CN104830746A - Large-scale plasmid preparation process - Google Patents
Large-scale plasmid preparation process Download PDFInfo
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- CN104830746A CN104830746A CN201510236995.6A CN201510236995A CN104830746A CN 104830746 A CN104830746 A CN 104830746A CN 201510236995 A CN201510236995 A CN 201510236995A CN 104830746 A CN104830746 A CN 104830746A
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Abstract
The invention discloses a large-scale plasmid preparation process which comprises the following steps: preparing production seeds, performing high-density fermentation, thalli harvesting, removing RNA and extracting and purifying the plasmids. The large-scale plasmid preparation process disclosed by the invention is simple in method and convenient to operate, lots of plasmids can be produced, and the production cost is reduced.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to the extensive preparation technology of a kind of plasmid.
Background technology
DNA vaccination is the new generation vaccine grown up the nineties in 20th century, be after attenuated vaccine, recombinant vaccine the 3rd generation vaccine.DNA vaccination (DNAvaccine) starts the elements such as regulatory gene by the plasmid DNA (from bacterium) and eucaryon of inserting one or more foreign genes to form, the plasmid DNA being loaded with exogenous antigen, under the control of a kind of eukaryotic promoter and the genetic unit such as tailing signal and relevant enhanser, can give expression to relevant antigen protein in mammiferous various types of cells.Restructuring is had the plasmid of exogenous antigen encoding gene, utilizes and directly import someway in the cell of human or animal, by the re-reading system of host cell, by the active somatic cell synthetic antigen albumen of immunization body, thus induction body produces immunne response.After DNA plasmid is imported into host cell, the gene fragment of pathogen antigen obtains expressing and synthetic antigen in host cell, then through processing, processing, modify submission to immunity system, excites immunne response.This process is similar to cause pathogeny imcrobe infection or attenuated live vaccine inoculation, so DNA vaccination can excite humoral immunization and cellular immunization effectively, especially it has the effect activating cytotoxic T lymphocyte.
Extraction of plasmid DNA purifying is most basic work in molecular biology.Obtaining certain purity and a certain amount of plasmid DNA, is ensure the successful precondition of follow-up study work.If plasmid only needs electroresis appraisal, by simple bacteria lysis and alcohol settling step, obtain a small amount of plasmid DNA and can be competent at.But under normal circumstances, plasmid DNA also needs enzyme to cut, qualification of checking order, and be applied to the work such as in-vitro transcription, cell transfecting, just require that the plasmid DNA extracted has enough content and purity.Impurity main in plasmid extraction is genomic dna, RNA, protein, granulose (intracellular toxin) etc.Also the tramp material such as salt, organism may be remained in extraction purification.Phenol/chloroform or cesium chloride ultracentrifugation are the classical means of laboratory a small amount of and a large amount of plasmid purification.But phenol/chloroform only partly can remove protein impurities; Cesium chloride ultracentrifugation needs particular instrument, time and effort consuming, and with high costs.
Commercial plasmid extraction purification kit, is generally by some substrate materials absorption plasmid DNA, removes other impurity.The substrate material difference of all ingredients box because of employing and the difference of cleaning liquid formula, the total amount, the purity that obtain plasmid DNA are different.Generally speaking, post centrifugal purification method, a small amount of for ordinary plasmids is extracted and general application (as enzyme is cut, checked order) should be competent at.
A large amount of plasmid extraction purifying, adopts the centrifugal method of post, often has that the rate of recovery is low, quality is unstable, troublesome poeration, the shortcoming that wastes time and energy.A large amount of plasmid extraction purification techniques current in the world, be the method adopting special substrate material to utilize accepted filtered absorption and cleaning, this method almost becomes the unique selection of external common laboratory.When at present domestic there is no homotype production throughput, " noble quality " price of imported product, becomes the obstacle that this " ordinary " technology popularizes domestic laboratory.
In recent years, along with the expansion of molecular biology research, the clinical trial of nucleic acid vaccine, has higher requirement to the amount of plasmid extraction and purity.Therefore to the purifying of the plasmid of extensive high yield, be no matter adopt the centrifugal or accepted filtered absorption of post, all there is the difficulty of operation, result is difficult to the plasmid obtaining purifying.
Summary of the invention
An object of the present invention prepares the problem of difficulty on a large scale, provides a kind of convenient and simple plasmid extensive preparation technology.
The invention provides the extensive preparation technology of a kind of plasmid, comprise the following steps:
Prepared by seeding;
High density fermentation;
Thalline is gathered in the crops;
Remove RNA;
Extract plasmid;
Plasmid purification.
Further, described seeding fermentation step comprises:
Get colonies typical line LB agar plate, 37 DEG C of overnight incubation, wash down to LB liquid nutrient medium by the PBS solution of 0.01mol/L, pH7.2, be sub-packed in 5mL sterile vial, add 10% glycerine mixing, as first order seed ,-70 DEG C of preservations, inoculation first order seed is to the LB substratum containing 30 μ g/mL kantlex, the bacterium of phase of hastening towards saturation extracts plasmid, PCR primer order-checking qualification, produces be qualified first order seed without sudden change and the restructuring of other non-purpose;
Be inoculated in by qualified first order seed in 50ml Oscillating bottle substratum, put shaking table 37 DEG C, 200r/min cultivates 12h, as secondary seed, can be used for small-sized fermentation tank inoculation.As comparatively large in inoculated volume, can three-class strain be prepared.
Further, described high density fermentation step comprises:
Inoculation: load semi-synthetic nutrient solution in fermentor tank, after autoclaving, be cooled to 37 DEG C, secondary seed, glucose and kantlex is added respectively according to inoculation working specification, secondary seed final volume is 2% of the semi-synthetic nutrient solution volume of fermentor tank, and glucose final concentration is 2g/L, kantlex final concentration is 50mg/L.
Constant speed feeding culture mode: setting culture temperature 37 DEG C, pH value 7.2, according to dissolved oxygen changing conditions between incubation period, improve mixing speed and air flow gradually, make dissolved oxygen remain between 30% ~ 40%, start feed supplement when cultivating about 4h, feed rate is 100ml/h.Be cultured to 17h to stop cultivating, to zymocyte liquid.By automatically controlling the pH of alkali pump maintain base all the time in 7.0 ~ 7.2 scopes between incubation period.
Further, described thalline results step is, treat zymocyte liquid cool to room temperature in fermentation tank, open release bacterium fluid valve, with high pressure sterile gas, bacterium hydraulic pressure is entered hold-up vessel, thrust-augmenting nozzle is communicated with whizzer, open vertical streaming whizzer, when centrifuge speed stablizes 4000rpm, coutroi velocity, circular centrifugal, stop centrifugal when withdrawing fluid is clarified, results thalline, weigh, 60g every part packing, packing thalline is dissolved in the PBS solution of 500mL respectively, be placed in the centrifugal rotor of 1L volume respectively, the centrifugal 5min of 3000rpm, washing thalline once.Pack ,-20 DEG C save backup.
Further, described removal RNA step is, is taken out by point bacterium block installed from-20 DEG C of refrigerator-freezers, puts into the clean plastic barrel of 1L, adds 100mL cell suspension damping fluid, fully mix; Slow horizontal oscillations 1h in shaking table is educated in 37 DEG C of water.
Further, described cell suspension damping fluid comprises 50mmol/L, the Tris-HCl of pH7.5; The EDTA of 10mmol/L and the RNaseA of 100 μ g/mL.
Further, described extraction plasmid procedure is, in the container after vibration, add 100mL cell pyrolysis liquid, limit edged slowly mixes, and turn upside down 8 times, room temperature places 5 minutes; Slowly add 125mL neutralizer immediately, limit edged slowly mixes, and turn upside down 8 times, room temperature places 5 minutes, the centrifugal 10min of 4000rpm.
Further, described cell pyrolysis liquid comprises the NaOH solution of 0.2mol/L and the SDS solution of 1%, and described neutralizer is the liquor kalii acetici of pH4.8,1.32mol/L.
Further, described plasmid purification procedures is, get centrifugal after supernatant liquor by 8 layers of filtered through gauze in the 1000mL centrifuge tube of a cleaning, add the Virahol of 0.6 times of volume, in 4 DEG C after mixing, the centrifugal 15min of 4000rpm; Abandoning supernatant, adds the TE buffer solution precipitation of 20mL, and is is slowly blown and beaten evenly by solution with suction pipe, place 4 DEG C and spend the night.Again in the centrifugal 10min of 13000rpm, get supernatant liquor, add isopyknic 40%PEG8000, the centrifugal 10min of 13000rpm after mixing, abandoning supernatant, add the TE buffer solution precipitation of 20mL, measure solution O D260.
Beneficial effect of the present invention is: the extensive preparation technology of the plasmid that the present invention relates to, and method is simple, easy to operate, can production of plasmid in a large number, reduction production cost.Utilize vertical streaming whizzer can obtain bacterium mud fast and efficiently, be applicable to the in enormous quantities of bacterium liquid in industry and concentrate, its Quality and yield reaches standard.
Embodiment
Hereafter will describe the present invention in detail in conjunction with specific embodiments.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
The invention provides the extensive preparation technology of a kind of plasmid, comprise the following steps:
The first step: the preparation of seed lot
1) will transform the separation and purification of K12DH-5 α freeze-drying lactobacillus line LB agar plate, restriction enzyme cuts identifies recombinant plasmid with PCR.Get colonies typical line LB agar plate, 37 DEG C of overnight incubation, wash down to LB liquid nutrient medium by the PBS solution of 0.01mol/L, pH7.2, packing 5mL sterile vial, add 10% glycerine mixing, as first order seed, and-70 DEG C of preservations.Inoculation first order seed is to the LB substratum containing 30 μ g/mL kantlex, and the bacterium of the phase that hastens towards saturation extracts plasmid, PCR primer order-checking qualification, should produce without sudden change and other non-purpose restructuring.
2) be inoculated in by first order seed in 50ml Oscillating bottle substratum, put shaking table 37 DEG C, 200r/min cultivates 12h, as secondary seed, can be used for small-sized fermentation tank inoculation.As comparatively large in inoculated volume, can three-class strain be prepared.
Second step: fermentation culture
1) inoculate: in fermentor tank, load semisynthetic medium, after autoclaving, be cooled to 37 DEG C, secondary seed, glucose and kantlex is added respectively according to inoculation working specification, the secondary seed volume added is 2% of semi-synthetic nutrient solution volume, and the final concentration adding glucose and kantlex is 2g/L, 50mg/L.
2) constant speed feeding culture mode: setting culture temperature 37 DEG C, pH value 7.2, according to dissolved oxygen changing conditions between incubation period, improve mixing speed and air flow gradually, make dissolved oxygen remain between 30% ~ 40%, start feed supplement when cultivating about 4h, feed rate is 100ml/h.Be cultured to 17h to stop cultivating.By automatically controlling the pH of alkali pump maintain base all the time in 7.0 ~ 7.2 scopes between incubation period.
3) fermenting process detects:, suitably dilute, make the OD600 of dilution between 0.1 ~ 0.7, depict the growth curve of bacterium according to measured data by purified water after cultivation 8h every 2h sampling once.
4) plasmid stability inspection: get fermented liquid 1mL, after dilution suitable multiple, get 0.1mL dibbling contain and do not contain on substratum two kinds of flat boards of kantlex, respectively in triplicate, calculate the bacterium number of two kinds of grow on plates, the ratio calculated with plating dilutions counting process compares, and the miss rate of checking plasmid, bacterium colony ratio is considered as stablizing higher than 99%.
3rd step: thalline is gathered in the crops
Treat zymocyte liquid cool to room temperature in fermentation tank, open release bacterium fluid valve, with high pressure sterile gas, bacterium hydraulic pressure is entered hold-up vessel, thrust-augmenting nozzle is communicated with whizzer, open vertical streaming whizzer, when centrifuge speed stablizes 4000rpm, coutroi velocity, circular centrifugal, stop centrifugal when withdrawing fluid is clarified, results thalline, weighs, 60g every part packing, respectively packing thalline is dissolved in the PBS solution of 500mL, be placed in the centrifugal rotor of 1L volume respectively, the centrifugal 5min of 3000rpm, washing thalline once.Pack ,-20 DEG C save backup.
The removal of the 4th step: RNA
Point bacterium block installed is taken out from-20 DEG C of refrigerator-freezers, puts into the clean plastic barrel of 1L, add the 100mL cell suspension damping fluid (Tris-HCl of 50mmol/L, pH 7.5; The EDTA of 10mmol/L; The RNaseA of 100 μ g/mL), fully mix; Slow horizontal oscillations 1h in 37 DEG C of shaking baths.
5th step: plasmid extraction purifying
Plasmid extraction: add 100mL cell pyrolysis liquid in the container after vibration, limit edged slowly mixes, and turn upside down 8 times, room temperature places 5 minutes; Slowly add 125mL neutralizer immediately, limit edged slowly mixes, and turn upside down 8 times, room temperature places 5 minutes, the centrifugal 10min of 4000rpm.Described cell pyrolysis liquid comprises the NaOH solution of 0.2mol/L and the SDS solution of 1%, and described neutralizer is the liquor kalii acetici of pH4.8,1.32mol/L.
Plasmid prepares the purification of liquid and concentrated: get centrifugal after supernatant liquor by 8 layers of filtered through gauze in the 1000mL centrifuge tube of a cleaning, add the Virahol of 0.6 times of volume, mix latter 4 DEG C, the centrifugal 15min of 4000rpm; Abandon supernatant liquor, respectively add the TE buffer solution precipitation of 20mL, and with suction pipe, solution is is slowly blown and beaten evenly, place 4 DEG C and spend the night.The centrifugal 10min of 13000rpm, gets supernatant, adds isopyknic 40%PEG8000, and the centrifugal 10min of 13000rpm after mixing, abandons supernatant liquor, adds the TE buffer solution precipitation of 20mL, measures solution O D260.
The extensive preparation technology of the plasmid that the present invention relates to, method is simple, easy to operate, can production of plasmid in a large number, reduction production cost.Utilize vertical streaming whizzer can obtain bacterium mud fast and efficiently, be applicable to the in enormous quantities of bacterium liquid in industry and concentrate, its Quality and yield reaches standard.
Although give some embodiments of the present invention, it will be understood by those of skill in the art that without departing from the spirit of the invention herein, can change embodiment herein.Above-described embodiment is exemplary, should using embodiment herein as the restriction of interest field of the present invention.
Claims (9)
1. the extensive preparation technology of plasmid, is characterized in that, comprise the following steps:
Prepared by seeding; High density fermentation; Thalline is gathered in the crops; Remove RNA; Extract plasmid; Plasmid purification;
Described seeding preparation process comprises: get colonies typical line LB agar plate, 37 DEG C of overnight incubation, wash down to LB liquid nutrient medium by the PBS solution of 0.01mol/L, pH7.2, be sub-packed in sterilizing bottle, add 10% glycerine mixing, as first order seed ,-70 DEG C of preservations, inoculation first order seed is to the LB substratum containing 30 μ g/mL kantlex, the bacterium of phase of hastening towards saturation extracts plasmid, PCR primer order-checking qualification, produces be qualified first order seed without sudden change and the restructuring of other non-purpose;
Be inoculated in by described qualified first order seed in 50ml Oscillating bottle substratum, put shaking table 37 DEG C, 200r/min cultivates 12h, as secondary seed.
2. the extensive preparation technology of plasmid as claimed in claim 1, it is characterized in that, described high density fermentation step comprises:
After semi-synthetic nutrient solution autoclaving, be cooled to 37 DEG C, add described secondary seed, glucose and kantlex respectively;
Setting culture temperature 37 DEG C, pH value 7.2, keep dissolved oxygen 30% ~ 40%, start feed supplement after cultivating 4h, feed rate is 100ml/h, and stop cultivating after cultivating 17h, obtain zymocyte liquid, between incubation period, the pH of maintain base is in 7.0 ~ 7.2 scopes.
3. the extensive preparation technology of plasmid as claimed in claim 2, it is characterized in that, described secondary seed add-on is 2% of described semi-synthetic nutrient solution volume, and glucose add-on is 2g/L for making its final concentration, and kantlex add-on is 50mg/L for making its final concentration.
4. the extensive preparation technology of plasmid as claimed in claim 3, is characterized in that, described thalline results step is, treat described zymocyte liquid cool to room temperature, 4000rpm circular centrifugal, stop centrifugal when withdrawing fluid is clarified, results thalline, weighs, 60g every part packing, packing thalline is dissolved in the PBS solution of 500mL respectively, be placed in the centrifugal rotor of 1L volume respectively, the centrifugal 5min of 3000rpm, then wash thalline once, pack ,-20 DEG C save backup.
5. the extensive preparation technology of plasmid as claimed in claim 4, it is characterized in that, described removal RNA step is that the bacterium block installed described point takes out from-20 DEG C of conditions, pours container into, adds 100mL cell suspension damping fluid, fully mixes; Slow horizontal oscillations 1h in shaking table is educated in 37 DEG C of water.
6. the extensive preparation technology of plasmid as claimed in claim 5, it is characterized in that, described cell suspension damping fluid comprises 50mmol/L, the Tris-HCl of pH7.5; The EDTA of 10mmol/L and the RNaseA of 100 μ g/mL.
7. the extensive preparation technology of plasmid as claimed in claim 6, it is characterized in that, described extraction plasmid procedure is, in the container after vibration, add 100mL cell pyrolysis liquid, limit edged slowly mixes, and turns upside down, and room temperature places 5 minutes; Slowly add 125mL neutralizer immediately, limit edged slowly mixes, and again turns upside down, and room temperature places 5 minutes, the centrifugal 10min of 4000rpm.
8. the extensive preparation technology of plasmid as claimed in claim 7, it is characterized in that, described cell pyrolysis liquid comprises the NaOH solution of 0.2mol/L and the SDS solution of 1%, and described neutralizer is the liquor kalii acetici of pH4.8,1.32mol/L.
9. the extensive preparation technology of plasmid as claimed in claim 8, it is characterized in that, described plasmid purification procedures is, get centrifugal after supernatant liquid filtering, add the Virahol of 0.6 times of volume, in 4 DEG C after mixing, the centrifugal 15min of 4000rpm; Abandoning supernatant, adds the TE buffer solution precipitation of 20mL, and is is slowly blown and beaten evenly by solution with suction pipe, place 4 DEG C and spend the night; Again in the centrifugal 10min of 13000rpm, get supernatant liquor, add isopyknic 40%PEG8000, the centrifugal 10min of 13000rpm after mixing, abandoning supernatant, add the TE buffer solution precipitation of 20ml, measure solution O D260.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106754885A (en) * | 2017-01-18 | 2017-05-31 | 河南师范大学 | A large amount of preparation methods of Tagln functional study related plasmids |
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| US20010034435A1 (en) * | 1996-07-19 | 2001-10-25 | Valentis, Inc. | Process and equipment for plasmid purification |
| CN1737137A (en) * | 2005-08-15 | 2006-02-22 | 朱鸿飞 | Plasmid DNA large scale purification process |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754885A (en) * | 2017-01-18 | 2017-05-31 | 河南师范大学 | A large amount of preparation methods of Tagln functional study related plasmids |
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