CN106589136A - Hybrid antibacterial peptide based on FV7 anti-bio-membrane, preparation method and application thereof - Google Patents
Hybrid antibacterial peptide based on FV7 anti-bio-membrane, preparation method and application thereof Download PDFInfo
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- CN106589136A CN106589136A CN201611054376.6A CN201611054376A CN106589136A CN 106589136 A CN106589136 A CN 106589136A CN 201611054376 A CN201611054376 A CN 201611054376A CN 106589136 A CN106589136 A CN 106589136A
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- peptide
- antibacterial peptide
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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
-
- 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
Abstract
The invention relates to a hybrid antibacterial peptide based on an FV7 anti-bio-membrane, and a preparation method and an application thereof. The sequence of the hybrid antibacterial peptide is represented as the SEQ ID No.1. According to main physical and chemical parameters and structural and functional relationship of an original peptide and a hybrid peptide, an anti-bio-membrane fragment FV7 and an active centre fragment LL of a natural antibacterial peptide LL-37 are synthesized through a chemical method to obtain the hybrid peptide FV-LL. Through a series of in-vitro test, the antibacterial activities of the original peptides FV7 and LL and the hybrid antibacterial peptide FV-LL are verified, and through comparison, it is found that the FV-LL is greatly improved in antibacterial activity and also has anti-bio-membrane activity, so that through functional fragment hybridization, the antibacterial activity of the antibacterial peptide can be improved, thereby improving cell selectivity. Furthermore, the antibacterial mechanism of the hybrid antibacterial peptide is verified, thereby providing theoretical basis for application of the antibacterial peptide.
Description
Technical field
This invention belongs to agriculture animal and veterinary application, and in particular to a kind of heterozygosis based on FV7 antibiont films
Antibacterial peptide and preparation method and application.
Background technology
As the abuse of feeding antibiotic causes the generation of environmental pollution, drug residue and bacterial drug resistance, this is to agricultural
Greatly harm is brought with animal husbandry, the maximum of animal productiong potentiality has been had a strong impact on and has been played and green safety animal products
Supply.Therefore find the new of alternative antibiotic, noresidue, efficiently immunity catalyst become asking for current urgent need to resolve
Topic.The small molecule polypeptide of external microbe infringement is resisted as animal body, antibacterial peptide has broad spectrum antibacterial, without drug resistance
Property, avirulence, pollution-free and noresidue the advantages of, and heat stability is generally preferably, and additive capacity is little, complies fully with livestock products
Product security needs, is adapted to used in feed preparation, has the potential quality as feed additive of new generation.
In nature, antibacterial peptide species is various, widely distributed, but is seldom applied to medicine and herding field.Limit anti-
The factor of bacterium peptide application is a lot, wherein for some antibacterial peptides antibacterial activity it is low be limit the main impact of its application because
Element.It is one of effective way of high-efficiency antimicrobial preparation development that molecular modification is carried out to existing natural antibacterial peptide.Antibacterial peptide changes
Great majority are made with natural antibacterial peptide as female peptide, after reaching design requirement, will have therapeutic or preventative fragments of peptides to be used in peptide
Source body, to keep body health.Hybrid design is a kind of good strategy for optimizing these antibacterial peptide molecules.This method
Can be good at retaining the biologic activity of required fragment, and do not affected by the change of a certain parameter.Further, since only protecting
Active center is stayed, therefore this method can be significantly shorter peptide chain length, reduce chemosynthesis cost.
The content of the invention
Based on above weak point, it is an object of the invention to provide a kind of heterozygous antibacterial peptide based on FV7 antibiont films and
Preparation method and application, improve its antibacterial activity.
The present invention is realized by following technology：A kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, its sequence is such as
Shown in sequence table SEQ ID No.1.
The present invention also has following technical characteristic：
1st, a kind of preparation method of the heterozygous antibacterial peptide FV-LL based on FV7 antibiont films is as follows：
(1) choosing polypeptide fragment FV7 and LL carries out heterozygosis, obtains T1249 FV-LL；
(2) physicochemical property of FV7, LL and FV-LL, spiral wheel construction are analyzed by peptide database analysis software and
Prediction；
(3) antibacterial peptide for obtaining design is synthesized using solid-state chemical reaction method method, that is, is completed by Peptide synthesizer
The preparation of polypeptide, sequence is as shown in sequence table SEQ ID No.1.
2nd, a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films as above, is preparing treatment Gram-positive
Application in bacterium or gram positive bacterial infection disease medicament.
The heterozygous antibacterial peptide FV7 that the present invention is obtained only remains the active center fragment of former peptide FV7, not because of the change of parameter
And its activity is affected, peptide chain length is shortened, the cost of chemosynthesis is reduced.The heterozygous antibacterial peptide for obtaining simultaneously has wide spectrum
Antibiotic property, and with developing into the potentiality of antibacterium biomembrane medicine.Illustrate to strengthen antibacterial peptide by functional fragment heterozygosis
Antibacterial activity, improve cell selective, further demonstrate its Antibacterial Mechanism, the application for antibacterial peptide provides theoretical base
Plinth.
Description of the drawings
Fig. 1 is the helical wheel prognostic chart of former peptide LL and T1249 FV-LL.
Fig. 2 is the antibiont film activity comparison diagram of former peptide FV7 and T1249 FV-LL.
Specific embodiment
The design synthesis of 1 antibacterial peptide of embodiment
LL：The 17-29 aminoacid functional sequences of people derived antimicrobial peptide LL-37, this fragment is rich in the amphipathic of cation
The peptide chain of helical structure.Be identified as the most short sequence with antibacterial and active anticancer, can effectively with bacterial cell membrane
Anionic phospholipid combines and presents selective cytotoxicity, does not have toxicity to human cell.
By one section of sequence FV7 with antibiont film activity and polypeptide fragment LL heterozygosis, heterozygous antibacterial peptide FV-LL is obtained.
The helical wheel prognostic chart of T1249 FV-LL is as shown in Figure 1.Using Peptide synthesizer, synthesize above-mentioned three using solid-phase synthesis
Antibacterial peptide.The sequence and physical and chemical parameter of three peptides is as shown in table 1.
The sequence and physical and chemical parameter of 1 three peptides of table
A molecular weight：Molecular weight is measured by mass spectrum.
The physical and chemical index analysis of 2 antibacterial peptide of embodiment
The secondary structure of antibacterial peptide is analyzed by CD spectrum, is as a result shown, find in water environment T1249 with
Former peptide is presented random coil structure, and in the hydrophobic environment (50%TFE) and negatively charged protokaryon of simulation microbial film
In cell-membrane environment (30mM SDS), former peptide LL and T1249 FV-LL show certain α-helixstructure, and FV7 then tables
Reveal certain beta sheet conformation.
Shown by the stability analyses to antibacterial peptide, T1249 FV-LL has preferable resistance to heat treatment.However,
Under the physiological concentration of different salt ions, there is reduction by a small margin in the antibacterial activity of peptide FV-LL.
The antibacterial and antibiont film activity of 3 antibacterial peptide of embodiment
1 bacteriostatic activity
Peptide is configured as into 2.56mM/L storing liquids standby.Using the minimum of several antibacterial peptides of micro-broth dilution method
Mlc.Graded series, as diluent, are configured successively using doubling dilution using 0.01% acetic acid (containing 0.2%BSA)
Antibacterial peptide solution.Take 100 μ L of above-mentioned solution to be placed in 96 porocyte culture plates, then add isopyknic bacterium solution to be measured respectively
(～105Individual/mL) in each hole.It is respectively provided with positive control (antibacterial peptide is not contained containing bacterium solution) and negative control is (neither
Peptide is not contained containing bacterium solution) yet.With naked eyes, 37 DEG C of constant temperature culture 20h, have no that there is the as minimal inhibitory concentration of research of chaotic phenomenon in bottom hole portion.
As a result it is as shown in table 2.
The antibacterial activity of 2 FV7 of table and FV-LL
Note:aMinimum inhibitory concentration is that antibacterial peptide can be with the least concentration of bacteria growing inhibiting.Test at least carry out three times it is heavy
It is multiple.
2 antibiont film activities
To determine the effect that low concentration antibacterial peptide suppresses to biomembrane, we are from static biosolids surface method (crystallization
Purple staining).Take frozen in -20 DEG C of Pseudomonas aeruginosa PAO1, be inoculated in TSB culture medium, at 37 DEG C, under the conditions of 220r/m
Culture.Thalline after overnight is forwarded in brand-new culture medium, cultivates 1-2h, is in exponential phase to antibacterial.Spectrophotometric
The lower brand-new TSB culture medium corrected concentrations to 0.4 Maxwell of meter make the clump count of gained thalline be adjusted to 10 than turbid standard6CUF/mL
Left and right；500 μ L are added separately in 12 orifice plates with the antibacterial peptide that TSB culture medium contains variable concentrations gradient.Tune is taken with liquid-transfering gun
Whole good 500 μ L of thalline are sequentially added in 12 orifice plates, and in mixed liquor, the concentration of antibacterial is 5 × 105CUF/mL or so, sealed membrane will
12 orifice plates are sealed and are incubated 20-24h under the conditions of 37 DEG C.Positive controls are set wherein to be not added with antibacterial peptide.Carefully discard and carry
The culture medium of planktonic bacteria, is then rinsed 2-3 time with PBS, it is ensured that established biomembrane is not destroyed.Hereafter, 12 are attached to
Biomembrane violet staining 20min of orifice plate bottom.After dyeing 20min, reject crystal violet solution is rinsed repeatedly with sterilized water
The bottom in hole and side wall, until the purple in sterilized water is invisible.Plus 100 μ L dehydrated alcohol shaking table vibration, be transferred to 96 holes
570nm ultraviolet light measured values are used in plate.As a result it is as shown in Figure 2.Show T1249 FV-LL with the antibiont with FV7 similarity degrees
Film activity.
<110>Northeast Agricultural University
<120>Heterozygous antibacterial peptide and preparation method and application based on FV7 antibiont films
<160>1
<210>1
<211>20
<212>PRT
<213>Artificial sequence
<400>1
Phe Arg Ile Arg Val Arg Val Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg-NH2
1 5 10 15 20
Claims (3)
1. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, it is characterised in that its sequence such as sequence table SEQ ID
Shown in No.1.
2. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films, it is characterised in that preparation method is as follows：
(1) choosing polypeptide fragment FV7 and LL carries out heterozygosis, obtains T1249 FV-LL；
(2) physicochemical property of FV7, LL and FV-LL, spiral wheel construction are analyzed by polypeptide database analysises software and in advance
Survey；
(3) antibacterial peptide for obtaining design is synthesized using solid-state chemical reaction method method, that is, is completed polypeptide by Peptide synthesizer
Preparation, its sequence is as shown in sequence table SEQ ID No.1.
3. a kind of heterozygous antibacterial peptide FV-LL based on FV7 antibiont films according to claim 1, is preparing treatment leather orchid
Application in family name's positive bacteria or gram positive bacterial infection disease medicament.
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| CN201611054376.6A CN106589136B (en) | 2016-11-25 | 2016-11-25 | Heterozygous antibacterial peptide and preparation method and application based on FV7 antibiont film |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266585A (en) * | 2017-07-13 | 2017-10-20 | 陕西科技大学 | A kind of MLH fusions antibacterial peptide and its preparation method and application |
| CN110066342A (en) * | 2019-04-03 | 2019-07-30 | 中国农业大学 | It is a kind of to clear up endotoxin and the hybrid peptide of anti-inflammatory properties and the preparation method and application thereof with immunological regulation, neutralization |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016007551A1 (en) * | 2014-07-11 | 2016-01-14 | C3 Jian, Inc. | Targeting peptides that bind s. mutans, constructs comprising such peptides and uses thereof |
| CN105801669A (en) * | 2016-03-25 | 2016-07-27 | 东北农业大学 | Hybrid anti-biology type antibacterial peptide and preparation method and application thereof |
-
2016
- 2016-11-25 CN CN201611054376.6A patent/CN106589136B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016007551A1 (en) * | 2014-07-11 | 2016-01-14 | C3 Jian, Inc. | Targeting peptides that bind s. mutans, constructs comprising such peptides and uses thereof |
| CN105801669A (en) * | 2016-03-25 | 2016-07-27 | 东北农业大学 | Hybrid anti-biology type antibacterial peptide and preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266585A (en) * | 2017-07-13 | 2017-10-20 | 陕西科技大学 | A kind of MLH fusions antibacterial peptide and its preparation method and application |
| CN107266585B (en) * | 2017-07-13 | 2019-08-06 | 陕西科技大学 | A kind of MLH fusion antibacterial peptide and its preparation method and application |
| CN110066342A (en) * | 2019-04-03 | 2019-07-30 | 中国农业大学 | It is a kind of to clear up endotoxin and the hybrid peptide of anti-inflammatory properties and the preparation method and application thereof with immunological regulation, neutralization |
| CN110066342B (en) * | 2019-04-03 | 2020-12-01 | 中国农业大学 | Hybrid peptide with functions of immunoregulation, endotoxin neutralization and digestion and anti-inflammation, and preparation method and application thereof |
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| CN106589136B (en) | 2018-11-20 |
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