CN102153631B

CN102153631B - Antimicrobial protein with high-efficiency sterilizing activity and application thereof

Info

Publication number: CN102153631B
Application number: CN 201010605662
Authority: CN
Inventors: 董世雷; 刘伟; 王欣
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2010-12-25
Filing date: 2010-12-25
Publication date: 2013-01-23
Anticipated expiration: 2030-12-25
Also published as: CN102153631A

Abstract

The invention provides antimicrobial protein with high-efficiency sterilizing activity and application thereof in the preparation of bacteriostat. The antimicrobial peptide has a amino acid sequence shown in SEQ ID NO:1. Sequence structure analysis and sequence comparison analysis indicate that protease is endolysin in bacteriophage. Confirmed by tests, the protein has good sterilizing activity. The protein mainly has the benefits of high efficiency, low toxicity, wide application, a notable effect of sterilizing a range of pathogenic bacteria in vitro after the expression and purification of the existing proper expression carrier in the market, and availability for preparing the bacteriostat.

Description

A kind of antibacterial protein and application thereof with high-efficiency sterilizing activity

(1) technical field

The present invention relates to a kind of antibacterial protein with high-efficiency sterilizing activity, and the application in the preparation fungistat.

(2) background technology

Far-famed authorities,medical periodical " lancet " has been published one piece of research report about " superbacteria " in the world, and report claims to have found a kind ofly have multiple drug resistance, almost can resist all antibiotic dangerous transgenation products---contain " superbacteria " of New Delhi metalloprotease-1 (NDM-1).At present, the researchist confirms that in India and Pakistan case exceedes hundred people, has confirmed 37 patients in Britain, and similarly " superbacteria " infects and also appear at the U.S., Canada, Australia and the state such as Dutch.Abuse of antibiotics is the reason that superbacteria occurs.Microbiotic once was the magical weapon of sterilization at the beginning of being born, the resistance but bacterium is also evolved out gradually.Because the research and development speed of new antibiotic is relatively slow, developing a kind of microbiotic approximately needed for 10 years, and produced the resistance rhzomorph within 2 years, and antibiotic development speed is unable to catch up with the reproduction speed of resistant organism far away.Tackle superbacteria and become the difficult problem that modern medicine faces.The appearance of superbacteria tells us to want Using adapted Antibios on the one hand, will accelerate on the one hand the paces of antibacterials research and development.The weapon of a kind of new, effective, safe resisting pathogenic microbes.Its unique mode of action makes it be expected to solve the bacterial drug resistance problem that traditional microbiotic life-time service brings, and all has wide practical use in fields such as medical and health, agriculture production, foodstuffs industry.As mentioned above, the R﹠D cycle of antibacterials is long, how to shorten the gap that resistant organism produces the resistance time, and the easiest method is exactly to carry out the molecular biology structure of modification on the basis of existing antibacterials, improves sterilization and tires.The benefit of doing like this is exactly " short, adaptable and fast "---and less investment, cycle are short, instant effect, high efficiency.

(3) summary of the invention

The object of the invention provides a kind of antibacterial protein and application thereof with remarkable fungistatic effect.

The technical solution used in the present invention is:

A kind of antibacterial protein with remarkable fungistatic effect has the aminoacid sequence shown in the SEQ ID NO:1.Through sequential structure analysis and sequence comparing analysis, show that this proteolytic enzyme is endolysin in the phage, confirm through test, this sequence all has preferably fungicidal activity.

Described antibacterial protein aminoacid sequence shown in SEQ ID NO:1, called after E _E17GAlbumen, its encoding gene nucleotide sequence is shown in SEQ ID NO:2.

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

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

(4) description of drawings

Fig. 1 is Electronic Speculum picture: LL37, E albumen and E _E17GAlbumen is on the impact of ne ar; A is that empty carrier contrasts at E.coli TOP10; B is that LL37 induces in E.coli TOP10; C is that E albumen is induced in E.coli TOP10; D is E _E17GIn Ecoli TOP10, induce; E is E _E17GIn P.aeruginosa, induce.

Fig. 2 is LL37 (A), E (B), E _E17G(C) expression in vivo is on the impact of Escherichia coli Growth;

Fig. 3 is LL37 (A), E albumen (B) and E _E17GAlbumen (C) expression in vivo is to the growth effect of Pseudomonas aeruginosa.

Fig. 4 is to endolysin E and E _E17GCarry out the prediction of structure and bindingsite assay relatively; A is the three-dimensional structure of endolysin E albumen, and B is the bindingsite assay of E albumen; C is E of the present invention _E17GThe three-dimensional structure of albumen, D is E of the present invention _E17GThe bindingsite assay of albumen; The hyaloplasmic sphere bar chart shows the binding site of albumen and part among figure B, the D;

(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: LL37 antibacterial peptide in take the false unit cell of intestinal bacteria and verdigris as the target bacteria body, endolysin carries out E for contrast _E17GTo the cellular form Electronic Speculum relatively

LL37 is the antibacterial peptide that contains 37 amino acid unique generations in human body, and is verified to multiple pathogenic bacteria at present, such as intestinal bacteria, Salmonellas, streptococcus aureus etc. widely fungistatic effect arranged.Among the present invention, at first (the GenBank accession number: Z38026), living worker has synthesized the LL37 gene to the LL37 nucleotide sequence of discovery of basis by Shanghai.The LL37 gene is connected to pHERD30t carrier (U.S. Marshall University is so kind as to give in grand professor), consists of the pHERD30t-ll37 recon.By electrization recon is imported in the intestinal bacteria.Filter out recon at the LB flat board that contains gentamicin.Recon adds 0.1% pectinose inductor when growing into logarithmic phase, same, has cloned endolysin E gene (SEQ ID NO:4), is transformed into intestinal bacteria according to same method and carries out the endolysin protein expression.Subsequently endolysin E gene is carried out fallibility PCR, by the impact on bacterial growth, filtered out the strongest endolysin albumen E of vigor _E17GWith empty carrier induce, LL37 and E is protein induced and E _E17GElectronic Speculum comparative observation after inducing, found that empty carrier is induced after, cell walls and cytolemma are perfect.And LL37, E albumen (SEQ ID NO:3) and E _E17GAlbumen (SEQ ID NO:1) is induced and rear cell walls and cytolemma has been caused very large impact, makes cell walls and cytolemma produce irregular variation.The results are shown in Figure 1.

Embodiment 2: the test endolysin E take intestinal bacteria as target bacteria _E17GThe albumen fungistatic effect

From the chicken fresh excreta, isolate the colibacillary virulent phage WL08 of a strain.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.Utilize the fallibility round pcr that endolysin albumen is carried out random mutation.By the observation to growth curve, choose the strongest albumen of sterilization, subsequently its Nucleotide and amino acid are compared, find that it compares with endolysin E albumen, a point mutation occurs, be mutated into Gly, called after E by 17 Glu in the aminoacid sequence _E17GTake it as the basis, test antibacterial protein of the present invention.Choose antibacterial peptide gene ll37 and original endolysin e gene in contrast, the encoding sequence of institute's DCRP is connected with the pHERD30T carrier, electrization imports in the intestinal bacteria the expression vector that builds.Recon adds 0.1% pectinose inductor when growing into logarithmic phase, each hour takes a sample at OD ₆₀₀Lower survey absorbancy, the result as shown in Figure 2.E _E17GThe albumen of genetic expression is the strongest to the intestinal bacteria inhibition in vivo, also is eager to excel than the antibacterial peptide of known good anti-bacterial effect.

Embodiment 3: the test endolysin E take Pseudomonas aeruginosa as target bacteria _E17GThe albumen antibacterial effect

In order further to seek out the antibacterial protein sequence that Pseudomonas aeruginosa is had obvious sterilization effect, with three fragment genes in above-described embodiment 2, ll37 and e (SEQ ID NO:4) and e _E17G(SEQID NO:2) is connected on the pHERD30t carrier, imports among the Pseudomonas aeruginosa PAO1 by electrization.Recon adds 1.8% pectinose inductor when growing into logarithmic phase, each hour takes a sample at OD ₆₀₀Lower survey absorbancy.Compare with the contrast recon, at Pseudomonas aeruginosa PAO1 expression in vivo E _E17GThe 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.

Embodiment 4: to the endolysin E of phage _E17GCarry out three-dimensional structure and the analysis of ligand binding site estimation with E albumen.

Because endolysin E _E17GDiffer an amino acid between albumen and the E albumen, but its albumen has higher bacteriostasis, even be higher than the positive ion antibacterial peptide of humanized LL37, so utilize Molecular Simulation Technique that the space structure of endolysin is analyzed, find out the difference between them.The structure of its simulation is seen Fig. 4, found that endolysin E of the present invention _E17GAlbumen is slightly different from the E protein structure, binding site also all changes, and illustrates that its space structure is very large on the impact of its activity.

E _E17GThe binding site of protein ligands has 13, and is as follows respectively: ILE:73, LEU:79, VAL:82, TYR:83, LEU:86, LEU:94, MET:97, VAL:98, VAL:106, PHE:109, LEU:113, LEU:116, PHE:148.

And 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.

SEQUENCE LISTING

＜110〉Zhejiang Academy of Agricultural Science

＜120〉a kind of antibacterial protein and application thereof with high-efficiency sterilizing activity

<130>

<160> 4

<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

Gly 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

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

85 90 95

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

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

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

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<212> DNA

<213> Unknown

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＜223〉artificial sequence

<400> 2

atgcttcgta atgacgaagg tctcagactg tctttatata aagacactgg 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

<210> 3

<211> 157

<212> PRT

<213> Unknown

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＜223〉artificial sequence

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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

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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

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<210> 4

<211> 474

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 4

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

1. antibacterial protein with efficient sterilizing activity, its aminoacid sequence is shown in SEQ ID NO:1.

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

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