CN102603883A - Antibacterial peptide, as well as preparation method and application thereof - Google Patents
Antibacterial peptide, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a novel antibacterial peptide, and the antibacterial peptide has an amino acid sequence as shown in SEQ ID NO: 1 (sequence identity number 1). The polypeptide is a protein which has the molecular weight of 5.3kDa and is secreted by haemaphysalis longicornis, the pI (isoelectric point) is 8.68, and the protein has 19 amino acids, does not contain a conserved domain and further has a relatively wide range of the antibacterial activity. In addition, the invention further provides a preparation method and application of the antibacterial peptide.
Description
Technical field
The invention belongs to biomedicine and biological technical field, particularly, the present invention relates to the preparation method and its usage of a kind of antibacterial peptide, said antibacterial peptide.
Background technology
Because being widely used of antibiotic medicine caused many pathogenic bacterium to produce resistance, biological medicine circle is put into the new ideas medicine with increasing energy at present---the antibacterial peptide field.
Antibacterial peptide is the one type of amphiphilic small molecules basic polypeptide that in whole organic sphere, extensively exists, and it is found in the various organisms, for example bacterium, fungi, plant, insect, fish, birds, crustacean, Amphibians and Mammals etc.At present existing antibacterial peptide surpasses 750 kinds more than, and wherein the overwhelming majority is a cationic antibacterial peptide, and a small amount of negatively charged ion antibacterial peptide is also arranged.
Antibacterial peptide shows broad-spectrum anti-microbial activity; And bacterial poison have neutralizing effect, its mechanism of action is unique, is difficult for producing resistance; And some antibacterial peptide only works to prokaryotic organism and mutant cell; Harmless to eukaryote, thereby produced under the obvious chemical sproof situation in many pathogenic bacterium, the discovery of antibacterial peptide and further investigation for the brand-new antibacterials of exploitation provide maybe.Yet; Though can be used as one type, antibacterial peptide has broad-spectrum high efficacy fungistatic effect, the very difficult new ideas medicine that produces patience of pathogenic bacterium,, the natural antibacterial peptide source is very limited; As new drug research with in developing, the acquisition of antibacterial peptide also is a problem demanding prompt solution at antibacterial peptide.
Summary of the invention
The object of the present invention is to provide a kind of novel antibacterial peptide.
Another object of the present invention provides the preparation method of said antibacterial peptide.
In addition, of the present invention have a purpose to provide the purposes of said antibacterial peptide in the preparation antibacterials.
The objective of the invention is to realize through following technical scheme:
On the one hand, the present invention provides a kind of antibacterial peptide, and said antibacterial peptide comprises the aminoacid sequence shown in the SEQ ID NO:1.
Preferably, said antibacterial peptide is the peptide of aminoacid sequence shown in the SEQ ID NO:1.
Wherein, said antibacterial peptide separates from haemaphysalis longicornis, and molecular weight is 5.3kDa, and iso-electric point is 8.68.
On the other hand, the present invention provides the method for the above-mentioned antibacterial peptide of preparation, and said method comprises: the saliva of 1) collecting haemaphysalis longicornis; 2) saliva of collecting in the step 1) being carried out ultracentrifugation filters; And 3) with step 2) in the filtrating component that obtains carry out gel permeation chromatography.
In addition, above-mentioned preparation method further comprises: 4) elutriant that obtains through gel permeation chromatography in the step 3) is carried out purifying through the HPLC RPLC.
Preferably, the step 1) of aforesaid method comprises:
The rabbit blood haemaphysalis longicornis of feeding makes it body weight and reaches 200-300 μ g; Then with this blood tick clean; Use the salivation inductor to induce the blood tick to salivate; After observing the blood tick and beginning to salivate, the blood tick is placed in 34.5-35.5 ℃, preferred 35 ℃ box 2-3 hour, collect this haemaphysalis longicornis excretory saliva afterwards and store for future use in-70 ℃;
Preferably; Said salivation inductor is a pilocarpine; Inductive dose is the pilocarpine of every haemaphysalis longicornis 0.8-1.1 μ g, preferred 1 μ g, be given to for the first time behind the blood tick every at a distance from 20 minutes interpolation 0.5 μ g pilocarpines, till observing the blood tick and secreting a small amount of saliva.
The step 2 of aforesaid method) preferably include:
Get the haemaphysalis longicornis saliva of collecting in the 200-300 μ l step 1), use molecular weight cut-off as the YM-10 filter membrane of 10kDa and molecular weight cut-off as the YM-3 filter membrane of 3kDa in 4 ℃ of ultracentrifugations, obtain component I I, i.e. the filtrating of molecular weight between 3-10kDa.
The step 3) of aforesaid method preferably includes:
With step 2) in the component I I that obtains be added on the Superdex Peptide 10/300 gel permeation chromatography post; Pillar specification: 10mm * 300mm; And (acetonitrile is a solute to Filter column with the acetonitrile methanol solution of 30% (volume ratio v/v); Methyl alcohol is solvent) and 0.1% (volume ratio v/v) formic acid (formic acid is water-soluble) pre-equilibration, collect the elutriant of the 3rd elution peak then with the flow velocity of 5 μ l/min.
The step 4) of aforesaid method preferably includes:
Adopt 0.3mm * 150mm C8 magnetic posts, ABI 140D pump and 785A Ultraviolet Detector, irritate post with the speed of 2 or 5 μ l/min; Solution A is the aqueous solution (volume ratio v/v) that contains 10% methyl alcohol and 0.1% formic acid, and the solution B component is 30% (volume ratio v/v) acetonitrile methanol solution and 0.1% (volume ratio v/v) formic acid solution;
Chromatographic column is with the preceding solution A pre-equilibration of using; Elutriant with the 3rd peak that obtains in the step 3) after the pillar balance is added on the pillar; And then with gradient 10%-95% adding solution B, flow rate control continues 20min at 5 μ l/min; Flow velocity is adjusted to 2 μ l/min then, collects an elutriant with run tank is every at a distance from 1min then.
Another aspect, the present invention provides the purposes of above-mentioned antibacterial peptide in the preparation antibacterials.
Below be detailed description of the present invention:
According to an embodiment of the present invention, adopt following step to obtain the target antibacterial peptide:
1) catches wild haemaphysalis longicornis (Haemaphysalis longicornis) from forest, under test conditions, raise through new zealand white rabbit.Before collecting blood tick saliva, let the blood tick suck blood more than 120 hours at the rabbit ear, blood tick body weight reaches 200-300 μ g.
2) collection of the saliva of haemaphysalis longicornis:
The blood tick that body weight is reached 200-300 μ g is clean with purified rinse water; Be placed on the clean slide glass; Be put into the hypostomal plate preparation collection saliva of blood tick then with a clean glass capillary; Use salivation inductor pilocarpine (claiming pilocarpine again) thus being added drop-wise to the tick hypostomal plate induces the blood tick to salivate, inductor dosage is the 0.5mg/ml pilocarpine ethanolic soln (the 5mg pilocarpine is prepared with 100ml 95% ethanolic soln) of 2 μ l.Every add 1 μ l pilocarpine ethanolic soln, till observing the blood tick and secreting a small amount of saliva at a distance from 20min.Then, the blood tick is placed in 35 ℃ the wet box 2-3 hour.Take out kapillary then, watch the saliva amount.Each blood tick approximately can be gathered the saliva of 15-30 μ l, then these saliva enrichments is got up, be placed on-70 ℃ subsequent use.
3) purifying of antibacterial peptide:
With the blood tick saliva of the 200-300 μ l that collects, in 4 ℃ of ultracentrifugations, centrifugal condition is 20000rpm, and 20min uses the filter membrane (molecular weight cut-off is 3kDa) of YM-10 (molecular weight cut-off is 10kDa) and YM-3 to filter when centrifugal.Filtrating is used to detect anti-microbial activity.Again the spissated antibacterial components of enrichment is separated through successive Superdex Peptide 10/300 gel permeation chromatography post, obtain the antibacterial peptide composition of purifying at last.
In brief, the filtrating that contains antibacterial peptide in the blood tick saliva is added on the Superdex Peptide 10/300 gel permeation chromatography post pillar specification: 10mm * 300mm.With before, Filter column is with 30% acetonitrile methanol solution and 0.1% formic acid pre-equilibration, then with the flow velocity collection elutriant of 0.25ml/min.Elutriant is monitored absorbance at 215nm, collects the elutriant of all peak values, and with micro-traditional vacuum concentrate eluant, the elutriant after concentrating is used to detect anti-microbial activity.
With above-mentioned elutriant, further pass through HPLC rp-hplc system purifying once more with higher anti-microbial activity.The specification of HPLC chromatographic column: 0.3mm * 150mm C8 magnetic posts.Irritate post through ABI 140D pump and 785A Ultraviolet Detector with the speed of 2 or 5 μ l/min.The solution A component is 0.1% formic acid solution, and the solution B component is 30% acetonitrile methanol solution and 0.1% formic acid solution.Chromatographic column is added to sample on the pillar after the pillar balance with preceding usefulness 10% methanol solution and 0.1% formic acid solution balance, and then adds solution B with gradient 10%-95%, and flow rate control continues 20min at 5 μ l/min, and flow velocity is adjusted to 2 μ l/min then.Use the gloomy 203B run tank of gill then, every at a distance from elutriant of 1min collection.Use nanometer Drop UV/vis spectrophotometer protein value then, and detect the anti-microbial activity of these cuts.
4) molecular weight of mass spectrometric detection antibacterial peptide:
With the MALDI/TOF-MS mass spectrometer antibacterial peptide molecular weight of above-mentioned purifying is measured.Matrix solution is: 1-cyanic acid-4-alcohol acid (Sigma company) 100% acetonitrile solution (analytical pure).
5) amino acid sequence analysis:
Above-mentioned antibacterial peptide is measured the-terminal amino acid order with the Edman edman degradation Edman.Detect the phenylthiohydantoin verivate with the pulse automatic DNA sequencer DNA.The aminoacid sequence of this antibacterial peptide is compared through BLASTP and the NR DB of NCBI.
In sum, the invention provides a kind of from the saliva of haemaphysalis longicornis isolating antibacterial peptide, this antibacterial peptide has the molecular weight of 5.3kDa, iso-electric point is 8.68, and its aminoacid sequence is made up of 19 amino-acid residues shown in SEQ IDNO:1.Experiment showed, that antibacterial peptide provided by the invention has anti-microbial property comparatively widely, and this antibacterial peptide of hemolytic experiment proof do not show significant hemolytic activity, pair cell toxicity is low, in the brand-new antibacterials of research and development, has bigger potentiality.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1 is the gel chromatography stratographic analysis result of component I I;
Fig. 2 is the stratographic analysis result of the HPLC at peak 3 among Fig. 1;
Fig. 3 is the mass spectrometry results of antibacterial peptide of the present invention;
Fig. 4 is the expression of antibacterial peptide of the present invention at the haemaphysalis longicornis different tissues, wherein: 1. standard protein lower molecular weight; 2. fatty body; 3. hemocyte; 4. sialisterium; 5. intestines.
Embodiment
The concrete embodiment of following reference explains the present invention.It will be appreciated by those skilled in the art that these embodiment only are used to explain the present invention, the scope that it does not limit the present invention in any way.
Embodiment 1: the separation of haemaphysalis longicornis saliva different components and anti-microbial activity detect
1: the separation of component
Catch wild haemaphysalis longicornis (Haemaphysalis longicornis) from forest, under the raising condition of standard clean level experimental animal room, raise through new zealand white rabbit (available from Beijing Experimental Animal Center).Before collecting blood tick saliva, let the blood tick suck blood more than 120 hours at the rabbit ear, blood tick body weight reaches 200-300 μ g.
The blood tick that body weight is reached 200-300 μ g is clean with purified rinse water; Be placed on the clean slide glass; Be put into the hypostomal plate preparation collection saliva of blood tick then with a clean glass capillary; Use salivation inductor pilocarpine (available from Switzerland Fluka & RDH company) (claiming pilocarpine again) to induce the blood tick to salivate, inductor dosage is 2 μ l pilocarpine ethanolic solns (the 5mg pilocarpine is with the preparations of 100ml 95% ethanolic soln).Every add this pilocarpine ethanolic soln of 1 μ l, till observing the blood tick and secreting a small amount of saliva at a distance from 20min.Then, the blood tick is placed in 35 ℃ the wet box 2-3 hour.Take out kapillary then, watch the saliva amount.Each blood tick approximately can be gathered the saliva of 15-30 μ l, then these saliva enrichments is got up, be placed on-70 ℃ subsequent use.
With the blood tick saliva of the 200-300 μ l that collects in 4 ℃ of ultracentrifugation (centrifugal conditions: 20000rpm; 20min); Use the filter membrane (molecular weight cut-off is 3kDa) of YM-10 (molecular weight cut-off is 10kDa) and YM-3 to filter when centrifugal; Thereby obtain 3 components: component I (molecular weight＞10kDa), component I I (molecular weight is between 3-10kDa) and component III (molecular weight＜3kDa).
2: anti-microbial activity detects
The anti-microbial activity that the selection standard bacterial isolates carries out above-mentioned 3 components detects.The bacterium of being adopted is following:
Gram-positive microorganism: Bacillus cereus (Bacillus cereus) (bacterial strain preserving number NKCCMR NK 7.G1808;); Subtilis (B.subtilis) (bacterial strain preserving number CCTCC AB205127;), streptococcus aureus (Staphylococcus aureus) (bacterial strain preserving number NKCCMR NK 7.G1758);
Gram-negative bacteria: intestinal bacteria EDL933 (bacterial strain preserving number GIM1.299) and intestinal bacteria MG/655 (bacterial strain preserving number GIM1.215).
Detection method is following:
With above-mentioned microbial culture in Luria-Bertain (LB) nutrient solution, when growing into OD
600nmValue is 0.8 o'clock, gets 10 μ l bacterium liquid and joins fresh the containing in the 0.7% agar LB substratum of 8ml, the bacterium liquid after the above-mentioned dilution is poured into fill the petri diss that 25ml contains the diameter 90mm of 1.5% agar LB substratum then.After treating that top-layer agar solidifies, 20 μ l test sample are rubbed into the top-layer agar surface, dry the back in 37 ℃ of overnight cultures.
The judgement of anti-microbial activity: observe the size that the bacterial growth media surface forms a clear and definite zone, represent the bacteriostatic level of test sample.Minimal inhibitory concentration (MIC) is meant that the minimum concentration of tangible bacterial growth does not appear in test sample.The concentration determination of test sample is calculated with the ultraviolet absorptivity value of 215nm and 225nm, and formula is: concentration (mg/ml)=(A215-A225nm) * 0.144.Each sample is done 3 repetitions.
Carried out the detection of above-mentioned bacteriostatic activity to separating from 3 components of haemaphysalis longicornis saliva, the result shows: the anti-microbial activity of three components of blood tick saliva there are differences (seeing table 1), and that wherein anti-microbial activity is the highest is component I I, and minimal inhibitory concentration is 3.5 μ g/ml.
The activity of the enterobacteria EDL933 of the Chinese People's Anti-Japanese Military and Political College of each component of table 1
| Component | Molecular weight ranges | Anti-microbial activity (minimal inhibitory concentration, μ g/ml) |
| Component I | ＞10kDa | 30.4±0.34 |
| Component I I | 3-10kDa | 3.5±0.05 |
| Component III | ＜3kDa | 8.5±0.10 |
Embodiment 2: component I I is through the anti-microbial activity of each elution peak of gel permeation chromatography
The antibacterial components II of enrichment is added on the Superdex Peptide 10/300 gel permeation chromatography post (Sigma company) pillar specification: 10mm * 300mm.Before using, Filter column is collected elutriant with the flow velocity of 0.25ml/min then with acetonitrile methanol solution and 0.1% formic acid (v/v) pre-equilibration of 30% (v/v).Elutriant is monitored absorbance at 215nm, collects the elutriant of all peak values.With micro-traditional vacuum concentrate eluant, adopt the method identical that the elutriant of each elution peak after concentrated is carried out the detection of anti-microbial activity then with embodiment 1.
Through the analysis revealed to component I I, gel chromatography is analyzed component II and 5 peak value (see figure 1)s, wherein peak 3 and 5 show antibacterial activities are occurred.Compare peak 5, peak 3 shows better anti-microbial activity, and minimal inhibitory concentration is merely 3.2 μ g/ml.So, confirm that peak 3 further analyzes.
Embodiment 3: the anti-microbial activity of antibacterial peptide of the present invention
Above-mentioned peak 3 with higher anti-microbial activity is further passed through HPLC rp-hplc system purifying once more.The specification of HPLC chromatographic column: 0.3mm * 150mm C8 magnetic posts.Irritate post through ABI 140D pump and 785A Ultraviolet Detector with the speed of 2 or 5 μ l/min.The solution A component is 0.1% formic acid solution, and the solution B component is 30% acetonitrile methanol solution and 0.1% formic acid solution (v/v).Chromatographic column is with preceding usefulness 10% methanol solution and 0.1% formic acid solution (v/v) balance; After the pillar balance sample is added on the pillar, and then adds solution B with gradient 10%-95%, flow rate control is at 5 μ l/min; Continue 20min, flow velocity is adjusted to 2 μ l/min then.Use the gloomy 203B run tank of gill then, every at a distance from elutriant of 1min collection.Use nanometer Drop UV/vis spectrophotometer protein value then.
Protein called after HLAMP with purifying obtains adopts then and carries out antibacterial tests with embodiment 1 identical method, and the result sees table 2.
The anti-microbial activity of table 2HLAMP
| Strain name | Anti-microbial activity (minimal inhibitory concentration, μ g/ml) |
| Bacillus cereus | 5.8±0.05 |
| Subtilis | 12.3±0.1 |
| Streptococcus aureus | 10.4±0.08 |
| Intestinal bacteria EDL 933 | 3.2±0.02 |
| Intestinal bacteria MG/655 | 4.2±0.03 |
The result shows that HLAMP all has anti-microbial activity to Gram-negative bacteria and gram-positive microorganism, but concerning different bacterium, minimal inhibitory concentration is different, and two kinds of Gram-negative bacterias are responsive more to HLAMP.
In addition, with the MALDI/TOF-MS mass spectrometer antibacterial peptide molecular weight of above-mentioned purifying is measured, matrix solution is: 1-cyanic acid-4-alcohol acid (Sigma company) 100% acetonitrile solution (analytical pure).And this antibacterial peptide measured-terminal amino acid order with the Edman edman degradation Edman, with pulse automatic DNA sequencer DNA detection phenylthiohydantoin verivate, the aminoacid sequence of this antibacterial peptide is compared through BLASTP and the NR DB of NCBI.
Interpretation of result shows that the molecular weight of this polypeptide is 5.3kDa, and sequence is PDPGQPWQVKAGRPPCYSI (SEQ ID NO:1), is made up of 19 amino-acid residues.BLASTP result shows that it possibly be the secreted polypeptides of the 5.3kDa of a blood tick, and 19 amino acid are formed, no conserved sequence, and pI is 8.68, belongs to a unknown function protein family.The molecular weight of this polypeptide of mass spectroscopy is the 5300.5Da (see figure 3).
The hemolytic experiment of embodiment 4:HLAMP
Hemolytic test adopts new zealand white rabbit red blood corpuscle liquid culture based assays.Carry out continuous doubling dilution to antibacterial peptide to be detected, hatch 30min for 37 ℃, centrifugal red corpuscle is measured absorbance with supernatant at 595nm.1%Triton X-100 is joined in the cell sample, confirm maximum haemolysis degree.
When showing 150 μ g/ml, the result do not show significant hemolytic activity.
Embodiment 4: the specificity analyses of antibacterial peptide gene tissue expression of the present invention
(the gene accession number: AF483734) (SEQID NO:2) design primer, the amplification different tissues comprises the cDNA fragment of fatty body, hemocyte, sialisterium and enteron aisle etc. according to blood tick HLAMP specificity est sequence.These tissues obtain through the adult haemaphysalis longicornis of dissecting full blood 48-72 hour.Extract total RNA of each tissue with total RNA separating kit (Ambion), and measure the concentration of RNA, ℃ preservation is subsequent use then-80.(Invitrogen) carries out reverse transcription to total RNA with the M-MLV ThermoScript II.Use following primer amplification cDNA then:
HLAMP-F 5`-AAGGTACTTCCTAATAGCCC GCCA-3`; With
HLAMP-R?5`-AGCCATTATTGCAACGTGAGCAGG-3`；
Na
+K
+-ATPase-F 5`-ACGAAACTGCCGAGAGCGACATTA-3` and 5`-ATCCT GAGACCTTTGTCCATGCCT-3` are as reference.
The PCR response procedures: 94 ℃, 4min; 94 ℃, 1min; 49 ℃, 1min; 72 ℃, 1min, 35 circulations.72℃，10min。
The result shows that the HLAMP antibacterial peptide can have expression at the different tissues of blood tick, can express at hemocyte, fatty body and sialisterium, but almost not express (see figure 4) at intestines.
Claims (10)
1. an antibacterial peptide is characterized in that, said antibacterial peptide comprises the aminoacid sequence shown in the SEQ ID NO:1.
2. antibacterial peptide as claimed in claim 1 is characterized in that, said antibacterial peptide is the peptide of aminoacid sequence shown in the SEQ ID NO:1.
3. according to claim 1 or claim 2 antibacterial peptide is characterized in that, said antibacterial peptide separates from haemaphysalis longicornis, and molecular weight is 5.3kDa, and iso-electric point is 8.68.
4. method for preparing like each said antibacterial peptide among the claim 1-3 is characterized in that said method comprises:
1) saliva of collection haemaphysalis longicornis;
2) saliva of collecting in the step 1) being carried out ultracentrifugation filters; And
3) with step 2) in the filtrating component that obtains carry out gel permeation chromatography.
5. method as claimed in claim 4 is characterized in that, said method further comprises:
4) elutriant that obtains through gel permeation chromatography in the step 3) is carried out purifying through the HPLC RPLC.
6. like claim 4 or 5 described methods, it is characterized in that said step 1) comprises:
The rabbit blood haemaphysalis longicornis of feeding makes it body weight and reaches 200-300 μ g; Then with this blood tick clean; Use the salivation inductor to induce the blood tick to salivate; After observing the blood tick and beginning to salivate, the blood tick is placed in 34.5-35.5 ℃, preferred 35 ℃ box 2-3 hour, collect this haemaphysalis longicornis excretory saliva afterwards and store for future use in-70 ℃;
Preferably; Said salivation inductor is a pilocarpine; Inductive dose is the pilocarpine of every haemaphysalis longicornis 0.8-1.1 μ g, preferred 1 μ g, be given to for the first time behind the blood tick every at a distance from 20 minutes interpolation 0.5 μ g pilocarpines, till observing the blood tick and secreting a small amount of saliva.
7. like each described method among the claim 4-6, it is characterized in that said step 2) comprising:
Get the haemaphysalis longicornis saliva of collecting in the 200-300 μ l step 1), use molecular weight cut-off as the YM-10 filter membrane of 10kDa and molecular weight cut-off as the YM-3 filter membrane of 3kDa in 4 ℃ of ultracentrifugations, obtain component I I, i.e. the filtrating of molecular weight between 3-10kDa.
8. like each described method among the claim 4-7, it is characterized in that said step 3) comprises:
With step 2) in the component I I that obtains be added on the Superdex Peptide 10/300 gel permeation chromatography post; Pillar specification: 10mm * 300mm; And Filter column is collected the elutriant of the 3rd elution peak then with 30% acetonitrile methanol solution and 0.1% formic acid pre-equilibration with the flow velocity of 5 μ l/min.
9. like each described method among the claim 4-8, it is characterized in that said step 4) comprises:
Adopt 0.3mm * 150mm C8 magnetic posts, ABI 140D pump and 785A Ultraviolet Detector, irritate post with the speed of 2 or 5 μ l/min; Solution A is the aqueous solution that contains 10% methyl alcohol and 0.1% formic acid, and the solution B component is 30% acetonitrile methanol solution and 0.1% formic acid solution;
Chromatographic column is with the preceding solution A balance of using; Elutriant with the 3rd peak that obtains in the step 3) after the pillar balance is added on the pillar; And then with gradient 10%-95% adding solution B, flow rate control continues 20min at 5 μ l/min; Flow velocity is adjusted to 2 μ l/min then, collects an elutriant with run tank is every at a distance from 1min then.
10. like the purposes of each said antibacterial peptide among the claim 1-3 in the preparation antibacterials.
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Cited By (5)
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| CN102940874A (en) * | 2012-12-12 | 2013-02-27 | 中国科学院昆明动物研究所 | Application of small molecular polypeptide CW14 in preparation of goat antibacterial medicament |
| CN102940877A (en) * | 2012-12-12 | 2013-02-27 | 中国科学院昆明动物研究所 | Application of polypeptide DM29 in preparing drugs for increasing natural immunity of goats |
| CN103642811A (en) * | 2013-07-18 | 2014-03-19 | 南昌大学 | DNA expressing new-type defensin antibacterial peptide |
| CN107266546A (en) * | 2017-06-16 | 2017-10-20 | 苏州大学 | Antibacterial peptide HlDFS1 and its application |
| CN113214377A (en) * | 2020-01-21 | 2021-08-06 | 四川大学 | Antibacterial peptide AP-64 and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102940874A (en) * | 2012-12-12 | 2013-02-27 | 中国科学院昆明动物研究所 | Application of small molecular polypeptide CW14 in preparation of goat antibacterial medicament |
| CN102940877A (en) * | 2012-12-12 | 2013-02-27 | 中国科学院昆明动物研究所 | Application of polypeptide DM29 in preparing drugs for increasing natural immunity of goats |
| CN102940877B (en) * | 2012-12-12 | 2013-12-18 | 中国科学院昆明动物研究所 | Application of polypeptide DM29 in preparing drugs for increasing natural immunity of goats |
| CN102940874B (en) * | 2012-12-12 | 2013-12-18 | 中国科学院昆明动物研究所 | Application of small molecular polypeptide CW14 in preparation of goat antibacterial medicament |
| CN103642811A (en) * | 2013-07-18 | 2014-03-19 | 南昌大学 | DNA expressing new-type defensin antibacterial peptide |
| CN103642811B (en) * | 2013-07-18 | 2015-03-11 | 南昌大学 | DNA expressing new-type defensin antibacterial peptide |
| CN107266546A (en) * | 2017-06-16 | 2017-10-20 | 苏州大学 | Antibacterial peptide HlDFS1 and its application |
| CN107266546B (en) * | 2017-06-16 | 2020-08-04 | 苏州大学 | Antibacterial peptide HlDFS1 and application thereof |
| CN113214377A (en) * | 2020-01-21 | 2021-08-06 | 四川大学 | Antibacterial peptide AP-64 and preparation method and application thereof |
| CN113214377B (en) * | 2020-01-21 | 2022-10-28 | 四川大学 | Antibacterial peptide AP-64 and preparation method and application thereof |
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Application publication date: 20120725 |