CN102391370B - Antimicrobial peptides, preparation method and application thereof - Google Patents

Abstract

The invention provides a new group of antimicrobial peptides, which have stronger antimicrobial activity than natural antimicrobial peptides and have good killing effect on various pathogenic bacteria. The invention also discloses a preparation method of the group of antimicrobial peptides, which can be synthesized by a solid phase chemical method and also can be obtained through genetic engineering expression. The synthesized antimicrobial peptides of the invention can be used for preparing medicaments for treating diseases caused by infection of Gram-positive bacteria, Gram-negative bacteria or fungi.

Description

A kind of antibacterial peptide and its preparation method and application

The application is that application number is 200910223409.9, the applying date be November 16, denomination of invention in 2004 be one group of new antibacterial peptide and its preparation method and application divide an application divide an application again.

Technical field

The present invention relates to a kind of antibacterial peptide, its preparation method and the application in preparation treatment bacterium, fungi, medicine for treating viral infections.

Background technology

Antibacterial peptide is the micromolecule polypeptide of a kind of biologically active of organism through inducing generation, generally is made up of 20-60 amino acid, and molecular weight is about 2000-7000D.Along with Medical Immunology and molecular biological developing rapidly, the research of antibacterial peptide more and more becomes the heat subject in biotechnology and the biomedicine field.Up to now, found to surpass more than 200 kind of antibacterial peptide on many animals (especially insect), plant, microorganism and human body, this class small peptide not only has the germicidal action of wide spectrum to bacterium, fungi, and virus, protozoon and cancer cells are also had attack function.Clinical trial also shows, maybe may cause under the situation of courses of infection the organism infection germ, and antibiotic Toplink is killed the germ of having invaded fast, and can stop germ continue infect.

Along with to the deepening continuously of antibacterial peptide primary structure and higher structure research, existing many investigators study the structure and function of these antibacterial peptides, find that alpha-helix degree and its fungicidal activity in the molecule are closely related under the hydrophobic environment of analogue membrane.Result of study shows that antibacterial peptide is to make bacterial cell membrane seepage and killing bacteria (NakajimaY.etal., J.Biol.Chem, 262:1665-1669 by the integrity of destroying film in addition; ZasloffM.Nature, 2002,415:389-395).Therefore there is the people to attempt by α-Luo Xuanjiegou in the increase molecule or improve in the polypeptide to contain polypeptide (the Broth W.B.etal. that the amino acid whose ratio of positive charge is sought stronger anti-microbial activity, Antimicrobial Agents Chemotherapy, 2001,45:1894-1895; HongS.Y.etal., Peptides, 2001,22:1669-1674).

United States Patent (USP) 6,316,594 disclose natural antibacterial peptide parasinI, it is a kind of new separating from catfish, be to be that its epithelium mucous layer of invasion that prevents microorganism produces a kind of polypeptide with very strong anti-microbial effect by catfish when the epidermis injury, belong to the alpha-helix antibacterial peptide, molecular weight is 2000.4Da, be made up of 19 amino-acid residues, sequence is: Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr Arg Ser Ser.Although the microorganism of Parasin I in the patent documentation shows very effective broad spectrum antibiotic activity, comprise gram-positive and gram-negative bacteria and fungi, and without any hemolytic.Its synthetic derivative also has effective activity.But find that but its fungicidal activity is lower in our experiment, native sequences is difficult to be applied to clinical, but Parasin I does not have hemolytic substantially, and it is less that the sequence transformation that it some are relevant and the exploitation of function application facet are also carried out.

So the present invention wants by the transformation to Parasin I sequence, obtains the stronger antibacterial peptide that has medicinal exploitation to be worth of fungicidal activity, namely can form new patented technology.

Summary of the invention

One of technical issues that need to address of the present invention provide one group of antibacterial peptide.

Two of the technical issues that need to address of the present invention provide the preparation method of one group of antibacterial peptide.

Three of the technical issues that need to address of the present invention are the application that disclose described antibacterial peptide.

Inventive concept of the present invention is such: we are through after finding to insert at interval hydrophobic amino acid and positively charged amino acid after the test of many times in Parasin I sequence, form two hydrophobic amino acids and two sequences that positive charge amino acid is alternately arranged, such structure reduced toxicity also improves fungicidal activity.Screen following 12 sequences after transforming, pressing down bactericidal assay proves that its fungicidal activity is greatly improved.And hemolytic toxicity does not have to change substantially, is hopeful very much to be used for the treatment of the exploitation of the new drug of infectation of bacteria.

Antibacterial peptide provided by the invention is synthetic in sequence, basis of structural analysis design to natural antibacterial peptide, and a series of sequences of they this are named as GKV, and sequence is as follows:

GKV-1：Lys?Gly?Ala?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Gly?Lys?Lys?Val?Ala?Arg?Lys?Ala?Leu?Lys?Arg?Ala?Gly?Lys

GKV-2：Lys?Gly?Gly?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Gly?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys?Arg?Ala?Gly?Arg

GKV-3：Lys?Gly?Gly?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Gly?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys

GKV-4：Lys?Gly?Val?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Cys?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys

GKV-5：Lys?Gly?Ala?Arg?Arg?Gly?Ala?Lys?Arg?Gly?Gly?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys

GKV-6：Lys?Gly?Ala?Arg?Arg?Leu?Ala?Lys?Arg?Ser?Gly?Lys?Lys?Val?Ala?Arg?Lys?Ala?Gly?Arg

GKV-7：Lys?Phe?Ala?Arg?Arg?Leu?Ala?Lys?Arg?Leu?Gly?Lys?Lys?Val?Ala?Arg?Lys?Leu?Gly?Arg

GKV-8：Lys?Phe?Leu?Arg?Arg?Leu?Ile?Lys?Arg?Leu?Val?Lys?Lys?Val?Leu?Arg?Lys?Leu?Gly?Arg

GKV-9：Lys?Ala?Ala?Lys?Lys?Ala?Ala?Lys?Arg?Ala?Ala?Lys?Lys?Ala?Thr?Arg

GKV-10：Lys?Ala?Leu?Lys?Lys?Ala?Leu?Lys?Arg?Ala?Leu?Lys?Lys?Ala?Leu?Arg

GKV-11：Lys?Gly?Leu?Lys?Lys?Gly?Leu?Lys?Arg?Gly?Leu?Lys?Lys?Gly?Leu?Arg

GKV-12：Lys?Ala?Thr?Lys?Lys?Ala?Leu?Lys?ArgAla?Gly?Lys?Lys?Ala?Thr?Arg

One group of antibacterial peptide provided by the invention, its preparation method can be mechanochemical method, also the encoding gene of antibacterial peptide can be cloned on the carrier, expresses the back then and obtain in host cell.Wherein expression vector can be a kind of in plasmid or the virus; host cell can be prokaryotic cell prokaryocyte; comprise intestinal bacteria, subtilis etc., host cell can be eukaryotic cell also, comprise yeast cell, vegetable cell, insect cell and mammalian cell etc.The antibacterial peptide of preparation can be identified by mass spectrum.

In order to further investigate the structure-function relationship of this quasi-biology active antibacterial peptide of the present invention, the Pioneer Peptide synthesizer that utilizes U.S. application system biotech firm to produce prepares one group of antibacterial peptide disclosed by the invention and antibacterial peptide in contrast, to study.

Utilize 96 well plate method to detect fungicidal activity (the In Yup Park etc of polypeptide; FEBS Letters; 437 (1998) 258-262) with synthetic in advance natural antibacterial peptide GramicidinS, parasinI, magaininII is contrast, carries out fungicidal activity and detects.The result shows that the fungicidal activity of antibacterial peptide of the present invention is better than the fungicidal activity of described three kinds of natural antibacterial peptides.

Antibacterial peptide also might act on high organism and comprise human body cell in efficient sterilizing, because the mode of action of antibacterial peptide all is to make the death of cell generation seepage in cytolemma perforation.So can make antibacterial peptide red corpuscle generation seepage as its virose standard whether, if antibiotic Toplink makes the oxyphorase generation seepage in the red corpuscle, just can be by detecting OD ₄₉₀Value is determined the size of toxicity.Therefore the present invention has also detected the hemolytic activity of antibacterial peptide to human erythrocyte, and experiment shows that antibacterial peptide hemolysis rate value is very low, confirms that the hemolytic toxicity of antibacterial peptide of the present invention is minimum.

In addition, again antibacterial peptide of the present invention is carried out acute toxicity test in the animal body, prove the antibacterial peptide free of toxic effects.Carry out antibacterial peptide again to the test of small white mouse acute infection aureus with inhibition, show that antibacterial peptide has significant inhibitory effect to infection of staphylococcus aureus.

The invention provides the synthetic antibacterial peptide of one group of new artificial design.Can adopt mechanochemical method preparation easily or with the gene clone of encoding antimicrobial peptide to carrier, enter then and express the back in the host cell and obtain.This antibacterial peptide has the wide spectrum killing activity to Gram-negative bacteria, gram-positive microorganism, fungi, virus, and than natural antibacterial peptide stronger fungicidal activity is arranged, but the animal and plant cell is not had any toxic action.And by antibacterial peptide the mouse test of streptococcus aureus acute infection fungicidal activity is shown that antibacterial peptide gives 0.25mg/kg dosage, just can reach 100% sterilization inhibiting rate, just can reach 100% sterilization inhibiting rate and will give 4.0mg/kg dosage as the vancomycin of killing the streptococcus aureus specifics, show that antibacterial peptide of the present invention has very significant sterilization effect to the streptococcus aureus acute infection, so antibacterial peptide of the present invention can be applicable to prepare the medicine of the disease that treatment gram-positive microorganism, Gram-negative bacteria or fungi infestation causes.

Description of drawings

Fig. 1 is the mass spectrum of antibacterial peptide GKV-4.

Embodiment

The synthetic preparation of the solid state chemistry of embodiment 1 antibacterial peptide and separation and purification

Prepare GKV-1 to GKV-12 by above-mentioned sequence, prepare GramicidinS simultaneously, parasinI and magaininII are in contrast.

The sequence of GramicidinS:

Val?Orn?Leu?Phe?Pro?Val?Orn?Leu?Phe?Pro

The sequence of ParasinI

Lys?Gly?Arg?Gly?Lys?Gln?Gly?Gly?Lys?Val?Arg?Ala?Lys?Ala?Lys?Thr?Arg?Ser?Ser

The sequence of magaininII:

Gly?Ile?Gly?Lys?Phe?Leu?His?Ser?Ala?Lys?Lys?Phe?Gly?Lys?Ala?Phe?Val?Gly?Glu

Ile?MetAsn?Ser

Present embodiment adopts mechanochemical method synthetic, the Pioneer Peptide synthesizer that used instrument is produced for u.s.a. applied biosystem company.Synthetic polypeptide is used reverse column purification after the TFA of excessive concentrations shears, the polypeptide behind the purifying is identified by mass spectrum.Concrete testing sequence is as follows:

1, the preparation of antibacterial peptide (GKV-1 with preparation 0.1mmol amount is example)

Below all the preparation antibacterial peptides reagent all purchase in u.s.a. applied biosystem company.

The GKV-1 peptide sequence of preparation is

N-Lys?Gly?Val?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Cys?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys-C

Preparation holds the N end to carry out one by one from C, is controlled automatically by synthesizer.At first weighing 0.1mmol's combines the resin that first amino acid is Arg (purchasing in u.s.a. applied biosystem company); the dress post; use 20% piperidines dimethyl formamide solution deprotection again; dimethyl formamide cleans; the total free aminoacids of 9-tablet held before the breast by officials methoxycarbonyl (Fmoc) protection is dissolved in carbodiimide (DCC); hydroxybenzotriazole (HOBt)/diisopropyl ethyl amine (DIPEA); solution after the dissolving is post cocycle coupled reaction 30 minutes, dimethyl formamide clean repeat above deprotection to the coupled reaction step up to preparation end (the concrete operations step is seen pioneer Peptide synthesizer operational guidance).

Polypeptide after the preparation is sheared through following steps:

Take off reacted resin, add Type B and shear liquid (88% trifluoroacetic acid, 5% phenol, 5% water, 2% tri isopropyl silane), room temperature reaction 2 hours filters, the precooling anhydrous diethyl ether that adds 10 times of volumes in the filtrate, 4000 rev/mins centrifugal 10 minutes, collecting precipitation and drying at room temperature.

2, antibacterial peptide purifying

The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is collected elution peak by oppositely post separation (elutriant is 0.1% trifluoroacetic acid that contains 80% second cyanogen).

Embodiment 2 expression of antibacterial peptide GKV-4 gene in intestinal bacteria

At first design the GKV-4 gene of composite coding antibacterial peptide, it is cloned into (available from Amersham Pharmcia Biotech company) on the pGEX-4T1 carrier, transformed into escherichia coli JM109 then, through IPTG abduction delivering GST-GK V-4 fusion rotein, after the zymoplasm cutting, get antibacterial peptide GKV-4 again.

The ATP that uses among the embodiment, IPTG, T ₄Polynueleotide kinase, T ₄Dna ligase, Klenow enzyme, restriction enzyme are BIOLAB company product except specializing, it is that worker company product is given birth in Shanghai that test kit is reclaimed in rubber tapping, and oligonucleotide is that Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic.Zymoplasm is sheared test kit and is purchased the company in SIGMA.

The separation of relative dna, purifying, pcr amplification, enzymolysis, plasmid transform host bacterium, fragment and reclaim, connect the equimolecular clone operations and all carry out according to " the molecular cloning experiment guide " that the husky nurse Brooker of J. etc. is write.E. coli jm109 is cultivated in the LB of liquid or solid substratum.

Adopt the dna sequence dna of the preferred antibacterial peptide GKV-4 gene of codon design coding of intestinal bacteria preference, sequence is as follows.5 ' end at antibacterial peptide GKV-4 gene is introduced restriction enzyme site BamHI (GGATCC), adds terminator codon (TAG) at 3 ' end, and the length that gained makes up gene is 72bp.

Directly synthesize this nucleotide fragment by dna synthesizer.At first with PCR method gene is increased.Design a pair of primer: 5 '-CCTAGGTTTCCACA-3 ' and 5 '-GATGAAGTTTCG-3 '.The PCR reaction conditions is as follows: 94 ℃, and 30 seconds; 45 ℃, 45 seconds; 72 ℃, 30 seconds; React 30 circulations.After the PCR reaction this segment being mended flat back through the Klenow enzyme cuts with the BamHI enzyme, enzyme section cracked ends is crossed rubber tapping recovery back (rubber tapping reclaimer operation reference reagent box operation instructions) and is connected back transformed into escherichia coli JM109 with the BamHI of pGEX-4T1 carrier and the double digestion segment of SmaI, and transformant is cut evaluation, screening by the SmaI enzyme.Transformant is through IPTG abduction delivering GST-GKV-4 fusion rotein.Fusion rotein obtains antibacterial peptide GKV-4, concrete operations reference reagent box operation instructions after cutting by zymoplasm with the GST affinity column purifying.

The GKV-4 protein sequence:

Lys?Gly?Val?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Cys?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys

The nucleotide sequence of coding GKV-4:

AAAGGTGTTCGTAAAGGCGCCAAACGCCAGGGTTGCAAGAAACTTGCCCGTAAGGCTTTGAAG

Embodiment 3 expression of antibacterial peptide GKV-4 gene in yeast cell

The ATP that uses in the present embodiment, IPTG, T ₄Polynueleotide kinase, T ₄Dna ligase, Klenow enzyme, restriction enzyme are BIOLAB company product except specializing, it is that worker company product is given birth in Shanghai that test kit is reclaimed in rubber tapping, and oligonucleotide is that Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic.Zymoplasm is sheared test kit and is purchased the company in SIGMA.

The GKV-4 gene of design composite coding antibacterial peptide, process BamHI enzyme is cut with the dna sequence dna of the GST that encodes and is connected, after be connected among the plasmid pBluescriptSKII (available from U.S. Stratagene company), transformed into escherichia coli DH5 α (available from DSMZ of Wuhan University), after the extraction plasmid is correct through order-checking proof sequence, plasmid is through EcoRI, after cutting, the XhoI enzyme is connected in the pPIC9 carrier (available from American I nvitrogen company), the back transforms pichia yeast bacterial strain KM71 (available from American I nvitrogen company), methanol induction expressed fusion protein GST-GKV-4.

The separation of relative dna, purifying, pcr amplification, enzymolysis, plasmid transform host bacterium, fragment and reclaim, connect the equimolecular clone operations and all carry out according to " the molecular cloning experiment guide " that the husky nurse Brooker of J. etc. is write.Pichia yeast bacterial strain KM71 cultivates in the BMGY of liquid or solid substratum, and yeast strain is cultivated in the BMMY substratum during methanol induction expressed fusion protein GST-GKV-4, and adding methyl alcohol to final concentration every 24 hours is 1%.

Adopt the codon design preferred antibacterial peptide GKV-4 of coding of yeast preference and the dna sequence dna of GST.5 ' end at antibacterial peptide GKV-4 gene is introduced restriction enzyme site BamHI (GGATCC), add terminator codon (TAG) and EcoRI restriction enzyme site (GAATCC) at 3 ' end, the length that gained makes up gene is 78bp, connect the dna sequence dna of coding GST simultaneously at GKV-4 gene leading portion, the 5 ' end of while at the dna sequence dna of GST added an XhoI restriction enzyme site.

The preparation of GKV-4 nucleotide fragment: by the direct composite coding GKV-4 of dna synthesizer nucleotide fragment.With PCR method gene is increased then.Design a pair of primer 5 '-CCTAGGTTTCCACA-3 and 5 ,-CTTAAGGATGAAGTTTCG-3 '.The PCR reaction conditions is as follows: 94 ℃, and 30 seconds; 45 ℃, 45 seconds; 72 ℃, 30 seconds; React 35 circulations.

The coding GST dna sequence dna preparation: design a pair of primer

5’-CTCGAGATGTCCCCTATACTAGGTT-3’

5’-CAGTGCTACGCCGGCGAG-3’

With above primer amplification pGEX-4T1 carrier.The PCR reaction conditions is as follows: 94 ℃, and 30 seconds; 45 ℃, 45 seconds; 72 ℃, 60 seconds; React 30 circulations.

Amplified fragments is connected with plasmid: the pcr amplification segment of GKV-4 is mended flat back through the Klenow enzyme cut with BamHI and EcoRI enzyme, enzyme section cracked ends is crossed BamHI that rubber tapping reclaims back (rubber tapping reclaimer operation reference reagent box operation instructions) and the DNA cloning sequence of the GST that encodes and XhoI enzyme and is cut back to close and cut back to close fragment with the XhoI of pBluescriptSKII with the EcoRI enzyme after fragment is connected and be connected.Connect product transformed into escherichia coli DH _{5 α}, transformant further determines that through order-checking expressed sequence is correct after cutting evaluation through resistance, blue hickie, enzyme.The extraction plasmid cuts back to close small segment through XhoI and EcoRI enzyme, cuts to be connected with XhoI and the EcoRI enzyme of pPIC9 to be built into expression plasmid pPIC9-gst-GKV-4, transformed into escherichia coli DH _{5 α}By amicillin resistance screening transformant.Press method (the Clare JJ of bibliographical information, etc., Gene, 1991,105:205-212) the competent cell of preparation pichia yeast KM71, and the linearizing recombinant plasmid pPIC9-gst-GKV-4 of SacI single endonuclease digestion transformed through electricity import competent cell, the condition that electricity transforms is 1.5KV, 22.5uF.In the YPD flat board, 30 ℃ of cultivations are screened the positive colony of expressed fusion protein two days later with electric transformed yeast cells coated plate.

Transformant is expressed the GST-GKV-4 fusion rotein through methanol induction.Fusion rotein obtains antibacterial peptide GKV-4, concrete operations reference reagent box operation instructions after cutting by zymoplasm with the GST affinity column purifying.

GST-GKV-4 fusion rotein sequence:

Leu?Glu?Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln?Pro?Thr?Arg?Leu?Leu?Leu

Glu?Tyr?Leu?Glu?Glu?Lys?Tyr?Glu?Glu?His?Leu?Tyr?Glu?Arg?Asp?Glu?Gly?Asp?Lys?Trp?Arg?Asn?Lys

Lys?Phe?Glu?Leu?Gly?Leu?Glu?Phe?Pro?Asn?Leu?Pro?Tyr?Tyr?Ile?Asp?Gly?Asp?Val?Lys?Leu?The?Gln

Ser?Met?Ala?Ile?Ile?Arg?Tyr?Ile?Ala?Asp?Lys?His?Asn?Met?Leu?Gly?Gly?Cys?Pro?Lys?Glu?Arg?Ala

Glu?Ile?Ser?Met?Leu?Glu?Gly?Ala?Val?Leu?Asp?Ile?Arg?Tyr?Gly?Val?Ser?Arg?Ile?Ala?Tyr?Ser?Lys

Asp?Phe?Glu?The?Leu?Lys?Val?Asp?Phe?Leu?Ser?Lys?Leu?Pro?Glu?Met?Leu?Lys?Met?Phe?Glu?Asp?Arg

Leu?Cys?His?Lys?The?Tyr?Leu?Asn?Gly?Asp?His?Val?The?His?Pro?Asp?Phe?Met?Leu?Tyr?Asp?Ala?Leu

Asp?Val?Val?Leu?Tyr?Met?Asp?Pro?Met?Cys?Leu?Asp?Ala?Phe?Pro?Lys?Leu?Val?Cys?Phe?Lys?Lys?Arg

Ile?Glu?Ala?Ile?Pro?Gln?Ile?Asp?Lys?Tyr?Leu?Lys?Ser?Ser?Lys?Tyr?Ile?Ala?Trp?Pro?Leu?Gln?Gly

Trp?Gln?Ala?The?Phe?Gly?Gly?Gly?Asp?His?Pro?Pro?Lys?Ser?Asp?Leu?Val?Pro?Arg?Gly?Ser

Lys?Gly?Val?Arg?Lys?Gly?Ala?Lys?Arg?Gln?Gly?Cys?Lys?Lys?Leu?Ala?Arg?Lys?Ala?Leu?Lys

GST-GK V-4 nucleotide sequence:

CTCGAGATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGC

AACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGC

ATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTG

AATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTT

AAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACA

ACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTG

AAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAG

TAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAA

ATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGG

TGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTG

TTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGT

TTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATC

CAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGT

GGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCGTAAAGGTGTTC

GTAAAGGCGCCAAACGCCAGGGTTGCAAGAAACTTGCCCGTAAGGCTT

TGAAGTAGGAATTC

The evaluation of embodiment 4 antibacterial peptides

As shown in Figure 1, the antibacterial peptide GKV-4 of preparation is through mass spectroscopy, and the molecular weight that GKV-4 shows in mass spectrum is 2324.0.The theoretical value that is calculated by peptide sequence is 2324.0.The polypeptide of proof preparation is the GKV-4 antibacterial peptide of design.The antibacterial peptide product of accreditation is standby.

Antibacterial peptide GKV-1, GKV-2 and other antibacterial peptide disclosed by the invention, natural antibacterial peptide GramicidinS, parasinI and magaininII can adopt preparation method's preparation of GKV-4 antibacterial peptide.

The fungicidal activity of embodiment 5 antibacterial peptides detects

Employed various bacterial strains are purchased in Chinese biological goods calibrating institute in following examples.

Adopt 96 well plate method that the fungicidal activity of antibacterial peptide is detected, and with the synthetic antibacterial peptide GramicidinS (S) of mechanochemical method, parasinI (I) and magaininII (II) are in contrast, and with 12 antibacterial peptide GKV-1 among the synthetic the present invention of solid state chemistry synthesis method to GKV-12, detect their fungicidal activity.

Measure the fungicidal activity of antibacterial peptide according to the following steps:

The bacterial classification recovery, the 37 ℃ of overnight incubation in inoculation inclined-plane are chosen bacterium in common LB substratum, 37 ℃ of overnight incubation, it is 10 that dilution bacterium liquid makes bacteria concentration ⁴-10 ⁵CFU/ml is inoculated in 96 orifice plates by every hole 100ul bacterium liquid, after polypeptide is diluted with certain proportion, adds 10ul in every hole, and 96 orifice plates are placed 37 ℃ of overnight incubation, and microplate reader detects OD ₆₂₀Value (In Yup Park etc; FEBS Letters; 437 (1998) 258-262).Detected result sees Table 1.

Growth concentration (the OD that contains the bacterium of antibacterial peptide ₆₂₀) be minimal inhibitory concentration (minimal inhibitory concentration (MIC) is defined as the minimum concentration of remarkable bacteria growing inhibiting) with the ratio of the growth concentration of the bacterium that does not add antibacterial peptide greater than 90% o'clock antibacterial peptide concentration.

Several antibacterial peptides of table 1 are to the comparison of the anti-microbial activity minimal inhibitory concentration (ug/ml) of different bacterium

In the last table the minimal inhibitory concentration value more little, it is more strong then to represent antibacterial ability, as can be seen from the above table, the MIC of antibacterial peptide of the present invention compares GramicidinS, parasinI and magaininII are all little, particularly to the effect of Gram-negative bacteria, the antibacterial ability of antibacterial peptide of the present invention is better than three antibacterial peptides of contrast greatly.

The fungicidal activity of embodiment 6 antibacterial peptide function equivalents---cyclisation derivative detects

Design synthetic antibacterial peptide function equivalent: cyclisation GK-20 derivative, synthetic method are seen u.s.a. applied biosystem company (Applied Biosystems) pioneer Peptide synthesizer operational guidance.After the synthetic product process separation and purification (with embodiment 1), detected the minimal inhibitory concentration of derivative, fungicidal activity detects with embodiment 4, and detected result sees Table 2.The sequence of cyclisation GKV-2 derivative is as follows:

GKV-2：

Table 2 antibacterial peptide GKV-2 function equivalent is to the minimal inhibitory concentration (MIC) of different bacterium

Embodiment 3 external hemolytic activities detect

Whether present embodiment has hemolytic activity for detection of antibacterial peptide to human erythrocyte, and with the synthetic antibacterial peptide GramicidinS of mechanochemical method, parasinI and magaininII are in contrast.Use blood sample be taken at normal human blood.

The detection step of antibacterial peptide hemolytic activity is:

The release of fresh red blood cell suspension oxyphorase under 414nm of 4% detects.HRBC is through PBS (PBS:35mM phosphoric acid buffer/0.15MNaCl, PH7.0) washing, get the 8% HRBC suspension of 100ul in 96 orifice plates, add 100ul antibacterial peptide solution in every hole, 37 ℃ after one hour, centrifugal 5 minutes of 1500rpm shifts the 100ul supernatant in 96 new orifice plates, by the absorption under the microplate reader detection 414nm.Negative control PBS, positive control 0.1%TritonX-100.Detected result sees Table 2

Five kinds of antibacterial peptide hemolytic activities of table 3 detected result

The hemolysis rate value of antibacterial peptide is more little in the table 2, and the hemolytic toxicity that then represents antibacterial peptide is more little.

Acute toxicity test in embodiment 7 animal bodies

Present embodiment is in order to detecting antibacterial peptide to the toxicity of animal, and with the synthetic antibacterial peptide GramicidinS of mechanochemical method, parasinI and magaininII measure antibacterial peptide GKV-1 provided by the invention, the toxicity of GKV-2 and GKV-4 in contrast.

Adopt 60 of Kunming small white mouses, male and female half and half, body weight 33.5 ± 0.25g, antibacterial peptide press 1mg/kg dosage, observe animal toxic reaction under maximal dose through the intramuscular injection small white mouse in continuous 7 days once a day.Experimental result shows that animal via intramuscular injection antibacterial peptide is after 7 days, and no abnormal reaction, activity are normally.Observed through 7 days, 60 small white mouses all survive.Proof antibacterial peptide free of toxic effects.

Embodiment 8 antibacterial peptides and vancomycin are to the comparison of mouse infection of staphylococcus aureus model inhibition

Use the streptococcus aureus acute infection model of Kunming small white mouse, experimental procedure is as follows:

Place the veal that contains 5% pig gastric mucoitin to inculcate in the meat soup streptococcus aureus CMCC26003, shaking culture is spent the night.Injection 10 in the mouse peritoneal of the about 32.6 ± 0.25g of body weight ⁶-10 ⁷Survivaling cell has 3 mouse in each treatment group.Intravenous injection antibacterial peptide GKV-1 and GKV-3 (being dissolved in respectively in 0.1 milliliter of 5% dextrose for injection), injection in 10 minutes.The subcutaneous injection vancomycin (but vancomycin is complete biological utilisation to the mouse subcutaneous administration, no matter subcutaneous give or vein has similar activity).

Table 4 antibacterial peptide GKV-1, GKV-3 and vancomycin are to the restraining effect of streptococcus aureus acute infection small white mouse model

As shown in table 3, intravenous injection gives 0.25mg/kg, antibiotic Toplink 100% protection infecting mouse.Vancomycin ability 100% under 4.0 milligrams/kg dosage is effective.All untreated mice are all dead in less than 24 hours.

This experimental example shows that antibacterial peptide is highly effective to killing streptococcus aureus with the chmice acute infection model of the fatal bacterium dosage of height.

Claims (2)

1. an antibacterial peptide is characterized in that, the aminoacid sequence of described antibacterial peptide is the SEQ ID No.2 shown in the sequence table.

2. the application of the described antibacterial peptide of claim 1 in the medicine of preparation treatment gram-positive microorganism, Gram-negative bacteria or fungi infestation.

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