CN109535227B - Antibacterial peptide, preparation method of antibacterial peptide, antibacterial composition, antibacterial method and application - Google Patents
Antibacterial peptide, preparation method of antibacterial peptide, antibacterial composition, antibacterial method and application Download PDFInfo
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- CN109535227B CN109535227B CN201811551219.5A CN201811551219A CN109535227B CN 109535227 B CN109535227 B CN 109535227B CN 201811551219 A CN201811551219 A CN 201811551219A CN 109535227 B CN109535227 B CN 109535227B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The scheme relates to an antibacterial peptide, a preparation method thereof, a composition containing the antibacterial peptide, an antibacterial method using the antibacterial peptide and application of the antibacterial peptide, wherein the amino acid sequence of the antibacterial peptide is Val Pro Tyr Ile Gln His Thr Pro Asn Leu Leu Leu Glu Gln Asn Leu Leu; wherein, the amino acids in the amino acid sequence are D-type amino acids. The scheme adopts a chemical synthesis means, can efficiently obtain a large amount of high-purity all-trans D-type antibacterial peptide, can ensure the integrity and purity of an antibacterial peptide sequence, and has higher thermal stability and protease degradation resistance.
Description
Technical Field
The invention belongs to the field of polypeptides, and particularly relates to an antibacterial peptide, a preparation method of the antibacterial peptide, an antibacterial composition, an antibacterial method and application of the antibacterial peptide.
Background
The antibacterial peptide is also called antimicrobial peptide or host defense peptide, is a kind of small molecular polypeptide with broad-spectrum antibacterial activity and immunoregulation activity produced by organism through induction, belongs to the inherent component of organism nonspecific defense system, is almost present in all life forms (insects, amphibians, birds, fishes, mammals, plants and human bodies), is composed of 12-60 amino acids, and has a relative molecular weight of about 4 kD. It has been found that antimicrobial peptides have rapid bactericidal and broad spectrum antimicrobial activity, including gram positive bacteria, gram negative bacteria, multidrug resistant bacteria, fungi, parasites, enveloped viruses and tumor cells, and by chemotaxis of dendritic cells, monocytes and memory T cells, bridge the innate and adaptive immune responses and enhance the body's ability to resist microbial infections by priming the adaptive immune system.
The production of antibacterial peptides is carried out in three ways: 1. natural extraction method. Because the natural extraction resources are limited and the cost is high, the method is mainly used for scientific research at present; 2. a genetic engineering method. The method can improve the yield through microbial and cell expression, can realize industrialization, and is the best way for large-scale production, but only can obtain natural L-type antibacterial peptide through the method, and the L-type antibacterial peptide is very sensitive to proteolysis, so the stability in vivo is very poor. 3. Chemical synthesis method. The method can be used for synthesizing amino acid during the production of antibacterial peptide.
beta-Casein-197 is an L-type polypeptide containing 17 amino acids and separated from human milk, has certain antibacterial activity, is a potential therapeutic antibacterial peptide, but has higher antimicrobial Minimum Inhibitory Concentration (MIC), namely lower antibacterial activity and stability, and does not meet the condition of becoming a clinical therapeutic drug.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the full-reverse D-type antibacterial peptide. The scheme is based on a beta-Casein-197 sequence, and a D-type transformation technology is adopted to synthesize a full reverse D-type beta-Casein-197, namely D-R-beta-Casein-197; the full-reverse D-type polypeptide not only uses D-type amino acid to replace L-type amino acid in the original polypeptide, but also overturns the polypeptide sequence; D-R-beta-Casein-197 not only has better tolerance to proteolysis, but also has greatly enhanced antibacterial activity, and can become a potential broad-spectrum antibacterial drug.
Another object of the present invention is to provide a method for preparing the antibacterial peptide.
It is still another object of the present invention to provide an antibacterial composition comprising such an antibacterial peptide.
It is still another object of the present invention to provide an antibacterial method using such antibacterial peptide.
It is a further object of the present invention to provide the use of such antimicrobial peptides in pharmaceutical or medical consumables.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an antibacterial peptide, the amino acid sequence of which is Val Pro Tyr Ile Gln His Thr Pro Asn Leu Leu Glu Gln Asn Leu Leu;
wherein at least one D-form amino acid is present in the amino acid sequence.
Preferably, the antibacterial peptide is a D-type amino acid.
A method for preparing the antibacterial peptide, which comprises the following steps:
1) taking Boc-leucine-PAM resin, deprotecting with TFA, dissolving another Boc-protected leucine in DMF and DIEA containing 0.5M HBTU, reacting at room temperature for 15min, and washing with DMF;
2) sequentially reacting with asparagine, glutamine, glutamic acid, leucine, asparagine, proline, threonine, histidine, glutamine, isoleucine, tyrosine, proline and valine according to the operation of the previous step;
3) adding DMF solution containing 10% of beta-mercaptoethanol and 5% of DIEA to wash twice, then removing Boc protection by using TFA, adding p-cresol serving as a protective agent with the same molar amount as the PAM resin by using hydrogen fluoride as a cutting agent, and reacting for 1h at 0 ℃;
4) removing hydrogen fluoride, precipitating with glacial ethyl ether, washing the precipitate with washing liquid to obtain crude polypeptide solution, purifying, and lyophilizing to obtain antibacterial peptide.
Preferably, in the preparation method, the washing solution comprises the following components: 50 wt% acetonitrile, 49.9 wt% water and 0.1 wt% TFA.
An antimicrobial composition comprising at least an antimicrobial peptide as described in any of the above.
An antibacterial method using an antibacterial peptide as described in any one of the above.
Use of an antimicrobial peptide as described in any of the above in medicine.
Use of an antimicrobial peptide as described in any of the above in a medical consumable.
The invention has the beneficial effects that: the scheme adopts a chemical synthesis means, can efficiently obtain a large amount of high-purity all-trans D-type antibacterial peptide, can ensure the integrity and purity of an antibacterial peptide sequence, and has higher thermal stability and protease degradation resistance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the purity and molecular weight of β -Casein-197.
FIG. 2 is a graph showing the purity and molecular weight of D-R-beta-Casein-197.
FIG. 3 is a diagram showing the secondary structure identification of β -Casein-197 and D-R- β -Casein-197.
FIG. 4 is a graph showing the measurement of thermal stability of β -Casein-197.
FIG. 5 is a graph showing the measurement of thermal stability of D-R-. beta. -Casein-197.
FIG. 6 is a graph comparing the survival rate of cells treated with different concentrations of the polypeptide.
FIG. 7 is a graph showing the measurement of the binding ability of β -Casein-197 to LPS.
FIG. 8 is a graph showing the measurement of the binding ability of D-R-. beta. -Casein-197 to LPS.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
Polypeptide sequence:
β-Casein-197：Leu Leu Asn Gln Glu Leu Leu Leu Asn Pro Thr His Gln IleTyr Pro Val；
D-beta-Casein-197: leu Leu Asn Gln Glu Leu Leu Leu Asn Pro Thr His GlnIle Tyr Pro Val; wherein each amino acid is a D-form amino acid;
D-R-beta-Casein-197: val Pro Tyr Ile Gln His Thr Pro Asn Leu Leu Leu GluGln Asn Leu Leu; wherein each amino acid is D-type amino acid.
The synthesis method of D-beta-Casein-197 is the same as that of D-R-beta-Casein-197, taking D-R-beta-Casein-197 as an example, the synthesis method of D-R-beta-Casein-197 is as follows:
1) weighing 0.2g Boc (tert-butyloxycarbonyl) -leucine (Leu) -PAM resin (polyacrylamide resin), deprotecting with TFA (trifluoroacetic acid), dissolving another Boc-protected 0.2mM leucine in 4mL of DMF (N, N-dimethylformamide) containing 0.5M HBTU (O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) and 1mL of N, N-Diisopropylethylamine (DIEA), reacting at room temperature for 15min, DMF washing;
2) sequentially reacting with 0.2mM of asparagine (Asn), glutamine (Gln), glutamic acid (Glu), leucine (Leu), asparagine (Asn), proline (Pro), threonine (Thr), histidine (His), glutamine (Gln), isoleucine (Ile), tyrosine (Tyr), proline (Pro) and valine (Val) according to the operation of the previous step;
3) adding 10mL of DMF solution containing 10% of beta-mercaptoethanol and 5% of DIEA for washing twice, removing a histidine Dnp protective group, removing Boc protection by using TFA, adding p-cresol (p-cresol) which is a protective agent and is equal to the PAM resin in molar quantity into hydrogen fluoride serving as a cutting agent, and reacting for 1h at 0 ℃;
4) removing hydrogen fluoride, precipitating with glacial ethyl ether, washing the precipitate with washing liquid to obtain crude polypeptide solution, purifying, and lyophilizing to obtain antibacterial peptide.
Among them, the washing liquid preferably contains, but is not limited to: 50 wt% acetonitrile, 49.9 wt% water and 0.1 wt% TFA.
Example 2
An antimicrobial composition comprising at least the antimicrobial peptide D-R- β -Casein-197 of example 1. Any other ingredients that do not conflict with D-R- β -Casein-197 may be included in the antimicrobial composition.
Example 3
An antibacterial method using the antibacterial peptide D-R- β -Casein-197 in example 1.
Example 4
An application of the antibacterial peptide D-R-beta-Casein-197 in example 1 in medicine.
Example 5
An application of the antibacterial peptide D-R-beta-Casein-197 in the embodiment 1 in a medical consumable.
Characterization test:
1.1 high Performance liquid chromatography and Mass Spectrometry characterization
The purity of the product is detected by adopting a liquid phase in the preparation process, and the chromatographic conditions are as follows:
a chromatographic column: XBridge BEH Shield RP18Column,
5 μm,4.6mm X250 mm; mobile phase: 5-65% (CH)3CN) gradient elution, mobile phase a: ddH2O(0.1％TFA)，B：CH3CN (0.1% TFA); column temperature: 40 ℃; a detector: DAD (λ 214nm and 280 nm); flow rate: 1 mL/min.
The molecular weight of each product was determined by mass spectrometry (ESI-MS).
1.2 high performance liquid chromatography purification
A chromatographic column: xbridge Peptide BEH C18 OBD Prep Column,
10 μm,19mm X250 mm; mobile phase 15-40% (CH)3CN) gradient elution, mobile phase a: ddH2O(0.1％TFA)，B：CH3CN (0.1% TFA); column temperature: 50 ℃; a detector: DAD (λ 214nm and 280 nm); flow rate: 40mL/min。、
FIG. 1 is a graph showing the purity and molecular weight characterization of β -Casein-197; FIG. 2 is a graph showing the purity and molecular weight of D-R-beta-Casein-197. As can be seen from FIGS. 1 and 2, through chemical synthesis and purification, we obtained beta-Casein-197 and D-R-beta-Casein-197 with actual molecular weights consistent with theoretical molecular weights, and the purities thereof were as high as 95% or more.
1.3 Secondary Structure determination
First, the polypeptide was folded, 10mg of the polypeptide was dissolved in 1ml of 6M guanidine hydrochloride, and 5ml of 20mM PB (sodium phosphate buffer: 23.02g Na per liter solution) was rapidly added2HPO4And 4.558g NaH2PO4pH7.4), and dialyzed against 2L of 20mM PB 3 times (10 mM of TCEP (tris (2-carboxyethyl) phosphine) in the dialysate).
After removing residual traces of trifluoroacetic acid by overnight dialysis, the polypeptide concentration was adjusted to 20. mu.M, and circular dichroism was measured at room temperature in a measuring dish having a diameter of 0.1 cm. The wavelength scanning range is 190nm to 260nm, and the scanning speed and the resolution are respectively 10nm/min and 1 nm. The resulting ellipticity raw data were converted to average residue molar ellipticity ([ theta ] R) according to the following formula:
[θ]R＝θ/(10×C×N×I)
wherein theta is ellipticity (millidegree), C is polypeptide molar concentration (M), I is measuring vessel diameter width (cm), and N is the number of polypeptide residues. The wavelength scan range is plotted as the abscissa and the average residue molar ellipticity is plotted as the ordinate to plot a circular dichroism plot of the polypeptide.
FIG. 3 is a diagram showing the secondary structure identification of β -Casein-197 and D-R- β -Casein-197; as can be seen from FIG. 3, the characteristic peaks at 192nm, 208nm and 222nm of beta-Casein-197 and D-R-beta-Casein-197 appear after 50% TFE addition, i.e., under the hydrophobic environment simulating microbial membranes or under the environment of negatively charged prokaryotic cells, and the spectra show that the two are both alpha helical structures, and D-R-beta-Casein-197 is a mirror image of beta-Casein-197.
1.4 measurement of thermal stability
As with the circular dichroism chromatogram characterization experiment, the polypeptide is first folded in 1xPBS, 10% glycerol, 200mM L-Arginine, pH 7.4.
The thermal stability was measured using a J815 circular dichroism chromatograph, Jasco, Japan, and 10. mu.M of the sample polypeptide was assayed in a buffer. The prepared 2.5 ml of PBS solution was loaded into a 3ml quartz cuvette with a rotator. The CD circular dichroism spectrum signal at 222nm at 25 ℃ to 90 ℃ was measured at a rate of 1 ℃ per minute with constant stirring at 360 rpm. And after heating every minute, waiting for 20 seconds to fully mix the solution, stabilizing the signal at the temperature, and then sampling and integrating for 16 seconds to obtain a signal value of a change point. The temperature and the collected CD signal data are automatically processed by control software provided by JASCO. The CD signal at each temperature sample is obtained.
FIG. 4 is a chart of determination of thermal stability of β -Casein-197, Tm48.7 deg.C, and FIG. 5 is the thermal stability determination chart of D-R- β -Casein-197, TmAs can be seen from fig. 4 and 5, the thermal stability of the modified D-type holopolypeptide, i.e., D-R- β -Casein-197, was greatly improved.
1.5 determination of the bacteriostatic Activity
Inoculating each drug-resistant pathogenic strain (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Enterococcus faecalis, Acinetobacter baumannii) to MH culture medium, culturing at 37 ℃ for 24h-48h until visible colonies are observed, and selecting a single colony to inoculate in the MH culture medium at 37 ℃ for overnight culture. Inoculating overnight thallus into fresh MH broth culture medium, culturing until thallus is in logarithmic growth phase, correcting concentration to 0.5 McLeod turbidity standard with MH broth culture medium, and adjusting colony count of thallus to about 105About cfu/mL.
Antimicrobial peptide was dissolved in sterile distilled water to prepare a stock solution of 2000. mu.M, and then diluted in sterile MH broth medium in two-fold proportions to give final concentrations of 1000. mu.M, 500. mu.M, 250. mu.M, 125. mu.M, 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.8. mu.M, 3.9. mu.M, and 1.95. mu.M.
Adding 90 mu L of diluted bacteria liquid into 1-10 rows of a 96-well plate, adding no medicine into the bacteria liquid in the 11 th row as a positive control, adding no bacteria liquid into the culture medium in the 12 th row as a negative control, and then adding 10 mu L of gradient diluted antibacterial peptide into the 1-10 rows one by one. After being uniformly mixed, the mixture is cultured for 18-24 hours in a shaking incubator at 37 ℃. As a result of observation, when the positive control grows well and the negative control does not show turbidity, the lowest antibacterial peptide concentration which can inhibit the bacterial growth is observed by naked eyes and is taken as the minimum inhibitory concentration of the antibacterial peptide on pathogenic bacteria.
TABLE 1 comparison of antibacterial Activity of Polypeptides against pathogenic bacteria
As shown in Table 1, the MIC of D-R-beta-Casein-197 against various pathogenic bacteria is greatly reduced, that is, the antibacterial activity is greatly enhanced, compared with that of beta-Casein-197, and D-beta-Casein-197 has no antibacterial activity.
1.6 cytotoxicity assays
The cytotoxicity of the antibacterial peptide on human embryonic fibroblast MRC-5 is determined by adopting an MTT method.
Recovering MRC-5, culturing in DMEM containing 10% fetal calf serum at 37 deg.C and 5% CO2Changing culture solution every other day, using trypsin digestion method for cell passage, when the cell number is 80% full of culture bottle bottom, washing with PBS solution for 2 times, adding 2mL of 0.25% pancreatin digestive juice, adding DMEM culture solution containing fetal bovine serum after 3min to stop digestion, removing digestive juice, adding 3mL of cell culture solution to blow and beat cells to form suspension, adding 50 muL of cell suspension into each hole of 96-hole plate, the concentration is about 1 × 104cells/well.
After the cells are attached to the wall, adding different gradient diluted antibacterial peptide into each hole, taking the hole without the antibacterial peptide and the hole as negative control, taking the hole without the antibacterial peptide as positive control, and culturing for 20 h.
Adding 50 mu L of MTT into each well, culturing for 4h, adding 150 mu L of DMSO (dimethyl sulfoxide), shaking for 10min to dissolve crystals, measuring absorbance at 492nm by using a multifunctional microplate reader, and repeating the experiment three times.
FIG. 6 is a graph comparing the survival rate of cells treated with different concentrations of the polypeptide. As can be seen from FIG. 6, the cell survival rate decreased to a different extent with the increase of the concentration of the antimicrobial peptide, but even when the concentration of β -Casein-197 reached 120. mu.M, the cell survival rate was still over 80%, indicating that β -Casein-197 is not very toxic to mammalian cells, while at the same maximum concentration, D-R- β -Casein-197 is less toxic to mammalian cells, and the cell survival rate is over 90%, indicating that D-type polypeptide has a lower cytotoxicity than L-type polypeptide, and has the advantage of low toxicity.
1.7 determination of resistance to proteolysis
And (3) determining the bacteriostatic activity of the antibacterial peptide after being treated by different types of protease.
Respectively treating the antibacterial peptide with Trypsin (Trypsin), Pepsin (Pepsin), Papain (Papain) and proteinase K (protease K) solutions with reaction concentration of 1mg/mL under the condition of 37 ℃ water bath for 1h, and then determining whether the minimum inhibitory concentration of the antibacterial peptide treated by each proteinase is changed or not according to an antibacterial activity determination method, wherein a control group is a group which is not treated by the antibacterial peptide.
TABLE 2 stability of polypeptides under different protease treatments
As shown in Table 2, the antibacterial activity of both beta-Casein-197 and D-R-beta-Casein-197 is reduced after being treated by various proteases, but the antibacterial activity of D-R-beta-Casein-197 is still much higher than that of beta-Casein-197, which shows that D-R-beta-Casein-197 has better protease degradation resistance.
1.8 isothermal calorimetry (iTC) determination of binding of antimicrobial peptides to Lipopolysaccharide (LPS)
The main component of the cell outer wall of gram-negative bacteria is LPS, and the antibacterial peptide is combined with the LPS to destroy the structure of a bacterial membrane to cause apoptosis, so that the antibacterial activity of the antibacterial peptide is determined.
The binding capacity of the antibacterial peptide to Pseudomonas aeruginosa LPS was determined. Polypeptide concentration setting: the antimicrobial peptide concentration was 300. mu.M and the LPS concentration was 30. mu.M. Setting experimental parameters: total information 20, Cell temperature 25 deg.c, Reference power 15 μ Cal/sec, Initial delay 60s, and standing speed 1000 rpm. Setting titration parameters: the first Volume is 0.5 μ L, Duration is 1.0s, the last 19 volumes is 2 μ L, Spacing is set to 120s and Filter period is set to 2 s. Titration and data analysis: titration was started according to the above parameters, and after titration was completed, data was analyzed by origine 9.0 software, and curve was fitted to obtain Kd.
FIG. 7 is a graph showing the measurement of the binding ability of β -Casein-197 with LPS, with a binding constant of 10.5. mu.M, FIG. 8 is a graph showing the measurement of the binding ability of D-R- β -Casein-197 with LPS, with a binding constant of 51.5nM, which is different by 200-fold, indicating that D-R- β -Casein-197 has a stronger binding ability with LPS, and further explaining the reason for its higher antibacterial activity.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Polypeptide sequence:
β-Casein-197：Leu Leu Asn Gln Glu Leu Leu Leu Asn Pro Thr His Gln IleTyr Pro Val
D-β-Casein-197：Leu Leu Asn Gln Glu Leu Leu Leu Asn Pro Thr His GlnIle Tyr Pro Val
D-R-β-Casein-197：Val Pro Tyr Ile Gln His Thr Pro Asn Leu Leu Leu GluGln Asn Leu Leu
Claims (4)
1. an antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is Val Pro Tyr Ile Gln His Thr Pro AsnLeu Leu Leu Glu Gln Asn Leu Leu;
wherein, the amino acids in the amino acid sequence are D-type amino acids.
2. A method for preparing the antimicrobial peptide of claim 1, comprising the steps of:
1) taking Boc-leucine-PAM resin, deprotecting with TFA, dissolving another Boc-protected leucine in DMF and DIEA containing 0.5M HBTU, reacting at room temperature for 15min, and washing with DMF;
2) sequentially reacting with asparagine, glutamine, glutamic acid, leucine, asparagine, proline, threonine, histidine, glutamine, isoleucine, tyrosine, proline and valine according to the operation of the previous step;
3) adding DMF solution containing 10% of beta-mercaptoethanol and 5% of DIEA to wash twice, then removing Boc protection by using TFA, adding p-cresol serving as a protective agent with the same molar amount as the PAM resin by using hydrogen fluoride as a cutting agent, and reacting for 1h at 0 ℃;
4) removing hydrogen fluoride, precipitating with glacial ethyl ether, washing the precipitate with washing liquid to obtain crude polypeptide solution, purifying, and lyophilizing to obtain antibacterial peptide.
3. The method according to claim 2, wherein the washing liquid comprises: 50 wt% acetonitrile, 49.9 wt% water and 0.1 wt% TFA.
4. An antimicrobial composition comprising at least the antimicrobial peptide of claim 1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102079777A (en) * | 2009-11-26 | 2011-06-01 | 浙江海洋学院 | Artificially synthesized antimicrobial peptide, preparation method and application thereof |
| CN102719477A (en) * | 2012-06-29 | 2012-10-10 | 兰州大学 | Non-viral gene vector constructed on basis of N-terminal octadecane acylated antibacterial peptide |
| CN106488929A (en) * | 2014-05-09 | 2017-03-08 | 乌得勒支大学控股有限公司 | Novel C ATH2 derivant |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102079777A (en) * | 2009-11-26 | 2011-06-01 | 浙江海洋学院 | Artificially synthesized antimicrobial peptide, preparation method and application thereof |
| CN102719477A (en) * | 2012-06-29 | 2012-10-10 | 兰州大学 | Non-viral gene vector constructed on basis of N-terminal octadecane acylated antibacterial peptide |
| CN106488929A (en) * | 2014-05-09 | 2017-03-08 | 乌得勒支大学控股有限公司 | Novel C ATH2 derivant |
Non-Patent Citations (2)
| Title |
|---|
| Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk;Yanrong Fu et al;《AMB Expr》;20171231;第7卷(第119期) * |
| 紫花苜蓿的抗菌肽基因Rev4遗传转化;武慧等;《江苏农业科学》;20171231;第45卷(第7期);第44-46页 * |
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