CN107266585A - A kind of MLH fusions antibacterial peptide and its preparation method and application - Google Patents
A kind of MLH fusions antibacterial peptide and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention discloses a kind of MLH fusion antibacterial peptides and its preparation method and application, using house fly antibiotic peptide, LL 37 and helicobacter pylori peptide as parent peptide, the amino acid sequence for obtaining MLH is analyzed using bioinformatics method, and according to E.coli codon preference encoding hybrid gene Ms LH genetic fragment.Target gene needed for being synthesized using SOE round pcrs, and be connected it with expression vector pET 32a (+), conversion to E. coli expression strains build recombination engineering bacteria pET 32a MLH/BL21 (DE3), through IPTG inducible protein amalgamation and expressions, bacteriostatic activity identification finally is carried out to the hybrid peptide of the amalgamation and expression of acquisition, as a result show, the hybrid peptide of amalgamation and expression has obvious fungistatic effect to staphylococcus aureus and bacillus subtilis.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of MLH merges antibacterial peptide and preparation method thereof and should
With.
Background technology
In the last few years, abuse of antibiotics phenomenon was increasingly serious, and increasing bacterium is developed into conventional antibiotic resistance
Property bacterial strain.Antibacterial peptide is because of its stable physicochemical property, the antibiotic property of wide spectrum and the features such as be not likely to produce drug resistance to target bacterial strain
Ideal substitute as antibiotic, it is of great interest.Antibacterial peptide is that a class is widely present in nature biotechnology body
Micromolecule polypeptide class material, be the important component of body congenital immunity.With the target spot and work different from antibiotic
With mechanism, also there is good fungistatic effect to antibiotic resistant bacteria, the antibiotic property with wide spectrum, to G+、G-Bacterium and fungi etc. are
There is certain inhibitory action.
The acquisition of current antibacterial peptide mainly has three kinds of approach：Extraction is directly separated from organism；Chemical method is directly closed
Into；Obtained with gene engineering method.Because content of the antibacterial peptide in biology is relatively low, directly extract that difficulty is larger and expense compared with
It is high, it is difficult to realize a large amount of productions；Chemical synthesis is also rarely used in large-scale production because cost is high.In the last few years, gene work
The fast development of journey technology, makes it possible large-scale production antibacterial peptide, has a good application prospect.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is that providing a kind of MLH fusions
Antibacterial peptide and its preparation method and application, produces Substitutes For Antibiotic, in medical and health, livestock-raising, food processing and agriculture
The fields such as industry, which are gathered around, to have broad application prospects.
The present invention uses following technical scheme：
A kind of MLH merges antibacterial peptide, and its amino acid sequence is as shown in SEQ ID NO.1.
It is preferred that, the nucleotide sequence of the MLH fusions antibacterial peptide is encoded as shown in SEQ.ID.NO.2.
A kind of MLH merges antibacterial peptide preparation method, comprises the following steps：
S1, using house fly antibiotic peptide, LL-37 and helicobacter pylori peptide as parent peptide, design heterozygous genes MLH encoding gene
Fragment；
S2, step S1 encoding gene segment is connected with expression vector pET-32a, builds recombinant expression carrier pET-
32a-MLH；
S3, recombinant expression carrier pET-32a-MLH is transferred in host cell E.coli BL21, builds recombination work
Journey bacterial strain pET-32a-MLH/BL21；
S4, to restructuring engineering strain pET-32a-MLH/BL21 carry out IPTG inducible protein amalgamation and expressions, through affine
Chromatograph the MLH fusion antibacterial peptides purified.
It is preferred that, in step S1, heterozygous antibacterial peptide MLH encoding gene is obtained according to the Preference of E.coli codons,
Full genome synthesis is carried out by SOE-PCR, three primers are designed, PCR primer is formed overlapping chain, three primers difference
For：Seq1 is as shown in SEQ ID NO.3 in sequence table；Seq2 is as shown in SEQ ID NO.4 in sequence table；In Seq3 such as sequence table
Shown in SEQ ID NO.5, seq1 and Seq3 5 ' end add EcoR I restriction enzyme sites, 3 ' end add Xho I restriction enzyme sites and
The common 146bp of protection base.
It is preferred that, according to the encoding gene segment sequences Design sense primer p1 such as sequence tables of the heterozygous antibacterial peptide MLH
Shown in middle SEQ ID NO.6, anti-sense primer p2 is carried out as shown in SEQ ID NO.7 in sequence table by template of the PCR primer
PCR is expanded, and PCR primer is carried out into gel extraction.
It is preferred that, purpose fragment and expression vector pET-32a skeletons are reclaimed, carries out connecting overnight in Solution I systems
Connect, competence E.coli DH5 α cells are prepared using CaCl2 methods, product overnight is imported into competent cell, build restructuring
Expression vector pET-32a-MLH.
It is preferred that, in step S4, using LB culture mediums, 37 DEG C of incubator overnight cultures to OD600 reach 0.6~0.8 addition
1.0mM IPTG, 37 DEG C of induced expression 3.5h.
It is preferred that, by the bacterium solution of step S4 induced expressions in 360W ultrasound 0.5s, stop 2s, 15min sonicated cells；4
DEG C, 12000rpm centrifuges 15min, collects supernatant；Wherein, 2mL bacterial lysates, 20 μ L albumen are added by every 100mg thalline
The thalline that Protease Inhibitor Cocktail is fully mixed and suspended centrifugal is collected.
It is preferred that, Ni is carried out to the supernatant of collection2+Ion affinity chromatography is purified, and is obtained MLH of the purity more than 90% and is melted
Close standby after antibacterial peptide, dialysis desalination and super filter tube concentration.
A kind of application of MLH fusions antibacterial peptide in feed addictive, antibacterial medicine preparation, health products, preservative is prepared.
Compared with prior art, the present invention at least has the advantages that：
1st, the invention using house fly antibiotic peptide, LL-37 and helicobacter pylori peptide as parent peptide, obtain with height
The fusion antibacterial peptide of antibacterial activity, to research and development novel antibacterial immune formulation, effectively substitutes or reduces antibiotic and added as feed
The use of agent, raising livestock product safety are significant.
2nd, because antibacterial peptide can produce toxicity in host cell, and expression quantity is relatively low, is easily degraded by host, and the present invention makes
PET-32a expression systems are used, the expression of control foreign protein that can be strictly, expression quantity is low in the presence of without derivant, leads to
The consumption for crossing control derivant IPTG controls the expression of fusion protein, so as to ensure that the steady of the foreign gene being harmful to host
It is fixed.
3rd, simple to operate, cost of the invention is low, by building engineering strain, is largely given birth to by the optimization of fermentation condition
Produce fusogenic peptide, it is easy to industrialize large-scale popularization application.
4th, fused polypeptide of the present invention is shown to staphylococcus aureus and withered grass gemma in E. coli
The inhibition of bacillus.
5th, to solve natural antibacterial peptide expression quantity low by the present invention, injures big to host, it is difficult to obtain by genetic engineering means
The problem obtained, itself or encoding gene can be applied in antibacterials preparation, be that the antibacterials of new generation are laid
Basis, is with a wide range of applications.
Below by drawings and examples, technical scheme is described in further detail.
Brief description of the drawings
Fig. 1 is of the invention using SOE-PCR products as masterplate, the electrophoretogram of the purpose fragment amplified with p1, p2 primer；
Fig. 2 is supernatant SDS-PAGE protein electrophoresis figures after sonicated cells of the present invention；
Fig. 3 is that fusogenic peptide of the present invention is determined to staphylococcus aureus biocidal property；
Fig. 4 is that fusogenic peptide of the present invention is determined to bacillus subtilis biocidal property；
Fig. 5 is implementation process schematic diagram of the present invention.
Embodiment
Antibacterial peptide preparation method is merged the invention provides a kind of MLH, with house fly antibiotic peptide, LL-37 and helicobacter pylori
Peptide is parent peptide, and the amino acid sequence for obtaining MLH is analyzed using bioinformatics method, and according to E.coli codon preferences
Encoding hybrid gene M LH genetic fragment, the target gene needed for being synthesized using SOE-PCR technologies, and by itself and expression vector
PET-32a (+) connection, conversion to E. coli expression strains builds recombination engineering bacteria pET-32a-MLH/BL21 (DE3),
Through IPTG inducible protein amalgamation and expression, bacteriostatic activity identification finally is carried out to the hybrid peptide of the amalgamation and expression of acquisition, as a result shown,
The hybrid peptide of amalgamation and expression has obvious fungistatic effect to staphylococcus aureus and bacillus subtilis.
Referring to Fig. 5, merging antibacterial peptide preparation method the invention discloses a kind of MLH, comprise the following steps：
The encoding gene segment synthesis of step 1, design heterozygous genes MLH；
The amino acid sequence of parent peptide is checked according to GenBank, is spliced by different fragments and is designed and utilize biological information
The amino acid sequence that method analysis obtains heterozygous genes MLH fusion antibacterial peptide is：
GWLKKIGKKIERVGQHTRKRIVQRIKAKKVFKRLEKLFSKIQN。
Heterozygous genes MLH coding gene sequence is obtained according to the Preference of E.coli codons, for the ease of carrier
Build, EcoR I restriction enzyme sites are added at 5 ' ends, 3 ' ends add Xho I restriction enzyme sites and protection base.
Encoding hybrid gene M LH fusion antibacterial peptide gene base sequence be：
CGGAATTCGGTTGGCTGAAAAAAATCGGTAAAAAAATCGAACGTGTTGGTCAGCACACCCGTAAACGTATCGTTCAG
CGTATCAAAGCTAAAAAAGTTTTCAAACGTCTGGAAAAACTGTTCTCTAAAATCCAGAACCTCGAGCGG；
The synthesis of full genome is carried out by SOE-PCR technologies, three primers seq1, seq2, seq3 are designed, by with mutual
The primer of end is mended, PCR primer is formed overlapping chain.
Three primer is：
seq1:5’-CGGAATTCGGTTGGCTGAAAAAAATCGGTAAAAAAATCGAACGTGTTGGTCAGCACACCCG
TAAA-3’；
Seq2:5’-AAAACTTTTTTAGCTTTGATACGCTGAACGATACGTTTACGGGTGTGCTGACCAACACG-
3’；
Seq3:5’-CCGCTCGAGGTTCTGGATTTTAGAGAACAGTTTTTCCAGACGTTTGAAAACTTTTTAGCTT
TGATACG-3’；
First by seq1 and seq2, masterplate carries out the amplification of first round PCR each other, amplified production T1 is obtained, then using T1 as mould
Version, seq1, seq3 are that primer carries out the second wheel amplification, and amplified production is MLH complete genome sequence after the second wheel amplification.
First round PCR amplification system (50 μ L) is：dd H238.5 μ L, 10 × PCR Buffer of O 5 μ L, MgCl2 3μL,
DNTPs2 μ L, the μ L of primer seq1 1, the μ L of primer seq2 1, the μ L of TaKaRa Taq 0.25.
Reaction condition：94 DEG C of preheating 5min；94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations；72 DEG C of extensions
5min。
Second, which takes turns amplification system (50uL), is：ddH237.5 μ L, 10 × PCR Buffer of O 5 μ L, MgCl2 3μL,dNTPs
2 μ L, the μ L of primer seq1 1, the μ L of primer seq3 1, the μ L of first round PCR primer T1 1, TaKaRaTaq 0.25 μ L.
Reaction condition：94 DEG C of preheating 5min；94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations；72 DEG C of extensions
5min。
Step 3, the primer according to heterozygous antibacterial peptide MLH gene order design correlation, referring to Fig. 1,
Sense primer p1：5’-CGGAATTCGGTTGGCTGAAAAAAAT-3’；
Anti-sense primer p2：5’-CCGCTCGAGGTTCTGGATTTTAGAGA-3’；
And PCR condition and system are finally determined, entering performing PCR as masterplate using the PCR primer that step 2 is obtained expands, and will
PCR primer carries out gel extraction.
PCR reaction systems (50uL) are：Pre mix taq 25uL, sense primer 1uL, anti-sense primer 1uL, template 1uL,
Water 22uL,
Wherein PCR reaction conditions are：94℃、5min；94 DEG C, 30s, 62 DEG C, 30s, 72 DEG C, 60s, totally 35 circulations；72
DEG C extension 10min.
Step 4, the PCR primer for respectively obtaining vector plasmid and step 3 carry out EcoR I and Xho I double digestions, reclaim
Purpose fragment and expression vector pET-32a skeletons.
Double digestion system (50uL) is：10 × Buffer 5uL, purpose fragment/carrier 35uL, water 5uL, EcoR I
2.5uL, Xho I 2.5uL, 37 DEG C of incubation 3h.
Step 5, the purpose fragment that step 4 is reclaimed and expression vector pET-32a skeletons enter in Solution I systems
Row is connected overnight.
Solution I systems (10uL) are：10 × DNA connection buffer 1uL, purpose after carrier 2uL, digestion after digestion
Genetic fragment 6uL, T4DNA ligase 1uL, 4 DEG C connect overnight.
Step 6, CaCl2 methods prepare competence E.coli DH5 α cells, and the product overnight in step 5 is imported into sense
By in state cell, the culture of positive colony is sent the sequence that sequencing company carries out recombinant plasmid by double digestion and bacterium colony PCR checkings
Determine, whether foreign gene is correctly inserted into analysis recombinant plasmid.
Bacterium colony PCR verification steps：Some bacterium colonies of picking are inoculated in 10mL LB culture mediums (blue or green containing 50 μ g/mL ammonia benzyls respectively
Mycin), after 37 DEG C of shaken overnight cultures, take culture PCR to react screening positive clone.50uL PCR systems are：Pre mix
Taq 25uL, sense primer 1uL, anti-sense primer 1uL, template 1uL, water 22uL, PCR condition are ibid.
Step 7, it will identify that correct positive plasmid is transferred in host cell E.coli BL21 (DE3) in step 6, picking
Positive single bacterium colony, 4mL LB culture mediums, 37 DEG C of incubated overnights；Then incubated overnight liquid is inoculated in 4mL according to 1% bacterium amount that connects
In LB culture mediums, 28 DEG C of 220rpm are cultivated to 1.0mM IPTG are added during OD600 values 0.6 or so, and 37 DEG C of 220rpm induce 3h.
12000rpm, centrifuges 15min, collects thalline, and sonicated cells collect broken rear cell supernatant.
Referring to Fig. 2,1 expression albumen Marker in Fig. 2,2 represent bacterial cell disruption supernatants, sonicated cells：By every
100mg thalline (weight in wet base) addition 2mL bacterial lysates (1 μ l DNase I (1,000U/mL) are added in 1mL bacterium extraction agents,
2 μ l Lysozyme (50mg/mL), per 1mL bacterial lysates in add 10 μ L protease inhibitor cocktails) ratio fully hangs
The floating thalline being collected by centrifugation；360W power, per circulating ultrasonic 0.5s, cools down 2s, crushes 15min；4 DEG C, 12000rpm centrifugations
15min, collects supernatant and carries out SDS-PAGE detections.
Step 8, to expression condition：Derivant IPTG concentration, expression time and expression temperature is optimized, it is determined that most preferably
Expression condition, carries out a large amount of induced expressions of fusion protein under optimum condition of the expression.
To expression condition：Derivant IPTG concentration, expression time and expression temperature is optimized, optimal expression condition
For：IPTG concentration is 1.0mM；The induced expression time is 3.5h；It is 37 DEG C to express temperature, is merged under optimum condition of the expression
A large amount of induced expressions of albumen.
Step 9, fusion protein are isolated and purified：In a large amount of induced expressions of optimal expression condition of step 8, supernatant is collected
Liquid.Carry out Ni2+Ion affinity chromatography is purified, and carries out Ni2+Ion affinity chromatography purifying is concretely comprised the following steps：
By supernatant after clasmatosis and Binding Buffer (Tris-HCl (pH7.9) 20mM, Imidazole 5mM,
NaCl 0.5M) isometric mixing back loading upper prop, flow velocity is 10 times of column volume/hours, and collection flows through liquid.
Pillar is rinsed using the Binding Buffer of 15 times of column volumes, foreign protein is washed away.
Use appropriate Elution Buffer (Tris-HCl (pH7.9) 20mM, Imidazole 500mM, NaCl 0.5M)
Elution, collects eluting peak, and the fusion protein purity of acquisition is more than 90%.The concentration of 10kDa super filter tubes is standby after dialysis desalination.
Step 10, by the fusion protein obtained in step 9 carry out biocidal property checking.
Bacteriostatic activity checking is carried out using agar hole diffusion method.
Refer in Fig. 3 and Fig. 4, figure, 1 is hybrid peptide, and 2 be water, and 3 be blank, and hybrid peptide after purification is to golden yellow Portugal
Grape coccus and bacillus subtilis have obvious bacteriostatic activity, and antibacterial circle diameter is respectively 12nm and 13nm.
The technological thought of above content only to illustrate the invention, it is impossible to which protection scope of the present invention is limited with this, it is every to press
According to technological thought proposed by the present invention, any change done on the basis of technical scheme each falls within claims of the present invention
Protection domain within.
Nucleotides sequence list
<110>Shaanxi Tech Univ
<120>A kind of MLH fusions antibacterial peptide and its preparation method and application
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<212> PRT
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25 30 35 40
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Claims (10)
1. a kind of MLH merges antibacterial peptide, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.1.
2. MLH according to claim 1 merges antibacterial peptide, it is characterised in that the coding MLH merges the nucleosides of antibacterial peptide
Acid sequence is as shown in SEQ.ID.NO.2.
3. a kind of MLH merges antibacterial peptide preparation method, it is characterised in that comprise the following steps：
S1, using house fly antibiotic peptide, LL-37 and helicobacter pylori peptide as parent peptide, design heterozygous genes MLH encoding gene piece
Section；
S2, step S1 encoding gene segment is connected with expression vector pET-32a, builds recombinant expression carrier pET-32a-
MLH；
S3, recombinant expression carrier pET-32a-MLH is transferred in host cell E.coli BL21, builds recombination engineering bacteria
Strain pET-32a-MLH/BL21；
S4, to restructuring engineering strain pET-32a-MLH/BL21 carry out IPTG inducible protein amalgamation and expressions, through affinity chromatography
The MLH fusion antibacterial peptides purified.
4. a kind of MLH fusions antibacterial peptide preparation method according to claim 3, it is characterised in that in step S1, according to
The Preference of E.coli codons obtains heterozygous antibacterial peptide MLH encoding gene, and full genome synthesis is carried out by SOE-PCR, if
Three primers are counted, PCR primer is formed overlapping chain, three primers are respectively：SEQ ID NO.3 institutes in seq1 such as sequence table
Show；Seq2 is as shown in SEQ ID NO.4 in sequence table；Seq3 is as shown in SEQ ID NO.5 in sequence table, seq1's and Seq3
5 ' ends add EcoR I restriction enzyme sites, and 3 ' ends add Xho I restriction enzyme sites and the common 146bp of protection base.
5. a kind of MLH fusions antibacterial peptide preparation method according to claim 4, it is characterised in that anti-according to the heterozygosis
Bacterium peptide MLH encoding gene segment sequences Design sense primer p1 is as shown in SEQ ID NO.6 in sequence table, and anti-sense primer p2 is such as
In sequence table shown in SEQ ID NO.7, performing PCR amplification is entered by template of the PCR primer, and glue is cut into PCR primer progress and is returned
Receive.
6. a kind of MLH fusion antibacterial peptide preparation method according to claim 5, it is characterised in that reclaim purpose fragment with
Expression vector pET-32a skeletons, carry out connecting overnight in Solution I systems, competence are prepared using CaCl2 methods
E.coli DH5 α cells, product overnight is imported into competent cell, builds recombinant expression carrier pET-32a-MLH.
7. a kind of MLH fusions antibacterial peptide preparation method according to claim 2, it is characterised in that in step S4, using LB
Culture medium, 37 DEG C of incubator overnight cultures to OD600 reach 0.6~0.8 addition 1.0mM IPTG, 37 DEG C of induced expression 3.5h.
8. a kind of MLH fusions antibacterial peptide preparation method according to claim 7, it is characterised in that step S4 is induced into table
The bacterium solution reached stops 2s, 15min sonicated cells in 360W ultrasound 0.5s；4 DEG C, 12000rpm centrifuges 15min, collects supernatant
Liquid；Wherein, by every 100mg thalline add 2mL bacterial lysates, 20 μ L protease inhibitor cocktails fully mix and suspend from
The thalline that the heart is collected.
9. a kind of MLH fusions antibacterial peptide preparation method according to claim 8, it is characterised in that to the supernatant of collection
Carry out Ni2+Ion affinity chromatography is purified, and obtains the MLH fusion antibacterial peptides that purity is more than 90%, dialysis desalination and super filter tube concentration
It is standby afterwards.
10. the MLH fusion antibacterial peptides described in claim 1 are preparing feed addictive, antibacterial medicine preparation, health products, anti-corrosion
Application in agent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110256570A (en) * | 2017-12-26 | 2019-09-20 | 杭州皇冠农业生物工程技术研究中心有限公司 | A kind of recombination fusion antibacterial peptide and application |
| CN110294809A (en) * | 2019-06-13 | 2019-10-01 | 东北农业大学 | Target staphylococcus aureus antibacterial peptide S2 and its preparation method and application |
| CN114853901A (en) * | 2022-03-15 | 2022-08-05 | 四川贝斯安贸易有限公司 | Construction and application of engineering bacteria for expressing antimicrobial peptide AFP1 fusion protein |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468109A (en) * | 2000-10-18 | 2004-01-14 | Ӣ����������ҽҩ�о���չ�ɷݹ�˾ | Vaccine composition |
| CN103467584A (en) * | 2013-09-03 | 2013-12-25 | 黑龙江八一农垦大学 | Method for obtaining and fermenting prokaryotic genetic-engineered hybrid cationic antimicrobial peptide CC |
| CN104448004A (en) * | 2014-12-03 | 2015-03-25 | 华侨大学 | Fused antibacterial peptide and preparation method thereof |
| CN104788568A (en) * | 2015-01-16 | 2015-07-22 | 黑龙江八一农垦大学 | Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof |
| CN105238809A (en) * | 2015-11-04 | 2016-01-13 | 黑龙江八一农垦大学 | Expressing method of antimicrobial peptide CC31 in bacillus subtilis |
| CN105255935A (en) * | 2015-11-04 | 2016-01-20 | 黑龙江八一农垦大学 | Expression method of antimicrobial peptide CC34 in bacillus subtilis |
| CN105693866A (en) * | 2016-03-03 | 2016-06-22 | 河南欧普生物科技有限公司 | Fusion antibacterial peptide and application thereof |
| CN105816912A (en) * | 2016-03-09 | 2016-08-03 | 上海交通大学医学院附属第九人民医院 | Infection preventing and osteoblast differentiation promoting antibacterial peptide modified titanium alloy prosthesis and making method thereof |
| CN106008718A (en) * | 2016-03-31 | 2016-10-12 | 西北民族大学 | Recombinant human-derived antimicrobial peptide C16LL-37 and application method thereof in streptococcus mutans bioactivity role |
| CN106366172A (en) * | 2016-10-17 | 2017-02-01 | 黑龙江八农垦大学 | Bigeminal peptide Cec Md2ys, preparation method and application |
| CN106432513A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Efficient hybrid antibacterial peptide LI and preparation method and application thereof |
| CN106478793A (en) * | 2016-10-17 | 2017-03-08 | 黑龙江八农垦大学 | A kind of three PEPC ec Md3cs, preparation method and application |
| CN106478792A (en) * | 2016-10-17 | 2017-03-08 | 黑龙江八农垦大学 | A kind of three PEPC ec Md3js, preparation method and application |
| CN106589136A (en) * | 2016-11-25 | 2017-04-26 | 东北农业大学 | Hybrid antibacterial peptide based on FV7 anti-bio-membrane, preparation method and application thereof |
-
2017
- 2017-07-13 CN CN201710572119.XA patent/CN107266585B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468109A (en) * | 2000-10-18 | 2004-01-14 | Ӣ����������ҽҩ�о���չ�ɷݹ�˾ | Vaccine composition |
| CN103467584A (en) * | 2013-09-03 | 2013-12-25 | 黑龙江八一农垦大学 | Method for obtaining and fermenting prokaryotic genetic-engineered hybrid cationic antimicrobial peptide CC |
| CN104448004A (en) * | 2014-12-03 | 2015-03-25 | 华侨大学 | Fused antibacterial peptide and preparation method thereof |
| CN104788568A (en) * | 2015-01-16 | 2015-07-22 | 黑龙江八一农垦大学 | Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof |
| CN105238809A (en) * | 2015-11-04 | 2016-01-13 | 黑龙江八一农垦大学 | Expressing method of antimicrobial peptide CC31 in bacillus subtilis |
| CN105255935A (en) * | 2015-11-04 | 2016-01-20 | 黑龙江八一农垦大学 | Expression method of antimicrobial peptide CC34 in bacillus subtilis |
| CN105693866A (en) * | 2016-03-03 | 2016-06-22 | 河南欧普生物科技有限公司 | Fusion antibacterial peptide and application thereof |
| CN105816912A (en) * | 2016-03-09 | 2016-08-03 | 上海交通大学医学院附属第九人民医院 | Infection preventing and osteoblast differentiation promoting antibacterial peptide modified titanium alloy prosthesis and making method thereof |
| CN106008718A (en) * | 2016-03-31 | 2016-10-12 | 西北民族大学 | Recombinant human-derived antimicrobial peptide C16LL-37 and application method thereof in streptococcus mutans bioactivity role |
| CN106366172A (en) * | 2016-10-17 | 2017-02-01 | 黑龙江八农垦大学 | Bigeminal peptide Cec Md2ys, preparation method and application |
| CN106478793A (en) * | 2016-10-17 | 2017-03-08 | 黑龙江八农垦大学 | A kind of three PEPC ec Md3cs, preparation method and application |
| CN106478792A (en) * | 2016-10-17 | 2017-03-08 | 黑龙江八农垦大学 | A kind of three PEPC ec Md3js, preparation method and application |
| CN106589136A (en) * | 2016-11-25 | 2017-04-26 | 东北农业大学 | Hybrid antibacterial peptide based on FV7 anti-bio-membrane, preparation method and application thereof |
| CN106432513A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Efficient hybrid antibacterial peptide LI and preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110256570A (en) * | 2017-12-26 | 2019-09-20 | 杭州皇冠农业生物工程技术研究中心有限公司 | A kind of recombination fusion antibacterial peptide and application |
| CN110294809A (en) * | 2019-06-13 | 2019-10-01 | 东北农业大学 | Target staphylococcus aureus antibacterial peptide S2 and its preparation method and application |
| CN114853901A (en) * | 2022-03-15 | 2022-08-05 | 四川贝斯安贸易有限公司 | Construction and application of engineering bacteria for expressing antimicrobial peptide AFP1 fusion protein |
| CN114853901B (en) * | 2022-03-15 | 2023-09-15 | 四川贝斯安贸易有限公司 | Construction and application of engineering bacteria for expressing antibacterial peptide AFP1 fusion protein |
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