CN108048456A - A kind of plasmid extraction method - Google Patents

Abstract

The present invention relates to a kind of plasmid extraction methods, include the following steps：（1）It by cellular lysate liquid the first chromatographic column of loading containing plasmid, is eluted using the first solution, collects the eluent containing plasmid, obtain the first eluent；（2）It by the second chromatographic column of the first eluent loading, is eluted using the second solution, collects the eluent containing plasmid, obtain the second eluent；（3）The second eluent loading third layer is analysed into column, is eluted using the 3rd solution, collects the eluent containing plasmid, obtains the 3rd eluent；（4）It by the 4th chromatographic column of the 3rd eluent loading, is eluted using the 4th solution, collects the eluent containing plasmid, obtain the 4th eluent.Plasmid extraction method of the present invention improves the rate of recovery of plasmid during plasmid extraction.

Description

A kind of plasmid extraction method

Technical field

The present invention relates to biological technical field, more particularly to a kind of plasmid extraction method.

Background technology

Plasmid (plasmid) is DNA points beyond chromosome (or nucleoid) in the biology such as bacterium, saccharomycete and actinomyces Son is present in cytoplasm, has autonomous replication capacity, supercoil, open loop and linear three kinds of structures is generally comprised, wherein super spiral shell Revolve conversion and expression efficiency highest of the Plasmid DNA of structure in cell.The length of plasmid is generally in 2-20kbp, molecular weight one As 10⁶More than, plasmid DNA molecule aobvious negative electricity when with phosphate group can also be specifically bound with some groups.

It is currently based on the gene editing of Plasmid DNA and control technique is widely used to scientific research, agriculture test, medical treatment In the fields such as diagnosis, application in disease treatment research or even clinically attracts tremendous attention.

Gene editing and gene therapy are to carry out the emerging skill of disease treatment from molecular level using gene correction means Art.Cure mechanism be mainly by gene transfer technique prize carry therapeutic genes foreign gene import sick cell in thus Modification blocks Disease-causing gene, so as to fulfill the treatment to disease.Because plasmid DNA molecule has better security and lower The advantages that toxicity and immunogenicity, the large-scale separating and purifying technology of high quality DNA plasmid by there is an urgent need to.

The content of the invention

It is an object of the invention to provide a kind of plasmid extraction method, for extracting different size of plasmid and improving plasmid The rate of recovery of extraction.

The present invention provides a kind of plasmid extraction methods, include the following steps：It (1) will be on the cellular lysate liquid containing plasmid The first chromatographic column of sample, is eluted using the first solution, collects the eluent containing plasmid, obtains the first eluent；Wherein, institute The first chromatography column packing is stated as Ago-Gel 6FF；(2) it is molten using second by the second chromatographic column of the first eluent loading Liquid is eluted, and collects the eluent containing plasmid, obtains the second eluent；Wherein, the second chromatography column packing is agar Sugared pearl and the 2- mercaptopyridine ligands being fixed on sepharose 4B；(3) the second eluent loading third layer is analysed into column, used 3rd solution is eluted, and collects the eluent containing plasmid, obtains the 3rd eluent；Wherein, the third layer analysis column packing For glucan；(4) by the 4th chromatographic column of the 3rd eluent loading, eluted using the 4th solution, collect washing containing plasmid De- liquid, obtains the 4th eluent；Wherein, the 4th chromatography column packing is with quaternary ammonium group single group styrene/divinylbenzene Bead.

In a kind of possible realization method, the 4th solution is 1.0M NaCl, 10mM EDTA, 100mM Tris- HCl,pH 7.5。

In a kind of possible realization method, in the step (4), the 4th solution elution is washed for linear gradient It is de-.

In a kind of possible realization method, the linear gradient elution be mobile phase in the 4th solution content by 0% rises to 100%.

In a kind of possible realization method, in the step (4), chromatographed by the 3rd eluent loading the 4th Before column, the 4th chromatographic column described in the 5th solution equilibria is used；Wherein, the 5th solution is 10mM EDTA, 100mMTris- HCl,pH 7.5。

In a kind of possible realization method, the blade diameter length ratio of the third layer analysis column is 13:40-13:60；Preferably, footpath Height is than being 13:50.

In a kind of possible realization method, in the step (1), applied sample amount is the described first chromatography column packing volume 20%-40%；Preferably, applied sample amount is the 30% of the described first chromatography column packing volume；It is highly preferred that applied sample amount is described The 20/53 of first chromatography column packing volume.

In a kind of possible realization method, first solution is 2.1M (NH4) 2SO4,10mM EDTA, 100mM Tris,pH 7.5。

In a kind of possible realization method, second solution is 0.3M NaCl, 1.7M (NH4) 2SO4,10mM EDTA,100mM Tris-HCl,pH 7.5。

In a kind of possible realization method, the 3rd solution is 10mM EDTA, 100mM Tris-HCl, pH 7.5.

Compared with prior art, the present invention has the advantages that：Improve the recycling of plasmid during plasmid extraction Rate improves the 65%-80% rate of recovery of the prior art to 88.5%；It can be adapted for the extraction of different size plasmid, from And eliminate the troublesome operation for groping extracting method for extraction different size plasmid in the prior art；Anion can also be extended The service life of exchange chromatography column.

Description of the drawings

Fig. 1 is the efflux test map of step 15 in embodiment 1；

Fig. 2 is the test map of the efflux of step 16 in embodiment 1；

Fig. 3 is the test map of the efflux of step 17 in embodiment 1；

Fig. 4 is the test map of the efflux of step 18 in embodiment 1；

Fig. 5 is the efflux test map of step 25 in embodiment 2；

Fig. 6 is the test map of the efflux of step 26 in embodiment 2；

Fig. 7 is the test map of the efflux of step 27 in embodiment 2；

Fig. 8 is the test map of the efflux of step 28 in embodiment 2；

Fig. 9 is the efflux test map of step 38 in comparative example 1；

Figure 10 is the test map of the efflux of step 48 in comparative example 2；

Figure 11 is the agarose gel electrophoretogram for the plasmid that embodiment 1 and comparative example 2 are extracted；

Figure 12 is the agarose gel electrophoretogram for the plasmid that embodiment 2 and comparative example 3 are extracted.

Specific embodiment

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.

When embodiment provides numberical range, it should be appreciated that except non-invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The process that isolates and purifies of Plasmid DNA mainly includes：Upstream fermentation cracks and downstream purification.Upstream fermentation mainly includes Bacterial fermentation, cell is collected and cracking, three steps of lysate clarification and concentration.At present, first, bacterial fermentation mainly has shaking flask Two methods of fermentation and ferment tank.Shake flask test can in a limited space, under conditions of time and manpower, obtain in a short time Substantial amounts of data are obtained, so being widely adopted in laboratory research.Shake flask test result can provide production bacterial strain simultaneously Essential information and zymotechnique data, subsequently can be after enlarged experiment for factory's fermentation tank production.

Consider with reference to factors such as current laboratory equipment condition and purifying scales, select shake flask fermentation as bacterial fermentation side Method.It is just collected next, bacterial cell is collected with centrifugal process, cleavage method is common at present alkaline lysis, pyrolysis method, machine Tool cracking process etc..Pyrolysis method is not suitable for a large amount of cellular lysates, exists simultaneously security risk and the shortcomings such as organic efficiency is low.And For mechanical lysis method, Mechanical Crushing cracking Escherichia coli show only microfluid chemical combination to discharge the research of complete plasmid Bead mill method can obtain complete plasmid molecule, and wherein highest yield is generally only 50% or so.Therefore, in the embodiment of the present invention In selected alkaline process crack bacterial cell.By bacterial suspension in the strong anionic detergent of high pH, break cell membrane It splits, chromosomal DNA and protein denaturation.Although basic solvent makes base pairing destroy completely, the Plasmid DNA due to closed loop is being opened up Flutter on is that mutual winding will not be still separated from each other.When pH is restored to neutrality, DNA double chain will be formed again, and large intestine bar The formation such as bacterium fragment, the most gene group DNA of bacterium, cell membrane, cell protein, lipid, polysaccharide precipitate.The method has Safety, it is economical, it is efficient the advantages of.Next, the method that centrifugal filtration is taken in lysate clarification, the concentration of cleared lysate are adopted The method concentrated with ultrafiltration cup, subsequently as the increase of scale is it is contemplated that flow through the method for filter using cut phase.It cracks at this time clear Mainly include RNA, other forms DNA, endotoxin, a small amount of bacterioprotein and purpose supercoiled plasmid DNA molecule in clear liquid.

The relatively common method of downstream purification is the ethidium bromide precipitation method and chromatographic separation and purification method.Ethidium bromide passes through embedding Enter between base and combined with DNA, and then double helix is made to untwist.Thus the length of linear DNA is caused to increased, as benefit It repays, supercoil unit will be introduced in closed circular form plasmid DNA.Finally, supercoil degree greatly increases, so as to prevent ethidium bromide point Son continues to be embedded in.But linear molecule is not limited, can be continuing with more ethidium bromides, until reaching saturation.Due to dyestuff Binding capacity difference, the buoyant density of wire and closed circular DNA molecules in the cesium chloride degree containing saturation capacity ethidium bromide Also it is different, so as to be separated.However the purification process is both expensive and time consuming, and still need other means place to go nucleic acid with Outer impurity.Chromatography has high selectivity, and specificity is good, and economy is convenient, it is quality controllable the advantages that；But there are the problem of Have, 1, low for the purification efficiency of the larger plasmid of molecular weight；2., the rate of recovery of plasmid DNA molecule it is relatively low；3rd, it is remaining more Endotoxin and other impurities.

The embodiment of the present invention improves chromatography method, and provides a kind of plasmid extraction method based on this, Combine gel permeation chromatography, affinity chromatography, anion exchange chromatography and carry out plasmid purification, can be summarized as following Step：The first step crosses gel permeation chromatography based on molecular size difference and realizes removal RNA.Second step, based on super spirial plasmid DNA molecular is different from affinity chromatography aglucon binding ability to realize specific capture supercoiled plasmid DNA.3rd step, based on impurity Removal endotoxins different from anion-exchange chromatography aglucon binding ability and other impurities realize final purifying.

Next in a particular embodiment, plasmid extraction method provided in an embodiment of the present invention is specifically described.

Embodiment 1

11st, Crispr/Cas9 plasmids are converted and to competent cell, is then inoculated in the LB solids training containing ammonia benzyl mycin It supports in base, chooses monoclonal and 2~5ml contains progress just culture in ammonia benzyl mycin culture medium, then by culture medium：50% glycerine= 1:1 adds in 50% glycerine, and to obtain strain, strain then is placed in -80 DEG C of preservations.Wherein, 50% glycerine refers to 10 volume of water In contain 5 volume glycerine.

12nd, the strain 50ul that step 11 preserves is taken to be inoculated in 7ml benzyls containing ammonia mycin culture medium, 37 DEG C of 220r/min cultures To OD in 0.4-0.6.Afterwards 1:1000 add in the mycin culture medium of benzyl containing ammonia, are subsequently placed in 37 DEG C, and 220r/min shaking tables are cultivated 14-16h.The collection of thalline is centrifuged by 6000r/min and realized, and claims weight in wet base.

13rd, the thalline that step 12 obtains is taken to be resuspended in solution 1, then adding in isometric solution 2, mildly overturns 5~10 It is secondary, 5 minutes are stood, the solution 3 of precooling is then added according to 13.4ml/ thalline weight in wet bases (g), mildly overturns 5~10 times, sees white Flocculent deposit is simultaneously placed 30 minutes on ice；It is then centrifuged for filtering, collects filtrate, i.e. cleared lysate.Wherein, solution 1 is 50mM Tris-HCl,10mM EDTA,PH8.0；Solution 2 is 200mM NaOH, 1%SDS；Solution 3 is 1M potassium acetates (potassium acetate),PH5.5。

14th, the cleared lysate obtained using cup concentration step 13 is concentrated by ultrafiltration concentrates 5~10 times.Ultrafiltration cup concentrates cup Concentration filter membrane molecule interception regard plasmid size decision.In the present embodiment, the molecule of the concentration filter membrane of ultrafiltration cup concentration cup Interception is 300KDa.

15th, RNA is removed.The filler for the gel chromatography column that the present embodiment uses is Ago-Gel 6FF (Sepharose 6Fast Flow；It is 6% highly cross-linked sepharose 4B of 90 μ m diameters)；The internal diameter of packed column is 26mm, a height of 20cm；I.e. The blade diameter length ratio of packed column is 13:100；Packing volume is 106ml.Then solution 4 balances.Solution 4 is 2.1M (NH₄)₂SO₄,10mM EDTA,100mM Tris,pH 7.5.Step 14 is obtained into concentration lysate loading gel chromatography column, applied sample amount 40ml compares Adsorbance (volume ratio of the sample of loading and chromatography column packing) is 4:15.It is eluted using solution 4, flow velocity 15ml/ min。

In step 15, when being eluted using solution 4, the substances such as larger DNA of molecular weight first flow out, and molecular weight is smaller It is flowed out after the substances such as RNA, so as to remove RNA.Specific eluent is collected as：The AKTA instruments of GE companies are used during elution Efflux collection of illustrative plates is detected, as shown in Figure 1；When having eluted 120ml, there is the first eluting peak 11, collect the first eluting peak 11 Efflux 75ml；Through nano drop detections and agarose gel electrophoresis qualitative analysis, it is known that the first eluting peaks of 75ml 11 of collection Efflux contain final purpose plasmid.When having eluted 230ml, there is the second eluting peak 12, through nano drop detections and fine jade The qualitative analysis of sepharose electrophoresis understands that the second eluting peak 12 corresponds to the substances such as RNA.

In step 15, the separation of the small molecules such as DNA and RNA is realized.

16th, the capture of supercoiled plasmid DNA.The capture of supercoiled plasmid DNA is by crossing the height that filler is a diameter of 34 μm The affinity column of crosslinked sepharose 4B and the 2- mercaptopyridine ligands being fixed thereon is realized.The internal diameter of affinity column is 26mm, is filled out Material height is 3.7cm, i.e., the blade diameter length ratio of affinity column is 26:37, the volume of filler is 20ml.The 75ml first that step 15 is collected The efflux loading of eluting peak 11 is into the affinity column prepared.Affinity column before use, is balanced with solution 5, and solution 5 is 2.1M (NH₄)₂SO₄,10mM EDTA,100mM Tris,pH 7.5.Loading, 5 releveling of solution.Solution 6 elutes, and solution 6 is 0.3M NaCl,1.7M(NH₄)₂SO₄,10mM EDTA,100mM Tris-HCl,pH 7.5.Elution flow rate is 10ml/min.

In elution, act on weaker other substances with filler affinity groups and first flow out, with the effect of filler affinity groups compared with Outflow is eluted after strong supercoiled plasmid DNA, so as to which plasmid is further purified.Specifically, with GE companies during elution AKTA detects efflux collection of illustrative plates, as shown in Figure 2；Collect the efflux 120ml of the 3rd eluting peak 21, through nano drop detection and Agarose gel electrophoresis qualitative analysis, it is known that, wherein containing final purpose Plasmid DNA.

The separation by Plasmid DNA and other structures form DNA is realized in step 16.

17th, desalting processing.The desalting processing of supercoiled plasmid DNA efflux is realized by the gel chromatographic columns independently filled. The filler of the gel chromatographic columns independently filled in this step is highly cross-linked 9% glucan of 90 μ m diameters, the pillar of use Internal diameter for 26mm, be highly 10cm, i.e., blade diameter length ratio is 13:50；Packing volume is 50ml.It is balanced using preceding with solution 7.Solution 7 be 10mM EDTA, 100mM Tris-HCl, pH 7.5.The efflux 120ml concentrations for the 3rd eluting peak that step 16 is collected To 15ml, loading, solution 7 elutes.

The substances such as the larger DNA of molecular weight first flow out, and are flowed out after the substances such as smaller salt of molecular weight.Specifically, the phase is eluted Between with the AKTA of GE companies detection efflux collection of illustrative plates, as shown in Figure 3；When having eluted 20ml, there is the 4th eluting peak 31, collect 4th peak efflux 15ml, through nano drop detections and agarose gel electrophoresis qualitative analysis, it is known that, wherein containing final mesh Plasmid DNA；Wherein, agarose gel electrophoresis the qualitative analysis is as shown in swimming lane 122 in Figure 11.Swimming lane 121 in Figure 11 For the electrophoresis result of 1KB DNA marker.

In step 17, further the small molecular weight impurities such as DNA and RNA are separated.

18th, endotoxin removal.The filler of chromatographic column used is purified again as SOURCE 30Q, SOURCE30Q be with Single styrene/the divinylbenzene beads of a diameter of 30 μm of quaternary ammonium group.The internal diameter that the blade diameter length ratio of chromatographic column is is 10mm, a height of 20cm, i.e. blade diameter length ratio are 1:20.Filling packing volume is 20ml.Solution 7 balances, molten by the sample loading after step 17 desalination Liquid 7 balances, 8 gradient elution of solution.Specially linear gradient elution, 8 content of solution is risen to by 0% in 0-200ml mobile phases 100%.Solution 8 is 1.0M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5.

It is first flowed out with other substances that filler cation group does not act, it is rear to be acted on according to filler cation group Substance gradients elution outflow of different strengths and weaknesses.As shown in Figure 4；Eluent composition is to occur the 5th stream when 8 content of solution is 30% Appearance 41, eluent composition is that 8 content of solution is the 6th eluting peak 42 of 80% appearance.Two peaks are collected to correspond to efflux and use nano Drop carries out content detection, and agarose gel electrophoresis carries out qualitative analysis.Wherein, the 5th eluting peak efflux 35ml of collection, Second eluting peak 10ml effluxes, through nano drop detections and agarose gel electrophoresis qualitative analysis, it is known that, wherein all including Final purpose Plasmid DNA.Wherein, the swimming in 41 corresponding agarose gel electrophoresis the qualitative analysis of the 5th eluting peak such as Figure 11 Shown in road 123；6th eluting peak, 42 corresponding agarose gel electrophoresis the qualitative analysis is as shown in the swimming lane 124 in Figure 11.

The efflux of efflux and the 6th eluting peak 42 to the 5th eluting peak 41 detects protein content, reagents inspection with BCA Endotoxin content is surveyed, the results show is without significant difference.

In step 18, before loading, the Plasmid DNA content in loading sample is treated using nano drop detections；Elution, then Measure the Plasmid DNA content containing Plasmid DNA eluent, it is known that, the Plasmid DNA rate of recovery of step 18 is 88.5%.

19th, Plasmid DNA preserves.The eluent containing plasmid obtained toward step 18 adds in appropriate isopropanol, to precipitate matter Grain DNA, is then washed with 30% ethyl alcohol.Then TE buffer solutions (Tris-EDTA buffer solution) redissolve, Ran Houbao It deposits.

Embodiment 2

21st, the Crispr/Cas9 plasmids in the step 11 of embodiment 1 are replaced with into PGL3 plasmids, other are the same as step 11.

22nd, with step 12.

23rd, with step 13.

24th, with step 14.

25th, with step 15；Wherein, the efflux collection of illustrative plates detected during elution with AKTA is as shown in Figure 5.

26th, with step 16；Wherein, the efflux collection of illustrative plates detected during elution with AKTA is as shown in Figure 6.

27th, with step 17；Wherein, the efflux collection of illustrative plates detected during elution with AKTA is as shown in Figure 7.Stream containing plasmid Go out the agarose gel electrophoresis the qualitative analysis of liquid as shown in swimming lane 132 in Figure 12.Swimming lane 131 in Figure 12 is 1KB DNA The electrophoresis result of marker.

28th, with step 18；Wherein, the difference collected during elution with efflux collection of illustrative plates such as Fig. 8 steps 28 of AKTA detections The agarose gel electrophoresis the qualitative analysis of the corresponding efflux containing plasmid in peak respectively as in Figure 12 swimming lane 133, swimming Shown in road 124.

Comparative example 1

31st, with step 11.

32nd, with step 12.

33rd, with step 13.

34th, with step 14.

35th, with step 15.

36th, with step 16.

37th, the efflux for being collected into step 36 dilutes twice.

38th, the solution 7 in step 18 is replaced with into solution 9.Solution 9 is 0.4M NaCl, 10mM EDTA, 100mMTris- HCl,pH 7.5.The sample of its loading is the solution after step 37 dilution.The elution of solution 8 is (direct during elution for directly elution Eluent is increased to 100%), not use linear gradient elution, other are with step 18, the outflow detected with AKTA during elution Liquid collection of illustrative plates is as shown in Figure 9.

Comparative example 2

41st, with step 11.

42nd, with step 12.

43rd, with step 13.

44th, with step 14.

45th, with step 15.

46th, with step 16.

47th, the efflux for being collected into step 46 dilutes 4 times.

48th, with step 38, the efflux collection of illustrative plates detected during elution with AKTA is as shown in Figure 10.Wherein, step 48 is collected To the efflux containing plasmid agarose gel electrophoresis the qualitative analysis as shown in the swimming lane 125 in Figure 11.Step The rate of recovery of 48 Plasmid DNA is 65%.

Comparative example 3

51st, with step 11.

52nd, with step 12.

53rd, with step 13.

54th, with step 14.

55th, with step 15.

56th, with step 16.

57th, the efflux for being collected into step 56 dilutes 2 times.

58th, with step 38.Wherein, the agarose gel electrophoresis for the efflux containing plasmid that step 58 is collected into is qualitative Analysis result is as shown in the swimming lane 135 in Figure 12.The rate of recovery of the Plasmid DNA of step 58 is 80.1%.

To sum up plasmid extraction method provided by the invention has the following advantages that compared with the prior art：

1st, the analysis of salt ionic concentration is reduced instead of water dilution method on gel chromatography desalination.It is specific as follows：

The prior art：For anion column into sample liquid (be mostly top purifying gained containing the with high salt molten of purposeful Plasmid DNA Liquid) processing, more common recommendation method be two times of water dilution methods.The method processing sample is not applied for all big miniplasmids (such as comparative example 2VS comparative examples 3).The plasmid of bigger needs the dilution (for example, comparative example 1VS comparative examples 2) of more high magnification numbe.

The drawbacks of prior art, has：

A. processing method is not fixed, when changing purification of target, it is necessary to grope condition again.

B. excess dilution can cause sample solution plasmid concentration low, low so as to cause efflux purpose plasmid DNA concentration, cause Efflux waste (be in charge of collection, too low is identified of nano drop detection plasmid concentrations exists without effective Plasmid DNA, without Next step concentration extraction), also can be subsequent concentration extraction increase difficulty (the too small Plasmid DNA of concentration can not by isopropanol from Gains in depth of comprehension to precipitation).

Present invention employs gel chromatographies to chromatograph desalination, and advantage is as follows：

C. the processing before anion-exchange chromatography is crossed suitable for all size Plasmid DNA, method is fixed general.

D. final sample salinity/buffer compositions can be controlled, it is dead by gel chromatography layer in Examples 1 and 2 Sample buffer is finally replaced as 10mM EDTA, 100mM Tris-HCl, pH 7.5；Next step anion can not only handed over Changing chromatography equilibrium liquid buffer composition salt ionic concentration, lower (salinity of anion-exchange chromatography equilibrium liquid buffer is not small Being completely combined for sample and anion-exchange column is ensured that in sample salt ionic concentration), so as to equilibrium liquid and eluent salt Ion concentration difference becomes larger, and can so that elution is more complete；It is unfavorable for moreover, because continuing mode of operation with high salt to anion The use state of exchange chromatography is kept and the service life keeps, and therefore, the method is also beneficial to chromatographic column life-time dilatation and cycling is held It is continuous to use.

E. the method is dilute sample, may be such that Plasmid DNA yield becomes higher, and is also convenient for subsequent operation processing.

2. the analysis that the gradient elution substitution of anion-exchange chromatography directly elutes

Elution protocol of the prior art：Equilibrium liquid is 0.4M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5, eluent is 1.0M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5.Directly elution (will directly wash during elution 100%) de- liquid is increased to.The prior art is limited as not knowing whether elution is complete, and whether do not know is optimal elution protocol.

Present invention employs linear gradient elution, 8 content of solution linearly rises to 100% by 0% in 0-200ml mobile phases. Solution 8 is 1.0M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5.It was found that：Two eluting peaks, pass through at present (gel electrophoresis analysis structure, BCA methods analysis residual protein, limulus reagent test detection residual endotoxin), two peak streams from the point of view of detection It is Plasmid DNA to go out liquid, and related check represents that the Plasmid DNA of two peaks outflow meets the plasmid quality needed for current laboratory It is required that.The Plasmid DNA collected by two peaks of (1) is analyzed compared with Plasmid DNA obtained by original technology, quality does not have difference, Yield increases.(2) gradient elutions can be used for purifying determining for new purpose plasmid elution requirement, buffer when observing appearance Condition, can obtain optimum washing engaging condition, and in Examples 1 and 2, when 80%B+20%A is eluted, the last one eluting peak goes out It is existing, you can be eluted later according to this condition, reduce the protection that eluent salt ion concentration is also advantageous for chromatographic column.

In conclusion the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.

The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Claims (10)

1. A kind of 1. plasmid extraction method, which is characterized in that include the following steps：

   (1) by cellular lysate liquid the first chromatographic column of loading containing plasmid, eluted using the first solution, collection contains plasmid Eluent, obtain the first eluent；Wherein, the first chromatography column packing is Ago-Gel 6FF；

   (2) by the second chromatographic column of the first eluent loading, eluted using the second solution, collect the elution containing plasmid Liquid obtains the second eluent；Wherein, the second chromatography column packing is sepharose 4B and the 2- sulfydryls being fixed on sepharose 4B Pyridine ligand；

   (3) the second eluent loading third layer is analysed into column, is eluted using the 3rd solution, collect the elution containing plasmid Liquid obtains the 3rd eluent；Wherein, the third layer analysis column packing is glucan；

   (4) by the 4th chromatographic column of the 3rd eluent loading, eluted using the 4th solution, collect the eluent containing plasmid, Obtain the 4th eluent；Wherein, the 4th chromatography column packing is with quaternary ammonium group single group styrene/divinylbenzene beads.
2. A kind of 2. plasmid extraction method according to claim 1, which is characterized in that the 4th solution is 1.0M NaCl, 10mM EDTA,100mM Tris-HCl,pH 7.5。
3. 3. plasmid extraction method according to claim 2, which is characterized in that in the step (4), the 4th solution It elutes as linear gradient elution.
4. 4. plasmid extraction method according to claim 3, which is characterized in that the linear gradient elution is in mobile phase The content of 4th solution rises to 100% by 0%.
5. 5. plasmid extraction method according to claim 1, which is characterized in that in the step (4), by the described 3rd Before the 4th chromatographic column of eluent loading, the 4th chromatographic column described in the 5th solution equilibria is used；Wherein, the 5th solution is 10mM EDTA,100mMTris-HCl,pH 7.5。
6. 6. plasmid extraction method according to claim 1, which is characterized in that the blade diameter length ratio of the third layer analysis column is 13: 40-13:60。
7. 7. plasmid extraction method according to claim 1, which is characterized in that in the step (1), applied sample amount is described The 20%-40% of first chromatography column packing volume.
8. 8. plasmid extraction method according to claim 1, which is characterized in that first solution is 2.1M (NH₄)₂SO₄, 10mM EDTA,100mM Tris,pH 7.5。
9. 9. plasmid extraction method according to claim 1, which is characterized in that second solution is 0.3M NaCl, 1.7M (NH₄)₂SO₄,10mM EDTA,100mM Tris-HCl,pH 7.5。
10. 10. plasmid extraction method according to claim 1, which is characterized in that the 3rd solution is 10mM EDTA, 100mM Tris-HCl,pH 7.5。