CN102120760B - Antibacterial peptide derived from phage and application thereof - Google Patents

Abstract

The invention discloses an antibacterial peptide derived from a phage and application thereof in preparing a bacteriostat. The antibacterial peptide has total or partial amino acid sequences shown in SEQ ID No:1 in the specification; sequence structure analysis and sequence comparison analysis show that the protease is an endolysin in a phage; and experiments demonstrate that protein formed by the total or partial amino acid sequences (such as 1-87aa, 34-157aa or 110-157aa) of the sequence has better bactericidal activity. The invention has the main beneficial effects that the antibacterial peptide has the advantages of high efficiency, low toxicity and wide application range, can be used for preparing the bacteriostat, and has an obvious bactericidal effect on a series of pathogenic bacteria in vitro after being expressed and purified by traditional proper expression carriers in the market.

Description

A kind of antibacterial peptide and application thereof that derives from phage

(1) technical field

The present invention relates to a kind of antibacterial peptide that derives from phage, and the application in the preparation fungistat.

(2) background technology

In recent years, the bacterial resistance phenomenon is at all parts of the world ubiquity, becomes one of serious problems that the world of medicine faces.The just listing of many new antibiotics has found namely that soon Resistant strain occurs; The resistance of bacterium causes the antibacterials curative effect more and more unable, has impelled people to seek with the development of new antibacterials and has tackled this phenomenon.The discovery of antibacterial peptide has brought new hope to people.

Antibacterial peptide (antimicrobial peptides, AMPs) is that a class that produces in the organism system of defense has the peptide matters of the activity efficiently killed to the external source germ.It is the general name with anti-microbial activity small peptide.The mankind have found the antibacterial peptide of kind more than 300 on earth various animals and plants bodies up to now.And along with to the understanding in depth of the antibacterial peptide mechanism of action, a greater variety of antibacterial peptides develop endlessly.If antibacterial peptide with origin classification, comprises antibacterial protein or the antibacterial peptide that contains in bacterium or viral genome, the antibacterial peptide of animal-origin and the antibacterial peptide of plant origin.The difference of these antibacterial peptide amino acid structures has determined the specificity of its antimicrobial spectrum aspect, but generally speaking antibacterial peptide to the multiple pathogenic bacterias such as bacterium, fungi, virus, spirochete even cancer cells all show certain activity of killing.In bacterial drug resistance day by day serious today, especially on market to unprecedented today of improving of demand of novel antibacterial medicine, the difficult generation resistance of antibacterial peptide and the characteristics of thermostability become the optimal candidate that green feed adds.

Although the domestic work that utilizes genetic engineering technique to build antibacterial peptide production bacterial strain has had certain progress, but the important problem that exists in natural antibacterial peptide research and production process is that the shown biological characteristics out of natural antibacterial peptide can not satisfy our requirement in use.For example, antibacterial peptide is usually high at the host animal activity in vivo, in case use at other target animals, because the environment that uses object changes, active obviously reduction the and toxicity raises.So, develop efficient, low toxicity and widely used antibacterial peptide or antibiotic protein gene are the keys of the antibacterial peptide marketization.

(3) summary of the invention

The object of the invention is to provide a kind of antibacterial peptide with remarkable fungistatic effect and application thereof from phage.

The technical solution used in the present invention is:

A kind of antibacterial peptide that derives from phage has all or part of aminoacid sequence shown in SEQ ID NO:1.Through sequential structure analysis and sequence comparing analysis, show that this proteolytic enzyme is endolysin in phage, confirm through test, the albumen that all or part of aminoacid sequence of this sequence (for example 1-87aa, 34-157aa or 110-157aa) forms all has fungicidal activity preferably.

Described antibacterial peptide aminoacid sequence as shown in SEQ ID NO:1, called after E albumen, its encoding gene nucleotide sequence is as shown in SEQ ID NO:5.By three-dimensional structural analysis, segmentation intercepting partial amino-acid carries out the analysis of bacteriostatic activity to the E albumen that contains 157 aminoacid sequences in the present invention, has obtained several bacteriostatic activities antibacterial peptide (following E for example preferably_1-87Albumen, E_34-157Albumen and E_110-157Albumen).

Perhaps, described antibacterial peptide aminoacid sequence is (the 1-87aa sequence of SEQ ID NO:1) as shown in SEQ ID NO:2, called after E_1-87Albumen.

Perhaps, described antibacterial peptide aminoacid sequence is (the 34-157aa sequence of SEQ ID NO:1) as shown in SEQ ID NO:3, called after E_34-157Albumen.

Perhaps, described antibacterial peptide aminoacid sequence is (the 110-157aa sequence of SEQ ID NO:1) as shown in SEQ ID NO:4, called after E_110-157Albumen.

The invention still further relates to the application of described antibacterial peptide in the preparation fungistat.

Beneficial effect of the present invention is mainly reflected in: antibacterial peptide of the present invention, efficiently, low toxicity, be widely used, express, after purifying, external a series of pathogenic bacterias shown significant sterilization effect by existing suitable expression vector on market, can be used for the preparation of fungistat.

(4) description of drawings

Fig. 1 is to endolysin E, E_1-87Carry out structure and bindingsite assay prediction with RB69E albumen; A is the three-dimensional structure of RB69E albumen, and B is the bindingsite assay of RB69E albumen; C is the three-dimensional structure of E albumen of the present invention, and D is the bindingsite assay of E albumen of the present invention; E is E_1-87The three-dimensional structure of albumen; In figure B, D, the hyaloplasmic sphere bar chart shows the binding site of albumen and part;

Fig. 2 is E, E_1-87, E_34-157, E_110-157The impact of expression in vivo on Escherichia coli Growth;

Fig. 3 is E albumen and E_110-157The growth effect of albumen expression in vivo to Pseudomonas aeruginosa.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: the test Bacteriophages bacteriolysin E albumen fungistatic effect take intestinal bacteria as target bacteria

Isolate a colibacillary virulent phage WL08 of strain from the chicken fresh excreta.Through Molecular Identification, this phage and the coliphage RB69 that has delivered have higher similarity, according to the predictive genes of the RB69 that has announced, clone the endolysin albumen (E albumen) of this phage.E albumen contains 157 aminoacid sequences, the albumen that clones and endolysin albumen in ncbi database phage RB69 have two amino acid whose different, be respectively that the amino acid of 11 becomes S by T, the amino acid of 104 becomes A by S.E albumen contains 157 aminoacid sequences, take it as the basis, and the illustrated antibacterial protein of test this patent.According to E albumen catalytic site and the zone that the antibacterial peptide activity may be arranged of prediction wherein, according to aminoacid sequence, E albumen is divided into three sections of 1-87aa, 34-157aa and 110-157aa, called after e_1-87, e_34-157And e_110-157Be a series of primers of the template design primer e that increase respectively according to e pyrenoids nucleotide sequence again_1-87, e_34-157And e_110-157Three fragment genes.The encoding sequence of gained subclone is connected recon called after pHERD-e, pHERD-e with the pHERD30T carrier_1-87, pHERD-e_34-157And pHERD-e_110-157Electrization imports in intestinal bacteria above-mentioned 4 expression vectors that build.Recon adds 0.1% pectinose inductor when growing into logarithmic phase, each hour takes a sample at OD₆₀₀Lower survey absorbancy, result as shown in Figure 2.The albumen of E genetic expression is the strongest to the intestinal bacteria inhibition in vivo, e_1-87Next, and e_110-157The inhibition of gene in the intestinal bacteria body is relatively relatively poor.

Utilize subsequently the Invitrogen IMPACT of company expression system to e, e_1-87, e_34-157And e_110-157Four fragment genes are expressed respectively, and further by the Affinity chromatography purification system, having obtained molecular weight is 18.9KDa, 13.8KDa, four purifying proteins of 9.6KDa and 5.5KDa.These four kinds of albumen are as shown in table 1 to the minimal bactericidal concentration (MIC) of intestinal bacteria and streptococcus aureus.Wherein, the antibacterial activity in vitro of E albumen (SEQ ID NO:1) is better than E_1-87(SEQ IDNO:2), E_34-157(SEQ ID NO:3) and E_110-157(SEQ ID NO:4), as shown in Figure 2.The proof antibacterial activity in vivo correctly reflects antibacterial activity in vitro, can filter out antibacterial protein by high throughput screening system.

Table 1: antibacterial peptide is to streptococcus aureus, colibacillary minimal inhibitory concentration

Annotate: LL37 is existing known humanized's cationic antibacterial peptide.

Embodiment 2: E albumen of the present invention, E_1-87Albumen and RB69E albumen carry out three-dimensional structure and the analysis of ligand binding site estimation

Owing to differing 2 amino acid (seeing Table 2) between endolysin E albumen and RB69E (being the endolysin albumen of phage RB69), E1-87 albumen has the bacteriostasis suitable with endolysin albumen E, so utilize Molecular Simulation Technique that the space structure of endolysin is analyzed, find out the difference between them.Result analysis to simulation is seen Fig. 1, finds endolysin E albumen and the RB69E structure differs widely, binding site also all changes, so its sterilization mechanism is fully different from RB69E.

The binding site of endolysin E protein ligands has 13, and is as follows respectively: GLU:6, ASP:15, GLU:17, GLY:25, HIS:26, LEU:27, ASP:65, VAL:98, PHE:99, GLN:100, MET:101, GLY:102, TRP:133.

And the binding site of RB69E protein ligands has 12, and is as follows respectively: ILE:73LEU:79VAL:82TYR:83LEU:86LEU:94MET:97VAL:98VAL:106L EU:113LEU:116PHE:148.

E_1-87The Argine Monohydrochloride number is few, and its binding site is out unpredictable.

Table 2:RB69E and E Argine Monohydrochloride sequence are relatively

Amino acid	11aa	104aa
			RB69E	T	S
E albumen	S	A

Embodiment 3: the test endolysin E albumen antibacterial effect take Pseudomonas aeruginosa as target bacteria

With three fragment genes in above-describedembodiment 1, e_1-87, e_34-157And e_110-157, be connected on the pHERD30t carrier, import in Pseudomonas aeruginosa PAO1 by electrization.Recon adds 1.8% pectinose inductor when growing into logarithmic phase, each hour takes a sample at OD₆₀₀Lower survey absorbancy.The recon of not inducing is compared, at Pseudomonas aeruginosa PAO1 expression in vivo E and e_110-157The gene pairs target bacteria has good restraining effect (seeing Fig. 3).Remaining proteins and peptides is not obvious to the fungistatic effect of Pseudomonas aeruginosa.

SEQUENCE LISTING

＜110〉Zhejiang Academy of Agricultural Science

＜120〉a kind of antibacterial peptide and application thereof that derives from phage

<130>

<160> 5

<170> PatentIn version 3.4

<210> 1

<211> 157

<212> PRT

<213> Unknown

<220>

＜223〉artificial sequence

<400> 1

Met Leu Arg Asn Asp Glu Gly Leu Arg Leu Ser Leu Tyr Lys Asp Thr

1 5 10 15

Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Val Thr Lys Asn Pro

20 25 30

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

35 40 45

Cys Asn Gly Thr Ile Thr Leu Asp Glu Ala Glu Lys Leu Phe Asn Glu

50 55 60

Asp Val Asp Lys Ala Val Arg Gly Ile Leu Gly Asn Ala Lys Leu Lys

65 70 75 80

Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Cys Ala Leu Val Asn

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Met Val Phe Gln Met Gly Val Ala Gly Val Ala Gly Phe Thr Asn Ser

100 105 110

Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala Val Asn Leu

115 120 125

Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg Ala Lys Arg Val

130 135 140

Ile Ser Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr Ile

145 150 155

<210> 2

<211> 87

<212> PRT

<213> Unknown

<220>

＜223〉artificial sequence

<400> 2

Met Leu Arg Asn Asp Glu Gly Leu Arg Leu Ser Leu Tyr Lys Asp Thr

1 5 10 15

Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Val Thr Lys Asn Pro

20 25 30

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

35 40 45

Cys Asn Gly Thr Ile Thr Leu Asp Glu Ala Glu Lys Leu Phe Asn Glu

50 55 60

Asp Val Asp Lys Ala Val Arg Gly Ile Leu Gly Asn Ala Lys Leu Lys

65 70 75 80

Pro Val Tyr Asp Ser Leu Asp

85

<210> 3

<211> 124

<212> PRT

<213> Unknown

<220>

＜223〉artificial sequence

<400> 3

Leu Ala Val Ala Lys Ala Glu Leu Asp Arg Met Ile Gly Arg Lys Cys

1 5 10 15

Asn Gly Thr Ile Thr Leu Asp Glu Ala Glu Lys Leu Phe Asn Glu Asp

20 25 30

Val Asp Lys Ala Val Arg Gly Ile Leu Gly Asn Ala Lys Leu Lys Pro

35 40 45

Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Cys Ala Leu Val Asn Met

50 55 60

Val Phe Gln Met Gly Val Ala Gly Val Ala Gly Phe Thr Asn Ser Leu

65 70 75 80

Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala Val Asn Leu Ala

85 90 95

Gln Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg Ala Lys Arg Val Ile

100 105 110

Ser Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr Ile

115 120

<210> 4

<211> 48

<212> PRT

<213> Unknown

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＜223〉artificial sequence

<400> 4

Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala

1 5 10 15

Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg Ala

20 25 30

Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr Ile

35 40 45

<210> 5

<211> 474

<212> DNA

<213> Unknown

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＜223〉artificial sequence

<400> 5

atgcttcgta atgacgaagg tcttagactg tctttatata aagacactga aggcttttgg 60

acgattggca taggccattt agtaacaaag aacccgtctt tagccgtagc taaagctgaa 120

cttgacagaa tgatcggacg taaatgcaac ggtacaatta cccttgatga ggccgaaaag 180

ctatttaatg aagacgttga taaagccgtt cgcgggatct tgggtaatgc taaacttaaa 240

ccggtatatg attctttaga tgcagttcgt cgatgtgcat tggtcaatat ggtcttccaa 300

atgggtgtag caggcgtcgc tggttttact aattctcttc gtatgcttca acagaaacgt 360

tgggatgaag cggcagtaaa tctagcccaa tctaaatggt atcgtcagac acctaatcgc 420

gcgaaacgcg taatctcaac atttaaaaca ggaacttgga aagcgtatat atga 474

Claims (3)

1. antibacterial peptide that derives from phage, its aminoacid sequence is all or part of aminoacid sequence shown in SEQ ID NO:1, described partial amino-acid series is as shown in SEQ ID NO:2 or SEQ ID NO:3.

2. the gene of the described antibacterial peptide of coding claim 1, is characterized in that the nucleotide sequence of described gene is as shown in SEQ ID NO:5.

3. the application of antibacterial peptide as claimed in claim 1 in the preparation fungistat.

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