CN107298706A - A kind of holstein cow spleen derived antimicrobial peptide and preparation method and application - Google Patents
A kind of holstein cow spleen derived antimicrobial peptide and preparation method and application Download PDFInfo
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- CN107298706A CN107298706A CN201710640324.5A CN201710640324A CN107298706A CN 107298706 A CN107298706 A CN 107298706A CN 201710640324 A CN201710640324 A CN 201710640324A CN 107298706 A CN107298706 A CN 107298706A
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- 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
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Abstract
The invention discloses the antibacterial peptide with antibacterial activity isolated in a kind of spleen from holstein cow, i.e. holstein cow spleen derived antimicrobial peptide, its amino acid sequence is as shown in SEQ ID NO.1.Antibacterial peptide of the present invention is located away from Cow Spleen, and spleen is one of animal immune organ, participate in blood storage and immunologic process, hemolysis rate is extremely low, and with has a broad antifungal spectrum, sterilization is quick, high selectivity, do not produce drug resistance, without teratogenesis, it is not likely to produce the biological characteristics for some safe without toxic side effect such as accumulating poisoning, and has a broad antifungal spectrum, to Gram-negative bacteria, gram-positive bacteria and fungi all have efficient antibacterial action, gram-positive bacteria is treated available for preparing, Gram-negative bacteria or/and the medicine of fungal infection, also act as feed addictive or food additives.
Description
Technical field
The invention belongs to biological technical field, and in particular to that is isolated in a kind of spleen from holstein cow has antibacterial
The antibacterial peptide and its coded sequence and purposes of activity.
Background technology
The application of antibiotic has brought great convenience to the disease treatment of human and animal.But, it is big with antibiotic
Amount is used or abused, and the appearance of multi-drug resistant bacteria brings huge challenge to the treatment of the mankind and Animal diseases, while antibiotic
The side effect of residue problem and antibiotic causes grave danger to human and animal's health.Therefore, antibiotic substitute is found
As inevitable choice.
Antibacterial peptide (antimicrobial peptides, AMPs) is the important composition in living organism innate immune system
Part.The molecular weight of antibacterial peptide is small, is typically made up of 10~60 amino acid residues.Antibacterial peptide is except anti-thin with wide spectrum
Bacterium, antimycotic, antiviral, anti parasitic isoreactivity, while also there is antitumor, immunological regulation, endotoxin isoreactivity is neutralized.It is anti-
The sterilization mechanism of bacterium peptide is different from antibiotic.Antibacterial peptide is typically positively charged, can be acted on negatively charged target cell membrane, destruction
Its integrality simultaneously produces perforated phenomenon so that intracellular is tolerant largely to ooze out, so as to reach antibacterial action.And pathogen is to antibacterial peptide
Drug resistance will not typically be produced.Therefore, antibacterial peptide is the outstanding person in antibiotic substitute.
Spleen is the important peripheral immune organ of humans and animals, participates in the humoral immunity and cellular immunity of body.In spleen
There are the immunity-associated cells such as T lymphocytes, bone-marrow-derived lymphocyte, BMDC, red blood cell, macrophage, there is also important
Functional molecular, such as stimulin (Tuftsin), immunoglobulin, complement.Cell and molecule in spleen resist external jointly
The invasion and attack of pathogenic microorganism, so as to ensure body health.There are some researches show have antibacterial activity thing in cattle spleen crude extract
Matter.
At present, there is not yet the research that antibacterial peptide separation and identification are carried out out of cattle spleen is reported.
The content of the invention
There is anti-gram positive bacteria, Ge Lanyin it is an object of the invention to provide what is separated in a kind of spleen from holstein cow
Property bacterium and disease fungus activity antibacterial peptide and preparation method and application.
To realize goal of the invention, the technical solution adopted by the present invention is：
A kind of holstein cow spleen derived antimicrobial peptide, the amino acid sequence of the antibacterial peptide is as follows：
Arg-Pro-Pro-Ile-Arg-Pro-Pro-Phe-Tyr-Pro-Pro-Phe-Arg-Pro-Pro-Ile-Arg-
Pro-Pro-Ile-Phe-Pro-Pro-Ile-Arg-Pro-Pro(SEQ ID NO.1)。
The preparation method of above-mentioned holstein cow spleen derived antimicrobial peptide, comprises the following steps：
(1) preparation of crude extractions from spleen：Healthy holstein cow spleen is taken, surface-coating is removed, tissue is carried out after shredding
Homogenate, ultrasonication, then add isometric concentration (mass fraction concentration) for 5% acetic acid, 6h are stirred in ice bath；Then
Supernatant is taken after centrifuging 30min, centrifugation under conditions of 4 DEG C, 8000 revs/min, supernatant is subjected to rotary evaporation in 38-40 DEG C to second
Untill acid is steamed completely, remaining liquid after rotary evaporation is collected, freeze-drying obtains crude extractions from spleen；
(2) purifying of antibacterial peptide：By crude extractions from spleen acetic acid, crude extractions from spleen solution is obtained, spleen is slightly carried
Thing solution is by cation exchange column, and the cation constituent in crude extractions from spleen solution is attached on cation exchange column, then
With 25mmol/L ammonium acetate buffer (pH5.4) with 50mL/h flow velocity by cation exchange column, wash away it is uncombined into
Point, then with concentration (mass fraction concentration) be 10% acetic acid eluted with 50mL/h flow velocity the sun that is combined on cation exchange column from
Subconstiuent, elution process measures light absorption value of the elution fraction in 230nm wavelength with Ultraviolet Detector, collects cation eluting peak；
The cation eluting peak component of collection is further separated using RPLC, the eluting peak of the 7th appearance is collected
Corresponding elution fraction, freeze-drying produces holstein cow spleen derived antimicrobial peptide.
According to above-mentioned preparation method, it is preferable that the chromatographic column that RPLC is used is the U.S.
Phenomenex Products, model Jupiter 5u C18
According to above-mentioned preparation method, it is preferable that the eluent that RPLC is used is buffer A and buffering
Liquid B, wherein, the buffer A is：5% acetonitrile, (compound method is 0.1% trifluoroacetic acid：By 5mL acetonitriles addition 95mL go from
In sub- water, mix, then add 0.1mL trifluoroacetic acids thereto, well mixed to produce buffer A)；The buffer B is：95%
Acetonitrile, (compound method is 0.1% trifluoroacetic acid：5mL deionized waters are added in 95mL acetonitriles, mixed, then add thereto
0.1mL trifluoroacetic acids, well mixed to produce buffer B)；Gradient is：In 30min, eluent A ratio by 100% by
Step is to 60%, and eluent B ratio is progressively adjusted to 40% by 0%；During 30.1min, eluent A ratio is changed into 100%, washes
De- liquid B ratio is changed into 0%.
According to above-mentioned preparation method, it is preferable that the flow velocity of the eluent is 1mL/min.
According to above-mentioned preparation method, it is preferable that the Detection wavelength that the RPLC is used is 230nm.
Antibacterial peptide of the present invention can also be artificial synthesized according to related coding sequences progress, and its preparation method can be solid
The encoding gene of antibacterial peptide, can also be cloned on carrier by phase chemical method, be obtained after then being expressed in host cell.Its
In, expression vector can be one kind in plasmid or virus, and host cell can be prokaryotic (including Escherichia coli, withered grass
Bacillus etc.) or eukaryotic (including yeast cells, plant cell, insect cell and mammalian cell etc.).
The antibacterial peptide of preparation can pass through Mass Spectrometric Identification.
The holstein cow spleen derived antimicrobial peptide can be used for prepare treatment gram-positive bacteria, Gram-negative bacteria or/
With the medicine of fungal infection.The medicine includes above-mentioned holstein cow spleen derived antimicrobial peptide, and be mixed with it is a kind of or it is a kind of with
Upper pharmaceutically acceptable carrier and/or additive.
The holstein cow spleen derived antimicrobial peptide also acts as feed addictive or food additives.
The positive beneficial effect that the present invention has：
(1) antibacterial peptide of the present invention is located away from Cow Spleen, and spleen is one of animal immune organ, participate in blood storage and
Immunologic process, hemolysis rate is extremely low, and has quick has a broad antifungal spectrum, sterilization, high selectivity, do not produce drug resistance, without teratogenesis
Act on, the biological characteristics for some safe without toxic side effect such as accumulating poisoning, and has a broad antifungal spectrum be not likely to produce, to gram-negative
Property bacterium, gram-positive bacteria and fungi all have efficient antibacterial action, available for prepare treatment gram-positive bacteria, gram
Negative bacterium or/and the medicine of fungal infection, also act as feed addictive or food additives.
(2) present invention has isolated antibacterial peptide from holstein cow spleen first, except with general antibacterial peptide antimicrobial spectrum
Extensively, outside sterilizing quick, high selectivity, not producing the general character of drug resistance, compared with the antibacterial polypeptide in other sources, the group resists
Bacterium polypeptide has simple in construction, artificial synthesized convenience, applied to mammal no antigen.
Brief description of the drawings
Fig. 1 is the result figure that crude extractions from spleen of the present invention carries out ion exchange chromatography.
Fig. 2 carries out RP-HPLC result figure for the cation peak component that the present invention is collected.
Fig. 3 is the Mass Spectrometer Method result figure of holstein cow spleen derived antimicrobial peptide of the present invention.
Fig. 4 is holstein cow spleen derived antimicrobial peptide of the present invention to Escherichia coli minimal inhibitory concentration measurement result.
Fig. 5 is that holstein cow spleen derived antimicrobial peptide of the present invention determines knot to staphylococcus aureus minimal inhibitory concentration
Really.
Fig. 6 is holstein cow spleen derived antimicrobial peptide of the present invention to Candida albicans minimal inhibitory concentration measurement result.
Fig. 7 is the hemolysis rate measurement result of holstein cow spleen derived antimicrobial peptide of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is further detailed explanation, but is not intended to limit the model of the present invention
Enclose.
Embodiment 1：The preparation of crude extractions from spleen
Healthy holstein cow spleen is taken, surface-coating is removed, spleen is cut into about 3mm3Fritter, be placed in sterilizing beaker
In, add the sterile saline of appropriate precooling, tissue mashing, homogenate；Homogenate is with Ultrasonic Cell Disruptor (8s-4s, 35%Amp)
In crush twice, 15min/ times.Isometric concentration is added into the spleen after homogenate for 5% acetic acid, 6h is stirred in ice bath；
Then 30min is centrifuged with 8000rpm in 4 DEG C, takes supernatant；It is complete to acetic acid by supernatant in 38~40 DEG C of rotary evaporation 6-8h
Untill pressure decatizing goes out；Remaining liquid after rotary evaporation is collected, freeze-drying obtains crude extractions from spleen.
Embodiment 2：The cation exchange chromatography of antibacterial peptide
By 0.01% acetic acid of crude extractions from spleen, crude extractions from spleen solution is obtained, crude extractions from spleen solution is passed through
Cation constituent in cation exchange column, crude extractions from spleen solution is attached on cation exchange column, first with 25mmol/L's
Ammonium acetate buffer (pH5.4), by cation exchange column, washes away uncombined composition, then be with concentration with 50mL/h flow velocity
10% acetic acid is with the cation constituent combined on 50mL/h flow velocity elution cation exchange column, elution process Ultraviolet Detector
Light absorption value of the elution fraction in 230nm wavelength is measured, and receives eluting peak (5ml/ peaks).After separation, two eluting peaks, peak A are had
For anion peak, peak B is cation peak (referring to Fig. 1).Detect that (antibacterial activity detection method is shown in embodiment 5) is sent out through antibacterial activity
Existing, peak A is without antibacterial activity, and peak B has antibacterial activity (referring to table 1).
Analysis of Antimicrobial Activity result (the unit of the ion-exchange purification product of table 1：mm)
Embodiment 3：The RPLC purifying of antibacterial peptide
The cation eluting peak component (peak B) of collection is further separated with RPLC (RP-HPLC) method.
The chromatographic column that the RPLC is used is U.S.'s Phenomenex Products, its model Jupiter 5u
C18The eluent used for buffer A and buffer B, wherein, the buffer A is：5% acetonitrile, 0.1% trifluoro second
(compound method is acid：5mL acetonitriles are added in 95mL deionized waters, mixed, then add 0.1mL trifluoroacetic acids, mixing thereto
Uniformly produce buffer A)；The buffer B is：95% acetonitrile, (compound method is 0.1% trifluoroacetic acid：By 5mL deionizations
Water is added in 95mL acetonitriles, is mixed, then adds 0.1mL trifluoroacetic acids thereto, well mixed to produce buffer B)；It elutes ladder
Spend and be：In 30min, eluent A ratio is progressively adjusted to 60% by 100%, and eluent B ratio is progressively adjusted to 40% by 0%；
During 30.1min, eluent A ratio is changed into 100%, and eluent B ratio is changed into 0%, and flow velocity is 1mL/min, Detection wavelength
For 230nm.RPLC separation process, collects each eluting peak component manually, and 7 eluting peak components are have collected altogether,
(referring to Fig. 2), detects the antibacterial activity of 7 eluting peak components respectively (antibacterial activity detection method is referring to embodiment 5).Detection knot
Fruit finds that the component of eluting peak 7 is respectively provided with antibacterial activity (referring to table 2) to 3 kinds of challenge organisms, collects the component of eluting peak 7, carries out
Freeze-drying, that is, obtain the holstein cow spleen derived antimicrobial peptide of the present invention.
Embodiment 4：The mass spectral analysis of holstein cow spleen derived antimicrobial peptide of the present invention：
(1) holstein cow spleen derived antimicrobial peptide molecular weight determination：
Using 5800 type MALDI-TOF/TOF (AB SCIEX, USA) mass spectrographs to holstein cow spleen derived antimicrobial peptide point
Son amount is measured analysis.Concrete operations are：0.5 μ L samples of point are spontaneously dried, then put 0.5 μ L 4mg/ on MALDI target plates
ML CHCA solution (50% acetonitrile solution, contain 0.1%TFA), is spontaneously dried, and then sample is with 5800 type MALDI-TOF/
TOF (AB SCIEX, USA) mass spectrograph is analyzed, and lasing light emitter is 355nm Nd:YAG laser, accelerating potential is 20kV, is used
Positive ion mode and automatic acquisition data pattern gathered data, external standard correction is carried out with myoglobin peptide hydrolysis.Sample one-level
Scanning of the mass spectrum scope is 700-3600Da, and then selection target peptide fragment ion carries out Tandem Mass Spectrometry Analysis.First mass spectrometric is used
Reflector Positive patterns, parameter：CID (OFF), mass rang (700-5000Da), Focus Mass
(1600Da), Fixed laser intensity (4500) Digitizer：Bin Size(0.5ns).
(2) holstein cow spleen derived antimicrobial peptide sequence analysis：
The amino acid sequence that instrument determines analysis holstein cow spleen derived antimicrobial peptide is used in conjunction using liquid chromatogram and mass spectrum.Its
In, the liquid phase systems of instrument are used in conjunction for 20AD HPLC system (Shimadzu) in liquid chromatogram and mass spectrum, including Micromass
C18 post (specifications：5μm,0.1*15mm).Mobile phase A liquid (water:Acetonitrile:Formic acid=98:2:0.1) with D liquid (water:Acetonitrile:
Formic acid=2:98:0.1) correcting fluid (Thermo Fisher Scientific) is added in.Applied sample amount is 2.25 μ g (9 μ l), uses A
Liquid elutes 15min with 2 μ L/min flow velocity, carries out peptide fragment absorption and desalination.With the mobile phase of the liquid containing 5%D with 200nL/min's
Flow velocity elutes 1min, then sets up gradient：D liquid gradient line style is risen in 35%, 5min from 35% liter from 5% in 65min
To 80%, then 80% 5min is persistently eluted, last 2min recovers post material.The mass spectrograph that instrument is used in conjunction in liquid chromatogram and mass spectrum is
TripleTOF 5600 (AB SCIEX, Concord, ON), its ion gun be Nanospray III source (AB SCIEX,
Concord, ON), emitter is the nozzle needle (New Objectives, Woburn, MA) that quartz material is drawn.Data acquisition opportunity
Device parameter：Ion gun spray voltage 2.5kv, nitrogen pressure 30psi (14.5psi ≈ 1bar), spray pressure 15psi, spraying connects
150 DEG C of temperature at mouthful；Scan pattern is reflective-mode；The accumulation 250ms ion from 2+ to 5+ is selected, wherein intensity product per second
Tire out first 50 more than 120 points to be scanned, 2.8s is a circulation；The transmission window of second quadrupole rod (Q2) is set to：
100Da is 100%；Pulse radiation frequency electricity frequency is 11kHz；Detector detection frequency is 40GHz；The particle signal scanned every time with
Four passages are recorded respectively, totally four times, and rear merging changes into data；Parent ion is dynamically excluded and is set to：In the appearance of half
Interior (about 18s), the fragmentation of identical parent ion is no more than 2 times.MS/MS data are obtained, MASCOT (V 2.3) is used by software
Retrieved, its search argument is：NCBI bovine proteins storehouse, pancreatin digestion, two leak enzyme sites, and first mass spectrometric tolerance is
0.05Da, second order mses tolerance is 0.1Da, fixed modification, and variable modification is set to methionine oxidized, and electric charge is set to
+2、+3、+4.The amino acid sequence of holstein cow spleen derived antimicrobial peptide is obtained by analysis.
By detecting and analyzing, holstein cow spleen derived antimicrobial peptide amino acid sequence and molecular weight of the present invention are respectively：
Arg-Pro-Pro-Ile-Arg-Pro-Pro-Phe-Tyr-Pro-Pro-Phe-Arg-Pro-Pro-Ile-Arg-
Pro-Pro-Ile-Phe-Pro-Pro-Ile-Arg-Pro-Pro, its molecular weight is 3214.89.
(3) isoelectric point is analyzed：EditSeq in DNAStar softwares is opened, file is opened, " new in " new " is clicked on
Protien ", inputs amino acid sequence, saves as the document of pro forms.Then the Protean in DNAStar softwares is opened again,
File is opened, is clicked on " open ", selection just saves as the document of pro forms, is clicked on " opening ", in selection " Analysis "
" Titration Curve ", you can obtain isoelectric point.The isoelectric point of holstein cow spleen derived antimicrobial peptide of the present invention is 12.18.
Embodiment 5：The application implementation of holstein cow spleen derived antimicrobial peptide of the present invention
1st, holstein cow spleen derived antimicrobial peptide antibacterial activity detection of the present invention
The eluting peak component and RPLC of ion-exchange chromatography separation are determined using lysoplate assay
The bacteriostatic activity of the absworption peak component of separation, its challenge organisms bacterial strain is：Escherichia coli ATCC8099, staphylococcus aureus
ATCC6538, Candida albicans ATCC10231, the challenge organisms bacterial strain are purchased from Nanjing Bian Zhen bio tech ltd.
Above-mentioned 3 kinds of challenge organisms are diluted in 50 DEG C of bottom agar culture medium (10mL tryptoses enzymolysis junket egg respectively
White soybean broth, agarose ultrapure 0.1g, pH 7.4) in, sterilizing flat board, solidification punches that (aperture is about with card punch
3mm), the slightly hot carry out back cover of alcolhol burner, the μ L of test sample 5 are added with pipettor into every hole, and each flat board includes positive right
According to and negative control.Positive control：Ampicillin is directed to gram-negative bacteria and gram positive bacteria, and nystatin is read for white
Pearl bacterium.Negative control：Aqua sterilisa.Flat board is stood into 1h, test fluid is diffused into agarose.Then, then one layer of agar is added
Culture medium (50 DEG C or so, nutritional ingredient is identical with bottom agar culture medium).By culture plate in 37 DEG C of overnight incubations, Ran Houji
The diameter of the transparent circle on hole periphery is recorded, each strain is repeated three times, calculate average value.
7 eluting peak components that RPLC separation is obtained carry out antibacterial activity detection, its testing result ginseng
It is shown in Table 2.
As shown in Table 2, holstein cow spleen derived antimicrobial peptide (component of eluting peak 7) of the present invention, tested micro- to above-mentioned three kinds
Biological bacterial strain all has stronger antibacterial activity.Therefore, holstein cow spleen derived antimicrobial peptide of the invention is expected dynamic in treatment
There is good application prospect in terms of thing bacteriosis and fungal disease.According to the general characteristic of known antibacterial peptide, this hair
Bright holstein cow spleen derived antimicrobial peptide the application of antibacterials and feedstuff mildew and it is fresh-keeping in terms of also have it is certain
Application potential.
Analysis of Antimicrobial Activity result (the unit of the RPLC purified product of table 2：mm)
2nd, the measure of holstein cow spleen derived antimicrobial peptide minimal inhibitory concentration of the present invention
Challenge organisms bacterial strain is：Escherichia coli ATCC8099, staphylococcus aureus ATCC6538, Candida albicans
ATCC10231, the challenge organisms bacterial strain is purchased from Nanjing Bian Zhen bio tech ltd.
Strain subject is expanded into culture in TSB fluid nutrient mediums, the OD of bacterium solution is surveyed600Value.When OD values are in 0.6-0.8,
Bacterium solution is centrifuged into 10min under the conditions of 6000r/min, supernatant discarding collects bacterial sediment, bacterial sediment is used in equal volume
PBS (0.01M) is resuspended, then bacterial suspension is diluted into 2 × 10 with MH culture mediums6CFU/mL.Diluted with deionized water
Holstein cow spleen derived antimicrobial peptide sample of the present invention, it is 0.58-300 μ g/mL to make its concentration.In 96 porocyte culture plates,
Add the antibacterial peptide of the present invention of 50 μ L various concentrations and the bacterial solution of 50 μ L dilutions respectively per hole, each concentration repeats three holes,
37 DEG C of culture 16-18h are placed in, the solution in then mixing per hole determines its OD600Value, with the corresponding antibacterial of OD value suddenly changes
Peptide concentration is its MIC, as a result sees Fig. 4, Fig. 5 and Fig. 6.
From Fig. 4, Fig. 5 and Fig. 6, minimal inhibitory concentration of the holstein cow spleen derived antimicrobial peptide to Escherichia coli
(MIC) it is 37.5 μ g/mL, the minimal inhibitory concentration (MIC) to staphylococcus aureus is 18.75 μ g/mL, to Candida albicans
Minimal inhibitory concentration (MIC) be 150 μ g/mL.As a result antibacterial peptide of the present invention reaches to the minimal inhibitory concentration of common bacterium
To for Gamma Magnitude, with stronger Antibacterial Activity.
3rd, the hemolytic activity detection of holstein cow spleen derived antimicrobial peptide of the present invention：
Fresh rabbit blood is taken, with 3.8% sodium citrate anti-freezing, anticoagulation is centrifuged into 10min through 3000r/min, phosphoric acid is used
Salt buffer (PBS) washing precipitation 3 times, is colourless, transparent to supernatant, then prepare 1% red blood cell with PBS.With PBS by class Bac5
The concentration of antibacterial peptide be adjusted to 300 μ g/mL, 150 μ g/mL, 75 μ g/mL, 37.5 μ g/mL, 18.75 μ g/mL, 9.375 μ g/mL,
4.6875 μ g/mL, 2.34375 μ g/mL, 1.17185 μ g/mL, 0.585938 μ g/mL, add isometric red cell suspension.With
PBS is negative control, using 1%Tritonx-100 as positive control, 37 DEG C of effect 1h, 1500r/min centrifugation 10min, by supernatant
Liquid sequentially adds 96 orifice plates, the OD values surveyed with ELIASA at 540nm.According to calculation formula：Hemolysis rate (%)=(detection hole OD
Value-negative hole OD values)/(positive hole OD values-negative hole OD values) × 100% calculating hemolysis rate, as a result see Fig. 7.
As shown in Figure 7, under 300 μ g/mL and 150 μ g/mL concentration conditions, holstein cow spleen derived antimicrobial peptide it is molten
Blood rate is 6.45% and 5.53%, slightly above 5%, but under 75 μ g/mL concentration, hemolysis rate is less than 5%, illustrates He Sitan milk
Cattle spleen derived antimicrobial peptide has preferable security in the range of finite concentration.
In summary, holstein cow spleen derived antimicrobial peptide has a broad antifungal spectrum of the invention, to gram-positive bacteria, gram
Negative bacterium and fungi all have efficient antibacterial action, and hemolysis rate is low, therefore, and holstein cow spleen source of the invention resists
Bacterium peptide can obtain preferably should in treatment gram-positive bacteria, Gram-negative bacteria or/and the medicine of fungal infection is prepared
With, and also act as feed addictive or food additives.
SEQUENCE LISTING
<110>Henan Science and Technology College
<120>A kind of holstein cow spleen derived antimicrobial peptide and preparation method and application
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> PRT
<213>Antibacterial peptide
<400> 1
Arg Pro Pro Ile Arg Pro Pro Phe Tyr Pro Pro Phe Arg Pro Pro Ile
1 5 10 15
Arg Pro Pro Ile Phe Pro Pro Ile Arg Pro Pro
20 25
Claims (9)
1. a kind of holstein cow spleen derived antimicrobial peptide, it is characterised in that the amino of the holstein cow spleen derived antimicrobial peptide
Acid sequence is as follows：
Arg-Pro-Pro-Ile-Arg-Pro-Pro-Phe-Tyr-Pro-Pro-Phe-Arg-Pro-Pro-Ile-Arg-Pro-
Pro-Ile-Phe-Pro-Pro-Ile-Arg-Pro-Pro。
2. the preparation method of holstein cow spleen derived antimicrobial peptide described in a kind of claim 1, it is characterised in that including following step
Suddenly：
(1) preparation of crude extractions from spleen：Take healthy holstein cow spleen, remove surface-coating, carried out after shredding tissue homogenate,
Ultrasonication, then adds the acetic acid that isometric concentration is 5%, 6h is stirred in ice bath；Then in 4 DEG C, 8000 revs/min
Under the conditions of centrifuge 30min, centrifugation after take supernatant, by supernatant in 38-40 DEG C carry out rotary evaporation untill acetic acid is steamed completely, receive
Collect remaining liquid after rotary evaporation, freeze-drying obtains crude extractions from spleen；
(2) purifying of antibacterial peptide：By crude extractions from spleen acetic acid, crude extractions from spleen solution is obtained, crude extractions from spleen is molten
Liquid is by cation exchange column, and the cation constituent in crude extractions from spleen solution is attached on cation exchange column, Ran Houyong
25mmol/L ammonium acetate buffer washes away uncombined composition, then washed for 10% acetic acid with concentration by cation exchange column
The cation constituent combined on decationized Y sieve exchange column, elution process measures elution fraction in 230nm wavelength with Ultraviolet Detector
Light absorption value, collect cation eluting peak component；The cation eluting peak component of collection is entered using RPLC
One step is separated, and collects the elution fraction corresponding to the eluting peak of the 7th appearance, and freeze-drying produces holstein cow spleen source and resisted
Bacterium peptide.
3. the preparation method of holstein cow spleen derived antimicrobial peptide according to claim 2, it is characterised in that the anti-phase height
The chromatographic column model Jupiter 5u C18 that effect liquid phase chromatogram is used
4. the preparation method of holstein cow spleen derived antimicrobial peptide according to claim 2, it is characterised in that the anti-phase height
The eluent that effect liquid phase chromatogram is used for eluent A and eluent B, wherein, the eluent A is：5% acetonitrile, 0.1% trifluoro
Acetic acid；The eluent B is：95% acetonitrile, 0.1% trifluoroacetic acid；Its gradient is：In 30min, eluent A ratio
60% is progressively adjusted to by 100%, eluent B ratio is progressively adjusted to 40% by 0%；During 30.1min, eluent A ratio becomes
For 100%, eluent B ratio is changed into 0%.
5. the preparation method of holstein cow spleen derived antimicrobial peptide according to claim 4, it is characterised in that the eluent
Flow velocity be 1mL/min.
6. the preparation method of holstein cow spleen derived antimicrobial peptide according to claim 2, it is characterised in that the anti-phase height
The Detection wavelength of effect liquid phase chromatogram is 230nm.
7. a kind of purposes of the holstein cow spleen derived antimicrobial peptide described in claim 1, it is characterised in that the He Sitan milk
Application of the cattle spleen derived antimicrobial peptide in treatment gram-positive bacteria, Gram-negative bacteria or/and the medicine of fungal infection is prepared.
8. purposes according to claim 7, it is characterised in that the medicine includes the He Sitan milk described in claim 1
Cattle spleen derived antimicrobial peptide, and it is mixed with one or more kinds of pharmaceutically acceptable carriers and/or additive.
9. a kind of the answering in feed addictive or food additives of the holstein cow spleen derived antimicrobial peptide described in claim 1
With.
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| CN111848739A (en) * | 2020-06-30 | 2020-10-30 | 河南科技学院 | Antibacterial peptide LJ-2 and application thereof |
| CN111848739B (en) * | 2020-06-30 | 2022-08-30 | 河南科技学院 | Antibacterial peptide LJ-2 and application thereof |
| CN114940698A (en) * | 2022-07-22 | 2022-08-26 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
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