CN101683524B - Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof - Google Patents
Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof Download PDFInfo
- Publication number
- CN101683524B CN101683524B CN2009100672879A CN200910067287A CN101683524B CN 101683524 B CN101683524 B CN 101683524B CN 2009100672879 A CN2009100672879 A CN 2009100672879A CN 200910067287 A CN200910067287 A CN 200910067287A CN 101683524 B CN101683524 B CN 101683524B
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- normal saline
- antibacterial
- bacterial ghost
- bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for preparing a bacterial ghost by the combination of an antibacterial peptide and ultrahigh pressure and application thereof, wherein the method comprises the following steps of: adding the antibacterial peptide when the bacterial strain is cultured until the logarithmic growth phase (the OD600 is 0.4 to 0.6) for action overnight at a temperature of 4 DEG C or for 2 to 4 hours at the room temperature; centrifugally collecting the thalli and washing the thalli twice by normal saline, adding normal saline for re-suspension, and performing ultra-high pressure treatment at a pressure of between 200 and 250MPa; and centrifugally collecting the thalli and washing the thalli twice by normal saline, and finally obtaining the bacterial ghost. The invention provides a novel method for preparing the bacterial ghost, which has the advantages that: the effect is good, the crackling efficiency is as high as over 99.9999999 percent, the operation is simple, and the method can be widely used for the preparation of the bacterial ghost; and by using the prepared bacterial ghost, the immunizing effect is good, and the bacterial ghost can also serve as an alternate vaccine.
Description
Technical field
The present invention relates to a kind of the associating utilize antibacterial peptide and superhigh pressure technique to prepare the method and the application of bacteria simulacrum, belong to the genetic engineering field.
Background technology
The bacterium shadow is a kind of new generation vaccine of carrying out abroad in recent years and target medicine carrier system; It is that lysis genes is expressed in gram negative bacteria; On bacterial cell membrane and cell wall, can form a plurality of film tunnels of wearing; After making antibacterial born of the same parents inner cell slurry and nucleic acid compositions be degraded to small pieces under the effect of osmotic pressure, be discharged from, form a kind of antibacterial shell of sky then, be " bacterial membrane shadow ".The bacterium shadow can produce specificity and nonspecific immunne response by the effective stimulus body; Comprise activation and mucosal immunity of T cell etc.; And can carry exogenous antigen, simultaneously nucleic acid delivery and other drug again are so it is considered to very useful candidate vaccine and deactivation carrier.The bacterium shadow can pass through fermentation and mass production, handling ease, and safe and reliable, there are a lot of receptors in bacterium shadow surface simultaneously, can be used for targeting in specific cell and tissue.At present, except that escherichia coli, a lot of gram negative bacterias all can produce the bacterium shadow under lysis genes E effect, as: Actinobacillus pleuropneumoniae, vibrio cholera, pasteurella haemolytica, helicobacter pylori and blunt tarda etc.Bacterium shadow immunogenicity is good, adjuvant effect obviously, still excellent drug and proteic targeting vector, method for preparing is simple, and is with low cost; Route of inoculation is various, and is safe in utilization, is easy to Industry Promotion; Make the bacterium shadow become the focus of current vaccine research, also shown the application prospect of very attractive.
Antibacterial peptide be in the organism through the micromolecule polypeptide of a kind of biologically active of inducing generation, molecular weight is made up of 20～60 amino acid residues about 2000～7000.This type active polypeptide majority has characteristics such as strong basicity, heat stability and broad-spectrum antiseptic.First found antibacterial peptide is to be induced through injection cloaca through rod bacterium and escherichia coli by people such as Sweden scientist GBoman in 1980 to cherish the polypeptide with antibacterial activity that guppy sky Pupa bombycis produces in the world, names to be Cecropins.After this between the several years, people find from antibacterial, fungus, amphibian, insecticide, higher plant, mammal and even the mankind in succession and separate the polypeptide that obtains to have antibacterial activity.Because it is found that this type active polypeptide has the broad-spectrum high efficacy bactericidal activity to antibacterial at first, thereby called after " antibactetial pepiides, ABP ", Chinese is translated into antibacterial peptide, its former antibacterium peptide that means.Carrying out in a deep going way of Along with people's research work; Find that some antibacterium peptide all has strong lethal effect to part fungus, protozoon, virus and cancerous cell etc., thereby the many scholars of the name of this type active polypeptide tended to be referred to as " peptide antibiotics " peptide antibiotics.At present, corresponding Drug resistance pathogenic strain system has all appearred in all conventional antibiotic, and the Drug resistance problem of pathogenic bacterium has day by day seriously threatened people's health.The antibiotic of seeking brand-new type is an effective way that solves the Drug resistance problem.Antibacterial peptide is high because of antibacterial activity, has a broad antifungal spectrum, and kind is many, and alternative scope is wide, and the target bacterial strain is difficult for producing reasons such as resistant mutation, and is considered to that wide application prospect will be arranged on medical industry.At present, existing multiple polypeptides antibiotic is carrying out preclinical feasibility study, and wherein magainins has got into the phase iii clinical trial stage.
Superhigh pressure technique is a kind of novel cold sterilization technology, and its ultimate principle is that the pressure with 100～1000MPa puts on antibacterial, changes the cells of microorganisms morphosis; Destroy microorganisms cell membrane and cell wall, the permeability of increase cell membrane causes cellular content to run off; Suppress biochemical reaction, checked the metabolic processes of cell, the antibacterial division is slowed down; Growth lags behind, even stops growing, and is usually used in the sterilization of food and fresh-keeping.
Not enough below at present traditional bacterium shadow method for preparing exists: the lysis efficiency of bacterium shadow is difficult to improve always, and the thalline inclusions discharges not thorough, and the thalline deactivation is not thorough; Making up the cracking carrier wastes time and energy, and operates complicated; Traditional bacterium shadow method for preparing is to be based upon on the basis of cracking carrier, and the bacterium shadow of preparation different bacterium needs different cracking carriers, if do not have corresponding carrier then can't prepare the bacterium shadow of this antibacterial.
At present both at home and abroad also combination is not used to prepare the report of bacterium shadow with superhigh pressure technique with antibacterial peptide.
Summary of the invention:
The present invention discloses and a kind of the associating utilizes antibacterial peptide and supertension to prepare the method for bacteria simulacrum, and a kind of new bacteria simulacrum method for preparing is provided.
The invention discloses and unite the bacteria simulacrum that utilizes antibacterial peptide and supertension preparation, can be used as a kind of new candidate vaccine and use.
The of the present invention associating utilizes antibacterial peptide and supertension to prepare the method for bacteria simulacrum, it is characterized in that: unite and utilize antibacterial peptide and the alternative traditional method of supertension to prepare bacteria simulacrum, a kind of new bacteria simulacrum method for preparing is provided.
Of the present inventionly unite that to utilize antibacterial peptide and supertension to prepare the method for bacteria simulacrum following:
Utilize the method for gene engineering expression, obtain antibacterial peptide; Bacterial isolates is cultured to exponential phase (OD
600Be 0.4～0.6) time, adding antibacterial peptide, 4 ℃ of effects are spent the night or room temperature effect 2～4h; Centrifugal collection thalline also repeats to wash twice with normal saline, and it is resuspended to add normal saline, carries out 200～250MPa ultra high pressure treatment; Centrifugal collection thalline also repeats to wash twice with normal saline, promptly is prepared into bacteria simulacrum, and it is subsequent use that lyophilization is stored in-20 ℃ or-80 ℃.
The of the present invention associating utilizes antibacterial peptide and supertension to prepare the application of bacteria simulacrum; May further comprise the steps: the preparation bacteria simulacrum; The immunization experiment animal can stimulate body to produce special humoral immunization and cellular immunization, and comparing with inactivated vaccine has identical protective rate; Have good immune effect, can be used as a kind of new candidate vaccine.
Good effect of the present invention is:
Not enough below traditional bacterium shadow method for preparing exists: the lysis efficiency of bacterium shadow is difficult to improve always, and the thalline inclusions discharges insufficient, and the thalline deactivation is not thorough; Making up the cracking carrier wastes time and energy, and operates complicated; Traditional bacterium shadow method for preparing is to be based upon on the basis of cracking carrier, and the bacterium shadow of preparation different bacterium needs different cracking carriers, if do not have corresponding carrier then can't prepare the bacterium shadow of this antibacterial.The present invention unites and utilizes antibacterial peptide and supertension to prepare bacteria simulacrum, and a kind of new bacteria simulacrum method for preparing is provided, and has obtained good effect; Lysis efficiency is high; Can reach more than 99.9999999%, and simple to operate, and the bacterium shadow that can be widely used in antibacterial prepares; Bacteria simulacrum to preparation is used, and has good immune effect, can be used as a kind of new candidate vaccine and uses.
Description of drawings
Fig. 1. the TEM photo (13000 *) of normal pig Actinobacillus pleuropneumoniae;
Fig. 2. the TEM photo (13000 *) of actinobacillus pleuropneumoniae bacterium shadow;
Fig. 3. the TEM photo (13000 *) of normal bacillus subtilis;
Fig. 4. the TEM photo (13000 *) of bacillus subtilis bacterium shadow;
Fig. 5. normal escherichia coli O
138TEM photo (13000 *);
Fig. 6. escherichia coli O
138The TEM photo (13000 *) of bacterium shadow;
The specific embodiment
Through following examples the present invention is described for example further; And do not limit the present invention in any way; Under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.Further specify essentiality content of the present invention with embodiment below, but content of the present invention is not limited thereto.
Embodiment 1
Utilize antibacterial peptide Tachyplesin I to prepare the bacterium shadow of actinobacillus pleuropneumoniae
The antibacterial peptide that present embodiment is selected for use is Tachyplesin I; The antibacterial of selecting for use is actinobacillus pleuropneumoniae (gram negative bacteria), for preserving this research department.
The DNA sequence of Tachyplesin I is:
aaatggtgct tccgtgtttg ctaccgtggt atctgctacc gtcgttgccg t 51
The aminoacid sequence of Tachyplesin I is:
Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys Arg
1. the expression of antibacterial peptide
Utilize yeast expression system that antibacterial peptide is carried out eukaryotic expression.
2. the preparation of bacteria simulacrum
With bacterial isolates spend the night increase bacterium after, change to be inoculated in the culture medium 37 ℃ of shaken cultivation OD by 0.5%
600Be 0.4～0.6 o'clock, adding the antibacterial peptide Tachyplesin I that expresses is 3.5ug/mL to final concentration, and 4 ℃ of effects are spent the night or room temperature effect 2～4h; After effect finishes, 4 ℃, the centrifugal 10min of 5000rpm, centrifugal collection thalline also repeats to wash twice with normal saline, is prepared into bacteria simulacrum; To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
3. the preservation of bacterium shadow
Centrifugal collection bacterium shadow cell is abandoned most supernatant, be dissolved in the proper volume normal saline at last, calculate bacterium shadow concentration, be sub-packed in 1.5mL vaccine bottle, (contain bacterium shadow 5.0 * 10 about every bottle 800 μ L
10Individual/as mL), highly to be no more than 0.5cm, be stored in-20 ℃ or-80 ℃ after the lyophilization.
Embodiment 2
Unite and utilize antibacterial peptide and supertension to prepare the bacterium shadow of actinobacillus pleuropneumoniae
The antibacterial peptide that present embodiment is selected for use is Tachyplesin I, and the antibacterial of selecting for use is actinobacillus pleuropneumoniae (gram negative bacteria), for preserving this research department.
The DNA sequence of Tachyplesin I is:
aaatggtgct tccgtgtttg ctaccgtggt atctgctacc gtcgttgccg t 51
The aminoacid sequence of Tachyplesin I is:
Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys Arg
1. the expression of antibacterial peptide
Utilize yeast expression system that antibacterial peptide is carried out eukaryotic expression.
2. antibacterial peptide is handled antibacterial
With bacterial isolates spend the night increase bacterium after, change to be inoculated in the culture medium 37 ℃ of shaken cultivation OD by 0.5%
600Be 0.4～0.6 o'clock, adding antibacterial peptide to the final concentration of expressing is 0.35ug/mL, and 4 ℃ of effects are spent the night or room temperature effect 2～4h; 4 ℃, the centrifugal 10min of 5000rpm, centrifugal collection thalline also repeats to wash twice with normal saline.To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
3. ultra high pressure treatment
To pack in the polyethylene complex pocket of aseptic process with the bacterium liquid that antibacterial peptide was handled, vacuum seal is put into ultra-high pressure apparatus; Through 200MPa, pressurize 10min carries out ultra high pressure treatment, and centrifugal collection thalline also repeats to wash twice with normal saline, is prepared into bacteria simulacrum; To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
4. the preservation of bacterium shadow
Centrifugal collection bacterium shadow cell is abandoned most supernatant, be dissolved in the proper volume normal saline at last, calculate bacterium shadow concentration, be sub-packed in 1.5mL vaccine bottle, (contain bacterium shadow 5.0 * 10 about every bottle 800 μ L
10Individual/as mL), highly to be no more than 0.5cm, be stored in-20 ℃ or-80 ℃ after the lyophilization.
Embodiment 3
Unite and utilize antibacterial peptide and supertension to prepare the bacterium shadow of bacillus subtilis
The antibacterial peptide that present embodiment is selected for use is Catesbeianin-1, and the antibacterial of selecting for use is bacillus subtilis (gram positive bacteria), for preserving this research department.
The DNA sequence of Catesbeianin-1 is:
atgatgaggg tgatgaggag gaagactaaa gttatttggg aaaaaaagga ctttattgga 60
ttatacagta ttgattga 78
The aminoacid sequence of Catesbeianin-1 is:
Met Met Arg Val Met Arg Arg Lys Thr Lys Val Ile Trp Glu Lys Lys
Asp Phe Ile Gly Leu Tyr Ser Ile Asp
1. the expression of antibacterial peptide
Utilize yeast expression system that antibacterial peptide is carried out eukaryotic expression.
2. antibacterial peptide is handled antibacterial
With bacterial isolates spend the night increase bacterium after, change to be inoculated in the culture medium 37 ℃ of shaken cultivation OD by 0.5%
600Be 0.4～0.6 o'clock, adding antibacterial peptide to the final concentration of expressing is 0.35ug/mL, and 4 ℃ of effects are spent the night or room temperature effect 2～4h; 4 ℃, the centrifugal 10min of 5000rpm, centrifugal collection thalline also repeats to wash twice with normal saline.To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
3. ultra high pressure treatment
To pack in the polyethylene complex pocket of aseptic process with the bacterium liquid that antibacterial peptide was handled, vacuum seal is put into ultra-high pressure apparatus; Through 250MPa, pressurize 10min carries out ultra high pressure treatment, and centrifugal collection thalline also repeats to wash twice with normal saline, is prepared into bacteria simulacrum; To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
4. the preservation of bacterium shadow
Centrifugal collection bacterium shadow cell is abandoned most supernatant, be dissolved in the proper volume normal saline at last, calculate bacterium shadow concentration, be sub-packed in 1.5mL vaccine bottle, (contain bacterium shadow 5.0 * 10 about every bottle 800 μ L
10Individual/as mL), highly to be no more than 0.5cm, be stored in-20 ℃ or-80 ℃ after the lyophilization.
Embodiment 4
Unite and utilize antibacterial peptide and supertension to prepare escherichia coli O
138The bacterium shadow
The antibacterial peptide that present embodiment is selected for use is Tachyplesin I, and the antibacterial of selecting for use is escherichia coli O
138(gram negative bacteria) is for preserving this research department.
The DNA sequence of Tachyplesin I is:
aaatggtgct tccgtgtttg ctaccgtggt atctgctacc gtcgttgccg t 51
The aminoacid sequence of Tachyplesin I is:
Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys Arg
1. the expression of antibacterial peptide
Utilize yeast expression system that antibacterial peptide is carried out eukaryotic expression.
2. antibacterial peptide is handled antibacterial
With bacterial isolates spend the night increase bacterium after, change to be inoculated in the culture medium 37 ℃ of shaken cultivation OD by 0.5%
600Be 0.4～0.6 o'clock, adding antibacterial peptide to the final concentration of expressing is 0.35ug/mL, and 4 ℃ of effects are spent the night or room temperature effect 2～4h; 4 ℃, the centrifugal 10min of 5000rpm, centrifugal collection thalline also repeats to wash twice with normal saline.To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
3. ultra high pressure treatment
To pack in the polyethylene complex pocket of aseptic process with the bacterium liquid that antibacterial peptide was handled, vacuum seal is put into ultra-high pressure apparatus; Through 250MPa, pressurize 10min carries out ultra high pressure treatment, and centrifugal collection thalline also repeats to wash twice with normal saline, is prepared into bacteria simulacrum; To the antibacterial before and after handling suitably dilution carry out count plate (CFU, individual/as ml), to calculate cleavage rate.
4. the preservation of bacterium shadow
Centrifugal collection bacterium shadow cell is abandoned most supernatant, be dissolved in the proper volume normal saline at last, calculate bacterium shadow concentration, be sub-packed in 1.5mL vaccine bottle, (contain bacterium shadow 5.0 * 10 about every bottle 800 μ L
10Individual/as mL), highly to be no more than 0.5cm, be stored in-20 ℃ or-80 ℃ after the lyophilization.
Experimental example 1
The lysis efficiency of bacteria simulacrum and transmission electron microscope (TEM) are observed
Press embodiment 1,2,3 and 4 preparation bacteria simulacrums, measure lysis efficiency, can reach more than 99.9999% through the lysis efficiency of measuring embodiment 1.Embodiment 2,3 and 4 lysis efficiencies can reach more than 99.9999999%.This shows, unite and use the bacterium shadow of antibacterial peptide and supertension preparation higher than the bacterium shadow lysis efficiency that uses the antibacterial peptide preparation merely.
Press embodiment 2,3 and 4 preparation bacteria simulacrums, with the fixing 4h of 2.5% glutaraldehyde, 1% osmic acid is 1h fixedly; 30% ethanol dehydration 10min, 50% ethanol dehydration 15min, 70% acetic acid uranium effect 12h; 80% ethanol dehydration 15min, 95% ethanol dehydration 20min, twice dehydrated alcohol dehydration 40min; With the expoxy propane 30min that dewaters, add 1: 1 expoxy propane and epoxy resin effect 2h then, add pure epoxy resin infiltration 3h; At 50 ℃ of following epoxy resin embedding 12h, continue embedding 48h with epoxy resin at last then.Ultramicrotome is processed section after the embedding, puts into copper mesh.Section is dyeed, and with acetic acid uranium effect 30min, washing adds lead citrate effect 20min then, washing.Observe down in transmission electron microscope (TEM).
Electronic Speculum result sees Fig. 1-Fig. 6, can be found out by figure, unites the method for utilizing antibacterial peptide and supertension and has successfully prepared actinobacillus pleuropneumoniae (gram negative bacteria), bacillus subtilis (gram positive bacteria) and escherichia coli O
138The bacterium shadow of (gram negative bacteria).
Experimental example 2
The immune effect evaluation of bacteria simulacrum
1. the preparation of bacteria simulacrum
Press the bacterium shadow of embodiment 1 preparation actinobacillus pleuropneumoniae, be resuspended in the proper volume normal saline, making bacterium shadow concentration is 5.0 * 10
10Individual/mL, abundant mixing.
2. the preparation of formalin-inactivated aluminium adjuvant Seedling
Actinobacillus pleuropneumoniae is inoculated in the culture medium, behind 37 ℃ of cultivation 18-24h, is prepared into kind of a daughter bacteria liquid, be inoculated in fluid medium; Take out behind 37 ℃ of cultivation 18-24h and carry out count plate on a small quantity; All the other bacterium liquid add formaldehyde by 0.1%, 37 ℃ of deactivation 24h, and 4 ℃ of preservations are subsequent use.5000rpm is centrifugal during use, and after washing twice with normal saline, adds aluminium hydroxide gel in 1: 5 ratio, and fully vibration is formalin-inactivated aluminium adjuvant Seedling.
3. steriling test
Bacterium shadow and formalin-inactivated aluminium adjuvant Seedling stochastic sampling with prepared actinobacillus pleuropneumoniae are inoculated in the LB flat board, cultivate 48h for 37 ℃, and inspection has or not bacterial growth.
4. safety examination
Get 10 18-22g mices, every lumbar injection bacteria simulacrum, ID are about 10 times of immunizing dose, observe 7-10d continuously; Formalin-inactivated aluminium adjuvant Seedling is the same.Mice does not have death and disease symptom behind lumbar injection bacteria simulacrum and the formalin-inactivated aluminium adjuvant Seedling, and liver organization contact microscopy does not have actinobacillus pleuropneumoniae, shows that bacteria simulacrum and formalin-inactivated aluminium adjuvant Seedling safety are qualified.
5. immunization experiment
The mice random packet, 20 every group, inoculate not that synantigen carries out immunity, the blank group is exempted from the same dose normal saline, sub-cage rearing under the same terms, immune programme for children sees the following form.
Immunization experiment divides into groups
6. the detection of immune level
Indirect elisa method detects antibody titer, and the splenocyte conversion test detects cellular immune level.
Serum antibody horizontal detection result: indirect ELISA regularly detects serum IgG antibody and tires behind the immune mouse, and three immunity back antibody titers reach 3200 *.
Splenocyte conversion test result: immune group has tangible cell increment than matched group.
7. counteracting toxic substances experiment
Three exempt from one week of back, and the viable bacteria of 3 times of minimum lethal doses of mouse peritoneal injection carries out heavy dose of infection experiment, and the mice survival is counted, and detects the protection effect of vaccine.
Three times immunity back immunoprotection result sees the following form, and the protective rate of the protective rate of bacteria simulacrum and formalin-inactivated Seedling all reaches 100%, and the blank group is all dead.
The result is measured in the vaccine immunity protection
conclusion: can find out by above result; Unite and utilize the bacteria simulacrum of antibacterial peptide and supertension preparation higher than the bacterium shadow cleavage rate of utilizing the antibacterial peptide preparation merely; Can stimulate body to produce special humoral immunization and cellular immunization; Comparing with inactivated vaccine has identical protective rate, has good immune effect, can be used as a kind of new candidate vaccine and uses.
Sequence table
< 110>Jilin University
< 120>unite and utilize antibacterial peptide and supertension to prepare the method and the application of bacteria simulacrum
<130>22
<160>4
<170>PatentIn version 3.5
<210>1
<211>51
<212>DNA
< 213>synthetic
<400>1
aaatggtgct tccgtgtttg ctaccgtggt atctgctacc gtcgttgccg t 51
<210>2
<211>17
<212>PRT
< 213>synthetic
<400>2
Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys
1 5 10 15
Arg
<210>3
<211>78
<212>DNA
< 213>synthetic
<400>3
atgatgaggg tgatgaggag gaagactaaa gttatttggg aaaaaaagga ctttattgga 60
ttatacagta ttgattga 78
<210>4
<211>25
<212>PRT
< 213>synthetic
<400>4
Met Met Arg Val Met Arg Arg Lys Thr Lys Val Ile Trp Glu Lys Lys
1 5 10 15
Asp Phe Ile Gly Leu Tyr Ser Ile Asp
20 25
Claims (1)
1. method of utilizing antibacterial peptide Tachyplesin I to prepare the bacterium shadow of actinobacillus pleuropneumoniae may further comprise the steps: with the actinobacillus pleuropneumoniae strain culturing to exponential phase OD
600Be 0.4～0.6 o'clock, add antibacterial peptide Tachyplesin I, 4 ℃ of effects are spent the night or room temperature effect 2～4h; Centrifugal collection thalline also repeats to wash twice with normal saline, and it is resuspended to add normal saline, carries out 200～250MPa ultra high pressure treatment; Centrifugal collection thalline also repeats to wash twice with normal saline, promptly is prepared into bacteria simulacrum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100672879A CN101683524B (en) | 2009-07-15 | 2009-07-15 | Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100672879A CN101683524B (en) | 2009-07-15 | 2009-07-15 | Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101683524A CN101683524A (en) | 2010-03-31 |
| CN101683524B true CN101683524B (en) | 2012-03-28 |
Family
ID=42047051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100672879A Expired - Fee Related CN101683524B (en) | 2009-07-15 | 2009-07-15 | Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101683524B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457231A (en) * | 2008-11-28 | 2009-06-17 | 吉林大学 | Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli |
| CN101658668A (en) * | 2009-07-15 | 2010-03-03 | 吉林大学 | Method and application for preparing bacteria simulacrum by using antibacterial peptide |
-
2009
- 2009-07-15 CN CN2009100672879A patent/CN101683524B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457231A (en) * | 2008-11-28 | 2009-06-17 | 吉林大学 | Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli |
| CN101658668A (en) * | 2009-07-15 | 2010-03-03 | 吉林大学 | Method and application for preparing bacteria simulacrum by using antibacterial peptide |
Non-Patent Citations (3)
| Title |
|---|
| 常月红等.猪传染性胸膜肺炎放线杆菌"菌影"的制备.《中国预防兽医学报》.2008,第30卷(第9期), |
| 李书光等.菌影疫苗的研究进展.《首届中国兽药大会——兽医生物制品学、兽医微生物学学术论坛论文集(2008)》.2008, * |
| 韩国全等.猪传染性胸膜肺炎新型疫苗的研究进展.《猪业科学》.2009,(第2期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101683524A (en) | 2010-03-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103045636B (en) | Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris | |
| CN102224237A (en) | Bacterial ghost (bg) production process using betapropiolactone (blp) for final inactivation | |
| CN104524564A (en) | Crucian herpes virus disease compound vaccine preparation, preparation method and application | |
| CN105462936A (en) | Porcine epidemic diarrhea virus strain MY01 and application thereof | |
| CN106834336A (en) | A kind of heterogenous expression and purification process of Trichoderma harzianum acid protease P6281 | |
| CN103319586B (en) | Antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus and preparation method and application thereof | |
| CN101457231A (en) | Construction of bacteria ghost vector PMAL-E-SN and its application in bacillus coli | |
| CN110408637A (en) | A kind of hemorrhagic disease of grass carp yeast oral vaccine and application | |
| CN104693310A (en) | Chimeric protein, virus-like particle and application thereof | |
| CN101880647A (en) | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application | |
| CN102796200B (en) | Hybrid peptide bursin adjuvant and preparation method and application thereof | |
| CN106478783A (en) | A kind of Porcine Circovirus genetic engineering subunit vaccine and its application | |
| CN106350527A (en) | Diphtheria toxin mutant with characteristic of soluble high expression in Escherichia coli | |
| CN104404057B (en) | A kind of giant salamander irido virus disease vaccine and preparation method and application | |
| CN110201153A (en) | A kind of rabbit hemorrhagic disease, pasteurellosis, Disease Caused By Bordetella Avium triple inactivated vaccine and preparation method thereof | |
| CN102274496A (en) | O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose | |
| CN101857870B (en) | HPV58 L1 gene, vector, strain and expression method | |
| CN101683524B (en) | Method for preparing bacterial ghost by combination of antibacterial peptide and ultrahigh pressure and application thereof | |
| CN104513826A (en) | Human papilloma virus genes, vector, strain, and expression method thereof | |
| CN102286408B (en) | Bacillus amyloliquefaciens WH3, and preparation method and application thereof | |
| CN108558998A (en) | Porcine IL-4/6 co-express the preparation and application of recombination yeast bacteria preparation with pig antibacterial peptide is merged | |
| CN102643756A (en) | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice | |
| CN104894045A (en) | Recombinant lactobacillus for coexpression of foot and mouth disease virus VP1 gene and immunoadjuvant cattle IL-6 gene, and preparation method and application of recombinant lactobacillus | |
| CN101658668B (en) | Method and application for preparing bacteria simulacrum by using antibacterial peptide | |
| CN103333912A (en) | Method for implementing constitutive expression of plectasin derivative MP1102 in Pichia pastoris |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120328 Termination date: 20150715 |
|
| EXPY | Termination of patent right or utility model |