CN104805046A - Bacterial ghost preparation method of independent lysis gene E - Google Patents
Bacterial ghost preparation method of independent lysis gene E Download PDFInfo
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- CN104805046A CN104805046A CN201510231093.3A CN201510231093A CN104805046A CN 104805046 A CN104805046 A CN 104805046A CN 201510231093 A CN201510231093 A CN 201510231093A CN 104805046 A CN104805046 A CN 104805046A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a bacterial ghost preparation method of an independent lysis gene E. According to the preparation method, a bacterium suspension solution is added with four chemical substances including NaOH, Triton X-100, Na2EDTA and H2O2 to prepare the bacterial ghost in order to satisfy the requirements of complete release of bacterium content and completeness of a bacterium ghost. The method is simple and rapid to operate; except lysis holes, the cell shape and surface structure of the prepared bacterial ghost are not obviously changed. The bacterial ghost prepared by the method disclosed by the invention can enter a specific cell in a targeted manner, is free of toxicity to the cell and can be used as a novel transferring carrier for a drug. The bacterial ghost prepared by the method disclosed by the invention can also be used for immunizing a BALB/c mouse by intraperitoneal injection or oral gavage, also has quite good protection efficiency to homologous counteracting toxic substances and can be used as a novel vaccine.
Description
Technical field
The present invention relates to a kind of ghost preparation method, particularly a kind of ghost preparation method of non-dependent Lysis gene E, the invention still further relates to the application of ghost prepared by application the method, belong to bioengineering field.
Background technology
Bacterium ghost (Bacterial ghost) is the empty bacterial not comprising the entocyte such as nucleic acid and tenuigenin, current ghost preparation strategy and the technology overwhelming majority express crack protein based on the Lysis gene E of Phage PhiX174, thus the formation of cracking Gram-negative bacteria.Ghost very intactly remains the bacterial membrane structure consistent with viable bacteria and related antigen composition, thus can induce humoral immunization and the cellullar immunologic response of body.The natural high conservative structure PAMP (pathogen-associated molecular patterns) of ghost adventitia, such as: lipopolysaccharides, peptidoglycan, pili etc., can by immunocyte by pattern recognition receptors identification, effectively by dendritic cell and macrophage phagocytic, and effectively promote maturation and the activation of dendritic cell.These Biological characteristics of ghost make it directly use as vaccine, also can be used as submission heterologous antigen recombiant vaccine and as albumen, the nucleic acid even delivery vector of medicine.
Application Lysis gene E prepares ghost, needs Lysis gene E to be cloned into expression regulation system, realizes the controlled expression of Lysis gene E.The expression regulation system of Lysis gene E has successfully been applied to various coli strain, Salmonella typhimurium, Salmonella enteritidis, vibrio cholerae, Klebsiella pneumonia, Hp, pleuropneumonia radiation bacillus, hemophilus influenzae, haemolysis pasteurellosis bacillus, pasteurella multocida, edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila etc.But still having a variety of pathogenic bacteria to there is significant restricted hereditary barrier, the expression regulation system of Lysis gene E cannot import these thalline and maybe cannot be identified and start expression, is therefore difficult to successfully prepare ghost.In addition, when preparing ghost, have also been introduced antibiotics resistance gene by while the expression regulation system introducing bacterium of Lysis gene E, easily cause the lateral propagation of antibiotics resistance gene, and the expression regulation system introducing thalline of Lysis gene E and abduction delivering need to consume the regular hour.Therefore, searching out a kind of method preparing ghost fast not relying on Lysis gene E is numerous pathogenic bacterias, especially there is the pathogenic bacteria of restricted hereditary barrier, successfully can prepare the key of ghost.
Summary of the invention
Prepare the deficiency of ghost aspect for current application Lysis gene E, the invention provides a kind of ghost preparation method of non-dependent Lysis gene E.The method adopts NaOH, Triton X-100, Na
2eDTA and H
2o
2these 4 kinds of compounds prepare ghost, breach the limitation relying on Lysis gene E and prepare ghost, can extensively be effectively applied to numerous pathogenic bacteria, especially there is the pathogenic bacteria ghost preparation of restricted hereditary barrier.
Technical scheme of the present invention is: a kind of ghost preparation method of non-dependent Lysis gene E, is characterized in that, by adding NaOH, Triton X-100, Na in bacterial suspension
2eDTA and H
2o
2four kinds of chemical substances prepare ghost, meet the release completely of thalline content and the requirement of the integrity of thalline ghost.
Further, aforesaid method is specially:
(1) minimal inhibitory concentration (MIC) and the smallest bacteria growth concentration (MGC) of the above-mentioned four kinds of chemical substances of micro-broth dilution method is applied;
(2) optimum concn that Plackett-Burman test design obtains the above-mentioned four kinds of chemical substances prepared required for ghost is applied;
(3) bacterial cultures is passed through collected by centrifugation thalline, rinse the resuspended thalline of rear distilled water; According to above-mentioned optimum concn by NaOH, Triton X-100, Na
2eDTA and H
2o
2add in bacterial suspension respectively and cultivate; After cultivation, the centrifugal post-flush bacterial sediment of mixture, then bacterial sediment is resuspended in sterilizing distilled water, (-20 DEG C) shaken at room temperature 4-6min after freezing, centrifugal post-flush bacterial sediment, the bacterial sediment finally obtained is ghost, and the ghost that takes a morsel carries out living stems, should grow without viable bacteria.Preserve after ghost lyophilize.
Further, described ghost is Escherichia coli O138 ghost, described NaOH, Triton X-100, Na
2eDTA and H
2o
2concentration can by the following technical solutions:
(1) NaOH, Na
2eDTA and H
2o
2concentration be the concentration of smallest bacteria growth concentration (MGC), Triton X-100 be minimal inhibitory concentration (MIC);
(2) NaOH and H
2o
2concentration be smallest bacteria growth concentration (MGC), Triton X-100 and Na
2the concentration of EDTA is minimal inhibitory concentration (MIC);
(3) NaOH, Na
2the concentration of EDTA and Triton X-100 is smallest bacteria growth concentration (MGC), H
2o
2concentration be minimal inhibitory concentration (MIC);
(4) NaOH and Na
2the concentration of EDTA is smallest bacteria growth concentration (MGC), Triton X-100 and H
2o
2concentration be minimal inhibitory concentration (MIC).
Further, described H
2o
2concentration is adopted to be the H of 30%
2o
2.
Further, NaOH, Na
2eDTA, 30%H
2o
2be 1.0mg/ml, 0.93mg/ml, 5.0 μ l/ml and 6.25 μ l/ml with the MIC of Triton X-100.NaOH, Na
2eDTA, 30%H
2o
2be 0.15mg/ml, 0.19mg/ml, 0.80 μ l/ml and 0.78 μ l/ml with the MGC of Triton X-100.
The preparation method of preferred Escherichia coli O138 ghost is further: by Escherichia coli O138 culture by centrifugal (5000rpm, 10min) collect thalline, with PBS damping fluid (0.01M, pH7.4) rinse the resuspended thalline of rear distilled water, adjustment bacterial concentration is (0.8-1.2) × l0
6cFU/ml; According to above-mentioned concentration by NaOH, Triton X-100, Na
2eDTA and H
2o
2add in bacterial suspension respectively and cultivate 50-70 minute, the centrifugal rear PBS wash buffer bacterial sediment of mixture, then bacterial sediment is resuspended in sterilizing distilled water, (-20 DEG C) shaken at room temperature 4-6min after freezing, centrifugal rear PBS wash buffer bacterial sediment.The bacterial sediment finally obtained is ghost, and the ghost that takes a morsel carries out living stems, should grow without viable bacteria.Preserve after ghost lyophilize.
The present invention proves by experiment: ghost prepared by the present invention, and except cracking hole, the cellular form of ghost and surface tissue have no obvious change, and the quality of ghost reaches 100%.The present invention proves further by experiment: ghost energy target prepared by the present invention enters specific cells (as Hela cell), does not have toxicity to cell, can be used as the Novel Delivery carrier of medicine.Ghost prepared by the present invention also by abdominal injection immunity or the immune BALB/c mouse of oral administration gavage, is attacked poison to homology and is had good protective efficacy, can be used as new generation vaccine.
Mechanism of the present invention is: bacterium ghost prepared by success will meet two conditions: the release completely of thalline content and the integrity of thalline ghost.The present invention adopts NaOH, Triton X-100, Na
2eDTA and H
2o
2these 4 kinds of compounds prepare ghost; Wherein NaOH can disturb the formation of bacteria cell wall, and the phospholipid bilayer that Triton X-100 can destroy cytolemma makes intracellular organic matter discharge, Na
2eDTA can connect the Ca of adjacent lipopolysaccharides by chelating
2+, Mg
2+thus destruction lipopolysaccharides, H
2o
2degradation of dna is carried out as oxygenant.Adopt above-mentioned four kinds of materials successfully to prepare bacterium ghost, meet the release completely of thalline content and the requirement of the integrity of thalline ghost.
The invention has the beneficial effects as follows: applied chemistry material of the present invention prepares ghost fast, breach the limitation relying on Lysis gene E and prepare ghost, can extensively be effectively applied to numerous pathogenic bacteria especially there is the pathogenic bacteria ghost preparation of restricted hereditary barrier.The method is simple to operate, quick, and the ghost of preparation is except cracking hole, and cellular form and surface tissue have no obvious change.Ghost energy target enters specific cells, does not have toxicity to cell, and has good immunogenicity, can be used as Novel Delivery carrier or the new generation vaccine of medicine.
Accompanying drawing explanation
Fig. 1 is the stereoscan photograph of Escherichia coli O138 ghost;
Fig. 2 is the transmission electron microscope photo of ghost Hela cell (a) of hatching and normal Hela cell (b).
Embodiment
Explain the present invention further below in conjunction with specific embodiment, the advantage of the ghost preparation method of a kind of non-dependent Lysis gene E provided by the invention will be more clear along with description.In following embodiment, method therefor if no special instructions, is ordinary method.The present invention is can cause intestinal bacteria (Escherichia coli) the O138 explanation of baby pig edema.
The mensuration of embodiment 1, chemical substance minimal inhibitory concentration (MIC) and smallest bacteria growth concentration (MGC)
The cultivation of 1.1 Escherichia coli O138
Escherichia coli O138 inoculation LB liquid nutrient medium, is placed in 37 DEG C of constant incubator quiescent culture 72h.
The mensuration of 1.2 chemical substance MIC
Application micro-broth dilution method NaOH, Triton X-100, Na
2eDTA and H
2o
2mIC.Get 96 porocyte culture plates, by the certain density above-mentioned chemical substance of LB liquid nutrient medium 2 multiple proportions serial dilution, the O138 bacterium liquid then adding equivalent in every hole (is about 1.0 × l0
6cFU/ml), the whole bacterial concentration of every Kongzui is made to be about 5 × 10
5cFU/ml.Culture plate is put in 37 DEG C of incubators and hatch about 16h ~ 20h judged result, test and parallelly carry out 3 times.With the minimum concentration of bacteria growing inhibiting complete in aperture for MIC.Result shows: NaOH is 1.0mg/ml, Na to the MIC of O138
2eDTA is 0.93mg/ml, 30%H to the MIC of O138
2o
2be 5.0 μ l/ml to the MIC of O138.Triton X-100 can not the growth of complete antibacterial O138, and when volumetric concentration is 6.25 more than μ l/ml, part suppresses the growth of O138, therefore the MIC of Triton X-100 to O138 is decided to be 6.25 μ l/ml.
The mensuration of 1.3 chemical substance MGC
MGC is the minimum concentration that under MIC, bacterium grows completely.Application micro-broth dilution method NaOH, Triton X-100, Na
2eDTA and H
2o
2mGC.Get 96 porocyte culture plates, with the certain density above-claimed cpd of LB liquid nutrient medium geometric ratio serial dilution, the O138 bacterium liquid then adding equivalent in every hole (is about 1.0 × l0
6cFU/ml), the whole bacterial concentration of every Kongzui is made to be about 5 × 10
5cFU/ml.Culture plate is put in 37 DEG C of incubators and hatch about 16h ~ 20h judged result.Test and parallelly carry out 3 times.The minimum concentration grown completely with bacterium in aperture is MGC.Result shows: NaOH is 0.15mg/ml, Na to the MGC of O138
2eDTA is 0.19mg/ml, 30%H to the MGC of O138
2o
2be 0.80 μ l/ml, Triton X-100 to the MGC of O138 be 0.78 μ l/ml to the MGC of O138.
The optimization of embodiment 2, Escherichia coli O138 ghost preparation method
2.1Plackett – Burman test design
Plackett-Burman test design is a kind of test design being principle with incomplete equilibrium block, more of paramount importance factors are filtered out fast and effectively in multivariate of can comforming, for further investigation further, and have data processing simple, be applicable to the advantages such as multiple factors, be generally used for the screening experiment of project commitment.
Select the Plackett-Burman test design of experiment number N=12, investigate 5 factors, each factor gets+1 and-1 two level, with ghost quality for response value.Four kinds of chemical substance NaOH, Triton X-100, Na
2eDTA and H
2o
2be respectively 4 factors, MIC is+1, MGC is-1.A factor of the speed physical parameter interaction formation different with temperature two is shaken in 5th factor representative, and+1 is 37 DEG C and 100rpm, and-1 is 30 DEG C and 50rpm.12 groups of experiments are carried out according to the Plackett-Burman test design in table 1.
Table 1 Plackett-Burman test design
Step method prepared by 2.2 ghosts
The Escherichia coli O138 culture of quiescent culture 72h collects thalline by centrifugal (5000rpm, 10min), rinses twice, then use the resuspended thalline of distilled water with PBS (0.01M, pH7.4), and adjustment bacterial concentration is about l0
6cFU/ml.The preparation of ghost has been come by 2 steps.1st step, contains all factors, by NaOH, Triton X-100, Na of 1ml
2eDTA and H
2o
25 times of storing solutions add to respectively in the bacterial suspension of 1ml and make final volume be 5ml, 1h is cultivated according to the design of Plackett-Burman, mixture (each experiment is different) the centrifugal 10min of 5000rpm, be transferred to by supernatant liquor to detect the DNA and protein that discharge in new centrifuge tube, bacterial sediment PBS rinses.2nd step, bacterial sediment is resuspended in sterilizing distilled water, freezing (-20 DEG C) shaken at room temperature 5min afterwards, and centrifugal transfer supernatant liquor also rinses bacterial sediment.
The mensuration of 2.3DNA and protein concentration
The DNA concentration discharged in ghost preparation process is measured by the absorption value measuring 260nm wavelength.Absorbancy E260=1 is equivalent to 50 μ g/ml dsDNA.The protein concentration discharged in ghost preparation process is analyzed by the absorption value measuring 280nm wavelength, and draws different protein concentrations according to the typical curve of bovine serum albumin.Because Triton X-100 has photoabsorption under ultraviolet band 275-283nm, therefore eliminated the impact of Triton X-100 in the absorption value of 280nm wavelength by the Triton X-100 solution that measures 1 times of concentration.In experiment, the measurement result of DNA and protein concentration is in table 2.
Table 2 Plackett-Burman test design result
The assessment of 2.4 ghost quality (BGQ) and the observation of scanning electron microscope
The cell finally obtained in each experiment carries out smear, dyeing, microscopic examination, and using the structure of bacterium, whether complete or distortion is as the evaluation index of ghost quality.Ghost quality is expressed as a percentage, each experiment the results are shown in Table 2.
In order to determine the quality of ghost further, phage surface observed by application scanning electron microscope.Be cell 0.1M phosphoric acid buffer (PB, pH7.4) 4 DEG C of fixing 2h containing 2.5% glutaraldehyde of 100% by ghost quality in experiment, rinse 3 times with 0.1M PB, then use 1% osmic acid, 4 DEG C of fixing 1.5h.Sample distilled water rinses subsequently, step by step dehydration of alcohol, isoamyl acetate displacement 30min, CO
2critical point drying, platinum plating in IB-5 ion sputtering instrument after sticky holder, Hitachi S-570 Electron microscope showed is except cracking hole, and the cellular form of ghost and surface tissue have no obvious change (Fig. 1).
The determination of 2.5 main effect factors and multiple linear regression analysis
The result application SPSS statistical software of Plackett-Burman test design analyzes.T-check analysis the results are shown in Table 3, and front 4 factors in experiment all produce negative sense impact to ghost quality, and wherein the negative sense of NaOH has the greatest impact, and statistical discrepancy is remarkable, are secondly Na respectively
2eDTA, Triton X-100, H
2o
2; The 5th factor in experiment is shaken speed-temperature and is had forward impact to ghost quality, but statistical discrepancy is not remarkable.
The analysis of table 3 main effect factor
With ghost quality for response value, adopt the method for screening backward to carry out multiple linear regression analysis to 5 factors, result display eliminating factor shakes speed-temperature and H
2o
2after, significantly, the results of analysis of variance is in table 4, and multiple linear regression analysis the results are shown in Table 5 for ghost quality and other 3 factor linear dependences.Multiple linear regression model describes the relation between ghost quality and 3 independent variables.Regression equation is: BGQ=72.5 – 19.375*NaOH – 9.1667*Triton X-100 – 9.375*Na
2eDTA, R=0.779.
Table 4 linear regression the results of analysis of variance
Table 5 multiple linear regression analysis result
The preparations and applicatio of embodiment 3, Escherichia coli O138 ghost
The preparation of 3.1 Escherichia coli O138 ghosts
According to Plackett-Burman design of experiment analysis result, design preparation 3 batches of Escherichia coli O138 ghosts according to experiment 1,4,9 respectively, ghost quality all reaches 100%.The ghost 0.01M PBS that takes a morsel respectively dilutes, and then coats LB flat board, and cultivate 5d for 37 DEG C, result is showed no bacterial growth, shows in the Escherichia coli O138 ghost that this legal system is standby not containing viable bacteria.The Escherichia coli O138 ghost freeze-drying prepared is preserved.
The particular target of 3.2 ghosts is to application
Hela cell is inoculated in DMEM perfect medium (containing 10% newborn calf serum, penicillin 100U/ml, Streptomycin sulphate 100U/ml), 37 DEG C, saturated humidity, 5%CO
2cultivate in incubator.The DMEM perfect medium of the Hela cell of taking the logarithm vegetative period containing Escherichia coli O138 ghost hatches 3h, and DMEM perfect medium continues after rinsing to cultivate 45h again.The centrifugal 5min of 2000rpm collects the Hela cell that normal Hela cell and ghost are hatched, glutaraldehyde phosphoric acid buffer with 2.5% 4 DEG C of fixing 2h, 0.1M PB rinses 3 times, use fixing 1.5h, the 0.1M PB of 1% osmic acid 4 DEG C to rinse 3 times again, at room temperature dewatering with serial acetone successively embeds and solidification, finally prepare section, thickness is about 50nm, the two dyeing of 3% acetic acid uranium-Chinese holly edge lead plumbate, transmission electron microscope observing.Result shows, and ghost energy particular target, to entering Hela cell, does not affect form and the internal structure of cell, do not have toxicity to cell.The Hela cell that ghost is hatched and normal Hela cell surface all have abundant microvillus, and the structure of mitochondria in tenuigenin is complete and have a lot of rough surfaced endoplasmic reticulum and rrna, and the chromatin in nucleus is euchromosome and kernel is clear, sees Fig. 2.
3.3 ghosts are as the application of vaccine
The BALB/c mouse applying 4 week age carries out Immunization test, evaluates the immune effect of Escherichia coli O138 ghost.30 BALB/c mouse, male and female are not limit, and are divided into 3 groups at random, often organize 10, sub-cage rearing under the same terms, fasting 24h before immunity.1 group is abdominal injection immune group, and every mouse peritoneal injecting immune 100 μ l O138 ghost PBS solution (is equivalent to 5 × l0
9individual Ghost cells); 1 group is oral administration gavage immune group, and before immunity, 10min irrigation stomach device gavages in 10% sodium carbonate solution 30 μ l and hydrochloric acid in gastric juice, and then gavages 100 μ l O138 ghost PBS solution; Another 1 group is PBS control group, and every mouse peritoneal injects 100 μ l PBS solution.Booster immunization 1 time behind interval 3 weeks.Two exempt from latter 2 weeks, and abdominal injection Escherichia coli O138 bacterium liquid carries out attacking poison, the bacterium liquid (about 4 × l0 of every injected in mice 5 times of minimum lethal doses
7cFU), record mouse survival situation, calculates Escherichia coli O138 ghost to the protective efficacy of mouse, the results are shown in Table 6.All dead in control group mice 72h, and the equal healthy survival of abdominal injection immune group mouse, protection ratio reaches 100%, and oral immunity group has the death in the 2nd day after attacking poison of 1 mouse, all healthy survival of all the other mouse, and protection ratio reaches 90%.This test-results shows that Escherichia coli O138 ghost has good immunogenicity, can use as vaccine.
The immune protective effect of table 6 Escherichia coli O138 ghost
Claims (10)
1. a ghost preparation method for non-dependent Lysis gene E, is characterized in that, by adding NaOH, Triton X-100, Na in bacterial suspension
2eDTA and H
2o
2four kinds of chemical substances prepare ghost.
2. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 1, is characterized in that,
(1) minimal inhibitory concentration and the smallest bacteria growth concentration of the above-mentioned four kinds of chemical substances of micro-broth dilution method is applied;
(2) optimum concn that Plackett-Burman test design obtains the above-mentioned four kinds of chemical substances prepared required for ghost is applied;
(3) bacterial cultures is passed through collected by centrifugation thalline, rinse the resuspended thalline of rear distilled water; According to above-mentioned optimum concn by NaOH, Triton X-100, Na
2eDTA and H
2o
2add in bacterial suspension respectively and cultivate; After cultivation, the centrifugal post-flush bacterial sediment of mixture, is then resuspended in bacterial sediment in sterilizing distilled water, freezing rear shaken at room temperature 4-6min, centrifugal post-flush bacterial sediment, the bacterial sediment finally obtained is ghost, the ghost that takes a morsel carries out living stems, should grow without viable bacteria.Preserve after ghost lyophilize.
3. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 1, is characterized in that, described ghost is Escherichia coli O138 ghost.
4. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 3, is characterized in that, described NaOH, Triton X-100, Na
2eDTA and H
2o
2concentration adopt following four kinds of modes:
(1) NaOH, Na
2eDTA and H
2o
2concentration be smallest bacteria growth concentration, the concentration of Triton X-100 is minimal inhibitory concentration;
(2) NaOH and H
2o
2concentration be smallest bacteria growth concentration, Triton X-100 and Na
2the concentration of EDTA is minimal inhibitory concentration;
(3) NaOH, Na
2the concentration of EDTA and Triton X-100 is smallest bacteria growth concentration, H
2o
2concentration be minimal inhibitory concentration;
(4) NaOH and Na
2the concentration of EDTA is smallest bacteria growth concentration, Triton X-100 and H
2o
2concentration be minimal inhibitory concentration.
5. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 4, is characterized in that, described H
2o
2concentration is adopted to be the H of 30%
2o
2.
6. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 5, is characterized in that, described NaOH, Na
2eDTA, 30%H
2o
2be 1.0mg/ml, 0.93mg/ml, 5.0 μ l/ml and 6.25 μ l/ml with the minimal inhibitory concentration of Triton X-100; Described NaOH, Na
2eDTA, 30%H
2o
2be 0.15mg/ml, 0.19mg/ml, 0.80 μ l/ml and 0.78 μ l/ml with the smallest bacteria growth concentration of Triton X-100.
7. the ghost preparation method of a kind of non-dependent Lysis gene E as claimed in claim 6, it is characterized in that, the preparation method of described Escherichia coli O138 ghost is: by Escherichia coli O138 culture by collected by centrifugation thalline, use the resuspended thalline of distilled water with after PBS wash buffer, adjustment bacterial concentration is (0.8-1.2) × l0
6cFU/ml; According to above-mentioned concentration by NaOH, Triton X-100, Na
2eDTA and H
2o
2add in bacterial suspension respectively and cultivate 50-70 minute, the centrifugal rear PBS wash buffer bacterial sediment of mixture, is then resuspended in bacterial sediment in sterilizing distilled water ,-20 DEG C of freezing rear shaken at room temperature 4-6min, centrifugal rear PBS wash buffer bacterial sediment.The bacterial sediment finally obtained is ghost, and the ghost that takes a morsel carries out living stems, should grow without viable bacteria.Preserve after ghost lyophilize.
8. the ghost in claim 1-7 prepared by any one.
9. ghost according to claim 8 enters specific cells at target, as the application of the Novel Delivery carrier aspect of medicine.
10. ghost according to claim 8 is as the application of vaccine aspect.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
| CN109852563A (en) * | 2019-03-06 | 2019-06-07 | 杨凌职业技术学院 | A kind of chemical preparation process of eggs crack detection bacterium shadow |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100203082A1 (en) * | 2007-07-27 | 2010-08-12 | Werner Lubitz | Virus-modified bacteria ghosts |
| KR101449628B1 (en) * | 2013-08-19 | 2014-10-22 | 배재대학교 산학협력단 | Method for preparing bacterial ghosts by the treatment with chemical compounds |
-
2015
- 2015-05-08 CN CN201510231093.3A patent/CN104805046B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100203082A1 (en) * | 2007-07-27 | 2010-08-12 | Werner Lubitz | Virus-modified bacteria ghosts |
| KR101449628B1 (en) * | 2013-08-19 | 2014-10-22 | 배재대학교 산학협력단 | Method for preparing bacterial ghosts by the treatment with chemical compounds |
Non-Patent Citations (1)
| Title |
|---|
| AMRO A. AMARA等: "Sponge-Like: A New Protocol for Preparing Bacterial Ghosts", 《THE SCIENTIFIC WORLD JOURNAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
| CN109852563A (en) * | 2019-03-06 | 2019-06-07 | 杨凌职业技术学院 | A kind of chemical preparation process of eggs crack detection bacterium shadow |
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| CN104805046B (en) | 2017-09-26 |
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