTP mixtures, 1.0 μ l PowerScript ThermoScript II.
5. reagent and of short duration centrifugal in the mixing centrifuge tube is incubated 1 hour at 42 ℃.
6. centrifuge tube is placed and end the synthetic of first chain on ice.
7. it is standby to get the 2 synthetic cDNA of μ l institute, first chain from centrifuge tube.
B. adopt long terminal polymerase chain reaction (LD-PCR) method second chain that increases
1.95 ℃ preheating PCR instrument.
2. 2 μ l cDNA, first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l, 10 * Advantage 2PCR buffering, 2 μ l, 50 * dNTP mixture, 2 μ l, 5 ' PCR primer, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted.
3. in the PCR instrument, increase by following program:
1. 95 ℃ of 20 second
2. 22 circulations:
95 ℃ of 5 second
68 ℃ 6 minutes
4. after the loop ends, with synthetic cDNA two strands-20 ℃ preservation in the centrifuge tube.
IV, Bungarus fasciatus antibacterial peptide cathelicidin-BF gene clone screening:
The cathelicidin-BF mature peptide sequences Design degenerate primer BFS that records according to the Edman edman degradation Edman 1Carry out pcr amplification, its sequence is (C/T) T (A/T/C/G) AA (A/G) AA (A/G) 3 ' of 5 ' AA (A/G) TT (T/C) TT (T/C) AG (A/G) AA (A/G), and another amplimer of PCR is the Creator of CLONTECH company TMSMART TM3 ' PCR Primer primer among the cDNA LibraryConstruction Kit. its sequence is 5 ' ATTCTACAGGCCGAGGCGGCCGACATG 3 '.PCR reaction is carried out under the following conditions: 94 ℃ 2 minutes, 92 ℃ of 10 second, 50 ℃ of 30 second and 72 ℃ of 40 second, 30 circulations.Reclaim test kit (day root biology) with glue after amplification is finished and carry out the recovery of purpose fragment.The purpose fragment that reclaims is connected to pGEM R(Promega, Madison WI), transform CaCl to-T Easyvector 2-MgCl 2The DH5 α competent cell that method prepares.Coated plate also carries out penbritin and the dual screening of blue hickie, and 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 DNAsequencer, and model ABI PRISM 377 carries out nucleotide sequencing.
V, Bungarus fasciatus antibacterial peptide cathelicidin-BF gene sequencing and result:
The gene of coding Bungarus fasciatus antibacterial peptide cathelicidin-BF from 5 ' end to 3 ' terminal sequence is:
atggaagggt?tcttctggaa?gaccttgctg?gtggttggag?ctcttgccat?tgctgggacc?60
tcctcacttc?cacacaaacc?cctgatctat?gaagaggctg?tggaccttgc?agtgagcatc?120
tacaacagca?aatctgggga?agactctctc?taccgtctcc?tggaggctgt?ttctccaccc?180
aagtgggatc?ctctttctga?aagcaaccaa?gagctgaact?tcaccatgaa?ggagacggtg?240
tgcctggtgg?ccgaagaacg?atccttggag?gaatgcgact?tccaggaaga?cggggtcgtc?300
atgggatgca?caggctacta?tttcttcggg?gagtcgcccc?cggtggtcgt?tctcacctgc?360
aagcctgtgg?gtgaagaagg?ggagcagaag?caggaggagg?ggaacgagga?ggagaaggaa?420
gtggaggagg?aggaacagga?ggaagacgag?aaggatcagc?ccaggagggt?caagagattc?480
aagaaatttt?tcaggaagct?gaagaagagc?gtgaagaaac?gtgccaagga?attcttcaag?540
aagccgaggg?tcatcggggt?ctccatcccc?ttctaagaga?ggggctcaga?aggacctgcg?600
gcctccgctc?tccgatccca?ggaaaaccgg?cagagaagac?gatgcgggat?gctccagtcc?660
gtcaaatcat?ttcccaatag?gatgctccgc?cattcaatcc?atgaataaat?aaatatatac?720
ttgaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 750
The sequence table of Bungarus fasciatus antibacterial peptide cathelicidin-BF gene nucleotide is: sequence length is 750 bases, sequence type: nucleic acid, chain number: strand, topology: straight chain shape, sequence kind: cDNA, source: Gold-banded Krait poison gland.What encode Bungarus fasciatus antibacterial peptide cathelicidin-BF mature peptide is the 484-573 Nucleotide, and its aminoacid sequence is:
Lys 1?Phe 2?Phe 3?Arg 4?Lys 5?Leu 6?Lys 7?Lys 8?Ser 9?Val 10?Lys 11?Lys 12?Arg 13?Ala 14?Lys 15
Glu 16?Phe 17?Phe 18?Lys 19?Lys 20?Pro 21?Arg 22?Val 23?Ile 24?Gly 25?Val 26?Ser 27?Ile 28?Pro 29?Phe 30
Bungarus fasciatus antibacterial peptide cathelicidin-BF gene prepares the application of Bungarus fasciatus antibacterial peptide cathelicidin-BF as genetically engineered.
Bungarus fasciatus antibacterial peptide cathel icidin-BF separation and purification:
I, Sephadex G-50 gel permeation chromatography:
The thick poison of the freeze dried Gold-banded Krait of 0.4g is dissolved in 50mM Tris-HCl, 50mM NaCl (pH 7.8) damping fluid, centrifugal 10 minutes of 12000rpm gets supernatant and is splined on the Sephadex G-50 gel-filtration column that balance is good (26cm * 100cm), use same buffer solution elution, and collect with automatic Fraction Collector, flow velocity is 2.8mL/ pipe/10min, the concentration of albumen or polypeptide in 280nm ultraviolet detection collection liquid, and merging has the part of anti-microbial activity, freeze-drying ,-20 ℃ of preservations are standby.
II, CM-Sephadex C-25 cation-exchange chromatography:
To go up the active peak of step gained sample dialyses with phosphoric acid salt (pH 6.0) damping fluid, dialysis back sample is splined on cationic exchange coloum CM-Sephadex C-25, and (1.6cm * 30cm), NaCl linear gradient 1ml/min wash-out is collected the peak with anti-microbial activity, freeze-drying ,-20 ℃ of preservations are standby.
III, reverse phase HPLC (RP-HPLC):
The resulting active peak of CM-Sephadex C-25 cation-exchange chromatography is dissolved again with ultrapure water, and 4 ℃, centrifugal 15 minutes of 12000rpm gets supernatant liquor, with 0.45 μ m membrane filtration, collects filtrate and is splined on anti-phase high-pressure liquid phase C 4Post, with water (containing 0.1% trifluoroacetic acid): the elution system that acetonitrile (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient elution, and elution speed is 0.7ml/min.Collection has the peak of anti-microbial activity, freeze-drying ,-20 ℃ of preservations.The Edman edman degradation Edman carries out N-end order-checking (model 491, ABI, the U.S.) to the pure product of the resulting antibacterial peptide of purifying.Electrospray ionization mass spectrometry (ESI-MS) is measured the antibacterial peptide molecular weight.
The chemosynthesis of Bungarus fasciatus antibacterial peptide cathelicidin-BF:
The chemical synthesis process of I, Bungarus fasciatus antibacterial peptide cathelicidin-BF: the aminoacid sequence that records according to the Edman edman degradation Edman, with automatic Peptide synthesizer (433A, Applied Biosystems) synthetic its complete sequence is 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).
Bungarus fasciatus antibacterial peptide cathelicidin-the BF of III, purifying identifies its purity with the high-efficient liquid phase chromatogram HPLC method, molecular weight determination adopts ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF), isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
Bungarus fasciatus antibacterial peptide cathelicidin-BF is a kind of straight-chain polypeptide of Gold-banded Krait cathelicidin genes encoding, contains 30 amino-acid residues, molecular weight 3637.54Da, iso-electric point 11.79.Bungarus fasciatus antibacterial peptide cathelicidin-BF total order is classified as: Methionin-phenylalanine-phenylalanine-arginine-Methionin-leucine-Methionin-Methionin-Serine-Xie Ansuan-Methionin-Methionin-arginine-L-Ala-Methionin-L-glutamic acid-phenylalanine-phenylalanine-Methionin-Methionin-proline(Pro)-arginine-Xie Ansuan-Isoleucine-glycine-Xie Ansuan-Serine-Isoleucine-proline(Pro)-phenylalanine.
The pharmacological evaluation of Bungarus fasciatus antibacterial peptide cathelicidin-BF:
1. Bungarus fasciatus antibacterial peptide cathelicidin-BF anti-microbial activity detects:
The test strain that picking is stored on the inclined-plane is evenly coated on the MH solid medium flat board, to place media surface through the filter paper of 0.5cm diameter of sterilization, drip certain density Bungarus fasciatus antibacterial peptide cathelicidin-BF sample solution 10 μ l, cultivated 18-20 hours in 37 ℃ of inversions, observe inhibition zone and whether form.
2. Bungarus fasciatus antibacterial peptide cathelicidin-BF minimal inhibitory concentration (Minimum InhibitoryConcentration) is measured:
Test strain is inoculated in the MH liquid nutrient medium, and 37 ℃ of shaking culture are diluted to 2 * 10 to logarithmic phase with fresh MH liquid nutrient medium 5Cfu/ml.
The certain density Gold-banded Krait cathelicidin-BF antibacterial peptide 0.1mL that adds in the 1.9mL substratum through 0.22 μ m aperture membrane filtration manages as first, get 1mL behind the mixing and add the 2nd pipe, doubling dilution discards from the 9th pipe sucking-off 1mL successively, the 10th piping control tube, as showing:
Table. dilution process
In each pipe, add and diluted good bacterium liquid 0.05mL, placed 37 ℃ of slow shaking culture 18 hours behind the mixing, measure photoabsorption in 600nm wavelength place.Minimal inhibitory concentration is to cannot see the minimum sample concentration of bacterial growth.The result is as shown in table 1.
By table 1 as seen, Bungarus fasciatus antibacterial peptide cathelicidin-BF has very strong anti-microbial activity to most test strains, has the characteristics of broad-spectrum antimicrobial.In 40 kinds of test strains, cathelicidin-BF has extremely strong germicidal action to most of gram negative bacteriums, its minimal inhibitory concentration some less than corresponding positive control (penicillin, penbritin, imipenum), the MIC to Klebsiella Pneumoniae 08040724 reaches 0.3 μ g/ml especially.And it is not obvious to most of gram positive bacterium effects.In addition, cathelicidin-BF also has very strong anti-mycotic activity, and the minimal inhibitory concentration of Candida albicans ATCC2002 and pichia spp is reached 4.7 μ g/ml and 0.3 μ g/ml respectively.
Table 1.cathelicidin-BF and the contrast of microbiotic anti-microbial activity
Figure A200810058976D00121
Figure A200810058976D00131
MIC: minimal inhibitory concentration, BF:cathlicidin-BF, BF-15:cathlicidin-BF15, Amp: penbritin, Ben: injection penicillin is received, ICS: imipenem for injection-cilastatin sodium, ND: do not detect activity,-: not experiment, IS: clinical separation strain, DR: penbritin and penicillin Resistant strain.Above result is three independent repeated experiments mean values.
3. Bungarus fasciatus antibacterial peptide cathelicidin-BF salt-dependent is measured:
Bungarus fasciatus antibacterial peptide cathelicidin-BF is dissolved in the sterilization deionized water respectively, and 150mM PBS and 150mM NaCl are mixed with the solution of same concentrations, detect the MIC difference of each sample to the different tests bacterial strain, and the result is as shown in table 2.
Cathelicidin-BF anti-microbial activity in table 2. different solutions
MIC: minimal inhibitory concentration.Above result is three independent repeated experiments mean values.
By table 2 as seen, compare with aqua sterilisa, cathelicidin-BF has stronger anti-microbial activity in 150mM PBS and 150mM NaCl.The existence that this shows the anti-microbial activity of cathelicidin-BF and does not rely on salt, but certain density salt can improve its activity.
4. Bungarus fasciatus antibacterial peptide cathelicidin-BF sterilization kinetic determination:
The test bacterial strain uses therefor is clinical separation strain intestinal bacteria 08A866, and imipenem for injection-cilastatin sodium is made positive control.Intestinal bacteria 08A866 is inoculated in the MH liquid nutrient medium, and 37 ℃ of shaking culture are diluted to 10 to logarithmic phase with fresh MH liquid nutrient medium 6-10 7CFU/ml.Cathelicidin-BF and imipenem for injection-cilastatin sodium is added the good bacterium liquid of dilution, make final concentration be 1 *, 5 * and 10 * MIC, 37 ℃, the 150rpm shaking culture was got bacterium liquid in 0,0.1,1,3 and 6 hour, dilution 10 3-10 4Doubly, get 50 μ l and be coated with MH flat board, enumeration.The result is as shown in table 3.
Table 3.cathelicidin-BF is to intestinal bacteria sterilization dynamic experiment result
Figure A200810058976D00141
BF:cathlicidin-BF, CFU: colony-forming unit, ICS: imipenem for injection-cilastatin sodium, * 1, * 5 and * 10:1,5 and 10 times of MIC.
By table 3, cathelicidin-BF plays a role extremely rapid, and concentration is 1 *, 5 * and during 10 * MIC, within 1 minute, can kill all bacteriums.By comparison, imipenem for injection-cilastatin sodium speed of action is slow, still has bacteria living when acting on 6 hours, and only just can kill all bacteriums under 10 * MIC concentration.
Bungarus fasciatus antibacterial peptide cathelicidin-BF and gene thereof and application _ ST25
SEQUENCE?LISTING
＜110〉Kunming Institute of Zoology, Chinese Academy of Sciences
＜120〉Bungarus fasciatus antibacterial peptide cathelicidin-BF and gene thereof and application
<130>1
<160>2
<170>PatentIn?version?3.4
<210>1
<211>750
<212>DNA
<213>Bungarus?fasciatus
<400>1
Figure A200810058976D00151
<210>2
<211>30
<212>PRT
<213>Bungarus?fasciatus
<400>2
Figure A200810058976D00152

Claims (3)

1, Bungarus fasciatus antibacterial peptide cathelicidin-BF, it is characterized in that Bungarus fasciatus antibacterial peptide cathelicidin-BF is a kind of straight-chain polypeptide of Gold-banded Krait cathelicidin genes encoding, contain 30 amino-acid residues, molecular weight 3637.54Da, iso-electric point 11.79; Bungarus fasciatus antibacterial peptide cathelicidin-BF total order is classified as: Methionin-phenylalanine-phenylalanine-arginine-Methionin-leucine-Methionin-Methionin-Serine-Xie Ansuan-Methionin-Methionin-arginine-L-Ala-Methionin-L-glutamic acid-phenylalanine-phenylalanine-Methionin-Methionin-proline(Pro)-arginine-Xie Ansuan-Isoleucine-glycine-Xie Ansuan-Serine-Isoleucine-proline(Pro)-phenylalanine.
2, the gene of Bungarus fasciatus antibacterial peptide cathelicidin-BF is characterized in that Bungarus fasciatus antibacterial peptide cathelicidin-BF gene is made up of 750 Nucleotide, from 5 ' end to 3 ' terminal sequence is:
atggaagggt?tcttctggaa?gaccttgctg?gtggttggag?ctcttgccat?tgctgggacc 60
tcctcacttc?cacacaaacc?cctgatctat?gaagaggctg?tggaccttgc?agtgagcatc 120
tacaacagca?aatctgggga?agactctctc?taccgtctcc?tggaggctgt?ttctccaccc 180
aagtgggatc?ctctttctga?aagcaaccaa?gagctgaact?tcaccatgaa?ggagacggtg 240
tgcctggtgg?ccgaagaacg?atccttggag?gaatgcgact?tccaggaaga?cggggtcgtc 300
atgggatgca?caggctacta?tttcttcggg?gagtcgcccc?cggtggtcgt?tctcacctgc 360
aagcctgtgg?gtgaagaagg?ggagcagaag?caggaggagg?ggaacgagga?ggagaaggaa 420
gtggaggagg?aggaacagga?ggaagacgag?aaggatcagc?ccaggagggt?caagagattc 480
aagaaatttt?tcaggaagct?gaagaagagc?gtgaagaaac?gtgccaagga?attcttcaag 540
aagccgaggg?tcatcggggt?ctccatcccc?ttctaagaga?ggggctcaga?aggacctgcg 600
gcctccgctc?tccgatccca?ggaaaaccgg?cagagaagac?gatgcgggat?gctccagtcc 660
gtcaaatcat?ttcccaatag?gatgctccgc?cattcaatcc?atgaataaat?aaatatatac 720
ttgaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 750
What encode Bungarus fasciatus antibacterial peptide cathelicidin-BF mature peptide is the 484-573 Nucleotide, and its aminoacid sequence is: Lys 1Phe 2Phe 3Arg 4Lys 5Leu 6Lys 7Lys 8Ser 9Val 10Lys 11Lys 12Arg 13Ala 14Lys 15Glu 16Phe 17Phe 18Lys 19Lys 20Pro 21Arg 22Val 23Ile 24Gly 25Val 26Ser 27Ile 28Pro 29Phe 30
3, Bungarus fasciatus antibacterial peptide cathelicidin-BF gene prepares the application of Bungarus fasciatus antibacterial peptide cathelicidin-BF as genetically engineered.
CNA2008100589769A 2008-09-27 2008-09-27 Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof Pending CN101412753A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNA2008100589769A CN101412753A (en) 2008-09-27 2008-09-27 Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNA2008100589769A CN101412753A (en) 2008-09-27 2008-09-27 Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof

Publications (1)

Publication Number Publication Date
CN101412753A true CN101412753A (en) 2009-04-22

Family

ID=40593515

Family Applications (1)

Application Number Title Priority Date Filing Date
CNA2008100589769A Pending CN101412753A (en) 2008-09-27 2008-09-27 Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof

Country Status (1)

Country Link
CN (1) CN101412753A (en)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101845092A (en) * 2010-04-29 2010-09-29 大连理工大学 Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof
CN102115496A (en) * 2010-11-03 2011-07-06 大连理工大学 Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof
CN102294019A (en) * 2011-09-07 2011-12-28 中国科学院昆明动物研究所 Application of cathelicidin-BF
CN102702333A (en) * 2012-05-29 2012-10-03 中国药科大学 Drug-resistant pathogen infection resistant polypeptide and uses thereof
CN103127493A (en) * 2013-02-20 2013-06-05 中国药科大学 Purpose of polypeptide Cbf-K16 anti-tumor drugs
CN103665111A (en) * 2012-09-07 2014-03-26 山东国际生物科技园发展有限公司 Transformed body HC-15 of antimicrobial peptide Hc-CATH (cathelicidins) of hydrophis cyanocinctus and preparation method and application of transformed body
CN104497119A (en) * 2013-12-09 2015-04-08 王义鹏 Natural antibacterial peptide and application thereof
CN104630213A (en) * 2014-12-22 2015-05-20 中国农业大学 Primer system and applications thereof
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
CN104830891A (en) * 2015-03-23 2015-08-12 广东药学院 An efficient preparing method of a banded krait antibacterial peptide Cath-BF30
CN104945494A (en) * 2015-07-08 2015-09-30 王爱丽 Sinonatrix annularis source antibacterial peptide SAC and application
CN104987377A (en) * 2015-06-19 2015-10-21 苏州大学 Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof
CN105012936A (en) * 2015-07-30 2015-11-04 中国药科大学 Application of antibacterial peptide BF-30 in preparation of medicines for treating or preventing thrombotic diseases
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
CN105273074A (en) * 2014-05-30 2016-01-27 山东国际生物科技园发展有限公司 Hydrophis cyanocinctus reforming body antibacterial peptide QHA3, preparation method and applications thereof
CN105273073A (en) * 2014-05-30 2016-01-27 山东国际生物科技园发展有限公司 Sea snake reforming body antibacterial peptide QHA2, preparation method and applications thereof
EP3255059A4 (en) * 2015-02-06 2017-12-13 Zhejiang University Antimicrobial peptide wy-21 and use thereof
CN109395060A (en) * 2018-11-05 2019-03-01 苏州大学 A kind of application of inherent immunity activating peptide
CN113940895A (en) * 2020-07-15 2022-01-18 南京星银药业集团有限公司 Antibacterial peptide hand sanitizer and preparation method thereof
CN115947814A (en) * 2022-11-25 2023-04-11 青岛农业大学 Antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101845092A (en) * 2010-04-29 2010-09-29 大连理工大学 Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof
CN102115496A (en) * 2010-11-03 2011-07-06 大连理工大学 Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof
CN102294019A (en) * 2011-09-07 2011-12-28 中国科学院昆明动物研究所 Application of cathelicidin-BF
CN102702333A (en) * 2012-05-29 2012-10-03 中国药科大学 Drug-resistant pathogen infection resistant polypeptide and uses thereof
CN103665111A (en) * 2012-09-07 2014-03-26 山东国际生物科技园发展有限公司 Transformed body HC-15 of antimicrobial peptide Hc-CATH (cathelicidins) of hydrophis cyanocinctus and preparation method and application of transformed body
CN103665111B (en) * 2012-09-07 2016-02-24 山东国际生物科技园发展有限公司 Variant HC-15 of a kind of chittue antibacterial peptide Hc-CATH and its preparation method and application
CN103127493A (en) * 2013-02-20 2013-06-05 中国药科大学 Purpose of polypeptide Cbf-K16 anti-tumor drugs
CN103127493B (en) * 2013-02-20 2014-10-29 中国药科大学 Purpose of polypeptide Cbf-K16 anti-tumor drugs
CN104497119A (en) * 2013-12-09 2015-04-08 王义鹏 Natural antibacterial peptide and application thereof
CN104497119B (en) * 2013-12-09 2017-11-03 王义鹏 A kind of natural antibacterial peptide and its application
CN105273074A (en) * 2014-05-30 2016-01-27 山东国际生物科技园发展有限公司 Hydrophis cyanocinctus reforming body antibacterial peptide QHA3, preparation method and applications thereof
CN105273073B (en) * 2014-05-30 2018-12-18 山东国际生物科技园发展有限公司 A kind of sea snake variant antibacterial peptide QHA2 and its preparation method and application
CN105273074B (en) * 2014-05-30 2018-12-18 山东国际生物科技园发展有限公司 A kind of sea snake variant antibacterial peptide QHA3 and its preparation method and application
CN105273073A (en) * 2014-05-30 2016-01-27 山东国际生物科技园发展有限公司 Sea snake reforming body antibacterial peptide QHA2, preparation method and applications thereof
CN104630213A (en) * 2014-12-22 2015-05-20 中国农业大学 Primer system and applications thereof
EP3255059A4 (en) * 2015-02-06 2017-12-13 Zhejiang University Antimicrobial peptide wy-21 and use thereof
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
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
CN104830891A (en) * 2015-03-23 2015-08-12 广东药学院 An efficient preparing method of a banded krait antibacterial peptide Cath-BF30
CN104987377A (en) * 2015-06-19 2015-10-21 苏州大学 Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof
CN104945494B (en) * 2015-07-08 2018-04-27 潍坊科技学院 A kind of Sinonatrix annularis source antibacterial polypeptide SAC and application
CN104945494A (en) * 2015-07-08 2015-09-30 王爱丽 Sinonatrix annularis source antibacterial peptide SAC and application
CN105012936A (en) * 2015-07-30 2015-11-04 中国药科大学 Application of antibacterial peptide BF-30 in preparation of medicines for treating or preventing thrombotic diseases
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
CN109395060A (en) * 2018-11-05 2019-03-01 苏州大学 A kind of application of inherent immunity activating peptide
CN113940895A (en) * 2020-07-15 2022-01-18 南京星银药业集团有限公司 Antibacterial peptide hand sanitizer and preparation method thereof
CN115947814A (en) * 2022-11-25 2023-04-11 青岛农业大学 Antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris

Similar Documents

Publication Publication Date Title
CN101412753A (en) Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof
JP4037525B2 (en) New antibacterial peptide
CN101386640A (en) Variant cathelicidin-BF15 of cathelicidin-BF and use thereof
CN106188265B (en) A kind of antimicrobial peptide Cm-CATH2 and its gene, preparation method and application
CN103665111B (en) Variant HC-15 of a kind of chittue antibacterial peptide Hc-CATH and its preparation method and application
CN101168563A (en) Anticancer active peptide
CN106749595B (en) Tree frog defense peptides cathelicidin-PP and its gene and application
CN104497119A (en) Natural antibacterial peptide and application thereof
CN108484747A (en) Japan frog skin repair peptide cathelicidin-NV and its gene and application
CN110156875B (en) Antibacterial peptide H5-p5, and preparation method and application thereof
CN104031135B (en) Rhacophorus defense peptides PopuDef and gene thereof and application
CN104761629B (en) A kind of broad-spectrum high efficacy antimicrobial peptide Pb CATH OH1 and its gene, preparation method and application
CN102250217A (en) Hyla simplex skin injury repair promotion polypeptides and genes as well as application thereof
CN102516382B (en) Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis
CN102796176B (en) Kunyuenin, preparation method and application
CN101724632B (en) Bullfrog skin active peptide, gene and application thereof in pharmacy
CN101058602A (en) Rana grahami boulenger antibiotic peptide, preparation method and application thereof
CN104910265B (en) A kind of gene and the application of Hejiang spine frog antibacterial peptide and its coded sequence
CN101845092A (en) Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof
CN104356216B (en) Version receives rope buffalo gnat defense peptides SibaDef and its gene and application
CN102115496A (en) Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof
CN103172721B (en) Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof
WO2022104863A1 (en) Antibacterial peptide derived from taihu white fish and application thereof
CN1142181C (en) Giant web bombinator polypeptide and its prepn. and pharmaceutical application
CN110627891B (en) Method for concealing hemolytic toxic and side effects of bee venom hemolytic peptide

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Open date: 20090422