CN105985407A - Hippocampus kuda-like antimicrobial peptide hkplpl-2 - Google Patents
Hippocampus kuda-like antimicrobial peptide hkplpl-2 Download PDFInfo
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- CN105985407A CN105985407A CN201510060607.3A CN201510060607A CN105985407A CN 105985407 A CN105985407 A CN 105985407A CN 201510060607 A CN201510060607 A CN 201510060607A CN 105985407 A CN105985407 A CN 105985407A
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Abstract
The invention relates to a hippocampus kuda-like antimicrobial peptide hkplpl-2. The hippocampus kuda-like antimicrobial peptide hkplpl-2 contains a sequence from a conserved region GIVHAGKT-NH2 of a hippocampus kuda antimicrobial peptide hkplp. The hkplpl-2 having a sequence of GFLFHGLLHAGKVIHGIVHRRG is designed and synthesized mainly by conducting homologous comparison on an antimicrobial peptide hkplp, conducting analysis to obtain a conserved region and hybridizing the conserved region with a conserved region IHRRR-NH2 of an antimicrobial peptide, namely piscidin-like antimicrobial peptide precursor [Epinephelus bleekeri] by using hkplp as a main body. The hippocampus kuda-like antimicrobial peptide hkplpl-2 has the beneficial effects of highly effective broad-spectrum antibacterial effects and low toxicity, and can be used as a substitute of antibiotics.
Description
Number of patent application:
Technical field: the present invention is a kind Hippocampus antibacterial peptide hkplpl-2.
Background technology: recently as the abuse of antibiotic, drug resistance bacterium gets more and more, and drug resistance is the most serious.According to data, the S. aureus L-forms being separated on U.S. clinical has 95% pair of Penicillin-resistant, more than 50% to methicillin resistance (Frindkin SK, Gaynes RP.Antimicrobial resistance in intensive care units.Clin Chest Med.1999,20 (2): 303-316)；Within 2012, China's Clinical detection finds, in S. aureus L-forms and coagulase negative staphylococcus, the recall rate of methicillin resistance strain (MRSA and MRCNS) is averagely respectively 47.9% and 77.1% (Chinese CHINET bacterial drug resistance monitoring Chin J Infect Chemother in 2012,2013,13 (5): 321-330).The productive life of the mankind in the gradually serious threat of this situation, thus cause awakening and the raising of food safety rank consciousness that this potential threat realized by people, create substitute old antibiotic and environmental protection, safe overriding resistance antibiotic of new generation in the urgent need to.
Antibacterial peptide is the important component part of immune defense system in organism, is small molecule peptide, is referred to as safe natural antibiotics, have has a broad antifungal spectrum, have no drug resistance, the advantage such as thermally-stabilised good, water solublity is high, nontoxic residue-free.According to statistics, current China still has up to 20 kinds people and animals to share or feed additive made by the special antibiotic of poultry, in addition, antibacterial peptide also by and will be widely used in cosmetics, food preservative, medicine, transgene expression etc. and live central with human being's production.By the product replacing conventional antibiotic of antibacterial peptide product development Cheng Xin, a pin cardiac tonic will be injected to the relevant industries of China, promote China's relevant industries constantly to advance.
Antibacterial peptide hkplpl-2 sequence: GFLFHGLLHAGKVIHGIVHRRG；Molecular formula: C113H177N37O23, molecular weight MW [AV]: 2421.8；Do not contain rare amino acid and exogenous chemical components, there is the advantages such as immunogenicity little, good water solubility, broad-spectrum sterilization, be resistant to protease and the degraded of peptidase in gastrointestinal tract.The most in acid condition, heat even high temperature high pressure process its antibacterial activity is not affected.Can persistently play its function during product preserves, keep product quality, extend the shelf life.Hkplpl-2 can be good conventional antibiotic succedaneum as the additive of a new generation, effectively can must solve the residual problem with pathogenic bacteria resistance to drugs of humans and animals medicine.
Summary of the invention: it is an object of the invention to provide a kind of artificial constructed antibacterial peptide sequence.We are on the basis of having the original sequence of antibacterial peptide hkplp of good antibacterial activity, and we carry out artificial reconstructed design to it, according to sequence analysis, analyze conserved sequence GIVHAGKT.Heterozygosis is carried out with the conserved sequence IHRRR of antibacterial peptide plapp (piscidin-like antimicrobial peptide precursor [Epinephelus bleekeri]), design ten kinds of artificial antimicrobial peptides, by screening, finally obtain sequence: GFLFHGLLHAGKVIHGIVHRRG.
The method of the present invention comprises the steps:
Step (1) present invention utilizes bioinformatics method antagonism thalline hkplp to carry out BLAST comparison, analyzes and select conservative region P1:GIVHAGKT；
Step (2) selects the conservative region P2:IHRRR of the antibacterial peptide plapp close with its function, carries out hybrid design with hkplp；
Step (3) uses chemical synthesis synthesis hkplpl series antibacterial peptide ten groups；
Step (4) uses plating method to screen synthetic antibacterial peptide hkplpl Series Design antibacterial peptide, and antibacterial peptide obvious to effect carry out to staphylococcus aureus, bacillus coli DH 5 alpha, Salmonella, escherichia coli that animal pathogenic escherichia coli separates with hospital clinical case, staphylococcus haemolyticus antibacterial experiment as it is shown in figure 1, result display antibacterial peptide hkplpl-2 all has good antibacterial activity to above each bacterium.
Step (5) uses the hkplpl-2 solution of the method preparation variable concentrations of doubling dilution, joins and survey OD after cultivation bacterium solution cultivates 16h630Value, determines synthetic hkplpl-2 minimal inhibitory concentration MIC to different strains.
Accompanying drawing illustrates:
Figure is synthetic antibacterial peptide hkplpl-2 to staphylococcus aureus, bacillus coli DH 5 alpha, Salmonella, escherichia coli that animal pathogenic escherichia coli separates with hospital clinical case, the antibacterial experiment result of staphylococcus haemolyticus, in figure: 1 represents the antibacterial activity of ampicillin；0 is negative control ddH2O；2,3,4 is the antibacterial activity of artificial synthetic antimicrobial peptide.
Detailed description of the invention:
The present invention is specifically described by embodiment given below, but is pointed out that and the invention is not restricted to specific embodiment, and embodiment is served only for further illustrating the present invention, it is impossible to be interpreted as limitation of the scope of the invention.
Specific embodiment illustrates:
The preparation of embodiment 1:Fmoc-Arg (pbf)-amino resins
By amino resins Fmoc-Rink Amide 10.03g, substitution degree 0.84mmol/g, join in solid phase reactor, add DMF to soak so that it is abundant swelling 30min, drain, add 20% piperidines/DMF solution and take off Fmoc 5min, 15min, by DMF, DCM, MeOH washes clean.Take 11.1g Fmoc-Arg (Pbf)-OH, 2.24gHOBt, 6.14gHBTU DMF joins balance 5min in reactor after dissolving, add 6.0ml DIPEA room temperature reaction 1 hour, drain, priority DMF, DCM, MeOH respectively wash 3 times, are dried 17.12g Fmoc-Arg (pbf)-Rink Amide resin of weighing, and detection substitution value is 0.67mmol/g.
The preparation of embodiment 2:hkplpl-1-amino resins
Above-mentioned resin is put in and connects in peptide bottle, add 20% piperidines/DMF solution and take off Fmoc 5min, 15min, by DMF, DCM, MeOH washes clean.Then by sequence GFLFHGLLHAGKVIHGIVHRRG-NH2Order connect the aminoacid of Fmoc protection successively, reaction system mol ratio is Fmoc-AA:DIC.HOBt throws in by 1: 1: 1, and Fmoc-AA is by 3 times of additions of amount of resin in this instance, and each DIC adds about 4.10g, HOBt and adds about 4.27g.Period 20% piperidines successively/DMF removing Fmoc blocking group 2 times, first set reaction 5min, second time reaction 15min；Each step amino acid condensation reacts one time 2-4 hour, all uses whether Kaise Test detection reaction is condensed completely, repeats to be condensed this aminoacid as chromogenic reaction is positive, and the last directly removing Fmoc blocking group of sequence condensation, DMF, DCM, MeOH wash several times.
The preparation of embodiment 3:hkplpl-1 crude product
Hkplpl-1 amino resins with TFA:Tis:Phenol by volume 95: 2.5: 2.5 mixed solution process peptide resin 3-4 hour; peptide is scaled off from resin and excises Side chain protective group; separating out solid with ice ether after being concentrated to dryness, centrifugal drying obtains crude product 29.51g.
The preparation of embodiment 4:hkplpl-1 finished product
After Hkplpl-1 crude product dissolves with 1% acetum, centrifuging and taking supernatant crosses G10 post, collects antibacterial peptide hkplpl-1 peak concentrate drying and obtains finished product 17.71g.Purity is more than 60%, and total recovery reaches 54%.
Embodiment 5: by the polypeptide of remaining hkplpl series of embodiment 1 to 4 synthesis.Sequence is as follows:
Hkplpl-2:GFLFHGLLHAGKVIHGIVHRRG
Hkplpl-3:GLIFKGIVHAGKT LHRVIHRRG
Hkplpl-4:GLFIKGIVHAGKT LHRVIHRRG
Hkplpl-5:GLIKFGIVHAGKT LHRVIHRRR
Hkplpl-6:GLIFKGIVHAGKT KHRVIHRRR
Hkplpl-7:GLIFKGIVHAGKT HLRVIHRRR
Hkplpl-8:GILKFGIVHAGKT LHRVIHRRR
Hkplpl-9:GILKFGIVHAGKT IKRVIHRRR
Hkplpl-10:GLIFHGIVHAGKT LKRVIHRRR
Embodiment 6:hkplpl series is screened
From YPD flat board, picking PCR is accredited as single colony inoculation of the positive in 100mlBMGY culture medium, 30 DEG C of shaken cultivation to OD6002～6, remove supernatant, add the resuspended thalline of 20mlBMMY culture medium, 30 DEG C of shaken cultivation 96 hours, took 1ml sample every 24 hours in Eppendorff pipe, take supernatant and detect for antibacterial activity.
Agar culture medium is added in a water bath heat fusing, is cooled to about 50 DEG C, draw the staphylococcus aureus (OD600=0.3) of 60 μ l by aseptic manipulation, add in 20ml agar culture medium, mixing, pours in the aseptic plane ware of a diameter of 9cm rapidly, and horizontal positioned is to be solidified.Agar is beaten the circular hole of a diameter of 2.7mm, respectively the synthesis hkplpl of addition phosphate buffered saline, sterilized water and 100mg/ml Amplicin in hole.Plate is inverted in 37 DEG C of overnight incubation, next day observed result.The results are shown in Table 1.
The table 1 staphylococcus aureus Hkplpl many peptide screenings of series
Embodiment 7: the mensuration of synthetic antibacterial peptide hkplpl-2 antimicrobial spectrum
From YPD flat board, picking PCR is accredited as single colony inoculation of the positive in 100mlBMGY culture medium, 30 DEG C of shaken cultivation to OD6002～6, remove supernatant, add the resuspended thalline of 20mlBMMY culture medium, 30 DEG C of shaken cultivation 96 hours, took 1ml sample every 24 hours in Eppendorff pipe, take supernatant and detect for antibacterial activity.
Agar culture medium is added in a water bath heat fusing, is cooled to about 50 DEG C, draw the raw of 60 μ l by aseptic manipulation and survey bacterium (OD600=0.3), add in 20ml agar culture medium, mixing, pours in the aseptic plane ware of a diameter of 9cm rapidly, and horizontal positioned is to be solidified.Agar is beaten the circular hole of a diameter of 2.7mm, respectively the synthesis hkplpl-2 of addition phosphate buffered saline, sterilized water and 100mg/ml Amplicin in hole.Plate is inverted in 37 DEG C of overnight incubation, next day observed result.
The antibacterial peptide hkplpl-2 of synthetic to staphylococcus aureus, bacillus coli DH 5 alpha, Salmonella, escherichia coli that animal pathogenic escherichia coli separates with hospital clinical case, staphylococcus haemolyticus antibacterial experiment as it is shown in figure 1, this antibacterial peptide all has good antibacterial activity to above each bacterium.
Embodiment 8: the mensuration of synthetic hkplpl-2 minimal inhibitory concentration (MIC)
MIC: test strain is cultivated the bacterium solution dilution about 5 × 10 to exponential phase6CFU/ml, adds in 96 well culture plates, and the every hole of instrument connection adds bacterium solution 90 μ l, is subsequently adding the synthetic antimicrobial peptide solution of the variable concentrations of doubling dilution, 10 μ l/ holes.Positive control is the bacterium solution in 100 μ l/ holes, and negative control is corresponding 100 μ l culture medium.Then shake slowly in 37 DEG C and cultivate about 16h, survey OD by microplate reader630.The least concentration of bacteria growing inhibiting is MIC, the results are shown in Table 2.
The MIC of table 2 recombinant antibacterial peptide HKPLPL-2
Embodiment 9: artificial synthetic antimicrobial peptide hkplpl-2 antibacterial activity unit determines
1mg purity is not less than the antibacterial activity of the antibacterial peptide hkplpl-1 of the solid-state chemical reaction method of 95% and is defined as 1000U by us.Therefore the antibacterial activity of the antibacterial peptide hkplpl-2 of synthetic is not less than 630U/mg.
Above content is the further description combining concrete preferred implementation to the present invention, it is impossible to assert the present invention be embodied as be confined to these explanations.Without departing from the inventive concept of the premise, those skilled in the art can make some deduction or replace, is regarded as protection scope of the present invention.
Claims (10)
1. the chemical synthesis process of a kind Hippocampus antibacterial peptide hkplpl-2 and part antimicrobial spectrum function thereof, mainly comprise the steps that
1) with Fmoc-amino resins as carrier, being sequentially connected with the aminoacid with blocking group according to the method for solid phase synthesis, it is thus achieved that hkplpl-2-amino resins, period removes Fmoc blocking group successively；
2) with trifluoroacetic acid mixed solution, peptide is cut from resin, process and be dried to obtain crude product；
3) crude product crosses G10 column purification, and concentrate drying obtains finished product；
4) antimicrobial spectrum of the antibacterial peptide hkplpl-2 of synthetic is measured；
5) minimal inhibitory concentration (MIC) of the antibacterial peptide hkplpl-2 of synthetic is measured.
Method the most according to claim 1; it is characterized in that: during solid phase synthesis connects aminoacid; Fmoc blocking group scavenger 20% piperidines/DMF; Fmoc-AA is added by the 2-5 times amount of condensation amount; wherein Fmoc-AA, condensing agent ratio are thrown in molar ratio at 1: 1, each 2-4 hour of condensation reaction.
3. according to method described in claim 1 and 2, it is characterized in that: in aminoacid connection procedure, each step amino acid condensation all uses whether Kaise Test detection reaction is condensed completely, repeats to be condensed this aminoacid as chromogenic reaction is positive.
4. according to method described in claim 1 and 2, it is characterized in that: condensing agent system uses DIC+A or A+B+C, and wherein A is HOBt or HOAt, and B is HBTU, HATU, PyBOP or TBTU, and C is DIPEA or TMP, can be mutually combined in the way of using permutation and combination.
5. according in claim 1 2) described method, it is characterized in that: trifluoroacetic acid mixed solution is TFA: Tis: Phenol by volume 95: 2.5: 2.5, and the response time is 3-4 hour.
6. according in claim 1 2) described method, it is characterized in that: process is dried and refers to that cutting liquid uses ice ether precipitation solid, centrifugal drying after concentrating or be blown to do.
7. according in claim 1 3) described method, it is characterized in that: the acetum with 1% processes hkplpl-2 crude product, and centrifuging and taking supernatant crosses G10 post.
8. according in claim 1 3) described method, it is characterized in that: it is concentrated rear dry that the hkplpl-2 of collection crosses post liquid.
9. according in claim 1 4) described content, it is characterized in that: show that the hkplpl-2 of synthetic is to staphylococcus aureus, escherichia coli K by plating method12D31, the escherichia coli that separates with hospital clinical case of bacillus coli DH 5 alpha, Salmonella, animal pathogenic escherichia coli, urine enterococcus, staphylococcus haemolyticus all have good antibacterial activity.
10. according in claim 1 5) described content, it is characterized in that: use the hkplpl-2 solution of the method preparation variable concentrations of doubling dilution, join and survey OD after cultivation bacterium solution cultivates 16h630Value, determines synthetic hkplpl-2 minimal inhibitory concentration MIC to different strains.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105837674A (en) * | 2016-03-08 | 2016-08-10 | 无限极（中国）有限公司 | Bombina orientalis polypeptide, and preparation method and application thereof |
| CN113698464A (en) * | 2021-08-31 | 2021-11-26 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp II-4 (63-88) and application thereof |
| CN116143882A (en) * | 2022-10-13 | 2023-05-23 | 广西中医药大学 | Hippocampus trimarans hepcidin antibacterial peptide and application thereof |
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|---|---|---|---|---|
| CN1654651A (en) * | 2004-12-27 | 2005-08-17 | 中山大学 | Novel natural antibacterial peptide Hippocampusin and its preparation and application |
| CN101988038A (en) * | 2009-07-31 | 2011-03-23 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding |
| CN102757490A (en) * | 2011-04-28 | 2012-10-31 | 广东中大南海海洋生物技术工程中心有限公司 | Chemical synthesis method of hippocampus antimicrobial peptide hkplp and application thereof in breeding industry |
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- 2015-02-04 CN CN201510060607.3A patent/CN105985407A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654651A (en) * | 2004-12-27 | 2005-08-17 | 中山大学 | Novel natural antibacterial peptide Hippocampusin and its preparation and application |
| CN101988038A (en) * | 2009-07-31 | 2011-03-23 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus antibiotic peptide pichia pastoris engineering strain and use thereof in breeding |
| CN102757490A (en) * | 2011-04-28 | 2012-10-31 | 广东中大南海海洋生物技术工程中心有限公司 | Chemical synthesis method of hippocampus antimicrobial peptide hkplp and application thereof in breeding industry |
Non-Patent Citations (1)
| Title |
|---|
| DANDAN SUN，等: "Identification, synthesis and characterization of a novel antimicrobial peptide HKPLP derived from Hippocampus kuda Bleeker", 《THE JOURNAL OF ANTIBIOTICS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105837674A (en) * | 2016-03-08 | 2016-08-10 | 无限极（中国）有限公司 | Bombina orientalis polypeptide, and preparation method and application thereof |
| CN113698464A (en) * | 2021-08-31 | 2021-11-26 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp II-4 (63-88) and application thereof |
| CN113698464B (en) * | 2021-08-31 | 2022-05-17 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp II-4 (63-88) and application thereof |
| CN116143882A (en) * | 2022-10-13 | 2023-05-23 | 广西中医药大学 | Hippocampus trimarans hepcidin antibacterial peptide and application thereof |
| CN116143882B (en) * | 2022-10-13 | 2023-08-29 | 广西中医药大学 | Hippocampus trimarans hepcidin antibacterial peptide and application thereof |
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