CN110283252A - Pig source heterozygous antibacterial peptide PP-1 and its preparation method and application - Google Patents
Pig source heterozygous antibacterial peptide PP-1 and its preparation method and application Download PDFInfo
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- CN110283252A CN110283252A CN201910629105.6A CN201910629105A CN110283252A CN 110283252 A CN110283252 A CN 110283252A CN 201910629105 A CN201910629105 A CN 201910629105A CN 110283252 A CN110283252 A CN 110283252A
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- antibacterial peptide
- peptide
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- polypeptide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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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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- 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 present invention provides boar source heterozygous antibacterial peptide PP-1 and its preparation method and application, and the sequence of antibacterial peptide is as shown in sequence table SEQ ID No.1.The present invention intercepts the segment heterozygosis of its segment and pig source marrow antibacterial peptide PMAP-23 or PMAP-36 and obtains 3 antibacterial peptide sequences of amino acid length 19 to 22 using PR-39 as target；Then Peptide synthesizer is used, solid-phase synthesis synthesis polypeptide is carried out；It is purified, and is identified using electron spray mass spectrometry；Finally, measuring the bacteriostatic activity and cytotoxicity of derivative antibacterial peptide by minimal inhibitory concentration and hemolytic test, screening has more satisfactory active polypeptide.Antibacterial peptide PP-1 safety of the invention is preferable, has broad spectrum antibiotic activity, hypotoxicity and low hemolytic；This antibacterial peptide is made of 19 amino acid, and peptide chain is shorter, and preparation method is simple and technology maturation, and synthesis cost is low, is suitably applied production.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to boar source heterozygous antibacterial peptide PP-1 and preparation method thereof and answer
With.
Background technique
Conventional antibiotic improves the production performance of animal, reduces disease incident, but also cause simultaneously medicament residue,
The generation of the problems such as endurance strain, environmental pollution.Especially day is presented in abuse of antibiotics in recent years and drug resistance problems
Increasingly acute trend, it can be seen that the efficient and safe novel fodder of research and development, which carries out substitution to conventional antibiotic, has important meaning
Justice.Antibacterial peptide is the important component of organism natural immune defence system, not only has and inhibits, kill various bacteria, is true
Bacterium, virus, helminth biological function, and animal body immunocompetence can be improved, secondary makees there is no residual, without any
With.Therefore, antibacterial peptide substitute antibiotics have become research hotspot in recent years.
Pig derived antimicrobial peptide type is more, wherein PR-39 research it is relatively broad, be a kind of Pro-rich-it is arginic by
The linear Endogenous antimicrobial polypeptide of 39 amino acid residues composition.But mainly there is antibacterial action to Gram-negative bacteria, it is right
Part gram-positive bacteria, such as staphylococcus aureus and verdigris add sporangium etc., without inhibiting effect.Therefore how to widen
The antimicrobial spectrum of PR-39 has great importance.
Summary of the invention
Based on the above shortcomings, the purpose of the present invention is to provide a boar source heterozygous antibacterial peptide PP-1, sequence is such as
Shown in sequence table SEQ ID No.1, the polypeptide is by pig derived antimicrobial peptide PR-39 and pig source marrow antibacterial peptide PMAP-23 heterozygosis, peace
Full property is preferable, has broad spectrum antibiotic activity, hypotoxicity and low hemolytic.
Another object of the present invention is that providing the preparation method of a boar source heterozygous antibacterial peptide PP-1, the preparation side
Method is simple and technology maturation, and synthesis cost is low, is suitably applied production, and method and step is as follows:
(1) according to the amino acid compositing characteristic of pig derived antimicrobial peptide PR-39, its segment and pig source marrow antibacterial peptide are intercepted
The segment heterozygosis of PMAP-23 or PMAP-36 and 3 heterozygosis antibacterial PP-1, PP-2 and PP-3 for obtaining amino acid length 19 to 22,
Its sequence is respectively as shown in sequence table SEQ ID No.1-3；
(2) solid-state chemical reaction method method is used, Peptide synthesizer synthesis polypeptide is passed through；
(3) polypeptide of synthesis is purified using reversed-phase high performance liquid chromatography, and using electrospray mass spectrometry to synthesis
Polypeptide identified, complete the preparation of polypeptide；
(4) method for the measurement minimal inhibitory concentration recommended using U.S. clinical Laboratory Standard research institute is found miscellaneous
The minimal inhibitory concentration average value for closing antibacterial PP-1 is 3.56 μM, and without hemolytic toxicity, comprehensive therapeutic index reaches 144, thus
Choose pig source heterozygous antibacterial peptide PP-1.
Leather orchid is treated in preparation another object of the present invention is to provide pig source heterozygous antibacterial peptide PP-1 as described above
Application in family name's positive bacteria or/and gram positive bacterial infection disease medicament.
Advantages of the present invention and the utility model has the advantages that the polypeptide by pig derived antimicrobial peptide PR-39 and pig source marrow antibacterial peptide PMAP-
23 heterozygosis, safety is preferable, has broad spectrum antibiotic activity, hypotoxicity and low hemolytic；The polypeptide is made of 19 amino acid, peptide
Chain is shorter, and preparation method is simple and technology maturation, and synthesis cost is low, is suitably applied production.
Detailed description of the invention
Fig. 1 is the mass spectrogram of antibacterial peptide PP-1.
Fig. 2 is the mass spectrogram of antibacterial peptide PP-2.
Fig. 3 is the mass spectrogram of antibacterial peptide PP-3.
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.
Embodiment 1: the design of pig source heterozygous antibacterial peptide
Pig derived antimicrobial peptide PR-39, PMAP-23 and PMAP-36 overall amino acid sequence is obtained by NCBI, passes through biological information
Credit analysis, obtains the nucleus of three.By heterozygosis mode, 3 antibacterial peptide sequences of amino acid length 19 and 22 are obtained
PP-1, PP-2, PP-3.As shown in table 1.
The amino acid sequence and molecular weight of 1 pig source heterozygous antibacterial peptide of table
Embodiment 2: the synthesis of pig source heterozygous antibacterial peptide
Three polypeptides shown in table 1 are synthesized using Peptide synthesizer, select Solid-phase organic synthesis, is protected and is closed using Fmoc
Cheng Fa, compound direction carry out one by one from C-terminal to N-terminal, the specific steps are as follows:
(1) the Wang resin connected with first amino acid of C-terminal, i.e. Fmoc-A (trt)-Wang (9- fluorenes first are chosen
Oxygen carboxyl-trimethyl-A, wherein A is first amino acid of C-terminal), using dimethylformamide (DMF) impregnate 15min or so with
Remove impurity；With the Fmoc protection on the DMF removing resin containing 20% piperidines, 20min is reacted, washing resin is until completely.
Piperidines is washed with DMF, remaining solid suspension is the A-Wang of deprotection.Check that A-Wang is de- with triketohydrindene hydrate detection agent
The quality of protection.
(2) by Fmoc-B (trt)-OH, (9- fluorenes methoxy carboxyl-trimethyl-B, B is second amino of each antibacterial peptide C-terminal
Acid) with the Wang resin of deprotection obtained above carry out condensation reaction；Then Fmoc group is sloughed again.According to this program
Successively extend one by one from C-terminal to N-terminal, until the synthesis of entire peptide chain is finished, after the deprotection of the last one amino acid, uses DMF
Washing 8 times, then with ethyl alcohol and methylene chloride (DCM) cross washing 8 times.By trifluoroacetic acid (TFA): tri isopropyl chlorosilane
(TIS): water=95:2.5:2.5 (volume ratio) is mixed, and preparing becomes at cutting reagent and 20 DEG C of polypeptide obtained above
2h is reacted, polypeptide is cut down from resin.Rotary Evaporators evaporate TFA, and the pre-cooling for adding 10 times or so volumes is anhydrous
White powdery solids are precipitated in ether precipitated polypeptide 3h.Vacuum drying, obtains crude product polypeptide.
(3) above-mentioned crude product polypeptide is dissolved using 90% acetonitrile solution, is purified using preparation chromatographic column, analytic type
Chromatography post detection purity.Semipreparative high performance liquid chromatography instrument is Waters Delta Prep 4000, and preparation chromatographic column is
Waters X-Bridge C18, 5 μm of reversed-phase columns.Eluent A is the aqueous solution containing 0.1%TFA, and B is to contain 0.1%TFA's
Acetonitrile solution；Detection wavelength 220nm, the linear concentration gradient that type of elution is 30%B~65%B elute, and flow velocity is
30mL/min.The fraction that purity is higher than 95% is collected, and is freeze-dried.Analytic type high performance liquid chromatograph is Agilent
1100, analytic type chromatographic column is SepaxGP-C18Reversed-phase column (4.6mm × 150mm, 5 μm), eluent A liquid are the water of 0.1%TFA
Solution, B liquid are the acetonitrile solution containing 0.1%TFA；Detection wavelength 220nm.Type of elution is the linear of 50%B~75%B
Concentration gradient elution, flow velocity 1.0mL/min.
(4) Mass Spectrometric Identification of polypeptide: polypeptide obtained above is analyzed by electrospray mass spectrometry, is shown in mass spectrogram
Molecular weight is consistent with theoretical molecular weight.As shown in the picture.It is purified using high performance liquid chromatography, so that the purity of antibacterial peptide is big
In 95%.
Embodiment 3: the bacteriostatic activity and cytotoxicity assay of pig source heterozygous antibacterial peptide
Using the side for the measurement minimal inhibitory concentration (MIC) that U.S. clinical Laboratory Standard research institute (CLSI) is recommended
Method, while the feature of the cationic for antibacterial peptide, are correspondingly improved.With 0.01% acetic acid (containing 0.2%BSA)
As dilution, the antibacterial peptide solution of graded series is configured in order using doubling dilution.Above-mentioned 50 μ L of solution is taken to be placed in 96 holes
In tissue culture plate, isometric bacterium solution to be measured (~105/mL) is then added respectively in each hole.It is positive right to be respectively set
According to (not containing antibacterial peptide containing bacterium solution) and negative control (be both free of bacterium solution or be free of antibacterial peptide).37 DEG C of constant temperature incubation 20h,
Visually to have no that the as minimal inhibitory concentration of research of chaotic phenomenon is arranged at hole bottom.The results are shown in Table 2, it can be seen that PP-1 is most
Small Mlc is 2-8 μM, and PP-2 is 1-4 μM, and PP-3 is 4-16 μM.From table 3 it can be seen that PP-1, PP-2, PP-3
Minimal inhibitory concentration be respectively 3.56,2,7.13 μM.
The bacteriostatic activity of 2 pig source heterozygous antibacterial peptide of table
In addition, by the hemolytic activity for measuring polypeptide using human red blood cells, as a result, it has been found that the minimum of PP-1, PP-2, PP-3
Hemolytic activity is respectively > 256,64, > 256 μM.Comprehensive minimal inhibitory concentration and minimum hemolytic concentration, discovery hybrid peptide PP-1,
The TI value of PP-2, PP-3 are followed successively by 143.82,32 and 71.81, and wherein the TI value of PP-1 is maximum, show that highest treatment refers to
Number, is more suitable for production application.
The hemolytic activity of 3 pig source heterozygous antibacterial peptide of table
Remarks:
GM: the geometric mean of minimal inhibitory concentration is represented
MHC: minimum hemolytic concentration is represented
TI: MHC/GM is represented.
Sequence table
<110>Northeast Agricultural University
<120>pig source heterozygous antibacterial peptide PP-1 and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Arg Arg Arg Pro Arg Pro Pro Tyr Gly Gly Gly Lys Phe Val Thr Val
1 5 10 15
Trp Val Arg
<210> 2
<211> 22
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Arg Arg Arg Pro Arg Pro Pro Tyr Gly Gly Gly Arg Ile Ile Arg Leu
1 5 10 15
Leu Trp Arg Val Arg Arg
20
<210> 3
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Arg Arg Arg Pro Arg Pro Pro Tyr Gly Gly Gly Ile Gly Lys Val Leu
1 5 10 15
Lys Trp Ile
Claims (3)
1. pig source heterozygous antibacterial peptide PP-1, it is characterised in that: its sequence is as shown in sequence table SEQ ID No.1.
2. the preparation method of pig source heterozygous antibacterial peptide PP-1 according to claim 1, which is characterized in that method is as follows:
(1) according to the amino acid compositing characteristic of pig derived antimicrobial peptide PR-39, its segment and pig source marrow antibacterial peptide PMAP-23 are intercepted
Or PMAP-36 segment heterozygosis and obtain 3 heterozygosis antibacterial PP-1, PP-2 and PP-3 of amino acid length 19 to 22, sequence
Respectively as shown in sequence table SEQ ID No.1-3；
(2) solid-state chemical reaction method method is used, Peptide synthesizer synthesis polypeptide is passed through；
(3) polypeptide of synthesis is purified using reversed-phase high performance liquid chromatography, and using electrospray mass spectrometry to the more of synthesis
Peptide is identified, the preparation of polypeptide is completed；
(4) method for the measurement minimal inhibitory concentration recommended using U.S. clinical Laboratory Standard research institute, discovery heterozygosis are anti-
The minimal inhibitory concentration average value of bacterium PP-1 is 3.56 μM, and without hemolytic toxicity, comprehensive therapeutic index reaches 144, to choose
Pig source heterozygous antibacterial peptide PP-1.
3. pig source heterozygous antibacterial peptide PP-1's according to claim 1 is blue in preparation treatment gram-positive bacteria or/and leather
Application in family name's negative microbial infections disease medicament.
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| CN113527513A (en) * | 2021-06-23 | 2021-10-22 | 青岛联吉生物医疗科技有限公司 | Preparation method and application of hybrid antibacterial peptide Spcrus-APP |
| CN113603748A (en) * | 2021-07-09 | 2021-11-05 | 东北农业大学 | Beta-folded antibacterial peptide HINGE-RV and preparation method and application thereof |
| CN113773367A (en) * | 2021-08-09 | 2021-12-10 | 东北农业大学 | I and i +4 fixed-point mutation high-activity derivative antibacterial peptide of porcine TP peptide, and preparation method and application thereof |
| CN113896767A (en) * | 2021-09-27 | 2022-01-07 | 中国农业大学 | Self-assembled antibacterial peptide nanoparticles and application thereof |
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Cited By (6)
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| CN113527513A (en) * | 2021-06-23 | 2021-10-22 | 青岛联吉生物医疗科技有限公司 | Preparation method and application of hybrid antibacterial peptide Spcrus-APP |
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| CN113603748A (en) * | 2021-07-09 | 2021-11-05 | 东北农业大学 | Beta-folded antibacterial peptide HINGE-RV and preparation method and application thereof |
| CN113773367A (en) * | 2021-08-09 | 2021-12-10 | 东北农业大学 | I and i +4 fixed-point mutation high-activity derivative antibacterial peptide of porcine TP peptide, and preparation method and application thereof |
| CN113773367B (en) * | 2021-08-09 | 2022-06-14 | 东北农业大学 | I and i +4 fixed-point mutation high-activity derivative antibacterial peptide of porcine TP peptide, and preparation method and application thereof |
| CN113896767A (en) * | 2021-09-27 | 2022-01-07 | 中国农业大学 | Self-assembled antibacterial peptide nanoparticles and application thereof |
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