Plasmid DNA extraction kit based on anion exchange column and application thereof

Abstract

The invention provides a plasmid DNA extraction kit based on an anion exchange column and application of the plasmid DNA extraction kit, and relates to the technical field of biology. The invention provides a plasmid DNA extraction kit based on an anion exchange column. The plasmid DNA extraction kit comprises the anion exchange column, a lysis buffer solution group, an equilibrium buffer solution, a washing buffer solution and an elution buffer solution, the anion exchange column comprises a solid-phase extraction hollow column, an upper sieve plate and a lower sieve plate which are arranged on the inner side of the hollow column and positioned at the bottom of the hollow column, and an anion exchange ligand filled between the upper sieve plate and the lower sieve plate; the anion exchange ligand is prepared from diethylaminoethyl sephadex (DEAE)-A50 and diethylaminoethyl cellulose (DE52. According to the present invention, the domestic filler and the optimized filler ratio are adopted as the anion exchange ligand, the cost is only 1 / 20-1 / 40 of the cost of the imported product, the operation time and the labor intensity are saved through the gravity flow purification process, the yield of the obtained plasmid is high, and the transfection effect is equivalent to the transfection effect of the commercially available kit.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a plasmid DNA extraction kit based on anion exchange columns and its applications. Background Technology

[0002] Plasmids are widely distributed in the biological world, possess autonomous replication capabilities, and can stably inherit and express the genes they carry within host cells. Due to their compact structure and ease of manipulation, plasmids have become a core tool in molecular biology research. Plasmid extraction is a fundamental technique in molecular biology and basic medical research, and is widely used in gene cloning, protein expression, gene therapy, and other fields.

[0003] At present, the laboratory mainly relies on imported or domestic reagent kits for large-scale plasmid extraction, but the existing methods have the following problems: (1) Imported anion exchange column method (such as products from German companies such as MACHEREY-NAGEL, Qiagen, and Thermo): Although the extraction effect is good, the cost of a single extraction is as high as 100-200 yuan, and the economic burden is heavy for long-term high-frequency use; (2) Domestic silica membrane method (such as Savill G3645-10T): The cost is slightly lower than that of imported reagent kits, but multiple high-speed centrifugations or manual squeezing and filtration are required, which is physically demanding and time-consuming; (3) Domestic magnetic bead method (such as Savill G3610-10T): The lack of a targeted endotoxin removal step results in high endotoxin residue and the high price of magnetic bead materials, resulting in a high overall cost.

[0004] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a plasmid DNA extraction kit based on anion exchange column and its application.

[0006] This invention uses domestically produced packing materials and optimized packing material ratios as anion exchange ligands, with costs only 1 / 20 to 1 / 40 of imported products. Furthermore, the gravity flow purification process saves operation time and labor intensity, resulting in high plasmid yield and transfection efficiency comparable to commercially available kits.

[0007] This invention employs an integrated column design, reducing operational procedures and consumable consumption, resulting in lower costs compared to existing silica membrane methods and significantly cheaper than magnetic bead adsorption-based plasmid DNA extraction kits. Based on the principle that plasmid DNA carries a strong negative charge on its phosphate backbone at specific pH levels (usually neutral or weakly alkaline), enabling efficient binding to anion exchange resins, while endotoxins have a lower negative charge density and may aggregate due to hydrophobic interactions, resulting in weaker binding. This invention utilizes gradient salt elution (e.g., NaCl or KCl). Plasmid DNA is selectively eluted at high salt concentrations, while endotoxins, due to their weak binding, may be washed away at lower salt levels or remain on the column. To further reduce the endotoxin content in the extracted plasmids, this invention also tested the effects of Triton X-100 concentration in the EQU Buffer and the EQU Buffer elution volume on endotoxin residue, optimizing the optimal Triton X-100 concentration for endotoxin removal. With its X-100 concentration and elution buffer volume, the kit of this invention eliminates the need for additional endotoxin removal. Furthermore, the invention utilizes gravity to allow the E. coli lysate to pass through the adsorption column, saving significant time and labor.

[0008] In a first aspect, the present invention provides a plasmid DNA extraction kit based on an anion exchange column, comprising: an anion exchange column, a lysis buffer group, an equilibration buffer, a washing buffer, and an elution buffer; the anion exchange column comprises: a solid-phase extraction empty column, an upper sieve plate and a lower sieve plate disposed inside the empty column at the bottom of the empty column, and an anion exchange ligand filling the space between the upper sieve plate and the lower sieve plate; the anion exchange ligand comprises diethylaminoethyl dextran gel DEAE-A50 and diethylaminoethyl cellulose DE52 (hereinafter referred to as DEAE-A50 and DE52, respectively) in a mass ratio of (0:1) to (2.5:1).

[0009] Optionally, the mass ratio of DEAE-A50 and DE52 is (1.2:1) to (1.6:1).

[0010] Optionally, the pretreatment method for the anion exchange ligands includes: adding ultrapure water to the mixture of DEAE-A50 and DE52 to obtain anion exchange ligand mixture, followed by activation with low pH buffer, washing with water, and buffer equilibration treatment.

[0011] Optionally, ultrapure water is added when DEAE-A50 and DE52 are mixed to obtain an anion exchange ligand mixture; the mass-to-volume ratio of the ligand mixture to ultrapure water is 1 g: (30-50) mL.

[0012] Optionally, the low pH buffer activation includes hydrochloric acid activation; the hydrochloric acid activation uses a 0.5 mol / L to 5.0 mol / L hydrochloric acid solution.

[0013] Optionally, the buffer equilibration process uses a phosphate buffer with a pH of 1.0-6.5 or an acetate buffer with a pH of 3.6-5.6.

[0014] Optionally, the lysis buffer group includes a P1 buffer containing RNase A, an alkaline P2 buffer, and an acidic P3 buffer.

[0015] Optionally, the pH of the equilibration buffer is 7.0-7.5, and it contains Tris-HCl, potassium chloride, ethanol and Triton X-100; preferably, the volume of the equilibration buffer (EQU Buffer) used each time is 6 mL, and when the concentration of Triton X-100 in the EQU Buffer is 1%, the endotoxin concentration in the extracted plasmid is less than 1 EU / μg.

[0016] Optionally, the washing buffer has a pH of 6.0-6.5 and contains Tris-HCl, potassium chloride, and ethanol.

[0017] Optionally, the elution buffer has a pH of 8.0-9.0 and contains Tris-HCl, potassium chloride, and ethanol.

[0018] Optionally, the equilibration buffer, washing buffer, and elution buffer all use phosphate as a pH adjuster.

[0019] Optionally, the upper and lower sieve plates are hydrophilic sieve plates.

[0020] Optionally, the apertures of the upper and lower sieve plates are independently set to 10μm-200μm.

[0021] Secondly, the present invention provides a method for extracting plasmid DNA using any of the above-mentioned optional kits, characterized by comprising the following steps: (1) Treat bacterial cells containing plasmids with lysis buffer; (2) Load the lysis product onto an anion exchange column equilibrated with equilibration buffer; (3) Rinse with washing buffer; (4) Elution with elution buffer to obtain plasmid DNA.

[0022] Optionally, steps (2), (3), and (4) can be performed using gravity flow.

[0023] Thirdly, the present invention provides an application of any of the above-mentioned optional kits in the preparation of transfection-grade plasmid DNA.

[0024] Optionally, the yield of the transfection-grade plasmid DNA is 300 ng / mL to 2000 ng / mL of bacterial culture. Attached Figure Description

[0025] Figure 1 This is a structural diagram of the anion exchange column of the present invention; Figure 2 The plasmid prepared in Example 1 of this invention, the plasmid purified using the Seville endotoxin-free plasmid DNA large-scale extraction kit, and Thermo PureLink TM Comparison of plasmid expression after purification using the HiPure plasmid large-scale extraction kit. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0027] This invention provides a plasmid DNA extraction kit based on anion exchange column, comprising: anion exchange column, lysis buffer group, equilibration buffer, washing buffer and elution buffer; the anion exchange column comprises: a solid-phase extraction empty column, an upper sieve plate and a lower sieve plate disposed inside the empty column at the bottom of the empty column, and anion exchange ligand filling the space between the upper sieve plate and the lower sieve plate; the anion exchange ligand comprises DEAE-A50 and DE52 in a mass ratio of (0:1)-(2.5:1).

[0028] In fact, diethylaminoethyl (DEAE) is the key group responsible for adsorbing DNA in a DNA weak anion exchange column. It can adsorb negatively charged substances such as DNA, RNA, and endotoxins through ionic bonds. This binding ability is regulated by salt ion concentration and is pH dependent.

[0029] At acidic pH (pH 2-7), the amino group of diethylaminoethyl is fully protonated, enabling it to adsorb the DNA phosphate backbone (-PO4) via ionic bonds. - At this point, the protein / RNA is washed away, while the DNA remains on the exchange column.

[0030] During the washing stage, the exchange column was rinsed with a washing solution of moderate salt ion concentration (neutral or weakly acidic pH). + / Cl - or K + / Cl -The endotoxins and RNA will competitively bind to the positively charged groups on the anion exchange column. At this point, the remaining endotoxins and RNA will be washed away by the washing solution because their binding force with the anion exchange column is weak.

[0031] During the DNA elution stage, the high-salt and alkaline (pH 8.5) elution buffer deprotonates the diethylaminoethyl group, thereby disrupting the interaction between the anion exchange column and the DNA, thus eluting the DNA from the exchange column.

[0032] By adding the plasmid-containing supernatant obtained by alkaline lysis to the anion exchange column of this patent invention, and then washing and eluting, transfection-grade plasmids can be obtained.

[0033] In some embodiments, the mass ratio of DEAE-A50 to DE52 in the anion exchange ligands used is (1.2:1) to (1.6:1).

[0034] In some embodiments, the anion exchange ligands need to be pretreated before using the kit to extract plasmid DNA. The pretreatment method includes: adding ultrapure water to the ligand mixture obtained by mixing DEAE-A50 and DE52 to obtain the anion exchange ligand mixture, followed by activation with low pH buffer, washing with water and buffer equilibration.

[0035] In fact, hydrochloric acid provides hydrogen ions to ensure complete protonation of the amino group of DEAE, enhances the positive charge density, and improves the efficiency of subsequent DNA binding. Washing with water is to remove residual hydrochloric acid, balance the charge environment, and prevent local over-acidity from causing excessive protonation of DEAE groups. Buffer balances pH to ensure proper protonation of DEAE while avoiding acidic damage to DNA. At the same time, pH adjustment makes the anion exchange ligand packing material compatible with subsequent lysis / washing buffers, reducing binding fluctuations caused by pH abrupt changes.

[0036] In fact, DEAE-A50, as a key component for fine purification, creates a unique molecular sieving environment through the small pore size formed by its cross-linked dextran matrix. This structural characteristic makes it particularly adept at retaining small molecule impurities, including acidic protein fragments and degraded RNA. The DEAE groups (positively charged) ionize at pH less than 7 and bind to negatively charged biomolecules (such as DNA and proteins) through electrostatic interactions. This selective adsorption mechanism effectively reduces the competitive pressure on DNA binding sites. At the same time, the high mechanical strength brought by its cross-linked structure can withstand various physical stresses during the purification process, ensuring the stability of the purification process.

[0037] DE52, with its microcrystalline cellulose matrix and moderate pore size, has become the main adsorption medium for plasmid DNA extraction. Its particle size ensures good hydrodynamic properties while fully exposing the DEAE functional groups. Under conditions of pH less than 7, the DEAE groups are fully protonated and positively charged, specifically capturing the negatively charged DNA phosphate backbone through strong electrostatic interaction. Furthermore, the three-dimensional network structure of cellulose has excellent molecular sieving effect, which can efficiently bind DNA while allowing plasmid macromolecules to pass through freely.

[0038] By using DEAE-A50 and DE52 packing materials and optimizing the mass ratio of the packing materials as anion exchange ligands, it is possible to obtain plasmid DNA with low endotoxin, high yield, and good transfection effect at a cost that is only 1 / 20 to 1 / 40 of that of imported products.

[0039] In some embodiments, ultrapure water is added when DEAE-A50 and DE52 are mixed to obtain an anion exchange ligand mixture; the mass-to-volume ratio of the ligand mixture to ultrapure water is 1 g: (30-50) mL.

[0040] In fact, adding ultrapure water allows the dried packing particles (DEAE-A50 and DE52) to fully swell through osmosis, restoring their porous structure; in addition, ultrapure water can provide an undisturbed media environment.

[0041] In some embodiments, activation with low pH buffer is performed using hydrochloric acid, with a 0.5 mol / L to 5.0 mol / L hydrochloric acid solution. Specifically, a 0.6 mol / L hydrochloric acid solution is preferred. Hydrochloric acid provides hydrogen ions, ensuring complete protonation of the amino group of DEAE, increasing the positive charge density, and improving subsequent DNA binding efficiency. Furthermore, moderate acidification causes the cellulose micropores to swell from a dry state, increasing the pore size and improving the accessibility of plasmid DNA (100 nm to 200 nm in diameter). During hydrochloric acid activation, it is necessary to control the appropriate hydrochloric acid concentration to avoid insufficient conversion of the DEAE group (-N(C2H5)2) to the positively charged -N group due to excessively low concentration. + H(C2H5)2 leads to decreased DNA binding force, weakened electrostatic interaction, and insufficient structural swelling; excessively high concentrations cause hydrolytic damage to the filler matrix and the shedding of DEAE groups.

[0042] In some embodiments, phosphate buffer with a pH of 1.0–6.5 or acetate buffer with a pH of 3.6–5.6 is used for buffer equilibration. In practice, the equilibration buffer maintains a pH in the range of 6.0–6.5 to allow the DEAE groups (-N) to equilibrate. + H(C2H5)2) maintains proper protonation to ensure proper bonding with the DNA phosphate backbone (-PO4). -It enhances the electrostatic binding capacity of the packing material, preventing DEAE deprotonation (a sudden drop in binding force) caused by excessively high pH (>7.0) or DNA acid damage caused by excessively low pH (<5.0). In addition, it provides a buffer transition for subsequent lysis buffer loading (usually at pH 7.0-8.0), preventing packing material shrinkage / expansion caused by sudden pH changes.

[0043] In some embodiments, the lysis buffer group includes a P1 buffer containing RNase A, an alkaline P2 buffer, and an acidic P3 buffer. Specifically, the P1 buffer suspends and pre-lyses the cells; the alkaline P2 buffer dissolves the cell wall or cell membrane, allowing for complete cell lysis and release of plasmid DNA; and the acidic P3 buffer rapidly neutralizes the alkaline environment after P2 buffer treatment, restoring the pH to 7.0-8.0, thus allowing the plasmid to renature.

[0044] In some embodiments, the equilibration buffer has a pH of 7.0-7.5 and contains Tris-HCl, potassium chloride, ethanol, and Triton X-100. Specifically, Tris-HCl maintains the stability of the buffer system, ethanol dissolves lipid impurities, and Triton X-100 significantly reduces endotoxin residue by disrupting the lipid structure of endotoxin (LPS). The pH of this buffer is precisely controlled at 7.0-7.5 to optimize the protonation of the DEAE group, thereby enhancing DNA binding efficiency.

[0045] In some embodiments, the wash buffer has a pH of 6.0-6.5 and contains Tris-HCl, potassium chloride, and ethanol. In practice, the purpose of the wash buffer is to selectively remove impurities. A weakly acidic environment enhances the positive charge of DEAE, which binds strongly to DNA, promoting the elution of RNA / endotoxins (which have a weaker negative charge). A wash solution with a moderate salt concentration (neutral or weakly acidic pH) rinses the exchange column. The salt ions in potassium chloride competitively bind to the positively charged DEAE groups on the anion exchange column. At this point, residual endotoxins and RNA are washed away by the wash solution because their binding force to the anion exchange column is weak.

[0046] In some embodiments, the elution buffer has a pH of 8.0-9.0 and contains Tris-HCl, potassium chloride, and ethanol. In effect, the alkaline environment deprotonates the DEAE groups (-NH(C2H5)2), weakening the electrostatic binding to DNA and protecting the DNA from acidic degradation; high salt completely competes for DEAE binding sites, releasing high-purity DNA; and the high-salt and alkaline (pH 8.5) elution buffer deprotonates the diethylaminoethyl group, thereby disrupting the interaction between the anion exchange column and the DNA, thus eluting the DNA from the exchange column.

[0047] In some embodiments, phosphate is used as the pH adjuster in the equilibration buffer, washing buffer, and elution buffer. In fact, the tertiary dissociation of phosphate gives it a wide buffering range, allowing it to function effectively at different pH stages: in the equilibration buffer (pH 7.0-7.5), H₂PO₄ is primarily utilized. - / HPO4 2- Buffer pair; biased towards H2PO4 in wash buffer (pH 6.0–6.5). - Dominantly enhances DEAE protonation; HPO4 in elution buffer (pH 8.0-9.0) 2- / PO4 3- Buffers maintain an alkaline environment. Furthermore, PO4 in phosphoric acid... 3- With the DNA phosphate backbone (-PO4) - Similar in structure but with lower charge density, it can gently compete for DEAE binding sites, avoiding sudden DNA desorption (gradient elution is smoother).

[0048] In some embodiments, the upper and lower sieve plates are hydrophilic sieve plates. In fact, hydrophilic sieve plates are selected to avoid sample solution flow deviation within the column and to ensure sufficient contact between the lysis buffer / buffer and the packing material; in addition, hydrophilic sieve plates can prevent specific adsorption, reduce plasmid DNA retention and loss on the sieve plate surface; and can also inhibit pore blockage caused by the accumulation of hydrophobic contaminants, maintaining long-term stability.

[0049] In some embodiments, the pore sizes of the upper and lower sieve plates are independently ranging from 10 μm to 200 μm. In practice, controlling the ideal pore size range prevents the loss of anion exchange ligand packing material, while a pore size ≥10 μm avoids flow rate limitation. If the pore size is too small, some packing particles may become stuck in the sieve openings, reducing the effective column volume; if the pore size is too large, cellulose / dextran gel particles may penetrate the sieve plate, leading to packing material loss.

[0050] The present invention also provides a method for extracting plasmid DNA using a kit from any of the above embodiments, characterized by comprising the following steps: (1) Treat bacterial cells containing plasmids with lysis buffer; (2) Transfer the supernatant of the lysis product to an anion exchange column equilibrated with equilibration buffer; (3) Rinse with washing buffer; (4) Elution with elution buffer to obtain plasmid DNA.

[0051] This invention provides an application of the kit according to any of the above embodiments in the preparation of transfection-grade plasmid DNA. In practice, the yield of transfection-grade plasmid DNA extracted using the kit according to any embodiment of this invention is 300 ng / mL-2000 ng / mL of bacterial culture.

[0052] Example 1

[0053] Example 1 provides a plasmid DNA extraction kit based on anion exchange column, comprising the following steps: (1) Preparation of anion exchange column: S1. Preparation of anion exchange ligand mixture: Weigh 1.2g of DEAE-A50 and 0.864g of DE52 and add them to 86mL of ultrapure water to obtain anion exchange ligand mixture; S2. Column Assembly: Take out an empty solid-phase extraction column, first add a hydrophilic sieve plate with a pore size between 10μm and 50μm to the bottom, then add 4.8mL of anion exchange ligand mixture, and then add another hydrophilic sieve plate with a pore size between 10μm and 50μm to form... Figure 1 The sandwich structure shown is "sieve plate-anion exchange ligand-sieve plate"; S3. Hydrochloric acid activation: Plug the bottom with a plug, add 3 mL of diluted 0.6 mol / L hydrochloric acid to soak the anion exchange ligand in the solid phase extraction column for more than 10 minutes, rinse the anion exchange ligand with 5 mL of pure water, and repeat the rinsing three times; S4. Equilibrium storage: Add a small amount of Tris-HCl (pH 6.3) to wet the anion exchange ligand in the solid phase extraction column, and then plug the top and bottom ports respectively for long-term storage.

[0054] (2) Dedicated buffer system: 1. Lysis buffer group: 250mL P1 buffer: 12.5mL of 1M Tris-HCl (pH 8.0) + 5mL of 0.5M EDTA, bring the volume to 250mL, then add 25mg of RNase A; 250mL P2 buffer: NaOH 2g + 10% w / v SDS 25mL, bring volume to 250mL; 250mL P3 buffer: Add 73.6g of potassium acetate to acetic acid to adjust the pH to 5.1-5.5, and bring the volume to 250mL.

[0055] 2. Column purification buffer: 500mL Equilibration Buffer (EQU Buffer): 6.06g Tris-HCl + 75mL EtOH + 33.55g KCl + 5 mL Triton X-100, adjust pH to 7.3 with H3PO4, and bring volume to 500mL; 500mL Wash Buffer: 6.06g Tris-HCl + 42.87g KCl + 75mL EtOH, adjust pH to 6.3 with H3PO4, and bring volume to 500mL; 500mL Elution Buffer: 6.06g Tris-HCl + 42.87g KCl + 75 mL EtOH, adjust pH to 8.5 with H3PO4, and bring volume to 500mL.

[0056] The anion exchange column prepared in (1) above and the special buffer system composed in (2) above together constitute a plasmid DNA extraction kit based on anion exchange column in this Example 1.

[0057] Example 2

[0058] Example 2 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 2g of DEAE-A50 and 0g of DE52 (10:0) are weighed and added to 86mL of ultrapure water to obtain anion exchange ligand mixture.

[0059] Example 3

[0060] Example 3 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.4g of DEAE-A50 and 0.6g of DE52 (7:3) are weighed and added to 86mL of ultrapure water to obtain anion exchange ligand mixture.

[0061] Example 4

[0062] Example 4 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing anion exchange ligands, 1g of DEAE-A50 and 1g of DE52 (5:5) are weighed and added to 86mL of ultrapure water to obtain anion exchange ligand mixture.

[0063] Example 5

[0064] Example 5 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of the preparation of anion exchange ligands, 0.6g of DEAE-A50 and 1.4g of DE52 (3:7) are weighed and added to 86mL of ultrapure water to obtain anion exchange ligand mixture.

[0065] Example 6

[0066] Example 6 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 0g of DEAE-A50 and 2g of DE52 (0:10) are weighed and added to 86mL of ultrapure water to obtain the anion exchange ligand.

[0067] Example 7

[0068] Example 7 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0069] Prepare an anion exchange ligand mixture by taking 4.8 mL of DEAE-agarose gel solution, 0 mL of DEAE-A50 solution and 0 mL of DE52 solution (10:0:0).

[0070] Example 8

[0071] Example 8 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0072] An anion exchange ligand mixture was prepared by taking 3.36 mL of DEAE-agarose gel solution, 1.44 mL of DEAE-A50 solution and 0 mL of DE52 solution (7:3:0).

[0073] Example 9

[0074] Example 9 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0075] An anion exchange ligand mixture was prepared by taking 3.36 mL of DEAE-agarose gel solution, 0 mL of DEAE-A50 solution and 1.44 mL of DE52 solution (7:0:3).

[0076] Example 10

[0077] Example 10 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0078] An anion exchange ligand mixture was prepared by taking 1.44 mL of DEAE-agarose gel solution, 3.36 mL of DEAE-A50 solution and 0 mL of DE52 solution (3:7:0).

[0079] Example 11

[0080] Example 11 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0081] Prepare an anion exchange ligand mixture by taking 1.44 mL of DEAE-agarose gel solution, 1.92 mL of DEAE-A50 solution and 1.44 mL of DE52 solution (3:4:3).

[0082] Example 12

[0083] Example 12 provides a plasmid DNA extraction kit based on anion exchange column. The difference from Example 1 is that in step S1 of preparing the anion exchange ligand, 1.2g of DEAE-A50 and 0.864g of DE52 are added to 86mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution, respectively. The DEAE-agarose gel itself is preserved in liquid and is DEAE-agarose gel.

[0084] An anion exchange ligand mixture was prepared by taking 1.44 mL of DEAE-agarose gel solution, 0 mL of DEAE-A50 solution and 3.36 mL of DE52 solution (3:0:7).

[0085] Plasmid DNA yield test: Plasmids were extracted using the plasmid DNA extraction kits obtained in Examples 1 to 12, including the following steps: (1) Collection and lysis of bacterial cells: Take 30-200 mL of overnight Escherichia coli culture containing a specific plasmid, collect the bacterial cells by centrifugation, add 4 mL of buffer P1 (confirm whether RNase A has been added) and resuspend thoroughly; add 4 mL of alkaline lysis buffer P2, mix quickly, and let stand at room temperature for 5 min (the time should not be too long); add 4 mL of acidic neutralization buffer P3 and mix thoroughly to obtain a suspension.

[0086] (2) Purification of plasmids by anion exchange column: Place the filter screen inside the anion exchange column of the plasmid DNA extraction kit obtained in any of the examples 1 to 12, and then rinse the filter screen and DNA exchange column with 12 mL EQU Buffer (6 mL each time, 2 times); add the suspension obtained in (1) above to the exchange column containing the filter screen; after the liquid has dried, add 12 mL EQU Buffer (6 mL each time, 2 times) along the filter screen again, and let it dry naturally; discard the filter screen, add 5 mL Wash Buffer each time, and wash twice in total; after the liquid in the previous step has dried naturally, add 3.75 mL Elution Buffer and collect the filtrate; add 3.75 mL Elution Buffer again, collect and combine the two filtrates.

[0087] (3) Plasmid concentration: Add 5 mL of isopropanol to the combined filtrate, centrifuge at 12000 g and 4 °C for 30 minutes; discard the supernatant, dry thoroughly, add 400 μL of sterile milliQ water to completely dissolve, and transfer to a 1.5 mL tube; add 40 μL of 10 mol / L ammonium acetate and 880 μL of anhydrous ethanol, and mix thoroughly; centrifuge at 12000 rpm for 3 minutes; wash twice with 1 mL of 75% ethanol in a fume hood; discard the supernatant, add 50-200 μL of TE to completely dissolve the plasmid to obtain a concentrated plasmid DNA solution.

[0088] The yield test results of plasmid DNA extraction in Examples 1 to 12 are shown in the table below.

[0089] Table 1: Yield test results of plasmid DNA extracted in Examples 1 to 12

[0090]

[0091] Plasmid yield: The amount of plasmid extracted per milliliter of bacterial culture (unit: ng / mL) Table 1 shows that plasmids can be extracted from any one, any two, or any three of the anion exchange ligands selected from DEAE-agarose gel, DEAE-A50, or DE52. The results of one experiment are shown above. The combination of DEAE-A50 and DE52 as anion exchange ligands results in a higher plasmid yield. It can also be seen that the matrix composition in Example 1 has a better plasmid extraction effect.

[0092] Plasmid DNA transfection efficiency and protein expression performance testing: Day 1: Inoculated cells

[0093] 1) Prepare 3 trays of HeLa cells, each containing 250,000 HeLa cells, and culture the cells in DMEM / 10% v / v FBS medium.

[0094] 2) Shake gently and place in an incubator at 37℃ and 5% CO2.

[0095] the next day: plasmid transfection

[0096] 1) Observe the cell status after 12 hours. When the cell density occupies 30%-50% of the bottom area of ​​the dish, perform the transfection operation.

[0097] 2) Perform plasmid transfection: 2.1) Reagent preparation

[0098] ① Dilute the concentrated plasmid DNA solution obtained using the plasmid DNA extraction kit of Example 1: Dilute 1 μg of the plasmid DNA extraction kit of Example 1, commercial kit Case #1 (Seville endotoxin-free plasmid DNA large-scale extraction kit), and commercial kit Case #2 (Thermo PureLink) with the concentrated plasmid DNA solution obtained using the plasmid DNA extraction kit of Example 1. TM The plasmid DNA extracted by the HiPure plasmid extraction kit was added to 150 μL of serum-free Opti-MEM medium.

[0099] ② Dilute the transfection reagent: Add 1.5 μL of 1.6 μg / μL linear polyethyleneimine (LPEI) to 150 μL of serum-free culture medium; prepare three tubes, each corresponding to plasmid DNA samples extracted from different kits.

[0100] ③ Mix the diluted sample thoroughly and let it stand for 5 minutes.

[0101] 2.2) Mixing of plasmids and transfection reagents and transfection

[0102] ① Mix the sample thoroughly with an equal volume of DNA and transfection reagent, and let stand at room temperature for 20 minutes to form a complex.

[0103] ② Add the obtained complex dropwise to HeLa cells and gently shake to mix; mark the corresponding HeLa dish lids to distinguish plasmid DNA from different kits.

[0104] ③ After culturing for 6 hours, replace the culture medium of HeLa cells with brand new DMEM / 10% v / v FBS medium.

[0105] Day 4: Total protein extraction from HeLa cells (before this operation, prepare 1×RIPA lysis buffer: 150mM NaCl, 5mM EDTA, 50mM Tris-HCl base, 1% v / v NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, and adjust the pH to 8.0): 1) 48 hours after transfection with plasmid DNA, scrape HeLa cells from the bottom of the dish with a cell scraper, transfer HeLa cells to centrifuge tubes with culture medium, and label the centrifuge tubes according to the source of the plasmid.

[0106] 2) Separate the cell pellet and culture medium solution by centrifugation. The centrifugation force is 1000 × g, the time is 5 min, and the centrifuge temperature is maintained at 4℃.

[0107] 3) Discard the culture medium in the centrifuge tube and fully lyse the HeLa cells with 120 μL of 1×RIPA lysis buffer containing the protease inhibitor Cocktail.

[0108] 4) Centrifuge the HeLa cell lysis products at 4℃ for 10 min with a centrifugal force of 12000×g.

[0109] 5) After centrifugation, transfer 60 μL of supernatant to a new centrifuge tube and label the tube according to the source of the plasmid.

[0110] 6) Add 30 μL of protein loading electrophoresis buffer 2×loading (Saiwell Biotech, G2031-1ML) to each tube.

[0111] Protein electrophoresis (SDS-PAGE electrophoresis)

[0112] 1) Sample loading and electrophoresis

[0113] ①The protein sample containing the above protein loading electrophoresis buffer is denatured by boiling at 95°C for 10 minutes.

[0114] ② Load 40 μg of protein per well and perform the sample loading operation. Then perform electrophoresis according to the electrophoresis program of 90V for 25 min and 150V for 55 min.

[0115] 2) Transfer membrane (wet transfer method)

[0116] ① After electrophoresis, soak the gel, PVDF membrane (activated with methanol), and filter paper together in the transfer buffer.

[0117] ② Assemble in the order of "cathode-filter paper-gel-membrane-filter paper-anode" to avoid air bubbles.

[0118] ③ Transfer the film at a constant current of 200mA -250mA (or 100V) for 1 hour.

[0119] 3) Sealing: Place the transferred PVDF membrane in 5% w / v skim milk powder (prepared in 1×TBST, TBST is from Savill Biotech, G2150-1L), and slowly shake on a shaker for more than 30 minutes at room temperature.

[0120] 4) Pour off 5% w / v skim milk powder, place the membrane in TBST cleaning solution, and wash it three times at a relatively fast speed on a shaker, 5 minutes each time.

[0121] 5) After absorbing the liquid from the membrane, place it in 5% w / v skim milk powder containing rabbit-derived anti-Flag antibody (1:5000 dilution, antibody from Xavier Biotech, GB11938-100) and incubate overnight at 4°C.

[0122] 6) The next day, take out the membrane and place it in 1×TBST. Wash it three times on a shaker for 5 minutes each time.

[0123] 7) Dilute HRP-labeled goat anti-rabbit IgG antibody (Saiwell Biotech, GB23303) 1:5000 in 5% w / v skim milk powder and incubate the membrane with the diluted solution for 1 hour.

[0124] 8) Wash three times with a 1×TBST shaker, 5 minutes each time.

[0125] 9) After cleaning, the PVDF film is exposed and imaged using a chemiluminescence imaging system to obtain... Figure 2 The results are shown.

[0126] from Figure 2 As can be seen from the results, the plasmids extracted in Example 1 of this invention have no difference in transfection and protein expression effects compared to plasmids extracted by other commercial kits.

[0127] To further reduce the endotoxin content in the extracted plasmid, this invention tested the effects of Triton X-100 concentration in EQU Buffer and EQU Buffer elution volume on endotoxin residue, and optimized the optimal Triton X-100 concentration and elution volume for endotoxin removal.

[0128] The optimized concentrations of Triton X-100 in EQU Buffer and the volume of EQU Buffer equilibration buffer are shown in the table below: Table 2: Results of Triton X-100 Concentration and EQU Buffer Equilibration Buffer Volume Optimization

[0129]

[0130] Endotoxin levels were detected using the horseshoe crab (LAL) gel electrophoresis method. A positive result was indicated if a gel formed in the tube and remained intact without deforming or slipping off the tube wall, meaning the endotoxin level was higher than the sensitivity of the test kit (1 EU / μg). A negative result was indicated if no gel formed, or if the formed gel was not firm, deformed, or slipped off the tube wall, meaning the endotoxin level was lower than the sensitivity of the test kit (1 EU / μg). The 1 EU / μg endotoxin standard meets the requirements of most basic scientific research experiments and in vitro cell transfection experiments.

[0131] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A plasmid DNA extraction kit based on anion exchange column, characterized in that, include: Anion exchange column, lysis buffer set, equilibration buffer, washing buffer and elution buffer; The anion exchange column includes: a solid-phase extraction empty column, an upper sieve plate and a lower sieve plate disposed inside the empty column at the bottom of the empty column, and anion exchange ligands filled between the upper sieve plate and the lower sieve plate; the anion exchange ligands include diethylaminoethyl dextran gel DEAE-A50 and diethylaminoethyl cellulose DE52 in a mass ratio of (0:1)-(2.5:1).

2. The plasmid DNA extraction kit according to claim 1, characterized in that, The mass ratio of the diethylaminoethyl dextran gel DEAE-A50 to the diethylaminoethyl cellulose DE52 is (1.2:1)-(1.6:1).

3. The plasmid DNA extraction kit according to claim 1, characterized in that, The pretreatment method for the anion exchange ligands includes: when mixing diethylaminoethyl dextran gel DEAE-A50 and diethylaminoethyl cellulose DE52 to obtain a ligand mixture, adding ultrapure water to obtain an anion exchange ligand mixture solution, and then sequentially performing low pH buffer activation, water washing, and buffer equilibration treatment.

4. The plasmid DNA extraction kit according to claim 3, characterized in that, The mass-to-volume ratio of the ligand mixture to ultrapure water is 1 g: (30-50) mL.

5. The plasmid DNA extraction kit according to claim 3, characterized in that, The activation of the low pH buffer solution includes hydrochloric acid activation; the hydrochloric acid activation uses a 0.5 mol / L to 5.0 mol / L hydrochloric acid solution; and / or, the buffer equilibration treatment uses a phosphate buffer solution with a pH of 1.0 to 6.5 or an acetate buffer solution with a pH of 3.6 to 5.

6.

6. The plasmid DNA extraction kit according to claim 1, characterized in that, The lysis buffer group includes a P1 buffer containing RNase A, an alkaline P2 buffer, and an acidic P3 buffer; and / or, the equilibration buffer has a pH of 7.0-7.5 and contains Tris-HCl, potassium chloride, ethanol, and Triton X-100; the volume concentration of Triton X-100 in the equilibration buffer is 1%; and / or, the washing buffer has a pH of 6.0-6.5 and contains Tris-HCl, potassium chloride, and ethanol; and / or, the elution buffer has a pH of 8.0-9.0 and contains Tris-HCl, potassium chloride, and ethanol; and / or, the equilibration buffer, washing buffer, and elution buffer all use phosphate as a pH adjuster; and / or, the upper and lower sieve plates are hydrophilic sieve plates; and / or, the pore sizes of the upper and lower sieve plates are independently 10 μm-200 μm.

7. A method for extracting plasmid DNA using the kit according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Treat bacterial cells containing plasmids with lysis buffer; (2) Transfer the supernatant of the lysis product to an anion exchange column equilibrated with equilibration buffer; (3) Rinse with washing buffer; (4) Elution with elution buffer to obtain plasmid DNA.

8. The method according to claim 7, characterized in that, Steps (2), (3), and (4) are performed using gravity flow.

9. The use of a kit as described in any one of claims 1 to 6 in the preparation of transfection-grade plasmid DNA.

10. The application according to claim 9, characterized in that, The yield of transfection-grade plasmid DNA was 300 ng / mL to 2000 ng / mL of bacterial culture.

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