CN114456940A - Method for improving plasmid stability in escherichia coli cracking process - Google Patents

Method for improving plasmid stability in escherichia coli cracking process Download PDF

Info

Publication number
CN114456940A
CN114456940A CN202210076041.3A CN202210076041A CN114456940A CN 114456940 A CN114456940 A CN 114456940A CN 202210076041 A CN202210076041 A CN 202210076041A CN 114456940 A CN114456940 A CN 114456940A
Authority
CN
China
Prior art keywords
solution
plasmid
liquid
chromatography
cracking
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210076041.3A
Other languages
Chinese (zh)
Other versions
CN114456940B (en
Inventor
胡海兵
韦厚良
潘讴东
由庆睿
贾国栋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Obio Technology (shanghai) Corp ltd
Original Assignee
Obio Technology (shanghai) Corp ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Obio Technology (shanghai) Corp ltd filed Critical Obio Technology (shanghai) Corp ltd
Priority to CN202210076041.3A priority Critical patent/CN114456940B/en
Publication of CN114456940A publication Critical patent/CN114456940A/en
Application granted granted Critical
Publication of CN114456940B publication Critical patent/CN114456940B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention provides a method for improving plasmid stability in an escherichia coli cracking process. The method comprises the following specific steps: 1) re-suspending thalli, wherein the re-suspension contains disaccharide chemical substances; 2) alkali cracking; 3) neutralizing; 4) clarifying; 5) concentrating and replacing liquid; 6) performing molecular sieve chromatography; 7) and (5) replacing the liquid preparation. The method has simple and convenient process, protects supercoiled plasmids in the alkaline cracking process of the escherichia coli by adding disaccharide (such as sucrose, trehalose and the like) with certain concentration into the thallus heavy suspension, simplifies the downstream chromatography purification process, improves the plasmid recovery rate, is convenient and quick, and is suitable for popularization and application.

Description

Method for improving plasmid stability in escherichia coli cracking process
Technical Field
The invention relates to a plasmid production method, in particular to a method for improving plasmid stability.
Background
Plasmids are small circular supercoiled double-stranded DNA, contain extrachromosomal genetic information, and can replicate autonomously in bacteria. Plasmids are one of the most important tools in gene therapy and vaccines, and there are mainly 3 application forms: 1) direct in vivo injection, such as DNA vaccines and naked plasmid gene vectors; 2) transforming target cells, such as T cells, by transfection in vitro, and then returning to the body; 3) in vitro, multi-plasmid co-transfections of cells, viral vectors such as adeno-associated virus (AAV) and Lentivirus (LV) are produced for subsequent use in vivo or in vitro gene therapy.
No matter naked plasmid or virus vector is packed, the target gene carried by plasmid is finally introduced into target cell to reach the aim of preventing and treating diseases. The supercoiled plasmid with high quality and high purity is a precondition for ensuring the safety and the effectiveness of gene vectors such as naked plasmid, AAV, LV and the like. The plasmid is produced by taking glycerol bacteria in an escherichia coli working library as a starting raw material, and performing resuscitation, fermentation, harvesting, cracking, clarification, purification, preparation liquid exchange, filtration and subpackaging to obtain the finished product plasmid. Plasmids have various topologies, including supercoiled, open-loop, and linear plasmids, wherein supercoiled plasmids are considered to be the most active component, while open-loop plasmids require strict control of their content in the finished plasmid due to low efficiency of transducing cells.
In the production process, especially in the alkaline lysis process, the open-loop plasmid is easily generated because the plasmid is exposed in the environment of pH 12-13. Two difficulties are brought to the plasmid production process by open-loop plasmids: 1) the complexity of the plasmid purification process is increased, and at least two steps of chromatography, such as various combinations of gel filtration, hydrophobic chromatography, affinity chromatography and ion exchange chromatography, are required to achieve the effect of removing the open-loop plasmids; 2) the recovery rate of plasmid is reduced, the open-loop plasmid is generated by the denaturation of supercoiled plasmid, and the generation of large amount of open-loop plasmid inevitably leads to the reduction of supercoiled plasmid content, and finally the supercoiled yield is reduced. Conventional methods for increasing the proportion of supercoiled plasmid in the lysis process include optimizing the lysis time, the sodium hydroxide concentration or the stirring speed, but these methods are difficult to scale up.
Disclosure of Invention
The invention aims to provide a simple method for improving the stability of supercoiled plasmid in the escherichia coli cracking process. The method can improve the recovery rate of the supercoiled plasmid, reduce the generation of open-loop plasmid in the cracking process, reduce the chromatography step of plasmid purification, is convenient and quick, and is suitable for popularization and application.
The invention discloses a method for improving the stability of supercoiled plasmid in the process of escherichia coli cracking, which is characterized by comprising the following steps:
(1) and (3) resuspending the thallus: adding the wet thalli obtained by fermentation into a heavy suspension, and uniformly stirring, wherein the heavy suspension contains sucrose or trehalose;
(2) cracking: mixing and stirring the resuspension and the lysate uniformly;
(3) neutralizing: adding a neutralizing solution into the cracking solution, and uniformly mixing and stirring;
(4) clarification: filtering or centrifuging the neutralized lysate to obtain a clarified harvest solution;
(5) concentrating and replacing liquid: performing tangential flow filtration on the clarified harvest liquid, and concentrating and replacing the harvest liquid into a heavy suspension;
(6) and (3) chromatographic purification: carrying out one-step chromatographic purification on the concentrated and liquid-changed sample to obtain a plasmid after chromatographic purification;
(7) and (3) replacing the preparation liquid: and (3) carrying out tangential flow filtration on the plasmids after chromatography purification, concentrating and changing the liquid into a buffer solution, thereby obtaining the purified plasmid finished product.
Preferably, the mass concentration of the sucrose or the trehalose is 1-10%. More preferably, the mass concentration of the sucrose or the trehalose is 2.5 to 5 percent.
Preferably, the ratio of the volume of the resuspension to the mass of the wet thallus in the step (1) is 10 ml: 1g of the total weight of the composition.
Preferably, the lysis solution is 0.2M NaOH, 1% SDS solution.
Preferably, the neutralization solution is a 3M KAc, 2M HAc solution.
Preferably, the clarification step is performed by using a depth filter or centrifugation at 5000 Xg for 30 min;
preferably, the tangential flow filtration of step (5) is selected from the group consisting of a hollow fiber or flat sheet membrane with a molecular weight cut-off of 50 kD, 100 kD or 300 kD.
Preferably, the resuspension solution of steps (1) and (5) comprises: 50 mM Tris, 10 mM EDTA, 2.5% (w/v) sucrose, pH 8.0.
Preferably, the chromatographic purification method of step (6) is selected from affinity chromatography, anion exchange chromatography, or gel filtration chromatography.
Preferably, the tangential flow filtration of step (7) is performed with hollow fiber or flat sheet membranes having a molecular weight cut-off selected from 50 kD, 100 kD or 300 kD.
Preferably, the escherichia coli (e.coli) includes JM109, DH5 α, Stbl3, Top10, DH10B, and the like.
Compared with the prior art, the invention has the following advantages:
1. improves the components of the resuspension, greatly improves the supercoiled proportion of the finished plasmid only by one-step chromatography purification, simplifies the process and improves the technical effect.
2. In addition, the improved heavy suspension is used for cracking thalli, the supercoiled proportion of intermediate products (cracking, concentration and liquid exchange and molecular sieve chromatography) in the purification step is also larger than that of a control group, and the stability in the plasmid purification process is greatly improved.
3. The improved raw materials are cheap and easy to obtain, and the method is economic and environment-friendly.
Drawings
FIG. 1 is a flow chart of the purification process of one-step molecular sieve chromatography in example 1 of the present invention.
FIG. 2 is a HPLC chart of the plasmid product purified by using sucrose-containing cell suspension in example 1 of the present invention.
FIG. 3 is a HPLC chart of the plasmid product purified by using a sucrose-free bacterial suspension (control group) in example 1 of the present invention.
FIG. 4 is a flow chart of a purification process of one-step ion exchange chromatography in example 2 of the present invention.
FIG. 5 is a HPLC chart of the plasmid product purified by using trehalose-containing cell resuspension solution (control group) in example 2 of the present invention.
FIG. 6 is a HPLC chart of the plasmid products purified by using the trehalose-free cell suspension (control) in example 2 of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
The bacterial lysis and one-step molecular sieve chromatography purification are carried out according to the process route shown in figure 1:
the specific implementation steps are as follows:
1) and (3) resuspending the thallus: adding 100 gDH5 alpha wet thalli obtained by fermentation into 1L of the heavy suspension according to the proportion of 10ml of the heavy suspension/g of the wet thalli, and uniformly stirring, wherein the heavy suspension comprises the following components: 50 mM Tris, 10 mM EDTA, 2.5% (w/v) sucrose, pH 8.0;
2) alkali cracking: mixing and stirring the bacterial suspension and 1L of lysate (0.2M NaOH, 1% SDS solution) uniformly for 3-5 min;
3) neutralizing: adding 1L of pre-cooled neutralizing solution (3M KAc, 2M HAc solution) at 4 deg.C into the lysate, mixing and stirring;
4) clarification: filtering the neutralized lysate by using a 1.0 mu m deep filter to obtain a clarified harvest solution;
5) concentrating and replacing liquid: performing tangential flow filtration on the clear harvest liquid by using a flat membrane package with the molecular weight cutoff of 100 kD and the flow rate of 0.1 m2, controlling the transmembrane pressure within 1 bar, firstly concentrating the clear harvest liquid by 30 times to 100 ml, and then washing the filtrate by 6 times to 50 mM Tris, 10 mM EDTA, 2.5% (w/v) sucrose and pH 8.0 solution;
6) molecular sieve chromatography: purifying the concentrated solution by molecular sieve chromatography, wherein Sepharose 6FF is used as a molecular sieve filler, the volume of a column bed is 350 ml, firstly 100 mM Tris, 10 mM EDTA, 2.0M ammonium sulfate and pH7.5 solution are used for balancing the chromatographic column, the balance volume is 700 ml, the flow rate is 15 ml/min, then the concentrated solution-changed sample is loaded into the molecular sieve chromatographic column, the loading flow rate is 15 ml/min, after loading is finished, the chromatographic column is washed by 100 mM Tris, 10 mM EDTA, 2.0M ammonium sulfate and pH7.5 solution, and the first peak of the loading and washing process is collected, namely a plasmid sample, and the volume is 120 ml;
7) and (3) replacing the preparation liquid: the molecular sieve chromatography harvest liquid is used for 50 cm2Tangential flow filtration is carried out by a membrane package with the molecular weight cutoff of 100 kD, the flow rate is 30 ml/min, the transmembrane pressure is controlled within 1 bar, firstly, the sample is washed to the concentration of 1 mg/ml, then, 6 times of volume of the sample is washed into a solution of 10 mM Tris, 1 mM EDTA and pH 8.0, and the purified supercoiled plasmid is obtained;
8) control experiments 100 g of cells were lysed in the same manner as described above and purified by ultrafiltration and chromatography using a suspension of 50 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0.
The purified supercoiled plasmid was examined and the results are given below. The thalli is cracked by using a heavy suspension containing 2.5 percent of sucrose, and only one-step chromatography purification is carried out, so that the supercoiled proportion of the finished product plasmid can reach 95.2 percent, the proportion of the open-loop plasmid is only 2.3 percent, and the quality standard of the regulation on the supercoiled plasmid proportion of more than 90 percent is met. And the conventional resuspension is used for cracking thalli, and only one-step chromatography purification is carried out, so that the supercoiled proportion of the finished product plasmid is only 86.4 percent and is lower than 90 percent, and the proportion of the open-loop plasmid reaches 7.3 percent, which means that further chromatography purification is needed, and the supercoiled proportion is improved. In addition, when the bacterial cells were lysed using a 2.5% sucrose-containing resuspension solution, the supercoiled fraction of the intermediate products of the purification step (lysis, concentration and exchange and molecular sieve chromatography) was also greater than that of the control group, indicating that the plasmids in the experimental group were more stable during the purification process.
TABLE 1 results of plasmid purification using sucrose-containing cell resuspension solution
Figure 158215DEST_PATH_IMAGE002
Example 2
The bacterial lysis and one-step ion exchange chromatography purification are carried out according to the process route shown in figure 4:
the specific implementation steps are as follows:
1) and (3) resuspending the thallus: adding 150 g of Stbl3 wet thalli obtained by fermentation into 1.5L of the heavy suspension according to the proportion of 10ml of the heavy suspension per g of the wet thalli, and uniformly stirring, wherein the heavy suspension comprises the following components: 50 mM Tris, 10 mM EDTA, 5% (w/v) trehalose, pH 8.0;
2) and (3) cracking of thalli: mixing and stirring the bacterial suspension and 1.5L of lysate (0.2M NaOH, 1% SDS solution) uniformly for 3-5 min;
3) neutralizing: adding 1.5L of 4 deg.C pre-cooled neutralizing solution (3M KAc, 2M HAc solution) into the lysate, mixing and stirring;
4) clarification: filtering the neutralized lysate by using a 1.0 mu m deep filter to obtain a clarified harvest solution;
5) concentrating and replacing liquid: the clear harvest was used at 0.1 m2Tangential flow filtration is carried out by a flat membrane package with the molecular weight cutoff of 100 kD, the flow rate is 500 ml/min, the transmembrane pressure is controlled within 1 bar, firstly, the clear harvest liquid is concentrated by 10 times to 450 ml, and then, the filtrate is washed by 6 times of volume to 50 mM Tris, 10 mM EDTA, 5% (w/v) trehalose, and the pH value is 8.0;
6) ion exchange chromatography: and (3) purifying the concentrated solution by ion exchange chromatography, wherein the Capto Q ImPres is used as an ion exchange filler, the volume of a column bed is 200 ml, the chromatographic column is balanced by using a solution of 100 mM Tris, 10 mM EDTA, 0.4M sodium chloride and pH7.5, the equilibrium volume is 500 ml, the flow rate is 20 ml/min, then the sample after the concentrated solution exchange is loaded into the ion exchange chromatographic column, the loading flow rate is 20 ml/min, and after the loading is finished, the chromatographic column is washed by using a solution of 100 mM Tris, 10 mM EDTA, 0.4M sodium chloride and pH7.5, and the washing volume is 400 ml. After washing, performing linear gradient elution by using 0-100% B and 10 column volumes (2000 ml) at an elution flow rate of 20 ml/min, wherein the phase B is 100 mM Tris, 10 mM EDTA, 1.0M sodium chloride and a pH7.5 solution, and collecting a plasmid peak in an elution process, namely a plasmid sample with the volume of 430 ml;
7) and (3) replacing the preparation liquid: the ion exchange chromatography harvest solution is used at 0.1 m2Tangential flow filtration is carried out by a membrane package with the molecular weight cutoff of 100 kD, the flow rate is 500 ml/min, the transmembrane pressure is controlled within 1 bar, firstly, the sample is washed to the concentration of 1 mg/ml, then, 6 times of volume of the sample is washed into a solution of 10 mM Tris, 1 mM EDTA and pH 8.0, and the purified supercoiled plasmid is obtained;
8) control experiments 150 g of cells were lysed in the same manner as described above and purified by ultrafiltration and chromatography using a suspension of 50 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0.
The purified supercoiled plasmid was examined, and the results are as follows. The recombinant containing 5% trehalose is used for cracking thalli, and only one-step ion exchange chromatography purification is carried out, so that the supercoiled proportion of the finished product plasmid can reach 94.7%, the proportion of the open-loop plasmid is only 1.4%, and the quality standard of the regulation on the supercoiled plasmid proportion of more than 90% is met. And the conventional resuspension is used for cracking thalli, and only one-step chromatography purification is carried out, so that the supercoiled proportion of the finished product plasmid is only 85.6 percent and is lower than 90 percent, and the proportion of the open-loop plasmid reaches 9.2 percent, which means that further chromatography purification is needed, and the supercoiled proportion is improved. In addition, when the bacterial cells were lysed using a 5% trehalose-containing resuspension solution, the supercoiled fraction of the intermediate products of the purification step (lysis, concentration and exchange of liquid and molecular sieve chromatography) was also greater than that of the control group, indicating that the plasmids in the experimental group were more stable during the purification process.
TABLE 2 results of plasmid purification using trehalose-containing cell resuspension solution
Figure 652519DEST_PATH_IMAGE004

Claims (10)

1. A method for improving the stability of supercoiled plasmid in the process of Escherichia coli cracking is characterized by comprising the following steps:
(1) and (3) resuspending the thallus: adding the wet thalli obtained by fermentation into a heavy suspension, and uniformly stirring, wherein the heavy suspension contains sucrose or trehalose;
(2) cracking: mixing and stirring the heavy suspension and the lysate uniformly;
(3) neutralizing: adding a neutralizing solution into the cracking solution, and uniformly mixing and stirring;
(4) clarification: filtering or centrifuging the neutralized lysate to obtain a clarified harvest solution;
(5) concentrating and replacing liquid: performing tangential flow filtration on the clarified harvest liquid, and concentrating and replacing the harvest liquid into a heavy suspension;
(6) and (3) chromatographic purification: carrying out one-step chromatographic purification on the concentrated and liquid-changed sample to obtain a plasmid after chromatographic purification;
(7) and (3) replacing the preparation liquid: and (3) carrying out tangential flow filtration on the plasmids after chromatography purification, concentrating and changing the liquid into a buffer solution, thereby obtaining the purified plasmid finished product.
2. The method according to claim 1, wherein the sucrose or trehalose is at a mass concentration of 1% to 10%.
3. The method according to claim 1, wherein the sucrose or trehalose is at a mass concentration of 2.5% to 5%.
4. The method according to claim 1, wherein the ratio of the volume of the resuspension to the mass of the wet thallus in step (1) is 10 ml: 1g of the total weight of the composition.
5. The method of claim 1, wherein the lysis solution is 0.2M NaOH, 1% SDS solution.
6. The method of claim 1, wherein the neutralizing solution is a 3M KAc, 2M HAc solution.
7. The process of claim 1, wherein the tangential flow filtration of steps (5) and (7) selects hollow fiber or flat sheet membranes with a molecular weight cut-off of 50 kD, 100 kD, or 300 kD.
8. The method of claim 1, wherein the resuspension composition of steps (1) and (5) comprises 50 mM Tris, 10 mM EDTA, 2.5% (w/v) sucrose or trehalose, pH 8.0.
9. The method of claim 1, wherein the chromatographic purification method of step (6) is selected from affinity chromatography, anion exchange chromatography, or gel filtration chromatography.
10. The method of claim 1, wherein the buffer of step (7) is 10 mM Tris, 1 mM EDTA, pH 8.0.
CN202210076041.3A 2022-01-23 2022-01-23 Method for improving plasmid stability in escherichia coli cracking process Active CN114456940B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210076041.3A CN114456940B (en) 2022-01-23 2022-01-23 Method for improving plasmid stability in escherichia coli cracking process

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210076041.3A CN114456940B (en) 2022-01-23 2022-01-23 Method for improving plasmid stability in escherichia coli cracking process

Publications (2)

Publication Number Publication Date
CN114456940A true CN114456940A (en) 2022-05-10
CN114456940B CN114456940B (en) 2024-05-03

Family

ID=81411745

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210076041.3A Active CN114456940B (en) 2022-01-23 2022-01-23 Method for improving plasmid stability in escherichia coli cracking process

Country Status (1)

Country Link
CN (1) CN114456940B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115572739A (en) * 2022-10-31 2023-01-06 天津科技大学 Method for preparing hydrogel by using fermentation thalli as raw material
CN116790578A (en) * 2023-08-22 2023-09-22 赛奥斯博生物科技(北京)有限公司 Production process for rapidly purifying plasmid based on alkaline cracking method
CN116855359A (en) * 2023-07-11 2023-10-10 江苏谱新生物医药有限公司 System and method for alkaline lysis of plasmid
CN116987581A (en) * 2023-08-08 2023-11-03 江苏耀海生物制药有限公司 Plasmid DNA preparation device and preparation process

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1190435A (en) * 1995-05-19 1998-08-12 麦克公司 Method for large scale plasmid purification
US6281349B1 (en) * 1999-08-18 2001-08-28 David E. Pulleyblank Method for nucleic acid purification using iodine
CN1914330A (en) * 2004-02-04 2007-02-14 默克公司 Process for large scale production of plasmid DNA by e. coli fermentation
CN101310022A (en) * 2005-01-31 2008-11-19 默克公司 Purification process for piasmid DNA
US20080299621A1 (en) * 2007-05-31 2008-12-04 Ge Healthcare Uk Limited Miniprep system for simple and rapid plasmid dna extraction
US20080300397A1 (en) * 2007-05-31 2008-12-04 Ge Healthcare Uk Limited Modified spin column for simple and rapid plasmid dna extraction
CN101717770A (en) * 2009-12-24 2010-06-02 上海市农业科学院 Method for extracting large plasmid DNA of bacillus thuringiensis
CN102220256A (en) * 2011-04-14 2011-10-19 暨南大学 Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof
CN109294968A (en) * 2017-07-25 2019-02-01 格兰柏生化科技有限公司 A kind of mass production techniques of animal nucleic acid vaccine
CN109468311A (en) * 2018-11-27 2019-03-15 河南普诺易生物制品研究院有限公司 A method of extracting the cellular lysate liquid, kit and extraction Plasmid DNA of Plasmid DNA

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1190435A (en) * 1995-05-19 1998-08-12 麦克公司 Method for large scale plasmid purification
US6281349B1 (en) * 1999-08-18 2001-08-28 David E. Pulleyblank Method for nucleic acid purification using iodine
CN1914330A (en) * 2004-02-04 2007-02-14 默克公司 Process for large scale production of plasmid DNA by e. coli fermentation
CN101310022A (en) * 2005-01-31 2008-11-19 默克公司 Purification process for piasmid DNA
US20080299621A1 (en) * 2007-05-31 2008-12-04 Ge Healthcare Uk Limited Miniprep system for simple and rapid plasmid dna extraction
US20080300397A1 (en) * 2007-05-31 2008-12-04 Ge Healthcare Uk Limited Modified spin column for simple and rapid plasmid dna extraction
CN101717770A (en) * 2009-12-24 2010-06-02 上海市农业科学院 Method for extracting large plasmid DNA of bacillus thuringiensis
CN102220256A (en) * 2011-04-14 2011-10-19 暨南大学 Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof
CN109294968A (en) * 2017-07-25 2019-02-01 格兰柏生化科技有限公司 A kind of mass production techniques of animal nucleic acid vaccine
CN109468311A (en) * 2018-11-27 2019-03-15 河南普诺易生物制品研究院有限公司 A method of extracting the cellular lysate liquid, kit and extraction Plasmid DNA of Plasmid DNA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WERNER PANSEGRAU等: "In vitro assembly of relaxosomes at the transfer origin of plasmid RP4", PROC. NATL. ACAD. SCI., vol. 87, pages 6555 - 6559 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115572739A (en) * 2022-10-31 2023-01-06 天津科技大学 Method for preparing hydrogel by using fermentation thalli as raw material
CN116855359A (en) * 2023-07-11 2023-10-10 江苏谱新生物医药有限公司 System and method for alkaline lysis of plasmid
CN116987581A (en) * 2023-08-08 2023-11-03 江苏耀海生物制药有限公司 Plasmid DNA preparation device and preparation process
CN116790578A (en) * 2023-08-22 2023-09-22 赛奥斯博生物科技(北京)有限公司 Production process for rapidly purifying plasmid based on alkaline cracking method
CN116790578B (en) * 2023-08-22 2023-12-12 赛奥斯博生物科技(北京)有限公司 Production process for rapidly purifying plasmid based on alkaline cracking method

Also Published As

Publication number Publication date
CN114456940B (en) 2024-05-03

Similar Documents

Publication Publication Date Title
CN114456940B (en) Method for improving plasmid stability in escherichia coli cracking process
AU2017391164B2 (en) Adeno-associated virus purification methods
US5561064A (en) Production of pharmaceutical-grade plasmid DNA
US20100273254A1 (en) Process for purification of plasmid dna
CN109456963B (en) Method for large-scale purification of plasmid DNA
CN111876393A (en) Method for large-scale rapid production of high-purity high-activity lentiviral vector
JP2024041758A (en) Method for large-scale preparation of purified preparations of recombinant lentiviral vectors at GMP grade
CN111304193A (en) Method for large-scale rapid purification of plasmid DNA
Joshi et al. Development of a Scalable and Robust Anion-Exchange Chromatographic Method for Enriched Recombinant Adeno-Associated Virus Preparations in Genome Containing Vector Capsids of Serotypes-5, 6, and 8
CN114317464B (en) Separation and purification method of adeno-associated virus rAAV9
CN115925890A (en) Method for purifying anti-new coronavirus neutralizing antibody
Franco-Medrano et al. Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography
McCarron et al. Scale-up of lentiviral vectors for gene therapy: advances and challenges
CN116790578B (en) Production process for rapidly purifying plasmid based on alkaline cracking method
CN117757759A (en) Method for purifying NK cell-directed lentivirus and application thereof
CN116200282A (en) Hepatitis B vaccine and its production method
CN113584106B (en) Preparation method and application of recombinant novel coronavirus NCP-RBD protein expressed by CHO (Chinese hamster ovary) cells
US11879139B1 (en) Scalable methods for purification of recombinant viruses
WO2023177639A1 (en) Lentiviral manufacturing platform
WO2023165831A1 (en) A method for separating supercoiled plasmid dna
CN118006688A (en) Purification method of recombinant adeno-associated virus vector
CN117660373A (en) Large-scale lentivirus purification method and application thereof
CN118006595A (en) Plasmid DNA purification method and kit
Kearney Check Chapter 4 updates for
CN115612684A (en) GMP-grade plasmid DNA large-scale simplified production method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant