CN106434631B - A kind of method and its application of large quantity extracting plasmid - Google Patents
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Abstract
The invention discloses a kind of method and its application of large quantity extracting plasmid.GoldenView is applied to the nucleic acid in the extraction of CsCl plasmid and developed the color by the method that the plasmid extracts, the ethidium bromide (EB) that substitution tradition uses.Compared with existing application EB carries out CsCl plasmid extracting technology, the amount for extracting plasmid using GoldenView is slightly higher, and it extracts endotoxin, protein residue in plasmid and is not detected, illustrate to guarantee the extracted amount for not influencing plasmid using GoldenView dyestuff in the extraction of CsCl plasmid and extracts quality, GoldenView can also substantially mitigate dyestuff to the toxic effect of experimenter simultaneously, have apparent advantage compared with prior art.
Description
Technical field
The present invention relates to molecular biology fields, more particularly to a kind of method and its application of a large amount of extracting plasmids.
Background technique
Plasmid be the hereditary unit independently replicated is able to carry out outside chromosome, including Eukaryotic organelle and bacterium it is thin
DNA (DNA) molecule in born of the same parents other than chromosome.Currently, plasmid usually refers exclusively to bacterium, saccharomycete and actinomyces etc.
DNA molecular in biology other than chromosome.
A useful target DNA fragment by recombinant DNA technology, it is sent into recipient cell and goes to be bred and be expressed
Tool carrier (Vector).Bacterial plasmid is one of most common carrier in recombinant DNA technology.Plasmid molecule itself is to contain
There is the genetic structure of copy function.Plasmid also has certain hereditary information, so can assign host cell some inhereditary feature ratios
Such as marker gene.It is right in (such as amplification, screening process) that its of self-replication capacity and entrained hereditary information are operated in recombinant DNA
Genetic engineering research and application downstream are all very useful and valuable.
In R & D of complex, demand of the research staff to plasmid amount is generally little, and the plasmid of μ g rank is sufficient for
The demand of a large amount of research and development experiments；But in actual industrial application, the usage amount of plasmid often than research and development when high 2-3
The order of magnitude reaches 100 μ g or even 1000 μ g ranks.With the anti-CD19 chimeric antigen for treating acute lymphoblastic leukemia by
For body T (CAR-T) cell, the process of the therapeutic scheme approximately as: extract the blood of patient first, separate periphery therein
Blood mononuclear cell, and Fiber differentiation T cell therein；Then it is slow passing through for anti-CD19 single-chain antibody (scFv) gene will to be expressed
Virus is imported into T cell, and continues to cultivate these cells；When the quality and quantity of these cells reaches the standard of clinical use
Afterwards, these cells can be fed back to patient's body and carry out treatment.In this process, the most key is exactly that channel genes are thin
Slow virus used intracellular.In the prior art, 293T cell and slow virus plasmid, auxiliary are mainly used for the slow virus for the treatment of
Plasmid carries out and packs and purify obtained.The step for it is more demanding to the amount of plasmid, generally can reach hundreds of μ g ranks.
In the prior art, the method for large quantity extracting plasmid mainly includes by plasmid adsorption column and by cesium chloride (CsCl)
Two kinds of ultracentrifugation.By the method for plasmid adsorption column, it is only necessary to which about 2 hours can extract from the bacterium solution of 400mL or so
The plasmid of 1000 μ g or so out；And centrifuged overnight is then needed by the ultracentrifugal method of CsCl, the time is relatively long.But CsCl
Supercentrifugation is much better than plasmid adsorption column method in other respects:
1, bacterial genomes DNA pollution is substantially absent.In contrast, the plasmid that plasmid adsorption column method is extracted is by bacterium
The potential risk of contaminating genomic DNA wants high many.
2, covalent, closure, ring-shaped DNA molecule (cccDNA) are that plasmid normally plays a role in the later period in the plasmid extracted
Principal mode, by the plasmid that CsCl supercentrifugation is extracted, the ratio of cccDNA is significantly higher than to be mentioned by plasmid adsorption column method
The plasmid taken.
3, by ultracentrifugation, bacterial endotoxin can be removed more thoroughly.
Therefore carrying out a large amount of extractions of plasmid by supercentrifugation is a kind of better method, this is by the art
Experimenter is received.
During CsCl supercentrifugation large quantity extracting plasmid, need that nucleic acid dye is added so that plasmid hypervelocity from
It develops the color after the heart, experimenter is facilitated to draw by utensils such as syringes to it.Nucleic acid dye commonly used in the prior art is
Ethidium bromide (EB), this is a kind of very high nucleic acid dye of sensitivity, can detect the DNA down to 10ng.But EB is a kind of
Strong mutagens, carcinogenicity with higher, therefore to being used for a long time for the experimenter of the nucleic acid dye, how both to protect
The health of oneself, obtaining ideal experimental result again, to become one for having to face in their routine works important
Problem.There has been no the methods that can solve the problems, such as very well this in the prior art.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of method of large quantity extracting plasmid, feature
As follows: the novel GoldenView dyestuff of application substitutes highly toxic EB dyestuff and carries out CsCl ultracentrifugation extraction plasmid.
The specific technical solution of the present invention is as follows:
S1, it will be seeded in 400mL TB culture medium containing the Escherichia coli for having purpose plasmid, 37 DEG C, 200rpm was cultivated
Night.Then 4 DEG C, 6000g is centrifuged 10min, abandons supernatant, and being inverted centrifuge tube makes remaining liquid outflow.
S2, the P1 buffer for being cooled to 4 DEG C in advance is added to final volume to 10mL, concussion, which is resuspended, divides the bacterium of precipitating uniformly
It dissipates.The ingredient of the P1 buffer is 50mmol/L Tris-HCl, 10mmol/L EDTA, 100ug/mL Rnase A, pH=
8.0。
S3,20mg solid lysozyme (hen egg white lysozyme) is added, precipitating is resuspended completely, is placed at room temperature for
10min。
S4, the freshly prepared P2 solution of 20mL is added, is mixed gently with pipette until solution transparent and homogeneous, is placed at room temperature for
5-10min.The ingredient of the P2 buffer is 200mmol/L NaOH, 1%SDS.
S5, the pre- P3 solution 10mL for being cooled to 4 DEG C is added, gently but fully concussion mixes for several times, until two liquid phases are not
It separates again, original yellow disappears, and White Flocculus occurs, and is subsequently placed in 10min on ice.The ingredient of the P3 solution is
3mol/L KAc, pH=4.8.
S6,4 DEG C, 8000rpm are centrifuged 10min, abandon precipitating, by supernatant through four layers of filtered through gauze into graduated cylinder.
S7, the isopropanol that 0.6 times of volume is added are mixed, are placed at room temperature for 10-15min, are subsequently placed in 50mL centrifuge tube.
, will be clean at supernatant abandoning after S8,8000rpm are centrifuged 15min, recycling nucleic acid precipitating.
S9, washing precipitating.At room temperature, tube bottom and tube wall are rinsed with 70% ethyl alcohol of 2mL.Then ethyl alcohol is poured out, and inhaled
Drop on main pipe wall；Or 8000rpm, room temperature are centrifuged 10min, abandon supernatant.Centrifuge tube nozzle 10-15min is opened wide, is made remaining
Ethyl alcohol vapors away completely.
S10,1 × TE of 7mL solution dissolution precipitating is added, and the 7mL solution is added and rinses tube wall.
S11, CsCl 12.5g is added, dissolution mixes.
S12, it is mixed after being added 10 microlitres of GoldenView nucleic acid dye.
S13, the above-mentioned solution of transfer are into ultracentrifugation pipe, full of centrifuge tube and balanced seal.
S14, at room temperature 55000rpm are centrifuged at least 12h or 65000rpm and are centrifuged 4h.
After S15, centrifugation, centrifuge tube is taken out, is placed on the rack for test tube of masking foil package, in the room of only low-light
It is interior, it is seen that two region of DNA bands, top regions band are chromosome and linear DNA, and lower layer is required cccDNA.
S16, with No. 18 needle tubings, careful collection cccDNA is placed in 50mL centrifuge tube.
S17, isometric water saturation isoamyl alcohol is added, covers tightly pipe lid, concussion mixing organic phase and water phase.
S18, at room temperature 450g are centrifuged 3min, separate water phase and organic phase, and upper organic phase (pink) is shifted
Into suitable waste liquid barrel.
S19, extracting above three step 4-6 times is repeated, until water phase and organic phase lose green.
S20, the water that 2.2 times of volumes are added, the isopropanol of 0.6 times of volume mix.
S21, at room temperature, 8000rpm are centrifuged 15min, completely abandoning supernatant, and recycle nucleic acid precipitating.
S22,8mL ultrapure water is added precipitating is resuspended, add 10mol/L ammonium acetate solution to final concentration 2mol/L, sufficiently
2.5-3 times of volume is added after mixing, is cooled to -20 DEG C of ethyl alcohol (- 20 DEG C) in advance, mixes, is placed in dry ice or -80 DEG C of refrigerators, incubates
Educate 10min.
S23,4 DEG C, 8000rpm are centrifuged 10min, abandon supernatant.
S24,70% ethyl alcohol of 20mL is added, mixing, 4 DEG C, 12000g is centrifuged 10min, abandons supernatant.
S25, centrifuge tube is placed in super-clean bench air-dried 2min, TE is added and dissolves plasmid.
Compared with prior art, beneficial effects of the present invention are as follows: the present invention extracts traditional CsCl supercentrifugation
Plasmid is transformed, and using novel GoldenView dyestuff instead of highly toxic EB dyestuff, is reducing experimental implementation pair
While the genotoxic potential risk of researcher, it is ensured that the yield and quality of extracted plasmid is without being decreased obviously.
Detailed description of the invention
Fig. 1 is to carry out the extraction of CsCl plasmid using GoldenView and EB, obtains the result figure of plasmid total amount.
Fig. 2 is the electrophoresis result figure that CsCl extracts plasmid.A. the result (3 of plasmid extraction is carried out using GolenView dyestuff
Secondary repetition)；B. the result (3 repetitions) of plasmid extraction is carried out using EB dyestuff.
Fig. 3 is BSA canonical plotting.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies specific material proportion, process conditions and its result described in example and is merely to illustrate the present invention, without that should will not limit
The present invention described in detail in claims processed.Method as used in the following examples is conventional side unless otherwise instructed
Method, specific steps can be found in: " Molecular Cloning:A Laboratory Manual " (Sambrook, J.,
Russell,David W.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,
Cold Spring Harbor)。
CsCl used in the present invention is purchased from Sigma Aldrich, and GoldenView dyestuff is purchased from Beijing SBS Genetech gene
Technology Co., Ltd..Other experiment reagents and consumptive material are as being commercial goods without clearly stating.
The culture of one Escherichia coli of embodiment
The 10 μ L of Escherichia coli containing plasmid is taken, is placed in 400mL TB culture medium, 37 DEG C, after 200rpm cultivates 14h, 4
DEG C, 6000g is centrifuged 10min and collects thallus.
Two CsCl ultracentrifugation of embodiment extracts plasmid
Plasmid is extracted using CsCl supercentrifugation according to the following steps:
1, the P1 buffer for being cooled to 4 DEG C in advance is added to 10mL, above-mentioned thallus is resuspended, concussion, which is resuspended, keeps the bacterium of precipitating uniform
Dispersion.
2,20mg solid lysozyme (hen egg white lysozyme) is added, precipitating is resuspended completely, is placed at room temperature for
10min。
3, the freshly prepared P2 solution of 20mL is added, is mixed gently with pipette until solution transparent and homogeneous, is placed at room temperature for
5-10min。
4, the pre- P3 solution 10mL for being cooled to 4 DEG C is added, gently but fully concussion mixes for several times, until two liquid phases are no longer
Separation, original yellow disappear, and White Flocculus occurs, and are subsequently placed in 10min on ice.
5,4 DEG C, 8000rpm is centrifuged 10min, abandons precipitating, by supernatant through four layers of filtered through gauze into graduated cylinder.
6, the isopropanol of 0.6 times of volume is added, mixes, is placed at room temperature for 10-15min, is subsequently placed in 50mL centrifuge tube.
7, will be clean at supernatant abandoning after 8000rpm is centrifuged 15min, recycling nucleic acid precipitating.
8, washing precipitating.At room temperature, tube bottom and tube wall are rinsed with 70% ethyl alcohol of 2mL.Then ethyl alcohol is poured out, and inhaled
Drop on main pipe wall；Or 8000rpm, room temperature are centrifuged 10min, abandon supernatant.Centrifuge tube nozzle 10-15min is opened wide, is made remaining
Ethyl alcohol vapors away completely.
9,1 × TE of 7mL solution dissolution precipitating is added, and the 7mL solution is added and rinses tube wall.
10, cesium chloride 12.5g is added, dissolution mixes.
11, it is mixed after being added 10 microlitres of GoldenView nucleic acid dye.
12, above-mentioned solution is shifted into ultracentrifugation pipe, full of centrifuge tube and balanced seal.
13,55000rpm is centrifuged at least 12h or 65000rpm and is centrifuged 4h at room temperature.
14, after being centrifuged, centrifuge tube is taken out, is placed on the rack for test tube of masking foil package, in the interior of only low-light,
It can be seen that two region of DNA bands, top regions band is chromosome and linear DNA, and lower layer is required cccDNA.
15, with No. 18 needle tubings, careful collection cccDNA is placed in 50mL centrifuge tube.
16, isometric water saturation isoamyl alcohol is added, covers tightly pipe lid, concussion mixing organic phase and water phase.
17,450g is centrifuged 3min at room temperature, separates water phase and organic phase, and upper organic phase (pink) is transferred to
In suitable waste liquid barrel.
18, extracting above three step 4-6 times is repeated, until water phase and organic phase lose green.
19, the water of 2.2 times of volumes is added, the isopropanol of 0.6 times of volume mixes.
20, at room temperature, 8000rpm is centrifuged 15min, completely abandoning supernatant, and recycles nucleic acid precipitating.
21,8mL ultrapure water is added and precipitating is resuspended, add 10mol/L ammonium acetate solution to final concentration 2mol/L, sufficiently mix
2.5-3 times of volume is added after even, is cooled to -20 DEG C of ethyl alcohol (- 20 DEG C) in advance, mixes, is placed in dry ice or -80 DEG C of refrigerators, is incubated for
10min。
22,4 DEG C, 8000rpm is centrifuged 10min, abandons supernatant.
23,70% ethyl alcohol of 20mL is added, mixing, 4 DEG C, 12000g is centrifuged 10min, abandons supernatant.
24, centrifuge tube is placed in super-clean bench and air-dries 2min, TE is added and dissolves plasmid.
The detection of three plasmid extracted amount of embodiment
The sample arm for lifting 2000 detector of Thermo Nanodrop takes 1 μ L TE solution to be added in detection pedestal, uses
It returns to zero in instrument, after the completion with clean cleansing tissue by pedestal wiped clean；Then the plasmid for taking 1 μ L to be dissolved in TE again is added to detection
On pedestal, then sample arm is put down, running control software reads light absorption value；Plasmid is estimated according to light absorption value by software again
Concentration.
It is calculated according to extracted amount of the following formula to plasmid: n=C × V.Wherein n is the extracted amount of plasmid, and C is matter
The concentration of grain, V are the volume for dissolving TE used in plasmid.
Inventor respectively repeats plasmid with GoldenView and ethidium bromide (EB) and extracts experiment three times, extracts situation such as following table
It is shown:
1 plasmid of table extracts situation
T check analysis is carried out to these data, as a result as shown in Figure 1.It can be seen that being extracted using GoldenView dyestuff
The total amount of gained plasmid is higher than using the extraction of EB dyestuff, but without significant difference (p > 0.05).
Example IV extracts the endotoxic detection of plasmid
The endotoxin in gel method reagents detection Plasmid samples produced using Zhanjiang Andusi Biology Co., Ltd. is residual
It stays.With ultrapure water by endotoxin standard be diluted to 10EU/mL, 1EU/mL, 0.5EU/mL, 0.25EU/mL, 0.125EU/mL,
6 concentration gradients of 0.0625EU/mL make standard curve；(MVD=is diluted to sample by gel method reagents specification again
CL/ λ), and carry out endotoxic detection.Each sample carries out 2 repetitions, and it is in interior that acquired results, which show whole Plasmid samples,
Toxin is negative, shows contained endotoxin Jun≤5EU/mL in Plasmid samples, i.e., is 5EU/mL wanting sample control endotoxin limit value
Range illustrates that endotoxin content is extremely low in Plasmid samples.
The detection of the extraction plasmid residual protein of embodiment five
The BCA detection kit produced using Beijing Quanshijin Biotechnology Co., Ltd is to the residual egg extracted in plasmid
White matter content is measured.With ultrapure water compound concentration be respectively 0,25ng/ μ L, 50ng/ μ L, 100ng/ μ L, 200ng/ μ L,
The BSA standard items of 300ng/ μ L, 400ng/ μ L and 500ng/ μ L, draw standard curve, as a result table 2 and as shown in Figure 3.It can see
It arrives, light absorption value and the linear pertinent trends of standard concentration, r2Value is greater than 0.99.
2 BSA Specification Curve of Increasing data of table
| Standard concentration | 0 | 25 | 50 | 100 | 200 | 300 | 400 | 500 |
| Light absorption value | 0.169 | 0.186 | 0.210 | 0.244 | 0.335 | 0.417 | 0.488 | 0.551 |
The inspection that sample carries out residual protein content is extracted to 6 plasmids obtained in embodiment three according to same steps
It surveys, light absorption value is as shown in table 3.It can be seen that it is 0 that 6 obtained absorbance values of sample detection, which are respectively less than standard concentration,
When corresponding absorbance value, do not detect protein residues, illustrate that the purity for extracting plasmid is very high.
36 Plasmid samples residual protein testing results of table
| Sample name | GoldenView-1 | GoldenView-2 | GoldenView-3 | EB-1 | EB-2 | EB-3 |
| Light absorption value | 0.164 | 0.165 | 0.166 | 0.163 | 0.165 | 0.163 |
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (1)
1. a kind of method of large quantity extracting plasmid, which comprises the steps of:
S1, it will be seeded in 400mL TB culture medium containing the Escherichia coli for having purpose plasmid, 37 DEG C, 200rpm overnight incubation；So
4 DEG C afterwards, 6000g centrifugation 10min, abandon supernatant, and being inverted centrifuge tube makes remaining liquid outflow；
S2, the P1 buffer for being cooled to 4 DEG C in advance is added to final volume to 10mL, concussion, which is resuspended, keeps the bacterium of precipitating evenly dispersed, institute
The RNase A of EDTA, 100ug/mL of Tris-HCl, 10mmol/L that P1 buffer includes 50mmol/L are stated, and the P1 is slow
The pH of fliud flushing is 8.0；
Precipitating is resuspended completely in S3, the lysozyme that 20mg is added, and is placed at room temperature for 10min, and the lysozyme is solid, and described molten
Bacterium enzyme is hen egg white lysozyme；
S4, the freshly prepared P2 solution of 20mL is added, is mixed gently with pipette until solution transparent and homogeneous, is placed at room temperature for 5min
~10min, the P2 solution includes the NaOH of 200mmol/L and mass percentage is 1%SDS；
S5, the pre- P3 solution 10mL for being cooled to 4 DEG C is added, gently but fully concussion mixes for several times, until two liquid phases are no longer divided
From original yellow disappears, and White Flocculus occurs, and is subsequently placed in 10min on ice, and the P3 solution is pH4.8,2mol/L
KAc aqueous solution；
S6,4 DEG C, 8000rpm centrifugation 10min, abandon precipitating, by supernatant through four layers of filtered through gauze into graduated cylinder；
S7, the isopropanol that 0.6 times of volume is added are mixed, are placed at room temperature for 10min~15min, are placed in 50mL centrifuge tube；
, will be clean at supernatant abandoning after S8,8000rpm are centrifuged 15min, recycling nucleic acid precipitating；
S9, the washing nucleic acid precipitating: tube bottom and tube wall are rinsed with 70% ethyl alcohol of 2mL at room temperature, then pours out ethyl alcohol simultaneously
Drop or 8000rpm room temperature the centrifugation 10min blotted on tube wall abandons supernatant；Centrifuge tube nozzle 10min~15min is opened wide, makes to remain
Remaining ethyl alcohol vapors away completely；
S10,1 × TE of 7mL solution dissolution precipitating is added, and 7mL 1 × TE solution is added and rinses tube wall；
S11, the cesium chloride that 12.5g is added, dissolution mix；
S12, the GoldenView nucleic acid dye mixing for being added 10 microlitres；
S13, the solution after mixing is transferred in ultracentrifugation pipe, full of centrifuge tube and balanced seal；
S14, at room temperature 55000rpm are centrifuged at least 12h or 65000rpm and are centrifuged 4h；
After S15, centrifugation, centrifuge tube is taken out and is placed on the rack for test tube of masking foil package, it, can in the interior of only low-light
The centrifuge tube after seeing centrifugation has two region of DNA bands, and top regions band is chromosome and linear DNA, and lower layer is required
cccDNA；
S16, with No. 18 needle tubing careful collection cccDNA, be placed in 50mL centrifuge tube；
S17, isometric water saturation isoamyl alcohol is added, covers tightly pipe lid, concussion mixing organic phase and water phase；
S18, at room temperature 450g are centrifuged 3min, separate water phase and organic phase, and the organic phase on upper layer is transferred to properly
Waste liquid barrel in, the organic phase pinkiness；
S19, step 4 time~6 time for repeating step S16~S17, until water phase and organic phase lose green；
The isopropanol of water and 0.6 times of volume that 2.2 times of volumes are added in S20, Xiang Suoshu water phase mixes；
S21, at room temperature 8000rpm are centrifuged 15min, abandon supernatant, and recycle nucleic acid precipitating；
S22,8mL ultrapure water resuspension precipitating is added, adds 10mol/L ammonium acetate solution to final concentration 2mol/L, mixes well
The ethyl alcohol that 2.5 times~3 times volumes are added afterwards, are cooled to -20 DEG C in advance, mixes, is placed in dry ice or -80 DEG C of refrigerators, is incubated for 10min；
S23,4 DEG C, 8000rpm centrifugation 10min, abandon supernatant；
S24, the mixing of 70% ethyl alcohol of 20mL is added, 4 DEG C, 12000g centrifugation 10min abandon supernatant；
S25, the centrifuge tube equipped with precipitating is placed in super-clean bench air-dried 2min, TE dissolution is added, obtains plasmid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5428896A (en) * | 1977-08-08 | 1979-03-03 | Mitsubishi Chem Ind Ltd | Preparation of plasmid |
| US6214586B1 (en) * | 1997-12-08 | 2001-04-10 | Genzyme Corporation | Method for purifying plasmid DNA and plasmid DNA substantially free of genomic DNA |
-
2016
- 2016-09-28 CN CN201610857441.2A patent/CN106434631B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5428896A (en) * | 1977-08-08 | 1979-03-03 | Mitsubishi Chem Ind Ltd | Preparation of plasmid |
| US6214586B1 (en) * | 1997-12-08 | 2001-04-10 | Genzyme Corporation | Method for purifying plasmid DNA and plasmid DNA substantially free of genomic DNA |
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| CN106434631A (en) | 2017-02-22 |
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Inventor after: Yi Jihui Inventor after: Chen Xin Inventor after: Xu Chunlian Inventor after: Mao Kanlang Inventor before: Chen Xin |
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