CN101935344A - Chinese housefly antibacterial peptides 1 and method for separating and purifying same - Google Patents
Chinese housefly antibacterial peptides 1 and method for separating and purifying same Download PDFInfo
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Abstract
The invention discloses Chinese housefly antibacterial peptides 1 and a method for separating and purifying the same. The Chinese housefly antibacterial peptide 1 comprises five peptide segments. The method for separating and purifying the Chinese housefly antibacterial peptides comprises the following steps of: 1, performing immune induction of houseflies; 2, performing tissue homogenate of immune-induced houseflies to obtain sample solution containing antibacterial peptides; 3, performing dialysis concentration of the sample solution to obtain a concentrated sample; 4, performing ultra-centrifugal filtration of the concentrated sample to obtain the semi-purified antibacterial peptides; and 5, performing HPLC separation and purification of the semi-purified antibacterial peptides to obtain Chinese housefly antibacterial peptides with pure chromatograph. The Chinese housefly antibacterial peptide 1 of the invention is a micromolecular peptide newly found, the activity of the antibacterial peptide is higher, and the Chinese housefly antibacterial peptide 1 is a novel antibacterial medicament which has development potential and can resist multiple resistant bacteria.
Description
Technical field
The present invention relates to the biological antibacterial peptide field, relate in particular to a kind of musca vicina antibacterial peptide 1 and separation purification method thereof.
Background technology
Antibiotic widespread use, Resistant strain and multiple antibiotic resistant strain increase day by day, and clinical anti-infective therapy faces a severe challenge, seeks antibacterium, the antiviral of broad-spectrum high efficacy, remains a very difficult task.Fly mainly is grown in rotten thing, ight soil and rubbish etc. and contains in the severe environment of a large amount of bacteriums, and its body surface has many pathogenic bacterias, can propagate multiple disease.But himself can not cause death because of infection pathogen, and this explanation fly should have powerful immunizing power to pathogenic micro-organism, and can produce potent antimicrobial substance.
Biological antibacterial peptide has broad spectrum antibiotic activity, to G
+, G
-Bacterium and fungi all have anti-microbial activity, particularly multi-drug resistant bacteria are also had anti-microbial activity.The effect that also has antitumor cell is in addition compared with traditional microbiotic, promotes effects such as wound healing and immunomodulatory in addition, is the novel antibacterial medicine that a class has development potentiality.
Research is comparatively active in the world at present in this field, finds and can be purified into antibiotic spectrum width, antibacterial peptide that anti-microbial activity is strong will have the wide development application prospect.
Different antibacterial peptide molecular sizes, biological property has certain difference, and the purification technique of peptide class is complicated at present, separation and purification single peptide class technical difficulty bigger.In the research of antibacterial peptide, the foundation of antibacterial peptide separation purification method is a technological difficulties, set up a method that the result is reliable and stable, simple to operate, needs extensive work, repetition test.
Summary of the invention
For addressing the above problem, main purpose of the present invention is to provide a kind of musca vicina antibacterial peptide 1, and this antibacterial peptide has wide spectrum, anti-microbial activity efficiently.
Second purpose of the present invention be to provide a kind of in the musca vicina body method of separation and purification musca vicina antibacterial peptide 1.
For achieving the above object, the present invention adopts following technical scheme:
A kind of musca vicina antibacterial peptide 1 comprises 5 peptide sections, and the aminoacid sequence of described 5 peptide sections is respectively shown in SEQID NO:1 to SEQ ID NO:5.
A kind of separation purification method of musca vicina antibacterial peptide 1 may further comprise the steps:
1) housefly is carried out immune induction, make housefly produce antibacterial peptide;
2) housefly behind the immune induction is carried out tissue homogenate, get the sample solution that contains antibacterial peptide;
3) sample solution that will contain antibacterial peptide is dialysed concentratedly, obtains spissated sample;
4) spissated sample is carried out 3TGCOO ultrafiltration post and carry out the ultracentrifugation filtration, obtain half pure product antibacterial peptide;
5) half pure product antibacterial peptide is carried out the HPLC separation and purification, obtain chromatographically pure musca vicina antibacterial peptide 1.
Described step 1) is specially: make housefly anesthesia earlier, have the saturating body wall of stainless steel acupuncture of bacterial suspension then, make its recovery again, stab the back and raise 24h for 28～30 ℃ in temperature, induce by the damage infection immunity, make housefly produce antibacterial peptide.
Described bacterial suspension is colon bacillus suspension, Pseudomonas aeruginosa suspension or streptococcus aureus suspension.
Described step 3) is specially: at first will contain 4 ℃ of centrifugal 60min of following 60000g of sample solution of antibacterial peptide, and remove precipitation and upper strata buoyant lipid layer; Getting the supernatant liquor molecular weight cutoff value of packing into again is in the dialysis tubing of 1500U, is embedded in the polyoxyethylene glycol liquid of dialysing under 4 ℃.
Described step 4) is specially: the centrifugal 30min of spissated sample 60000g that dialyses, get supernatant with UFC3TGCOO ultrafiltration post, with the 60000g centrifugal ultrafiltration.
Described step 5) is specially: the sample ultrafiltrated adopts RT-HPLC semipreparative column purifying, and the semipreparative column applied sample amount is 1.0ml～2.0ml, and flow velocity is 1.0ml/min, detects under the wavelength at 214nm, when beginning the peak, carries out the part of sample and collects.Collection when retention time is 18.008min is a chromatographically pure musca vicina antibacterial peptide 1
Musca vicina antibacterial peptide 1 with method for preparing.
Musca vicina antibacterial peptide 1 of the present invention is a kind of small-molecular peptides of latest find, and the activity of antibacterial peptide is stronger, and non-specific ground of energy antibacterium is that a class has development potentiality, multi-drug resistant bacteria is had the novel antibacterial medicine of resistant function.
Musca vicina antibacterial peptide 1 separation purification method of the present invention, have simple and easy to do, advantages such as stability, good reproducibility, the anti-microbial activity of different batches purifying is measured content of peptides with the Lowry method, its protein concn scope is 0.20g～0.25g/L, is a kind of brand-new antibacterial peptide separating and purifying technology with practical value.
Figure of description
Fig. 1 is that 3TGCOO ultrafiltration post carries out the ultracentrifugation filtration, obtains the RP-HPLC color atlas of the musca vicina anti-microbial activity component of half pure product;
Fig. 2 is that the RP-HPLC analytical column of purifying musca vicina antibacterial peptide 1 is analyzed color atlas;
Fig. 3 is the anti-microbial activity test-results figure of 1 pair of Pseudomonas aeruginosa of musca vicina antibacterial peptide of Sephadex G50 chromatography collection, 15 (((90min～95min) is the sample that different retention time are collected to 85min～90min), 17 to 80min～85min), 16, application of sample amount: 20 μ g protein/scraps of paper;
Fig. 4 is the mass spectrum of the peptide section 1 of musca vicina antibacterial peptide 1 trypsin hydrolyzing;
Fig. 5 is the mass spectrum of the peptide section 2 of musca vicina antibacterial peptide 1 trypsin hydrolyzing;
Fig. 6 is musca vicina antibacterial peptide 1Tricine SDS one polyacrylamide gel electrophoresis figure as a result.
Embodiment
1 one kinds of musca vicina antibacterial peptides 1 of embodiment
Musca vicina antibacterial peptide 1 obtains 5 enzymatic fragments behind trypsin hydrolyzing, enzymatic fragment carries out mass spectroscopy with the Nano-ESI-MS/MS technology, the mass spectrum information that determines is carried out sequential analysis by the special software MasSeq of Micromass, and the aminoacid sequence that obtains 5 peptide sections is respectively shown in SEQ ID NO:1-5:
(1)YDNVNLDELLNSDR(TOF?MSMS?840.47ES+，Query?4)
Tyr-Asp-Asn-Val-Asn-Leu-Asp-Glu-Leu-Leu-Asn-Ser-Asp-Arg
(2)YDLLNVDEEVRFR(TOF?MSMS?826.95ES+，Query?5)
Tyr-Asp-Leu-Leu-Asn-Val-Asp-Glu-Leu-Val-Arg-Phe-Arg
(3)QPNLYYNK(TOF?MSMS?520.30ES+，Query?2)
Gln-Pro-Asn-Leu-Tyr-Tyr-Asn-Lys
(4)PNNVYYTK(TOF?MSMS?499.76ES+，Query?1)
Pro-Asn-Asn-Val-Tyr-Tyr-Thr-Lys
(5)DYPNNVYYTK(TOF?MSMS?639.34ES+，Query?3)
Asp-Tyr-Pro-Asn-Asn-Val-Tyr-Tyr-Thr-Lys
Mass spectrum goes out peptide section sequence by the special software MasSeq direct derivation of Micromass after the special software MaxEnt3 of Micromass handles.Inquire about the NCBInr database with Spectrum List by Mascot, find no the protein that sequence with it is complementary by retrieval, can prove separation and purification of the present invention to house fly antibiotic peptide be a new antibacterial peptide, called after house fly antibiotic peptide 1 (Musca domestica antibacterial peptide 1, MdAP1).The mass spectrum of peptide section 1 is seen Fig. 4, and the mass spectrum of peptide section 2 is seen Fig. 5.
The separation purification method of 2 one kinds of musca vicina antibacterial peptides 1 of embodiment
May further comprise the steps:
1. immune induction
Housefly is divided into test group and control group, and control group does not damage induces processing.Test group is placed on the culture dish that fills ether in the fly cage, makes fly anesthesia, with the saturating body wall of stainless steel acupuncture that speckles with bacterial suspension, puts into another cage immediately and makes its recovery then.Stab the back and raise 24h for 28～30 ℃ in temperature.Induce by the damage infection immunity, make housefly produce antibacterial peptide.Bacterium is colon bacillus, Pseudomonas aeruginosa or streptococcus aureus,
2. tissue homogenate
Housefly 1.0g adds the mixed of liquid nitrogen 7g, grind with mortar rapidly, or larva is put-80 ℃ of freeze overnight, taking-up is put and grind to form thin foam shape in the mortar under ice bath, add a small amount of 4 ℃ of buffer A (containing urea 2.0mol/L, 0.2mol/L pH6.8Tris-HCl) and continue to grind to form pasty state, add the ratio mixing of 10ml bufferA with the 1g tissue, 4 ℃ of centrifugal 60min of following 60000g, it is standby to get supernatant liquor-80 ℃ preservation.
3. dialysis concentrates
The sample solution of-80 ℃ of storages is taken out, melt 4 ℃ of centrifugal 60min of following 60000g under the room temperature, remove precipitation and upper strata buoyant lipid layer, getting the supernatant liquor molecular weight cutoff value of packing into is in the dialysis tubing of 1500U, is embedded in the polyoxyethylene glycol liquid of dialysing under 4 ℃.
4. the centrifugal 30min of spissated sample 60000g that dialyses uses UFC 3TGCOO ultrafiltration post, again with the 60000g centrifugal ultrafiltration.
5.HPLC separation and purification
5.1 chromatographic column is loaded onto pre-column, inserts the tubing system of 1100 series of high efficiency liquid chromatographs, main frame should be in opened condition when connecting, and flow rate of mobile phase is set to 0.3ml/min, in order to avoid in the air admission post, the two ends pipeline should connect closely in case leakage.
5.2 chromatographic column balance, chromatographic column is a Precerving liquid with 100% methyl alcohol, time spent 100% methyl alcohol is the moving phase flushing, monitor under the 214nm wavelength to the baseline level off, methanol concentration progressively is reduced to 0% by 100% simultaneously, distilled water washes to baseline when steady, PROTEIN PAR 300 chromatographic columns are used buffer B (0.2mol/L pH6.8PBS instead, PMSF 0.2mmol/L, EDTA 0.05mol/L) makes moving phase, Lichrospher C-18 chromatographic column is made moving phase with buffer C (0.2mol/L pH6.8PBS), and concentration is incremented to 100% gradually from 30%, goes up sample when steady to baseline.
5.3 last sample and wash-out, the half pure product anti-microbial activity component that Sephadex G50 chromatography is collected is with the further separation and purification of semipreparative column.The purifying anti-microbial activity component of collecting is analyzed with analytical column again.According to chromatographic column model different decision applied sample amount and flow velocity, analytical column is generally 10 μ l～100 μ l, and flow velocity is 0.5ml/min; Semipreparative column is 1.0ml～2.0ml, and flow velocity is 1.0ml/min, and the detection wavelength is 214nm.
5.4 sample collection, according to the chromatographic peak in the chromatographic curve, substep is collected the sample at each peak, and writing time.
5.5 sample concentration: the sample of will substep collecting carries out vacuum lyophilization, adds the distilled water dissolving with 1/10 ratio of sample volume before the lyophilize, carry out protein quantification with the Lowry method after, measure usefulness for anti-microbial activity.
6. sample concentration drying
With the sample vacuum lyophilization of collecting, add the distilled water dissolving after concentrating, making volume is 1/10～1/20 of original volume, does anti-microbial activity and detects, the moving phase of identical cycles of concentration is done the reagent contrast.
The housefly tissue extract is through ultrafiltration, resulting anti-microbial activity composition, and through RT-HPLC semipreparative column purifying, each component separating is respond well.Each peak collection is carried out the anti-microbial activity test, and the collection when retention time is 18.008min as a result has tangible anti-microbial activity (Fig. 1).This component is analyzed with the RT-HPLC analytical column again, has only an absorption peak in the color atlas, and retention time is 36.140min, has reached chromatographically pure (Fig. 2).
After musca vicina antibacterial peptide 1 lyophilize that will obtain through above-mentioned purification process 100: 1 by volume heavy molten, get 15 μ l and carry out Tricine SDS-PAGE electrophoresis, this antibacterial peptide molecular weight is 9.0kU, the results are shown in Figure 6.
Embodiment 3 musca vicina antibacterial peptides 1 molecular structure is measured
(1) musca vicina antibacterial peptide 1 is carried out Tricine SDS-polyacrylamide gel electrophoresis:
Adopt discontinuous system, resolving gel concentration 18%, squeegee concentration 8% concentrates gum concentration 4%, 1.5 volts/cm of voltage, behind the electrophoresis 80min, changing voltage is 10 volts/cm electrophoresis 120min again.Methyl alcohol-the alcohol mixeding liquid of the R-250 Xylene Brilliant Cyanine G with 0.2% (50% methyl alcohol+10% ethanol) dyeing, destainer is methyl alcohol-alcohol mixeding liquid (50% methyl alcohol+10% ethanol).
(2) amino acid sequencing:
The PAGE blob of viscose decolouring of antibacterial peptide is placed on dry 20min in the vacuum drier, adds the trypsin solution of 5～1010mg/L then, behind the placement 40min, adds 10 μ l 25mmol/L ammonium bicarbonate buffers in 4 ℃ of refrigerators, is incubated overnight in 37 ℃.Extract enzyme with 5% trifluoroacetic acid (TFA) in 40 ℃ and cut the peptide section; Use vinyl cyanide (ACN)-TFA solution in 30 ℃ of extractions again with volume; Use the ACN supersound extraction at last once; Merge No. 3 times extracting solution, the vacuum drier drying.Dried sample dissolves with TFA solution.With being equipped with capillary liquid chromatography and receiving liter the mass spectrum that rises electron spray(ES)-quadrupole-flight time tandem mass spectrum (Nano-ESI-Q-TOF2-MS/MS) technical measurement peptide section of receiving in spraying source, determine the aminoacid sequence of house fly antibiotic peptide.
(3) searching database:
The mass spectrum of measuring goes out peptide section sequence with the special software MasSeq direct derivation of Micromass, inquires about ncbi database with Spectrum List by Mascot according to the aminoacid sequence of peptide section, establishes its primary structure.
Experimental example 1 musca vicina antibacterial peptide 1 anti-microbial activity is measured
Adopt flat band method,, adopt the LB substratum with reference to national Clinical Laboratory working specification, with inoculation in the LB substratum, 35 ℃ are cultured to logarithmic phase, are diluted to stroke-physiological saline solution to be equivalent to 0.5 Maxwell standard unit (Mc Farland standard unit is equivalent to 1.5 * 10
8CFU/ml), isolating each composition of SephalexG50 chromatography and high performance liquid chromatography (HPLC) is the composition that K-B method (Kirby-Bauer method) detects anti-microbial activity with agar diffusion method of the paper, use micro-dilution method (microdilution test) to measure minimal inhibitory concentration (minimal inhibitory concentration again, MIC), penbritin compares.By measuring the antibacterial vigor of antibacterial circle diameter qualitative detection.
Experimental result shows that the house fly antibiotic peptide that is separated to has tangible broad spectrum antibiotic activity, and gram negative bacilli, gram positive coccus and fungi are all had restraining effect.(minimal inhibitory concentration is MIC) between 2.22 μ mol/L～13.33 μ mol/L (20 μ g/ml～120 μ g/ml) for the minimal inhibitory concentration of 1 pair of 11 kinds of clinical isolates strain of this musca vicina antibacterial peptide.House fly antibiotic peptide is stronger relatively to colibacillary anti-microbial activity, and to the anti-microbial activities of positive coccus and other enterobacterias relative a little less than, the anti-microbial activity of dialogue candidiasis is the most weak.
At the penbritin control group, penbritin to the MIC of 10 kinds of clinical isolates strains between 21.54 μ mol/L～＞86.16 μ mol/L (8 μ g/ml～＞32 μ g/ml).Clinical isolates strain salmonella typhi, shigella flexneri sensitivity, quick in colon bacillus, special-shaped citrobacter, the enterobacter cloacae, Pseudomonas aeruginosa, proteus vulgaris, Klebsiella pneumonia, staphylococcus epidermidis and methicillin-resistant staphylococcus aureus (MRSA) resistance.The anti-microbial activity of antibacterial peptide 1 the results are shown in Figure 3, and antimicrobial spectrum the results are shown in Table 1.
Table 1 house fly antibiotic peptide is to the MIC of clinical isolates strain
The anti-infective curative effect of 1 pair of mouse surface of a wound of experimental example 2 musca vicina antibacterial peptides
1. the foundation of enterobacter cloacae infection model
30 of healthy male cleaning level ICR mouse, body weight (21 ± 2) * 10
-3Kg.1d intends the field of operation skin depilatory with 80g/L sodium sulphite with mouse back before the modeling, use etherization before the operation, fixing, in mouse back backbone both sides, excise square holostrome skin and the subcutis of 1cm * 1cm with scalpel, on the surface of a wound, evenly smear enterobacter cloacae bacterium liquid 0.1ml (1 * 10
8CFU/ml), wrap up with sterile gauze.
2. experiment grouping
Adopt the table of random number method that 30 mouse are divided into control group, mafenide group, antibacterial peptide group at random, 10 every group, sub-cage rearing is in the laboratory of cleaning, dry, room temperature (25 ± 1) ℃.Hinder back 2h and respectively organize the mouse surface of a wound and wait 4 layers of soak of sterilization aseptic filter paper of oozing physiological saline, 100g/L mafenide solution and 0.1g/L antibacterial peptide solution with containing respectively, wrap up with sterile gauze again, change dressings every day 1 time.Observe the mouse traumatic infection situation and the mental status, get blood with back 3 days mouse tail points of wound before the wound and observe the blood picture variation; Hemoculture is done in fast dead mouse blood drawing, calculated the survival rate of mouse behind the 3d.
3. the muscle tissue bacteria quantified detects under white blood cell count(WBC) and the scab
Hindered the back the 3rd day, the cervical vertebra dislocation method is put to death and is respectively organized survival mice, under aseptic technique, get muscle tissue under the scab, weigh, add isotonic saline solution 1ml, homogenate, by being seeded in behind the serial dilution on the agar plate, after placing 37 ℃ of incubators to hatch 24h, the counting colony number also calculates the colony-forming unit of every gram tissue, and the selection colonies typical is identified.
4. result
4.1 anti-infectious function to infected animal model
Experimental result shows that control group mice is compared other two groups of activities and reduced, and wound secretion is many; Mafenide group and antibacterial peptide are formed the face incrustation, drying, and secretory product is few.Hindering proleukocyte is (9.65 ± 1.87) * 109/L, hinders back 3d white blood cell count(WBC) and all reduces.Control group (3.36 ± 0.45) * 109/L obviously is less than mafenide group (4.29 ± 0.33) * 109/L and antibacterial peptide group (4.13 ± 0.26) * 109/L (all P＜0.05), and there was no significant difference (P＞0.05) between back two groups sees Table 1.The control group mice survival is 6 after three days, 9 of antibacterial peptide group mouse survivals, and 9 of mafenide group survivals, survival rate is respectively 60%, 90% and 90%, and control group is significantly less than other two groups (all P＜0.05), and dead mouse hemoculture result is enterobacter cloacae.
4.2 the result of quantification of subeschar bacteria
The muscle tissue bacterial cultures is mainly enterobacter cloacae through morphology, growth characteristics and biochemical identification under the scab.Scab undertissue is carried out bacteria quantified, and the result shows: control group (504 ± 338) * 103CFU/g, mafenide group (87 ± 31) * 102CFU/g, antibacterial peptide group (99 ± 32) * 102CFU/g; Mafenide group and antibacterial peptide group are starkly lower than control group (P＜0.01), and do not have marked difference (P＞0.05) between antibacterial peptide group and the mafenide group.See Table 1.
Bacterial count under three days whole blood white corpuscles and the scab after the table 1 mouse wound
● P＜0.05 and control group ratio; * P＜0.01 and control group ratio
The toxic action of 1 pair of mouse of embodiment 3 musca vicina antibacterial peptides
Musca vicina antibacterial peptide 1 solution is through sterile filtration, and 100 ℃ of sterilization pneumoretroperitoneums are injected 100 μ L, observes toxic action to mouse liver function and renal function (experiment has proved that newfound house fly antibiotic peptide 1 can anti-100 ℃ of water-baths boils the 10min anti-microbial activity and do not reduce).
Pluck eyeball and extract blood testing ALT, AST, TBill, DBil, BUN, Cr content, see Table 2, statistical study control group and test group there was no significant difference illustrate that 1 pair of mouse of musca vicina antibacterial peptide does not have the effect of tangible liver renal toxicity.
The influence of 1 pair of Mouse Liver renal function index of table 2 musca vicina antibacterial peptide
Claims (8)
1. a musca vicina antibacterial peptide 1 is characterized in that, comprises 5 peptide sections, and the aminoacid sequence of described 5 peptide sections is respectively shown in SEQ ID NO:1 to SEQ ID NO:5.
2. the separation purification method of a musca vicina antibacterial peptide 1 may further comprise the steps:
1) housefly is carried out immune induction, make housefly produce antibacterial peptide;
2) housefly behind the immune induction is carried out tissue homogenate, get the sample solution that contains antibacterial peptide;
3) sample solution that will contain antibacterial peptide is dialysed concentratedly, obtains spissated sample;
4) spissated sample is carried out UFC 3TGCOO ultrafiltration post ultracentrifugation and filter, obtain half pure product antibacterial peptide;
5) half pure product antibacterial peptide is carried out the HPLC separation and purification, obtain chromatographically pure musca vicina antibacterial peptide 1.
3. the separation purification method of a kind of musca vicina antibacterial peptide 1 according to claim 2, it is characterized in that, described step 1) is specially: make housefly anesthesia earlier, the saturating body wall of stainless steel acupuncture that has bacterial suspension then, make its recovery again, stab the back and raise 24h for 28～30 ℃, induce, make housefly produce antibacterial peptide by the damage infection immunity in temperature.
4. the separation purification method of a kind of musca vicina antibacterial peptide 1 according to claim 3 is characterized in that, described bacterial suspension is colon bacillus suspension, Pseudomonas aeruginosa suspension or streptococcus aureus suspension.
5. the separation purification method of a kind of musca vicina antibacterial peptide 1 according to claim 2 is characterized in that, described step 3) is specially: at first will contain 4 ℃ of centrifugal 60min of following 60000g of sample solution of antibacterial peptide, and remove precipitation and upper strata buoyant lipid layer; Getting the supernatant liquor molecular weight cutoff value of packing into again is in the dialysis tubing of 1500U, is embedded in the polyoxyethylene glycol liquid of dialysing under 4 ℃.
6. the separation purification method of a kind of musca vicina antibacterial peptide 1 according to claim 2, it is characterized in that described step 4) is specially: adopt UFC 3TGCOO ultrafiltration post to carry out ultracentrifugation and filter, detect under the wavelength at 214nm, when beginning the peak, carry out the part of sample and collect.
7. the separation purification method of a kind of musca vicina antibacterial peptide 1 according to claim 2, it is characterized in that, described step 5) is specially: adopt RT-HPLC semipreparative column purifying, semipreparative column application of sample amount is 1.0ml～2.0ml, flow velocity is 1.0ml/min, the detection wavelength is 214nm, and the collection when retention time is 18.008min is a chromatographically pure musca vicina antibacterial peptide 1.
8. with any musca vicina antibacterial peptide 1 that described method prepares of claim 2-7.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321164A (en) * | 2011-05-25 | 2012-01-18 | 绍兴市曙光科技开发有限公司 | Preparation method and application of insect natural antibacterial peptide products |
| CN106432404A (en) * | 2016-10-25 | 2017-02-22 | 蚌埠学院 | Method for separating antibacterial peptides |
| CN113528600A (en) * | 2021-07-08 | 2021-10-22 | 石河子大学 | Method for preparing antibacterial peptide by inducing fly maggots with salmonella |
| CN114634553A (en) * | 2022-04-27 | 2022-06-17 | 贵州医科大学 | Cationic peptide C9 and application thereof |
-
2010
- 2010-08-04 CN CN2010102448870A patent/CN101935344A/en active Pending
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| Title |
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| 周义文: "家蝇抗菌肽分离纯化抗菌活性及分子结构研究", 《中国优秀博硕士学位论文全文数据库（博士）医药卫生科技辑》 * |
| 周义文等: "天然抗菌肽分离纯化方法的建立", 《国际检验医学杂志》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321164A (en) * | 2011-05-25 | 2012-01-18 | 绍兴市曙光科技开发有限公司 | Preparation method and application of insect natural antibacterial peptide products |
| CN106432404A (en) * | 2016-10-25 | 2017-02-22 | 蚌埠学院 | Method for separating antibacterial peptides |
| CN113528600A (en) * | 2021-07-08 | 2021-10-22 | 石河子大学 | Method for preparing antibacterial peptide by inducing fly maggots with salmonella |
| CN114634553A (en) * | 2022-04-27 | 2022-06-17 | 贵州医科大学 | Cationic peptide C9 and application thereof |
| CN114634553B (en) * | 2022-04-27 | 2024-03-08 | 贵州医科大学 | Cationic peptide C9 and application thereof |
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Application publication date: 20110105 |