CN101054408A - Method for separating housefly secretion type antibacterial peptide, product and application thereof - Google Patents
Method for separating housefly secretion type antibacterial peptide, product and application thereof Download PDFInfo
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Abstract
The present invention discloses a housefly-secreted antibacterial peptide separation method, its product, and uses. The method comprise: dialyzing the antibacterial peptide crude extract, carrying out solid phase extraction of dialysis liquid, ultrafiltering for separating elution ingredient, purifying two antibacterial activity ingredient using anti-phase high performance liquid phase chromatography, obtaining housefly-secreted antibacterial peptide. The present invention provides a convenient and highly effective and quick method for extracting antibacterial peptide of high protein yield, stable antibacterial activity and high activity which shorten the preparation time of antibacterial peptide, promote the separation and purification efficiency. Test prove that the antibacterial peptide can promote specific and nonspecific immune function, prevent bacterial infection, and establish a foundation for preparing drug, health product and animal feedstuff or preservative.
Description
Technical field: the present invention relates to a kind of separation method and products thereof of housefly secretion type antibacterial peptide and use, belong to the separating and purifying technology field of insect antimicrobial peptide.
Background technology: insect all can directly contact every day, eat, suck thousands of pathogenic micro-organism and be unlikely to suffer from infectious diseases, and the ability of the external pathogenic microorganism of this opposing mainly is to be achieved by autarcetic defense mechanism.Antibacterial peptide is the important component part of natural immunology defense in all species, and this class polypeptide can be intrinsic expression, also can be by pathogenic micro-organism and product abduction delivering thereof.
Insect antimicrobial peptide is that the molecule amount is little, the polypeptide of good water solubility, stable, has a broad antifungal spectrum and antibacterial mechanisms uniqueness, is the material that a class that Immune System produces is resisted extraneous pathogenic infection.Many scientists think that insect has surprising natural immunity, and with the main component of antibacterial peptide as its host defense mechanism.Antibacterial peptide also has the effect of antitumor cell, and the normal cell of human body is not had destruction except that nonspecific resisting pathogenic microbes is arranged.Therefore, the development and utilization of antibacterial peptide is a focus in the present resisting pathogenic microbes drug development.
Housefly is the important medical insect, can the multiple pathogenic bacteria of mechanical inoculation people, animal such as bacterium, virus and parasitic ovum etc.Report has been separated to multiple antibacterium, fungi, protozoacide biological activity antibacterial protein/polypeptide from its larva hemolymph both at home and abroad at present, has found the material of anti-phytopathogen, plant root-knot nematode and the parasitic ovum of wide spectrum in the external secretory product of larva.But these researchs focus mostly in the inducing and active detecting stage of antibacterial peptide, and external varying environment does not all have report to the influence of its anti-microbial activity.At present, the purification process of antibacterial peptide uses the gel chromatography of character gentleness more, but this method required time is longer, and the yield of each applied sample amount and target protein is less.
Summary of the invention:
The objective of the invention is to: a kind of separation method and products thereof of housefly secretion type antibacterial peptide is provided and uses.The present invention adopts dialysis, Solid-Phase Extraction, ultrafiltration and RPLC method to be separated to the peptide matters of tool broad spectrum antibiotic activity from Musca domestica larva secretory product, the antibacterial peptide preparation time is shortened, improved the efficient of separation and purification, and this antibacterial peptide its anti-microbial activity in external different environment has stronger stability, can by destroy the bacterial cell membrane permeability, suppress the bacterial body division, obstruction is relevant with cell fission, and some proteinic approach such as synthesize is brought into play anti-microbial effect; Have the effect that improves specificity and non-specific immune function, infectation of bacteria is had the certain protection effect.
The present invention is achieved in that the separation method of housefly secretion type antibacterial peptide is: the antibacterial peptide crude extract is dialysed, dialyzed solution is through Solid-Phase Extraction, elution fraction is through ultra-filtration and separation, active ingredient further is purified into two anti-microbial activity components with RPLC, promptly gets housefly secretion type antibacterial peptide.
Described antibacterial peptide crude extract, i.e. being prepared as of Musca domestica larva ectocrine crude extract: get three age Musca domestica larva place clean large beaker, fully clean and be dipped in an amount of distilled water jolting frequently behind the body surface 4 hours; Draw the centrifugal 10000g * 10min of immersion liquid, the supernatant liquor lyophilize concentrates, and promptly gets the antibacterial peptide crude extract, and 4 ℃ of preservations are standby.
Concrete separation method is: (1) dialysis: with spissated antibacterial peptide crude extract molecular weight cut-off is 3500 daltonian dialysis tubing dialysed overnight, changes twice of outer liquid therebetween; Get the sterile filters filtration sterilization of interior liquid with 0.22 μ m ,-20 ℃ of preservations are standby; (2) Solid-Phase Extraction: get Sep-pak C on the antibacterial peptide crude extract of dialysis back
18The Cartridge solid phase extraction column contains 0.05% trifluoroacetic acid (TFA) the solution stepwise elution of 0%～80% methyl alcohol successively with 5ml, flow velocity is 1ml/min; After methyl alcohol (ACN) and trifluoroacetic acid (TFA) were removed in each elution fraction lyophilize, ultrapure water dissolved, and detected the anti-microbial activity of this elution fraction; Collect, concentrate, freeze-drying has active component, the ultrapure water dissolving, the centrifugal 10min of 12000rpm, it is standby to collect supernatant liquor-20 ℃ preservation; (3) ultrafiltration: will be added in the ultrafiltration pipe that molecular weight cut-off is 3KD after the merging of Solid-Phase Extraction active ingredient, the centrifugal 30min of 5000 * g separates; The component that continuation will not see through the ultrafiltration pipe is added in the ultrafiltration pipe that molecular weight cut-off is 30KD, the same centrifugal, collect ultrafiltration pipe neutralization pipe liquid down respectively, with active substance be divided into three part: molecular weight＞30KD, 3～30KD,＜3KD, get each component and detect protein concentration and anti-microbial activity; (4) RPLC purifying: active ingredient is with 0.22 μ m filtering with microporous membrane, C on the 100 μ l after the ultrafiltration
18Reverse-phase chromatographic column, mobile phase A: 0.1% trifluoroacetic acid (the TFA)-aqueous solution; B:0.1% trifluoroacetic acid (TFA)-methyl alcohol (ACN) solution; Chromatographic column is with 5% methyl alcohol (CAN)-0.1% trifluoroacetic acid (TFA) solution equilibria, and elution requirement is: own nitrile concentration rises to 60% flow velocity 1ml/min from 5% in the gradient elution, 30min, and the manual 214nm wavelength elution peak of collecting promptly obtains two antibacterial peptide components.
More than used Sep-pak C
18The Cartridge solid phase extraction column is pre-with the activation of 4ml methyl alcohol, 4ml0.05% trifluoroacetic acid-water balance before application of sample; Used C
18Reverse-phase chromatographic column is Beckman, System Gold, U.S.A., ODS 0.5 μ m, 0.46 * 25cm.
The separation method of above-mentioned housefly secretion type antibacterial peptide separates the housefly secretion type antibacterial peptide that obtains.
Above-mentioned housefly secretion type antibacterial peptide improves specificity or non-specific immune function, the medicine of bacterial-infection resisting or the application in the healthcare products in preparation.
The application of above-mentioned housefly secretion type antibacterial peptide in animal-feed or sanitas.
The present invention is a raw material with the external secretory product of Musca domestica larva, adopts modern separating and purifying technology, separation, purifying and the character thereof of larva external secretion type antibacterial peptide is studied, and tentatively inquired into the antifungal mechanism of this antibacterial peptide.Simultaneously, be applied to normal antibacterial peptide and infecting mouse, study its influence, for the exploitation and the application of insect antimicrobial peptide provides theoretical basis immune function of mice, blood parameters and vitals pathological change.
One, the separation and purification of antibacterial peptide of the present invention
The contriver gropes a purifying time weak point by research, protein yield height, stable and the activity antibacterial peptide extraction preferably route of anti-microbial activity: be that salt and the small molecular weight impurity in the crude extract removed in 3500 daltonian dialysis tubings dialysis at first with molecular weight cut-off, dialyzed solution is further carried out the Solid-Phase Extraction gradient elution with the methyl alcohol of 0%-80%, the elution fraction lyophilize concentrates, again through 30KD ultrafiltration pipe uf processing, remove macromolecular non-activity composition, remaining component is further used C18 RPLC purifying, finally obtain two activated protein peaks at 15.5min and 18.5min place, ultraviolet absorption spectroscopy proves the albumen peptide matters.Whole purge process purifying multiple is 27.93 times, activity recovery 9.62%.
Antibacterial peptide usually is present in the cytoplasmic granule, and its molecular weight is lower.Generally speaking, the method for extraction polypeptide sample mainly contains: water extract method, method of organic solvent extraction and organic acid extraction process etc.The antibacterial peptide extracting method that this research is adopted is based on contriver's long-felt, and several purification process are carried out the detailed comparatively ripe and easy method that obtains relatively later on.
At first, consider in the Musca domestica larva secretory product crude extract except its excretory antibacterial peptide, some contents, salt and other small-molecule substances that also have many enteron aisles, therefore, we utilize molecular weight cut-off for the dialysis tubing of 3500D crude extract to be dialysed, and remove materials such as micromolecular impurity and salt with this.Solid-Phase Extraction is present a kind of common method to bio-molecular separation, and that the present invention uses is Sep-Pak C
18The Cartridge post, this post can reduce the time of specimen preparation, and is easy and simple to handle, quick.Can finish purifying, sample rich long-pending or concentrate, work such as fractional separation and exchange of solvent.In the fractional separation of sample,, adopt the way of gradient increase solvent strength, optionally wash-out and separate analytes according to analyte polar difference.Dialyse later larval secreta after Solid-Phase Extraction, 0%, 10%, 20%, 30% component plate method and liquid culture method all do not show anti-microbial activity, and the 40%-50% composition activity is weak and unstable, only 60%～80% component has strong anti-microbial activity, and active highly stable.The contriver also adopts the out-of-date methods of gel chromatography to separate the secretory product antibacterial peptide when groping the Solid-Phase Extraction condition early stage, but it is relatively comprehensive, effect is good not as Solid-Phase Extraction all the time, mainly be that latter's required time is long, sample also needs desalination behind the chromatography, and active stability also extraction is high, therefore, the present invention has finally selected Solid-Phase Extraction to separate antibacterial peptide.
Ultrafiltration is a kind of unit operation of membrane sepn, it is a selectivity of utilizing film, each component of solution sees through the mobility difference of film and separates, what transmitted in separation is solvent, the mass transfer process that belongs to rate-controlling, have easy to operate, no phase transformation, no chemical transformation, advantage such as processing efficiency is high and energy-conservation.After the contriver will extract the active ingredient merging, substep used the ultrafiltration pipe of molecular weight cut-off as 3KD and 30KD, found that the molecules of active components amount between between 3-30KD, meets the conclusion of forefathers' research.
High performance liquid chromatography has unique advantage in the separation and purification of biomolecules, for the compartment analysis of biomolecules provides good bio-compatibility, strong to the separating power of complex mixture.Its resolving power is high, and stationary phase is that nonpolar C-8 or C-18 non-polar group have fixedly been gone up in the surface that has the small silica gel particle of porous, moving phase polar solvent such as own nitrile, methyl alcohol, water or low salt buffer etc.The strong macromolecular substance of separated component Semi-polarity is at first by wash-out, and by wash-out, then retention time is longer for the little material of polarity behind the small-molecule substance.Antibacterial peptide belongs to polar material, is easy to be distributed in the polarity moving phase.
The contriver detects discovery with agar diffusion method and viable bacteria counting method to the antibacterial peptide activity that the present invention extracted, this antibacterial peptide has restraining effect to conditioned pathogen such as intestinal bacteria, pathogenic bacterium such as streptococcus aureus, and the non-condition bacterium subtilis of curing the disease also there is restraining effect, to the anti-microbial activity no significant difference of reference culture and clinical isolates strain, point out its restraining effect may not have selectivity to pathogenic bacterium and non-pathogenic bacteria.From antibacterial result, at first this antibacterial peptide has restraining effect to digestive tube infectious bacteria escherichia coli, points out this antibiotic peptide matters to have the effect of colony balance in treatment escherichia coli digestive tract diseases and the adjusting digestive tube; Secondly, its external sentimental microbiological contamination such as streptococcus aureus, Pseudomonas aeruginosa etc. have restraining effect, point out it can be used for prevention and treatment fowl poultry trauma infection contamination, promote wound healing.In the antibacterial effect detection of antibacterial peptide to various bacteria, for the bacterium of not homophyletic of the same race, action effect is basic identical as can be seen, but in general, this antibacterial peptide is better than gram-positive microorganism to the antibacterial effect of Gram-negative bacteria.According to the study, the antibacterial peptide of having reported mostly is to Gram-negative bacteria (G
-) antibacterial effect be better than gram-positive microorganism (G
+), this be since most of antibacterial peptides belong to neutrality or meta-alkalescence albumen, can by with G
-Electronegative species such as lipopolysaccharides on the bacterium adventitia interact, thereby destroy membrane structure with the performance anti-microbial effect.And G
+Bacterium does not have the lipopolysaccharides film, can only be with a small amount of negative charge by the teichoic acid in the polypeptide surface glycan, uronic acid etc.The antibacterial peptide that the present invention extracts is to G
-Antibacterial effect be better than G
+, infer also to belong to neutrality or meta-alkalescence albumen.
Pseudomonas aeruginosa, colon bacillus, methicillin-resistant staphylococcus aureus (MRSA) etc. are modal clinically pathogenic bacterias, easily develop immunity to drugs and cause clinical intractable infection.After house fly antibiotic peptide developed and utilized, will be to clinical anti-infective therapy, particularly the treatment to drug-fast bacteria infection plays a significant role, and also will have a tremendous social and economic benefits simultaneously.
Two, the biochemical characteristic of antibacterial peptide of the present invention
The contriver carries out Tris-TricineSDS-PAGE with reference to the method for (1987) such as Hermann Schagger and Wu Guanyun etc., has detected the purity of separation and purification gained purification thing of the present invention.With molecular weight 2,512,6,210,8,159,10,700,14,404,16,949Da is a standard protein, measures the electrophoresis relative mobility (Rf) of various standard proteins earlier, and the logarithm with various standard protein molecular weight is an ordinate zou then, its Rf value is an X-coordinate, and mapping obtains typical curve.At last, measure the Rf value of agnoprotein, find this proteinic molecular weight from typical curve.
At first detect the purity of antibacterial substance with low-molecular-weight Mark, finding has a clear protein band slightly down at standard lowest molecular weight (14.4KD) band, and molecular weight is less than 14.4KD.The molecular weight of considering target protein is less than normal, selects ultra-low molecular amount Mark electrophoresis once more again for use, finds that the main component of this active ingredient is following slightly at 14.4KD really, but simultaneously, 8,159D and 6, occurred a band that color is more shallow between 210D, protein content is starkly lower than a band.This phenomenon prompting, through the anti-microbial activity component that the 60%-80% methanol-eluted fractions obtains, the molecular weight of major protein is below the 14.4KD, promptly the anti-microbial activity composition is not an one matter.Measure the electrophoresis relative mobility (Rf) of various standard polypeptide in the above-mentioned running gel, the logarithm with various standard polypeptide molecular weights is an ordinate zou then, and its Rf value is an X-coordinate, and mapping can obtain typical curve.The Rf that records Musca domestica larva secretory product antibacterial peptide with quadrat method is respectively 0.7467 and 0.9067.(y=-1.6959x+5.4173 R=0.9899), calculates its molecular weight and is respectively 14157D and 7579D according to typical curve.
The segmental mass spectrometry results of the trypsin hydrolyzing of antibacterial peptide: antibacterial peptide I is hydrolyzed into 38 peptide sections on the electrophoretic band behind trypsin hydrolyzing, uses the peptide fingerprint pattern technology, and the fragment of selecting at random behind the enzymolysis is carried out mass spectroscopy.By Mascot inquiry NCBInr database, (number of landing: aminoacid sequence qi47117014) has certain homology to the precursor of this antibacterial peptide and N,O-Diacetylmuramidase, but the sequence fraction of coverage only is 20%, and both have notable difference by molecular weight, the accurate molecular weight of mass spectroscopy antibacterial peptide I is 14264D, and the latter (antibacterial peptide II) molecular weight is 16179D.
Peptide section 1 after the antibacterial peptide I hydrolysis is: K.SQQGWTAWSTWK.Y
Lys.Ser-Gln-Gln-Gly-Trp-Thr-Ala-Trp-Ser-Thr-Trp-Lys.Tyr
Methionin. Serine-glutamine-glutamine-glycine-tryptophane-Threonine-L-Ala-tryptophane-serine-threonine-tryptophane-Methionin. tyrosine
Peptide section 2 is: K.SELPQWTCIAEHESSYR.T
Lys.Ser-Glu-Leu-Pro-Gln-Trp-Thr-Cys-Ile-Ala-Glu-His-Glu-Ser-Ser-Tyr-Arg.Thr
Methionin. Serine-L-glutamic acid-leucine-proline(Pro)-glutamine-tryptophane-Threonine-halfcystine-Isoleucine-L-Ala-L-glutamic acid-Histidine-L-glutamic acid-Serine-Serine-tyrosine-arginine. Threonine
Among all the other peptide sections among the antibacterial peptide I and the peptide section among the antibacterial peptide II are studied further, are tested.
By detecting the antimicrobial spectrum of antibacterial peptide, tube dilution method is measured minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) to many strains bacterium; Observation is the active variation of antibacterial peptide in different pH values, cation concn, animal serum environment; Detect the blood coagulation and the haemolysis effect of antibacterial peptide.The result shows: housefly secretion type antibacterial peptide all has good antibacterial effect to many strains Gram-positive and negative bacterium, MIC to reference culture escherichia coli, salmonella typhi, dysentery bacterium, subtilis is 32.74 μ g/ml, MIC to pseudomonas aeruginosa is 16.37 μ g/ml, is 65.48 μ g/ml to the MIC of streptococcus aureus, micrococcus luteus.It is better at neutrality anti-microbial activity to weakly alkaline (pH6-9) condition; Monovalence and divalent cation do not have obvious influence to anti-microbial activity; This antibacterial peptide had not both had the blood coagulation effect and had not also had the haemolysis effect external.
Three, the in-vitro antibacterial mechanism of antibacterial peptide of the present invention
By observing antibacterial peptide in minimal inhibitory concentration (MIC) variation of escherichia coli (ATCC 25922) growth curve, intracellular enzyme content and bacterium protein SDS-PAGE collection of illustrative plates down; Transmission electron microscope observing antibacterial peptide metamorphosis; Flow cytometer was analyzed the somatic cells cycle.Results suggest: antibacterial peptide to the antifungal mechanism of escherichia coli may be: the permeability that 1, changes bacterial cell membrane; 2, suppress the synthetic of regulation and control bacterial growth, division associated protein, hinder its division and growth.
Four, antibacterial peptide of the present invention is to effect of immunologic function
Detecting macrophage function and thymus gland, spleen weight is to observe the important indicator of medicine to whole immunity influence.The contriver is testing under the effective prerequisite Musca domestica larva secretory product antibacterial peptide in-vitro antibacterial, and it is interior to effect of immunologic function to have detected the antibacterial peptide body, provides experimental basis for further verifying the effect of antibacterial peptide in the natural immunity.
For inquiring into antibacterial peptide to normal effect of immunologic function, with the small white mouse is experimental animal, adopts different concns (5mg/kg, 10mg/kg, 20mg/kg) to study the influence of antibacterial peptide to small white mouse immune organ index, non-specific immune function and specific immune function respectively.The result shows: 1, the antibacterial peptide amount of 10mg/kg.d and 20mg/kg.d does not have obvious influence to the mouse body weight, but thymus gland and spleen growth are had certain promoter action, and wherein, middle and high dosage group is apparently higher than control group.Illustrate that antibiotic Toplink promotes the growth of immune organ, improves the immune organ index.2, peritoneal macrophage is engulfed in the experiment of chicken red blood cell and carbon clearance, the middle and high dosage group of antibacterial peptide macrophage phagocytic index, engulfs percentage ratio and mouse carbon clearance index all apparently higher than control group.Illustrate that antibacterial peptide has the function of the non-specific immunity that improves the mouse body.3, the middle and high dosage of antibacterial peptide can strengthen the specificity defence capability that ConA inductive mouse spleen lymphocyte transformation efficiency, dinitrofluorobenzene (DNFB) inductive delayed allergy cellular immune functions such as (DTH) improve body.
(1) safety evaluation of antibacterial peptide
With the small white mouse is experimental animal, adopts the improvement karber's method, calculates the medium lethal dose (LD50) of this antibacterial peptide, does not all have and poisons and the phenomena of mortality but maximum dosage-feeding is the 200mg/kg.d animal, points out under this dosage antibacterial peptide for nontoxic substantially.With the experimental rabbit is object, adopts thermal source test method(s) and irritation test method that antibacterial peptide is carried out pyrogen and pungency inspection, and the result shows that this antibacterial peptide does not have thermal source and pungency, is safe with the administration of abdominal injection method.
(2) antibacterial peptide is to the influence of mouse non-specific immune function
1. antibacterial peptide is to mouse body weight and the influence of immune organ exponential
Index and spleen index, the thymus index of antibacterial peptide low dose group (5mg/kg) mouse are compared with control group, have improved 5% and 18% respectively, and body weight has reduced by 3%, but there was no significant difference (P＞0.05).Middle dosage group is compared obviously with control group with index and spleen index, the thymus index of high dose group (10mg/kg and 20mg/kg) mouse and is increased, learn check by statistics, difference has significance (P＜0.05), being antibacterial peptide has certain growth promoting function to thymus gland, the spleen of mouse, can obviously improve the immune organ weight of mouse, this effect has certain dose-effect relationship, along with consumption increases and the weight increase.The body weight of experimental mice is low slightly than control group, especially experimental group I, but adopt pairing T check that the body weight of experimental group and control group is carried out significance test of difference, get P＞0.05, so the body weight of the basic, normal, high dosage group of antibacterial peptide mouse is compared there was no significant difference with control group.
| Group | Body weight (g) x ± SD | Thymus index (mg/g) x ± SD | Index and spleen index (mg/g) x ± SD |
| Group I | 24.66±1.88 | 2.68±0.35 | 5.37±0.64 |
| Group II | 26.71±1.50 | 3.80±0.69 | 7.41±0.93 |
| Group III | 25.12±0.79 | 3.89±0.24 | 7.59±0.56 |
| Control group | 27.48±3.56 | 1.98±0.18 | 5.09±0.71 |
2. antibacterial peptide is to the influence of macrophage phagocytosis of mice
By following table as can be known, the ability that the antibacterial peptide low dosage is engulfed chicken red blood cell to mouse macrophage does not have castering action, but middle and high dosage then can obviously improve peritoneal macrophage engulf percentage ratio, phagocytic index.Simultaneously, the antibacterial peptide of basic, normal, high dosage all can improve carbon clearance index K and the α of mouse, and is wherein, comparatively obvious with middle and high dosage group effect.
Antibacterial peptide is to the influence of mouse macrophage phagocytic activity
| Group | Engulf chicken red blood cell rate (%) x ± SD | Phagocytic index x ± SD |
| Group I | 38.6±5.26 | 0.55±0.96 |
| Group II | 47.8±5.83 | 0.65±0.47 |
| Group III | 50.1±8.41 | 0.68±0.57 |
| Control group | 40±5.45 | 0.45±0.64 |
Antibacterial peptide is to the active influence of mouse carbon clearance
| Group | Clean up index K (x ± SD) | Clean up index α (x ± SD) |
| Group I | 4.90±0.17* | 5.06±0.28 |
| Group II | 5.73±0.19* | 5.17±0.32 |
| Group III | 5.99±0.30** | 6.27±0.38 |
| Control group | 4.43±0.19 | 6.34±0.41 |
* expression and physiological saline group comparing difference significance (P＜0.05), * * represents and the poor heteropole of physiological saline group remarkable (P＜0.01).
3. antibacterial peptide is to the active influence of spleen ACP
Antibacterial peptide low dose group mouse spleen ACP activity is 213.75 ± 29.27, and control group is that 186.66 ± 17.16 liang of groups are compared P＞0.05, does not have statistical significance.In high dosage antibacterial peptide group mouse spleen ACP activity be respectively 268.60 ± 36.64 and 270.48 ± 54.63, compare with control group and low dose group, P value is equal＜0.05, difference has significance.After the antibacterial peptide effect of high dosage, can improve the activity of mouse spleen ACP in the prompting.
4. antibacterial peptide is to the influence of mice serum lysozyme activity
Antibacterial peptide is to the influence of mice serum lysozyme content
| Group | Lysozyme content (μ g/ml) |
| Experimental group I | 13.73±3.85 |
| Experimental group II | 18.14±3.17 |
| Experimental group III | 27.48±5.20 |
| Control group | 8.98±1.49 |
(3) antibacterial peptide is to the influence of mouse specific immune function
1. experimental result
1.1 antibacterial peptide is to the influence of mice spleen T lymphocyte transformation rate
Through the conversion test of ConA inductive mouse spleen lymphocyte, found that, compare with control group, the basic, normal, high dosage group of antibacterial peptide can significantly strengthen ConA inductive spleen lymphocyte proliferation ability (seeing the following form), wherein better with middle and high dosage group reinforced effects, the antibacterial peptide of prompting 5-20mg/kg all has enhancement to the mouse cell immunologic function.
Antibacterial peptide is to the influence of mice spleen T lymphocyte transformation rate
| Group | Dosage (mg/kg) | Spleen T lymphocyte transformation rate x ± SD |
| Experimental group I | 5 | 0.2340±0.0130 |
| Experimental group II | 10 | 0.3111±0.0133 |
| Experimental group III | 20 | 0.3208±0.0095 |
| Control group | 0 | 0.2095±0.0071 |
1.2 utilize the ear swelling method to measure the inducing action of dinitrofluorobenzene (DNFB) to delayed allergy (DTH), the result shows: high dose group significantly strengthens the DTH reaction (seeing the following form) that mouse is brought out DNFB in the antibacterial peptide.
Antibacterial peptide causes the influence of mouse DTH reaction to DNFB
| Group | Dosage (mg/kg) | Auricular concha swelling degree (%) x ± SD |
| Experimental group I | 5 | 10.96±1.49 |
| Experimental group II | 10 | 12.15±1.22 |
| Experimental group III | 20 | 13.31±1.70 |
| Control group | 0 | 9.97±0.87 |
1.3 antibacterial peptide is to the influence of mice serum hemolysin
The results are shown in Table, the serum hemolysin comparing difference does not have significance between each experimental group and control group, and the use of prompting antibacterial peptide is to the not obviously influence of mice serum hemolysin.
Antibacterial peptide is to the influence of mice serum hemolysin
| Group | Dosage (mg/kg) | Serum hemolysin x ± SD |
| Experimental group I | 5 | 134.57±5.42 |
| Experimental group II | 10 | 132.16±6.14 |
| Experimental group III | 20 | 136.87±6.93 |
| Control group | 0 | 135.83±7.07 |
2 conclusions
2.1 immune organ index
Spleen and thymus gland are the important immune organs of animal.Because the intravital immunocyte great majority of machine are distributed in thymus gland and the spleen, the thymic weight of adult animals descends gradually.Therefore, spleen and thymic weight increase, and the then relative body's immunological function that shows strengthens.Thymus gland is immunifacient central lymphoid organs, is the place of T lymphocyte differentiation and maturation, and the T lymphocyte mainly participates in cell immune function of human body, plays a leading role in body homeostasis and immunosurveillance.Spleen is a lymphoid organ maximum in the body, and based on bone-marrow-derived lymphocyte (accounting for 50%-60%), bone-marrow-derived lymphocyte mainly participates in humoral immunization in its immunocyte.In addition, spleen can also be removed the hemocyte and the immunocomplex of self aging.The weight of immune organ can reflect the quantity of organ endolymph cell to a certain extent, thus the indirect aggregate level of disintegration endolymph cell.If both coefficients all increase, illustrate that immunity of medicine pair cell and humoral immunization all have promoter action.
This experimental result shows, high dosage all has significantly the immune organ index of mouse and increases in the antibacterial peptide, has reflected that to a certain extent antibacterial peptide of the present invention can strengthen the immunologic function of animal.From the antibacterial peptide exercising result of different dosage, the enhancing immunity effect of antibacterial peptide has the doses dependency, the low dosage DeGrain, and along with the increase of dosage, the immune organ index increases obviously, but is not that the high dosage effect always is better than middle dosage.
2.2 macrophage phagocytic function
Scavenger cell is a kind of non-specific immunity cell, and it can engulf and eliminate the cell of external foreign matter and self aging death.In addition, it has also played processing antigen in immune response, transmits antigenic information, regulates vital role such as immune response.The mensuration of macrophage function adopts carbon clearance phagocytic activity and the detection of engulfing the chicken red blood cell ability.
The size of carbon clearance ability is to estimate body nonspecific immunity function important indicator.Engulf the critical function that the particulate state foreign matter is monokaryon-macrophage system.Liver and spleen are the major organs of carrying out this function, and the liver Kupffer accounts for 90% of the amount of engulfing, and spleen cell accounts for about 10%.A certain amount of carbon granules suspension of injection in blood, its clearance rate in blood has reflected the state of native system cytophagy.In the finite concentration scope, the clearance rate of carbon granules and its dosage are exponential function relation, promptly engulf speed and are directly proportional with the blood carbon concentration, and be inversely proportional to the carbon granule of having engulfed.What clean up index K reflection is the carbon granules clearance rate.And in a single day monokaryon-macrophage system is activated, and often with liver, spleen M φ hyperplasia, phagocytic index α is a judgement criteria with the activate the phagocytic capacity of unit weight tissue, has got rid of the influence of hyperplasia factor, only estimates the active degree of phagocytic function.Mouse tail vein injection india ink, its carbon granules are engulfed by organ endothelium reticuloendothelial cells such as liver, spleens rapidly and its plasma concentration are descended, thus its Cl reflect indirectly mononuclear phagocyte system phagocytic function.
The result of this research shows that high dosage can obviously improve the index of cleaning up of mouse in the antibacterial peptide, though and low dose group increases to engulfing the chicken red blood cell ability, compare the meaning (P＞0.05) on the no statistics with control group.Experiment of mouse carbon clearance and peritoneal macrophage are engulfed the chicken red blood cell experiment, though can both react the phagocytic function of body immune system, but because of the two experimental technique difference, the place of immunization is also different, engulfed the kind difference of foreign matter, the mechanism that causes the phagocytic cell migration and kill foreign matter is also different, and both results conform to substantially in this experiment, points out antibacterial peptide can strengthen the non-specific immune function of mouse.
2.3 the activity of spleen ACP
The power of scavenger cell digestibility, mainly by how much the deciding of lysosome quantity in the scavenger cell and tens kinds of enzyme content thereof, wherein, acid phosphatase (ACP) is lysosomal marker enzyme, is playing an important role aspect digestion foreign matter and the cellular metabolism.The active enhancing of ACP is an item key of macrophage activation.Strengthen the digestive function of engulfing of scavenger cell, just mean the non-specific immunity that improves body.Scavenger cell and lymphocyte are in close relations, and the former has the function of antigen uptaking, and the latter is difficult for direct antigen uptaking, transmits antigenic immunologic information by scavenger cell.Therefore, scavenger cell is being brought into play important effect in the process of body opposing cause of disease invasion, and the power that it handles antigenic action can have influence on the specific immunity of body.
Spleen is the lymphoid organ of body maximum, and macrophage derived myeloid stem cell in marrow wherein constantly flows into by blood flow, is called migration and fixed macrophage.Monocyte in the blood enters spleen, and differentiation and maturation under the influence of local microenvironment becomes scavenger cell.The scavenger cell volume increases, and the cell surface ridge projections increases, and perfect organoid is arranged in the born of the same parents, and lysosome, plastosome increase, by engulf with cell in kill and wound and bring into play anti-infectious function, but have only the activatory scavenger cell that the powerful ability of killing is just arranged.This experimental selection the activity of the marker enzyme ACP in the spleen observe the height of macrophage activity, thereby further estimate the power of body non-specific immune function.
This experimental result shows, the antibacterial peptide of middle dosage and high dosage is significantly increased to normal mouse spleen ACP activity, improved 43% and 44% than control group respectively, illustrated that the two can both strengthen the activity of scavenger cell in the normal mouse spleen, thereby promoted the non-specific immune function of mouse.
2.4 the vigor of N,O-Diacetylmuramidase
It mainly is to rely on the enzyme system in the born of the same parents to finish that the exogenous material that mouse macrophage is engulfed decomposes in intracellular digestion, and N,O-Diacetylmuramidase is exactly to have one of enzyme of vital role in this enzyme system.
Mouse serum lysozyme level after bacille Calmette-Guerin vaccine (BCG) immunity obviously raises, further discover, when the serum lysozyme activity reaches the peak, minimum through the mouse death rate of infection of staphylococcus aureus, illustrate that the serum lysozyme level has certain relation with resistance infection.N,O-Diacetylmuramidase is except antibiosis plays an important role, can also the activated mononuclear cell, increase PMNS and the phagocytic function of scavenger cell and the antigen presentation function of scavenger cell.
Therefore, Musca domestica larva secretory product antibacterial peptide of the present invention strengthens the result that cytophagous phagocytic function may be multiple reason comprehensive action, and its approach may be: 1. activating macrophage; 2. promote functioning cell secretion N,O-Diacetylmuramidase; 3. improve activity of lysozyme etc.
2.5 spleen T lymphopoiesis ability
Spleen is the peripheral immune organ of body maximum, also be T, bone-marrow-derived lymphocyte is settled down and accept the important place that antigenic stimulation produces immunne response, with concanavalin A (ConA) is the nonspecific inducer T lymphocyte activation of mitotic division proper energy of representative, cause cytokine synthetic, cytokine receptor is expressed and final activation and proliferation.After the T lymphocyte is stimulated by ConA etc. in the vitro culture process, can occur that metabolism is vigorous, protein and nucleic acid is synthetic increases, and then be converted into bigger lymphoblast.The high low reaction body cell immune level of transformation efficiency.In the ConA inductive mouse lymphocyte transformation experiment, T lymphocyte receptor ConA stimulates the back that blast transformation takes place, viable cell particularly proliferative cell can be decomposed into the bluish voilet crystallization with MTT (a kind of flaxen azoles nitrogen salt) by the plastosome lytic enzyme and develops the color, the propagation situation that the variation of its optical density value can reacting cells.From experimental result as can be known, each dosage group all has the effect of raising T lymphopoiesis ability in various degree, and is more obvious with middle high dose group effect.
2.6 delayed allergy
After delayed allergy is the antigenic stimulation of T lymphocyte receptor, be divided into specific primed lymphocyte, when same antigen is invaded once more, cause that local primed lymphocyte discharges multiple lymphokine, cause inflammatory reaction based on monocyte infiltration, show as red swelling of the skin, scleroma.The degree of inflammatory reaction can be weighed the size of body to this antigen-specific cellular immunity.
2.7 the mensuration of serum hemolysin
Serum hemolysin is an index that detects the body humoral immune function, is that antibody-producting cell detects one of test.Behind antigen (SRBC) immune animal, bone-marrow-derived lymphocyte is divided into plasmocyte after being subjected to the SRBC stimulation, plasmocyte produces anti-SRBC antibody (hemolysin), when hatching with SRBC once more, in the presence of complement, can make sheep red blood cell generation hemolytic reaction, and discharge oxyphorase.The antibacterial peptide D that Yasuji etc. extract from antherea pernyi, can promote growth, function and the Differentiation of mouse hematopoietic cell, can also regulate the synthetic of lymphocyte DNA, and the colony of granulocyte and scavenger cell forms, also can influence the output of B cell antibody.
This experimental result shows that antibacterial peptide is to the not significantly influence of content of mice serum hemolysin.
Sum up The above results, the antibiotic Toplink of Musca domestica larva secretory product of the present invention significantly strengthens the normal mouse non-specific immune function, and helps regulating immunologic balance.
Five, antibacterial peptide of the present invention is to the intervention study of infectation of bacteria mouse
This experiment utilizes the disease animal model of infection due to Escherichia coli mouse, after the intervention of Musca domestica larva secretory product antibacterial peptide several angle preliminary study such as physiology, biochemistry and pathological change of animal act in the body of antibacterial peptide, for antibacterial peptide effect in animal body provides experimental basis.
By setting up the animal model that mouse peritoneal infects escherichia coli, give different treatment by the preventative intervention group of antibacterial peptide (infecting the preceding 3 days preventative antibacterial peptide 10mg/kg.d of giving), therapeutic intervention group (giving antibacterial peptide 10mg/kg after the infection) and control group (giving physiological saline).At first, observe the generalized case that infects the back animal, detect and infect back different time sections (4h, 8h, 12h, 24h) peritoneal fluid bacterial count; Secondly, detect mice serum intracellular toxin, N,O-Diacetylmuramidase, nitrogen protoxide (NO) content, cytokine levels such as mouse peripheral blood TNF-α, IL-6; At last, compared the morphological changes of various tissue components of respectively organizing important organs such as Mouse Liver, lung, kidney.
(1) antibacterial peptide is to the influence of infectation of bacteria mice serum TNF-α, IL-6, NO, level of endotoxin
1. antibacterial peptide is to the influence of the infectation of bacteria mouse cell factor
Behind the infectation of bacteria mouse, the content of serum TNF-α sharply rises in the 4h, be increased to 158.56pg/ml from the 23.3pg/ml of normal mouse, and after giving the Musca domestica larva antibacterial peptide, the TNF-alpha content all has decline in various degree, especially comparatively obvious with the preventive administration group, learn check by statistics, the TNF-alpha content of each time period is compared difference with model group all have significance (P＜0.05).
Antibacterial peptide is to the influence of mice serum TNF-alpha content
| Group I (pg/ml) | Group II (pg/ml) | Model group (pg/ml) | |
| 4h | 113.78±8.90 ** | 131.02±7.77 * | 158.56±13.30 |
| 8h | 122.20±14.41 * | 141.92±12.71 * | 168.36±9.67 |
| 12h | 104.96±11.19 * | 124.41±10.22 | 139.49±11.32 |
| 24h | 96.97±10.14 ** | 98.07±10.74 ** | 126.83±9.72 |
| Normal mouse TNF-α | 23.30+4.39 | ||
Antibacterial peptide is to the influence of mice serum IL-6 content
| Group I (pg/ml) | Group II (pg/ml) | Model group (pg/ml) | |
| 4h | 81.33±9.28 ** | 93.13±10.67 ** | 110.43±8.78 |
| 8h | 83.36±5.85 ** | 98.21±8.89 ** | 130.95±9.15 |
| 12h | 86.75±12.90 ** | 95.80±9.98 * | 114.47±17.41 |
| 24h | 84.31±7.52 ** | 85.62±7.31 ** | 101.18±10.71 |
| Normal mouse IL-6 | 57.26±11.27 | ||
2. antibacterial peptide is to the influence of mice serum NO content
Antibacterial peptide is to the influence of mice serum NO content
| Group I (μ mol/l) | Group II (μ mol/l) | Model group (μ mol/l) | |
| 4h | 55.64±10.28 * | 73.37±11.13 | 89.39±15.34 |
| 8h | 68.80±13.96 * | 98.92±14.41 | 116.99±12.08 |
| 12h | 56.98±11.25 ** | 85.85±16.40 * | 95.33±13.80 |
| 24h | 45.64±11.96 ** | 74.34±10.61 | 82.09±12.41 |
| The normal mouse serum NO level | 24.33±4.28 | ||
3. antibacterial peptide is to the influence of mice serum endotoxin content
Antibacterial peptide is to the influence of mice serum endotoxin content
| Group I (pg/ml) | Group II (pg/ml) | Model group (pg/ml) | |
| 4h | 19.85±3.77 ** | 22.68±4.15 ** | 32.29±4.97 |
| 8h | 24.43±3.84 ** | 28.34±4.18 | 34.09±4.76 |
| 12h | 20.53±4.83 ** | 21.72±3.51 ** | 29.95±3.30 |
| 24h | 13.38±4.35 ** | 18.66±3.51* | 26.39±5.57 |
| Normal mouse serum intracellular toxin | 4.77±0.91 | ||
(2) antibacterial peptide is to the influence of mouse peritoneal liquid bacterial count and organ-tissue structure
1. peritoneal fluid bacterial count
To 4h behind the bacterium, the enumeration of model group mouse peritoneal liquid can reach 4949CFU/ μ l, be reduced to 1594CFU/ μ l behind the 12h, and the same period, preventative and therapeutic obviously was less than model group to the bacterial count of antibacterial peptide group mouse, wherein particularly evident with preventive administration group effect, both equal P＜0.01 of comparing with model group, difference has utmost point significance.
Antibacterial peptide is to the influence of infectation of bacteria mouse peritoneal liquid bacterial count
| Group | 4h ascites bacteria number (CFU/ μ l) | 12h ascites bacteria number (CFU/ μ l) |
| Group I | 2776±471 ** | 252±57 ** |
| Group II | 3464±334 ** | 384±77 ** |
| Model group | 4949±563 | 1594±238 |
| Control group | 0 |
This experimental result shows, behind the bacterial infection, model group mouse peritoneal liquid amount of bacteria is from the 4949CFU/ μ l of 4h, drop to the 1594CFU/ μ l of 12h, illustrate that mouse relies on the immunizing power of self can eliminate certain bacterium, and the amount of bacteria of antibacterial peptide group mouse is starkly lower than model group, after the prompting abdominal cavity gives antibacterial peptide, antibacterial peptide has been brought into play certain antibacterium effect in the mouse body, can kill bacteria or suppress bacterial reproduction, this effect combines with the autoimmunity of body, has finally strengthened the germ-resistant effect of body.The amount of bacteria of preventive administration group mouse is minimum, may be because the antibacterial peptide that gives before infecting has improved due to the anti-infection ability of body to a certain extent.
2. respectively organize the histological variation of mouse lung
Behind the infection due to Escherichia coli 8h, the mouse interalveolar septum thickens, and inflammatory cell infiltration is arranged, visible lung tissue extravasated blood, hemorrhage and pulmonary emphysema; Infect back 24h, the mouse lung tissue injury is more obvious, and alveolar septum obviously thickens, and a large amount of inflammatory cell infiltrations are arranged, visible significantly pulmonary apoplexy and pulmonary emphysema.Antibacterial peptide prevention group, the mouse alveolar also has inflammatory cell infiltration, alveolar is separated with thickening to a certain degree, but all obviously alleviates than model group, therapeutic to antibacterial peptide group inflammation degree between antibacterial peptide prevention group and model group.
The organ-tissue structure is carried out observations show, Musca domestica larva secretory product antibacterial peptide has alleviated the tissue injury of the lung that bacterial endotoxin causes, liver.
Above-mentioned experimental result shows: after infecting mouse is given the antibacterial peptide intervention, can reduce the bacterial number of peritoneal fluid, and better with effect behind the preventive administration.Simultaneously antibiotic Toplink reduces infecting mouse serum TNF-a, IL-6, NO, endotoxin content; The infringement of infecting important organ can also reduced in varying degrees.
Compared with prior art, the present invention adopts dialysis, Solid-Phase Extraction, ultrafiltration and RPLC method separation and purification Musca domestica larva secretion type antibacterial peptide, easy, quick, efficient, a protein yield height is provided, anti-microbial activity is stable and activity preferably antibacterial peptide extract route, the antibacterial peptide preparation time is shortened, improved the efficient of separation and purification.And this antibacterial peptide has the effect that improves specificity and non-specific immune function, infectation of bacteria had the certain protection effect, for the application of antibacterial peptide in preparation medicine, healthcare products and animal-feed or sanitas laid a good foundation.
Embodiment:
Embodiments of the invention 1:
Get three age Musca domestica larva place clean large beaker, fully clean and be dipped in an amount of distilled water jolting frequently behind the body surface 4 hours; Draw the centrifugal 10000g * 10min of immersion liquid, the supernatant liquor lyophilize concentrates, and promptly gets the antibacterial peptide crude extract, and 4 ℃ of preservations are standby
(1) dialysis: with spissated antibacterial peptide crude extract molecular weight cut-off is 3500 daltonian dialysis tubing dialysed overnight, changes twice of outer liquid therebetween; Get the sterile filters filtration sterilization of interior liquid with 0.22 μ m ,-20 ℃ of preservations are standby.(2) Solid-Phase Extraction: Sep-pak C
18After the Cartridge solid phase extraction column activates with 4ml methyl alcohol, 4ml0.05% trifluoroacetic acid balance.Get dialysis back antibacterial peptide crude extract 1.5ml upper prop, contain 0.05% trifluoroacetic acid (TFA) the solution stepwise elution of 0%～80% methyl alcohol successively with 5ml, flow velocity is 1ml/min; After methyl alcohol (ACN) and trifluoroacetic acid (TFA) were removed in each elution fraction lyophilize, ultrapure water dissolved, and detected the anti-microbial activity of this elution fraction; Collect, concentrate, freeze-drying has active component, the ultrapure water dissolving, the centrifugal 10min of 12000rpm, it is standby to collect supernatant liquor-20 ℃ preservation.(3) ultrafiltration: will be added in the ultrafiltration pipe that molecular weight cut-off is 3KD after the merging of Solid-Phase Extraction active ingredient, the centrifugal 30min of 5000 * g separates, and the part that sees through the ultrafiltration pipe is the component of molecular weight less than 3KD; The component that continuation will not see through the ultrafiltration pipe is added in the ultrafiltration pipe that molecular weight cut-off is 30KD, the same centrifugal, collect ultrafiltration pipe neutralization pipe liquid down respectively, with active substance be divided into three part: molecular weight＞30KD, 3～30KD,＜3KD, get each component and detect protein concentration and anti-microbial activity.(4) RPLC purifying: active ingredient is with 0.22 μ m filtering with microporous membrane, C18 reverse-phase chromatographic column on the 100 μ l (Beckman, System Gold, U.S.A., ODS 0.5 μ m, 0.46 * 25cm) after the ultrafiltration.Mobile phase A: 0.1% trifluoroacetic acid (the TFA)-aqueous solution; B:0.1% trifluoroacetic acid (TFA)-methyl alcohol (ACN) solution; Chromatographic column is with 5% methyl alcohol (CAN)-0.1% trifluoroacetic acid (TFA) solution equilibria, elution requirement is as follows: own nitrile concentration rises to 60% by 5%, flow velocity 1ml/min in the gradient elution, 30min, the manual 214nm wavelength elution peak of collecting promptly obtains two antibacterial peptide components.The gained antibacterial peptide is the white powder solid after freeze-drying.
Embodiments of the invention 2: the antibacterial peptide 1g of embodiment 1 gained and the common broiler fodder 1Kg of city beast are mixed, feed fryer 2-3 every day, fed 30 days continuously, compare with the fryer that adds the nursing of herbal medicine microbiotic, there are no significant in aspect differences such as the rate of animals delivered to the slaughter-house, mean body weight, feed efficiencies, shows that antibacterial peptide of the present invention has the effect that promotes growth and improve immunizing power.
Embodiments of the invention 3: the antibacterial peptide 1g of embodiment 1 gained and the pork pig feed 1Kg of city beast are mixed, feed pork pig every day 23 times, fed 30 days continuously, compare with the pork pig of adding the nursing of herbal medicine antibiotic feed, there are no significant in aspect differences such as the rate of animals delivered to the slaughter-house, mean body weight, feed efficiencies.
Embodiments of the invention 4: with the antibacterial peptide of embodiment 1 gained with dissolved in distilled water after, add the edible surface activator solution, dissolving fully, quantitatively join with pump in the deionized water of high-shear stirring, stir ripe then, filter, obtain emulsion, this emulsion can be used as aseptic applications anticorrosion in food, medicine etc.
Embodiments of the invention 5: antibacterial peptide 20% of the present invention, hydroxy ester class sanitas 1%, poly(oxyethylene glycol) 400 or propylene glycol 2.5%, sodium-chlor 0.6%, surplus are water (described per-cent all is weight percentage)
Get hydroxy ester class sanitas and be dissolved in the suitable quantity of water, be heated to and make it whole dissolvings about 45 ℃; Add poly(oxyethylene glycol) 400 or propylene glycol, sodium-chlor again, stir and make whole dissolvings, high-temperature sterilization is made auxiliary material solution; The antibacterial peptide of embodiment 1 gained is added in the auxiliary material solution, add adding water to full dose, mixing is sub-packed in the quantitative atomizer, promptly gets spray agent.Said preparation can be used for treating trauma infection contamination.
Claims (7)
1. the separation method of a housefly secretion type antibacterial peptide, it is characterized in that: the antibacterial peptide crude extract is dialysed, dialyzed solution is through Solid-Phase Extraction, elution fraction is through ultra-filtration and separation, active ingredient further is purified into two anti-microbial activity components with RPLC, promptly gets housefly secretion type antibacterial peptide.
2. according to the separation method of the described housefly secretion type antibacterial peptide of claim 1, it is characterized in that: described antibacterial peptide crude extract, be being prepared as of Musca domestica larva ectocrine crude extract: get three age Musca domestica larva place clean large beaker, fully be dipped in an amount of distilled water jolting frequently behind the cleaning body surface 4 hours; Draw the centrifugal 10000g * 10min of immersion liquid, the supernatant liquor lyophilize concentrates, and promptly gets the antibacterial peptide crude extract, and 4 ℃ of preservations are standby.
3. according to the separation method of the described housefly secretion type antibacterial peptide of claim 1, it is characterized in that: (1) dialysis: with spissated antibacterial peptide crude extract molecular weight cut-off is 3500 daltonian dialysis tubing dialysed overnight, changes twice of outer liquid therebetween; Get the sterile filters filtration sterilization of interior liquid with 0.22 μ m ,-20 ℃ of preservations are standby; (2) Solid-Phase Extraction: get Sep-pak C18 Cartridge solid phase extraction column on the antibacterial peptide crude extract of dialysis back, contain 0.05% trifluoroacetic acid solution stepwise elution of 0%～80% methyl alcohol successively with 5ml, flow velocity is 1ml/min; After methyl alcohol and trifluoroacetic acid were removed in each elution fraction lyophilize, ultrapure water dissolved, and detected the anti-microbial activity of this elution fraction; Collect, concentrate, freeze-drying has active component, the ultrapure water dissolving, the centrifugal 10min of 12000rpm, it is standby to collect 20 ℃ of preservations of supernatant liquor; (3) ultrafiltration: will be added in the ultrafiltration pipe that molecular weight cut-off is 3KD after the merging of Solid-Phase Extraction active ingredient, the centrifugal 30min of 5000 * g separates; The component that continuation will not see through the ultrafiltration pipe is added in the ultrafiltration pipe that molecular weight cut-off is 30KD, the same centrifugal, collect ultrafiltration pipe neutralization pipe liquid down respectively, with active substance be divided into three part: molecular weight＞30KD, 3～30KD,＜3KD, get each component and detect protein concentration and anti-microbial activity; (4) RPLC purifying: active ingredient is with 0.22 μ m filtering with microporous membrane, C18 reverse-phase chromatographic column on the 100 μ l, mobile phase A: the 0.1% trifluoroacetic acid-aqueous solution after the ultrafiltration; B:0.1% trifluoroacetic acid-methanol solution; Chromatographic column is with 5% methyl alcohol-0.1% trifluoroacetic acid solution balance, and elution requirement is: gradient elution, in the 30min nitrile concentration rise to 60% from 5%; Flow velocity 1ml/min, the manual 214nm wavelength elution peak of collecting promptly obtains two antibacterial peptide components.
4. according to the separation method of the described housefly secretion type antibacterial peptide of claim 3, it is characterized in that: used Sep-pak C18 Cartridge solid phase extraction column is pre-with the activation of 4ml methyl alcohol, 4ml0.05% trifluoroacetic acid-water balance before application of sample; Used C18 reverse-phase chromatographic column is Beckman, System Gold, U.S.A., ODS 0.5 μ m, 0.46 * 25cm.
As among the claim 1-3 as described in each separation method of housefly secretion type antibacterial peptide separate the housefly secretion type antibacterial peptide that obtains.
6. improve specificity or non-specific immune function, the medicine of bacterial-infection resisting or the application in the healthcare products as housefly secretion type antibacterial peptide as described in the claim 5 in preparation.
7. as the application of housefly secretion type antibacterial peptide in animal-feed or sanitas as described in the claim 5.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104327175A (en) * | 2014-07-03 | 2015-02-04 | 浙江工业大学 | Method for separating antimicrobial peptide |
| CN104740599A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Compound housefly antimicrobial peptide preparation |
| CN106065027A (en) * | 2016-06-17 | 2016-11-02 | 贵州医科大学 | Housefly MSP albumen is in the application of antibiosis |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
| CN108522812A (en) * | 2018-03-16 | 2018-09-14 | 广东盈亨生物科技有限公司 | A kind of extracting method of fly maggot extra-corporeal secretions and application |
| CN110724175A (en) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | Preparation method for extracting antibacterial peptide from Mytilus edulis processing leftovers by utilizing ultrasonic homogenization |
| CN113528600A (en) * | 2021-07-08 | 2021-10-22 | 石河子大学 | Method for preparing antibacterial peptide by inducing fly maggots with salmonella |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1312292A (en) * | 2001-02-23 | 2001-09-12 | 辽宁天成生物制药研究所 | Preparation process of gene antibiotic peptide medicine from transgenic antibiotic peptide fly maggot |
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| CN104327175A (en) * | 2014-07-03 | 2015-02-04 | 浙江工业大学 | Method for separating antimicrobial peptide |
| CN104327175B (en) * | 2014-07-03 | 2017-12-29 | 浙江工业大学 | A kind of method for separating antibacterial peptide |
| CN104740599A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Compound housefly antimicrobial peptide preparation |
| CN106065027A (en) * | 2016-06-17 | 2016-11-02 | 贵州医科大学 | Housefly MSP albumen is in the application of antibiosis |
| CN106065027B (en) * | 2016-06-17 | 2019-04-30 | 贵州医科大学 | Application of the housefly MSP albumen in antibiosis |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
| CN108522812A (en) * | 2018-03-16 | 2018-09-14 | 广东盈亨生物科技有限公司 | A kind of extracting method of fly maggot extra-corporeal secretions and application |
| CN110724175A (en) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | Preparation method for extracting antibacterial peptide from Mytilus edulis processing leftovers by utilizing ultrasonic homogenization |
| CN113528600A (en) * | 2021-07-08 | 2021-10-22 | 石河子大学 | Method for preparing antibacterial peptide by inducing fly maggots with salmonella |
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