CN105777889B - A kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application - Google Patents
A kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application Download PDFInfo
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- CN105777889B CN105777889B CN201610177981.6A CN201610177981A CN105777889B CN 105777889 B CN105777889 B CN 105777889B CN 201610177981 A CN201610177981 A CN 201610177981A CN 105777889 B CN105777889 B CN 105777889B
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- C07—ORGANIC CHEMISTRY
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- 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
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Abstract
The present invention provides a kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application.Heterozygosis α screw type pig derived antimicrobial peptide, sequence is as shown in sequence table SEQ No.1.The present invention is first by 16 amino acid residues before the end N- in pig derived antimicrobial peptide PMAP-36 by transformation, then antibacterial peptide PR-FO is obtained with 15 amino acid heterozygosis before Fowlicidin-2, and by the terminal-carboxy amidation of PR-FO to improve a positive charge and increase the stability of peptide, sequence is as shown in SEQ No.1.Then Peptide synthesizer is used, solid-phase synthesis synthetic antibacterial peptide PR-FO is carried out.Antibacterial peptide of the invention has higher antibacterial activity, and low cytotoxicity substantially increases cell selective.The method becomes antibiotic substitute for antibacterial peptide and has established feasible theory and practice basis.
Description
Technical field
This invention belongs to agriculture animal and veterinary application field, and in particular to a kind of heterozygosis α screw type pig derived antimicrobial peptide and
Preparation method and application.
Background technique
Antibiotic is always human treatment because of the powerful mean of disease caused by cause pathogeny imcrobe infection, but with tradition
A large amount of uses of antibiotic, the drug resistance of pathogenic bacteria gradually increase, and the antibiotic epoch just gradually are walked to be at the end.Best at present is anti-
Raw element also gradually loses effect, so finding new antibacterials has become the focus being concerned.Especially recently about super
The report of grade bacterium, more people have beaten alarm bell.Find a kind of feed addictive of novel green with substitute antibiotics,
The task urgent as one.
Antibacterial peptide is many kinds of, is distributed in nature extremely wide, but is really applied to medicine and herding field
Example is seldom, and there are many factor of limitation antibacterial peptide application, wherein it is to limit it that antibacterial activity is low for certain antibacterial peptides
The major influence factors of application.Therefore, under the premise of guaranteeing not generate toxicity to normal cell, a kind of effective raising is found
The method of antibacterial peptide antibacterial activity is imperative.Hybrid design be it is a kind of it is good strategy be used to optimize these antibacterial peptide molecules.
This method can be good at the biological activity for retaining required segment, without being influenced by the change of a certain parameter.In addition,
Since only retentive activity center, this method can be significantly shorter peptide chain length, chemical synthesis cost is reduced.
Summary of the invention
The purpose of the present invention is to provide a kind of heterozygosis α screw type pig derived antimicrobial peptides and its preparation method and application.This hair
The bright pig derived antimicrobial peptide that will be transformed and alpha-helix type antibacterial peptide Fowlicidin-2 carry out heterozygosis, to obtain completely new antibacterial
Peptide, to improve its antibacterial effect.
The technology that the present invention uses is as follows: a kind of heterozygosis α screw type pig derived antimicrobial peptide, sequence such as sequence table SEQ No.1
It is shown.
Invention also has following technical characteristic: 1, a kind of preparation side of heterozygosis α screw type pig derived antimicrobial peptide as described above
Method is as follows:
(1) 16 amino acid residues obtain antibacterial peptide RI16 before the end N- in interception former peptide PMAP-36, will wherein the 7th
Amino acid substitution with the 11st obtains antibacterial peptide PRW4 at tryptophan, as shown in sequence table SEQ No.2；
(2) preceding 15 amino acid heterozygosis of PRW4 and alpha-helix type antibacterial peptide Fowlicidin-2 are obtained into antibacterial peptide PR-
FO, and by the terminal-carboxy amidation of PR-FO to improve a positive charge and increase the stability of peptide；
(3) solid-state chemical reaction method method is used, above-mentioned antibacterial peptide is synthesized by Peptide synthesizer.
2, a kind of heterozygosis α screw type pig derived antimicrobial peptide as described above is in preparation treatment gram-positive bacteria or gram-negative
Application in property bacterium infectious disease medicament.
Antibacterial peptide of the invention has higher antibacterial activity, and low cytotoxicity substantially increases cell selective.This side
Method becomes antibiotic substitute for antibacterial peptide and has established feasible theory and practice basis.
Detailed description of the invention
Fig. 1 is the permeabilization figure of antibacterial peptide of the invention to bacterial cell outer membrane.
Fig. 2 is the permeabilization figure of antibacterial peptide of the invention to bacterial cell inner membrance.
Fig. 3 is the hemolytic activity figure of antibacterial peptide of the invention.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The design and synthesis of 1 heterozygosis α screw type pig derived antimicrobial peptide sequence of embodiment
16 amino acid residues obtain antibacterial peptide RI16 before the end N- in interception pig derived antimicrobial peptide PMAP-36, will wherein the 7th
Position and the 11st amino acid substitution obtain antibacterial peptide PRW4 at tryptophan, then by PRW4 and alpha-helix type antibacterial peptide
Preceding 15 amino acid heterozygosis of Fowlicidin-2 obtain antibacterial peptide PR-FO, by the terminal-carboxy amidation of PR-FO to improve one
A positive charge and the stability for increasing peptide, the amino acid sequence of antibacterial peptide are as shown in table 1.
The amino acid sequence and molecular weight of 1 antibacterial peptide of table
Using Peptide synthesizer, above-mentioned antibacterial peptide is synthesized using solid-phase synthesis.
The destruction of 2 bacterial cell membrane of embodiment
The destruction of 1 antibacterial peptide external membrane
Escherichia coli bacterium was cultivated to the logarithm growth stage, thallus is centrifuged and collects, with the 5mM HEPES buffer solution (Portugal 5mM
Grape sugar, pH 7.4) it washs and is suspended in the buffer solution.It takes the bacterial solution of 2mL to be put in quartz cuvette, and is added
1mM NPN solution is mixed with, so that final concentration of 10 μM of NPN.It is strong using F-4500 sepectrophotofluorometer detection fluorescence
Degree (excitation 350nm emits 420nm), is added the antibacterial peptide of various concentration, the variation of fluorescence intensity.As a result as shown in Figure 1.
As seen from Figure 1, PRW4 and PR-FO all external membranes generate certain permeabilization, but the permeabilization intensity of PR-FO is stronger, table
The bright antibacterial peptide after tryptophan substitutes enhances the destruction of bacterial cell outer membrane.
Destruction of 2 antibacterial peptides to inner membrance
Escherichia coli bacterium was cultivated to the logarithm growth stage, thallus is centrifuged and collects, 5mM HEPES buffer solution (20mM is added
Glucose, pH 7.4), bacterial sediment is resuspended, final concentration of 0.4 μM of DiSC3 (5) is added into configured bacterial suspension
Storing liquid, room temperature are protected from light concussion and are incubated for 1-1.5h；The KCl storing liquid of final concentration of 4M is added, room temperature is protected from light concussion and is incubated for 15-
30min.It takes the bacterial suspension of 2mL to be added in the quartz cuvette of F-4500 sepectrophotofluorometer, antibacterial peptide to be measured is added,
The fluorescence intensity under 622nm excitation wavelength, 670nm launch wavelength, slit width 10nm, fluorescence burst size are 360s.
As a result as shown in Figure 2.As seen from Figure 2, the fluorescence intensity that the antibacterial peptide PR-FO compared with former peptide after heterozygosis is generated obviously increases
By force, show that PR-FO is stronger to the unpolarizing of bacterial cell plasma membrane, and time-concentration dependence is presented.
The bacteriostatic activity and hemolytic activity of 3 antibacterial peptide of embodiment
The bacteriostatic activity of 1 antibacterial peptide
Antibacterial peptide PRW4, FO and PR-FO are configured as a certain concentration storing liquid in case using.Utilize micro broth dilution
Method measures the minimal inhibitory concentration of several antibacterial peptides.It is dilute using two times using 0.01% acetic acid (containing 0.2%BSA) as dilution
Interpretation of the law configures in order the antibacterial peptide solution of graded series.It takes above-mentioned 100 μ L of solution to be placed in 96 porocyte culture plates, then distinguishes
The isometric bacterium solution to be measured (~10 of addition5A/mL) in each hole.Positive control is respectively set (containing bacterium solution without containing anti-
Bacterium peptide) and negative control (be both free of bacterium solution or be free of peptide).37 DEG C of constant temperature incubation 20h, visually to have no that it is muddy existing that hole bottom has
Elephant is minimal inhibitory concentration.The results are shown in Table 2.From Table 2, it can be seen that the minimal inhibitory concentration of PR-FO is 0.5-2
μM, show the activity of very strong inhibition Gram-negative and positive bacteria growth.
The minimal inhibitory concentration (MIC) of 2 former peptide of table and hybrid peptide
Minimal inhibitory concentration (MIC): antibacterial peptide inhibits the Cmin of bacterial growth, visually observes in this concentration less than thin
The growth of bacterium.
The hemolytic activity of 2 antibacterial peptides
The new blood 1mL of people is acquired, is dissolved into 2mL PBS solution after anticoagulant heparin, 1000g is centrifuged 5min, collects
Red blood cell；It is washed 3 times with PBS, then be resuspended with 10mLPBS；100 μ L red cell suspensions are taken to dissolve from 100 μ L PBS different dense
The antibacterial peptide solution of degree is uniformly mixed, and is taken out after constant-temperature incubation 1h in 37 DEG C of incubators, 4 DEG C, and 1000g is centrifuged 5min；It takes out
Supernatant microplate reader surveys absorbance value at 540nm；Every group is averaged, and comparative analysis.Wherein 100 μ LPBS are as yin
Property control；100 μ L0.2%Tritonx-100 are as positive control.As a result as shown in Figure 3.
It can be seen that the antibacterial activity of PR-FO compared with PRW4 in conjunction with table 2 and Fig. 3 to be remarkably reinforced, keep again at the same time
Lower hemolytic activity.
The definition of therapeutic index is the ratio of the minimum hemolytic concentration of antibacterial peptide and the geometric mean of minimum bactericidal concentration.
Therapeutic index is higher, and cell selective is stronger, and PR-FO has higher cell selective.
Claims (3)
1. a kind of heterozygosis α screw type pig derived antimicrobial peptide PR-FO, which is characterized in that its sequence such as sequence table SEQ ID No.1 institute
Show.
2. a kind of heterozygosis α screw type pig derived antimicrobial peptide PR-FO according to claim 1, which is characterized in that preparation method is such as
Under:
(1) intercept in former peptide PMAP-36 that 16 amino acid residues obtain antibacterial peptide RI16 before the end N-, it will wherein the 7th and the
11 amino acid substitutions obtain antibacterial peptide PRW4 at tryptophan, as shown in sequence table SEQ No.2；
(2) preceding 15 amino acid heterozygosis of PRW4 and alpha-helix type antibacterial peptide Fowlicidin-2 are obtained into antibacterial peptide PR-FO, and
By the terminal-carboxy amidation of PR-FO to improve a positive charge and increase the stability of peptide；
(3) solid-state chemical reaction method method is used, the antibacterial peptide, sequence such as sequence table SEQ ID are synthesized by Peptide synthesizer
Shown in No.1.
3. a kind of heterozygosis α screw type pig derived antimicrobial peptide PR-FO according to claim 1 treats gram-positive bacteria in preparation
Or the application in gram positive bacterial infection disease medicament.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106496333A (en) * | 2016-11-25 | 2017-03-15 | 东北农业大学 | One kind expresses hybrid peptide and preparation method and application using bacillus subtilis |
| CN106432513B (en) * | 2016-11-29 | 2018-03-13 | 东北农业大学 | A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application |
| CN107043428A (en) * | 2017-05-02 | 2017-08-15 | 东北农业大学 | A kind of anti-inflammatory heterozygous antibacterial peptide of αhelix and its preparation method and application |
| CN107141338B (en) * | 2017-05-02 | 2018-08-28 | 东北农业大学 | A kind of antibacterial peptide RW-P and preparation method thereof and application |
| CN108570103B (en) * | 2018-04-03 | 2019-07-30 | 东北农业大学 | One kind is rich in tryptophan antibacterial peptide WK12 and its preparation method and application |
| CN109021112B (en) * | 2018-06-22 | 2019-06-25 | 青岛蔚蓝生物股份有限公司 | Heterozygous antibacterial peptide PO-CH34 and its preparation method and application |
| CN110256573A (en) * | 2019-05-08 | 2019-09-20 | 吉林农业大学 | A kind of heterozygous antibacterial peptide BM16R and its preparation method and application |
| CN110283252B (en) * | 2019-07-12 | 2020-04-10 | 东北农业大学 | Pig-derived hybrid antibacterial peptide PP-1 and preparation method and application thereof |
| CN110283245B (en) * | 2019-07-12 | 2020-04-03 | 东北农业大学 | Pig marrow derived PMAP-23 derived antibacterial peptide, preparation method and application |
| CN117164722B (en) * | 2023-08-09 | 2024-04-09 | 东北农业大学 | Spiral bundle DAMP4-PR-FO fusion protein D2L and preparation method and application thereof |
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