CN117025585A - Preparation method of high-standard plasmid - Google Patents
Preparation method of high-standard plasmid Download PDFInfo
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- CN117025585A CN117025585A CN202310918483.2A CN202310918483A CN117025585A CN 117025585 A CN117025585 A CN 117025585A CN 202310918483 A CN202310918483 A CN 202310918483A CN 117025585 A CN117025585 A CN 117025585A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
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- 238000000034 method Methods 0.000 claims abstract description 34
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- 238000000855 fermentation Methods 0.000 claims abstract description 26
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- 238000005336 cracking Methods 0.000 claims abstract description 10
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- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims abstract description 8
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims abstract description 8
- 239000001099 ammonium carbonate Substances 0.000 claims abstract description 8
- 238000011097 chromatography purification Methods 0.000 claims abstract description 7
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- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 4
- 238000005273 aeration Methods 0.000 claims description 4
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- 239000012526 feed medium Substances 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
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- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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- 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
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Abstract
The invention discloses a preparation method of a high-standard plasmid, which belongs to the technical field of genetic engineering, and comprises the following steps: s1, culturing seed liquid; s2, fermenting; s3, cracking and clarifying; s4, chromatographic purification; compared with the traditional fermentation method and the feeding method, the method can further improve the thallus density, and the method comprises the following steps: (1) the fermentation operation process is stable, the purification method is simple and convenient, the supercoiled plasmid with higher supercoiled plasmid content more than 90% can be obtained by using only two-step chromatography purification, and the host DNA, protein and endotoxin content is lower. (2) The large intestine alkaline pyrolysis liquid is simple and convenient to clarify, and the liquid is clarified by adding the ammonium bicarbonate and a positive pressure filter, so that the clarification time of the complex pyrolysis liquid is greatly shortened.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of a high-standard plasmid.
Background
Plasmids are a class of double-stranded circular DNA molecules that carry genetic information outside of the chromosome. Since the plasmid has a self-replication function, one cell may contain multiple copies of the plasmid. The production of plasmid DNA on a laboratory scale firstly constructs a proper expression vector and screens an optimal host bacterium (generally, escherichia coli), then screens and optimizes a fermentation medium and fermentation conditions, separates and purifies products, and along with the development of biotechnology, particularly molecular cloning technology, recombinant escherichia coli is increasingly applied to the mass production of high-added-value products such as protein medicines, enzyme preparations, amino acids, biological additives and the like.
However, the stability of recombinant escherichia coli is poor, strain decay easily occurs in long-term production, and the conventional strain preservation method cannot keep the growth and metabolism activities of the strain for a long time, so that the problems of autolysis of the strain, plasmid loss, yield reduction and the like easily occur in production. In addition, in the fermentation process, the traditional fermentation method and the feeding method cannot reach high thallus density, so that the improvement of the product yield is limited.
Disclosure of Invention
The invention aims to provide a high-standard plasmid preparation method to solve the problem that the traditional fermentation method is unfavorable for improving the yield and quality in the plasmid preparation process.
The aim of the invention can be achieved by the following technical scheme:
the preparation method of the high-standard plasmid comprises the following steps:
s1, culturing seed liquid:
transferring the pMD2.G plasmid into NEB E.Coli Stable competent cells by using a heat shock method, and coating a plate for culturing at 30 ℃ to obtain a plate monoclonal colony;
taking a flat monoclonal colony to culture in an LB culture medium, and culturing at 30 ℃ until the OD600 is 0.6-0.8 as primary seed liquid; transferring the first-stage seed liquid into a second-stage seed culture medium shake flask for culture, wherein the inoculum size is 0.5% -1%, and culturing at 30 ℃ until the OD600 is 0.8-1, wherein the first-stage seed liquid is used as the second-stage seed liquid;
s2, fermenting:
inoculating: inoculating the secondary seed liquid into a fermentation culture medium with an inoculum size of 1-5%; fermenting and culturing at 30 ℃, wherein the aeration rate is 2L/min, the initial stirring speed is 50rpm, and the associated stirring maintains the dissolved oxygen DO at 30%;
and (3) fermentation regulation: the initial temperature is 30-35 ℃, the initial pH value is 6.7-6.8, acid (50% phosphoric acid) and alkali (50% ammonia water) are used for maintaining the pH value of 6.8-7.0 in the fermentation process, when the dissolved oxygen DO in the fermentation process rises suddenly and is more than 60%, a feeding culture medium is added, the value of the dissolved oxygen DO after feeding is reduced, and feeding is stopped when the dissolved oxygen DO is less than 40%; when the dissolved oxygen DO is more than 40%, and the pH is increased, continuously adding a feed culture medium, repeatedly feeding, heating to 37 ℃ after the first feeding for 30min, and continuously culturing for 4-6 h; then raising the temperature to 42 ℃ for 2 hours, and finally stopping feeding to ensure that the escherichia coli is kept starved for 10 minutes;
and (3) thallus collection: hollow fibers with an inner diameter of 750KD are used for collection and washing filtration, and the strains are weighed after collection.
S3, cracking and clarifying:
weighing strain, suspending in buffer solution, slowly adding alkaline lysis solution, slowly mixing, lysing for 8min, adding neutralization solution, and slowly mixing; transferring the solid-liquid separation device, adding ammonium bicarbonate solution, performing solid-liquid separation, standing for 10min, and retaining bottom clear liquid;
deep filtering the bottom clarified liquid with 0.22 mu m polypropylene filter paper, concentrating by using 300KD hollow fiber for 10-20 times, and washing and filtering by using buffer solution for 6 times (adding 6 times of buffer solution into the clarified liquid, and concentrating into the volume before the buffer solution);
adding ammonium sulfate to make the concentration of the ammonium sulfate be 2.1M, and filtering and clarifying by using 0.45um PES material filter paper to obtain a lysate;
s4, chromatographic purification:
sequentially carrying out Sepharose6Fast Flow molecular sieve chromatography and Capto Q ion exchange chromatography to obtain plasmid stock solution; precipitating plasmid stock solution with isopropanol, washing with ethanol, drying with sterile air, filtering, and sterilizing.
Further, in S1, the second seed culture medium in shake flask contains peptone 40g/L, yeast powder 20g/L, and potassium dihydrogen phosphate 2.22g/L, and pH is adjusted to 7 with 10M NaOH.
Further, 12g/L of soybean peptone, 20g/L of yeast powder and 7.5g/L, na of glycerol are contained in the fermentation medium in the step S2 2 HPO 4 ·12H 2 O 8.25g/L、KH 2 PO 4 3g/L、MgSO 4 ·7H 2 O2g/L, ammonium sulfate 4.2g/L, thiamine with 2% concentration 0.2mL/L and trace element solution 1mL/L.
Wherein the microelement solution is CuCl 2 ·2H 2 O 1.5g、CoCl 2 1.4g、ZnSO 4 ·7H 2 O 2g、H 3 BO 3 3g、NaMoO 4 ·H 2 O 2.5g、MnSO 4 ·H 2 O 1.5g、FeSO 4 ·7H 2 O10 g and HCl100 mL, and is obtained after filtration sterilization.
Further, mgSO in the feed medium in S2 4 ·7H 2 20g/L of O, 500g/L of glycerol, 50g/L of soybean peptone and 30g/L of yeast powder.
Further, the feeding speed in S2 is 0.2-0.5 mL/min.
Further, EDTA 10mM, sucrose 2%, tris-HCl50mM, pH 8.0 in the buffer in S3; naOH 0.2M, SDS percent in the alkaline cracking solution in S3; the neutralization solution was potassium acetate 3M and pH was adjusted to 5 using glacial acetic acid.
Further, the concentration of the ammonium bicarbonate solution in the S3 is 5g/L, and the dosage ratio of the strain, the buffer solution, the alkaline cracking solution and the neutralizing solution is 100g:1.5L:1.5L:1.5L.
Further, in S4, the balance liquid A (NH 4 ) 2 SO 4 2.1M, EDTA mM, tris-HCl100mM, pH 7.5;
equilibration solution B0.4M NaCl, EDTA 10mM, tris-HCl100mM, pH 7.5;
the balance C was NaCl 1.0M, EDTA mM, tris-HCl100mM, pH 7.5.
Further, sepharose6Fast Flow molecular sieve chromatography: balancing solution A balances 3 column volumes; sample loading amount: 30% of column volume, and purifying the lysate by molecular sieve chromatography to obtain plasmids;
capto Q ion exchange chromatography: the plasmid obtained by molecular sieve chromatography and purification is diluted by 3.85 times by pure water, and then NaCl is added so that the final concentration of NaCl is 0.1M to be used as a sample to be purified; the loading was 1.5mg plasmid DNA/mL of filler; and after the sample loading is finished, washing the mixed column volume by using the balance liquid B for 3 times to remove endotoxin, and finally eluting the plasmid by using the balance liquid C to obtain plasmid stock solution.
The invention has the beneficial effects that:
the method comprises the following steps: (1) the fermentation operation process is stable, the purification method is simple and convenient, the supercoiled plasmid with higher supercoiled plasmid content more than 90% can be obtained by using only two-step chromatography purification, and the host DNA, protein and endotoxin content is lower. (2) The large intestine alkaline pyrolysis liquid is simple and convenient to clarify, and the liquid is clarified by adding the ammonium bicarbonate and a positive pressure filter, so that the clarification time of the complex pyrolysis liquid is greatly shortened.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a diagram showing solid-liquid separation and standing in example 1 of the present invention;
FIG. 2 is a graph showing the analysis result of molecular sieves in example 1 of the present invention;
FIG. 3 is a gel electrophoresis detection chart in example 1 of the present invention;
FIG. 4 is a graph of HPLC results in example 1 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a high-standard plasmid, which comprises the following steps:
s1, culturing seed liquid:
transferring pMD2.G plasmid (virus packaging plasmid, pMD2.G, size 5822bp. Commercial plasmid, manufacturer Addgene, cat. No. 12259) into NEB E.Coli Stable competent cells by heat shock method, plating at 30deg.C, and culturing to obtain plate monoclonal colony;
taking a flat monoclonal colony to culture in an LB culture medium, and culturing at 30 ℃ until the OD600 is 0.6 as primary seed liquid; 10g/L tryptone, 5g/L, naCl g/L yeast extract in LB culture medium;
transferring the first-stage seed solution into a second-stage seed culture medium (peptone 40g/L, yeast powder 20g/L, potassium dihydrogen phosphate 2.22g/L, pH is regulated to 7 by 10M NaOH) for culturing, wherein the inoculation amount is 0.5%, and culturing at 30deg.C until OD600 is 0.8 to obtain a second-stage seed solution;
s2, fermenting:
inoculating: inoculating the secondary seed solution into fermentation medium (soybean peptone 12g/L, yeast powder 20g/L, glycerol 7.5g/L, na) 2 HPO 4 ·12H 2 O 8.25g/L、KH 2 PO 4 3g/L、MgSO 4 ·7H 2 O2g/L, ammonium sulfate 4.2g/L, thiamine with 2% concentration 0.2mL/L and trace element solution 1mL/L. Wherein the microelement solution is CuCl 2 ·2H 2 O 1.5g、CoCl 2 1.4g、ZnSO 4 ·7H 2 O 2g、H 3 BO 3 3g、NaMoO 4 ·H 2 O 2.5g、MnSO 4 ·H 2 O 1.5g、FeSO 4 ·7H 2 O10 g and HCl100 mL, and obtained after filtration sterilization), and the inoculum size is 1%; fermenting and culturing at 30 ℃, wherein the aeration rate is 2L/min, the initial stirring speed is 50rpm, and the associated stirring maintains the dissolved oxygen DO at 30%;
and (3) fermentation regulation: the initial temperature is 30 ℃, the initial pH value is 6.7-6.8, and when the dissolved oxygen DO rises suddenly and is more than 60% in the fermentation process, the feed medium (MgSO) is added 4 ·7H 2 O20 g/L, glycerin 500g/L, soytone 50g/L, yeast powder 30 g/L), after the feeding, the DO value of the dissolved oxygen is reduced, and when DO of the dissolved oxygen is less than 40%, the feeding is stopped; when the dissolved oxygen DO is more than 40%, and the pH is increased, continuously adding a feed culture medium, repeatedly feeding, wherein the feed speed is 0.2-0.5 mL/min, and after the primary feeding is performed for 30min, raising the temperature to 37 ℃, and continuously culturing for 4h; then raising the temperature to 42 ℃ for 2 hours, and finally stopping feeding to ensure that the escherichia coli is kept starved for 10 minutes;
and (3) thallus collection: the hollow fibers with an inner diameter of 750KD are used for collection and washing filtration, and the hollow fibers are weighed after collection.
S3, cracking and clarifying:
get 100g of strain, re-suspending in 1.5L of buffer (EDTA 10mM, sucrose 2%, tris-HCl50mM, pH 8.0), slowly adding 1.5L of alkaline lysate (NaOH 0.2M, SDS% in alkaline lysate), slowly mixing, cracking for 8min, adding neutralization solution (potassium acetate 3M, pH is adjusted to 5 by glacial acetic acid) 1.5L, and slowly mixing; transferring the solid-liquid separation device, adding 5g/L ammonium bicarbonate solution to generate a large amount of CO 2 Waiting for gas, so as to separate solid from liquid, and standing for 10min, as shown in figure 1; retaining a bottom clarified liquid;
deep filtering the bottom clarified liquid with 0.22 μm polypropylene filter paper, concentrating with 300KD hollow fiber for 10 times, and washing with buffer solution for 6 times (adding 6 times buffer solution into the clarified liquid, and concentrating to obtain pre-buffer volume);
adding ammonium sulfate to make the concentration of the ammonium sulfate be 2.1M, and filtering and clarifying by using 0.45um PES material filter paper to obtain a lysate;
s4, chromatographic purification:
SepHarose6Fast Flow molecular sieve chromatography:
balance liquid A ((NH) 4 ) 2 SO 4 2.1M, EDTA mM, tris-HCl100mM, pH 7.5) 3 column volumes were equilibrated; sample loading amount: 30% of column volume, purifying the lysate by molecular sieve chromatography to obtain plasmid, and the molecular sieve chromatography result is shown in figure 2;
capto Q ion exchange chromatography:
the plasmid obtained by molecular sieve chromatography and purification is diluted by 3.85 times by pure water, and then NaCl is added so that the final concentration of NaCl is 0.1M to be used as a sample to be purified; the loading was 1.5mg plasmid DNA/mL of filler; after the sample loading is finished, washing the mixed solution for 3 column volumes by using balance liquid B (0.4M NaCl, 10mM EDTA, 100mM Tris-HCl and pH 7.5) to remove endotoxin, and finally eluting the plasmid by using balance liquid C (NaCl 1.0M, EDTA mM, 100mM Tris-HCl and pH 7.5) to obtain plasmid stock solution;
plasmid stock solution is prepared according to the volume ratio of 1:0.8 adding isopropyl alcohol, precipitation of plasmid DNA, with 75% ethanol washing 2 times, and sterile air drying, according to the required solvent and the required concentration of plasmid DNA re-dissolved, and filter sterilization.
And (3) quality detection:
1. ScaI-HindIII cleavage and gel electrophoresis were performed to obtain the results shown in FIG. 3.
HPLC results are shown in FIG. 4, with supercoiled plasmid content determination, supercoiled content of about 95%.
3. Endotoxin content is less than 5EU/mg by limulus reagent gel method.
The LISA method is used for measuring the host protein content to be less than 0.1 percent.
The qPCR method measured 0.23% of host DNA residues.
Example 2
The embodiment provides a preparation method of a high-standard plasmid, which comprises the following steps:
s1, culturing seed liquid:
transferring pMD2.G plasmid (virus packaging plasmid, pMD2.G, size 5822bp. Commercial plasmid, manufacturer Addgene, cat. No. 12259) into NEB E.Coli Stable competent cells by heat shock method, plating at 30deg.C, and culturing to obtain plate monoclonal colony;
taking a flat monoclonal colony to culture in an LB culture medium, and culturing at 30 ℃ until the OD600 is 0.8 as primary seed liquid; 10g/L tryptone, 5g/L, naCl g/L yeast extract in LB culture medium;
transferring the first-stage seed solution into a second-stage seed culture medium (peptone 40g/L, yeast powder 20g/L, potassium dihydrogen phosphate 2.22g/L, regulating pH to 7 with 10M NaOH), culturing at 30deg.C until OD600 is 1 to obtain second-stage seed solution;
s2, fermenting:
inoculating: inoculating the secondary seed solution into fermentation medium (soybean peptone 12g/L, yeast powder 20g/L, glycerol 7.5g/L, na) 2 HPO 4 ·12H 2 O 8.25g/L、KH 2 PO 4 3g/L、MgSO 4 ·7H 2 O2g/L, ammonium sulfate 4.2g/L, thiamine with 2% concentration 0.2mL/L and trace element solution 1mL/L. Wherein the microelement solution is CuCl 2 ·2H 2 O 1.5g、CoCl 2 1.4g、ZnSO 4 ·7H 2 O 2g、H 3 BO 3 3g、NaMoO 4 ·H 2 O 2.5g、MnSO 4 ·H 2 O 1.5g、FeSO 4 ·7H 2 O10 g and HCl100 mL, and is obtained after filtration sterilization), and the inoculum size is 5%; fermenting and culturing at 30 ℃, wherein the aeration rate is 2L/min, the initial stirring speed is 50rpm, and the associated stirring maintains the dissolved oxygen DO at 30%;
and (3) fermentation regulation: the initial temperature is 35 ℃, the initial pH value is 6.7-6.8, and when the dissolved oxygen DO rises suddenly and is more than 60% in the fermentation process, the feed medium (MgSO) is added 4 ·7H 2 O20 g/L, glycerin 500g/L, soytone 50g/L, yeast powder 30 g/L), after the feeding, the DO value of the dissolved oxygen is reduced, and when DO of the dissolved oxygen is less than 40%, the feeding is stopped; when the dissolved oxygen DO is more than 40%, and the pH is increased, continuously adding a feed culture medium, repeatedly feeding, wherein the feed speed is 0.2-0.5 mL/min, and after the primary feeding is performed for 30min, raising the temperature to 37 ℃, and continuously culturing for 6h; then raising the temperature to 42 ℃ for 2 hours, and finally stopping feeding to ensure that the escherichia coli is kept starved for 10 minutes;
and (3) thallus collection: the hollow fibers with an inner diameter of 750KD are used for collection and washing filtration, and the hollow fibers are weighed after collection.
S3, cracking and clarifying:
taking 100g of strain, re-suspending in 1.5L buffer (EDTA 10mM, sucrose 2%, tris-HCl50mM, pH 8.0), slowly adding 1.5L alkaline lysate (NaOH 0.2M, SDS% in alkaline lysate), slowly mixing, cracking for 8min, adding neutralization solution (potassium acetate 3M, pH is adjusted to 5 by glacial acetic acid) 1.5L, and slowly mixing; transferring the solid-liquid separation device, adding 5g/L ammonium bicarbonate solution to generate a large amount of CO 2 Waiting for gas, separating solid from liquid, standing for 10min, and keeping clear liquid at the bottom;
deep filtering the bottom clarified liquid with 0.22 μm polypropylene filter paper, concentrating with 300KD hollow fiber for 20 times, and washing with buffer solution for 6 times (adding 6 times buffer solution into the clarified liquid, and concentrating to obtain pre-buffer volume);
adding ammonium sulfate to make the concentration of the ammonium sulfate be 2.1M, and filtering and clarifying by using 0.45um PES material filter paper to obtain a lysate;
s4, chromatographic purification:
SepHarose6Fast Flow molecular sieve chromatography:
balance liquid A ((NH) 4 ) 2 SO 4 2.1M, EDTA mM, tris-HCl100mM, pH 7.5) 3 column volumes were equilibrated; sample loading amount: 30% of column volume, and purifying the lysate by molecular sieve chromatography to obtain plasmids;
capto Q ion exchange chromatography:
the plasmid obtained by molecular sieve chromatography and purification is diluted by 3.85 times by pure water, and then NaCl is added so that the final concentration of NaCl is 0.1M to be used as a sample to be purified; the loading was 1.5mg plasmid DNA/mL of filler; after the sample loading is finished, washing the mixed solution for 3 column volumes by using balance liquid B (0.4M NaCl, 10mM EDTA, 100mM Tris-HCl and pH 7.5) to remove endotoxin, and finally eluting the plasmid by using balance liquid C (NaCl 1.0M, EDTA mM, 100mM Tris-HCl and pH 7.5) to obtain plasmid stock solution;
plasmid stock solution is prepared according to the volume ratio of 1:0.8 adding isopropyl alcohol, precipitation of plasmid DNA, with 75% ethanol washing 2 times, and sterile air drying, according to the required solvent and the required concentration of plasmid DNA re-dissolved, and filter sterilization.
Example 3
In the embodiment, the relation between the fermentation time and the bacterial liquid OD600 is regulated to record the plasmid extraction yield, and the other conditions are kept the same as those in the embodiment 2; the relationship between fermentation time and bacterial liquid OD600 and plasmid extraction yield is shown in the following Table 1:
TABLE 1
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. The preparation method of the high-standard plasmid is characterized by comprising the following steps:
s1, culturing seed liquid:
transferring the pMD2.G plasmid into NEB E.Coli Stable competent cells by using a heat shock method, and coating a plate for culturing at 30 ℃ to obtain a plate monoclonal colony;
taking a flat monoclonal colony to culture in an LB culture medium, and culturing at 30 ℃ until the OD600 is 0.6-0.8 as primary seed liquid; transferring the first-stage seed liquid into a second-stage seed culture medium shake flask for culture, wherein the inoculum size is 0.5% -1%, and culturing at 30 ℃ until the OD600 is 0.8-1, wherein the first-stage seed liquid is used as the second-stage seed liquid;
s2, fermenting:
inoculating: inoculating the secondary seed liquid into a fermentation culture medium with an inoculum size of 1-5%; fermenting and culturing at 30 ℃, wherein the aeration rate is 2L/min, the initial stirring speed is 50rpm, and the associated stirring maintains the dissolved oxygen DO at 30%;
and (3) fermentation regulation: the initial temperature is 30-35 ℃, the initial pH value is 6.7-6.8, acid and alkali are used for maintaining the pH value at 6.8-7.0 in the fermentation process, when the dissolved oxygen DO rises suddenly and is more than 60% in the fermentation process, a feeding culture medium is added, the value of the dissolved oxygen DO drops after feeding, and feeding is stopped when the dissolved oxygen DO is less than 40%; when the dissolved oxygen DO is more than 40%, and the pH is increased, continuously adding a feed culture medium, repeatedly feeding, heating to 37 ℃ after the first feeding for 30min, and continuously culturing for 4-6 h; then raising the temperature to 42 ℃ for 2 hours, and finally stopping feeding to ensure that the escherichia coli is kept starved for 10 minutes;
and (3) thallus collection: collecting and washing with hollow fiber with 750KD inside diameter;
s3, cracking and clarifying:
weighing strain, suspending in buffer solution, slowly adding alkaline lysis solution, slowly mixing, lysing for 8min, adding neutralization solution, and slowly mixing; transferring the solid-liquid separation device, adding ammonium bicarbonate solution, performing solid-liquid separation, standing for 10min, and retaining bottom clear liquid;
deep filtering the bottom clarified liquid with 0.22 mu m polypropylene filter paper, concentrating by 10-20 times with 300KD hollow fiber, and washing with buffer solution for 6 times;
adding ammonium sulfate to make the concentration of the ammonium sulfate be 2.1M, and filtering and clarifying by using 0.45um PES material filter paper to obtain a lysate;
s4, chromatographic purification:
sequentially carrying out Sepharose6Fast Flow molecular sieve chromatography and Capto Q ion exchange chromatography to obtain plasmid stock solution; precipitating plasmid stock solution with isopropanol, washing with ethanol, drying with sterile air, filtering, and sterilizing.
2. The method for preparing a high-standard plasmid according to claim 1, wherein the pH of the medium in the shake flask of the secondary seed medium in S1 is adjusted to 7 with 10M NaOH, wherein the medium comprises 40g/L peptone, 20g/L yeast powder and 2.22g/L potassium dihydrogen phosphate.
3. The method for preparing a high-standard plasmid according to claim 1, wherein the fermentation medium in S2 comprises 12g/L of soybean peptone, 20g/L of yeast powder and 7.5g/L, na of glycerol 2 HPO 4 ·12H 2 O8.25g/L、KH 2 PO 4 3g/L、MgSO 4 ·7H 2 O2g/L, ammonium sulfate 4.2g/L, thiamine with 2% concentration 0.2mL/L and trace element solution 1mL/L; wherein the microelement solution is CuCl 2 ·2H 2 O 1.5g、CoCl 2 1.4g、ZnSO 4 ·7H 2 O 2g、H 3 BO 3 3g、NaMoO 4 ·H 2 O 2.5g、MnSO 4 ·H 2 O1.5g、FeSO 4 ·7H 2 O10 g and HCl100 mL, and is obtained after filtration sterilization.
4. The method for preparing a high-standard plasmid according to claim 1, wherein MgSO is contained in the S2 feed medium 4 ·7H 2 20g/L of O, 500g/L of glycerol, 50g/L of soybean peptone and 30g/L of yeast powder.
5. The method for preparing a high-standard plasmid according to claim 1, wherein the feeding speed in S2 is 0.2-0.5 mL/min.
6. The method for preparing a high-standard plasmid according to claim 1, wherein EDTA 10mM, sucrose 2%, tris-HCl50mM, pH 8.0 in the buffer solution in S3; naOH 0.2M, SDS percent in the alkaline cracking solution in S3; the neutralization solution was potassium acetate 3M and pH was adjusted to 5 using glacial acetic acid.
7. The method for preparing high-standard plasmid according to claim 1, wherein the concentration of ammonium bicarbonate solution in S3 is 5g/L, and the ratio of the amount of the strain, buffer solution, alkaline lysis solution and neutralization solution is 100g:1.5L:1.5L:1.5L.
8. The method for preparing a high-standard plasmid according to claim 1, wherein the equilibrium solution A (NH 4 ) 2 SO 4 2.1M, EDTA mM, tris-HCl100mM, pH 7.5;
balancing solution B0.4M NaCl, EDTA 10mM, tris-HCl100mM, pH 7.5
The balance C was NaCl 1.0M, EDTA mM, tris-HCl100mM, pH 7.5.
9. The method for preparing a high-standard plasmid according to claim 1, wherein the Sepharose6Fast Flow molecular sieve chromatography: balancing solution A balances 3 column volumes; sample loading amount: 30% of column volume, and purifying the lysate by molecular sieve chromatography to obtain plasmids;
capto Q ion exchange chromatography: the plasmid obtained by molecular sieve chromatography and purification is diluted by 3.85 times by pure water, and then NaCl is added so that the final concentration of NaCl is 0.1M to be used as a sample to be purified; the loading was 1.5mg plasmid DNA/mL of filler; and after the sample loading is finished, washing the mixed column volume by using the balance liquid B for 3 times to remove endotoxin, and finally eluting the plasmid by using the balance liquid C to obtain plasmid stock solution.
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| CN117431149B (en) * | 2023-12-22 | 2024-03-08 | 北京艺妙神州医药科技有限公司 | Method for washing thalli |
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