CN103923189A - Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof - Google Patents
Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 150000001413 amino acids Chemical group 0.000 claims abstract description 14
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 12
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 5
- 239000004475 Arginine Substances 0.000 claims abstract description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 12
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 12
- 101000730859 Sus scrofa Protegrin-1 Proteins 0.000 claims description 12
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- 125000003275 alpha amino acid group Chemical group 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
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- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 description 1
- 125000003345 AMP group Chemical group 0.000 description 1
- CYXCAHZVPFREJD-LURJTMIESA-N Arg-Gly-Gly Chemical compound NC(=N)NCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O CYXCAHZVPFREJD-LURJTMIESA-N 0.000 description 1
- NIUDXSFNLBIWOB-DCAQKATOSA-N Arg-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NIUDXSFNLBIWOB-DCAQKATOSA-N 0.000 description 1
- BSGSDLYGGHGMND-IHRRRGAJSA-N Arg-Phe-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N BSGSDLYGGHGMND-IHRRRGAJSA-N 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
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- 241000607132 Salmonella enterica subsp. enterica serovar Gallinarum Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- KLGFILUOTCBNLJ-IHRRRGAJSA-N Tyr-Cys-Arg Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O KLGFILUOTCBNLJ-IHRRRGAJSA-N 0.000 description 1
- XJFXZQKJQGYFMM-GUBZILKMSA-N Val-Cys-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)O)N XJFXZQKJQGYFMM-GUBZILKMSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a derived peptide IR2 of a pig-derived antibacterial peptide as well as a preparation method and application thereof. The sequence of the derived peptide IR2 of the pig-derived antibacterial peptide is as shown in SEQ ID No.1. Antibacterial peptides IR1 and IR2 are obtained by using a fixed point amino acid fragment interception and binary amino acid sequence superimposing method for intercepting six amino acid fragments of a pig-derived PG-1 corner part and symmetrically and circularly arranging charged amino acid arginine and hydrophobic amino acid isoleucine serving as repeated binary sequence units at the two sides of the corner. The antibacterial activity of the antibacterial peptide IR2 is higher than that of the antibacterial peptide IR1. The therapeutic index of the antibacterial peptide IR2 reaches up to 43.2 and is 216 times of that of the pig-derived antibacterial peptide RG-1. By virtue of the method, the hemolytic activity of the antibacterial peptide is greatly reduced under the condition of increasing the antibacterial activity of the antibacterial peptide, the selectivity of the antibacterial peptide between bacterial cells and mammalian cells is increased, and the development potential of the antibacterial peptide serving as antibiotic substitutes is improved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to derived peptide IR2 of a boar derived antimicrobial peptide and its preparation method and application.
Background technology
Antibacterial peptide (Antimicrobial peptides, AMPs) is the small molecule polypeptide being present in organism innate immune defence system, participates in the immune defense function of body, is the first barrier that body is resisted pathogenic micro-organism invasion and attack.Since Boman in 1972 etc. are cherishing the peptide material of finding to have for the first time germicidal action in guppy giant silkworm (Hyalophora cecropia), and since called after cecropin, people in succession from multiple biology isolation identification go out to have the polypeptide of bacteriostatic activity, and be referred to as antibacterial peptide.Up to now, the antibacterial peptide of including in the antibacterial peptide database (APD:the Antimicrobial Peptide Database) that U.S. Nebraska University Medical Center is set up has exceeded 2000 kinds, and constantly has new antibacterial peptide to be added into.Antibacterial peptide is made up of 12-50 amino-acid residue conventionally, molecular weight is less than 10kDa, most of antibacterial peptides contain positively charged amino acid, there is amphipathic and cationic, various bacteria, fungi, virus, parasite or even cancer cells are had to restraining effect, and very low to the toxicity of body self, extremely strong stability is conducive to large-scale industrial production.Antibacterial peptide shows very strong anti-microbial activity, and the especially restraining effect to microbiotic Resistant strain becomes the focus in present microbiotic substitute products research.And the antibacterial mechanism of antibacterial peptide is different from microbiotic, bacterium is difficult for producing resistance, and noresidue in animal body, environmentally safe, meets the needs that livestock product are kept the safety in production, be adapted at using in fodder production process, there is the potential quality as new generation of green fodder additives.At present, the exploitation of antibacterial peptide and application are extensively carried out in herding production and pharmaceutical sanitary field.
As everyone knows, antibiotic being applied in when bringing the very big interests of people, its abuse has also brought increasing harm to human health.At present, all there is the corresponding resistance strain of causing a disease in all conventional microbiotic, and the kind of pathogenic bacterium is also in continuous increase.The antibiont of finding brand-new type is an effective way that solves resistance problem.Antibacterial peptide is high because have anti-microbial activity, has a broad antifungal spectrum, and kind is many, and alternative scope is wide, and target bacterial strain is difficult for producing the features such as resistant mutation, and is considered to have broad application prospects in fields such as medicine, food and livestock industry.But compared with traditional microbiotic, the anti-microbial activity of natural antibacterial peptide is desirable not enough, cytotoxicity is higher.Therefore, the existing natural antibacterial peptide of brand-new design antibacterial peptide or molecular modification is the effective way of creating high vigor antibacterial peptide.
Summary of the invention
The object of the present invention is to provide a kind of antibacterial peptide IR2 derived from pig source Protegrin-1 and its preparation method and application; This antibacterial peptide activity is compared with high and cytotoxicity is relatively low.
Object of the present invention realizes by following technology:
1, the derived peptide IR2 of a boar derived antimicrobial peptide, sequence is as shown in SEQ ID No.1.
2, the derived peptide IR2 of a boar derived antimicrobial peptide as above, preparation method is as follows:
(1) taking the β-rotation angle position aminoacid sequence CRRRFC in pig derived antimicrobial peptide PG-1 sequence as basis, select charged arginine R and hydrophobic amino acid I to extend the two ends lamella of PG-1 rotation angle position, design has obtained IR series derivatives peptide: (IR)
nh (RI)
n-NH
2, n=1,2; H is β-rotation angle position aminoacid sequence CRRRFC of PG-1, in the time of n=1, and derived peptide called after IR1, sequence is as shown in SEQ No.2; In the time of n=2, derived peptide called after IR2, sequence is as shown in SEQ No.1;
(2) adopt solid state chemistry synthesis method to obtain peptide resin by Peptide synthesizer, the peptide resin obtaining, after TFA cutting, is obtained to two polypeptide;
(3), after RPLC purifying and Mass Spectrometric Identification, complete the preparation of polypeptide.
3, the application of the derived peptide IR2 of a boar derived antimicrobial peptide as above, the application in preparation treatment gram-positive microorganism or gram positive bacterial infection disease medicament.
The experimental technique of the antibacterial peptide of preparing by present method is simple, the antibacterial peptide obtaining is carried out to antibacterial and hemolytic activity detection, find that IR2 is not only to intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus, staphylococcus epidermidis, Salmonella typhimurium, Salmonella gallinarum, subtilis, eight kinds of bacterial classifications of streptococcus faecium have obvious restraining effect, and have very low hemolytic activity.In sum, IR2 is a kind of antibacterial peptide with higher using value.
Brief description of the drawings
Fig. 1 is the mass spectrum of antibacterial peptide IR1;
Fig. 2 is the mass spectrum of antibacterial peptide IR2.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
The design of antibacterial peptide
The aminoacid sequence of pig derived antimicrobial peptide PG-1 is:
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val Gly Arg-NH
2
1 5 10 15 18
By intercepting β-rotation angle position aminoacid sequence CRRRFC (8-13 amino acid) of PG-1, select charged arginine R and hydrophobic amino acid I to extend the two ends lamella of PG-1 rotation angle position.By intercepting PG-1 β-rotation angle position aminoacid sequence, make these corner two ends connect respectively IR or RI, design has obtained IR series peptide: (IR)
nh (RI)
n-NH
2, (n=1,2; H is β-rotation angle position aminoacid sequence CRRRFC of PG-1), in the time of n=1, derived peptide called after IR1; In the time of n=2, derived peptide called after IR2.The aminoacid sequence of derived peptide is as shown in table 1.
The aminoacid sequence of table 1 derived peptide
The charge number of IR1 and IR2 is respectively+and 6 and+8, hydrophobic value is respectively-0.56 and-0.40.By the C-terminal amidation of PG-1, IR1, tri-peptides of IR2 to improve a positive charge and to increase the stability of peptide.In the situation that improving antibacterial peptide fungicidal activity, reduced the hemolytic activity of antibacterial peptide by the method, improved the selectivity of antibacterial peptide between bacterial cell and mammalian cell, having improved it becomes the development potentiality of antibiotic substitute.
Embodiment 2
The synthetic IR1 of solid state chemistry synthesis method and two antibacterial peptides of IR2
1, the preparation of antibacterial peptide holds N end to carry out one by one from C, completes by Peptide synthesizer.First Fmoc-X (X is that the C of each antibacterial peptide holds first amino acid) is linked into Wang resin, obtains X-Wang resin after then sloughing Fmoc group; Again by Fmoc-Y-Trt-OH (9-fluorenes methoxy carboxyl-trimethylammonium-Y, Y is second amino acid of each antibacterial peptide C end); Be synthesized to N end from C end successively according to this program, until synthetic complete, obtain the resin of the side chain protected of sloughing Fmoc group;
2, in peptide resin obtained above, add cutting reagent, under 20 DEG C of lucifuges, react 2h, filter; Precipitation TFA (trifluoroacetic acid) washing, washing lotion is mixed with above-mentioned filtrate, and Rotary Evaporators is concentrated, then adds the precooling anhydrous diethyl ether of 10 times of left and right volumes,-20 DEG C of precipitation 3h, separate out white powder thing, with the centrifugal 10min of 2500g, collecting precipitation, use again anhydrous diethyl ether washing precipitation, vacuum-drying, obtains polypeptide, and wherein cutting reagent is mixed according to mass ratio by TFA, water and TIS (tri isopropyl chlorosilane) at 95: 2.5: 2.5;
3, use 0.2mol/L sodium sulfate (phosphoric acid is adjusted to pH7.5) to carry out column equilibration 30min, with 90% acetonitrile solution dissolving polypeptide, filter, the anti-phase normal pressure post of C18, adopt gradient elution (eluent is that methyl alcohol and aqueous sodium persulfate solution are to mix for 30: 70～70: 30 according to volume ratio), flow velocity is 1mL/min, and detection ripple is 220nm, collect main peak, freeze-drying; Recycle anti-phase C18 post and be further purified, elutriant A is the 0.1%TFA/ aqueous solution; Elutriant B is 0.1%TFA/ acetonitrile solution, and wash-out concentration is 25%B～40%B, and elution time is 12min, and flow velocity is 1mL/min, more the same collection main peak, freeze-drying;
4, the qualification of antibacterial peptide: through electron spray mass spectrometry analysis, the molecular weight showing in mass spectrum (as shown in Figure 1, 2) is basically identical with the theoretical molecular in table 1 by antibacterial peptide obtained above, and the purity of antibacterial peptide is greater than 95%.
Embodiment 3: the mensuration of antibacterial peptide anti-microbial activity
1, the mensuration of anti-microbial activity: peptide is configured as to certain storage liquid in order to using.Utilize the minimal inhibitory concentration of several antibacterial peptides of micro-broth dilution method.Using 0.01% acetic acid (containing 0.2%BSA) as diluent, use doubling dilution to configure successively the antibacterial peptide solution of serial gradient.Get above-mentioned solution 100 μ L and be placed in 96 porocyte culture plates, then add respectively isopyknic bacterium liquid (～10 to be measured
5individual/mL) in each hole.Positive control (contain bacterium liquid and do not contain antibacterial peptide) and negative control (neither containing bacterium liquid also containing peptide) are set respectively.37 DEG C of constant temperature culture 20h, having no bottom, hole with naked eyes has the minimal inhibitory concentration that is of research of chaotic phenomenon.Detected result is in table 2.
The bacteriostatic activity of table 2 antibacterial peptide
Can find out by table 2, IR1 and IR2 show the bacteriostatic activity of different depths for Gram-negative and positive bacteria, and IR2 bacteriostatic activity, higher than PG-1, illustrates the bacteriostatic activity that can improve antibacterial peptide by connecting 2 amino-acid residues in PG-1 β-bend end symmetrical.
2, the mensuration of hemolytic activity: gather people's fresh blood 1mL, be dissolved in 2mLPBS solution after anticoagulant heparin, the centrifugal 5min of 1000g, collects red corpuscle; With PBS washing 3 times, then use 10mL PBS resuspended; Get 50 μ L red cell suspensions and mix with the antibacterial peptide solution of the different concns of 50 μ L PBS dissolvings, constant-temperature incubation 1h in 37 DEG C of incubators; After 1h, take out 4 DEG C, the centrifugal 5min of 1000g; Take out supernatant liquor microplate reader in 570nm place photometry absorption value; Average for every group, and comparative analysis.Wherein 50 μ L red corpuscle add 50 μ LPBS as negative control; 50 μ L red corpuscle add 50 μ L0.1%Tritonx-100 as positive control.Minimum hemolytic concentration is the antibacterial peptide concentration of antibacterial peptide while causing 10% hemolysis rate.Detected result is in table 3.
The mensuration of table 3 antibacterial peptide hemolytic activity
Can find out by table 3, IR1 does not show hemolytic activity in sensing range, and IR2 just shows lower hemolytic activity in the time of 128 μ M concentration.
Above result demonstration, in derived peptide, along with ArgIle (RI) or IleArg (IR) increase symmetrical tip length, its anti-microbial activity significantly improves.Comprehensively analyze the antibacterial and hemolytic activity of antibacterial peptide, can more fully evaluate by therapeutic index (ratio of hemolytic concentration and Mlc) biologic activity of each antibacterial peptide.As can be seen from Table 3, IR2 has higher therapeutic index, shows that the IR2 antibacterial peptide that obtains of design has the development potentiality of higher substitute antibiotics.
Claims (3)
1. the derived peptide IR2 of a boar derived antimicrobial peptide, is characterized in that, sequence is as shown in SEQ ID No.1.
2. the derived peptide IR2 of a boar derived antimicrobial peptide according to claim 1, is characterized in that, preparation method is as follows:
(1) taking the β-rotation angle position aminoacid sequence CRRRFC in pig derived antimicrobial peptide PG-1 sequence as basis, select charged arginine R and hydrophobic amino acid I to extend the two ends lamella of PG-1 rotation angle position, design has obtained IR series derivatives peptide: (IR)
nh (RI)
n-NH
2, n=1,2; H is β-rotation angle position aminoacid sequence CRRRFC of PG-1, in the time of n=1, and derived peptide called after IR1, sequence is as shown in SEQ No.2; In the time of n=2, derived peptide called after IR2, sequence is as shown in SEQ No.1;
(2) adopt solid state chemistry synthesis method to obtain peptide resin by Peptide synthesizer, the peptide resin obtaining, after TFA cutting, is obtained to two polypeptide;
(3), after RPLC purifying and Mass Spectrometric Identification, complete the preparation of polypeptide.
3. the application of the derived peptide IR2 of a boar derived antimicrobial peptide according to claim 1, is characterized in that, the application in preparation treatment gram-positive microorganism or gram positive bacterial infection disease medicament.
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