CN109266655A - A kind of antibacterial peptide and its prokaryotic expression method and application - Google Patents
A kind of antibacterial peptide and its prokaryotic expression method and application Download PDFInfo
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- CN109266655A CN109266655A CN201811038571.9A CN201811038571A CN109266655A CN 109266655 A CN109266655 A CN 109266655A CN 201811038571 A CN201811038571 A CN 201811038571A CN 109266655 A CN109266655 A CN 109266655A
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- cecropin
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention discloses a kind of antibacterial peptide and its prokaryotic expression method and applications.The recombinant vector of encoding gene of the present invention using Prokaryotic expression vector construction comprising antibacterial peptide cecropin-BM, realize the prokaryotic expression of antibacterial peptide cecropin-BM, method is simple and easy, and the recombinant antibacterial peptide of acquisition has apparent In Vitro Bacteriostasis effect without any processing after purification.
Description
Technical field
The invention belongs to gene engineering technology fields, more particularly to a kind of antibacterial peptide and its prokaryotic expression method and answer
With.
Background technique
Antibacterial peptide (antimicrobial peptide) is a kind of small molecule being naturally present in almost all creatures body
Polypeptide is the important component of the organism innate immunity, not only have broad-spectrum antibacterial action, to some fungies, helminth and
The tunicate virus in part also has killing effect.Different from Conventional antibiotic, antibacterial peptide is not easy to cause bacterium and generates drug resistance,
And also there is killing effect to the resistance to bacterial strain of antibiotic, huge potentiality are shown in novel antibacterial course of drug development.But day
The deficiencies of there is also sources to be limited for right antibiotic, potency is lower, longer polypeptide sequence leads to artificial synthesized higher cost.Cause
This, exploitation and preparation can overcome the antibacterial peptide of new generation of disadvantages mentioned above to be of great significance.
Natural antibacterial peptide is mostly cationic antibacterial peptide (charge number is between+2~+9), and can form stable alpha-helix
Structure.Alpha-helix can be located at the end N- or the end C- of peptide chain, have water rouge amphipathic, be the conformation base that antibacterial peptide plays antibacterial activity
Plinth.It is generally believed that positively charged antibacterial peptide is adsorbed to negatively charged bacterium plasma membrane surfaces by electrostatic attraction effect, into
And amphipathic helix conformation is formed, this conformation facilitates the hydrophobic surface insertion cell membrane phospholipid bilayer of antibacterial peptide, thus
Bacterium plasma membrane is destroyed, bactericidal effect is played.Existing research shows, improve in a certain range antibacterial peptide hydrophobicity and net positive electricity
Lotus number can enhance its bactericidal activity, this is because hydrophobicity increases the energy that can enhance the hydrophobic grouping insertion bacterium plasma membrane of antibacterial peptide
Power, and the binding ability of antibacterial peptide Yu bacterium plasma membrane can be enhanced by increasing net charges.In addition, enhancing hydrophobicity and net charges
It is more advantageous to the stability for improving alpha-helix.
Cecropin A (cecropin A) is a kind of cationic antibacterial peptide found from insect earliest, and precursor peptide is by 61
Amino acid profile.Cathelicidins antibacterial peptide is most important one kind cationic antimicrobial in mammal Endogenous antimicrobial polypeptide
Peptide, BMAP-27 are the representatives of Cathelicidins family, and mature peptide is made of 129 amino acid residues.It is living to obtain antibacterial
Property strong, novel antimicrobial peptide that peptide chain is shorter, the present invention analyzing both peptides using natural cecropin A and BMAP-27 as source
On the basis of alpha-helix, net positive charge number, hydrophobicity and the physicochemical properties such as amphipathic, one is devised by 32 amino acid residues
The cationic antibacterial peptide of composition, and establish the prokaryotic expression method of the novel antimicrobial peptide.Bacteriostatic test is the result shows that the antibacterial
Peptide has good antibacterial activity.
Summary of the invention
The purpose of the present invention is to provide a kind of antibacterial peptide and its prokaryotic expression method and applications.Antibacterial peptide of the invention is
A kind of novel cationic antibacterial peptide is made of 32 amino acid residues, and the recombination that prokaryotic expression method of the invention obtains
Antibacterial peptide has apparent In Vitro Bacteriostasis effect without any processing after purification.
To achieve the above object, the present invention takes following technical scheme:
A kind of encoding gene of antibacterial peptide cecropin-BM, nucleotide sequence is as shown in SEQ ID No.1.
Preferably, carrying out the primer sequence of PCR amplification to the expression vector is 8:
P1:CGCGGATCCATGAGCTATGGCCAGGGCCAGTTTTTTCGTGAAATTGAAAATCT GAAAGAATATTT
CA;
P2:CCGCCTTTCGCCACATCCGGGCTGCTCGCGTTGAAATATTCTTTCAGATTTTC AATTTCA;
P3:GATGTGGCGAAAGGCGGTCCTCTGTTTAGCGAAATTCTGAAAAATTGGAAAGA TGAAAGC;
P4:AAGCTCACAACTGGCTCTGAATAATCTTTTTATCGCTTTCATCTTTCCAATTT TTCAGA;
P5:CAGAGCCAGATTGTGAGCTTTTACTTCAAACTGTTTGAGAACTTGAAAGATGA GGAGTAA;
P6:CGCAAACACAAAGCTCAGAATACGCACAAATGTCATTACTCCTCATCTTTCAA GTTCTCA;
P7:ATTCTGAGCTTTGTGTTTGGCTGGTGCTGGCGCTGCGTTTTAAGCGTTTTCGT AAAAAG;
P8:CGGAATTCGCTCAGCTTCTTAAACAGTTTTTTGAACTTTTTACGAAAACGCTT AAAACG.
The present invention also provides the recombinant vector of antibacterial peptide cecropin-BM a kind of, which includes such as SEQ ID
The encoding gene of cecropin-BM shown in No.1.
The present invention also provides a kind of antibacterial peptide cecropin-BM, as the nucleotide sequence as shown in SEQ ID No.10
Coding.
The present invention also provides a kind of methods of antibacterial peptide cecropin-BM prokaryotic expression, method includes the following steps:
Obtain the encoding gene of antibacterial peptide cecropin-BM;
Building recombinant vector: the pcr amplification product of the encoding gene of antibacterial peptide cecropin-BM is connected to pMD19-T
In cloning vector, building obtains recombinant plasmid pMD19-INF-cecropin-BM;Digestion recombinant plasmid pMD19-INF- respectively
Cecropin-BM and prokaryotic expression carrier pET-28a (+), the recombination that building obtains the encoding gene comprising cecropin-BM carry
Body pET28-INF-cecropin-BM;
Expression antibacterial peptide cecropin-BM: recombinant vector pET28-INF-cecropin-BM is converted to BL21 bacterial strain
In, the bacterium comprising antibacterial peptide cecropin-BM is collected after inducing using IPTG.
Preferably, the method also includes: using containing the PBS re-suspension liquid of urea and imidazoles handle centrifugal breaking after cell bacterium
Liquid is then centrifuged and collects precipitating, obtains supernatant;
The supernatant is purified using Ni column affinity chromatography, obtains antibacterial peptide cecropin-BM after purification.
The present invention also provides antibacterial peptide cecropin-BM to inhibit answering in Escherichia coli and Staphylococcus aureus growth
With.
The present invention has following technical characterstic:
The recombinant vector of encoding gene of the present invention using Prokaryotic expression vector construction comprising antibacterial peptide cecropin-BM,
Realize the prokaryotic expression of antibacterial peptide cecropin-BM, method is simple and easy, the recombinant antibacterial peptide of acquisition after purification without appoint
Where reason has apparent In Vitro Bacteriostasis effect.
Detailed description of the invention
The amphipathic prediction of Fig. 1 Cecropin-BM.
Fig. 2 SOE-PCR gel electrophoresis result.
Fig. 3 Tricine-SDS-PAGE gel electrophoresis result (1:pET-Cecropin-BM plasmid expression holoprotein;2:
PET-28a (+) plasmid expression holoprotein;3: pET-Cecropin-BM plasmid expression albumen after purification;4: pET- after purification
28a (+) empty plasmid expresses albumen).
Fig. 4 recombinant protein bacteriostatic test (A:E.coli, B: S. aureus L-forms;1: kanamycins (100 μ g);2:Cecropin-BM
(100μg);3:Cecropin-BM (50 μ g);4::Cecropin-BM (25 μ g);5:pET-28a (+) empty carrier plasmid control)
Specific embodiment
Following specific embodiments are the further explanations to method provided by the invention and technical solution, but are not construed as
Limitation of the present invention.
One, the material and source used in the specific embodiment of the invention is as follows:
1 strain and plasmid
DH5 α Chemically Competent Cell (Beijing Qing Kexin industry Bioisystech Co., Ltd);BL21(DE3)
Chemically Competent Cell (Shanghai Wei Di Bioisystech Co., Ltd);(TaKaRa is public by pMD19-T Vector
Department);PET-28a (+) carrier, E.Coli, staphylococcus aureus are all from the preservation of this laboratory.
2 reagents
High fidelity enzyme PrimeSTAR HS DNA Polymerase, Taq enzyme, 10 × Loading of agarose gel electrophoresis
Buffer, DL1000DNA Marker, restriction endonuclease EcoR I connect DNA Ligation with BamH I, plasmid
Nickel column His60Ni Gravity Columns used in Kit, purifying protein is TaKaRa Products;AxyPrep Plasmid DNA
Small volume of reagent box is Axygen Products;PCR product purification kit, BCA protein quantification kit are purchased from U.S. logical sequence biology
Company;Protein Marker used in Tricine-SDS-PAGE is not moral biological product;On Tricine-SDS-PAGE albumen
Sample buffer (5 ×) is Beyotime product.
Two, the prokaryotic expression method of antibacterial peptide cecropin-BM is as follows in the specific embodiment of the invention:
The design and physicochemical property of 1 novel antimicrobial peptide cecropin-BM is predicted
Cecropin A (accession number: NP_ is analyzed using online tool SOPMA (https: //npsa-prabi.ibcp.fr/)
001037462) with the secondary structure of BMAP-27 (accession number: NP_001037462), alpha-helix amino acid in two kinds of peptide chains is obtained
Sequence.Choose the alpha-helix segment from above two peptide be combined, using online tool ExPASy (https: //
Web.expasy.org/protparam/) the charge number and hydrophilic and hydrophobic of the peptide fragment after analysis combination, using Antheprot
6.9.3 software carries out amphipathic prediction.Comprehensive alpha-helix, charge, hydrophobicity and the parameters such as amphipathic, determine the ammonia of novel antimicrobial peptide
The novel antimicrobial peptide is named as cecropin-BM by base acid sequence.
The prokaryotic expression of 2 Cecropin-BM
The design of 2.1 nucleotide sequences
Cecropin-BM is expressed using two-cistron expression vector, with the 21-85 ammonia of INF- γ (NP_776511)
Base acid sequence is as the first cistron, using the nucleotide sequence of cecropin-BM as the second cistron.It is close according to Escherichia coli
After the inclined preferendum of numeral carries out genetic modification, two gene orders are connected with 5 '-GAGGAGTAATGACA-3 ', merge gained INF-
Cecropin-BM gene order is as follows:
(single underscore is the first cistron, and overstriking letter is catenation sequence, and double underline is
Second cistron sequence).
2.2 PCR amplification
8 primers are designed, BamH I and I restriction enzyme site of EcoR is introduced respectively at the 5 '-ends of two primers of head and the tail, passes through weight
Folded extension PCR method synthesizes INF-cecropin-BM gene.Primer synthesizes (table by Hangzhou Qing Ke Zi Xi Bioisystech Co., Ltd
1)。
PCR amplification point 2 steps carry out: the first step, be separately added into 20 μ L PCR systems primer P1~P4, primer P5~
P8, amplification obtain two kinds of PCR products, are respectively designated as P14 and P58;PCR product is added in 20 μ L PCR systems in second step
P14 and P58 expands INF-Cecropin-BM full-length gene using P1 and P8 as upstream and downstream primer.Reaction system is shown in Table 2.PCR is anti-
Answer condition are as follows: 94 DEG C of 5min, (98 DEG C of 30s, 57 DEG C of 5s, 72 DEG C of 15s~1min) × 25 circulations, 72 DEG C of extension 10min.PCR
Product is detected with 1.5% agarose gel electrophoresis.
Table 1.PCR primer
Note: underscore is restriction enzyme site
Table 2.PCR reaction system
The building of 2.3 pMD19-INF-cecropin-BM plasmids
Using PCR product Purification Kit cecropin-BM product, carry out adding A end reaction, reaction using Taq enzyme
Condition is 72 DEG C, 30min, and reaction system is shown in Table 3.Cecropin-BM with A tail is subcloned into pMD19-T carrier, is converted
DH5 α competent cell, picking positive bacterium colony expand culture, and bacterium solution is sent to Hangzhou Qing Ke Zi Xi Bioisystech Co., Ltd and is sequenced.
Table 3.cecropin-BM product adds A end reaction system
Expand culture in LB liquid medium and correct DH5 α is sequenced, is mentioned using AxyPrep Plasmid DNA small volume of reagent box
Take Plasmid DNA.Extracting method by specification carries out.
The building of 2.4 expression vector pET28-INF-cecropin-BM
2.4.1 plasmid and carrier double digestion
PMD19-INF-cecropin-BM plasmid and pET-28a (+) carrier are used into restriction enzyme BamH I and EcoR I respectively
Double digestion is carried out in 10 × K buffer, 37 DEG C of digestions are stayed overnight.By pMD19-INF-cecropin-BM plasmid and pET-28a
The double enzyme digestion product of (+) carrier 10:1 mixing in molar ratio (total volume is 5 μ L), sets in 65 DEG C of water-baths and reacts 2min, after taking-up
I 5 μ L of Solution is added in ice bath 2min immediately, and 16 DEG C of connections are overnight.
2.4.2 plasmid converts
PET28-INF-cecropin-BM plasmid is added in BL21 competence, after ice bath stands 25min, sets 42 DEG C of water
Heat shock 45s in bath, and be immediately inserted into ice bath and stand 2min.It is added the not antibiotic sterile LB medium of 700 μ L, 37
DEG C, 200rpm shaking culture 60min.100 μ L bacterium solution even spreads are drawn to the solid LB media surface containing kanamycins, are set
10~14h is cultivated in 37 DEG C of incubators.Picking single colonie is inoculated in LB liquid medium of the 3mL containing kanamycins, 37 DEG C,
200rpm shaking culture to bacterium solution muddiness, company of Song Qing section is sequenced.
2.4.3 destination protein inducing expression
Picking positive transformant is inoculated in LB culture medium, and 37 DEG C, 200rpm shaking overnight incubation take 50 μ L bacterium solutions to be inoculated in
In 500mL LB liquid medium, 37 DEG C, 230rpm shaking culture to OD570Up to 0.6, IPTG to final concentration of 0.75mM is added,
37 DEG C of inducing expression 4h.There is the BL21 (DE3) of pET28a (+) empty carrier plasmid for control conversion.
After inducing expression, thalline were collected by centrifugation (4 DEG C, 10000g be centrifuged 30min), with urea containing 8M, 20mM imidazoles
Thallus, ultrasonication thallus (4 DEG C, power 40%, ultrasound opens 2s, closes 4s, total 30min), 10000g centrifugation is resuspended in PBS liquid
30min collects supernatant.
2.4.4 Ni column affinity chromatography
Above-mentioned supernatant is taken, destination protein is purified using Ni column affinity chromatography, the specific steps are as follows: (1) tie using 5mL
Close buffer (urea containing 8M, 20mM imidazoles) rinse Ni column;(2) be added 5mL recombinant protein supernatant, be mixed by inversion so that albumen with
Ni column sufficiently combines;(3) Ni column is placed vertically, after filling settlement behind bottom, the liquid in net pillar is flowed, after collecting column
Liquid;(4) step 2 and 3 is repeated, until having filtered all supernatants;(5) 5mL rinsing liquid is added into pillar and (contains 8M
Urea, 40mM imidazoles), it is mixed by inversion, stands, after filling settlement, then liquid in net column is flowed, so that foreign protein is sufficiently eluted;
(6) 1mL eluent (urea containing 8M, 300mM imidazoles) is added into pillar, mixing of turning upside down stands, after filling settlement, fills
Divide liquid in collection column, obtains destination protein.Step 1~6 are repeated sufficiently to collect destination protein.
2.4.5 Tricine-SDS-PAGE gel electrophoresis
The destination protein of purifying is after BCA kit carries out protein quantification, and in Tricine-SDS-PAGE gel, (4% is dense
Contracting glue, 10% spacing glue, 20% separation gel) on carry out electrophoresis, the bright dyeing of coomassie photographs to record result.
The identification of 3 recombinant protein antibacterial activities
Antibacterial activity identification is carried out using agarose plate diffusion method, E.coli and S. aureus L-forms used are that this laboratory is protected
The clinical separation strain deposited.Specific step is as follows: (1) cultivating E.coli and S. aureus L-forms to logarithm in LB liquid medium respectively
Growth period (OD570For 0.4-0.6);(2) bacterium solution is subjected to 10 times of doubling dilutions, and 50 μ L bacterium solutions is taken to be respectively coated in solid LB
On plate, bacterium colony counting is carried out after 37 DEG C of culture 16h;(3) bacterium solution of E.coli and S. aureus L-forms are diluted to 0.5 Maxwell standard list
Position (1.5 × 108CFU/mL), 50 μ L bacterium solutions difference even spread is taken on LB plate;(4) using aseptic card punch above-mentioned flat
It is punched on ware, every hole is separately added into 25 μ g, 50 μ g and 100 Cecropin-BMs of the μ g through Ni column purification.It is positive that kanamycins is set
Control wells (100 hole μ g/), negative control hole (pET-28a (+) empty plasmid expression product, 100 holes μ g/ are added in every hole), 37 DEG C of trainings
Fungistatic effect is observed after supporting 14h.
Three, the physicochemical property and antibacterial activity of antibacterial peptide cecropin-BM
The prediction of 1 recombinant protein c ecropin-BM physicochemical property
Bioinformatic analysis the result shows that, the present invention designed by cecropin-BM alpha-helix degree with higher, only
Charge number is+11, is hydrophobic proteins, and property stablizes (table 4).It is amphipathic analysis the result shows that, cecropin-BM peptide chain has
Good amphipathic (Fig. 1).
Table 4.Cecropin-BM secondary facility and physicochemical property prediction
2 PCR product gel electrophoresis results
As shown in Fig. 2, the cecropin-BM genetic fragment size for using Overlap extension PCR method to synthesize is 320bp, and it is pre-
Phase size is consistent.Sequencing result shows that sequence is completely correct.
3 Tricine-SDS-PAGE electrophoresis results
Destination protein after purification forms 2 band on Tricine-SDS-PAGE gel, and molecular size range is respectively
8.23kDa and 4.8kDa is consistent (Fig. 3) with expected IFN-His and cecropin-BM-His molecular size range, shows
Cecropin-BM successful expression.
4 recombinant protein c ecropin-BM bacteriostatic activity test results
As shown in figure 4, the sample-adding of 25~100 μ g recombination cecropin-BM to E.coli and S. aureus L-forms culture plate is added
Kong Houjun can form apparent inhibition zone, and additional amount is higher, and inhibition zone is bigger, and pET-28a (+) empty plasmid expression product is without suppression
Bacterium effect.The antibacterial circle diameter that 100 μ g cecropin-BM are formed on E.coli and S. aureus L-forms agarose plate is respectively
2.9cm and 2.8cm, and the antibacterial circle diameter that 100 μ g are formed on E.coli and S. aureus L-forms agarose plate be respectively 3.2cm and
3.1cm (table 5).The above results show that cecropin-BM of the present invention has preferable In Vitro Bacteriostasis.
Table 5.Cecropin-BM bacteriostatic test result
The method of the present invention that the above embodiments are only used to help understand and its core concept.It should be pointed out that for
For those skilled in the art, without departing from the principle of the present invention, if can also be carried out to the present invention
Dry improvement and modification, these improvement and modification are also fallen into the claims in the present invention protection scope.
Sequence table
<110>Zhejiang University
<120>a kind of antibacterial peptide and its prokaryotic expression method and application
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tttgagaact tgaaagatga ggagtaatga catttgtgcg tattctgagc tttgtgtttg 240
cgctggtgct ggcgctgcgt tttaagcgtt ttcgtaaaaa gttcaaaaaa ctgtttaaga 300
agctgagc 308
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Claims (7)
1. a kind of encoding gene of antibacterial peptide cecropin-BM, which is characterized in that the nucleotide sequence of the encoding gene such as SEQ
Shown in ID No.1.
2. the encoding gene of antibacterial peptide cecropin-BM according to claim 1 a kind of, which is characterized in that the table
The primer sequence that PCR amplification is carried out up to carrier is 8:
P1:CGCGGATCCATGAGCTATGGCCAGGGCCAGTTTTTTCGTGAAATTGAAAATCT GAAAGAATATTTCA;
P2:CCGCCTTTCGCCACATCCGGGCTGCTCGCGTTGAAATATTCTTTCAGATTTTC AATTTCA;
P3:GATGTGGCGAAAGGCGGTCCTCTGTTTAGCGAAATTCTGAAAAATTGGAAAGA TGAAAGC;
P4:AAGCTCACAACTGGCTCTGAATAATCTTTTTATCGCTTTCATCTTTCCAATTT TTCAGA;
P5:CAGAGCCAGATTGTGAGCTTTTACTTCAAACTGTTTGAGAACTTGAAAGATGA GGAGTAA;
P6:CGCAAACACAAAGCTCAGAATACGCACAAATGTCATTACTCCTCATCTTTCAA GTTCTCA;
P7:ATTCTGAGCTTTGTGTTTGGCTGGTGCTGGCGCTGCGTTTTAAGCGTTTTCGT AAAAAG;
P8:CGGAATTCGCTCAGCTTCTTAAACAGTTTTTTGAACTTTTTACGAAAACGCTT AAAACG.
3. a kind of recombinant vector of antibacterial peptide cecropin-BM, which is characterized in that the recombinant vector includes such as SEQ ID No.1
Shown in cecropin-BM encoding gene.
4. a kind of antibacterial peptide cecropin-BM, which is characterized in that the antibacterial peptide cecropin-BM is by such as SEQ ID No.10
Shown in it is nucleotide sequence coded.
5. a kind of method of antibacterial peptide cecropin-BM prokaryotic expression, which is characterized in that method includes the following steps:
Obtain the encoding gene of antibacterial peptide cecropin-BM;
Building recombinant vector: the pcr amplification product of the encoding gene of antibacterial peptide cecropin-BM is connected to pMD19-T clone
In carrier, building obtains recombinant plasmid pMD19-INF-cecropin-BM;Digestion recombinant plasmid pMD19-INF- respectively
Cecropin-BM and prokaryotic expression carrier pET-28a (+), the recombination that building obtains the encoding gene comprising cecropin-BM carry
Body pET28-INF-cecropin-BM;
Expression antibacterial peptide cecropin-BM: recombinant vector pET28-INF-cecropin-BM is converted into BL21 bacterial strain, benefit
The bacterium comprising antibacterial peptide cecropin-BM is collected after being induced with IPTG.
6. a kind of method of antibacterial peptide cecropin-BM prokaryotic expression according to claim 5, which is characterized in that described
Method further include: heavy using containing the cell bacterium solution after the PBS re-suspension liquid of urea and imidazoles processing centrifugal breaking, being then centrifuged and collecting
It forms sediment, obtains supernatant;
The supernatant is purified using Ni column affinity chromatography, obtains antibacterial peptide cecropin-BM after purification.
7. antibacterial peptide cecropin-BM according to claim 1-6 is inhibiting Escherichia coli and golden yellow grape ball
Application in bacterium growth.
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