CN106432513B - A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application - Google Patents
A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application Download PDFInfo
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- CN106432513B CN106432513B CN201611075909.9A CN201611075909A CN106432513B CN 106432513 B CN106432513 B CN 106432513B CN 201611075909 A CN201611075909 A CN 201611075909A CN 106432513 B CN106432513 B CN 106432513B
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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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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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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Abstract
The present invention relates to a kind of efficiently hybridization antibacterial peptide LI and its preparation method and application, its sequence is as shown in sequence table SEQ ID No.1；Its preparation method comprises the following steps：According to pinpoint amino acid fragment intercept method, the amino acids residue segments of interception people's derived antimicrobial peptide LL37 the 14th 21 and the amino acid residue segments of ox derived antimicrobial peptide Indolicidin the 5th 13, above-mentioned two fragment is sequentially connected, obtains LI antibacterial peptides；2) solid-phase synthesis synthesis peptide resin is carried out using Peptide synthesizer；Polypeptide, RPLC purifying are obtained after TFA is cut.Antibacterial peptide LI obtained by the present invention has stronger bacteriostatic activity and weaker hemolytic activity, and therapeutic index is high, has very big development potentiality.
Description
Technical field
The present invention relates to a kind of efficiently hybridization antibacterial peptide LI and its preparation method and application.
Background technology
Antibacterial peptide is the first line of defence that animal resists foreign pathogens.It not only has the function of killing pathogen, also
Can be with biological functions such as antimycotic, viral, parasite and inhibiting tumor cells.Antibacterial peptide has unique bactericidal mechanism, make its into
For most promising antibiotic substitute.Antibacterial peptide molecule has positively charged amino acid, such as arginine, lysine and group ammonia
Acid and with hydrophobic residue hydrophobic amino acid, such as leucine, isoleucine, valine, tryptophan, phenylalanine,
Alanine etc..Antibacterial peptide and the mutual electrostatic attraction of negatively charged bacterial cell membrane with quiet positive charge, antibacterial peptide is close to bacterium
Cell, hydrophobic residue probe into the lipid bilayer of somatic cells, somatic cells film is formed micropore or cavernous structure, cause
Intracellular matter outflows, so as to cause bacterial death.Antibacterial peptide is hindered to be applied to medical science, cosmetics, food and herding etc. at present
, there are following two aspects the reason for industry：First, the toxicity of antibacterial peptide.Natural antibacterial peptide has higher cytotoxicity.Find one
The design method that kind improves antibacterial peptide cell selective is imperative.The cell selective of antibacterial peptide refers to that antibacterial peptide is thin to cause of disease
Bacterium, which has, to be killed function and not to have toxic side effect to normal animal somatic cell, shows the selectively killing effect to cell.It is anti-
The therapeutic index of bacterium peptide is to evaluate the critical index of its cell selective.2nd, the production cost of antibacterial peptide.Antibacterial peptide at present
Prepare is mainly two methods of chemical method and genetic engineering.Chemical method production antibacterial peptide symthesis is accurate, but yield is few, cost
It is high.During the method for genetic engineering production antibacterial peptide is tested, the screening of engineering bacteria and the condition of gene expression process
Control is always to restrict the principal element of this method production antibacterial peptide.The method using genetic engineering of blindness expresses antibacterial peptide,
The 26S Proteasome Structure and Function to antibacterial peptide and in vitro test are not groped comprehensively, cause the antibacterial peptide that expression obtains to be lived mostly
Property it is not high, or with the cytotoxicity such as haemolysis.
Antibacterial peptide molecular weight is minimum, and species is various, complicated.At present, a variety of methods study antibacterial peptide
Relation between structure and function, for example, being transformed natural antibacterial peptide and the research method such as brand-new design antibacterial peptide.
The content of the invention
Based on above weak point, the present invention provides a kind of efficiently hybridization antibacterial peptide LI and its preparation method and application, should
Antibacterial activity is high and cytotoxicity is low.
The technology used in the present invention is as follows：A kind of efficiently hybridization antibacterial peptide LI, its sequence such as sequence table SEQ ID No.1
It is shown.
The present invention also has following technical characteristic：
1st, a kind of efficiently hybridization antibacterial peptide LI as described above preparation method comprises the following steps：
1) according to pinpoint amino acid fragment intercept method, interception people's derived antimicrobial peptide LL37 14-21 amino acids residues and
Ox derived antimicrobial peptide Indolicidin 5-13 amino acid residue segments, above-mentioned two fragment is sequentially connected, and obtains LI antibacterials
Peptide；
2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method；Obtained peptide resin is cut by TFA
After cutting, polypeptide products are tentatively obtained；Then after RPLC purifying and Mass Spectrometric Identification, that is, the antibacterial peptide is completed
Preparation.
2nd, a kind of efficiently hybridization antibacterial peptide LI as described above, is preparing treatment gram-positive bacteria or Gram-negative bacteria
Application in infectious disease medicament.
The present invention is by intercepting natural antibacterial peptide LL37 14-21 amino acids residues
(GlyLysGluPheLysArgIleVal) and Indolicidin (In13) characteristic amino acid residue segment
(LysTrpProTrpTrpProTrpArgArg), hybrid design obtains brand-new antibacterial peptide LI (GlyLysGluPheLysArgIle
ValLysTrpProTrpTrpProTrpArgArg-NH2).Antibacterial peptide LL37 is resisted by the protein derived α spirals of people source hCAP18
Bacterium peptide, there is the bioactivity such as the antibacterium, fungi, virus of wide spectrum.Indolicidin is derived from the one of ox neutrality grain Fine born of the same parents
The small peptide of 13 amino acid of the kind rich in tryptophan and proline.Tryptophan is hydrophobic amino acid, the phosphorus that can show with bacterium
Hydrophobicity effect occurs for fat, the immobilized artificial membrane of bacterium is destroyed, so as to produce biological activity.The two is respectively provided with certain haemolysis cell
Toxicity.It yet there are no both at home and abroad using the two characteristic fragment hybridization to study the report of antibacterial peptide structure-function relationship.Pass through this
The experimental technique of antibacterial peptide prepared by method is simple, carries out antibacterial to obtained antibacterial peptide and hemolytic activity detects, the antibacterial is lived
Property it is high and cytotoxicity is low, find LI not only to Escherichia coli, Pseudomonas aeruginosa, Salmonella gallinarum, it is salmonella typhimurium, golden yellow
Color staphylococcus, MRSE, streptococcus fecalis, bacillus subtilis, eight kinds of strains have obvious inhibitory action, and have
There is very low hemolytic activity.Thus, in general, LI is a kind of antibacterial peptide with higher application value.
Brief description of the drawings
Fig. 1 is the three-dimensional prediction figure for the antibacterial peptide that design obtains；
Fig. 2 is the mass spectrogram for the antibacterial peptide that design obtains.
Embodiment
Below according to Figure of description citing, the present invention will be further described：
Embodiment 1
People's derived antimicrobial peptide LL37 amino acid sequence is：
Ox derived antimicrobial peptide In13 amino acid sequence is：
Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg-NH2
1 5 10 13
According to pinpoint amino acid fragment intercept method, people's derived antimicrobial peptide LL37 14-21 amino acids residues are intercepted
And ox derived antimicrobial peptide In13 5-13 amino acid residue segments (GlyLysGluPheLysArgIleVal)
(LysTrpProTrpTrpProTrpArgArg)。
Above-mentioned two fragment is sequentially connected, obtains LI (GlyLysGluPheLysArgIleValLysTrpProTrpTrp
ProTrpArgArg-NH2) antibacterial peptide.
Embodiment 2
Above-mentioned two antibacterial peptide is synthesized using Peptide synthesizer, method is solid-state chemical reaction method method, specific steps
For：
1) preparation of antibacterial peptide is carried out one by one from C-terminal to N-terminal, is completed by Peptide synthesizer.First by Fmoc-X (X
It is first amino acid of C-terminal of each antibacterial peptide) Wang resins are linked into, obtain X-Wang trees after then sloughing Fmoc groups
Fat；Again by Fmoc-Y-Trt-OH (9- fluorenes methoxy carboxyl-trimethyl-Y, Y is each second amino acid of antibacterial peptide C-terminal)；According to
This program is synthesized to N-terminal from C-terminal successively, until synthesis finishes, obtains sloughing the resin of the side chain protected of Fmoc groups；
In peptide resin obtained above, cutting reagent is added, is reacted 2 hours under 20 DEG C of lucifuges, is filtered；Precipitate TFA (three
Fluoroacetic acid) washing, washing lotion is mixed with above-mentioned filtrate, Rotary Evaporators concentration, the precooling for adding 10 times or so volumes is anhydrous
Ether, -20 DEG C precipitate 3h, separate out white powder thing, and 10min is centrifuged with 2500g, collect precipitation, then washed and sunk with absolute ether
Form sediment, vacuum drying, obtain polypeptide, wherein cutting reagent by TFA, water and TIS (tri isopropyl chlorosilane) according to mass ratio 95:
2.5:2.5 mix；
Column equilibration 30min is carried out using 0.2mol/L sodium sulphate (phosphoric acid is adjusted to pH7.5), it is molten with 90% acetonitrile solution
Polypeptide is solved, filtering, the anti-phase normal pressure posts of C18, using gradient elution, (eluant, eluent is according to volume ratio for methanol and aqueous sodium persulfate solution
30:70~70:30 mixing), flow velocity 1ml/min, detection ripple is 220nm, collects main peak, is freezed；Anti-phase C18 posts are recycled to enter
One step purifies, and eluent A is the 0.1%TFA/ aqueous solution；Eluent B is 0.1%TFA/ acetonitrile solutions, wash-out concentration 25%B
~40%B, elution time 12min, flow velocity 1ml/min, then main peak is ibid collected, freeze；
The identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed by electron spray mass spectrometry, point shown in mass spectrogram
Son amount (see photo) and the theoretical molecular in table one are basically identical, and the purity of antibacterial peptide is more than 95%.
The design of the antibacterial peptide of table 1
Embodiment 3
Detection is compared by In Vitro Bacteriostasis and Hemolytic activity assays to the antibacterial peptide for designing and synthesizing to obtain；
The measure of antibacterial activity：Peptide is configured as certain storing liquid in case using.Utilize micro-broth dilution method
The minimal inhibitory concentration of several antibacterial peptides.Using 0.01% acetic acid (containing 0.2%BSA) as dilution, using doubling dilution according to
The antibacterial peptide solution of secondary configuration graded series.The above-mentioned μ l of solution 100 are taken to be placed in 96 porocyte culture plates, then addition etc. respectively
The bacterium solution to be measured (~10 of volume5Individual/ml) in each hole.Respectively set positive control (antibacterial peptide is not contained containing bacterium solution) and
Negative control (both without bacterium solution or be free of peptide).37 DEG C of incubated 20h, with visually have no bottom hole portion have research of chaotic phenomenon be
Minimal inhibitory concentration.
Testing result is shown in Table 2.It can be seen from Table 2 that LI shows stronger suppression for Gram-negative and positive bacteria
Bacterium activity, its bacteriostatic activity are more than LL37 and In13., illustrate to carry by heterozygosis LL37 and In13 characteristic amino acid residue segment
The high bacteriostatic activity of antibacterial peptide.
The antibacterial and hemolytic activity of the antibacterial peptide of table 2
The measure of hemolytic activity：The new blood 1mL of people is gathered, is dissolved into after anticoagulant heparin in 2mlPBS solution, 1000g
5min is centrifuged, collects red blood cell；Washed 3 times with PBS, then be resuspended with 10ml PBS；Take 50 μ L red cell suspensions and 50 μ L PBS
The antibacterial peptide solution of the various concentrations of dissolving is well mixed, the constant-temperature incubation 1h in 37 DEG C of incubators；Taken out after 1h, 4 DEG C,
1000g centrifuges 5min；Take out supernatant ELIASA light-metering absorption value at 570nm；Every group is averaged, and comparative analysis.
Wherein 50 μ L red blood cells add 50 μ l PBS as negative control；50 μ L red blood cells add 50 μ l 0.1%Tritonx-100 as sun
Property control.Minimum hemolytic concentration is antibacterial peptide concentration when antibacterial peptide causes 10% hemolysis rate.
Testing result is shown in Table 2.Hemolytic concentration is bigger, shows that hemolytic activity is smaller；It can be seen from Table 2 that LI is being detected
In the range of there is no hemolytic activity, and LL37 and In13 show certain hemolytic activity.
Result above shows that two characteristic fragments of interception people's derived antimicrobial peptide LL37 and ox derived antimicrobial peptide Indolicidin are simultaneously
Being sequentially connected obtained antibacterial peptide LI has higher antibacterial activity, and does not have toxicity to haemocyte.Comprehensive analysis antibacterial
The antibacterial and hemolytic activity of peptide, can more fully it be evaluated by therapeutic index (ratio of hemolytic concentration and Mlc) each
The cell selective of individual antibacterial peptide.As can be seen from Table 2, LI has higher therapeutic index, shows the LI antibacterials that design obtains
Peptide has the development potentiality of higher substitute antibiotics.
<110>Northeast Agricultural University
<120>A kind of efficiently hybridization antibacterial peptide LI and its preparation method and application
<160> 1
<210> 1
<211> 17
<212> PRT
<213>Artificial sequence
<400> 1
Gly Lys Glu Phe Lys Arg Ile Val Lys Trp
1 5 10
Pro Trp Trp Pro Trp Arg Arg-NH2
11 15 17
Claims (3)
1. a kind of efficiently hybridization antibacterial peptide LI, it is characterised in that its sequence is as shown in sequence table SEQ ID No.1.
A kind of 2. method for preparing the efficient hybridization antibacterial peptide LI according to claim requirement 1, it is characterised in that including such as
Lower step：
1) according to pinpoint amino acid fragment intercept method, people's derived antimicrobial peptide LL37 14-21 amino acids residues and Niu Yuan are intercepted
Antibacterial peptide Indolicidin 5-13 amino acid residue segments, above-mentioned two fragment is sequentially connected, and obtains LI antibacterial peptides；
2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method；By obtained peptide resin after TFA is cut,
Tentatively obtain polypeptide products；Then after RPLC purifying and Mass Spectrometric Identification, that is, the system of the antibacterial peptide is completed
It is standby.
3. efficiently hybridization antibacterial peptide LI according to claim 1 is preparing treatment gram-positive bacteria or Gram-negative bacteria
Application in infectious disease medicament.
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| 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 |
| CN107312095A (en) * | 2017-07-13 | 2017-11-03 | 陕西科技大学 | A kind of Melittin of antibacterial peptide LL 37 and its application and the preparation method in bacillus subtilis |
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| CN102827255B (en) * | 2012-08-09 | 2014-04-16 | 东北农业大学 | Antibacterial peptide GW13 and its preparation method and use |
| CN104017086B (en) * | 2014-06-24 | 2016-03-09 | 科美博瑞科技(北京)有限公司 | A kind of fusion antibacterial peptide and preparation method thereof |
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