CN104805046B - A kind of ghost preparation method of non-dependent Lysis gene E - Google Patents
A kind of ghost preparation method of non-dependent Lysis gene E Download PDFInfo
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- CN104805046B CN104805046B CN201510231093.3A CN201510231093A CN104805046B CN 104805046 B CN104805046 B CN 104805046B CN 201510231093 A CN201510231093 A CN 201510231093A CN 104805046 B CN104805046 B CN 104805046B
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Abstract
The invention discloses a kind of ghost preparation method of non-dependent Lysis gene E.It is by adding NaOH, Triton X 100, Na in bacterial suspension2EDTA and H2O2Four kinds of chemical substances prepare ghost, meet the complete release of thalline content and the requirement of the integrality of thalline ghost.This method is simple to operate, quick, and the ghost of preparation is in addition to cracking hole, and cellular morphology and surface texture have no obvious change.Ghost prepared by the present invention, which can be targetted, enters specific cells, does not have toxicity to cell, can as medicine Novel Delivery carrier.Ghost prepared by the present invention can also be immunized by intraperitoneal injection or BALB/c mouse is immunized in oral administration gavage, had good protective efficacy to homologous poison of attacking, can be used as new generation vaccine.
Description
Technical field
The present invention relates to a kind of ghost preparation method, more particularly to a kind of ghost preparation method of non-dependent Lysis gene E,
The application of the ghost prepared the invention further relates to application this method, belongs to bioengineering field.
Background technology
Bacterium ghost (Bacterial ghost) is the empty bacterial for not including the cellular contents such as nucleic acid and cytoplasm,
Current ghost prepares strategy and the technology overwhelming majority is the Lysis gene E expression crack protein based on Phage PhiX174, so that
Crack Gram-negative bacteria formation.Ghost extremely intactly remains the bacterial membrane structure consistent with viable bacteria and related antigen
Composition, so as to induce the humoral immunity and cellullar immunologic response of body.The natural highly conserved structure of ghost outer membrane
PAMP (pathogen-associated molecular patterns), for example：Lipopolysaccharides, peptide glycan, pili etc., can be exempted from
Epidemic disease cell is recognized by pattern recognition receptors, is effectively swallowed by BMDC and macrophage, and be effectively facilitated dendron
The maturation of shape cell and activation.These Biological characteristics of ghost make it to be used directly as vaccine, are alternatively arranged as submission different
The recombinant vaccine of source antigen and the delivery vector for being used as albumen, nucleic acid or even medicine.
Ghost is prepared using Lysis gene E, it is necessary to which Lysis gene E is cloned into expression regulation system, realizes Lysis gene E
Controlled expression.The expression regulation system of Lysis gene E has been applied successfully to various coli strains, mouse typhus sramana
Salmonella, Bacterium enteritidis, comma bacillus, Friedlander's bacillus, helicobacter pylori, pleuropneumonia radiation bacillus, influenza
Haemophilus, haemolysis Pasteurella, pasteurella multocida, edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila etc..But still
There are a variety of pathogens to there is significant restricted hereditary barrier, the expression regulation system of Lysis gene E can not import these
Thalline can not be identified and start expression, and therefore, it is difficult to successfully prepare ghost.In addition, when preparing ghost, by Lysis gene E
Antibiotics resistance gene is have also been introduced while expression regulation system introducing bacterium, the lateral biography of antibiotics resistance gene is easily caused
Broadcast, and the expression regulation system introducing thalline and induced expression of Lysis gene E need to consume the regular hour.Therefore, find
It is numerous pathogens to a kind of quick method for preparing ghost independent of Lysis gene E, especially in the presence of restricted heredity
The pathogen of barrier, can successfully prepare the key of ghost.
The content of the invention
Deficiency in terms of preparing ghost for application Lysis gene E at present, base is cracked the invention provides a kind of non-dependent
Because of E ghost preparation method.This method uses NaOH, Triton X-100, Na2EDTA and H2O2This 4 kinds of compound prepares bacterium
Slough off, breach and rely on the limitation that Lysis gene E prepares ghost, numerous pathogens can be widely and effectively applied to, especially deposited
Prepared in the pathogen ghost of restricted hereditary barrier.
The technical scheme is that：A kind of ghost preparation method of non-dependent Lysis gene E, it is characterized in that, by
NaOH, Triton X-100, Na are added in bacterial suspension2EDTA and H2O2Four kinds of chemical substances prepare ghost, meet in thalline
Tolerant complete release and the requirement of the integrality of thalline ghost.
Further, the above method is specially：
(1) minimal inhibitory concentration (MIC) and minimum of the application above-mentioned four kinds of chemical substances of micro-broth dilution method are thin
Bacteria growing concentration (MGC)；
(2) above-mentioned four kinds of chemical substances required for ghost are prepared using Plackett-Burman experimental design acquisitions
Optium concentration；
(3) by bacterial cultures by the way that thalline is collected by centrifugation, thalline is resuspended with distilled water after flushing；According to above-mentioned optimal dense
Spend NaOH, Triton X-100, Na2EDTA and H2O2It is respectively added to cultivate in bacterial suspension；After culture is finished, mixture
Bacterial sediment is rinsed after centrifugation, then bacterial sediment is resuspended in sterilizing distilled water, (- 20 DEG C) shaken at room temperature 4- after freezing
Bacterial sediment is rinsed after 6min, centrifugation, the bacterial sediment finally obtained as ghost takes a small amount of ghost to carry out living stems, should
Without viable bacteria growth.Preserved after ghost is freeze-dried.
Further, the ghost is Escherichia coli O138 ghost, described NaOH, Triton X-100, Na2EDTA and
H2O2Concentration can use following technical scheme：
(1)NaOH、Na2EDTA and H2O2Concentration be smallest bacteria growth concentration (MGC), Triton X-100 concentration
For minimal inhibitory concentration (MIC)；
(2) NaOH and H2O2Concentration be smallest bacteria growth concentration (MGC), Triton X-100 and Na2EDTA concentration
For minimal inhibitory concentration (MIC)；
(3)NaOH、Na2EDTA and Triton X-100 concentration is smallest bacteria growth concentration (MGC), H2O2Concentration
For minimal inhibitory concentration (MIC)；
(4) NaOH and Na2EDTA concentration is smallest bacteria growth concentration (MGC), Triton X-100 and H2O2Concentration
For minimal inhibitory concentration (MIC).
Further, the H2O2Use concentration for 30% H2O2。
Further, NaOH, Na2EDTA, 30%H2O2Be 1.0mg/ml with Triton X-100 MIC, 0.93mg/ml,
5.0 μ l/ml and 6.25 μ l/ml.NaOH、Na2EDTA, 30%H2O2Be 0.15mg/ml with Triton X-100 MGC,
0.19mg/ml, 0.80 μ l/ml and 0.78 μ l/ml.
The preparation method of further preferred Escherichia coli O138 ghost is：Escherichia coli O138 culture is passed through into centrifugation
(5000rpm, 10min) collects thalline, and thalline is resuspended with distilled water after being rinsed with PBS (0.01M, pH7.4), and adjustment is thin
Bacteria concentration is (0.8-1.2) × l06CFU/ml；According to above-mentioned concentration by NaOH, Triton X-100, Na2EDTA and H2O2Respectively
It is added in bacterial suspension and cultivates 50-70 minutes, PBS rinses bacterial sediment after mixture centrifugation, and then thalline sinks
Shallow lake is resuspended in sterilizing distilled water, and PBS rinses bacterial sediment after (- 20 DEG C) shaken at room temperature 4-6min, centrifugation after freezing.
The bacterial sediment finally obtained as ghost, takes a small amount of ghost to carry out living stems, should be grown without viable bacteria.Ghost is freeze-dried
After preserve.
The present invention is experimentally confirmed：Ghost prepared by the present invention, in addition to cracking hole, the cellular morphology and table of ghost
Face structure has no obvious change, and the quality of ghost reaches 100%.The present invention is further experimentally confirmed：Prepared by the present invention
Ghost can target into specific cells (such as Hela cells), there is no toxicity to cell, can as medicine Novel Delivery carrier.This
BALB/c mouse can also be immunized by the way that immune or oral administration gavage is injected intraperitoneally by inventing the ghost prepared, be had very well to homologous poison of attacking
Protective efficacy, new generation vaccine can be used as.
The present invention mechanism be：Bacterium ghost prepared by success will meet two conditions：The complete release of thalline content
With the integrality of thalline ghost.The present invention is using NaOH, Triton X-100, Na2EDTA and H2O2It is prepared by this 4 kinds of compound
Ghost；Wherein NaOH can disturb the formation of bacteria cell wall, and the phospholipid bilayer that Triton X-100 can destroy cell membrane makes
Intracellular organic matter discharges, Na2EDTA can chelate the Ca for connecting adjacent lipopolysaccharides2+、Mg2+So as to destroy lipopolysaccharides, H2O2As oxygen
Agent carrys out degradation of dna.Bacterium ghost is successfully prepared using above-mentioned four kinds of materials, meet the complete release of thalline content with
The requirement of the integrality of thalline ghost.
The beneficial effects of the invention are as follows：Applied chemistry material of the present invention quickly prepares ghost, breaches dependence Lysis gene E
The limitation of ghost is prepared, numerous pathogens can be widely and effectively applied to, especially in the presence of the cause of disease of restricted hereditary barrier
It is prepared by bacterium ghost.This method is simple to operate, quick, and the ghost of preparation is in addition to cracking hole, and cellular morphology and surface texture are not
See obvious change.Ghost, which can be targetted, enters specific cells, does not have toxicity to cell, and with good immunogenicity, can conduct
The Novel Delivery carrier or new generation vaccine of medicine.
Brief description of the drawings
Fig. 1 is the stereoscan photograph of Escherichia coli O138 ghost；
Fig. 2 is the Hela cells (a) that ghost is incubated and the transmission electron microscope photo of normal Hela cells (b).
Embodiment
The present invention, a kind of non-dependent Lysis gene E that the present invention is provided further are explained with reference to specific embodiment
The advantage of ghost preparation method will be relatively sharp with description.In following embodiments method therefor unless otherwise instructed,
For conventional method.Present invention explanation by taking Escherichia coli (Escherichia coli) O138 that can cause hydropsy for baby pigs as an example.
The measure of embodiment 1, chemical substance minimal inhibitory concentration (MIC) and smallest bacteria growth concentration (MGC)
The culture of 1.1 Escherichia coli O138s
Escherichia coli O138 is inoculated with LB fluid nutrient mediums, is placed in 37 DEG C of constant incubator quiescent culture 72h.
1.2 chemical substance MIC measure
Using micro-broth dilution method NaOH, Triton X-100, Na2EDTA and H2O2MIC.Take 96 hole cells
Culture plate, is serially diluted certain density above-mentioned chemical substance with the multiple proportions of LB fluid nutrient mediums 2, then adds equivalent into every hole
O138 bacterium solutions (about 1.0 × l06CFU/ml), it is about 5 × 10 to make the final bacterial concentration in every hole5CFU/ml.Culture plate is put 37
About 16h~20h judged results are incubated in DEG C incubator, parallel carry out 3 times is tested.To completely inhibit bacterial growth in aperture
Least concentration is MIC.As a result show：NaOH is 1.0mg/ml, Na to O138 MIC2EDTA is 0.93mg/ to O138 MIC
Ml, 30%H2O2MIC to O138 is 5.0 μ l/ml.Triton X-100 can not completely antibacterial O138 growth, in volumetric concentration
Part suppresses O138 growth during for more than 6.25 μ l/ml, therefore Triton X-100 are set into 6.25 μ l/ to O138 MIC
ml。
1.3 chemical substance MGC measure
The least concentration that MGC grows completely for bacterium under MIC.Using micro-broth dilution method NaOH, Triton X-
100、Na2EDTA and H2O2MGC.96 porocyte culture plates are taken, with LB fluid nutrient mediums etc. than being serially diluted on certain density
Compound is stated, O138 bacterium solutions (about 1.0 × l0 of equivalent is then added into every hole6CFU/ml), make the final bacterium solution in every hole dense
Degree about 5 × 105CFU/ml.Culture plate is put in 37 DEG C of incubators and is incubated about 16h~20h judged results.Experiment is parallel to carry out 3
It is secondary.Using bacterium grows completely in aperture least concentration as MGC.As a result show：NaOH is 0.15mg/ml to O138 MGC,
Na2EDTA is 0.19mg/ml, 30%H to O138 MGC2O2MGC to O138 is 0.80 μ l/ml, Triton X-100 couple
O138 MGC is 0.78 μ l/ml.
The optimization of embodiment 2, Escherichia coli O138 ghost preparation method
2.1Plackett-Burman experimental designs
Plackett-Burman experimental designs are a kind of experimental designs using incomplete equilibrium block as principle, can be comformed
Some mostly important factors are fast and effectively filtered out in multivariable, for further further investigation, and with data processing
Simply, suitable for Multiple factors the advantages of, it is generally used for the screening experiment of project early stage.
From experiment number N=12 Plackett-Burman experimental designs, 5 factors are investigated, each factor takes+1
With -1 two level, using ghost quality as response.Four kinds of chemical substances NaOH, Triton X-100, Na2EDTA and H2O2Point
Not Wei 4 factors, MIC is that+1, MGC is -1.5th factor, which is represented, shakes the two different physical parameter interactions of speed and temperature
The factor formed ,+1 is 37 DEG C and 100rpm, and -1 is 30 DEG C and 50rpm.12 groups of experiments are according to the Plackett- in table 1
Burman experimental designs are carried out.
The Plackett-Burman experimental designs of table 1
Step method prepared by 2.2 ghosts
Quiescent culture 72h Escherichia coli O138 culture collects thalline by centrifuging (5000rpm, 10min), uses PBS
(0.01M, pH7.4) is rinsed twice, thalline then is resuspended with distilled water, adjustment bacterial concentration is about l06CFU/ml.The system of ghost
It is standby to be completed by 2 steps.1st step, contains all factors, by 1ml NaOH, Triton X-100, Na2EDTA and H2O2
5 times of storing solutions be respectively added to cause final volume to be 5ml in 1ml bacterial suspension, according to Plackett-Burman's
Design culture 1h, mixture (each experiment different) 5000rpm centrifugation 10min, by supernatant be transferred in new centrifuge tube with
Just detect that the DNA and protein discharged, bacterial sediment PBS are rinsed.2nd step, bacterial sediment is resuspended in sterilizing distilled water, cold
Freeze (- 20 DEG C) shaken at room temperature 5min afterwards, centrifugation transfer supernatant simultaneously rinses bacterial sediment.
2.3DNA and protein concentration measure
The DNA concentration discharged in ghost preparation process is determined by determining the absorption value of 260nm wavelength.Absorbance
E260=1 is equivalent to 50 μ g/ml dsDNA.The protein concentration discharged in ghost preparation process is by determining 280nm wavelength
Absorption value is analyzed, and draws according to the standard curve of bovine serum albumin(BSA) different protein concentrations.Due to Triton X-100
There are light absorbs under ultraviolet band 275-283nm, therefore by determining the Triton X-100 solution of 1 times of concentration in 280nm ripples
Long absorption value eliminates Triton X-100 influence.The measurement result of DNA and protein concentration is shown in Table 2 in experiment.
The Plackett-Burman experimental design results of table 2
The assessment of 2.4 ghost quality (BGQ) and the observation of ESEM
Whether the cell finally obtained in each experiment carries out smear, dyeing, micro- sem observation, complete with the structure of bacterium
Or deformation is used as the evaluation index of ghost quality.Ghost quality is expressed as a percentage, and that each tests the results are shown in Table 2.
In order to further determine that the quality of ghost, application scanning electron microscope observes phage surface.Bacterium in testing
0.1M phosphate buffer (PB, pH7.4) 4 DEG C fixed 2h of the cell containing 2.5% glutaraldehyde that quality is 100% is sloughed off, 0.1M is used
PB is rinsed 3 times, then with 1% osmic acid, 4 DEG C of fixed 1.5h.Subsequent sample is rinsed with distilled water, step by step dehydration of alcohol, isoamyl acetate
Replace 30min, CO2Critical point drying, glues platinum plating in IB-5 ion sputtering instruments after support, Hitachi's S-570 Electron microscope showeds except
Crack outside hole, the cellular morphology and surface texture of ghost, which have no, substantially changes (Fig. 1).
2.5 determination and the multiple linear regression analysis of main effect factor
The result application SPSS statistical softwares of Plackett-Burman experimental designs are analyzed.T- check analysis results
3 are shown in Table, preceding 4 factors in experiment produce negative sense influence to ghost quality, wherein NaOH negative sense influence is maximum, statistical difference
It is different notable, it is secondly Na respectively2EDTA、Triton X-100、H2O2；The 5th factor in experiment shakes speed-temperature to ghost matter
Measurer has positive influence, but statistical discrepancy is not notable.
The analysis of the main effect factor of table 3
Using ghost quality as response, multiple linear regression analysis, knot are carried out using the method screened backward to 5 factors
Fruit shows that exclusion factor shakes speed-temperature and H2O2Afterwards, ghost quality and other 3 factor linear correlations are notable, the results of analysis of variance
4 are shown in Table, multiple linear regression analysis the results are shown in Table 5.Multiple linear regression model is described between ghost quality and 3 independent variables
Relation.Regression equation is：BGQ=72.5-19.375*NaOH-9.1667*Triton X-100-9.375*Na2EDTA, R=
0.779。
The linear regression the results of analysis of variance of table 4
The multiple linear regression analysis result of table 5
The preparation and application of embodiment 3, Escherichia coli O138 ghost
The preparation of 3.1 Escherichia coli O138 ghosts
According to Plackett-Burman design of experiment analysis results, 3 batches of large intestines are prepared according to the design of experiment 1,4,9 respectively
Bacillus O138 ghosts, ghost quality reaches 100%.Take a small amount of ghost 0.01M PBS to dilute respectively, be then coated on LB and put down
Plate, 37 DEG C of culture 5d, is as a result showed no bacterial growth, shows not containing viable bacteria in Escherichia coli O138 ghost prepared by the method.
The lyophilized preservation of Escherichia coli O138 ghost prepared.
The specific targeting application of 3.2 ghosts
Hela cells are inoculated in DMEM complete mediums and (contain 10% newborn calf serum, penicillin 100U/ml, strepto-
Plain 100U/ml), 37 DEG C, saturated humidity, 5%CO2Cultivated in incubator.The Hela cells in growth period of taking the logarithm are used containing big
The DMEM complete mediums of enterobacteria O138 ghosts are incubated 3h, and DMEM complete mediums are cultivated for 45h after rinsing.
2000rpm centrifugations 5min collects the Hela cells that normal Hela cells and ghost are incubated, with 2.5% glutaraldehyde phosphate buffer
4 DEG C of fixed 2h, 0.1M PB are rinsed 3 times, then are rinsed 3 times with 1% osmic acid, 4 DEG C of fixed 1.5h, 0.1M PB, and serial acetone is used successively
Dehydration embedding and solidification at room temperature, finally prepares section, thickness about 50nm, the double dyeing of 3% acetic acid uranium-Chinese holly edge lead plumbate, transmission
Electron microscopic observation.As a result show, ghost, which specific can be targetted, enters Hela cells, the form and internal structure of cell is not influenceed, to thin
Born of the same parents do not have toxicity.The Hela cells and normal Hela cell surfaces that ghost is incubated have in abundant microvillus, cytoplasm
Structure of mitochondria is complete and has the chromatin in many rough surfaced endoplasmic reticulum (RER)s and ribosomes, nucleus to be autosome and kernel
Clearly, Fig. 2 is seen.
3.3 ghosts as vaccine application
Immunization experiment is carried out using the BALB/c mouse of 4 week old, the immune effect of Escherichia coli O138 ghost is evaluated.
30 BALB/c mouses, male and female are not limited, and are randomly divided into 3 groups, every group 10, sub-cage rearing under the same terms, fasting before being immunized
24h.1 group is intraperitoneal injection immune group, every mouse peritoneal injection be immunized 100 μ l O138 ghosts PBS solutions (equivalent to 5 ×
l09Individual Ghost cells)；1 group is oral administration gavage immune group, and immune preceding 10min gavages the μ l of 10% sodium carbonate liquor 30 with irrigation stomach device
Hydrochloric acid in gastric juice is neutralized, 100 μ l O138 ghost PBS solutions are then gavaged again；Another 1 group is PBS control group, every mouse peritoneal injection 100
μ l PBS solutions.It is spaced booster immunization 1 time after 3 weeks.Two exempt from 2 weeks afterwards, and intraperitoneal injection Escherichia coli O138 bacterium solution carries out attacking poison, often
Mouse injects bacterium solution (about 4 × l0 of 5 times of minimal lethal doses7CFU), mouse survival situation is recorded, Escherichia coli are calculated
O138 ghosts the results are shown in Table 6 to the protective efficacy of mouse.It is all dead in control group mice 72h, and it is small that immune group is injected intraperitoneally
Mouse health survival, protective rate reaches 100%, and oral immunity group has the death in the 2nd day after poison is attacked of 1 mouse, and remaining mouse is equal
Health survival, protective rate reaches 90%.The result of the test shows that Escherichia coli O138 ghost has good immunogenicity, can make
Used for vaccine.
The immune protective effect of the Escherichia coli O138 ghost of table 6
Claims (2)
1. a kind of ghost preparation method of non-dependent Lysis gene E, it is characterized in that, by added in bacterial suspension NaOH,
Triton X-100、Na2EDTA and H2O2Four kinds of chemical substances prepare ghost；
The ghost is Escherichia coli O138 ghost, and preparation method specifically includes following steps：
（1）Minimal inhibitory concentration of the above-mentioned four kinds of chemical substances to Escherichia coli O138 is obtained using micro-broth dilution method
With smallest bacteria growth concentration；
The H2O2Use concentration for 30% H2O2；
Described NaOH, Na2EDTA、30% H2O2Minimal inhibitory concentration with Triton X-100 is 1.0 mg/ml, 0.93 mg/
Ml, 5.0 μ l/ml and 6.25 μ l/ml；Described NaOH, Na2EDTA、30% H2O2Given birth to Triton X-100 smallest bacteria
Long concentration is 0.15 mg/ml, 0.19 mg/ml, 0.80 μ l/ml and 0.78 μ l/ml；
（2）The optimal of above-mentioned four kinds of chemical substances required for ghost is prepared using Plackett-Burman experimental design acquisitions
Concentration；The optium concentration of acquisition is any one in following four mode：
①NaOH、Na2EDTA and 30%H2O2Concentration be smallest bacteria growth concentration, Triton X-100 concentration presses down to be minimum
Bacteria concentration；
2. NaOH and 30%H2O2Concentration be smallest bacteria growth concentration, Triton X-100 and Na2EDTA concentration is minimum
Mlc；
③NaOH、Na2EDTA and Triton X-100 concentration is smallest bacteria growth concentration, 30%H2O2Concentration press down to be minimum
Bacteria concentration；
4. NaOH and Na2EDTA concentration is smallest bacteria growth concentration, Triton X-100 and 30%H2O2Concentration for minimum
Mlc；
（3）By bacterial cultures by the way that thalline is collected by centrifugation, thalline is resuspended with distilled water after flushing；Will according to above-mentioned optium concentration
NaOH、Triton X-100、Na2EDTA and H2O2It is respectively added to cultivate in bacterial suspension；After culture is finished, mixture centrifugation
After rinse bacterial sediment, then by bacterial sediment be resuspended in sterilizing distilled water in, shaken at room temperature 4-6 min after freezing, after centrifugation
Bacterial sediment is rinsed, the bacterial sediment finally obtained as ghost takes a small amount of ghost to carry out living stems, should grown without viable bacteria；
Preserved after ghost is freeze-dried.
2. a kind of ghost preparation method of non-dependent Lysis gene E as claimed in claim 1, it is characterized in that, the large intestine bar
The preparation method of bacterium O138 ghosts is：By Escherichia coli O138 culture by the way that thalline is collected by centrifugation, after being rinsed with PBS
Thalline is resuspended with distilled water, adjustment bacterial concentration is（0.8-1.2）×l06CFU/ml；According to above-mentioned concentration by NaOH,
Triton X-100、Na2EDTA and H2O2It is respectively added to cultivate 50-70 minutes in bacterial suspension, PBS delays after mixture centrifugation
Fliud flushing rinses bacterial sediment, and then bacterial sediment is resuspended in sterilizing distilled water, shaken at room temperature 4-6 min after -20 DEG C of freezings,
PBS rinses bacterial sediment after centrifugation；The bacterial sediment finally obtained as ghost, takes a small amount of ghost to carry out viable bacteria inspection
Survey, should be grown without viable bacteria；Preserved after ghost is freeze-dried.
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