Anion exchange column-based plasmid DNA extraction kit and application thereof

Abstract

The application provides a plasmid DNA extraction kit based on an anion exchange column and application thereof, and relates to the technical field of biology.The plasmid DNA extraction kit based on the anion exchange column comprises an anion exchange column, a lysis buffer group, a balance buffer, a washing buffer and an elution buffer; the anion exchange column comprises a solid-phase extraction empty column, upper and lower sieve plates arranged on the inner side of the empty column and located at the bottom of the empty column, and an anion exchange ligand filled between the upper and lower sieve plates; the anion exchange ligand comprises diethylaminoethyl dextran gel DEAE-A50 and diethylaminoethyl cellulose DE52.The application uses domestic fillers and optimizes the ratio of the fillers as the anion exchange ligand, the cost of which is only 1 / 20-1 / 40 of that of imported products, and the operation time and labor intensity are saved through the gravity flow purification process, and the obtained plasmid has high yield, and the transfection effect is equivalent to that of commercially available kits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a plasmid DNA extraction kit based on an anion exchange column and application thereof. BACKGROUND

[0002] Plasmids exist widely in the biological world, have the ability of autonomous replication, can be stably inherited in host cells and express the genes carried. Due to its compact structure and easy operation, the plasmid has become a core tool for molecular biology research. Plasmid extraction is a basic technology in molecular biology and basic medical research, and is widely used in the fields of gene cloning, protein expression, gene therapy, etc.

[0003] At present, laboratories mainly rely on imported or domestic kits for plasmid extraction, but the existing methods have the following problems: (1) imported anion exchange column method (such as products of MACHEREY-NAGEL, Qiagen, Thermo, etc.): although the extraction effect is good, the cost of single extraction is as high as 100-200 yuan, and the long-term high-frequency use is economically burdensome; (2) domestic silica gel membrane method (such as SAVIL G3645-10T): the cost is slightly lower than that of imported kits, but multiple high-speed centrifugation or manual extrusion filtration is required, which is labor-intensive and time-consuming; (3) domestic magnetic bead method (such as SAVIL G3610-10T): the lack of steps for removing endotoxins leads to high endotoxin residues, and the high price of magnetic bead material leads to high overall cost.

[0004] Therefore, it is urgent to provide a solution to improve the above problems. SUMMARY

[0005] The present application relates to the technical field of biotechnology, and particularly relates to a plasmid DNA extraction kit based on an anion exchange column and application thereof.

[0006] The present application uses domestic filler and optimized filler ratio as anion exchange ligand, and the cost is only 1 / 20-1 / 40 of imported products. The gravity flow purification process saves operation time and labor intensity, and the obtained plasmid has high yield and equivalent transfection effect to commercial kits.

[0007] The application adopts an integrated column design, reduces the operation process and consumable consumption, and has a lower cost than the existing silica gel membrane method and is much cheaper than the magnetic bead adsorption method; based on the principle that the phosphate skeleton of plasmid DNA has strong negative charge at a specific pH (usually neutral or weak alkaline) and can be efficiently combined with anion exchange resin, while the negative charge density of endotoxin is low and may form aggregates due to hydrophobic interaction, the combination capacity is weak, the application elutes plasmid DNA at high salt concentration by gradient salt elution (such as NaCl or KCl), and endotoxin may be washed away at a low salt stage or remain on the column and not be eluted due to weak binding force, in order to further reduce the endotoxin content in the extracted plasmid, the application also tests the influence of Triton X-100 concentration in EQU Buffer and the eluent volume of EQU Buffer on endotoxin residue, and optimizes the optimal Triton X-100 concentration and eluent volume for endotoxin removal, therefore, the kit of the application does not need additional endotoxin removal operation; in addition, the application allows the E. coli lysate to pass through the adsorption column by gravity, which saves a lot of operation time and labor intensity.

[0008] In a first aspect, the application provides a plasmid DNA extraction kit based on an anion exchange column, comprising: an anion exchange column, a lysis buffer set, an equilibrium 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 arranged inside the empty column at the bottom of the empty column, and an anion exchange ligand filled 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) with a mass ratio of (0:1)-(2.5:1).

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

[0010] Optionally, the pretreatment method of the anion exchange ligand comprises: adding ultrapure water to obtain an anion exchange ligand mixture when DEAE-A50 and DE52 are mixed, and then sequentially performing low-pH buffer activation, water washing and buffer equilibrium 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 1g:(30-50)mL.

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

[0013] Optionally, the phosphate buffer with pH of 1.0-6.5 or the acetate buffer with pH of 3.6-5.6 is used in the buffer equilibration process.

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

[0015] Optionally, the pH of the equilibration buffer is 7.0-7.5, and the equilibration buffer 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 the endotoxin concentration in the obtained plasmid is less than 1 EU / μg when the concentration of Triton X-100 in the EQU Buffer is 1%.

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

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

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

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

[0020] Optionally, the pore size of the upper sieve plate and the lower sieve plate is independently 10 μm-200 μm.

[0021] In a second aspect, the present application provides a method for extracting plasmid DNA using any of the above optional kits, characterized in that the method comprises the following steps:

[0022] (1) treating bacterial cells containing plasmids with the lysis buffer set;

[0023] (2) loading the lysis product into a cation exchange column equilibrated with the equilibration buffer;

[0024] (3) washing with the washing buffer;

[0025] (4) eluting to obtain plasmid DNA with the elution buffer.

[0026] Optionally, steps (2), (3) and (4) are operated in a gravity flow mode.

[0027] In a third aspect, the present application provides use of any of the above optional kits in the preparation of transfection-grade plasmid DNA.

[0028] Optionally, the yield of the transfected plasmid DNA is 300-2000 ng / mL of bacterial solution. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Structure diagram of the anion exchange column of the present application;

[0030] Figure 2 Plasmid obtained by Example 1 of the present application, plasmid purified by Sevag endotoxin-free plasmid DNA extraction kit, and Thermo PureLink TM Comparison chart of expression of plasmid purified by HiPure plasmid extraction kit. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Unless otherwise defined, the technical terms or scientific terms used herein should be understood as the usual meanings understood by those skilled in the art in the field of the present application.

[0032] The present application provides a plasmid DNA extraction kit based on an anion exchange column, comprising: an anion exchange column, a lysis buffer set, an equilibrium buffer, a washing buffer and an elution buffer; the anion exchange column comprises: a solid-phase extraction empty column, upper and lower sieve plates arranged inside the empty column at the bottom of the empty column, and an anion exchange ligand filled between the upper and lower sieve plates; the anion exchange ligand comprises DEAE-A50 and DE52 in a mass ratio of (0:1)-(2.5:1).

[0033] In fact, diethylaminoethyl (DEAE) is the key group responsible for adsorbing DNA in the weak anion exchange column of DNA, which can adsorb negatively charged substances such as DNA, RNA and endotoxin through ionic bonds. This binding capacity shows salt ion concentration regulation and pH dependence.

[0034] When the pH is acidic (pH 2-7), the amino group of diethylaminoethyl is fully protonated, and can adsorb DNA phosphate skeleton (-PO4 - ) through ionic bonds. At this time, proteins / RNA are washed away, and DNA is retained on the exchange column.

[0035] In the washing stage, a washing solution with moderate salt ion concentration (pH neutral or weakly acidic) is used to flush the exchange column. The salt ions (Na + / Cl -or K + / Cl - ) will compete with the positive groups of the anion exchange column. At this time, the residual endotoxin and RNA will be washed away by the washing solution because of the weak binding force with the anion exchange column.

[0036] In the DNA elution stage, the high salt and alkaline (pH 8.5) elution solution will deprotonate the diethylaminoethyl, thereby destroying the interaction between the anion exchange column and the DNA, and thus eluting the DNA from the exchange column.

[0037] By adding the plasmid-containing supernatant obtained by alkaline lysis to the anion exchange column of the present application, and then washing and eluting, transfection-grade plasmid can be obtained.

[0038] In some embodiments, the mass ratio of DEAE-A50 and DE52 in the anion exchange ligand used is (1.2:1) to (1.6:1).

[0039] In some embodiments, the anion exchange ligand needs to be pretreated before using the kit to extract plasmid DNA. The pretreatment method includes: when mixing DEAE-A50 and DE52 to obtain a ligand mixture, adding ultrapure water to obtain an anion exchange ligand mixture, and then sequentially performing low-pH buffer activation, water washing, and buffer equilibration treatment.

[0040] In fact, hydrochloric acid can provide hydrogen ions to ensure that the amino groups of DEAE are completely protonated, increase the positive charge density, and improve the subsequent DNA binding efficiency; water washing is to remove residual hydrochloric acid, balance the charge environment, and prevent local over-acidification from causing excessive protonation of DEAE groups; buffer equilibration pH ensures moderate protonation of DEAE, while avoiding acid damage to DNA, and adjusts the pH to make the anion exchange ligand filler compatible with the subsequent lysis solution / washing solution, reducing the binding fluctuations caused by pH mutations.

[0041] In fact, DEAE-A50, as a key component of fine purification, has a small pore size formed by its cross-linked dextran matrix, creating a unique molecular sieving environment. This structural characteristic makes it particularly good at trapping small molecular impurities, including acidic protein fragments and degraded RNA; the DEAE group (positively charged) ionizes at a pH less than 7, and binds to negatively charged biomolecules (such as DNA, proteins) through electrostatic interaction. This selective adsorption mechanism effectively reduces the competition pressure on the DNA binding site, and 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.

[0042] DE52, with its matrix of microcrystalline cellulose and moderate pore size, has become the main adsorption medium for plasmid DNA extraction. The particle size of DE52 ensures good hydrodynamic characteristics and fully exposes the DEAE functional groups. At a pH less than 7, the DEAE groups are fully protonated and positively charged, and through strong electrostatic interactions, DE52 specifically captures the negatively charged DNA phosphate backbone. The three-dimensional network structure of cellulose has excellent molecular sieving effect, which can efficiently bind DNA while allowing the plasmid macromolecules to freely penetrate.

[0043] By using DEAE-A50 and DE52 fillers and optimizing the mass ratio of the fillers as anion exchange ligands, plasmid DNA with low endotoxin, high yield, and good transfection effect can be obtained at a cost of only 1 / 20-1 / 40 of imported products.

[0044] In some embodiments, when DEAE-A50 and DE52 are mixed, ultrapure water is added to obtain an anion exchange ligand mixture; the mass-volume ratio of the ligand mixture to ultrapure water is 1g: (30-50)mL.

[0045] In fact, the addition of ultrapure water swells the dry filler particles (DEAE-A50 and DE52) through osmotic action, restoring their porous structure. In addition, ultrapure water can provide an interference-free medium environment.

[0046] In some embodiments, the low-pH buffer activation uses hydrochloric acid activation, and 0.5mol / L-5.0mol / L hydrochloric acid solution is used for hydrochloric acid activation. Specifically, a hydrochloric acid solution with a concentration of 0.6mol / L is preferred. In fact, hydrochloric acid can provide hydrogen ions to ensure complete protonation of the DEAE amino groups, increase the positive charge density, and improve the subsequent DNA binding efficiency. In addition, moderate acidification causes the cellulose micropores to swell from the dry state, increasing the pore size and improving the accessibility of plasmid DNA (100nm-200nm in diameter). When hydrochloric acid is activated, the appropriate concentration of hydrochloric acid needs to be controlled to avoid the DEAE group (-N(C2H5)2) failing to be fully converted to the positively charged -N + H(C2H5)2, resulting in decreased DNA binding force, weakened electrostatic interaction, and insufficient structure swelling; and a concentration that is too high causes hydrolysis damage to the filler matrix and the DEAE group to fall off.

[0047] 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 fact, by controlling the pH in the range of 6.0-6.5, the DEAE group (-N + H(C2H5)2) is kept moderately protonated, ensuring that it can specifically capture the negatively charged DNA phosphate backbone (-PO4 -The electrostatic binding capacity of the DEAE group is avoided by avoiding a pH that is too high (> 7.0) to cause the DEAE to deprotonate (sudden loss of binding capacity) or too low (< 5.0) to cause DNA acid damage. In addition, the buffer transition is provided for subsequent loading of the lysis solution (usually at a pH of 7.0-8.0) to prevent filler shrinkage / expansion caused by pH mutations.

[0048] In some embodiments, the lysis buffer set includes a P1 buffer containing RNase A, a basic P2 buffer, and an acidic P3 buffer. In practice, the P1 buffer suspends the cells and pre-lyses them; the P2 buffer is basic and dissolves the cell wall or cell membrane to completely lyse the cells and release the plasmid DNA; and the P3 buffer is acidic and quickly neutralizes the alkaline environment after P2 buffer treatment, so that the pH returns to 7.0-8.0, allowing the plasmid to renature.

[0049] 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. In practice, Tris-HCl in the equilibration buffer maintains the stability of the buffer system, ethanol dissolves lipid impurities, and Triton X-100 significantly reduces endotoxin residues by destroying the lipid structure of endotoxin (LPS). The pH of this buffer is precisely controlled at 7.0-7.5 to optimize the degree of protonation of the DEAE group, thereby enhancing DNA binding efficiency.

[0050] In some embodiments, the washing buffer has a pH of 6.0-6.5 and contains Tris-HCl, potassium chloride, and ethanol. In practice, the purpose of the washing buffer is to selectively remove impurities. The weakly acidic environment enhances the positive charge of DEAE, firmly binds DNA, and promotes the elution of RNA / endotoxin (with weaker negative charge); the washing solution with a moderate salt ion concentration (pH neutral or weakly acidic) flushes the exchange column, and the salt ions in potassium chloride competitively bind the positive DEAE group of the anion exchange column. At this time, the residual endotoxin and RNA are washed away because of the weak binding force between them and the anion exchange column.

[0051] In some embodiments, the elution buffer has a pH of 8.0-9.0 and contains Tris-HCl, potassium chloride, and ethanol. In practice, the alkaline environment causes the DEAE group to deprotonate (-NH(C2H5)2), weakening the electrostatic binding with DNA and protecting DNA from acid degradation; high salt completely competes for the DEAE binding site, 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, thereby eluting the DNA from the exchange column.

[0052] In some embodiments, the equilibration buffer, the washing buffer and the elution buffer all use phosphoric acid as the pH regulator. In fact, the tertiary dissociation of phosphoric acid makes it have a wide buffer range, which can play a role at different pH stages: in the equilibration buffer (pH 7.0-7.5), the H2PO4 - / HPO4 2- buffer pair is mainly used, which enhances the protonation of DEAE; in the washing buffer (pH 6.0-6.5), the H2PO4 - buffer pair is dominant, which enhances the protonation of DEAE; in the elution buffer (pH 8.0-9.0), the HPO4 2- / PO4 3- buffer pair maintains an alkaline environment. In addition, PO4 3- in phosphoric acid is similar in structure to the DNA phosphate backbone (-PO4 - ), but has a lower charge density, which can gently compete for the DEAE binding site and avoid the sudden desorption of DNA (the gradient elution is more gentle).

[0053] In some embodiments, the upper sieve plate and the lower sieve plate are hydrophilic sieve plates. In fact, the hydrophilic sieve plate is selected to avoid the formation of a partial flow of the sample solution in the column, to ensure that the lysate / buffer is in full contact with the filler; in addition, the hydrophilic sieve plate can prevent specific adsorption and reduce the loss of plasmid DNA on the surface of the sieve plate; it can also inhibit the clogging of the sieve plate pores due to the accumulation of hydrophobic contaminants, thereby maintaining long-term stability.

[0054] In some embodiments, the pore size of the upper sieve plate and the lower sieve plate is independently 10 μm-200 μm. In fact, controlling the ideal pore size range can prevent the loss of anion exchange ligand filler, and the pore size ≥10 μm can avoid the limitation of flow rate. If the pore size is too small, part of the filler particles may be stuck in the sieve holes, reducing the effective column volume; if the pore size is too large, the cellulose / dextran gel particles may penetrate the sieve plate, causing the loss of the filler.

[0055] The present application also provides a method for extracting plasmid DNA using the kit of any of the above embodiments, characterized in that it comprises the following steps:

[0056] (1) treating bacterial cells containing plasmids with a lysis buffer set;

[0057] (2) transferring the supernatant of the lysis product to an anion exchange column equilibrated with an equilibration buffer;

[0058] (3) washing with a washing buffer;

[0059] (4) eluting to obtain plasmid DNA with an elution buffer.

[0060] The application provides application of the kit of any one of the embodiments in preparation of transfection level plasmid DNA. In fact, the yield of the transfection level plasmid DNA extracted by the kit of any one of the embodiments of the application is 300 ng / mL-2000 ng / mL of bacterial solution.

[0061] Embodiment 1

[0062] The embodiment 1 provides a plasmid DNA extraction kit based on an anion exchange column, which comprises the following steps:

[0063] (1) Preparation of an anion exchange column:

[0064] S1. Preparation of an anion exchange ligand mixture: 1.2 g of DEAE-A50 and 0.864 g of DE52 are weighed and added to 86 mL of ultrapure water to obtain an anion exchange ligand mixture;

[0065] S2. Column assembly: an empty solid phase extraction column is taken out, a hydrophilic screen plate with a pore size of 10-50 μm is first added at the bottom, then 4.8 mL of the anion exchange ligand mixture is added, and then another hydrophilic screen plate with a pore size of 10-50 μm is added, to form a "sandwich structure of screen plate-anion exchange ligand-screen plate" as shown in the figure; Figure 1

[0066] S3. Hydrochloric acid activation: the lower opening is blocked with a plug, 3 mL of 0.6 mol / L hydrochloric acid diluted with water is added to soak the anion exchange ligand in the solid phase extraction column for more than 10 minutes, and the anion exchange ligand is washed with 5 mL of pure water, and the washing is repeated three times;

[0067] S4. Equilibrium preservation: a small amount of Tris-HCl with a pH value of 6.3 is added to moisten the anion exchange ligand in the solid phase extraction column, and the upper and lower openings are blocked, so that the column can be stored for a long time.

[0068] (2) Special buffer system:

[0069] 1. Lysis buffer set:

[0070] 250 mL of P1 buffer (buffer P1): 1 M Tris-HCl (pH 8.0) 12.5 mL + 0.5 M EDTA 5 mL, and then 25 mg of RNase A is added to make up to 250 mL;

[0071] 250 mL of P2 buffer (buffer P2): NaOH 2 g + 10% w / v SDS 25 mL, and then make up to 250 mL;

[0072] ​250 mL P3 buffer: 73.6 g of potassium acetate is added to acetic acid to adjust pH to 5.1-5.5, and then diluted to 250 mL.

[0073] 2. Column purification buffer:

[0074] 500 mL EQU buffer: 6.06 g of Tris-HCl + 75 mL of EtOH + 33.55 g of KCL + 5 mL of Triton X-100, adjusted to pH 7.3 with H3PO4, and diluted to 500 mL;

[0075] 500 mL Wash buffer: 6.06 g of Tris-HCl + 42.87 g of KCL + 75 mL of EtOH, adjusted to pH 6.3 with H3PO4, and diluted to 500 mL;

[0076] 500 mL Elution buffer: 6.06 g of Tris-HCl + 42.87 g of KCL + 75 mL of EtOH, adjusted to pH 8.5 with H3PO4, and diluted to 500 mL.

[0077] The anion exchange column prepared in the above (1) and the special buffer system composed of the above (2) together constitute a plasmid DNA extraction kit based on anion exchange column of Example 1.

[0078] Example 2

[0079] Example 2 provides a plasmid DNA extraction kit based on anion exchange column, which is different from Example 1 in that 2 g of DEAE-A50 and 0 g of DE52 (10:0) are weighed and added to 86 mL of ultrapure water in step S1 of preparing anion exchange ligand to obtain anion exchange ligand mixture.

[0080] Example 3

[0081] Example 3 provides a plasmid DNA extraction kit based on anion exchange column, which is different from Example 1 in that 1.4 g of DEAE-A50 and 0.6 g of DE52 (7:3) are weighed and added to 86 mL of ultrapure water in step S1 of preparing anion exchange ligand to obtain anion exchange ligand mixture.

[0082] Example 4

[0083] This example 4 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1 g of DEAE-A50 and 1 g of DE52 (5:5) are weighed in step S1 of preparation of anion exchange ligand and added to 86 mL of ultrapure water to obtain an anion exchange ligand mixture.

[0084] Example 5

[0085] This example 5 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 0.6 g of DEAE-A50 and 1.4 g of DE52 (3:7) are weighed in step S1 of preparation of anion exchange ligand and added to 86 mL of ultrapure water to obtain an anion exchange ligand mixture.

[0086] Example 6

[0087] This example 6 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 0 g of DEAE-A50 and 2 g of DE52 (0:10) are weighed in step S1 of preparation of anion exchange ligand and added to 86 mL of ultrapure water to obtain an anion exchange ligand.

[0088] Example 7

[0089] This example 7 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2 g of DEAE-A50 and 0.864 g of DE52 are respectively added to 86 mL of ultrapure water in step S1 of preparation of anion exchange ligand to obtain DEAE-A50 solution and DE52 solution, and the DEAE-agarose gel itself is stored in liquid, which is DEAE-agarose gel.

[0090] 4.8 mL of DEAE-agarose gel solution, 0 mL of DEAE-A50 solution and 0 mL of DE52 solution (10:0:0) are configured to obtain an anion exchange ligand mixture.

[0091] Example 8

[0092] This example 8 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2 g of DEAE-A50 and 0.864 g of DE52 are respectively added to 86 mL of ultrapure water in step S1 of preparation of anion exchange ligand to obtain DEAE-A50 solution and DE52 solution, and the DEAE-agarose gel itself is stored in liquid, which is DEAE-agarose gel.

[0093] Take 3.36 mL DEAE-agarose gel solution, 1.44 mL DEAE-A50 solution and 0 mL DE52 solution (7:3:0) to prepare the anion exchange ligand mixture solution.

[0094] Example 9

[0095] This example 9 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2 g of DEAE-A50 and 0.864 g of DE52 are respectively added to 86 mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution in step S1 of preparation of anion exchange ligand, and DEAE-agarose gel is stored in liquid as DEAE-agarose gel.

[0096] Take 3.36 mL DEAE-agarose gel solution, 0 mL DEAE-A50 solution and 1.44 mL DE52 solution (7:0:3) to prepare the anion exchange ligand mixture solution.

[0097] Example 10

[0098] This example 10 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2 g of DEAE-A50 and 0.864 g of DE52 are respectively added to 86 mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution in step S1 of preparation of anion exchange ligand, and DEAE-agarose gel is stored in liquid as DEAE-agarose gel.

[0099] Take 1.44 mL DEAE-agarose gel solution, 3.36 mL DEAE-A50 solution and 0 mL DE52 solution (3:7:0) to prepare the anion exchange ligand mixture solution.

[0100] Example 11

[0101] This example 11 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2 g of DEAE-A50 and 0.864 g of DE52 are respectively added to 86 mL of ultrapure water to obtain DEAE-A50 solution and DE52 solution in step S1 of preparation of anion exchange ligand, and DEAE-agarose gel is stored in liquid as DEAE-agarose gel.

[0102] Take 1.44 mL DEAE-agarose gel solution, 1.92 mL DEAE-A50 solution and 1.44 mL DE52 solution (3:4:3) to prepare the anion exchange ligand mixture solution.

[0103] Example 12

[0104] This example 12 provides a plasmid DNA extraction kit based on anion exchange column, which is different from example 1 in that 1.2g DEAE-A50 and 0.864g DE52 are added to 86mL ultrapure water respectively in step S1 of preparation of anion exchange ligand to obtain DEAE-A50 solution, DE52 solution, while DEAE-agarose gel is stored in liquid itself, which is DEAE-agarose gel.

[0105] Take 1.44mL DEAE-agarose gel solution, 0mL DEAE-A50 solution and 3.36mL DE52 solution (3:0:7) to configure anion exchange ligand mixed solution.

[0106] Plasmid DNA yield test:

[0107] The plasmid DNA extraction kit obtained by using example 1 to example 12 is used to extract plasmid, which includes the following steps:

[0108] (1) Bacterial cell collection and lysis: take 30mL-200mL of E. coli containing specific plasmid overnight culture after centrifugal collection of bacterial cells, add 4mL buffer P1 (confirm whether to add RNase A) to resuspend thoroughly; add 4mL of alkaline lysis buffer P2, mix quickly, and stand at room temperature for 5min (the time should not be too long); add 4mL of acid neutralizing buffer P3, mix thoroughly to obtain a suspension.

[0109] (2) Purification of plasmid by anion exchange column: put the filter screen in the anion exchange column of the plasmid DNA extraction kit obtained in any one of example 1 to example 12, and then rinse the filter screen and DNA exchange column with 12mL EQU Buffer (6mL each time, 2 times); add the suspension obtained in the above (1) to the exchange column containing the filter screen; after the liquid flow is dry, add 12mL EQU Buffer along the filter screen (6mL each time, 2 times), and let it flow naturally; discard the filter screen, add 5mL Wash Buffer each time, and wash twice in total; after the liquid of the previous step is naturally dry, add 3.75mL Elution Buffer, and collect the filtrate; add 3.75mL Elution Buffer again, and collect and combine the filtrate of two times.

[0110] (3) Plasmid concentration: add 5 mL isopropanol to the combined filtrate, centrifuge at 12000 g for 30 min at 4°C; discard the supernatant, dry completely, add 400 μL sterilized milliQ water to dissolve completely, transfer to a 1.5 mL tube; add 40 μL 10 mol / L ammonium acetate and 880 μL anhydrous ethanol, mix thoroughly; centrifuge at 12000 rpm for 3 min; wash twice with 1 mL 75% ethanol in a fume hood; discard the supernatant, add 50-200 μL TE to dissolve the plasmid completely to obtain the concentrated plasmid DNA solution.

[0111] The yield test results of the plasmid DNA extracted in Examples 1 to 12 are shown in the following table

[0112] Table 1: Yield test results of the plasmid DNA extracted in Examples 1 to 12

[0113]

[0114] Plasmid yield: the amount of plasmid extracted per milliliter of bacterial solution (unit: ng / mL)

[0115] As can be seen from Table 1, the plasmid can be extracted from the anion exchange column with the anion exchange ligand filler selected from any one, any two or any three of DEAE-agarose gel, DEAE-A50 or DE52, and the above is the result of one experiment. The plasmid yield is higher when DEAE-A50 and DE52 fillers are combined as anion exchange ligands. It can be seen that the matrix with the ratio of Example 1 has better plasmid extraction effect.

[0116] Transfection efficiency and protein expression effect test of plasmid DNA:

[0117] Day 1:

[0118] Inoculate cells

[0119] 1) Prepare 3 plates of HeLa cells, each containing 250,000 HeLa cells, and culture the cells in DMEM / 10% v / v FBS medium.

[0120] 2) After shaking evenly, place in a 37°C, 5% CO2 incubator.

[0121] Day 2:

[0122] Plasmid transfection

[0123] 1) Observe the cell state after 12 hours, and when the cell density occupies 30-50% of the area of the dish bottom, perform the transfection operation.

[0124] 2) Perform the plasmid transfection operation:

[0125] 2.1) Reagent preparation

[0126] 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.

[0127] 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.

[0128] ③ Mix the diluted sample thoroughly and let it stand for 5 minutes.

[0129] 2.2) Mixing of plasmids and transfection reagents and transfection

[0130] 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.

[0131] 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.

[0132] ③ After culturing for 6 hours, replace the culture medium of HeLa cells with brand new DMEM / 10% v / v FBS medium.

[0133] Day 4:

[0134] 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):

[0135] 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.

[0136] 2) Centrifuge the cell pellet and media solution at 1000 x g for 5 min at 4°C.

[0137] 3) Discard the media in the centrifuge tube and resuspend the HeLa cells in 120 μΐ of 1 x RIPA lysis buffer containing protease inhibitor cocktail.

[0138] 4) Centrifuge the HeLa cell lysate at 12000 x g for 10 min at 4°C.

[0139] 5) After centrifugation, transfer 60 μΐ of the supernatant to a new centrifuge tube and label the tube according to the source of the plasmid.

[0140] 6) Add 30 μΐ of 2 x loading buffer (Savillex, G2031-1ML) to each tube.

[0141] Protein electrophoresis (SDS-PAGE electrophoresis)

[0142] 1) Loading and electrophoresis

[0143] 1) Denature the protein sample containing the loading buffer by boiling at 95°C for 10 min.

[0144] 2) Load 40 μg of protein per well and run the electrophoresis at 90 V for 25 min and 150 V for 55 min.

[0145] 2) Membrane transfer (wet transfer method)

[0146] 1) After the electrophoresis is complete, immerse the gel, PVDF membrane (activated with methanol), and filter paper in the transfer buffer.

[0147] 2) Assemble the components in the order of "cathode - filter paper - gel - membrane - filter paper - anode" to avoid air bubbles.

[0148] 3) Transfer the membrane at a constant current of 200 mA - 250 mA (or 100 V) for 1 hour.

[0149] 4) Discard the 5% w / v skim milk powder and wash the membrane in TBST for 5 min at a fast speed on a shaker.

[0150] 4) Discard the 5% w / v skim milk powder and wash the membrane in TBST for 5 min at a fast speed on a shaker.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 8) Wash three times with a 1×TBST shaker, 5 minutes each time.

[0155] 9) After cleaning, the PVDF film is exposed and imaged using a chemiluminescence imaging system to obtain... Figure 2 The results are shown.

[0156] 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.

[0157] 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.

[0158] The optimized concentration of Triton X-100 in EQU Buffer and the volume of EQU Buffer equilibration buffer are shown in the table below:

[0159] Table 2: Results of Triton X-100 Concentration and EQU Buffer Equilibration Buffer Volume Optimization

[0160]

[0161] 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.

[0162] While the embodiments of the application have been illustrated and described in detail, it will be readily apparent to those skilled in the art that various modifications and changes can be made to the embodiments without departing from the scope and spirit of the application, as described in the claims. Moreover, the application described is not limited in its application to the details set forth in the description or illustrated in the drawings. The application is capable of other embodiments and of being practiced 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 an anion exchange ligand filling the space between the upper sieve plate and the lower sieve plate; the anion exchange ligand is composed of diethylaminoethyl dextran gel DEAE-A50 and diethylaminoethyl cellulose DE52 in a mass ratio of (1.2:1)-(1.6:1).

2. 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.

3. The plasmid DNA extraction kit according to claim 2, characterized in that, The mass-to-volume ratio of the ligand mixture to ultrapure water is 1 g: (30-50) mL.

4. The plasmid DNA extraction kit according to claim 2, 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.

5. 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.

6. A method for extracting plasmid DNA using the plasmid DNA extraction kit according to any one of claims 1 to 5, 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.

7. The method according to claim 6, characterized in that, Steps (2), (3), and (4) are performed using gravity flow.

8. The use of a plasmid DNA extraction kit as described in any one of claims 1 to 5 in the preparation of transfection-grade plasmid DNA.

9. The application according to claim 8, 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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