CN105153281B - Antibacterial peptide HP10 and its preparation method and application - Google Patents
Antibacterial peptide HP10 and its preparation method and application Download PDFInfo
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- CN105153281B CN105153281B CN201510600706.6A CN201510600706A CN105153281B CN 105153281 B CN105153281 B CN 105153281B CN 201510600706 A CN201510600706 A CN 201510600706A CN 105153281 B CN105153281 B CN 105153281B
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Abstract
Antibacterial peptide HP10 of the present invention and its preparation method and application, belong to peptide symthesis technology field and the particular treatment active technologies field of compound and pharmaceutical preparation containing 5 20 amino acid.The amino acid sequence of the antibacterial peptide HP10 is HHHLHHIHKP；Its molecular weight is 1292.4Da, isoelectric point 8.79.Its preparation comprises the steps：(1), protect synthetic method that the antibacterial peptide HP10 that amino acid sequence is HHHLHHIHKP is entered into line activating and crosslinking one by one from C-terminal to N-terminal by FMOC；(2), the product of step (1) obtains antibacterial peptide HP10 through HPLC reversed phase column chromatography desalting and purifyings.Antibacterial peptide HP10 in the present invention has the advantages that small molecular weight, artificial synthesized convenience, heat endurance is strong, bactericidal action is strong, has no drug resistance；Also with being applied in pig starter feed also with intestine of young pigs health, enhancing immunity of organisms, raising piglet growth performance can be significantly improved the advantages of.
Description
Technical field
The invention belongs to the peptide symthesis technology field containing 5-20 amino acid and compound and the specific of pharmaceutical preparation to control
Treat active technologies field, and in particular to antibacterial peptide HP10 and its preparation method and application.
Background technology
Feeding antibiotic has played vital effect in the evolution of global animal husbandry.In recent years, in order to chase after
Seek bigger economic benefit, the abuse phenomenon of antibiotic is on the rise in animal productiong, and this is not only caused in animal food
Residue of veterinary drug problem, also results in the generation of antibody-resistant bacterium, the sustainable development to animal husbandry brings serious puzzlement.Cause
This, the developed country such as European Union and South Korea has completely forbidden and antibiotic is used in animal and fowl fodder, finds Substitutes For Antibiotic and becomes
For the big focus in Animal nutrition research, particularly piglet nutrition, because piglet immunological systematic growth and imperfection, it is easy to
Infect harmful bacteria and influence its production performance, severe patient is even dead.
Antibacterial peptide is the small peptide material synthesized by organism gene code and in ribosomes, is that animal non-specificity is exempted from
One important component of epidemic disease, it has the various biological work(such as antibacterium, antiviral, antimycotic and killing parasite
Can, particularly there is powerful lethal effect to multi-drug resistant bacteria.Antibacterial peptide has no toxic side effect to body, noresidue and is not easy to produce
Raw drug resistance, therefore, exploitation antimicrobial peptide preparation substitute the research heat that feeding antibiotic has become domestic and international animal productiong field
Point.Have immunogenicity, antibacterial activity not high, thin to animal greatly however, the antibacterial peptide of natural origin has molecular weight mostly
The defects of born of the same parents have toxic side effect or can cause haemolysis, limiting popularization of the antibacterial peptide in pig-breeding to a certain extent should
With.By the way that MOLECULE DESIGN and transformation acquisition molecular weight are small, antibacterial activity is high and is to the antibacterial peptide that zooblast has no toxic side effect
The effective means for promoting antibacterial peptide to be applied in pig-breeding.
The content of the invention
It is an object of the invention to disclose a kind of antibacterial peptide HP10.
Second object of the present invention is the preparation method for disclosing antibacterial peptide HP10.
Third object of the present invention is the application for disclosing antibacterial peptide HP10.
The purpose of the present invention is achieved through the following technical solutions：
Antibacterial peptide HP10, wherein, the amino acid sequence of the antibacterial peptide HP10 is HHHLHHIHKP.
Antibacterial peptide HP10 described in above-mentioned technical proposal, wherein, the molecular weight of the antibacterial peptide HP10 is 1292.4Da, etc.
Electricity point is 8.79.
Applications of the antibacterial peptide HP10 in weaning pigs described in above-mentioned technical proposal.
Antibacterial peptide HP10 preparation method, comprises the steps：
(1), by FMOC protect synthetic method by amino acid sequence be HHHLHHIHKP antibacterial peptide HP10 from C-terminal to N-terminal
Enter line activating and crosslinking one by one；
(2), the product of step (1) obtains antibacterial peptide HP10 through HPLC reversed phase column chromatography desalting and purifyings.
Preparation method described in above-mentioned technical proposal prepares applications of the antibacterial peptide HP10 of gained in weaning pigs.
The invention has the advantages that：
(1), the antibacterial peptide HP10 in the present invention has that small molecular weight, artificial synthesized convenience, heat endurance be strong, bactericidal action
By force, the advantages that having no drug resistance；
(2), the present invention in antibacterial peptide HP10 in pig starter feed apply have can significantly improve intestine of young pigs health,
Strengthen immunity of organisms, improve the advantages of piglet growth performance.
Brief description of the drawings：
1st, Fig. 1 is influences of the antibacterial peptide HP10 to 24 age in days diarrhea of weaned piglets rates.
Embodiment：
To readily appreciate technical scheme, below in conjunction with specific embodiment to antibacterial peptide HP10 and its preparation side
Method and application are further described, and are operated in procedure below using convenient technical process and instrument description.
Embodiment 1：Antibacterial peptide HP10 preparation：
Antibacterial peptide HP10 is at natural antibacterial peptide PMAP36 (GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGCG)
On the basis of interception transformation, because histidine is a kind of essential amino acid of growing animal, and its antibacterial activity ratio
Arginine and lysine are strong, so substituting arginine and lysine with histidine.
Antibacterial peptide HP10 chemical synthesis process, comprises the steps：
(1), according to antibacterial peptide HP10 amino acid sequence HHHLHHIHKP, synthetic method is protected by antibacterial peptide by FMOC
HP10 enters line activating and crosslinking one by one from C-terminal to N-terminal, is led to by Shanghai gill (GL) biochemical Co., Ltd solid-state chemical reaction method method
Cross full-automatic polypeptide synthetic instrument (ABI433) synthetic antibacterial peptide complete sequence；
(2) it is, anti-phase with high performance liquid chromatography (HPLC) to the product of step (1) by Shanghai gill (GL) biochemical Co., Ltd
Column chromatography desalting and purifying；
Electron spray mass spectrometry is recycled (when using the Agilent6520Q-TOF level Four bar flights of Agilent companies of the U.S.
Between mass spectrograph) antibacterial peptide is identified, purity be more than 95%；
2nd, the antibacterial peptide HP10 molecular weight of purifying uses MALDI-TOF-MS (MALDI-
TOF) determine, isoelectric point is determined using isoelectric focusing electrophoresis, and amino acid sequence is solved using automatic determined amino acid sequence instrument
Analysis.
Antibacterial peptide HP10 contains 10 amino acid residues, molecular weight 1292.4Da, isoelectric point 8.79, its amino acid sequence
For HHHLHHIHKP.
Illustrate that antibacterial peptide HP10 of the present invention is had in being applied in weanling pig feed below by way of specific experiment example
Beneficial effect.
Experimental example 1：Antibacterial peptide HP10 bacteriostatic experiments：
(1), test strain：Escherichia coli, salmonella, staphylococcus aureus, MRSE, bacillus subtilis
Bacterium and bacillus megaterium.
(2), polypeptide mother liquor (0.2%BSA+0.01% acetic acid)：Weigh 0.2g bovine serum albumin(BSA)s (BSA) addition
80mLddH2Mixed in O water, add 10 μ L acetic acid solution, then use ddH2O water is settled to 100mL, with 0.22 μm of aperture
After membrane filtration is degerming, 4 DEG C save backup.
By determining antibacterial peptide HP10 minimal inhibitory concentrations (minimal inhibitory concentration, MIC)
To reflect its antibacterial effect, minimal inhibitory concentration is that can't detect the minimum sample concentration of bacterial growth.By test organisms in 37 DEG C
16h is incubated overnight, then draws the bacterium solution that 100 μ L are incubated overnight and is transferred in fresh MHB broth bouillons, cultivates 2.5~3h, directly
Exponential phase is in thalline, then with 10mM sodium phosphate buffers (pH 7.4) suspension thalline cell, and thalline is diluted, makes
Its clump count is 106CFU/mL or so.90 μ L 10mM sodium phosphate buffers (pH are added in often arrange No. 1 hole of 96 orifice plates
7.4) 50 μ L buffer solutions are added in other holes, 10 μ L candidate polypeptide mother liquors are added in No. 1 hole, then by No. 1 hole
Polypeptide solution suctions out 50 μ L and added in No. 2 hole, the like, after doubling dilution to No. 10 hole, suction out 50 μ L and discard；With adding
Sample device is drawn the above-mentioned 50 μ L thalline adjusted and is sequentially added in preceding 11 holes of 96 orifice plates, and it is every to make final inoculated bacteria concentration
Hole 5 × 105CFU/mL.96 orifice plates are put to vibrate on microoscillator and mixed, microwell plate is capped to reduce the liquid during being incubated
Evacuator body, 2h is incubated at 37 DEG C.Wherein the 11st hole is set as positive controls：Only add 50 μ L buffer solutions and 50 μ L bacterium solutions；
12nd hole is negative control group：Only add 100 μ L buffer solutions.It will now be passed successively from the 1st hole to the concentration of the 10th hole antibacterial peptide
Subtract；50 μ L are drawn from each hole, serial dilution to 10-1,10-2 and 10-3, take 20 μ L point samples respectively to MHA flat boards
Respective regions.Flat board takes out counting after 8~10h is incubated at 37 DEG C.
As a result judge：Negative control group after incubation terminates should not long bacterium, Positive contrast bacteria well-grown.In a series of peptides
During concentration thalline counts, the treatment group of the Cmin peptide of not long bacterium is LC.Experiment repeats three times.As shown in table 1.From table
In it can be seen that antibacterial peptide HP10 to Escherichia coli, salmonella, staphylococcus aureus, MRSE, withered grass gemma
Bacillus and bacillus megaterium have very strong lethal effect.
The antibacterial peptide HP10 of table 1 minimum bactericidal concentration (μM)
Experimental example 2：Influences of the antibacterial peptide HP10 to Growth Performance of Weaning Piglets, intestinal health and immunologic function：
Weanling pig 320 after 21 ages in days transport is chosen, is designed using single factor experiment, is assigned at random by body weight and sex
In 4 treatment groups of experimental design as shown in table 2, wherein daily ration based on negative control group, day based on HP10-50 groups
50mg/kg antibacterial peptides HP10 is added in grain；100mg/kg antibacterial peptide HP10 are added based on HP10-100 groups in daily ration, it is positive right
According to 100mg/kg aureomycin is added in daily ration based on group, every group of 10 repetitions, 8 pigs, 14 days experimental periods are each repeated.
The experimental design of table 2
Influences of the antibacterial peptide HP10 to Production Performance of Weaning Pigs, diarrhea rate and immunoglobulin is shown in Table 3, Fig. 1 and table 4.
It can be seen that antibacterial peptide HP10 can significantly improve the growth performance of weanling pig from the experimental result of table 3；From Fig. 1 experiment knot
Fruit can be seen that antibacterial peptide HP10 can improve weanling pig intestinal health, reduce diarrhea of weaned piglets rate；From the experimental result of table 4
It can be seen that antibacterial peptide HP10 can improve the immunoglobulin concentrations of piglet, strengthen the immunocompetence and resistance against diseases of body.
Influences of the antibacterial peptide HP10 of table 3 to 24 age in days Growth Performance of Weaning Piglets
Influences (g/L) of the antibacterial peptide HP10 of table 4 to 24 age in days weanling pig immunoglobulins
It is described above, only presently preferred embodiments of the present invention, not the present invention is made it is any in form and substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made, it is the equivalent embodiment of the present invention；Meanwhile it is all according to
The variation, modification and evolution for any equivalent variations made according to the substantial technological of the present invention to above example, still fall within this
In the range of the technical scheme of invention.
SEQUENCE LISTING
<110>Guangzhou Ao Nong bio tech ltd
<120>Antibacterial peptide HP10 and its preparation method and application
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213>Antibacterial peptide HP10
<400> 1
His His His Leu His His Ile His Lys Pro
1 5 10
Claims (4)
1. antibacterial peptide HP10, it is characterised in that：The amino acid sequence of the antibacterial peptide HP10 is HHHLHHIHKP.
2. antibacterial peptide HP10 according to claim 1, it is characterised in that the antibacterial peptide HP10 is applied in weaning pigs
In.
3. antibacterial peptide HP10 preparation method, comprises the steps：
(1) synthetic method is protected to enter the antibacterial peptide HP10 that amino acid sequence is HHHLHHIHKP one by one from C-terminal to N-terminal by FMOC
Line activating and crosslinking；
(2) product of step (1) obtains antibacterial peptide HP10 through HPLC reversed phase column chromatography desalting and purifyings.
4. antibacterial peptide HP10 according to claim 3 preparation method, it is characterised in that the step (2) obtains anti-
Bacterium peptide HP10 is applied in weaning pigs.
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| CN105541970B (en) * | 2016-02-02 | 2020-06-26 | 福建傲农生物科技集团股份有限公司 | Polypeptide PR21, preparation thereof and application thereof in preparation of sow feed |
| CN106883288B (en) * | 2016-02-03 | 2020-08-14 | 辽宁傲农饲料有限公司 | Antibacterial peptide BV21 and application thereof |
| CN110257314B (en) * | 2019-07-02 | 2021-06-01 | 华中农业大学 | Recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, construction method and application thereof |
| CN112868668B (en) * | 2021-03-19 | 2021-09-14 | 常州英诺升康生物医药科技有限公司 | Fe3O4-DA-AMP nano composite antibacterial material and preparation method and application thereof |
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| CN104017087A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | Pig-source antimicrobial peptide and preparation method thereof |
| CN104341497A (en) * | 2014-11-05 | 2015-02-11 | 青岛农业大学 | Novel pig-source antibacterial peptide mutant, preparation method and application thereof |
| CN104356222A (en) * | 2014-11-20 | 2015-02-18 | 青岛宏昊生物科技有限公司 | Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof |
| CN104447976A (en) * | 2014-12-13 | 2015-03-25 | 青岛宏昊生物科技有限公司 | Swine-derived antibacterial peptide cecropin P1 mutant as well as preparation method and application of swine-derived antibacterial peptide cecropin P1 mutant |
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| CN104017087A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | Pig-source antimicrobial peptide and preparation method thereof |
| CN104341497A (en) * | 2014-11-05 | 2015-02-11 | 青岛农业大学 | Novel pig-source antibacterial peptide mutant, preparation method and application thereof |
| CN104356222A (en) * | 2014-11-20 | 2015-02-18 | 青岛宏昊生物科技有限公司 | Swine antibacterial peptide PR-39 mutant as well as preparation method and application thereof |
| CN104447976A (en) * | 2014-12-13 | 2015-03-25 | 青岛宏昊生物科技有限公司 | Swine-derived antibacterial peptide cecropin P1 mutant as well as preparation method and application of swine-derived antibacterial peptide cecropin P1 mutant |
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