CN104292324B - One group of HRP5 analog and preparation method thereof - Google Patents

One group of HRP5 analog and preparation method thereof Download PDF

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CN104292324B
CN104292324B CN201410524880.2A CN201410524880A CN104292324B CN 104292324 B CN104292324 B CN 104292324B CN 201410524880 A CN201410524880 A CN 201410524880A CN 104292324 B CN104292324 B CN 104292324B
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lys
leu
arg
phe
preparation
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CN104292324A (en
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冯军
路建光
张喜全
徐宏江
吴勇
朱裕辉
杨洁
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Shanghai Duomirui Biotechnology Co ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Abstract

The invention belongs to field of medicaments, in particular to one group of HRP5 analog and preparation method thereof.The amino acid sequence formula of HRP5 analogs involved in the present invention such as SEQ ID NO:Shown in 2：Xaa1‑Xaa2‑Xaa3‑Xaa4‑Xaa5‑Xaa6‑Xaa7‑Xaa8‑Xaa9‑Leu‑Xaa11‑Lys‑Tyr‑Leu‑Arg‑Xaa16‑Xaa17‑Xaa18.Active determination in vitro shows, the polypeptide that the present invention is provided is respectively provided with significant bactericidal effect to gram-positive bacteria, Gram-negative bacteria and fungi, equally there is significant bactericidal effect to the resistant gram-positive bacterium separating clinically, resistant Gram negative bacterium and the drug resistant fungal that clinically separate, with wide spectrum, it is efficient the characteristics of.

Description

One group of HRP5 analog and preparation method thereof
The application is Application No. 201110116643.9, and the applying date is on May 6th, 2011, and entitled " one group new The divisional application of HRP5 analogs of type and preparation method thereof ".
Technical field
The invention belongs to field of medicaments, in particular to one group of new HRP5 analog with broad spectrum antibiotic activity And preparation method thereof.
Background technology
Since penicillin finds, antibiotic is always the powerful mean of human treatment's cause pathogeny imcrobe infection, but with The abuse of conventional antibiotic, increasing pathogen starts to produce drug resistance to conventional antibiotic, so being badly in need of finding one The brand-new antibacterials of class are carried out substitute antibiotics and are used.
Cationic antibacterial peptide (cationic antimicrobial peptides) be plant and animal produce it is general by The 12-50 cationic of amino acid residue composition (rich in arginine and lysine) polypeptide, can protect host not by the external world Cause pathogeny imcrobe infection.The antimicrobial spectrum of cationic antibacterial peptide is wider than conventional antibiotic, from insect, pig, the frog and people sun from Sub- antibacterial peptide not only has antibacterial action to gram-positive bacteria and Gram-negative bacteria, but also with antimycotic and antiviral work Property.Conventional antibiotic such as makes enzyme denaturation to reach the mesh of sterilization by eliminating growth of microorganism or existence essential condition , as long as but can just resist the attack of such antibiotic after a kind of gene mutation of bacterium.And during cationic antibacterial peptide then passes through With the method and bacterial cell membrane interaction of electric charge, kill bacterium is penetrated with this, considerably reduce bacterium and produce drug resistance Possibility.But natural cationic antibacterial peptide is not flawless, part antibacterial peptide has certain toxicity to eucaryote, to disease It is usually associated with to Eukaryotic haemocylolysis while original killing activity high.Therefore its activity and maximum journey how to be improved Where degree reduces the difficult point that its toxicity is current antimicrobial peptide medicaments exploitation and wishes.The current main both sexes by alpha-helix of research Antibacterial peptide, around cationic amino acid and hydrophobic amino acid quantity and position, is tied as research object to antibacterial peptide Structure transform, such as residue replace, interception natural antibacterial peptide partial sequence and increase peptide chain positive charge content, achieve compared with Big progress.
Histatins (histidine rich protein, HRPs or histatins) be a class be present in primate move Cationic polypeptide rich in histidine in thing primate Parotid and submandibular gland secretion.At least 12 kinds people HRPs (HRP1- are isolated 12), wherein HRP1, HRP3 and HRP5 are main histatinses, account for the 85%-90% of total HRPs, respectively by 38,32,24 ammonia Base acid composition.By different gene codes, HRP5 is products of the HRP3 through protease hydrolytic to HRP1 and HRP3, is included in HRP3's In 24 residues of N-terminal.Coding HRP1,3 gene has been sequenced and has been positioned on mankind's rice chromosome q13.Existing research Show：HRPs has various active biological functions, and its anti-microbial effect is particularly important, such as suppresses, kills deformation hammer Bacterium and Candida albicans etc..
HRP5 contains 24 amino acid residues, and molecular weight is 3037D.Its amino acid sequence is Asp-Ser-His-Ala- Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly- Tyr(SEQ ID NO:1).The scholars such as Raj think that the characteristic of HRP5 anti-candida albicanses is relevant with its amino acid sequence, Ta Menfen Not by HRP5 and its residue segment N16 (1-16 amino acids), C16 (9-24 amino acids), C14 (11-24 amino acids), C12 (13-24 amino acids), C10 (15-24 amino acids) and M10 (7-16 amino acids) respectively with the white of various concentrations Coccus effect is read, the ability of its anti-Candida albicans is detected.Result of the test shows that C16 and C14 have stronger suppression Candida albicans The ability of bacteria growing, and the anti-Candida albicans of HRP5 and C16 is active basically identical, and it is same containing 16 amino acid The activity of N16 anti-candida albicanses is well below C16.The functional areas of the anti-candida albicanses of this result prompting HRP5 are located at carboxylic Cardinal extremity.In order to further study the relation of the 26S Proteasome Structure and Function of HRP5, many scholars are replaced by residue and have obtained a series of Variant, wherein that more important is dhvar1, dhvar2, dhvar4, dhvar5.They are HRP5 active regions (11-24 bit aminos Acid) variant.
The content of the invention
Polypeptide or its pharmaceutical salts, the polypeptide have following amino acid sequence：
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Leu-Xaa11-Lys-Tyr-Leu- Arg-Xaa16-Xaa17-Xaa18(SEQ ID NO:2)
Wherein Xaa1 is Cys or missing；
Xaa2 is Lys, D-Lys, Arg, D-Arg, HoR or missing；
Xaa3 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe, Thi or missing；
Xaa4 is Lys, D-Lys, Arg, D-Arg, Aib or HoR；
Xaa5 is Lys, D-Lys, Arg, D-Arg or HoR；
Xaa6 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi；
Xaa7 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi；
Xaa8 is Lys, D-Lys, Arg, D-Arg or HoR；
Xaa9 is Lys, D-Lys, Arg, D-Arg or HoR；
Xaa11 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi；
Xaa16 is Lys, D-Lys, Arg, D-Arg or HoR；
Xaa17 is Leu, Ile, Nle, Val, Trp, Phe, D-Phe or Thi；
Xaa18 is Cys or missing, and when Xaa1 and Xaa18 are all Cys, the polypeptide can form cyclic peptide.
Cys is all as a preferred embodiment of the present invention Xaa1 and Xaa18 or while lack；Xaa2 preferred Arg, D- Arg or missing；Xaa3 preferred Phe, D-Phe or missing；Xaa4 preferred Lys, D-Lys, Arg, Aib or HoR；The preferred Lys of Xaa5, Arg or D-Arg；Xaa6 preferred Leu, Ile or Nle；Xaa7 preferred Trp, Phe, D-Phe or Thi；Xaa8 preferred Lys, D-Lys Or Arg；Xaa9 preferred Lys, D-Lys or Arg；Xaa11 preferred Leu, D-Phe or Phe；Xaa16 preferred Lys, D-Lys or Arg； Xaa17 preferred Trp, Phe, D-Phe or Thi.
Amino acid residue involved in the present invention both includes natural amino acid, also including alpha-non-natural amino acid.Institute of the present invention As shown in table 1, alpha-non-natural amino acid institute involved in the present invention is right for three-letter codes table corresponding to the natural amino acid being related to The three-letter codes table and structure answered are as shown in table 2.
If all amino acid residues involved in the present invention are not particularly limited its configuration, L-type amino acid is represented.
Polypeptide involved in the present invention, both can be linear peptides, or cyclic peptide.When Xaa1 and Xaa18 are all Cys When, can be by adding oxidant so that forming disulfide bond between Xaa1 and Xaa18, now the peptide becomes cyclic peptide by linear peptides.
When polypeptide involved in the present invention is linear peptides, its C-terminal both can be the form, or acid amides of carboxylic acid Form, exists preferably in the form of acid amides.
Table 1：Natural amino acid three-letter codes table
Table 2：Alpha-non-natural amino acid three-letter codes table and structure
A kind of linear polypeptide (AMP-1) is disclosed in a currently preferred implementation method, its amino acid sequence is：
Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH2(SEQ ID NO:3)
Disclosed in presently preferred implementation method one group of linear polypeptide (AMP-2, AMP-3, AMP-4, AMP-5, AMP-6, AMP-7, AMP-8, AMP-9, AMP-10, AMP-11, AMP-12), its amino acid sequence is respectively：
Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH2(SEQ ID NO:4)
D-Lys-D-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH2 (SEQ ID NO:5)
D-Lys-D-Arg-Leu-Phe-D-Lys-D-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe- NH2(SEQ IDNO:6)
Phe-Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH2(SEQ ID NO:7)
Lys-Arg-Leu-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH2(SEQ ID NO:8)
Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH2(SEQ ID NO:9)
Arg-Leu-Arg-Arg-Ile-Phe-Lys-Arg-Leu-Phe-Lys-Tyr-Leu-Arg-Arg-Phe-NH2 (SEQ ID NO:10)Lys-Arg-Leu-Thi-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH2(SEQ ID NO:11)
Lys-Arg-Nle-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH2(SEQ ID NO:12)
Aib-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH2(SEQ ID NO:13)
HoR-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Thi-NH2(SEQ ID NO:14)
Of the invention another preferred embodiment discloses a kind of polypeptide of ring-type (AMP-13), its amino acid sequence For：
(SEQ ID NO:15)
The present invention also has a purpose to be just to provide the preparation method of above-mentioned linear and ring type polypeptide, can use this area skill Solid-state chemical reaction method technology familiar to art personnel is prepared linear peptides and is made on the basis of linear peptides by hydrogen peroxide oxidation method Standby cyclic peptide.
The preparation process of solid-state chemical reaction method technology is as follows：
(1) solid-phase synthetic peptide on resin；
(2) product of step (1) is cracked in trifluoroacetic acid or hydrofluoric acid, preferably trifluoroacetic acid, and joining side Chain protection group scavenger, then with the 5-20 times of ice ether precipitated polypeptide of volume, centrifugation abandons supernatant, then washed repeatedly with ice ether Wash precipitation 4-5 times, vacuum drying obtains thick peptide；
Step (1) is synthesized using Wang resins if peptide C end involved in the present invention is carboxylic acid form；If this For amide form thereof, then step (1) is synthesized the involved peptide C end of invention using Rink Amide mbha resins.
Step (1) is carried out in liquid phase environment, is specifically included：Immersion resin-removing amino protecting group-washing-idol Hydrazino acid-monitoring-washing-removing amino protecting group (order coupling remaining amino acid)-dry resin.
Wherein amino protecting group refers to the chemical group introduced for protection participates in the amino of condensation reaction.Described amino Protection group is selected from：Tertbutyloxycarbonyl (Boc), benzyloxycarbonyl group (Z) and 9- fluorenyls-methyl carbonyl (Fmoc), preferably 9- fluorenyls-methyl Carbonyl (Fmoc).
Used as an advantage of Solid phase peptide synthssis technology, the side chain to partial amino-acid can be by introducing chemical group Protected, such as Arg and HoR can use pentamethyl benzofuran -5- sulfonyls (Pbf)；Cys can use trityl (Trt)；Lys and Trp can use tertbutyloxycarbonyl (Boc)；Tyr can use the tert-butyl group (tBu).Described blocking group is not It is limited to this, reasonable selection can be carried out according to this area conventional scheme.
The liquid phase environment solvent for use of step (1) is selected from：Dimethylformamide (DMF) or dichloromethane (DCM), preferably DMF。
Removing amino protecting group needs to add the scavenger of amino protecting group, the scavenger of amino protecting group in step (1) From piperidines (PIP) solution, concentration 10-40% (PIP/DMF), the removing time is 20-50min.Preferred concentration is 20-25% (PIP/DMF) time 25-35min, is removed.
In step (1) coupling of amino acid need add coupling reagent, coupling reagent by：Carbodiimide type reagent or benzene And triazole salt form reagent is constituted with I-hydroxybenzotriazole (HOBt).
Carbodiimide type reagent includes dicyclohexylcarbodiimide (DCC), DIC (DIC) and N- diaminos Base propyl group-N- ethyl carbodiimides (EDC).
BTA salt form reagent includes 2- (1H- benzo trisazo- L-1- yls) -1,1,3,3- tetramethylurea tetrafluoros Borate (TBTU), O- BTAs-N, N, N', N'- tetramethylurea hexafluorophosphate (HBTU), hexafluorophosphoric acid BTA- 1- epoxides three (dimethylamino) phosphorus (BOP), hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus (PyBOP) etc..
The preferred DIC of coupling reagent (DIC) and I-hydroxybenzotriazole (HOBt), 2- (1H- benzos three Azo L-1- yls) -1,1,3,3- tetramethylurea tetrafluoro boric acid ester (TBTU) and I-hydroxybenzotriazole (HOBt), most preferably DIC (DIC) and I-hydroxybenzotriazole (HOBt).
" monitoring " in step (1) monitors the condensation reaction of polypeptide using ninhydrin detection method.
The amino acid that is linked in sequence in step (1) refers to connect amino one by one to N-terminal from C-terminal according to polypeptid acid sequence Acid.
Side chain protective group scavenger described in step (2) is to be selected from such as THIOANISOLE, tri isopropyl silane, phenol, water, 1, 2- dithioglycols, metacresol etc. two or more combination and enter by 5-20% (V/V) with trifluoroacetic acid or hydrofluoric acid Row preparation is obtained.It is preferred that trifluoroacetic acid (TFA):THIOANISOLE:1,2- dithioglycols:75% phenol:Water=85:5:5:4:1.
It will be particularly beneficial that the quality requirement to meet medical usage, polypeptide production methods provided by the present invention also include Purified using conventional meanses.The purification process for being used can be RP chromatography or ion-exchange chromatography, preferably instead Phase chromatography.
The antibacterial activity in vitro of polypeptide involved in the present invention can be reflected by determining its minimum inhibitory concentration (MIC) It is fixed.National Committee of Clinical Laboratory Standards (NCCLS) recommends to determine each antibacterial peptide using constant broth dilution method most Low Mlc (MIC), culture medium uses Mueller-Hinton (MH) broth bouillon.With amphotericin B, polymyxin e And vancomycin is used as positive control.Active determination in vitro shows that the polypeptide that the present invention is provided is to gram-positive bacteria, gram Negative bacterium and fungi are respectively provided with significant bactericidal effect, to the resistant gram-positive bacterium separating clinically, the drug resistance leather orchid that clinically separate Family name's negative bacterium and drug resistant fungal equally have significant bactericidal effect, with wide spectrum, it is efficient the characteristics of.Therefore the present invention is involved And polypeptide can clinically as active component substitute conventional antibiotic be treated.
Following examples only represent the one side of present invention elaboration, are not the limitations of present subject matter.
Specific embodiment
Embodiment 1：The preparation and purifying of AMP-1
Sequence：Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH2(SEQ ID NO:3)
(1) material and reagent
Rink Amide mbha resins, substitution value 0.41mmol/g.
It is required with protection amino acid for Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)- OH, Fmoc-L-Phe-OH and Fmoc-L-Tyr (tBu)-OH.
Reagent：HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 types Peptide synthesizer, Waters600 semipreparative high performance liquid chromatographies instrument, magnetic stirring apparatus.
(3) operating procedure (by taking 0.25mmol as an example)
A. solid-state chemical reaction method polypeptide
Rink Amide mbha resin 0.61g are weighed, is placed in the reactor of Peptide synthesizer, add 10mLDMF, leaching Bubble 2h, be subsequently adding 20%PIP/DMF solution 15mL, and mixing 30min removes amino protecting agent, with DMF washing resins 7 times, Then to addition 387.4mg Fmoc-L-Phe-OH, equimolar coupling reagent DIC (0.33mol/L) and HOBt in reactor (0.33mol/L) is reacted, and reaction temperature is room temperature, monitors reaction process with ninhydrin reaction, it is ensured that amino acid couplings are arrived On resin, with DMF washing resins 7 times.After on first amino acid couplings to resin, you can proceed according to the method described above The coupling reaction of next amino acid, so circulation, until whole amino acid couplings are completed.
B. crack and precipitate
After peptide symthesis terminate, resin is vacuum dried, weighed.Ratio according to 1g resin 10mL lytic reagents adds cracking examination Agent, reagent proportioning is TFA:THIOANISOLE:1,2- dithioglycols:75% phenol:Water=85:5:5:4:1, reaction 3 is stirred at room temperature Hour, suction filtration.Then to 10 times of ice ether precipitated polypeptides of volume are added in cracking smoke filtrate, centrifugation abandons supernatant, then use ice second Ether cyclic washing is precipitated 4~5 times, vacuum drying, thick peptide of weighing.
C. Reverse phase chromatography
With preparation HPLC, using RP chromatography, above-mentioned thick peptide is purified.
HPLC conditions are as follows：
Chromatographic column：XBridgeTM Prep C185μm OBDTM 19×150mm
Flow velocity：10mL/min
Detection wavelength：210nm
Mobile phase：A：The aqueous solution containing 0.1%TFA
B：Acetonitrile containing 0.1%TFA
Gradient elution program such as table 3:
The gradient elution table of table 3
Collect the part of target peptide purity more than 99%, then 50 DEG C be concentrated under reduced pressure into it is dry.Determined through ESI-MS, the peptide Molecular weight is 1880.28, theoretical value 1880.43.
Embodiment 2：The preparation and purifying of HRP5 analogs in table 4
(1) material and reagent
Rink Amide mbha resins, substitution value 0.41mmol/g.
Required amino acid Fmoc-L-Arg (Pbf)-OH, Fmoc-D-Arg (Pbf)-OH, Fmoc-L-Leu- with protection OH、Fmoc-L-HoR(Pbf)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-L-Phe-OH、 Fmoc-D-Phe-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Nle-OH, Fmoc-L-Thi-OH and Fmoc-L-Aib-OH。
Reagent：HOBt, DIC, DMF, piperidines.
(2) instrument
PSI300 types Peptide synthesizer, Waters600 semipreparative high performance liquid chromatographies instrument, magnetic stirring apparatus.
(3) operating procedure (by taking 0.25mmol as an example)
Prepared in the method for operating procedure a-c in similar embodiment 1 and purify the polypeptide in table 4, collected purity and be more than 99% part, then 50 DEG C be concentrated under reduced pressure into it is dry.ESI-MS measured values are as shown in table 4.
The HRP5 analogs of table 4
Embodiment 3：The preparation and purifying of cyclic peptide AMP-13
Sequence： (SEQ ID NO:15)
(1) material and reagent
Rink Amide mbha resins, substitution value 0.41mmol/g.
It is required with protection amino acid for Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)- OH, Fmoc-L-Phe-OH, Fmoc-L-Tyr (tBu)-OH and Fmoc-L-Cys (Trt)-OH.
Reagent：HOBt, DIC, DMF, piperidines, 30%H2O2。
(2) instrument
PSI300 types Peptide synthesizer, Waters600 semipreparative high performance liquid chromatographies instrument, magnetic stirring apparatus.
(3) operating procedure
A. prepared in the method for operating procedure a-c in similar embodiment 1 and purifying has following sequence (Cys-Lys-Arg- Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-Cys-NH2) polypeptide, collect purity be more than 99% part, then 50 DEG C be concentrated under reduced pressure into it is dry.Determined through ESI-MS, the molecular weight of the peptide is 2086.24, theoretical value 2086.72。
B. the formation of intramolecular disulfide bond
Polypeptide 100.0mg in the present embodiment operating procedure a is weighed, 200ml water is dissolved in, it is 0.5mg/ml's to be configured to concentration Solution, adds the hydrogen peroxide that 3.33ml concentration is 30%, at room temperature stirring reaction, and the carrying out of reaction is monitored with HPLC, until It is fully converted to oxidized form.
C. reverse chromatograms purifying
Purified in the method for operating procedure c in similar embodiment 1, collect part of the cyclic peptide AMP-13 purity more than 99%, Then 50 DEG C be concentrated under reduced pressure into it is dry.Determined through ESI-MS, the molecular weight of the peptide is 2084.25, theoretical value 2084.72.
Embodiment 4：Antibacterial activity in vitro is determined
According to each antibacterial peptide of constant broth dilution method determination that National Committee of Clinical Laboratory Standards (NCCLS) is recommended Minimum inhibitory concentration (MIC), culture medium use Mueller-Hinton (MH) broth bouillon.
Concretely comprise the following steps：
(1) prepared by antibacterials storage liquid：
Accurate formulation concentration is the above-mentioned HRP5 analogs AMP 1~13 and positive reference substance anphotericin of 1280 μ g/mL B, polymyxin e and vancomycin.Each storage liquid for preparing is placed in -20 DEG C of environment and saves backup.
(2) preparation of culture medium：
MH broth bouillon 21g are weighed, 1L, 121 DEG C of high-temperature sterilization 30min is dissolved in distilled water and is settled to.
(3) preparation of inoculum：
With the similar bacterium colony to be checked of oese picking form 3~5, be inoculated in the MH meat soups of 4~5mL, 35 DEG C be incubated 2~ 6h.Increase exponential phase bacterium solution physiological saline or MH meat soups corrected concentrations after bacterium to 0.5 Maxwell than turbid standard, containing about 1~2 ×108CFU/mL.Above-mentioned bacteria suspension is carried out 1 with MH meat soups：It is standby after 100 dilutions.
(4) preparation of dilution antibacterials and bacterium solution inoculation：
Sterile test tube (13 × 100mm) 13 is taken, is arranged in a row, 1.6mL MH meat soups are added in the 1st pipe, in 2-13 pipes It is each to add 1mL MH meat soups, antibacterials stoste (1280 μ g/mL) 0.4mL then is added to the 1st pipe, mix, then draw 1 ML to the 2nd is managed, and absorption 1mL to the 3rd is managed from the 2nd pipe again after mixing, and so continuous doubling dilution is managed to the 11st pipe from the 11st Middle absorption 1mL is discarded, then to above-mentioned each 1mL of the inoculum for preparing is added in 1-11 pipes and the 13rd pipe, make every pipe final Bacterial concentration is about 5 × 105CFU/mL.1-11 pipe drug concentrations are respectively 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ G/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, the 12nd pipe are without anti- The blank of bacterium medicine and inoculum, the 13rd pipe is the negative control without antibacterials.
(5) it is incubated：The dilution tube that will be inoculated with is stoppered stopper, puts 16~20h of incubation in 35 DEG C of normal air incubators.
(6) result：To visually observe, the lowest concentration of drug without bacterial growth is the minimum inhibitory concentration of the sample (MIC).The MIC measurement results of each antibacterial peptide are as shown in table 5.
The MIC measurement results of the HRP5 analogs of table 5

Claims (11)

1. polypeptide or its pharmaceutical salts, its amino acid sequence is：
Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH2,
Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH2,
D-Lys-D-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH2Or
D-Lys-D-Arg-Leu-Phe-D-Lys-D-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-D-Lys-D-Phe-NH2。
2. the polypeptide of claim 1, its amino acid sequence is：
Lys-Arg-Leu-Phe-Lys-Lys-Leu-Leu-Lys-Tyr-Leu-Arg-Lys-Phe-NH2。
3. the preparation method of the polypeptide of any one of claim 1-2, it is comprised the following steps：
(1) solid-phase synthetic peptide on resin；
(2) product of step (1) is cracked in trifluoroacetic acid or hydrofluoric acid, addition Side chain protective group scavenger, and with The 5-20 times of ice ether mixing of volume ratio, precipitated polypeptide, centrifugation is dried to obtain thick peptide.
4. the preparation method of claim 3, wherein step (1) is carried out in liquid phase environment, is specifically included：Immersion resin-de- Except amino protecting group-washing-coupling amino acid-monitoring-washing-removing amino protecting group-be linked in sequence remaining amino Acid-dry resin.
5. the preparation method of claim 4, wherein the amino protecting group be selected from tertbutyloxycarbonyl, benzyloxycarbonyl group or 9- fluorenyls- Methyl carbonyl.
6. the preparation method of claim 4, wherein the solvent that the liquid phase environment is used is selected from dimethylformamide or dichloro Methane.
7. the preparation method of claim 4, the coupling reagent that wherein coupling amino acid is used is by carbodiimide type reagent and 1- Hydroxybenzotriazole is constituted, or is made up of BTA salt form reagent and I-hydroxybenzotriazole.
8. the preparation method of claim 7, wherein the coupling reagent is by DIC and I-hydroxybenzotriazole Composition, or by 2- (1H- benzo trisazo- L-1- yls) -1,1,3,3- tetramethylurea tetrafluoro boric acid esters and I-hydroxybenzotriazole Composition.
9. the preparation method of claim 3, it is further included the step of by thick peptide purification, and wherein purification process is selected from anti-phase color Spectrometry or ion-exchange chromatography.
10. the polypeptide of any one of claim 1-2 is preparing anti-candida albicanses, staphylococcus aureus, Pseudomonas aeruginosa, big Application in the medicine of enterobacteria, resistance to ampicillin enterococcus and imipenem-resistant Acinetobacter bauamnnii.
Application described in 11. claims 10, wherein the Candida albicans is the Candida albicans of resistance to Fluconazole, the golden yellow Staphylococcus is methicillin-resistant staphylococcus aureus, and the Pseudomonas aeruginosa is imipenem-resistant Pseudomonas aeruginosa.
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