CN106434631A

CN106434631A - Method for extracting large number of plasmids and applications of method

Info

Publication number: CN106434631A
Application number: CN201610857441.2A
Authority: CN
Inventors: 陈鑫
Original assignee: Shenzhen Gentarget Medical Technology Co Ltd
Current assignee: Shenzhen Gentarget Medical Technology Co Ltd
Priority date: 2016-09-28
Filing date: 2016-09-28
Publication date: 2017-02-22
Anticipated expiration: 2036-09-28
Also published as: CN106434631B

Abstract

The invention discloses a method for extracting a large number of plasmids and applications of the method. According to the method for extracting the large number of plasmids, GoldenView is applied to nucleic acid color development during the CsCl plasmid extraction, and replaces ethidium bromide (EB) used in the prior art. Compared with the existing CsCl plasmid extraction technology adopting EB, the number of the plasmids extracted by adopting GoldenView is larger, and no residual endotoxin or protein is detected in the extracted plasmids, which shows that during the CsCl plasmid extraction, with the adoption of the GoldenView dye, the extracting number and quality of the plasmids are not influenced, meanwhile, due to the GoldenView, the toxic effects of dyes to experimenters can be greatly alleviated, and therefore, compared with the prior art, the method has obvious advantages.

Description

A kind of method of large quantity extracting plasmid and application thereof

Technical field

The present invention relates to biology field, more particularly to method and the application thereof of a kind of a large amount of extracting plasmids.

Background technology

Plasmid is the hereditary unit that can independently replicate outside chromosome, including Eukaryotic organelle and bacterium are thin DNA beyond chromosome (DNA) molecule in born of the same parents.At present, plasmid generally refers exclusively to bacterium, saccharomycete and actinomyces etc. DNA molecular beyond chromosome in Sheng Wu.

One useful target DNA fragment is passed through recombinant DNA technology, is sent in recipient cell and goes to breed and express Instrument carrier (Vector).Bacterial plasmid is one of carrier the most frequently used in recombinant DNA technology.Plasmid molecule itself is to contain There is the genetic structure of copy function.Plasmid is also with some hereditary information, so can give host cell some inhereditary feature ratios Such as marker gene.It is right that its of self-replication capacity and entrained hereditary information operate in (such as amplification, screening process) at recombinant DNA Genetic engineering research and application downstream are all very useful and valuable.

In R ＆ D of complex, research staff is typically little to the demand of plasmid amount, and the plasmid of μ g rank is sufficient for The demand of a large amount of research and development experiments；But in actual commercial application, the usage amount of a plasmid often ratio high 2-3 when researching and developing The order of magnitude, reaches 100 μ g or even 1000 μ g ranks.Anti-CD19 chimeric antigen for treating ALL is subject to As a example by body T (CAR-T) cell, the flow process of this therapeutic scheme approximately as：First extract the blood of patient, separate periphery therein Blood mononuclear cell, and Fiber differentiation T cell therein；Then passing through slowly of anti-CD19 single-chain antibody (scFv) gene will be expressed Virus imports in T cell, and continues to cultivate these cells；When the quality and quantity of these cells reaches the standard of Clinical practice After, these cells can be fed back to carry out in the patient treatment.In this flow process, the most key is exactly that channel genes is thin Slow virus used by intracellular.In prior art, the slow virus for treatment is mainly by 293T cell and slow virus plasmid, auxiliary Plasmid is carried out and obtained by packing and purifying.The step for, require higher to the amount of plasmid, typically can reach hundreds of μ g rank.

In prior art, the method for large quantity extracting plasmid mainly includes by plasmid adsorption column with by cesium chloride (CsCl) Ultracentrifugation two kinds.Method by plasmid adsorption column, it is only necessary to about 2 hours can be extracted from the bacterium solution of about 400mL Go out the plasmid of 1000 μ about g；Then needing centrifuged overnight 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：

1st, it is substantially absent from bacterial genomes DNA pollution.By comparison, the plasmid that plasmid adsorption column method is extracted is by bacterium The potential risk of contaminating genomic DNA wants high many.

2nd, in the plasmid extracting, covalency, Guan Bi, ring-shaped DNA molecule (cccDNA) are that plasmid normally played a role in the later stage Principal mode, the plasmid extracting by CsCl supercentrifugation, the ratio of cccDNA is significantly higher than and is carried by plasmid adsorption column method The plasmid taking.

3rd, by ultracentrifugation, bacterial endotoxin can be removed more up hill and dale.

Therefore carrying out extracting in a large number of plasmid by supercentrifugation is a kind of better method, and this is by this area Experimenter is accepted.

During CsCl supercentrifugation large quantity extracting plasmid, need add nucleic acid dye so that plasmid hypervelocity from Develop the color after the heart, facilitate experimenter to draw it by utensils such as syringes.Nucleic acid dye commonly used in the prior art is Ethidium bromide (EB), this is the very high nucleic acid dye of a kind of sensitivity, can detect the DNA of as little as 10ng.But EB is a kind of Strong mutagens, have higher carcinogenicity, therefore for the experimenter of this nucleic acid dye of Long-Time Service, how both to protect Oneself healthy, obtain again preferable experimental result become one faced by their routine work is had to important Problem.Prior art not yet has the method that can solve this problem very well.

Content of the invention

It is an object of the invention to, for deficiency of the prior art, provide a kind of method of large quantity extracting plasmid, its feature As follows：Apply novel GoldenView dyestuff to substitute highly toxic EB dyestuff and carry out CsCl ultracentrifugation extraction plasmid.

The concrete technical scheme of the present invention is as follows：

S1, being seeded to the Escherichia coli containing purpose plasmid in 400mL TB culture medium, 37 DEG C, 200rpm cultivated Night.Then 4 DEG C, 6000g centrifuges 10min, abandons supernatant, and being inverted centrifuge tube makes the liquid of remnants flow out.

S2, add and be cooled to the P1 buffer solution of 4 DEG C in advance to final volume to 10mL, shake the resuspended bacterium making precipitation and uniformly divide Dissipate.The composition of described P1 buffer solution is 50mmol/L Tris-HCl, 10mmol/L EDTA, 100ug/mL Rnase A, pH= 8.0.

S3, addition 20mg solid lysozyme (hen egg white lysozyme), complete resuspended precipitation, room temperature is placed 10min.

S4, the P2 solution adding the fresh preparation of 20mL, mixed by pipette gently until solution transparent and homogeneous, and room temperature is placed 5-10min.The composition of described P2 buffer solution is 200mmol/L NaOH, 1%SDS.

S5, add and be cooled to the P3 solution 10mL of 4 DEG C in advance, gently but fully concussion mixes for several times, until two liquid phases are not Separating, original yellow disappears again, and White Flocculus occurs, is subsequently placed in 10min on ice.The composition of described P3 solution is 3mol/L KAc, pH=4.8.

S6,4 DEG C, 8000rpm centrifuges 10min, abandons precipitation, by supernatant by four layers of filtered through gauze to graduated cylinder.

S7, the isopropanol adding 0.6 times of volume, mix, and room temperature places 10-15min, is subsequently placed in 50mL centrifuge tube.

After S8,8000rpm centrifuge 15min, the supernatant place of abandoning is clean, reclaim nucleic acid precipitation.

S9, washing precipitation.At room temperature, rinse at the bottom of pipe and tube wall with 2mL 70% ethanol.Then ethanol is poured out, and inhale Drop on main wall；Or 8000rpm, normal temperature centrifuges 10min, abandons supernatant.Open wide centrifuge tube mouth of pipe 10-15min, make remaining Ethanol vapors away completely.

S10, addition 7mL 1 × TE solution dissolution precipitation, and add this solution of 7mL to rinse tube wall.

S11, addition CsCl 12.5g, dissolve and mix.

Mix after S12, addition GoldenView nucleic acid dye 10 microlitre.

S13, shift above-mentioned solution in ultracentrifugation pipe, be full of centrifuge tube balanced seal.

Under S14, room temperature, 55000rpm centrifuges at least 12h, or 65000rpm centrifuges 4h.

S15, centrifugal terminate after, centrifuge tube is taken out, is placed on the rack for test tube of masking foil parcel, in the room of only low-light In, it is seen that two region of DNA bands, upper strata zone is chromosome and linear DNA, and lower floor is required cccDNA.

S16, with No. 18 needle tubings, careful collection cccDNA, be placed in 50mL centrifuge tube.

S17, add isopyknic water saturation isoamyl alcohol, cover tightly lid, concussion mixing organic phase and aqueous phase.

Under S18, room temperature, 450g centrifuges 3min, makes aqueous phase separate with organic phase, and shifts upper organic phase (pink) To suitable waste liquid barrel.

S19, repeat extracting above three step 4-6 time, all lose green until aqueous phase and organic phase.

S20, the water adding 2.2 times of volumes, the isopropanol of 0.6 times of volume, mix.

Under S21, room temperature, 8000rpm centrifuges 15min, abandons supernatant completely, and reclaims nucleic acid precipitation.

S22, add the resuspended precipitation of 8mL ultra-pure water, add 10mol/L ammonium acetate solution to final concentration 2mol/L, fully Add 2.5-3 times of volume after mixing, be cooled to the ethanol (-20 DEG C) of-20 DEG C in advance, mix, be placed in dry ice or-80 DEG C of refrigerators, incubate Educate 10min.

S23,4 DEG C, 8000rpm centrifuges 10min, abandons supernatant.

S24, addition 20mL 70% ethanol, mixing, 4 DEG C, 12000g centrifuges 10min, abandons supernatant.

S25, centrifuge tube is placed in super-clean bench air-dry 2min, add TE dissolve plasmid.

Compared with prior art, beneficial effects of the present invention is as follows：Traditional CsCl supercentrifugation is extracted by the present invention Plasmid is transformed, and applies novel GoldenView dyestuff to instead of highly toxic EB dyestuff, is reducing experimental implementation pair While the genotoxic potential risk of researcher, it is ensured that the yield of extracted plasmid and quality are all without being decreased obviously.

Brief description

Fig. 1 carries out CsCl plasmid extraction for application GoldenView and EB, it is thus achieved that the result figure of plasmid total amount.

Fig. 2 is the electrophoresis result figure that CsCl extracts plasmid.A. GolenView dyestuff is applied to carry out the result (3 of plasmid extraction Secondary repetition)；B. EB dyestuff is applied to carry out the result (repeating for 3 times) of plasmid extraction.

Fig. 3 is BSA canonical plotting.

Detailed description of the invention

According to following embodiment, the present invention may be better understood.But, as it will be easily appreciated by one skilled in the art that reality Execute the concrete material proportion described by example, process conditions and result thereof and be merely to illustrate the present invention, and should also will not limit The present invention described in detail in claims processed.Method used in following embodiment is routine side if no special instructions Method, concrete 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 without clearly stating, being commercial goods.

The colibacillary cultivation of embodiment one

Take the Escherichia coli 10 μ L containing plasmid, be placed in 400mL TB culture medium, 37 DEG C, after 200rpm cultivates 14h, 4 DEG C, 6000g centrifuges 10min and collects thalline.

Embodiment two CsCl ultracentrifugation extracts plasmid

Extract plasmid according to following steps application CsCl supercentrifugation：

1st, add and be cooled to the P1 buffer solution of 4 DEG C in advance to 10mL, resuspended above-mentioned thalline, shakes the resuspended bacterium making precipitation uniform Dispersion.

2nd, adding 20mg solid lysozyme (hen egg white lysozyme), complete resuspended precipitation, room temperature is placed 10min.

3rd, adding the P2 solution of the fresh preparation of 20mL, mixing gently with pipette until solution transparent and homogeneous, room temperature is placed 5-10min.

4th, add and be cooled to the P3 solution 10mL of 4 DEG C in advance, gently but fully concussion mixes for several times, until two liquid phases are no longer Separating, original yellow disappears, and White Flocculus occurs, is subsequently placed in 10min on ice.

5th, 4 DEG C, 8000rpm centrifuges 10min, abandons precipitation, by supernatant by four layers of filtered through gauze to graduated cylinder.

6th, adding the isopropanol of 0.6 times of volume, mixing, room temperature places 10-15min, is subsequently placed in 50mL centrifuge tube.

7th, after 8000rpm centrifuges 15min, the supernatant place of abandoning is clean, reclaim nucleic acid precipitation.

8th, washing precipitation.At room temperature, rinse at the bottom of pipe and tube wall with 2mL 70% ethanol.Then ethanol is poured out, and inhale Drop on main wall；Or 8000rpm, normal temperature centrifuges 10min, abandons supernatant.Open wide centrifuge tube mouth of pipe 10-15min, make remaining Ethanol vapors away completely.

9th, add 7mL 1 × TE solution dissolution precipitation, and add this solution of 7mL to rinse tube wall.

10th, add cesium chloride 12.5g, dissolve and mix.

11st, mix after adding GoldenView nucleic acid dye 10 microlitre.

12nd, shift above-mentioned solution in ultracentrifugation pipe, be full of centrifuge tube balanced seal.

13rd, the centrifugal at least 12h of 55000rpm under room temperature, or 65000rpm centrifuges 4h.

14th, after centrifuging end, centrifuge tube is taken out, be placed on the rack for test tube of masking foil parcel, in the indoor of only low-light, Visible two region of DNA bands, upper strata zone is chromosome and linear DNA, and lower floor is required cccDNA.

15th, it with No. 18 needle tubings, careful collection cccDNA, is placed in 50mL centrifuge tube.

16th, add isopyknic water saturation isoamyl alcohol, cover tightly lid, concussion mixing organic phase and aqueous phase.

17th, under room temperature, 450g centrifuges 3min, makes aqueous phase separate with organic phase, and is transferred to upper organic phase (pink) In suitable waste liquid barrel.

18th, extracting above three step 4-6 time is repeated, until aqueous phase and organic phase all lose green.

19th, add the water of 2.2 times of volumes, the isopropanol of 0.6 times of volume, mix.

20th, under room temperature, 8000rpm centrifuges 15min, abandons supernatant completely, and reclaims nucleic acid precipitation.

21st, add the resuspended precipitation of 8mL ultra-pure water, add 10mol/L ammonium acetate solution to final concentration 2mol/L, fully mixed Add 2.5-3 times of volume after even, be cooled to the ethanol (-20 DEG C) of-20 DEG C in advance, mix, be placed in dry ice or-80 DEG C of refrigerators, hatch 10min.

22nd, 4 DEG C, 8000rpm centrifuges 10min, abandons supernatant.

23rd, adding 20mL 70% ethanol, mixing, 4 DEG C, 12000g centrifuges 10min, abandons supernatant.

24th, centrifuge tube is placed in super-clean bench and air-dries 2min, add TE to dissolve plasmid.

The detection of embodiment three plasmid extraction amount

Lift the sample arm of Thermo Nanodrop 2000 detector, take 1 μ L TE solution and be added on detection pedestal, use In instrument zeroing, with clean cleansing tissue by pedestal wiped clean after completing；Then take 1 μ L again to be dissolved in the plasmid of TE and be added to detection On pedestal, then putting down sample arm, running control software reads light absorption value；Again software is estimated plasmid according to light absorption value Concentration.

Calculate according to the extracted amount to plasmid for the below equation：N=C × V.Wherein n is the extracted amount of plasmid, and C is matter The concentration of grain, V is for dissolving the volume of the TE used by plasmid.

Inventor GoldenView and ethidium bromide (EB) respectively repeat plasmid extraction and test three times, extract situation such as following table Shown in：

Table 1 plasmid extraction situation

Carrying out T check analysis to these data, result is as shown in Figure 1.It will be seen that application GoldenView dyestuff extracts The total amount of gained plasmid is extracted higher than application EB dyestuff, but without significant difference (p>0.05).

Embodiment four extracts the endotoxic detection of plasmid

The endotoxin that the gel method TAL using Zhanjiang Andusi Biology Co., Ltd. to produce detects in Plasmid samples is residual Stay.With ultra-pure water endotoxin standard is diluted to 10EU/mL, 1EU/mL, 0.5EU/mL, 0.25EU/mL, 0.125EU/mL, 6 concentration gradients of 0.0625EU/mL make calibration curve；By gel method TAL specification, (MVD=is diluted to sample again CL/ λ), and carry out endotoxic detection.Each sample carries out 2 times and repeats, and acquired results shows, whole Plasmid samples are all in interior Toxin is negative, the equal 5EU/mL of contained endotoxin in showing Plasmid samples, is i.e. 5EU/mL wanting sample to control endotoxin limit value Scope, in Plasmid samples is described, endotoxin content is extremely low.

Embodiment five extracts the detection of plasmid residual protein

Use the BCA detection kit that Beijing Quanshijin Biotechnology Co., Ltd produces to the residual egg extracting in plasmid White matter content is measured.With ultra-pure water compound concentration be respectively the 0th, 25ng/ μ L, 50ng/ μ L, 100ng/ μ L, 200ng/ μ L, 300ng/ μ L, 400ng/ μ L and the BSA standard items of 500ng/ μ L, draw calibration curve, result table 2 and as shown in Figure 3.Can see Arrive, light absorption value and standard concentration linearly pertinent trends, r²Value is more than 0.99.

Table 2BSA Specification Curve of Increasing data

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

Carry out the inspection of residual protein content according to same steps to 6 the plasmid extraction samples obtaining in embodiment three Surveying, its light absorption value is as shown in table 3.It will be seen that it is 0 that the absorbance obtained by 6 sample detection is respectively less than standard concentration When corresponding absorbance, do not detect protein residues, illustrate extract plasmid purity 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 the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, on the premise of without departing from the inventive method, can also make some improvement and supplementary, and these improve and supplement and also should be regarded as Protection scope of the present invention.

Claims

1. the method for a large quantity extracting plasmid, it is characterised in that employ novel nucleic acid dye.

2. the method for a kind of large quantity extracting plasmid according to claim 1, it is characterised in that the novel nucleic acids of described use Dyestuff is GoldenView.

3. the method for a kind of large quantity extracting plasmid according to claim 1, it is characterised in that include：P1 buffer solution, P2 delay Rushing liquid, P3 solution, its component is as follows：

P1 buffer solution：25-70mmol/L Tris-HCl, 5-30mmol/L EDTA, 100ug/mL Rnase A, pH=7.0- 8.5；

P2 buffer solution：100-350mmol/L NaOH, 0.1-3%SDS；

P3 solution：1-6mol/L KAc, pH=4.0-5.5.

4. the method for a kind of large quantity extracting plasmid according to claim 1, it is characterised in that comprise the steps：

Step 1, the preparation of plasmid.Escherichia coli containing purpose plasmid are seeded in 400mL TB culture medium, 37 DEG C, 200rpm overnight incubation.Then 4 DEG C, 6000g centrifuges 10min, abandons supernatant, and being inverted centrifuge tube makes the liquid of remnants flow out.Add Being cooled to the P1 buffer solution of 4 DEG C in advance to final volume to 10mL, it is dispersed to shake the resuspended bacterium making precipitation, is subsequently adding 20mg solid Body lysozyme (hen egg white lysozyme), complete resuspended precipitation, room temperature places 10min；Add the fresh system of 20mL Standby P2 solution, is mixed by pipette gently until solution transparent and homogeneous, and room temperature places 5-10min；It is eventually adding and pre-be cooled to 4 DEG C P3 solution 10mL, gently but fully concussion mixes for several times, until two liquid phases no longer separate, original yellow disappears, in vain Look floccule occurs, is subsequently placed in 10min on ice.4 DEG C, 8000rpm centrifuges 10min, abandons precipitation, and supernatant is passed through four layers of gauze Being filled in graduated cylinder, being subsequently adding the isopropanol of 0.6 times of volume, mix, room temperature places 10-15min, is placed in 50mL centrifuge tube In, then after 8000rpm centrifuges 15min, the supernatant place of abandoning is clean, reclaim nucleic acid precipitation.

Step 2, the purifying of plasmid.Washing nucleic acid precipitation：At room temperature, rinse at the bottom of pipe and tube wall with 2mL 70% ethanol.Then Ethanol is poured out, and blots the drop on tube wall；Or 8000rpm, normal temperature centrifuges 10min, abandons supernatant.Open wide the centrifuge tube mouth of pipe 10-15min, makes remaining ethanol vapor away completely.It is subsequently added into 7mL 1 × TE solution dissolution precipitation, and add this solution of 7mL Rinse tube wall.Add cesium chloride 12.5g, dissolve and mix after adding GoldenView nucleic acid dye 10 microlitre after mixing.In transfer State solution in ultracentrifugation pipe, be full of centrifuge tube balanced seal.The centrifugal at least 12h of 55000rpm under room temperature, or 65000rpm centrifuges 4h.After centrifugal end, centrifuge tube is taken out, be placed on the rack for test tube of masking foil parcel, at only low-light Indoor, it is seen that two region of DNA bands, upper strata zone is chromosome and linear DNA, and lower floor is required cccDNA.With No. 18 needle tubings, Careful collection cccDNA, is placed in 50mL centrifuge tube.Adding isopyknic water saturation isoamyl alcohol, covering tightly lid, concussion is mixed with Machine phase and aqueous phase.Under room temperature, 450g centrifuges 3min, makes aqueous phase separate with organic phase, and is transferred to upper organic phase (pink) In suitable waste liquid barrel.Repeat extracting above three step 4-6 time, until aqueous phase and organic phase all lose green.Add 2.2 times The water of volume, the isopropanol of 0.6 times of volume, mix.Under room temperature, 8000rpm centrifuges 15min, abandons supernatant completely, and reclaims nucleic acid Precipitation.Add the resuspended precipitation of 8mL ultra-pure water, add 10mol/L ammonium acetate solution to final concentration 2mol/L, add after fully mixing Enter 2.5-3 times of volume, be cooled to the ethanol (-20 DEG C) of-20 DEG C in advance, mix, be placed in dry ice or-80 DEG C of refrigerators, hatch 10min.4 DEG C, 8000rpm centrifuges 10min, abandons supernatant.Adding 20mL 70% ethanol, mixing, 4 DEG C, 12000g centrifuges 10min, abandons supernatant, Finally centrifuge tube is placed in super-clean bench and air-dries 2min, add TE to dissolve plasmid.