CN107827988A - It is chimeric to wear film antibacterial peptide T11N2 and its application - Google Patents
It is chimeric to wear film antibacterial peptide T11N2 and its application Download PDFInfo
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
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Abstract
New chimeric film antibacterial peptide T11N2, amino acid sequence such as SEQ ID NO are worn the invention discloses a kind of:Shown in 1, it is the chimeric polyeptides that cell-penetrating peptide Tat of the connection with 11 amino acid is formed on the basis of high activity antibacterial peptide N2.Antibacterial peptide T11N2 is simple in construction, safety low-poison, cost is relatively low, synthesis is relatively easy, is that a kind of Novel chimeric that can efficiently wear film and kill intracellular salmonella wears membrane sterilization peptide.The antibacterial peptide T11N2 of the present invention can be applied to the fields such as antibacterials, food additives, cosmetics, feed addictive, have wide application value and market prospects.
Description
Technical field
The present invention relates to biomedicine field, specifically, is related to a kind of be fitted together to and wears film antibacterial peptide T11N2 and its application.
Background technology
The appearance of the toxic side effect and its antibody-resistant bacterium of conventional antibiotic, people are promoted to look for new antimicrobial system
Agent.Natural antibacterial peptide is mostly the small peptide of 13-45 amino acid residue composition, has immune, suppression tumour of broad-spectrum antiseptic, regulation etc.
Various biological function, mechanism of action are unique, are one of the focuses of current Substitutes For Antibiotic research.But because its is excessive
Molecular weight and the hydrophilic nmature of itself, limit their applications in vivo to a certain extent.In addition, in animal husbandry, by Portugal
The disease such as incidence of disease such as mammitis, arthritis, typhoid fever, enteroidea, gastroenteritis and septicemia that grape coccus, salmonella trigger
Height, duration length, is easily propagated, and after healing easily repeatedly, huge economic losses is caused to aquaculture.Which part reason is this
A little bacteriums belong to facultative intracellular bacterial parasite, can infect into host cell intracellular, so as to host immune system and the externally applied drug of escaping
The lethal effect of thing, on the one hand, when feeding environment mutation or in animal body immunity degradation caused by other distresses,
Subinfection again can be caused；On the other hand, the transfer characteristic after pathogenic bacterial infection immunocyte also using immunocyte accelerates
Thalline shifts, and aggravates the state of an illness；In addition, even if persistent infection animal does not show symptom, it, which takes bacterium excreta, can also cause big face
The generation of hoarding fowl infectious disease.Due to being hindered by biofilm system, antibacterials especially polypeptide drugs molecule be difficult into
Enter into the cell of target organs and tissue and play a role.For problem above, people have developed a variety of coping styles, such as micro-pipe
Injection, electroporation and perforin method.Aforesaid way, though it is advantageous, it is big there is also deficiency, such as to cytositimulation, lead
Enter rate is relatively low and targeting is not strong etc..
In recent years, with micromolecule polypeptide such as MPG, PEP-1 of penetration cell film ability, TAT, Penetration,
BLFcin6 etc. is increasingly becoming the focus utilized in drug development.They can be with penetration cell film, therefore referred to as cell-penetrating peptide (cell-
Permeable peptides, CPP), CPP is usually the small peptide containing 5-30 amino acid, by natural, non-natural or chimeric egg
It is derived in vain, two major classes can be divided into：It is a kind of that thing is imported by chemical key connection, it is another kind of to form the non-of stabilization with importing thing
Covalent complex plays a role.CPP has the characteristics that hypotoxicity, to importing species type without specifically limited, by the extensive of people
Concern.The materials such as oligonucleotides, DNA, small peptide, albumen, nano particle, virus can be imported and played a role into the cell by it
(Heitz F etc., Twenty years of cell-penetrating peptides:from molecular
mechanisms to therapeutics.British journal of pharmacology,2009,157(2):195-
206).Had a good application prospect in fields such as disease target treatment, enhancing drug absorptions.
It is extra large earthworm antimicrobial peptide NZ17074 derived peptide by high activity antibacterial peptide N2, to G- bacteriums, especially Escherichia coli
There is good bactericidal effect with salmonella.Antibacterial peptide N2 can penetrate Escherichia coli outer membrane, be tied using intercalation mode and DNA
Close, and influence DNA replication dna, but the salmonella cell film after N2 processing is complete, and cell membrane penetrance is only after handling 2h
5.84% (Antibacterial and detoxifying activity of NZ17074analogues with multi-
layers of selective antimicrobial actions against Escherichia coli and
Salmonella enteritidis.Scientific reports,2017,7(1):3392.), this is largely limited
The elimination of its salmonella fallen to intracellular escape acts on.
The content of the invention
Chimeric film antibacterial peptide T11N2 and its application are worn it is an object of the invention to provide new.
In order to realize the object of the invention, Novel chimeric provided by the invention wears film antibacterial peptide T11N2, and its amino acid sequence is such as
SEQ ID NO:Shown in 1.
CPP used in the present invention is Tat (T11), and chimeric peptide is made up of two parts：Cell penetrating peptide Tat and antibacterial peptide N2 (SEQ ID
NO:2) Tat-N2, abbreviation T11N2, are represented by.
Polypeptide solid-state reaction method or Liquid phase peptides synthesis method synthetic antibacterial peptide T11N2 can be used.
At least one amino acid that the present invention is also provided in above-mentioned antibacterial peptide T11N2 is taken by its corresponding D types amino acid
Generation and formed antibacterial peptide.
Antibacterial peptide T11N2 provided by the invention can be pharmaceutically acceptable salt form.
Antibacterial peptide with modification group falls within protection scope of the present invention.It can be total in the C-terminal or N-terminal of the antibacterial peptide
Valency or non-covalent linking modification group, the modification group include but is not limited to acetyl group, amide groups, methyl, aldehyde radical, ester group,
N- alkyl.
The present invention also provides expression cassette, expression vector, cloning vector, transgenic cell line or engineering bacteria, it include comprising
Encode the nucleic acid of the antibacterial peptide T11N2 gene orders.
The present invention also provides applications of the antibacterial peptide T11N2 in extensive pedigree antibiotic or composition is prepared.
The present invention also provides the extensive pedigree antibiotic or composition prepared by the antibacterial peptide T11N2.
The bacterium is Gram-negative bacteria, including Escherichia coli, salmonella etc..
The antibacterial peptide T11N2 of the present invention can pass through the salmonella typhimurium inside cell membrane kill.
The present invention also provides the antibacterial peptide T11N2 and is preparing promotion skin wound healing medicine, suppressing Multidrug resistant bacteria
Application in medicine.
The medicine of the present invention or the formulation of composition can be pill, tablet, granule, capsule, sustained release preparation, injection
Agent, microparticle formulation, oral enteric agent or controlled release preparation etc..
The present invention also provides the antibacterial peptide T11N2 answering in food additives, cosmetics, feed additive field
With.
The present invention further provides food additives, cosmetics, the feed addictive containing the antibacterial peptide T11N2.
The present invention at least has following advantages and beneficial effect：
The present invention is cell-penetrating peptide Tat of the connection with 11 amino acid on the basis of high activity antibacterial peptide N2, into
Product T11N2 after work(coupling has significantly similar to its parent peptide N2 antibacterial to salmonella typhimurium ATCC14028
Activity (2 μM of MIC) 2 μM of MIC.In addition, by dose-effect curve ratio compared with T11N2 and parent peptide N2, under subinhibitory concentration still
The effect of higher is presented.Intracellular sterilization test proves that 20 μM of N2 sterilizing rates of the T11N2 with respect to 100 μM improves 38.6%.Altogether
The ratio that focusing microscope display T11N2 enters born of the same parents is significantly larger than N2.MTT experiment shows that T11N2 only has smaller toxicity.
It is provided by the invention chimeric to wear that film antibacterial peptide T11N2 is simple in construction, safety low-poison, cost is relatively low, synthesis is relative holds
Easily, it is that a kind of Novel chimeric that can efficiently wear film and kill intracellular salmonella wears membrane sterilization peptide.The antibacterial peptide T11N2 of the present invention
The fields such as antibacterials, food additives, cosmetics, feed addictive are can be applied to, there is wide application value and market
Prospect.
Brief description of the drawings
Fig. 1 is the peptide T11N2 MALDI-TOF MS analysis charts after being synthesized in the embodiment of the present invention 1.
Fig. 2 is that T11N2 is real to RAW264.7 cells (Turnover of Mouse Peritoneal Macrophages cell line) toxicity in the embodiment of the present invention 2
Test result.Ciprofloxacin:Ciprofloxacin.
Amount effect curve experimental results of the Fig. 3 for T11N2 in the embodiment of the present invention 4 to extracellular salmonella.
Sterilization test results of the Fig. 4 for T11N2 in the embodiment of the present invention 5 to intracellular salmonella.Cip:Ciprofloxacin.
It is common with RAW264.7 cell incubations, fluorescence after Fig. 5 carries out FITC fluorescence labelings for T11N2 in the embodiment of the present invention 6
Focus on microscope experiment image.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The culture medium and buffer formulation being related in following examples：
MH culture mediums：Casein hydrolysate 17.5g/L, beef extract powder 5g/L, starch 1.5g/L.
MHA culture mediums：2% agar powder is added in MH culture mediums.
The protein molecular method for determination of amount being related in following examples is MALDI-TOF MS methods.
The method for the protein purification being related in following examples is efficient liquid phase HPLC methods.
The strain being related in following examples is shown in Table 1, and these strains are available to the public, without carrying out preservation.
Table 1 is for examination strain
The chimeric preparation for wearing film antibacterial peptide of embodiment 1
In high activity antibacterial peptide N2 (SEQ ID NO:2) cell-penetrating peptide of the connection with 11 amino acid on the basis of
Tat, design Novel chimeric and wear film antibacterial peptide T11N2, its amino acid sequence such as SEQ ID NO:Shown in 1.
Antibacterial peptide T11N2 can be prepared using Fmoc solid-phase synthesis.
1st, synthesis order：From sequence C end to N-terminal, step is as follows：
1) n equivalent resins are weighed and are put into reactor, DCM swelling half an hour is added, then takes out DCM, add in sequence
First amino acid (Fmoc-Asn (Trt)-OH) 2n equivalent, add the DIEA of 2n equivalents, (refer to make in right amount by appropriate DMF, DCM
Resin fully expands), DIEA (diisopropylethylamine), DMF (DMF), DCM, nitrogen advertises 60min,
Then 5n equivalents of methanol is added, half an hour is reacted, filtrate is taken out, with DCM, methanol cleaning.
2) second amino acid (Fmoc-Cys (Trt) -0H) 2n equivalent in sequence, 2n equivalents HBTU are added into reactor
(1- hydroxyls, benzo, three chlorazol tetramethyl hexafluorophosphates), DIEA, nitrogen advertises 60min, takes out filtrate and is detected with ninhydrin,
Then blocked with pyridine and acetic anhydride, finally cleaned, added appropriate liquid of raising one's hat and remove Fmoc protection groups, clean, ninhydrin inspection
Survey.
3) different aminoacids in sequence are sequentially added according to the mode of step 2) and carries out reaction until last amino acid
Fmoc-Tyr(tbu)-OH。
4) remove, pour into flask from reaction column after resin is drained with vacuum, cutting liquid (cutting is added into flask
Liquid addition is 10ml/ grams), cutting liquid composition is：TFA 95%, two mercaptan 2%, tri isopropyl silane 2%, water 1%.Concussion
Resin is filtered after reaction.
5) a large amount of ether centrifugations are added in the filtrate obtained and obtain crude product.
6) air oxidation send MS to detect to thick peptide after 24 hours in the basic conditions, and oxidation obtains step oxidation crude product completely.
7) second oxidation (iodine oxidation) is carried out after the completion of step oxidation, is purified after obtaining second oxidation crude product.
2nd, peptide purification：
Crude product is purified to high performance liquid chromatography and requires purity, purity used is in test>90%.
3rd, polypeptide freezes
Purified liquid, which is put into freeze dryer, to be freeze-dried, and is lyophilized into white powder.
Molecular weight determination uses MALDI-TOF-MS (MALDI-TOF), as a result sees Fig. 1.
Antibacterial peptide T11N2 is made up of 32 amino acid, molecular weight 4111.8Da, isoelectric point 11.65.
The chimeric cytotoxicity experiment for wearing film antibacterial peptide of embodiment 2
1st, logarithmic phase RAW264.7 cells (Turnover of Mouse Peritoneal Macrophages cell line) are collected, adjust concentration of cell suspension, often
Hole adds 7 × 103Individual cell, 5%CO2, 37 DEG C of incubations, bottom hole (96 hole flat underside) is paved with to cell monolayer.
2nd, the antibacterial peptide T11N2 solution (being dissolved with 0.01M phosphate buffer) of various concentrations is prepared, while sets three
Negative control hole, 37 DEG C, 5%CO2Cultivate 1-5h.
3rd, 20ul MTT solution is added per hole, continues to terminate culture after cultivating 4h, carefully sucks nutrient solution in hole.
4th, 150ul dimethyl sulfoxide (DMSO)s are added per hole, put low-speed oscillation 10min on shaking table.In enzyme-linked immunosorbent assay instrument
The light absorption value in each hole is measured at OD490nm.
5 while zeroing hole (culture medium, MTT, dimethyl sulfoxide (DMSO)) is set, (cell, the medicine of same concentrations are molten for control wells
Solve medium, nutrient solution, MTT, dimethyl sulfoxide (DMSO)).
As a result as shown in Fig. 2 figure it is seen that T11N2 toxicity is right with respect to for parent peptide N2 after connection CPP
The toxicity of RAW264.7 cells somewhat strengthens, and when concentration is 256ug/ml, cell survival rate is 91%.
The anti-salmonella Activity determination of embodiment 3
Antibacterial peptide T11N2 is to the minimal inhibitory concentration (MIC) of salmonella with reference to (Tian etc., Expression such as Tian
of antimicrobial peptide LH multimers in Escherichia coli C43(DE3).Applied
Microbiology and Biotechnology,2009,83(1):143-149) the micro broth dilution method established, according to tool
Body situation is slightly changed, and concrete operations are as follows：
1) picking strains tested monoclonal is to MH culture mediums, 37 DEG C, 250rpm, shaken overnight culture；
2) antibacterials are diluted in 1.5mL sterile centrifugation tubes by 2 times of gradient series, concentration is the 10 of final concentration respectively
Times；
3) strains tested is forwarded in MH fluid nutrient mediums 37 DEG C respectively with 1% inoculum concentration, 250rpm shaken cultivations
To 0.5 Maxwell standard than turbid；
4) will for examination bacteria culture fluid dilute 1000 times (final cell concentration is about 105CFU/mL), it is transferred to steril cell
In culture plate, per hole containing the μ L of bacterium solution 90 after dilution；
5) the μ L of medicine 10 added after dilution, each 3 Duplicate Samples of concentration, and it is reserved without drug-negative control wells.Add nothing
Bacterium growth plate lid, 37 DEG C of static gas wave refrigerator to negative controls vacate now macroscopic obvious muddy bacterium solution after sealing membrane closure.
Tissue Culture Plate is taken out, observed result, MIC value is the Cmin that can substantially suppress strain subject growth.Such as jump
The inconsistent situation of result, then retest between hole or Duplicate Samples.
T11N2 Determination of Antibacterial Activity results are as shown in table 2.
The T11N2 of table 2 antibacterial activity
As shown in Table 2, T11N2 has to salmonella acts on compared with high inhibition, to most strong pathogenic salmonella typhimurium
ATCC 14028 MIC value is 2 μM.
Amount effect curves of the T11N2 of embodiment 4 to extracellular salmonella
Method is similar to MIC determination methods.It is specific as follows：
1) picking strains tested monoclonal is to MH culture mediums, 37 DEG C, 250rpm, shaken overnight culture；
2) antibacterials are diluted in 1.5mL sterile centrifugation tubes by 2 times of MIC concentration series, concentration is final concentration respectively
10 times；
3) strains tested is forwarded in MH fluid nutrient mediums 37 DEG C respectively with 1% inoculum concentration, 250rpm shaken cultivations
To 0.5 Maxwell standard than turbid；
4) for 1000 times of bacteria culture fluid of examination, (final cell concentration is 10 for dilution5CFU/mL or so), it is transferred to steril cell
In culture plate, per hole containing the μ L of bacterium solution 90 after dilution；
5) the μ L of medicine 10 added after dilution, each 3 Duplicate Samples of concentration, and it is reserved without drug-negative control wells.Add nothing
Bacterium growth plate lid, the clone's number contained is counted after 37 DEG C of static gas wave refrigerator 18-24h using plate dilution method after sealing membrane closure.
As a result as shown in figure 3, from figure 3, it can be seen that T11N2 is to mouse in the range of 0.125~1 times of MIC subinhibitory concentration
The bactericidal effect of salmonella typhi is higher than N2.
Sterilization tests of the T11N2 of embodiment 5 to intracellular salmonella typhimurium (S.t)
Cell culture is to concentration 5 × 105Cells/ml, 3000 × g/min centrifuge 10min, and PBS washings in triplicate, will
Cultivate to the S.t of mid-log phase with infection multiplicity (multiplicity of infection, MOI) 10:1 infection cell, in
37 DEG C contain 5%CO20.5h is incubated in incubator altogether.PBS rinses cell, adds fresh containing 150 μ g/L gentamicins
Nutrient solution, 37 DEG C are incubated 2h to kill extracellular bacteria.PBS is washed twice, and replaces medium to the T11N2 solution containing various concentrations,
Intracellular survival thalline number coated plate counts after 24h, and wherein cell addition 0.1%BSA and 0.1%Triton-X hanks delay
Salting liquid (Hanks buffered salinesolution) cracking is rushed, lysate is molten in the BSA containing 0.05%Tween-20
Coated plate counts after being diluted step by step in liquid.As a result Fig. 4 and table 3 are seen.
The T11N2 of table 3 intracellular bactericidal activity
From 3,10 μM of T11N2 of table 53.7% is improved relative to 50 μM of N2 intracellular bactericidal activities；20μM T11N2
80.5% and 38.6% has been respectively increased relative to the N2 of 50 μM and 100 μM；50 μM of T11N2 are relative to 50 μM and 100 μM of N2
99.6% and 98.7% has been respectively increased.N2 membrane efficiency of wearing significantly is lifted after fully indicating coupling T11.
The T11N2 of embodiment 6 cell-penetrating experiment
Six porocyte culture plates, are inoculated with RAW264.7 cells, and density is 2 × 105Individual/hole, overnight incubation (about 16h),
After cell attachment, fresh medium is changed, after 30min, removes nutrient solution, is changed containing finite concentration FITC marks
T11N2 DMEM culture mediums (serum-free, without dual anti-) 1.5mL.After hatching certain time, 1.5uM Hoechst 33342 are added
Dyeing.Remove nutrient solution, cell is washed 3 times with PBS, adds new DMEM culture mediums.Fluorescence microscopy Microscopic observation.
As a result as shown in figure 5, from fig. 5, it can be seen that the T11N2 with FITC marks is more into cytoplasm, on the contrary, N2
Into only having fragmentarily for cell, this demonstrate that connection Tat11 promotes N2 endocytosis.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>It is chimeric to wear film antibacterial peptide T11N2 and its application
<130> KHP171113993.5
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Phe Cys Trp Asn
1 5 10 15
Val Cys Val Tyr Arg Asn Ala Val Arg Val Cys His Arg Arg Cys Asn
20 25 30
<210> 2
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 2
Ala Phe Cys Trp Asn Val Cys Val Tyr Arg Asn Ala Val Arg Val Cys
1 5 10 15
His Arg Arg Cys Asn
20
Claims (10)
1. one kind is chimeric to wear film antibacterial peptide T11N2, it is characterised in that amino acid sequence such as SEQ ID NO:Shown in 1.
2. antibacterial peptide according to claim 1, it is characterised in that also including at least one amino acid by its corresponding D type
The antibacterial peptide that amino acid substitutes and formed.
3. antibacterial peptide according to claim 1, it is characterised in that the antibacterial peptide is pharmaceutically acceptable salt form.
4. antibacterial peptide according to claim 1 or 2, it is characterised in that covalent or non-in the C-terminal or N-terminal of the antibacterial peptide
Covalent attachment has modification group, and the modification group includes acetyl group, amide groups, aldehyde radical, methyl, ester group, alkyl.
5. expression cassette, expression vector, cloning vector, transgenic cell line or engineering bacteria, it is included comprising coding claim 1 institute
State the nucleic acid of antibacterial peptide gene sequence.
6. application of any one of the claim 1-4 antibacterial peptides in extensive pedigree antibiotic or composition is prepared.
7. the extensive pedigree antibiotic or composition that are prepared by any one of the claim 1-4 antibacterial peptides.
8. medicine according to claim 7 or composition, it is characterised in that the bacterium is Gram-negative bacteria, including big
Enterobacteria, salmonella.
9. medicine or composition according to claim 7 or 8, it is characterised in that formulation is pill, tablet, granule, glue
Wafer, sustained release preparation, injection, microparticle formulation, oral enteric agent or controlled release preparation.
10. application of any one of the claim 1-4 antibacterial peptides in food additives, cosmetics, feed additive field.
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| CN111171159A (en) * | 2020-01-21 | 2020-05-19 | 上海交通大学医学院附属仁济医院 | Antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacteria infection as well as preparation method and application thereof |
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| CN107266547A (en) * | 2017-06-26 | 2017-10-20 | 北京生泰云科技有限公司 | Extra large earthworm antimicrobial peptide NZ17074 derived peptides N2 and its application |
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| CN107266547A (en) * | 2017-06-26 | 2017-10-20 | 北京生泰云科技有限公司 | Extra large earthworm antimicrobial peptide NZ17074 derived peptides N2 and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171159A (en) * | 2020-01-21 | 2020-05-19 | 上海交通大学医学院附属仁济医院 | Antibacterial peptide TAT-KR-12 for resisting planktonic bacteria and intracellular bacteria infection as well as preparation method and application thereof |
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