CN1245414C - A kind of plasmid DNA extraction and purification method and production process - Google Patents

Abstract

Translated fromChinese

一种从原核生物中提取和纯化质粒DNA的方法及其生产工艺，其方法含有原核生物菌液浓缩、菌体裂解、质粒DNA分离和质粒DNA纯化；其生产工艺是：过滤分离法菌体、在溶菌酶作用下热裂解菌体、膜过滤法分离和纯化质粒DNA。本发明的技术效果：利用本发明提供的方法及生产工艺不仅能够有效的提高质粒提取产量和质量，而且制备过程简单、易于实施。A method for extracting and purifying plasmid DNA from prokaryotic organisms and its production process, the method includes prokaryotic bacterial liquid concentration, bacterial cell lysis, plasmid DNA separation and plasmid DNA purification; the production process is: filtration separation method bacterial cells, The bacteria were thermally lysed under the action of lysozyme, and the plasmid DNA was separated and purified by membrane filtration. The technical effect of the present invention: the method and production process provided by the present invention can not only effectively improve the yield and quality of plasmid extraction, but also the preparation process is simple and easy to implement.

Description

A kind of plasmid DNA extraction and purification process and production technique

Technical field

The present invention relates to a kind of from prokaryotic organism, the extraction and the method and the production technique thereof of plasmid DNA purification, be one of the most basic molecular biological step, related to fields such as gene clone, gene sequencing, nucleic acid vaccine, gene therapy and range gene recombinant technology.

Background technology

The extraction of plasmid DNA and purifying are one of the most basic molecular biological steps, have related to fields such as gene clone, gene sequencing, nucleic acid vaccine, gene therapy and range gene reorganization.Classical extracting with plasmid DNA purification from prokaryotic organism is to be divided into three steps: cellular lysate, plasmid separate and purifying.The most frequently used method of cellular lysate has two at present: alkaline lysis (Birnboim and Doly, Nucleic Acids Res., 7:1513-1523,1979) and boiling lysis.(Holmes?and?Quigley，Anal.Biochem.，114：193-197，1981)。The extraction scale generally can obtain the plasmid DNA of 1ug to the 1-3 milligram from preparing (5 milliliters of nutrient solutions) in a small amount to a large amount of preparations (1 liter of nutrient solution), and is enough to the demand that satisfies laboratory molecular cloning work.Yet along with the fast development of dna vaccination and gene recombination drug research and application, in continuous increase, conventional prepared in laboratory and method thereof have been difficult to satisfy to the demand of recombinant plasmid; The another one deficiency be to use these two kinds of methods not only the cost height, yield poorly, outer but also difficult expansion scale of poor quality.Thereby influenced the efficient of later stage work, be called dna vaccination and gene recombination drug research at present and use bottleneck.Also having some extensive methods of extracting at present is to use various types of affinity columns, and extraction effect is better but expense is very high, also is difficult for the expansion scale.There is reagent contamination in alkaline lysis owing to will add plurality of reagents in the leaching process in the finished product, output and quality are not high; Though and boiling lysis output height, complicated operation, cracking process be difficult for to be grasped, unstable result pollutes and is difficult for removing.So many nucleic acid technical developments are limited by a large amount of preparation plasmid DNA technical deficiencies at present.

Summary of the invention

Problem at above-mentioned existence; we have done improvement to the method for boiling big upgrading grain targetedly; wherein improved cellular lysate temperature, plasmid separation and each link of purifying, finally solved above-mentioned problem, and made it can successfully be applied to production practice and large-scale production.Our purpose is to prepare the plasmid DNA that meets quality requirements in a large number with easy method.

Technical scheme of the present invention reaches by following measure

1, after engineering bacteria carries out high density fermentation, uses the hollow-fibre membrane post to concentrate, collect and concentrate bacterium liquid; Rather than conventional high speed centrifugation is finished;

2, working concentration is 10mM Tris-HCL, 50mM EDTA, and the TE solution of pH=8.0 (TE solution is the abbreviation of the mixing solutions of Tris-HCl and EDTA) will concentrate bacterium liquid and be diluted to OD₆₀₀After=100, add the TritonX-100 that final concentration is preferably 0.5～2% (weightmeasurement ratio), the N,O-Diacetylmuramidase of 0.1～0.5M sodium-chlor and 50～300 mcg/ml.

3, be preferably 25～37 ℃, incubation under 35～37 ℃ the temperature more preferably, the incubation time is preferably 20～60 minutes, more preferably 20～30 minutes, it is reacted effectively;

4, above-mentioned reaction thalline is reduced to 50～80 ℃ that are easier to control by the temperature when the cellular lysate from 100 ℃ of boiling temps, is preferably 70～80 ℃ of cracking 20～60 seconds, and should improve still cracking bacterium well.

5, the bacterium liquid utilization after the cracking is preferably 400～1000 purpose filter membrane separation of supernatant and cracked bacterial chip and bacterial chromosome;

6, add the 3M NaAc (pH5.2) of 1/10 cumulative volume and the isopropanol precipitating nucleic acid of 0.6 times of cumulative volume in the supernatant liquor after separation, and utilize to be preferably in 100～400 order filter membranes and carry out filtering separation.

7, after cleaning this throw out and with 70% ethanol.

8, behind the dry sediment, add an amount of TE solution (being the mixing solutions of Tris-HCl and EDTA) and dissolve.

The present invention be by utilize the aperture be 0.2～0.4 μ the hollow-fibre membrane post with volume greater than 5 liters fermentation after engineering bacteria concentrate, collect to concentrate bacterium liquid, also can to volume greater than 5 liters fermentation after engineering bacteria to use the aperture be that tangential flow filter membrane and the device of 0.2～0.4 μ concentrates.Rather than conventional high speed centrifugation is finished.

Be the suitable scission reaction of pH that makes above-mentioned concentrated bacterium liquid, (10mMTris-HCL, 50mM EDTA pH=8.0) in above-mentioned concentrated bacterium liquid, make its balance and inhibition DNase activity wherein by adding TE solution in the present invention.

For making the abundant cracking of above-mentioned concentrated bacterium, before cracking, the present invention is Triton X-100 by its composition of lysate that adds, and final concentration is preferably 0.5～2% (weightmeasurement ratio); NaCl, final concentration is preferably 0.1～0.5M; N,O-Diacetylmuramidase, final concentration are preferably 50～300 mcg/ml, and react under proper temperature and reaction times, make it become protoplastis.

Optimal reactive temperature and time that the present invention will prepare protoplastis are preferably 25～37 ℃, incubation under 35～37 ℃ the temperature more preferably, the reaction times is preferably 20～60 minutes, more preferably 20～30 minutes and protoplastis, make it be easy to abundant cracking.

For making the abundant cracking of thalline, in cracking process, the temperature of the present invention by with cellular lysate the time is reduced to 50～80 ℃ that are easier to control from 100 ℃ of boiling temps, is preferably 70～80 ℃.

For reaching the even purpose of temperature, the present invention passes through thalline is passed through in the heating tube, and the time of thalline in heating tube is preferably 10～60 seconds, and more preferably 20～30 seconds, this improved still cracking bacterium well, was easy to accomplish in production practice simultaneously.

The present invention is by being preferably 200～1000 purpose filter membranes with the bacterium liquid utilization after the cracking, and more preferably 400～500 purpose filter membranes are isolated supernatant liquor under pressure, remove cracked bacterial chip and bacterial chromosome.

The bacterium liquid of the present invention after with cracking utilizes whizzer to go out supernatant liquor in the 12000g centrifugation, removes cracked bacterial chip and bacterial chromosome.

The present invention passes through the 3M NaAc (pH5.2) of addingcumulative volume 1/10 and the isopropanol precipitating nucleic acid of 0.6 times of volume in the supernatant liquor after separating, and at 100～400 purpose filter membranes, is preferably in 100～200 purpose filter membranes, filters to isolate the nucleic acid throw out.

The present invention carries out drying after isolating the nucleic acid throw out by the cleaning of 70% ethanol.

The present invention carries out drying after preferably isolating the nucleic acid throw out by chloroform and the cleaning of 70% ethanol.

The present invention adds an amount of TE solution and dissolves by behind the dry nucleic acid throw out, and obtains the plasmid DNA product of purifying.

Therefore, invention is done great improvement to the method for boiling big upgrading grain, has wherein improved cellular lysate temperature, plasmid separation and each link of purifying, obtains high yield and highly purified plasmid DNA product.

The present invention utilizes the hollow-fibre membrane post that the back engineering bacteria that ferments is carried out effectively concentrating, and does not use supercentrifuge, thereby has avoided industrial production to be difficult for the problem of amplifying.

The present invention effectively handles the generation protoplasma to concentrating thalline, makes the cracking temperature of one step of back thalline be reduced to the scope that is easier to control, thereby avoids boiling temps restive in industrial production, has also saved the energy.

The present invention effectively utilizes heating tube that protoplasma is carried out heat treated, and it is even to produce temperature, and sufficient reacting has effectively overcome the inadequate deficiency of the cracking that exists in the conventional boiling method.

The present invention can by two kinds of different filter membranes after to cracking bacterium liquid and the nucleic acid of isopropanol precipitating effectively separate, and do not use supercentrifuge, thereby avoided industrial production to be difficult for the problem of amplifying, improved output and the quality of extracting the back plasmid simultaneously.

Technique effect of the present invention: utilize production provided by the invention and using method not only to make to separate and purification step is easy to control and operation, output and the quality of extracting the back plasmid have also been improved simultaneously, and avoided complex apparatus in separation and the purge process, so easy to implement and production amplification.

Description of drawings

Fig. 1: 37 ℃ of incubations influence the bacterium cracked

Without 37 ℃ of incubations (the 1st road) with through 37 ℃ of incubations 10 (the 2nd road), 20 (the 3rd roads), 30 (the 4th roads), 40 minutes (the 5th road) boils 30 seconds cracking XL1-blue bacteriums at 70 ℃ respectively, finds that 20 minutes lytic effect of 37 ℃ of incubations is best.

Fig. 2: the cracking bacterium obtains plasmid relatively under the differing temps

37 ℃ of incubations are after 20 minutes, in 50 ℃ of different temperature (the 1st road), and 60 ℃ (the 2nd road), 70 ℃ (the 3rd road), 80 ℃ (the 4th road), 90 ℃ (the 5th road), 100 ℃ (the 6th road) be lysing cell respectively, through comparing, between 70 ℃～80 ℃, bacterium is cracking well.Along with the raising of cracking temperature, the amount of the RNA that produces after the bacterium cracking increases gradually.

Fig. 3: the comparison of N,O-Diacetylmuramidase consumption

After with TE concentrated solution OD600 being diluted to 100, by every milliliter of 0.05 milligram of bacterium liquid (the 1st road), 0.1 milligram (the 2nd road), 0.2 milligram (the 3rd road), 0.3 the amount of milligram (the 4th road) adds N,O-Diacetylmuramidase, discovery is that 0.05 milligram of N,O-Diacetylmuramidase can carry out effective cracking to protoplastis under 100 the situation at OD600.

Fig. 4: utilize the filter membrane washing to remove the RNA effect

Supernatant after separating is added isopropanol precipitating nucleic acid, and in 70% ethanol, impurity such as RNA are removed in the concussion washing repeatedly.Through 70% washing with alcohol (the 1st road), without 70% washing with alcohol (the 2nd road).

Fig. 5: big upgrading grain quantitatively reaches enzyme and cuts qualification result

The big upgrading grain of gradient dilution (the 1st to 7 road) is with standard Marker quantitative (M road), every liter of about 95 milligrams of Fig. 5 A of broth extraction plasmid.The pc3d-N plasmid double digestion to carrying greatly with EcoRI and XhoI obtains the band of 1200bp size, and identical with N gene size (the 2nd road), the 1st road is Marker.Carry out purity testing with OD260/OD280, recording ratio is 1.91 Fig. 5 B.

Embodiment

By following examples the present invention is further described, below these embodiment be exemplary, rather than restrictive, can determine concrete according to the technical scheme and the practical situation of the invention described above

Embodiment.

Embodiment 1: the fermentation of engineering bacteria

Plasmid pc3d-N is transformed the XL1-blue competent cell, and picking one single bacterium colony is cultured to logarithmic phase for 37 ℃ in 5 milliliters of LB (adding an amount of microbiotic) from the AMP agar plate.Get 1.5 milliliters of cultures in 37 ℃ of overnight incubation of 150 milliliters of LB (adding an amount of microbiotic).150 milliliters of overnight culture are added 37 ℃ of fermentation culture in the fermentor tank that fills 3 liters of LB substratum (adding an amount of microbiotic), fermentation 400 milliliters of flow feedings after 5～6 hours.OD behind the fermentation 15h₆₀₀When no longer including remarkable increase, stop fermentation, record OD₆₀₀Be 45.

Embodiment 2: microorganism collection

Fermented liquid 3.5L pumped in the hollow-fibre membrane post with peristaltic pump concentrate, (10mM Tris-HCL, 50mM EDTA pH=8.0) wash thalline with 200 milliliters of TE to concentrate the back.Concentrate back isopyknic TE of adding in concentrated solution and continue to concentrate, the 3.5L fermented liquid finally concentrates and obtains 350 milliliters of concentrated solutions.

Embodiment 3: microorganism collection

Fermented liquid 15L pumped in the hollow-fibre membrane post with peristaltic pump concentrate, (10mM Tris-HCL, 50mM EDTA pH=8.0) wash thalline with 500 milliliters of TE to concentrate the back.Concentrate back isopyknic TE of adding in concentrated solution and continue to concentrate, the 15L fermented liquid finally concentrates and obtains 750 milliliters of concentrated solutions.

Embodiment 4: the plasmid extraction and purification

Adding final concentration in the concentrated solution is the NaCl of 0.1M, and final concentration is 2% Triton X-100, and every milliliter of concentrated solution adds 0.1 milligram of N,O-Diacetylmuramidase.

37 ℃ of incubator incubations 10～40 minutes are used the magnetic stir bar stirring at low speed.

With peristaltic pump reaction solution is pumped into 50～70 ℃ of heating metal pipes in the water-bath, control peristaltic pump rotating speed heated reaction solution 20～30 seconds in heating metal pipe, and fairlead places ice bath, makes to react termination reaction immediately.

The dope that reaction solution is produced after as for 400～1000 order membrane filtration lysises.

The 3M NaAc (pH5.2) that addscumulative volume 1/10 in the supernatant, the Virahol of 0.6 times of volume of adding, fully mixing was placed 10 minutes under the room temperature.

Reaction solution is poured in 100～400 purpose filter membranes, and the filtering supernatant liquor is put then as in the beaker, adds 70% ethanol concussion washing, reclaims the nucleic acid precipitation, and is dry under the room temperature.With 2ml～5ml TE (10mMTris-HCL, 1mM EDTA, pH=7.0) dissolving nucleic acid precipitation.Nucleic acid to purifying in 1% agarose gel carries out the electrophoresis evaluation.

Embodiment 5: the plasmid extraction and purification

Adding final concentration in the concentrated solution is the NaCl of 0.1M, and final concentration is 2% Triton X-100, and every milliliter of concentrated solution adds 0.1 milligram of N,O-Diacetylmuramidase.

37 ℃ of incubator incubations 10～40 minutes are used the magnetic stir bar stirring at low speed.

With peristaltic pump reaction solution is pumped into 50～70 ℃ of heating metal pipes in the water-bath, control peristaltic pump rotating speed heated reaction solution 20～30 seconds in heating metal pipe, and fairlead places ice bath, makes to react termination reaction immediately.

The dope that reaction solution is produced after as for 400～1000 order membrane filtration lysises.

The 3M NaAc (pH5.2) that addscumulative volume 1/10 in the supernatant, the Virahol of 0.6 times of volume of adding, fully mixing was placed 10 minutes under the room temperature.

Reaction solution is poured in 100～400 purpose filter membranes, and the filtering supernatant liquor is put then as in the beaker, adds chloroform and 70% ethanol concussion washing, reclaims the nucleic acid precipitation, and is dry under the room temperature.With 2ml～5ml TE (10mM Tris-HCL, 1mM EDTA, pH=7.0) dissolving nucleic acid precipitation.Nucleic acid to purifying in 1% agarose gel carries out the electrophoresis evaluation.

Claims (7)

1, a kind ofly will ferment that the back engineering bacteria concentrates, Pintsch process, plasmid separate and the method for the production of plasmid DNA of purifying, comprising following steps:

1) the back engineering bacteria that ferments is concentrated, collect and concentrate bacterium liquid;

2) adding lysate reacts;

3) above-mentioned be reflected at 25～37 ℃ of incubations 20～60 minutes and protoplastis;

4) make the protoplastis temperature rise to 50～80 ℃ by heating tube above-mentioned protoplastis solution and make the thorough cracking of its protoplasma;

5) with the thalline after the cracking by bacterial chip and bacterial chromosome in 400～1000 purpose filter membrane separation of supernatant and the lysate;

6) pass through the supernatant liquor after separating is added an amount of NaAc and its nucleic acid of isopropanol precipitating, and in 100～400 order filter membranes, filter to isolate the nucleic acid throw out;

7) clean this nucleic acid throw out by 70% ethanol;

8) dry this nucleic acid throw out; Do not repay mistake

9) add an amount of TE solution it is dissolved, and obtain the plasmid DNA product, wherein said TE solution is the mixing solutions of Tris-HCl and EDTA.

2, the method for claim 1, concentrated bacterium obtains by the following method: to volume greater than 5 liters of fermentations after engineering bacteria to use the aperture be that the hollow-fibre membrane post of 0.2～0.4 μ concentrates.

3, the method for claim 1, concentrated bacterium obtains by the following method: to volume greater than 5 liters of fermentations after engineering bacteria to use the aperture be that the tangential flow filter membrane of 0.2～0.4 μ concentrates.

4, the method for claim 1, the cracking of thalline is carried out by the following method: add an amount of 10mMTris-HCL, 50mM EDTA, the TE solution of pH=8.0 will concentrate bacterium liquid and be diluted to OD₆₀₀After=100, add the final concentration weightmeasurement ratio again and be 0.5～2% Triton X-100,0.1 the N,O-Diacetylmuramidase of～0.5M sodium-chlor and 50～300 mcg/ml reacts, be reflected at 25～37 ℃ of incubations 20～60 minutes and get protoplastis, make the protoplastis temperature rise to 50～80 ℃ of cracking 20～60 seconds by heating tube, make the thorough cracking of its protoplasma.

5, the method of claim 1, the plasmid DNA product obtains by the following method: the thorough bacterial chip and the bacterial chromosome and obtain supernatant liquor of cracked protoplastis in 400～1000 purpose membrane filtration separation of supernatant and lysate, add 1/10 cumulative volume, the pH value is that 5.2 the 3M NaAc and the Virahol of 0.6 times of cumulative volume precipitate its nucleic acid in supernatant, in 100～400 order filter membranes, filter to isolate the nucleic acid throw out, and after cleaning this throw out with 70% ethanol, carry out drying, add an amount of TE solution and dissolve, and obtain the plasmid DNA product of purifying.

6, the method for claim 1, the plasmid DNA product obtains by the following method: thoroughly the cracking protoplastis separates the bacterial chip removed in the lysate and bacterial chromosome and obtains supernatant liquor through 400～1000 purpose filter membranes, add 1/10 cumulative volume, pH value and be 5.2 the 3M NaAc and the Virahol of 0.6 times of cumulative volume and in supernatant, precipitate its nucleic acid, in 200～400 order filter membranes, filter to isolate the nucleic acid throw out, and after cleaning this throw out with 70% ethanol, carry out drying, add an amount of TE solution and dissolve, and obtain the plasmid DNA product of purifying.

7, the method of claim 1, the plasmid DNA product obtains by the following method: thoroughly the cracking protoplastis separates the bacterial chip removed in the lysate and bacterial chromosome and obtains supernatant liquor through 400～1000 purpose filter membranes, add 1/10 cumulative volume, the pH value is that 5.2 the 3M NaAc and the Virahol of 0.6 times of cumulative volume precipitate its nucleic acid in supernatant, in 200～400 order filter membranes, filter to isolate the nucleic acid throw out, and after cleaning this throw out with chloroform and 70% ethanol respectively, carry out drying, add an amount of TE solution and dissolve, and obtain the plasmid DNA product of purifying.

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