CN103755797A

CN103755797A - Antimicrobial peptide BG-CATH6 (5-29) of Bufo bufo gargarizans as well as encoding genes and application of antimicrobial peptide BG-CATH6 (5-29) of Bufo bufo gargarizans

Info

Publication number: CN103755797A
Application number: CN201410034879.1A
Authority: CN
Inventors: 孙同毅; 李文辉
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2014-01-24
Filing date: 2014-01-24
Publication date: 2014-04-30
Anticipated expiration: 2034-01-24
Also published as: CN103755797B

Abstract

The invention provides antimicrobial peptides BG-CATH6 (5-29) of Bufo bufo gargarizans as well as encoding genes and application of the antimicrobial peptide BG-CATH6 (5-29) of Bufo bufo gargarizans. The antimicrobial peptide has amino acid sequences shown in SEQ ID NO: 3. The antimicrobial peptide BG-CATH6 (5-29) of the Bufo bufo gargarizan has a remarkable anti-tumor effect and has the advantages of simple structure, convenient artificial synthesis and wide antibacterial spectrum. The antimicrobial peptide can be applied to the preparation of anti-microbial infection drugs and anti-tumor drugs and is used for the treatment of people and animals.

Description

Bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) and encoding gene and application

Technical field

The invention belongs to biomedical sector, be specifically related to bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) and encoding gene and application.

Background technology

Bufo gargarizans Cantor is Anura Bufo animal, is distributed widely in China's most area, is the main base source animal of the traditional Chinese medicine dried venom of toads and toad skin, has and has detoxicating and resolving a mass, the effect of the Ji Lishui that disappears, destroying parasites for curing malnutrition.From the dry skin of bufo gargarizans Cantor Bufo bufo gargarizans Cantor, extract the refining water soluble preparation-cinobufagin forming, be widely used at present multiple middle and advanced stage tumour, as primary hepatocarcinoma, cancer of the stomach, lung cancer, colorectal carcinoma, carcinoma of the pancreas etc.

Antibacterial peptide (Cathelicidin) is the changeable antimicrobial peptide of a class formation of mainly finding in mammalian body, because of the cathelin peptide section family of having a style of one's own of containing a high conservative between the signal peptide expressing and mature peptide, ripe antibacterial peptide is positioned at the C-end of precursor molecule, has very high variability.Cathelicidin is the same with alexin, is also the important component part of host immune system of defense, have wide spectrum antimicrobial acivity, antitumor, in conjunction with several functions such as intracellular toxin, immunomodulatory, the healing of participation wound and the generations of blood vessel.

Summary of the invention

A kind of bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) and encoding gene and application have been the object of this invention is to provide.The antibacterial peptide BG-CATH6 (5-29) of Reptilia bufo gargarizans Cantor has the effect of significant inhibition tumor cell, and has beneficial features simple in structure, that synthetic convenient, antibacterial pedigree is wide.Can be applied to the preparation of antitumor cell preparation, for the treatment of humans and animals.

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

Bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29), it has the aminoacid sequence shown in SEQ ID NO:3.

Described antibacterial peptide BG-CATH6 (5-29) contains a pair of disulfide linkage, by Cys9 and Cys15, is formed.

Contain described bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29)) amyloid protein precursor, it has the aminoacid sequence shown in SEQ ID NO:2.

The encoding gene of the amyloid protein precursor of described bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29), it has the nucleotide sequence shown in SEQ ID NO:1.

Use T7 promoter sequence and the screening of SP6 promoter sequence to obtain described encoding gene,

Described T7 promoter sequence is 5 '-TAATACGACTCTATAGGGA-3 ';

Described SP6 promoter sequence 5 '-CATACGATTTAGGTGACACTATAG-3 '.

The invention provides described bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) in the application for the preparation of in anti-tumor drug.

Described antibacterial peptide BG-CATH6 (5-29) is when 6.25ug/ml, remarkable to the inhibition of tumor cells of hepatocellular carcinoma.

Described antibacterial peptide BG-CATH6 (5-29) is when 100 μ g/ml, remarkable to the inhibition of breast cancer cell.

Described antibacterial peptide BG-CATH6 (5-29) is when 50 μ g/ml, remarkable to the inhibition of cranial nerve oncocyte.

Compared with prior art, advantage of the present invention and technique effect are: the present invention has obtained cathelicidin antibacterial peptide BG-CATH6 (5-29) in bufo gargarizans Cantor venom gland cDNA library.Member's high conservative of its signal peptide and cathelin peptide section and other cathelicidin family, and its mature peptide height variation.Bufo gargarizans Cantor cathelicidin antibacterial peptide has shown the inhibition tumor cell activity of wide spectrum.Cathelicidin antibacterial peptide BG-CATH6 (5-29) the complete sequence aminoacid sequence of bufo gargarizans Cantor of the present invention is searched relatively through Protein Data Bank, found no any identical cathelicidin family mature peptide.

Therefore, bufo gargarizans Cantor cathelicidin family's antibacterial peptide in Reptilia source and derivative thereof have the effect of significant inhibition tumor cell growth, and have wide range of applications.The beneficial features that this antibacterial peptide has is simple in structure, synthetic convenient, antibacterial pedigree is wide.Can be applied to the preparation of anti-microbial infection and anti-tumor drug, for the treatment of humans and animals.There is good market application foreground.

Accompanying drawing explanation

Fig. 1 is cDNA and the protein sequence thereof of the bufo gargarizans Cantor cathelicidin antibacterial peptide BG-CATH6 of family of the present invention (5-29), and wherein italic adds the mature sequence that frame sequence is the antibacterial peptide BG-CATH6 of bufo gargarizans Cantor cathelicidin family (5-29) of derivation.

Fig. 2 shows that antibacterial peptide BG-CATH6 of the present invention (5-29) is to liver cancer cell HepG2 effect.

Fig. 3 shows that antibacterial peptide BG-CATH6 of the present invention (5-29) is to breast cancer cell MCF-7 effect.

Fig. 4 shows that antibacterial peptide BG-CATH6 of the present invention (5-29) is to cranial nerve oncocyte Neuro-2a effect.

Embodiment

Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is further described in detail.

The gene clone of embodiment 1, bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29)

1, the extraction of total RNA

Bufo gargarizans Cantor is put in glass beaker, adds several ether, collects venom glandular secretion thing, puts into immediately liquid nitrogen.After weighing, get 20mg secretory product is ground to Powdered in liquid nitrogen.Add the total RNA Extraction buffer of 10ml (Trizol solution, U.S. GIBCO/BRL product), homogenate 30 minutes in 20ml glass homogenizer.Then add isopyknic phenol/chloroformic solution, acutely mix.Room temperature was placed after 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, abandons precipitation, and supernatant liquor adds isopyknic Virahol, and room temperature is placed 10 minutes.4 ℃, centrifugal 10 minutes of 12000rpm, precipitation is washed once with 75% ethanol, dries, and pipe end throw out is total RNA.

2, the separation of mRNA

Adopt the mRNA separation and purification test kit of U.S. Promega company.Get the total RNA500 g of bufo gargarizans Cantor poison gland and be dissolved in 500 l and process then in autoclaved water through DEPC, put into 65 ℃ of water-baths 10 minutes.Add the Oligo(dT of 3 l) probe and 13 l20 × SSC solution, mix, placement room temperature is cooling, is called A liquid.The washing of magnetic bead (SA-PMP): magnetic bead (with mRNA separation and purification test kit) is mixed gently, to magnetic frame absorption 30 seconds, abandon supernatant, add 0.5 × SSC0.3ml, to magnetic frame absorption 30 seconds, finally add 0.1ml0.5 × SSC and suspend, be called B liquid.A liquid is added in B liquid, and room temperature is placed 10 minutes, to magnetic frame absorption 30 seconds, abandons supernatant, with 0.1 × SSC washing 4 times.Then abandoning supernatant, add 0.1ml DEPC aqueous suspension, to magnetic frame absorption 30 seconds, supernatant is moved in new test tube, add 0.15ml DEPC water Eddy diffusion, to magnetic frame absorption 30 seconds, supernatant is moved to above-mentioned test tube, is the mRNA of purifying in supernatant.Add the 3M sodium acetate of 1/10 volume, pH5.2, equal-volume primary isoamyl alcohol, in-70 ℃ of placements 30 minutes, then in 4 ℃, centrifugal 10 minutes of 12000rpm, abandoned supernatant, and precipitation is dissolved in 10 μ l DEPC water.

3, cDNA library builds

Adopt the U.S. SuperScriptTM of GIBCO/BRL company Construction of Plasmid cDNA Library test kit.

3.1, cDNA the first chain synthetic (mRNA reverse transcription): add 2.0 μ l NotI primers and 7 μ l mRNA in 1.5ml test tube, 3 μ l DEPC water, 70 ℃ are incubated 10 minutes, put into immediately ice bath cooling, then add 4 μ l5 × the first chains to synthesize damping fluid, 2 μ l0.1M DTT, 1 μ l10mM dNTP mixture, add again 1 μ l SuperScript II ThermoScript II, in 42 ℃ of insulations, after 1 hour, put into ice bath.

3.2, cDNA the second chain is synthetic: in the synthetic test tube of the first chain, add: 95 μ l DEPC water, 30 μ l5 × the second chains synthesize damping fluid, 3 μ l10mM dNTP mixtures, 1 μ l e. coli dna ligase, 4 μ l e. coli dna polymerase I, 1 μ l e. coli rna enzyme, reaction cumulative volume 150 μ l, are incubated 2 hours in 16 ℃ after mixing; Add 2 μ l T4DNA polymerases to continue insulation 5 minutes.

3.3, the extracting of DNA and ethanol precipitation: add equal-volume phenol/chloroform/primary isoamyl alcohol (25/24/1) mixture extracting, centrifugal 5 minutes of 12000rpm, getting 140 μ l upper solution transfers in clean test tube, add 70 μ l7.5M ammonium acetates, 0.5ml dehydrated alcohol, centrifugal 20 minutes of 12000rpm, abandons supernatant, precipitation is washed once with 75% ethanol, dries.

3.4, the connection of Sal I adapter: above-mentioned precipitation is dissolved in 25 μ l DEPC water, adds 10 μ l5 × T4DNA ligase enzyme damping fluids, 10 μ l SalI adapter, and 5 μ l T4DNA ligase enzymes, reaction cumulative volume 50 μ l, in 16 ℃ of insulations 16 hours.Repeat extracting and the ethanol precipitation process of above-mentioned DNA, be precipitated and dissolved in 41 μ l DEPC water.

3.5, Not I enzymic hydrolysis: in cDNA solution, add 5 μ l React3 damping fluids, 4 μ l NotI enzymes, reaction volume 50 μ l, in 37 ℃ of insulations 2 hours.Repeat extracting and the ethanol precipitation process of above-mentioned DNA, be precipitated and dissolved in 100 μ l TEN damping fluids.

3.6, DNA fractional separation: cDNA sample is crossed after post (containing in test kit), removal is less than the cDNA of 300bp Nucleotide, and the component that cDNA is greater than 300bp merges, and volume is 200 μ l, add 5 μ l yeast tRNA, 100 μ l7.5M thanomins, 0.6ml dehydrated alcohol, centrifugal 20 minutes of 12000rpm, abandon supernatant, precipitation is washed once with 75% ethanol, dries, and precipitation is dissolved in 20 μ l TEN damping fluids.

3.7, synthetic cDNA is connected to pSPORT1 plasmid: get 10 μ l and be dissolved in the cDNA in TEN damping fluid, add 4 μ l5 × T4DNA ligase enzyme damping fluids, 1 μ l pSPORT1 plasmid (Not I-Sal I enzymic hydrolysis, 50ng), 4 μ l DEPC water, 1 μ l T4DNA ligase enzyme, reaction volume 20 μ l, room temperature 3 hours.

3.8, the preparation of Escherichia coli HB101 competent cell: the single HB101 bacterium colony of picking, be inoculated in 3ml not containing in the LB substratum of penbritin, 37 ℃ of overnight incubation, get next day above-mentioned bacterium liquid in proportion 1:100 be inoculated in 50ml LB nutrient solution, 37 ℃ vibration 2 hours, until bacterium liquid, in the OD of 540nm value, be 0.4 o'clock, 4 ℃, centrifugal 8 minutes of 2000rpm, abandons supernatant, precipitation 0.1M CaCl ₂resuspended, then with 2000rpm centrifugal 8 minutes, abandon supernatant, precipitation is with appropriate 0.1M CaCl ₂resuspended, standby in the rearmounted ice bath of packing.

3.9, connect the conversion of product: get above-mentioned connection product 5 μ l and add 100 μ l competent cells, ice bath 60 minutes, 42 ℃ of heat-shockeds 60 seconds, put again ice bath 5 minutes, add the SOC substratum 0.9ml without penbritin, 37 ℃ of shaking culture 1 hour, get 200 μ l and coat (15cm diameter) on the LB plate containing penbritin.Cultivate 16 hours for 37 ℃, 5ml LB liquid nutrient medium washing bacterium colony for each LB plate, adds constructed bufo gargarizans Cantor skin venom gland cDNA library that 10% glycerine preserves in liquid nitrogen approximately containing 1.8 × 10 ⁵individual clone.

4, the gene clone of bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29).

Universal primer T7 promoter sequence (5 '-TAATACGACTCTATAGGGA-3 ') (SEQ ID No:4) and the SP6 promoter sequence (5 '-CATACGATTTAGGTGACACTATAG-3 ') (SEQ ID No:5) that adopt cDNA library to build plasmid pSPORT1 are identified each clone's Insert Fragment size.By the order-checking of the clone to more than 2000 Insert Fragment >400bp, the sequence comparison of bind nucleic acid and protein, obtained a bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) encoding gene, its cDNA is comprised of 642 Nucleotide, from 5 ' end to 3 ' terminal sequence is (SEQ ID NO:1):

The antibacterial peptide precursor protein aminoacid sequence of its coding is (SEQ ID NO:2):

MRSWWLSLLLVSAVTLHGCLSDTAEPEVQDGRSVGDVIDLYNQREGVTYL 50

YKSLDQLPPVPMEEDENPNRRGFIIKETVCLKSENPDLTQCDFKPDGDVK 100

ICSLDLDDEDPEDIMCTSLNKNVRIKRNGKK KRKKPEKLCMKPGACSVIF 150

DASVNE

From the sequential analysis of above-mentioned precursor protein, according to the current knowledge to cathelicidin antibacterial peptide mature peptide Processing position, be that ripe antibacterial peptide is all generally to cut from basic aminoacids enrichment region, in conjunction with the analysis of the cathelin peptide section to high conservative between signal peptide and mature peptide, can inference in this precursor protein, have two kinds of basic cutting modes, one is that Lys131-Glu156 is the mature sequence (SEQ ID NO:3KRKKPEKLCMKPGAC SVIFDASVNE) of bufo gargarizans Cantor cathelicidin antibacterial peptide BG-CATH6 (5-29).The theoretical pI9.24 of BG-CATH6 (5-29), molecular weight Mw2779.33Da.

The preparation of embodiment 2, bufo gargarizans Cantor skin antibacterial peptide

1, the aminoacid sequence of deriving ripe antibacterial peptide according to the nucleotide sequence of coding bufo gargarizans Cantor skin antibacterial peptide cathelicidin antibacterial peptide, the aminoacid sequence of derivative is take this maturation antibacterial peptide as template, and integrated structure simulation determines.With full-automatic polypeptide synthetic instrument, synthesize respectively the complete sequence of their correspondences.And by a pair of to Cys9 and Cys15 cyclisation formation disulfide linkage.By the desalination of the anti-phase C18 column chromatography of HPLC, purifying.

2, molecular weight determination adopts fast atom bombardment mass spectroscopy(FABMS) method (Fast atom bombardment mass spectrometry, FAB-MS), with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1:1:1, V:V:V, volume ratio) be substrate, Cs+ is as projectile, and electric current is 1 μ A, and emitting voltage is 25Kv.

3, the bufo gargarizans Cantor antibacterial peptide of purifying is identified its purity by high-efficient liquid phase chromatogram HPLC method, and molecular weight determination adopts fast atom bombardment mass spectroscopy(FABMS) method, by full-automatic Protein Sequencer mensuration aminoacid sequence structure.

Title and the aminoacid sequence of synthesized bufo gargarizans Cantor antibacterial peptide are as follows: BG-CATH6 (5-29) (SEQ ID NO:1): KRKKPEKLCMKPGAC SVIFDASVNE (Lys Arg Lys Lys Pro Glu Lys Leu Cys Met Lys Pro Gly Ala Cys Ser Val Ile Phe Asp Ala Ser Val Asn Glu)

Embodiment 3

Liver cancer cell HepG2 is incubated at the RPMI-1640 substratum containing 10% foetal calf serum.Growth of Cells is conventional digestion during to logarithmic phase, suspends with the RPMI-1640 substratum containing 10% foetal calf serum, and counting cells density, adjusts cell density to 3 × 10 ⁴/ ml will.Cell is inoculated in 96 orifice plates (100 μ l/ hole), after adherent spending the night, adds antibacterial peptide BG-CATH6 (5-29) 6.25 μ g/ml respectively, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, and control wells and zeroing hole are set.Each dosage arranges 5 multiple holes, cultivates and after 48 hours, adds 20 μ l MTT(5g/L).37 degree, after 4 hours, add 150 μ l DMSO, and microplate reader detects absorbance A 490, and for referencial use with A630.

Experimental result as shown in Figure 2, relatively cell survival rate/%=experimental group A490/ control group A 490 × 100%.6 concentration are set: 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, the corresponding HepG2 cell survival rate of 200 μ g/ml is respectively 42.25%, 64.54%, 73.57%, 94.09%, 102.15%, 83.67%.Therefore, antibacterial peptide BG-CATH6 (5-29) is when 6.25ug/ml, remarkable to liver cancer cell HepG2 inhibition, IC50=4.80 μ g/ml.

Embodiment 4

Breast cancer cell MCF-7 is incubated at the DMEM high glucose medium containing 10% foetal calf serum.Growth of Cells is conventional digestion during to logarithmic phase, suspends with the DMEM high glucose medium containing 10% foetal calf serum, and counting cells density, adjusts cell density to 3 × 10 ⁴/ ml will.Cell is inoculated in 96 orifice plates (100 μ l/ hole), after adherent spending the night, adds antibacterial peptide BG-CATH6 (5-29) 3.125 μ g/ml respectively, 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, and control wells and zeroing hole are set.Each dosage arranges 5 multiple holes, cultivates and after 48 hours, adds 20 μ l MTT(5g/L).37 degree, after 4 hours, add 150 μ lDMSO, and microplate reader detects absorbance A 490, and for referencial use with A630.

Experimental result as shown in Figure 3, relatively cell survival rate/%=experimental group A490/ control group A 490 × 100%.6 concentration are set: 3.125 μ g/ml, 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, corresponding MCF-7 is relative, and cell survival rate is respectively 80.32%, 72.81%, and 61.99%, 55.68%, 51.98%, 50.38%.Therefore known, antibacterial peptide BG-CATH6 (5-29) is when 100 μ g/ml, remarkable to breast cancer cell MCF-7 inhibition, IC ₅₀=65.11 μ g/ml.

Embodiment 5

Cranial nerve oncocyte Neuro-2a is incubated at the DMEM high glucose medium containing 10% foetal calf serum.Growth of Cells is conventional digestion during to logarithmic phase, suspends with the DMEM high glucose medium containing 10% foetal calf serum, and counting cells density, adjusts cell density to 3 × 104/ml will.Cell is inoculated in 96 orifice plates (100 μ l/ hole), after adherent spending the night, adds antibacterial peptide BG-CATH6 (5-29) 3.125 μ g/ml respectively, 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, and control wells and zeroing hole are set.Each dosage arranges 5 multiple holes, cultivates and after 48 hours, adds 20 μ l MTT(5g/L).37 degree, after 4 hours, add 150 μ lDMSO, and microplate reader detects absorbance A 490, and for referencial use with A630.

Experimental result as shown in Figure 4, relatively cell survival rate/%=experimental group A490/ control group A 490 × 100%.6 concentration are set: 3.125 μ g/ml, 6.25 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, the relative cell survival rate of the corresponding Neuro-2a of 100 μ g/ml is respectively 108.57%, 92.50%, 81.72%, 78%, 66.83%, 71.24%.Therefore known, antibacterial peptide BG-CATH6 (5-29) is when 50 μ g/ml, remarkable to cranial nerve oncocyte Neuro-2a inhibition.

In sum, the present invention has obtained bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) by serial experiment, and prove that by anti-tumor experiment it has remarkable restraining effect to tumour cell, and have wide range of applications, can be applied to liver cancer cell, breast cancer cell and cranial nerve oncocyte etc., effect is remarkable, can be applied to the preparation of antitumor drug.

Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, the technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.

SEQUENCE LISTING

<110> Weifang Medical College

<120> bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29) and encoding gene and application

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 642

<212> DNA

<213> bufo gargarizans Cantor

<400> 1

agtggagtgc ggagaggaca tttcctctaa gacaggccaa gatgaggagc tggtggctgt 60

ctctgctgct cgtctctgct gtcacattac acggctgtct ctctgacact gcagagcctg 120

aggtccaaga tggaagatct gtaggagatg tcatcgacct ctacaaccag agggaggggg 180

tcacatactt atataaatcc ctggaccagc tgccccctgt tccaatggag gaagatgaaa 240

atccgaacag aagaggcttt atcattaaag agacggtgtg cctcaaatcc gagaatcctg 300

atttaaccca gtgtgatttc aagcccgacg gagatgtgaa gatctgttct ctggatttgg 360

atgatgagga tcctgaggac ataatgtgca ccagtctgaa caagaatgtc cgtatcaaaa 420

ggaacgggaa aaagaaacgt aagaagcctg aaaaattgtg tatgaaacca ggagcatgta 480

gtgtcatctt tgatgcgtcc gtcaatgagt gaaaccatct gtcctgtttg tagcaaagtc 540

agagacgtag cagagctaga gacgtagagc gcccacatca tgtaaattct gtttaagtaa 600

tgattcggcc tgatatccat aaataaaatg ctcatatcaa ac 642

<210> 2

<211> 156

<212> PRT

<213> bufo gargarizans Cantor

<400> 2

Met Arg Ser Trp Trp Leu Ser Leu Leu Leu Val Ser Ala Val Thr Leu

1 5 10 15

His Gly Cys Leu Ser Asp Thr Ala Glu Pro Glu Val Gln Asp Gly Arg

20 25 30

Ser Val Gly Asp Val Ile Asp Leu Tyr Asn Gln Arg Glu Gly Val Thr

35 40 45

Tyr Leu Tyr Lys Ser Leu Asp Gln Leu Pro Pro Val Pro Met Glu Glu

50 55 60

Asp Glu Asn Pro Asn Arg Arg Gly Phe Ile Ile Lys Glu Thr Val Cys

65 70 75 80

Leu Lys Ser Glu Asn Pro Asp Leu Thr Gln Cys Asp Phe Lys Pro Asp

85 90 95

Gly Asp Val Lys Ile Cys Ser Leu Asp Leu Asp Asp Glu Asp Pro Glu

100 105 110

Asp Ile Met Cys Thr Ser Leu Asn Lys Asn Val Arg Ile Lys Arg Asn

115 120 125

Gly Lys Lys Lys Arg Lys Lys Pro Glu Lys Leu Cys Met Lys Pro Gly

130 135 140

Ala Cys Ser Val Ile Phe Asp Ala Ser Val Asn Glu

145 150 155

<210> 3

<211> 25

<212> PRT

<213> artificial sequence

<400> 3

Lys Arg Lys Lys Pro Glu Lys Leu Cys Met Lys Pro Gly Ala Cys Ser

1 5 10 15

Val Ile Phe Asp Ala Ser Val Asn Glu

20 25

<210> 4

<211> 19

<212> DNA

<213> artificial sequence

<400> 4

taatacgact ctataggga 19

<210> 5

<211> 24

<212> DNA

<213> artificial sequence

<400> 5

catacgattt aggtgacact atag 24

Claims

1. bufo gargarizans Cantor antibacterial peptide BG-CATH6 (5-29), it has the aminoacid sequence shown in SEQ ID NO:3.

2. bufo gargarizans Cantor antibacterial peptide BG-CATH6 according to claim 1 (5-29), is characterized in that described antibacterial peptide BG-CATH6 (5-29) contains a pair of disulfide linkage, is formed by Cys9 and Cys15.

3. contain bufo gargarizans Cantor antibacterial peptide BG-CATH6 claimed in claim 1 (5-29)) amyloid protein precursor, it has the aminoacid sequence shown in SEQ ID NO:2.

4. the encoding gene of the amyloid protein precursor of bufo gargarizans Cantor antibacterial peptide BG-CATH6 claimed in claim 3 (5-29), it has the nucleotide sequence shown in SEQ ID NO:1.

5. the encoding gene of the amyloid protein precursor of bufo gargarizans Cantor antibacterial peptide BG-CATH6 according to claim 4 (5-29), is characterized in that: use T7 promoter sequence and the screening of SP6 promoter sequence to obtain described encoding gene,

Described T7 promoter sequence is 5 '-TAATACGACTCTATAGGGA-3 ';

Described SP6 promoter sequence 5 '-CATACGATTTAGGTGACACTATAG-3 '.

6. bufo gargarizans Cantor antibacterial peptide BG-CATH6 claimed in claim 1 (5-29) is in the application for the preparation of in anti-tumor drug.

7. bufo gargarizans Cantor antibacterial peptide BG-CATH6 according to claim 6 (5-29) is in the application for the preparation of in anti-tumor drug, it is characterized in that: described antibacterial peptide BG-CATH6 (5-29) is when 6.25 ug/ml, remarkable to the inhibition of tumor cells of hepatocellular carcinoma.

8. bufo gargarizans Cantor antibacterial peptide BG-CATH6 according to claim 6 (5-29) is in the application for the preparation of in anti-tumor drug, it is characterized in that: described antibacterial peptide BG-CATH6 (5-29) is when 100 μ g/ml, remarkable to the inhibition of breast cancer cell.

9. bufo gargarizans Cantor antibacterial peptide BG-CATH6 according to claim 6 (5-29) is in the application for the preparation of in anti-tumor drug, it is characterized in that: described antibacterial peptide BG-CATH6 (5-29) is when 50 μ g/ml, remarkable to the inhibition of cranial nerve oncocyte.