CN101328207A - Citrus fruit fly antibacterial peptide, preparation and use thereof - Google Patents
Citrus fruit fly antibacterial peptide, preparation and use thereof Download PDFInfo
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- CN101328207A CN101328207A CNA2008100279565A CN200810027956A CN101328207A CN 101328207 A CN101328207 A CN 101328207A CN A2008100279565 A CNA2008100279565 A CN A2008100279565A CN 200810027956 A CN200810027956 A CN 200810027956A CN 101328207 A CN101328207 A CN 101328207A
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Abstract
The invention discloses a citrus fruit fly antibacterial peptide, which is a polypeptide obtained from separation of a citrus fruit fly pupa, with a molecular weight of 2.3 daltons and an isoelectric point of 11.43, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1. The invention simultaneously discloses a method for preparing the citrus fruit fly antibacterial peptide, comprising the following steps that: the citrus fruit fly pupa is induced by adoption of a mixed bacteria solution; a crude extract is obtained through tissue homogenate; and the citrus fruit fly antibacterial peptide is obtained after purification through ion exchange chromatography, reversed fast protein liquid phase chromatography and reversed high pressure liquid phase chromatography of the crude extract. The citrus fruit fly antibacterial peptide provided by the invention has inhibition on gram positive bacteria, gram negative bacteria and fungi, has no hemolytic activity, can be taken as a raw material for developing human and poultry biotechnical products with pathogenic microorganism infection resistance, and can be taken as a disease-resistant gene to be used for breeding transgenic plants and transgenic animals by means of gene transformation and other biotechnologies.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of citrus fruit fly antibacterial peptide and preparation method thereof, and in the application of biomedicine, agronomy, bromatology and makeup technical field.
Background technology
Since microbiotic such as penicillin are found, the control of pathogenic bacterial infection disease has been produced revolutionary leap.Meanwhile, people recognize that also pathogenic micro-organism will be finally can produce resistance to microbiotic such as penicillin, but the popularity of the fast and resistance of the speed that produces of resistance surprisingly.Along with the widespread use and the abuse of antibacterials, in less than the time in 50 years, originally easy killed bacterium begins to have had Resistant strain and multiple antibiotic resistant strain.
In order to tackle drug-fast bacteria infection, people are are constantly researching and developing the novel antibacterial medicine.The antibacterial peptide of Chu Xianing is exactly the alternative object of novel antibacterial medicine that a class has the great development potentiality in recent years.Insect antimicrobial peptide is the small molecule polypeptide that insect cell specific gene coding produces, great majority have resistance than wide spectrum to bacterium, what have also has restraining effect to fungi, virus, protozoon etc., especially Resistant strain there is tangible lethal effect, and do not destroy the cell of most eukaryotes, non-immunogenicity.
Citrus fruit fly is the important pests on vegetables and the fruit.Financial loss and expenses for prevention and control that annual this kind insect is caused all are quite surprising, also are to cause part province one of the insect of especially severe of causing harm when especially the fruit trees such as oranges and tangerines, loquat in Guangdong Province bear fruit at present.The adult of citrus fruit fly, larva and pupa are grown in the severe environment that contain bacterium, but not influenced by these pathogenic bacterias, show that it has stronger immunological competence.Correlation technique research about the citrus fruit fly antibacterial substance is significant, and has not yet to see the correlation technique report of delivering.
The present invention utilizes the method for bacterium acupuncture, immune induction citrus fruit fly pupa secretion antibacterial substance, separation and purification obtains citrus fruit fly antibacterial peptide Bactrocerin from citrus fruit fly pupa excretory antibacterial substance, and this antibacterial peptide amino acid complete sequence relatively finds no any similar polypeptide through the albumen database search.
Summary of the invention
An object of the present invention is to fill up the deficiencies in the prior art, a kind of new citrus fruit fly antibacterial peptide (Bactrocerin) is provided; Another object of the present invention provides the preparation method of described citrus fruit fly antibacterial peptide; A further object of the invention provides the application of described citrus fruit fly antibacterial peptide.
The invention provides a kind of citrus fruit fly antibacterial peptide, is a peptide species that obtains from the separation and purification of citrus fruit fly pupa, and molecular weight is 2.3 dalton, and iso-electric point is 11.43; The total order of described antibacterial peptide is classified NH2-VGKTWIKVIRGIGKSKIKWQ-COOH as, aminoacid sequence shown in SEQ ID NO:1, i.e. a word used in person's names propylhomoserin-glycine-Methionin-Threonine-tryptophane-Isoleucine-Methionin-a word used in person's names propylhomoserin-Isoleucine-arginine-glycine-Isoleucine-glycine-Methionin-Serine-Methionin-Isoleucine-Methionin-tryptophane-glutamine.
The present invention provides the preparation method of described citrus fruit fly antibacterial peptide simultaneously:
Method one, the present invention utilizes the method for bacterium acupuncture, immune induction citrus fruit fly pupa secretion antibacterial substance, separation and purification obtains citrus fruit fly antibacterial peptide Bactrocerin from citrus fruit fly pupa excretory antibacterial substance, and this antibacterial peptide amino acid complete sequence relatively finds no any similar polypeptide through the albumen database search.
Method one may further comprise the steps:
(1) with mixed bacteria liquid immune induction citrus fruit fly pupa;
(2) citrus fruit fly pupa tissue homogenate obtains crude extract;
(3) crude extract obtains described antibacterial peptide after purified.
The described method with mixed bacteria liquid immune induction citrus fruit fly pupa of step (1) is to adopt No. 1 insect needle to dip in the middle part of getting the resuspended mixed bacteria liquid acupuncture citrus fruit fly pupa of physiological saline; Described physiological saline is for containing 150mM NaCl and 5mM KCl; Described mixed bacteria liquid is OD
600Be 0.3 streptococcus aureus (Staphylococcus aureus) and bacillus coli DH 5 alpha (Escherichia coli DH5 α) mixed solution.
The described thick extracting method of step (2) is to take by weighing a certain amount of citrus fruit fly pupa after mixed bacteria liquid is induced, volume ratio is the extraction damping fluid of 1: 4～1: 6 ratio adding respective volume by weight, fully grind on the ice bath, centrifugal removal hemocyte and other tissue precipitation are got supernatant.
Described extraction damping fluid is that to add final concentration in the 50mmol/L ammonium acetate be that proteinase inhibitor Aprotinin and the final concentration of 1.5 μ mol/L is 20 μ mol/L phenylthioureas; Described centrifugal condition is to be under 4 ℃, 10,000 * g centrifugal 30 to 35 minutes in temperature.
The described purification process of step (3) is through ion-exchange chromatography, anti-phase fast protein liquid chromatography and anti-phase high pressure liquid chromatography purifying.
The method of described ion-exchange chromatography is that the crude extract with citrus fruit fly pupa tissue homogenate is splined on 50mmol/L ammonium acetate pH5.0 damping fluid equilibrated CM Sepharose FastFlow weak cation exchange column, exchange column length 260mm, diameter 16mm, with the ammonium acetate pH5.0 buffer solution elution of 50mmol/L～1mol/L linear gradient, collect the III peak;
Described anti-phase fast protein liquid chromatography is that the active ingredient peak (III peak) that ion-exchange chromatography obtains is splined on 2% acetonitrile-0.05% trifluoroacetic acid aqueous solution equilibrated RESOURCE RPC 3mL reversed-phase column, column length 100mm, diameter 6.4mm, with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid from 2%～80% linear gradient elution 60 minutes, collect the component between 29～36 minutes, per 1.5 minutes 1 components are collected altogether and are obtained a, b, a c and d4 component;
Described anti-phase high pressure liquid chromatography is that the highest component peaks of activity (b peak) that anti-phase fast protein liquid chromatography obtains is splined on 18% acetonitrile-0.05% trifluoroacetic acid aqueous solution equilibrated Hypersil ODS reversed-phase column, column length 250mm, diameter 4mm, from 18%～22% linear gradient elution 32 minutes, collect the peak of locating in 34 minutes with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid.
Method two is for passing through genetically engineered, protein engineering, fermentation engineering expression after ion-exchange chromatography, anti-phase fast protein liquid chromatography and the acquisition of anti-phase high pressure liquid chromatography purifying.
Described ion-exchange chromatography, anti-phase fast protein liquid chromatography and anti-phase high pressure liquid chromatography purification process are with method one.
The invention provides described citrus fruit fly antibacterial peptide as the application of raw material in the biological technology products that resisting pathogenic microbes infects is produced, biological technology products comprises medicine, agricultural-food and food preservative freshness retaining agent, animal feedstuff additive, makeup etc.
And citrus fruit fly antibacterial peptide as disease-resistant gene by transgenosis and other conventional biotechnology means in the application aspect transgenic plant and the transgenic animal breeding.
The present invention adopts conventional substance assistant laser desorpted/ionization time of flight mass spectrometry method to measure the molecular weight of citrus fruit fly antibacterial peptide, and software analysis calculates its iso-electric point, and automatic edman degradation method protein sequencing instrument is measured this antibacterial peptide aminoacid sequence.
The invention has the beneficial effects as follows:
(1) the invention provides a kind of citrus fruit fly antibacterial peptide, no hemolytic activity the experiment proved that, to various bacteria, for example gram-positive microorganism, Gram-negative bacteria and fungi have bacteriostatic activity, can be used for the biological technology products research and development that resisting pathogenic microbes infects; Based on citrus fruit fly antibacterial peptide Bactrocerin aminoacid sequence or gene order, by genetically engineered, protein engineering, fermentation engineering, transgenosis and other biotechnology means, can obtain citrus fruit fly antibacterial peptide Bactrocerin albumen on a large scale, be used for the biological technology products research and development that resisting pathogenic microbes infects;
(2) the present invention searches out effective channel of research citrus fruit fly antibacterial substance, and has set up feasible preparation method and purification condition, for technical foundation is laid in the application of further investigation citrus fruit fly antibacterial substance.
Description of drawings
The ion-exchange chromatography figure of Fig. 1 citrus fruit fly antibacterial peptide
The anti-phase fast protein liquid chromatography figure of Fig. 2 citrus fruit fly antibacterial peptide
The anti-phase high pressure liquid chromatography of Fig. 3 citrus fruit fly antibacterial peptide
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.
Separate the method for preparing Bactrocerin by immune citrus fruit fly pupa and comprise following examples 1,2,3 described processes; Express Bactrocerin by the genetically engineered cloning process and do not comprise immune citrus fruit fly pupa crude extract preparation process among the embodiment 1.
Embodiment 1: the preparation process of citrus fruit fly antibacterial peptide is as follows:
(1) with mixed bacteria liquid immune induction citrus fruit fly pupa;
Adopt No. 1 insect needle to dip in the middle part of getting the resuspended mixed bacteria liquid acupuncture citrus fruit fly pupa of physiological saline; Described physiological saline is for containing 150mM NaCl and 5mM KCl; Described mixed bacteria liquid is OD
600Be 0.3 streptococcus aureus and bacillus coli DH 5 alpha mixed solution.
(2) citrus fruit fly pupa tissue homogenate obtains crude extract;
Take by weighing a certain amount of citrus fruit fly pupa of inducing, 1: 4～1: 6 ratio of volume ratio adds the extraction damping fluid of respective volume by weight, extracting damping fluid is to add 1.5 μ mol/L (final concentration) proteinase inhibitor Aprotinin and 20 μ mol/L (final concentration) phenylthioureas in the 50mmol/L ammonium acetate, fully grind on the ice bath, 4 ℃, 10, centrifugal 30 to 35 minutes of 000 * g, supernatant is the crude extract of citrus fruit fly pupa tissue homogenate, low temperature, and preferred-80 ℃ of preservations are standby.
(3) crude extract obtains described antibacterial peptide after purified.
The first step of purifying is carried out ion-exchange chromatography.Zhi Bei crude extract is splined on 50mmol/L ammonium acetate pH5.0 damping fluid equilibrated CM Sepharose FastFlow weak cation exchange column as stated above, column length 260mm, diameter 16mm, ammonium acetate pH5.0 buffer solution elution with 50mmol/L～1mol/L linear gradient, collect the III peak, see accompanying drawing 1, the ordinary method freeze-drying ,-80 ℃ of preservations are standby;
In second step, carry out anti-phase fast protein liquid chromatography.The III peak freeze-dried component that ion-exchange chromatography obtains heavily is dissolved in 0.05% trifluoroacetic acid aqueous solution, be splined on 2% acetonitrile, 0.05% trifluoroacetic acid aqueous solution (volume ratio) equilibrated RESOURCERPC 3mL reversed-phase column, column length 100mm, diameter 6.4mm, with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid from 2%-80% linear gradient elution 60 minutes, collect the component between 29.5-35.5 minute, per 1.5 minutes 1 components are collected altogether and are obtained a, b, 4 components of c and d are seen accompanying drawing 2.Cryopreservation is standby after each component freeze-drying, preferred-80 ℃ of low temperature.
In the 3rd step, carry out anti-phase high pressure liquid chromatography.The highest component peaks of activity (b peak) freeze-dried component that anti-phase fast protein liquid chromatography obtains heavily is dissolved in 0.05% trifluoroacetic acid aqueous solution, be splined on 18% acetonitrile, 0.05% trifluoroacetic acid aqueous solution (volume ratio) equilibrated Hypersil ODS reversed-phase column, column length 250mm, diameter 4mm, with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid from 18%～22% linear gradient elution 32 minutes, collect the peak of locating in 34 minutes, see the peak of arrow indication in the accompanying drawing 3, freeze-drying, cryopreservation is standby, preferred-80 ℃ of low temperature.
Embodiment 2: the evaluation of the citrus fruit fly antibacterial peptide of purifying
Adopt substance assistant laser desorpted/ionization time of flight mass spectrometry method to measure the molecular weight of citrus fruit fly antibacterial peptide, software analysis calculates its iso-electric point, utilizes automatic edman degradation method protein sequencing instrument to measure this antibacterial peptide aminoacid sequence structure.
Citrus fruit fly antibacterial peptide by the embodiment preparation is that we separate a peptide species that obtains first from the citrus fruit fly pupa, its molecular weight is 2.3 dalton, iso-electric point 11.43, the polypeptide total order is classified as: a word used in person's names propylhomoserin-glycine-Methionin-Threonine-tryptophane-Isoleucine-Methionin-a word used in person's names propylhomoserin-Isoleucine-arginine-glycine-Isoleucine-glycine-Methionin-Serine-Methionin-Isoleucine-Methionin-tryptophane-glutamine.
Embodiment 3: citrus fruit fly antibacterial peptide is to the restraining effect experiment of microorganism
Citrus fruit fly antibacterial peptide 1/2 times of gradient dilution of sterile purified water with purifying.The bacterium LB substratum bacterium liquid 90 μ L that take the logarithm vegetative period add the citrus fruit fly antibacterial peptide of different concns 10 μ L again, mixing, and 30 ℃ of night incubation 24h on 96 hole flat boards are with the absorbancy of microplate reader at 600nm place each pipe of mensuration.Absorbancy do not have the minimum concentration of the antibacterial peptide of considerable change be minimal inhibitory concentration (Minimal Inhibitory concentration, MIC).Fungi is with the fungal spore (10 of 80 μ L
4Spore/ml) on the potato nutrient agar, carry out, the citrus fruit fly antibacterial peptide of adding different concns 20 μ L in each hole, mixing, 25 ℃ of night incubation 24h on 96 hole flat boards are with the absorbancy of microplate reader at 595nm place each pipe of mensuration.It is MIC that absorbancy does not have the minimum concentration of the antibacterial peptide of considerable change.The results are shown in Table 1, the result shows, citrus fruit fly antibacterial peptide is to bacterium, and especially gram positive bacterium (as genus bacillus, streptococcus aureus) has significant bacteria growing inhibiting effect, and fungi (as sickle-like bacteria, Penicillium notatum, black-koji mould) is also had higher fungistatic effect.
Table 1 citrus fruit fly antibacterial peptide is to the restraining effect of bacterium and fungi
SEQUENCE?LISTING
＜110〉Agricultural University Of South China
＜120〉a kind of citrus fruit fly antibacterial peptide and its production and application
<130>
<160>1
<170>PatentIn?version?3.2
<210>1
<211>20
<212>PRT
＜213〉citrus fruit fly pupa
<400>1
Val?Gly?Lys?Thr?Trp?Ile?Lys?Val?Ile?Arg?Gly?Ile?Gly?Lys?Ser?Lys
1 5 10 15
Ile?Lys?Trp?Gln
20
Claims (10)
1, a kind of citrus fruit fly antibacterial peptide is characterized in that described antibacterial peptide is a peptide species that obtains from the separation and purification of citrus fruit fly pupa, and molecular weight is 2.3 dalton, and iso-electric point is 11.43; The aminoacid sequence of described antibacterial peptide is shown in SEQ ID NO:1.
2, the preparation method of the described citrus fruit fly antibacterial peptide of a kind of claim 1 is characterized in that may further comprise the steps:
(1) with mixed bacteria liquid immune induction citrus fruit fly pupa;
(2) citrus fruit fly pupa tissue homogenate obtains crude extract;
(3) crude extract obtains described antibacterial peptide after purified.
3,, it is characterized in that the described method with mixed bacteria liquid immune induction citrus fruit fly pupa of step (1) is to adopt No. 1 insect needle to dip in the middle part of getting the resuspended mixed bacteria liquid acupuncture citrus fruit fly pupa of physiological saline according to the preparation method of the described citrus fruit fly antibacterial peptide of claim 2; Described physiological saline is for containing 150mM NaCl and 5mM KCl; Described mixed bacteria liquid is OD
600Be 0.3 streptococcus aureus and bacillus coli DH 5 alpha mixed solution.
4, according to the preparation method of the described citrus fruit fly antibacterial peptide of claim 2, it is characterized in that the described thick extracting method of step (2) is to take by weighing a certain amount of citrus fruit fly pupa after mixed bacteria liquid is induced, volume ratio is the extraction damping fluid of 1: 4～1: 6 ratio adding respective volume by weight, fully grind on the ice bath, centrifugal removal hemocyte and other tissue precipitation are got supernatant.
5,, it is characterized in that described extraction damping fluid is to add 1.5 μ mol/L (final concentration) proteinase inhibitor Aprotinin and 20 μ mol/L (final concentration) phenylthioureas in the 50mmol/L ammonium acetate according to the preparation method of the described citrus fruit fly antibacterial peptide of claim 5; Described centrifugal condition is to be under 4 ℃, 10,000 * g centrifugal 30 to 35 minutes in temperature.
6,, it is characterized in that the described purification process of step (3) is through ion-exchange chromatography, anti-phase fast protein liquid chromatography and anti-phase high pressure liquid chromatography purifying according to the preparation method of the described citrus fruit fly antibacterial peptide of claim 2.
7, according to the preparation method of the described citrus fruit fly antibacterial peptide of claim 6, the method that it is characterized in that described ion-exchange chromatography is that the crude extract with citrus fruit fly pupa tissue homogenate is splined on 50mmol/L ammonium acetate pH 5.0 damping fluid equilibrated CM Sepharose Fast Flow weak cation exchange columns, exchange column length 260mm, diameter 16mm, with ammonium acetate pH 5.0 buffer solution elution of 50mmol/L～1mol/L linear gradient, collect the III peak; Described anti-phase fast protein liquid chromatography is that the active ingredient peak (III peak) that ion-exchange chromatography obtains is splined on 2% acetonitrile-0.05% trifluoroacetic acid aqueous solution equilibrated RESOURCE RPC 3mL reversed-phase column, column length 100mm, diameter 6.4mm, with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid from 2%～80% linear gradient elution 60 minutes, collect the component between 29～36 minutes, per 1.5 minutes 1 components are collected altogether and are obtained a, b, 4 components of c and d; Described anti-phase high pressure liquid chromatography is that the highest component peaks of activity (b peak) that anti-phase fast protein liquid chromatography obtains is splined on 18% acetonitrile-0.05% trifluoroacetic acid aqueous solution equilibrated Hypersil ODS reversed-phase column, column length 250mm, diameter 4mm, from 18%～22% linear gradient elution 32 minutes, collect the peak of locating in 34 minutes with the acetonitrile damping fluid that contains 0.05% trifluoroacetic acid.
8, the preparation method of the described citrus fruit fly antibacterial peptide of a kind of claim 1 is characterized in that by genetically engineered, protein engineering, fermentation engineering expression after ion-exchange chromatography, anti-phase fast protein liquid chromatography and the acquisition of anti-phase high pressure liquid chromatography purifying.
9, the application of the citrus fruit fly antibacterial peptide for preparing of a kind of claim 2 or 8 described preparation methods is characterized in that being applied in the biological technology products that resisting pathogenic microbes infects as raw material.
10, the application of the citrus fruit fly antibacterial peptide for preparing of a kind of claim 2 or 8 described preparation methods is characterized in that being applied to transgenic plant and transgenic animal breeding aspect as disease-resistant gene.
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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 |
| CN102816769A (en) * | 2012-08-13 | 2012-12-12 | 华中农业大学 | Hermetia illucens L antibacterial peptide, preparation method and application thereof |
| CN104327175A (en) * | 2014-07-03 | 2015-02-04 | 浙江工业大学 | Method for separating antimicrobial peptide |
| CN104356215A (en) * | 2014-10-08 | 2015-02-18 | 广州格拉姆生物科技有限公司 | Antibacterial peptide of bactrocera dorsalis hendel and derivatives and application of antibacterial peptide |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
-
2008
- 2008-05-08 CN CN2008100279565A patent/CN101328207B/en not_active Expired - Fee Related
Cited By (6)
| 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 |
| CN102816769A (en) * | 2012-08-13 | 2012-12-12 | 华中农业大学 | Hermetia illucens L antibacterial peptide, preparation method and application thereof |
| 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 |
| CN104356215A (en) * | 2014-10-08 | 2015-02-18 | 广州格拉姆生物科技有限公司 | Antibacterial peptide of bactrocera dorsalis hendel and derivatives and application of antibacterial peptide |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
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