CN107298706B - Spleen-derived antibacterial peptide of Holstein cow, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an antibacterial peptide with antibacterial activity separated from spleens of Holstein cows, namely an antibacterial peptide derived from spleens of Holstein cows, wherein the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1. The antibacterial peptide is separated from the spleen of a milk cow, the spleen is one of animal immune organs and participates in blood storage and immune processes, the hemolytic rate is extremely low, the antibacterial peptide has the biological characteristics of safety and no toxic or side effect, such as wide antibacterial spectrum, quick sterilization, high selectivity, no drug resistance, no teratogenic action, difficult accumulation poisoning and the like, the antibacterial peptide has a wide antibacterial spectrum, has high-efficiency antibacterial action on gram-negative bacteria, gram-positive bacteria and fungi, can be used for preparing medicines for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection, and can also be used as a feed additive or a food additive.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to antibacterial peptide with antibacterial activity separated from spleens of Holstein cows, and a coding sequence and application thereof.
Background
The use of antibiotics provides great convenience for the treatment of diseases in humans and animals. However, with the heavy use or abuse of antibiotics, the emergence of multidrug-resistant bacteria poses a great challenge to the treatment of human and animal diseases, and the problem of antibiotic residues and the side effects of antibiotics pose a great threat to human and animal health. Therefore, the search for antibiotic alternatives is a necessary choice.
Antimicrobial peptides (AMPs) are important components of the innate immune system of living organisms. The antibacterial peptide has a small molecular weight and generally consists of 10 to 60 amino acid residues. The antibacterial peptide not only has broad-spectrum antibacterial, antifungal, antiviral, antiparasitic and other activities, but also has the activities of resisting tumors, regulating immunity, neutralizing endotoxin and the like. The bactericidal mechanism of antimicrobial peptides is different from that of antibiotics. The antibacterial peptide is generally positively charged and can act with a target cell membrane with negative charge to destroy the integrity of the target cell membrane and generate a perforation phenomenon, so that the cell contents are greatly exuded, thereby achieving the antibacterial effect. Pathogenic bacteria generally do not develop resistance to antimicrobial peptides. Therefore, the antibacterial peptides are superior substitutes for antibiotics.
The spleen is an important peripheral immune organ of human and animals and is involved in humoral immunity and cellular immunity of the body. The spleen contains immune-related cells such as T lymphocytes, B lymphocytes, dendritic cells, erythrocytes, macrophages and the like, and important functional molecules such as Tuftsin (Tuftsin), immunoglobulin, complement and the like exist. Cells and molecules in the spleen resist invasion of external pathogenic microorganisms together, so that the health of the organism is guaranteed. The research shows that antibacterial active substances exist in the crude extract of the bovine spleen.
At present, no research report on the separation and identification of antibacterial peptide from bovine spleen is found.
Disclosure of Invention
The invention aims to provide an antibacterial peptide which is separated from spleens of Holstein cows and has the activities of resisting gram-positive bacteria, gram-negative bacteria and pathogenic fungi, and a preparation method and application thereof.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the spleen-derived antibacterial peptide of the Holstein cow has the following amino acid sequence:
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 the spleen-derived antibacterial peptide of the Holstein cow comprises the following steps:
(1) preparation of crude spleen extract: taking the spleen of a healthy Holstein cow, removing a surface envelope, performing tissue homogenization and ultrasonic crushing after shearing, then adding acetic acid with the concentration (mass fraction concentration) of 5% with the same volume, and stirring for 6 hours in an ice bath; centrifuging at 4 deg.C and 8000rpm for 30min, collecting supernatant, performing rotary evaporation at 38-40 deg.C until acetic acid is completely evaporated, collecting the liquid remaining after rotary evaporation, and freeze drying to obtain spleen crude extract;
(2) and (3) purifying the antibacterial peptide: dissolving the spleen crude extract with acetic acid to obtain a spleen crude extract solution, passing the spleen crude extract solution through a cation exchange column, binding cation components in the spleen crude extract solution to the cation exchange column, then passing the spleen crude extract solution through the cation exchange column with 25mmol/L ammonium acetate buffer solution (pH5.4) at the flow rate of 50mL/h, washing away unbound components, eluting the bound cation components on the cation exchange column with 10% acetic acid at the flow rate of 50mL/h, measuring the absorbance value of the eluted components at the wavelength of 230nm by using an ultraviolet detector during the elution process, and collecting a cation elution peak; and further separating the collected cation elution peak components by using a reversed phase high performance liquid chromatography, collecting the elution component corresponding to the 7 th appearing elution peak, and freeze-drying to obtain the spleen-derived antibacterial peptide of the Holstein cow.
According to the above preparation method, preferably, the color used for reversed-phase high performance liquid chromatographyThe spectral column is manufactured by Phenomenex company of America, and is of Jupiter 5u C18
According to the preparation method, preferably, the eluents used by the reversed-phase high performance liquid chromatography are buffer A and buffer B, wherein the buffer A is: 5% acetonitrile, 0.1% trifluoroacetic acid (preparation method is, adding 5mL acetonitrile into 95mL deionized water, mixing, adding 0.1mL trifluoroacetic acid, mixing to obtain buffer solution A); the buffer solution B is: 95% acetonitrile, 0.1% trifluoroacetic acid (preparation method is, 5mL deionized water is added into 95mL acetonitrile, mixing, then 0.1mL trifluoroacetic acid is added into it, mixing to obtain buffer B); the elution gradient was: within 30min, the proportion of the eluent A is gradually adjusted from 100% to 60%, and the proportion of the eluent B is gradually adjusted from 0% to 40%; at 30.1min, the ratio of eluent A was 100% and the ratio of eluent B was 0%.
According to the above preparation method, preferably, the flow rate of the eluent is 1 mL/min.
According to the above production method, preferably, the detection wavelength used for the reversed-phase high performance liquid chromatography is 230 nm.
The antibacterial peptide can also be artificially synthesized according to related coding sequences, the preparation method can be a solid phase chemical method, and the antibacterial peptide can also be obtained by cloning the coding gene of the antibacterial peptide onto a vector and then expressing the coding gene in host cells. Wherein, the expression vector can be one of plasmids or viruses, and the host cell can be prokaryotic cells (including escherichia coli, bacillus subtilis and the like) or eukaryotic cells (including yeast cells, plant cells, insect cells, mammalian cells and the like). The prepared antibacterial peptide can be identified by mass spectrometry.
The spleen-derived antibacterial peptide of the Holstein cow can be used for preparing a medicament for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection. The medicine comprises the spleen-derived antibacterial peptide of the Holstein cow and is mixed with one or more than one pharmaceutically acceptable carriers and/or additives.
The spleen-derived antibacterial peptide of the Holstein cow can also be used as a feed additive or a food additive.
The invention has the following positive beneficial effects:
(1) the antibacterial peptide is separated from the spleen of a milk cow, the spleen is one of animal immune organs and participates in blood storage and immune processes, the hemolytic rate is extremely low, the antibacterial peptide has the biological characteristics of safety and no toxic or side effect, such as wide antibacterial spectrum, quick sterilization, high selectivity, no drug resistance, no teratogenic action, difficult accumulation poisoning and the like, the antibacterial peptide has a wide antibacterial spectrum, has high-efficiency antibacterial action on gram-negative bacteria, gram-positive bacteria and fungi, can be used for preparing medicines for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection, and can also be used as a feed additive or a food additive.
(2) The invention separates the antibacterial peptide from the spleen of the Holstein cow for the first time, and compared with the antibacterial peptides from other sources, the group of the antibacterial peptides have the advantages of wide antibacterial spectrum, quick sterilization, high selectivity and no drug resistance, and the antibacterial peptides have simple structure and convenient artificial synthesis and are applicable to mammals without antigenicity.
Drawings
FIG. 1 is a graph showing the results of ion exchange chromatography of a crude spleen extract of the present invention.
FIG. 2 is a graph showing the results of RP-HPLC on the collected cation peak components of the present invention.
FIG. 3 is a diagram showing the results of mass spectrometric detection of spleen-derived antibacterial peptides of Holstein cows.
FIG. 4 shows the results of the measurement of the minimum inhibitory concentration of spleen-derived antimicrobial peptide of Holstein cow against Escherichia coli.
FIG. 5 shows the result of the determination of the minimum inhibitory concentration of the spleen-derived antibacterial peptide of a Holstein cow on Staphylococcus aureus.
FIG. 6 shows the result of the measurement of the minimum inhibitory concentration of Candida albicans by the spleen derived antibacterial peptide of Holstein cow.
FIG. 7 shows the results of measuring the hemolysis rate of spleen-derived antibacterial peptide of Holstein cow.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1: preparation of crude spleen extract
Taking spleen of healthy Holstein cow, removing surface envelope, and cutting spleen to about 3mm3Placing the small blocks in a sterilization beaker, adding a proper amount of precooled sterilization normal saline, and mashing and homogenizing tissues; the homogenate was broken twice for 15 min/time in an ultrasonicator (8s-4s, 35% Amp). Adding acetic acid with the concentration of 5% and the same volume into the homogenized spleen, and stirring for 6 hours in an ice bath; centrifuging at 8000rpm at 4 deg.C for 30min, and collecting supernatant; carrying out rotary evaporation on the supernatant at 38-40 ℃ for 6-8h until acetic acid is completely evaporated; collecting the liquid left after rotary evaporation, and freeze-drying to obtain a spleen crude extract.
Example 2: ion exchange chromatography purification of antibacterial peptides
Dissolving the spleen crude extract with 0.01% acetic acid to obtain a spleen crude extract solution, passing the spleen crude extract solution through a cation exchange column, binding cation components in the spleen crude extract solution to the cation exchange column, firstly passing 25mmol/L ammonium acetate buffer solution (pH5.4) through the cation exchange column at a flow rate of 50mL/h, washing off unbound components, then eluting the cation components bound on the cation exchange column at a flow rate of 50mL/h with 10% acetic acid, measuring the absorbance value of the eluted components at a wavelength of 230nm by using an ultraviolet detector during the elution process, and collecting an elution peak (5 mL/peak). After separation, there were two elution peaks, peak a being an anionic peak and peak B being a cationic peak (see fig. 1). The antibacterial activity test (the antibacterial activity test method is shown in example 5) shows that the peak A has no antibacterial activity, and the peak B has antibacterial activity (see table 1).
TABLE 1 antibacterial Activity analysis results of ion-exchange purified product (unit: mm)
Example 3: reversed-phase high performance liquid chromatography purification of antibacterial peptide
The collected cation eluting peak component (peak B) was further separated by reverse phase high performance liquid chromatography (RP-HPLC). The chromatographic column adopted by the reversed phase high performance liquid chromatography is a product of Phenomenex company in America, and the model number of the chromatographic column is Jupiter 5uC18
The adopted eluents are a buffer solution A and a buffer solution B, wherein the buffer solution A is: 5% acetonitrile, 0.1% trifluoroacetic acid (preparation method is, adding 5mL acetonitrile into 95mL deionized water, mixing, adding 0.1mL trifluoroacetic acid, mixing to obtain buffer solution A); the buffer solution B is: 95% acetonitrile, 0.1% trifluoroacetic acid (preparation method is, 5mL deionized water is added into 95mL acetonitrile, mixing, then 0.1mL trifluoroacetic acid is added into it, mixing to obtain buffer B); the elution gradient is as follows: within 30min, the proportion of the eluent A is gradually adjusted from 100% to 60%, and the proportion of the eluent B is gradually adjusted from 0% to 40%; at 30.1min, the ratio of eluent A was 100%, the ratio of eluent B was 0%, the flow rate was 1mL/min, and the detection wavelength was 230 nm. In the reversed-phase high performance liquid chromatography separation process, all elution peak components are manually collected, 7 elution peak components are collected in total, (see figure 2), and the antibacterial activity of the 7 elution peak components is detected respectively (the antibacterial activity detection method is seen in example 5). The detection result shows that the 7 components of the elution peak have antibacterial activity on 3 tested microorganisms (see table 2), and the 7 components of the elution peak are collected and freeze-dried to obtain the spleen-derived antimicrobial peptide of the Holstein cow.
Example 4: the mass spectrometry of the spleen-derived antibacterial peptide of the Holstein cow comprises the following steps:
(1) and (3) determining the molecular weight of the spleen-derived antibacterial peptide of the Holstein cow:
molecular weight of spleen-derived antibacterial peptide of Holstein cow was measured and analyzed by MALDI-TOF/TOF (AB SCIEX, USA) mass spectrometer 5800. The specific operation is as follows: spotting 0.5. mu.L of the sample on a MALDI target plate, drying naturally, spotting 0.5. mu.L of 4mg/mL CHCA solution (50% acetonitrile in water, containing 0.1% TFA), drying naturally, and analyzing the sample with a 5800 type MALDI-TOF/TOF (AB SCIEX, USA) mass spectrometer, wherein the laser source is a 355nm Nd: YAG laser, the acceleration voltage is 20kV, data are collected in a positive ion mode and an automatic data acquisition mode, and peptide fragments are subjected to external standard correction by using myoglobin enzymolysis. The scanning range of the primary mass spectrum of the sample is 700-3600Da, and then target peptide fragment ions are selected for tandem mass spectrometry. The primary mass spectrum adopts a Reflector Positive mode, and parameters are as follows: CID (OFF), Mass ranging (700 and 5000Da), Focus Mass (1600Da), Fixed laser intensity (4500) Digitizer: bin Size (0.5 ns).
(2) Analyzing the sequence of spleen-derived antibacterial peptide of Holstein cows:
and (3) determining and analyzing the amino acid sequence of the spleen-derived antibacterial peptide of the Holstein cow by adopting a liquid chromatography and mass spectrometry instrument. Wherein the liquid phase system of the liquid chromatography-mass spectrometer is 20AD HPLC system (Shimadzu), and comprises a MicromasssC 18 column (specification: 5 μm,
0.1 x 15 mm). Mobile phase a (water: acetonitrile: formic acid 98:2:0.1) and D (water: acetonitrile: formic acid: 2:98:0.1) were added with calibration solution (Thermo Fisher Scientific). The sample was loaded at 2.25. mu.g (9. mu.l), and eluted with solution A at a flow rate of 2. mu.L/min for 15min for peptide adsorption and desalting. Eluting with a mobile phase containing 5% D solution at a flow rate of 200nL/min for 1min, and then establishing an elution gradient: the gradient of D liquid is increased from 5% to 35% in 65min, from 35% to 80% in 5min, then 80% is continuously eluted for 5min, and finally the column material is recovered in 2 min. The mass spectrometer used for both liquid chromatography and mass spectrometry was TripleTOF 5600(AB SCIEX, Concord, ON), the ion source was Nanospray III source (AB SCIEX, Concord, ON), and the emitter was a quartz material drawn needle (New objects, Woburn, MA). Machine parameters during data acquisition: ion source spray voltage 2.5kv, nitrogen pressure 30psi (14.5psi ≈ 1bar), spray pressure 15psi, and temperature at spray interface 150 deg.C; the scanning mode is a reflection mode; accumulating 250ms of 2+ to 5+ ion picks, with intensity accumulating over the first 50 scans at 120 minutes per second, with 2.8s being one cycle; the transmission window of the second quadrupole (Q2) is arranged to: 100Da is 100%; the pulse radio frequency electric frequency is 11 kHz; the detection frequency of the detector is 40 GHz; particle signal per scan in order toRecording four channels respectively for four times, and then merging and converting into data; the dynamic exclusion of parent ions was set as: within half the time of peak (about 18s), the same parent ion is fragmented no more than 2 times. Obtaining MS/MS data, and searching by software by adopting MASCOT (V2.3), wherein the searching parameters are as follows: NCBI cattle protein library, pancreatin enzyme digestion, two missed cutting sites, the tolerance of a primary mass spectrum is 0.05Da, the tolerance of a secondary mass spectrum is 0.1Da, no fixed modification is carried out, the variable modification is methionine oxidation, and the charge is set to be +2, +3 and + 4. And analyzing to obtain the amino acid sequence of the spleen-derived antibacterial peptide of the Holstein cow.
Through detection and analysis, the spleen-derived antibacterial peptide of the Holstein cow has the amino acid sequence and the molecular weight 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-Pro, and has a molecular weight of 3214.89.
(3) Isoelectric point analysis: opening EditSeq in DNAStar software, opening file, clicking 'newprotien' in 'new', inputting an amino acid sequence, and storing the sequence as a document in a pro format. And then opening a Protean in DNAStar software, opening a file, clicking "open", selecting the document just stored in the pro format, clicking "open", and selecting "transformation dark" in "Analysis", thus obtaining the isoelectric point. The isoelectric point of the spleen-derived antibacterial peptide of the Holstein cow is 12.18.
Example 5: application implementation of spleen-derived antibacterial peptide of Holstein cows
1. The invention relates to a method for detecting the antibacterial activity of spleen-derived antibacterial peptide of Holstein cows
The antibacterial activity of an elution peak component separated by ion exchange chromatography and an absorption peak component separated by reversed-phase high performance liquid chromatography is determined by adopting an agar plate diffusion method, and tested microbial strains are as follows: coli ATCC8099, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231, said test strains of microorganisms were purchased from Nanjing Biotech Ltd.
The above 3 test microorganisms were each diluted in a 50 ℃ lower agar medium (10mL trypticase soy broth, 0.1g ultrapure agarose, pH 7.4), plates were poured and sterilized, coagulated, punched with a punch (pore size about 3mm), bottom-sealed with slight heating with an alcohol lamp, and 5. mu.L of test sample was added to each well with a pipette, each plate including a positive control and a negative control. Positive control: ampicillin is directed against gram-negative and gram-positive bacteria, and nystatin is directed against candida albicans. Negative control: and (5) sterilizing water. The plate was allowed to stand for 1h to allow the test solution to diffuse into the agarose. Then, a layer of agar medium (about 50 ℃ C., the same nutrient content as the lower layer of agar medium) was added. The plates were incubated overnight at 37 ℃ and then the diameter of the transparent circle around the well was recorded and the measurement was repeated three times for each strain and the average was calculated.
7 elution peak components obtained by reversed-phase high performance liquid chromatography are subjected to antibacterial activity detection, and the detection results are shown in table 2.
As can be seen from Table 2, the spleen-derived antimicrobial peptide (elution peak 7 fraction) of Holstein cows of the present invention has strong antimicrobial activity against all of the three tested microbial strains. Therefore, the spleen-derived antibacterial peptide of the Holstein cow is expected to have good application prospect in the aspect of treating animal bacterial diseases and fungal diseases. According to the general characteristics of known antibacterial peptides, the spleen-derived antibacterial peptide of the Holstein cow has certain application potential in the aspects of application of antibacterial drugs, feed mildew prevention, fresh keeping and the like.
TABLE 2 antibacterial Activity analysis results of the purified product by reversed-phase high-performance liquid chromatography (unit: mm)
2. The invention relates to a method for measuring the minimal inhibitory concentration of spleen-derived antimicrobial peptide of Holstein cows
The tested microbial strains were: coli ATCC8099, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231, said test strains of microorganisms were purchased from Nanjing Biotech Ltd.
The tested strain is enlarged and cultured in TSB liquid culture medium, and OD of the bacterial liquid is measured600The value is obtained. When OD value is largerCentrifuging the bacterial solution at 0.6-0.8 r/min for 10min, discarding supernatant, collecting thallus precipitate, re-suspending thallus precipitate with PBS buffer (0.01M) of the same volume, and diluting the bacterial suspension with MH culture medium to 2 × 106CFU/mL. The spleen-derived antimicrobial peptide sample of the Holstein cow is diluted by deionized water to ensure that the concentration of the spleen-derived antimicrobial peptide sample is 0.58-300 mu g/mL. Adding 50 μ L of the antibacterial peptide of the present invention and 50 μ L of diluted bacterial solution to each well of 96-well cell culture plate, repeating three wells for each concentration, culturing at 37 deg.C for 16-18h, mixing the solutions in each well, and determining OD600Values were given as their MIC in terms of antimicrobial peptide concentration corresponding to sudden changes in OD values, and the results are shown in fig. 4, 5 and 6.
As can be seen from FIGS. 4, 5 and 6, the Minimal Inhibitory Concentration (MIC) of spleen-derived antibacterial peptide of Holstein cows against Escherichia coli was 37.5. mu.g/mL, the Minimal Inhibitory Concentration (MIC) against Staphylococcus aureus was 18.75. mu.g/mL, and the Minimal Inhibitory Concentration (MIC) against Candida albicans was 150. mu.g/mL. The result shows that the minimum inhibitory concentration of the antibacterial peptide reaches microgram level to common bacteria, and the antibacterial peptide has strong inhibitory activity.
3. The hemolytic activity detection of the spleen-derived antibacterial peptide of the Holstein cow comprises the following steps:
fresh rabbit blood is taken, anticoagulated by 3.8% sodium citrate, the anticoagulated blood is centrifuged for 10min at 3000r/min, and is washed and precipitated for 3 times by Phosphate Buffer Solution (PBS) until the supernatant is colorless and transparent, and then 1% erythrocyte is prepared by PBS. Bac 5-like antibacterial peptide was adjusted to a concentration of 300. mu.g/mL, 150. mu.g/mL, 75. mu.g/mL, 37.5. mu.g/mL, 18.75. mu.g/mL, 9.375. mu.g/mL, 4.6875. mu.g/mL, 2.34375. mu.g/mL, 1.17185. mu.g/mL, 0.585938. mu.g/mL with PBS, and an equal volume of the red blood cell suspension was added. PBS is used as a negative control, 1% Tritonx-100 is used as a positive control, the reaction is carried out for 1h at 37 ℃, centrifugation is carried out for 10min at 1500r/min, supernatant is sequentially added into a 96-well plate, and the OD value at 540nm is measured by a microplate reader. According to the calculation formula: the hemolysis ratio (%) was calculated as (detection well OD value-negative well OD value)/(positive well OD value-negative well OD value) × 100%, and the results are shown in fig. 7.
As can be seen from FIG. 7, under the concentration conditions of 300. mu.g/mL and 150. mu.g/mL, the hemolysis rates of the spleen-derived antibacterial peptide of Holstein cow are 6.45% and 5.53%, which are slightly higher than 5%, but under the concentration condition of 75. mu.g/mL, the hemolysis rate is lower than 5%, which indicates that the spleen-derived antibacterial peptide of Holstein cow has better safety in a certain concentration range.
In conclusion, the spleen-derived antibacterial peptide of the Holstein cow has a wide antibacterial spectrum, has high-efficiency antibacterial action on gram-positive bacteria, gram-negative bacteria and fungi, and is low in hemolysis rate, so that the spleen-derived antibacterial peptide of the Holstein cow can be better applied to preparation of medicaments for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection, and can also be used as a feed additive or a food additive.
SEQUENCE LISTING
<110> institute of science and technology of Henan
<120> holstein cow spleen source antibacterial peptide and preparation method and application thereof
<130>1
<160>1
<170>PatentIn version 3.5
<210>1
<211>27
<212>PRT
<213> antimicrobial peptides
<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 (4)
1. The spleen-derived antibacterial peptide of the Holstein cow is characterized in that the amino acid sequence of the spleen-derived antibacterial peptide of the Holstein cow 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 use of the spleen-derived antibacterial peptide of the Holstein cow as defined in claim 1, wherein the spleen-derived antibacterial peptide of the Holstein cow is used for preparing a medicament for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection, the gram-positive bacteria are Staphylococcus aureus, the gram-negative bacteria are Escherichia coli, and the fungi are Candida albicans.
3. The use according to claim 2, wherein the medicament comprises the spleen-derived antibacterial peptide of Holstein cow as claimed in claim 1, in admixture with one or more pharmaceutically acceptable carriers and/or additives.
4. The use of the spleen-derived antimicrobial peptide of Holstein cow as defined in claim 1 in the preparation of feed additives or food additives.
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| CN108148111B (en) * | 2017-12-19 | 2020-06-05 | 浙江丰安生物制药有限公司 | Method for extracting polypeptide in spleen aminopeptide |
| CN108129549B (en) * | 2017-12-19 | 2020-06-05 | 浙江丰安生物制药有限公司 | Method for extracting polypeptide in spleen aminopeptide |
| CN108264539B (en) * | 2017-12-28 | 2020-12-25 | 河南科技学院 | Antibacterial peptide RL-18 and application thereof |
| CN111848739B (en) * | 2020-06-30 | 2022-08-30 | 河南科技学院 | Antibacterial peptide LJ-2 and application thereof |
| CN114940698B (en) * | 2022-07-22 | 2022-10-21 | 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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| CN101607992B (en) * | 2009-07-27 | 2011-09-07 | 河南科技学院 | Antibacterial peptide separated from cow blood and code sequence and application thereof |
| CN104672302A (en) * | 2014-12-18 | 2015-06-03 | 山东深海生物科技股份有限公司 | Polypeptin, and preparation method as well as application thereof |
| CN105777875A (en) * | 2016-05-26 | 2016-07-20 | 青岛农业大学 | Antibacterial peptide CSTC24 and application thereof |
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2017
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|---|---|---|---|---|
| CN101607992B (en) * | 2009-07-27 | 2011-09-07 | 河南科技学院 | Antibacterial peptide separated from cow blood and code sequence and application thereof |
| CN104672302A (en) * | 2014-12-18 | 2015-06-03 | 山东深海生物科技股份有限公司 | Polypeptin, and preparation method as well as application thereof |
| CN105777875A (en) * | 2016-05-26 | 2016-07-20 | 青岛农业大学 | Antibacterial peptide CSTC24 and application thereof |
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| Title |
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| Amino Acid Sequences of Two Proline-rich Bactenecins;Rainer W.Frank等;《The journal of biological chemistry》;19900423;第265卷(第31期);第18871-18874页 * |
| 抗菌肽在现代养殖业应用现状及进展;傅志远;《经济动物学报》;20160930;第20卷(第3期);第179-183页 * |
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| CN107298706A (en) | 2017-10-27 |
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