An endotoxin removal kit and its application in nucleic acid extraction

Abstract

The present invention relates to the technical field of biomaterial purification, and specifically to an endotoxin removal kit and its application in nucleic acid extraction. The endotoxin removal kit provided by the present invention has a good endotoxin removal effect, can efficiently remove endotoxins in biological materials such as nucleic acids in vitro, and does not require repeated liquid phase extraction after treatment, which simplifies the endotoxin removal process and significantly improves the endotoxin removal efficiency. The endotoxin removal kit is used for plasmid extraction, and high-quality plasmids with low endotoxin content can be obtained without relying on nucleic acid adsorption columns and complex liquid phase extraction. The proportion of supercoiled plasmids reaches more than 90%, and the endotoxin residue is less than 0.1 EU / µg. It is suitable for large-scale plasmid extraction and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterial purification, in particular to an endotoxin removal kit and application thereof in nucleic acid extraction. Background Art

[0002] Endotoxin is a component of the cell wall of Gram-negative bacteria and a pyrogen. Its presence will reduce the quality of biological materials such as nucleic acids and affect subsequent cell transfection and other operations. For example, plasmids containing endotoxins will seriously reduce the transfection efficiency of the plasmids and the expression rate of cells, and have a great impact on cell growth. Therefore, in the preparation of biological materials such as nucleic acids, endotoxins need to be removed as much as possible. In addition, in the biopharmaceutical industry, the level of endotoxin residues is also strictly controlled.

[0003] Plasmids are a type of nucleic acid molecules that are inherent in biological cells, can replicate autonomously and be stably inherited independently of the host chromosome. They are widely present in bacteria, actinomycetes, filamentous fungi, large fungi, yeast, plants, etc., and are different from DNA molecules outside chromosomes. They are mainly located in the cytoplasm. From the perspective of molecular configuration, plasmids can be divided into linear and circular plasmids. Natural plasmids are generally circular, covalent, and closed.

[0004] Recombinant DNA technology can deliver DNA containing the target fragment to the recipient cell for reproduction and expression through plasmids as a carrier transport tool. This DNA carrier is called a plasmid vector. Bacterial plasmids are one of the most widely used vectors in recombinant DNA technology. The earliest plasmid extraction method was the traditional alkaline lysis method, which uses alkali and SDS to lyse bacterial cultures and isolate plasmid DNA. This method is also the most widely used plasmid extraction method. However, this method cannot separate and remove endotoxins. Therefore, in molecular biology and cell biology, plasmids extracted by traditional alkaline lysis methods are not suitable for cell transfection-related experiments.

[0005] At present, there are three main technical means for endotoxin removal: the first is to use alkali, lysozyme, ionic surfactants, etc. to lyse bacteria and then use nucleic acid adsorption columns to specifically adsorb plasmids. This method is mainly in the form of a kit and is limited by the capacity of the adsorption column and cannot be applied to large-scale plasmid extraction; the second is to use strong anion exchange resin to perform anion exchange chromatography on a large number of plasmids. Based on the principle that endotoxins are negatively charged and can be specifically adsorbed on the chromatography filler, the endotoxin residues in the plasmids are removed, but this method causes a large loss of plasmids and has a low recovery rate; the third is to use endotoxin removal reagents TritonX-114 and sodium deoxycholate. These substances can bind to the lipid part of endotoxins, and then through repeated liquid phase extraction methods, an endotoxin-free supernatant solution is obtained, and then the purified plasmid is obtained through an adsorption column. This method is also the best endotoxin removal technology among the current endotoxin-free plasmid extraction methods, but its process involves liquid phase extraction, which needs to be repeated many times to achieve the endotoxin removal effect, which takes a long time and has low efficiency. Summary of the invention

[0006] The invention provides an endotoxin scavenger composition, an endotoxin scavenging kit and applications of the same in nucleic acid extraction.

[0007] In view of the problems existing in the current endotoxin removal technology, the present invention attempts to improve the removal efficiency of endotoxins. During the research and development process, the present invention found that the combination of polymyxin B with neutral salt and PEG as an endotoxin scavenger, combined with high salt ion concentration conditions, can significantly improve the removal efficiency of endotoxins while ensuring the quality and yield of the biological material to be processed, simplify the endotoxin removal steps, and is independent of processing materials such as adsorption columns, and can be applied to large-scale extraction and endotoxin removal of biological materials such as nucleic acids.

[0008] Specifically, the present invention provides the technical solution described below.

[0009] In a first aspect, the present invention provides an endotoxin removal kit, the kit comprising an endotoxin removal agent composition and a salt ion supplement; wherein the endotoxin removal agent composition comprises the following components: 15-30 mM neutral salt, 20%-40% PEG, 0.5%-1% polymyxin B; the average molecular weight of the PEG is 7500-8500;

[0010] The salt ion supplement includes neutral salt, which is used to increase the salt ion concentration in the endotoxin removal treatment system. The working concentration of the salt ion supplement in the endotoxin removal treatment system is 0.5-1M in terms of neutral salt concentration.

[0011] In the above-mentioned endotoxin scavenger composition, polymyxin B cooperates with PEG and neutral salt to have a good endotoxin removal effect, and does not require repeated liquid phase extraction. After reacting with a solution containing nucleic acids such as plasmids, endotoxins can be effectively separated from nucleic acids such as plasmid DNA to obtain nucleic acid precipitates such as plasmid DNA from which endotoxins are removed. While ensuring a high nucleic acid yield and quality, the endotoxin removal effect is significantly improved.

[0012] The endotoxin scavenger composition of the present invention needs to play a better role in removing endotoxins in the presence of high concentrations of salt ions. The role of the above-mentioned salt ion supplement is to provide high concentrations of salt ions for the endotoxin scavenger composition to play an endotoxin removal role.

[0013] In some embodiments of the present invention, the PEG is PEG8000.

[0014] In the present invention, the neutral salt includes but is not limited to magnesium salt, sodium salt, potassium salt, ammonium salt and the like.

[0015] Preferably, the neutral salt in the endotoxin scavenger composition and the neutral salt in the salt ion supplementer are neutral salts having different cations.

[0016] Preferably, the neutral salt in the endotoxin scavenger composition is a magnesium salt, and the neutral salt in the salt ion supplement is a sodium salt.

[0017] Wherein, the magnesium salt can be any neutral magnesium salt, including but not limited to MgCl2 and the like.

[0018] The sodium salt may be any neutral sodium salt, including but not limited to NaCl and the like.

[0019] The above-mentioned endotoxin removing composition further comprises a solvent; the solvent is preferably water.

[0020] In some embodiments of the present invention, the endotoxin scavenger composition comprises the following components: 15-30 mM MgCl2, 20%-40% PEG8000, and 0.5%-1% polymyxin B.

[0021] For salt ion supplementation, neutral salts can be provided in liquid or solid form.

[0022] Preferably, the salt ion supplement is a 3-5 M neutral salt solution.

[0023] In some embodiments of the present invention, the salt ion supplement is a 3-5 M NaCl solution.

[0024] In some embodiments of the present invention, an endotoxin removal kit is provided, which includes an endotoxin removal agent composition and a salt ion supplement, wherein the endotoxin removal agent composition includes the following components: 15-30mM MgCl2, 20%-40% PEG8000, and 0.5%-1% polymyxin B; the salt ion supplement is a 3-5M NaCl solution.

[0025] When the above kit is used for endotoxin removal treatment, the volume ratio of the endotoxin scavenger composition added to the sample to be treated is (0.4-1):1, preferably (0.4-0.6):1; the volume ratio of the salt ion supplement added to the sample to be treated is (0.3-0.5):1, preferably (0.3-0.4):1.

[0026] In a second aspect, the present invention provides any one of the following uses of the above-mentioned endotoxin removal kit:

[0027] (1) Preparing a nucleic acid extraction kit;

[0028] (2) In vitro endotoxin clearance;

[0029] (3) Remove endotoxins from nucleic acids extracted in vitro.

[0030] In the present invention, the nucleic acid includes DNA and RNA, wherein DNA includes plasmid DNA, genomic DNA, genomic DNA fragments and the like.

[0031] In the above applications, in vitro endotoxin removal refers to endotoxin removal outside the body, and the target of action may be biological materials, including but not limited to nucleic acids.

[0032] In a third aspect, the present invention provides a nucleic acid extraction kit, which includes the endotoxin removal kit described above.

[0033] Preferably, the nucleic acid extraction kit also includes other reagents for nucleic acid extraction, such as: a suspension, a lysis solution, a nucleic acid precipitant, a dissolving solution, etc.

[0034] In some embodiments of the present invention, a plasmid extraction kit is provided, which includes the endotoxin removal kit described above, and one or more selected from the following reagents: a suspension, a lysis solution, a neutralization solution, a nucleic acid precipitant, a high salt ion solution, a dissolving solution, and a rinsing solution.

[0035] Preferably, the plasmid extraction kit extracts plasmids based on alkaline lysis method.

[0036] The above suspension is used to suspend the collected bacteria, and a common suspension can be used. Preferably, it includes the following components: glucose (preferably 40-60mM), Tris-HCl (preferably 20-30mM, pH 7.8-8.2), and EDTA (8-12mM, pH 7.8-8.2).

[0037] The above lysis solution is used for lysing bacteria and can be a common lysis solution for extracting plasmids by alkaline lysis method. It preferably includes the following components: NaOH (preferably 0.15-0.25M), SDS (preferably 0.8-1.2%).

[0038] The neutralizing solution is used to neutralize the lysate obtained by the lysing solution treatment, so that the plasmid DNA is renatured and the chromosome DNA is precipitated. Preferably, it includes the following components: potassium acetate (preferably 2.5-3.5M), acetic acid (11-13%).

[0039] The nucleic acid precipitant may include a DNA precipitant and an RNA precipitant, which are used to precipitate DNA and RNA respectively, wherein the DNA precipitant may be isopropanol or ammonium acetate (preferably 9-11 M); the RNA precipitant is preferably lithium chloride (preferably 4-6 M).

[0040] The above-mentioned rinse solution is used to wash the plasmid DNA precipitate and can be a 70%-80% ethanol solution.

[0041] The above-mentioned dissolving solution is used to dissolve the plasmid DNA precipitate, and can be TE buffer or water.

[0042] In a fourth aspect, the present invention provides a method for removing endotoxins from nucleic acid, the method comprising: using the endotoxin removal kit described above to treat a nucleic acid sample to be treated.

[0043] Preferably, the nucleic acid sample to be processed is mixed with the salt ion supplement and endotoxin remover composition in the endotoxin removal kit and then incubated.

[0044] Preferably, in the treatment, the volume ratio of the endotoxin scavenger composition to the nucleic acid sample to be treated is (0.4-1): 1. The volume ratio of the salt ion supplement to the nucleic acid sample to be treated is (0.3-0.5): 1.

[0045] Preferably, the salt ion supplement is added in an amount such that the final concentration of the salt ions contained therein in the treatment system is 0.5-1M.

[0046] Preferably, the treatment is incubation at 0-4°C.

[0047] Preferably, the treatment time is 5-15 min.

[0048] Preferably, after the treatment is completed, the supernatant and the precipitate are separated and the precipitate is collected.

[0049] In the above method, after treatment with the endotoxin scavenger composition, the removed endotoxin is present in the supernatant, while the nucleic acid is present in the precipitate. The endotoxin and the nucleic acid can be separated by centrifugation or the like, thereby achieving the effect of removing the endotoxin.

[0050] The collected precipitate can be further purified to remove impurities such as small molecules such as salts.

[0051] The nucleic acids mentioned above include DNA and RNA, wherein DNA includes plasmid DNA, genomic DNA, genomic DNA fragments and the like.

[0052] In a fifth aspect, the present invention provides a method for extracting plasmid DNA, the method comprising: isolating plasmid DNA from bacterial lysate to obtain a solution containing plasmid DNA, and mixing the solution containing plasmid DNA with the salt ion supplement and endotoxin scavenger composition in the above-mentioned endotoxin removal kit to perform endotoxin removal treatment.

[0053] Preferably, the volume ratio of the endotoxin scavenger composition added to the solution containing plasmid DNA is (0.4-1):1.

[0054] Preferably, the volume ratio of the salt ion supplement to the solution containing the plasmid DNA is (0.3-0.5):1.

[0055] Preferably, the treatment is incubation at 0-4°C, and after the treatment is completed, the supernatant and the precipitate are separated and the precipitate is collected.

[0056] Preferably, the treatment time is 5-15 min.

[0057] Preferably, the method comprises: lysing the bacteria, crudely extracting plasmid DNA, dissolving the plasmid DNA, and removing RNA by precipitation to obtain a solution containing plasmid DNA, mixing the solution containing plasmid DNA with the salt ion supplement and the endotoxin scavenger composition to perform endotoxin removal treatment, separating the supernatant and the precipitate after the treatment is completed, and further purifying the precipitate.

[0058] The bacterial lysis adopts alkaline lysis method, which includes the following steps: after the bacterial cells are heavily suspended, NaOH and SDS are added for treatment, potassium acetate and acetic acid are added for neutralization, and the supernatant is collected after centrifugation.

[0059] The crude plasmid DNA is precipitated by isopropanol.

[0060] The RNA is removed by precipitation using LiCl, which can precipitate RNA and remove protein residues at the same time.

[0061] In the above method, after removing endotoxin, the collected precipitate mainly contains plasmid DNA.

[0062] The further purification of the precipitate may be: after dissolving the precipitate, precipitate and wash again to improve the purity of the plasmid DNA. The precipitation may be performed again using ammonium acetate and / or ethanol precipitation; and the washing may be performed using 70%-80% ethanol aqueous solution.

[0063] The method for extracting plasmid DNA provided by the present invention is a large-scale extraction method for low-endotoxin plasmids. The method is based on the traditional alkaline lysis method. After using alkali-SDS to lyse bacteria, the crude plasmid DNA is first precipitated with isopropanol, and after the precipitate is dissolved in water, the RNA and protein residues are precipitated with LiCl, and then an endotoxin scavenger composition is added at a high salt ion concentration, and the endotoxin is removed by centrifugation. The plasmid DNA precipitate is collected, and a mixture of ammonium acetate and anhydrous ethanol is added to precipitate the plasmid DNA. The precipitate is washed, dried, and dissolved to obtain a plasmid with an ultra-low endotoxin content. This method does not require repeated liquid phase extraction, and does not require the addition of RNase digestion, and high-quality plasmids with low endotoxin content can be obtained. The proportion of supercoiled plasmids reaches more than 90%, and the endotoxin residue is less than 0.1 EU / µg, which is very suitable for large-scale plasmid extraction.

[0064] In some embodiments of the present invention, the method for extracting plasmid DNA comprises the following steps:

[0065] (1) Resuspend the cells in suspension solution;

[0066] (2) Add lysis buffer to lyse the bacteria;

[0067] (3) After adding neutralizing solution, centrifuge for 20 minutes, discard the precipitate, and collect the supernatant;

[0068] (4) mixing the supernatant solution of step (3) with isopropanol, precipitating, centrifuging, discarding the supernatant, and dissolving the precipitate with water;

[0069] (5) Add RNA precipitant, precipitate, centrifuge, discard the precipitate, and collect the supernatant;

[0070] (6) mixing the supernatant solution of step (5) with a salt ion supplement and an endotoxin scavenger composition, subjecting the mixture to precipitation treatment, centrifuging, discarding the supernatant, and collecting the precipitate;

[0071] (7) washing the precipitate from step (6) with 70%-80% ethanol;

[0072] (8) dissolving the precipitate washed in step (7) with water, adding a DNA precipitant and anhydrous ethanol, performing precipitation treatment, and collecting the precipitate after centrifugation;

[0073] (9) The precipitate from step (8) is washed with 70%-80% ethanol and then dried.

[0074] The above suspension is used to suspend the collected bacteria, and a common suspension can be used. Preferably, it includes the following components: glucose (preferably 40-60mM), Tris-HCl (preferably 20-30mM, pH 7.8-8.2), and EDTA (8-12mM, pH 7.8-8.2).

[0075] The above lysis solution is used for lysing bacteria and can be a common lysis solution for extracting plasmids by alkaline lysis method. It preferably includes the following components: NaOH (preferably 0.15-0.25M), SDS (preferably 0.8-1.2%).

[0076] The neutralizing solution is used to neutralize the lysate obtained by the lysing solution treatment, so that the plasmid DNA is renatured and the chromosome DNA is precipitated. Preferably, it includes the following components: potassium acetate (preferably 2.5-3.5M), acetic acid (11-13%).

[0077] The DNA precipitant is preferably ammonium acetate (preferably 9-11 M). The RNA precipitant is preferably lithium chloride (preferably 4-6 M).

[0078] The beneficial effects of the present invention include at least: the endotoxin removal kit provided by the present invention has a good endotoxin removal effect, can efficiently remove endotoxins in biological materials such as nucleic acids in vitro, and does not require repeated liquid phase extraction to remove endotoxins after treatment, which simplifies the endotoxin removal process and significantly improves the endotoxin removal efficiency. The endotoxin removal kit is used for plasmid extraction, which significantly improves the endotoxin removal effect while ensuring a high plasmid DNA yield and quality. High-quality plasmids with low endotoxin content can be obtained without relying on nucleic acid adsorption columns and complex liquid phase extraction. The proportion of supercoiled plasmids reaches more than 90%, and the endotoxin residue is less than 0.1 EU / µg, which is suitable for large-scale plasmid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0080] Figure 1 This is a diagram showing the electrophoresis result of the PUC18 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Example 1 of the present invention.

[0081] Figure 2 This is a diagram showing the electrophoresis result of the pET-32a plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Example 1 of the present invention.

[0082] Figure 3 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Example 1 of the present invention.

[0083] Figure 4 This is a diagram showing the electrophoresis result of the pcDNA3.1-EGFP plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Example 1 of the present invention.

[0084] Figure 5 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Example 2 of the present invention.

[0085] Figure 6 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Comparative Example 1 of the present invention.

[0086] Figure 7 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Comparative Example 2 of the present invention.

[0087] Figure 8 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Comparative Example 3 of the present invention.

[0088] Fig. 9 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Comparative Example 4 of the present invention.

[0089] Fig.10 This is a diagram showing the electrophoresis result of the pEGFP-N1 plasmid extracted by performing endotoxin removal treatment during plasmid extraction using the endotoxin removal kit of Comparative Example 5 of the present invention.

[0090] Figure 1-Figure 10 In the figure, M represents DNA marker, and the other three lanes are three parallel extracted plasmid samples. DETAILED DESCRIPTION

[0091] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0092] Example 1

[0093] This embodiment provides an endotoxin removal kit, which includes an endotoxin removal agent composition and a high salt ion solution (i.e., a salt ion supplement), wherein the formula of the endotoxin removal agent composition is as follows: 30mM MgCl2, 40% PEG8000, 1% polymyxin B; the high salt ion solution is 5M NaCl.

[0094] Example 2

[0095] This embodiment provides an endotoxin removal kit, which includes an endotoxin removal agent composition and a high salt ion solution (i.e., a salt ion supplement), wherein the formula of the endotoxin removal agent composition is as follows: 15mM MgCl2, 20% PEG8000, 0.5% polymyxin B; the high salt ion solution is 3M NaCl.

[0096] Comparative Example 1

[0097] This comparative example provides an endotoxin removal kit, which includes an endotoxin remover and a high salt ion solution. The only difference between the kit and the kit of Example 1 is that the formula of the endotoxin remover composition is as follows: 30mM MgCl2, 40% PEG8000.

[0098] Comparative Example 2

[0099] This comparative example provides an endotoxin removal kit, which includes an endotoxin remover and a high salt ion solution. The difference between the kit and the kit of Example 1 is that the formula of the endotoxin remover composition is as follows: 30mM MgCl2, 1% polymyxin B.

[0100] Comparative Example 3

[0101] This comparative example provides an endotoxin removal kit, which differs from the kit of Example 1 only in that it does not contain a high salt ion solution.

[0102] Comparative Example 4

[0103] This comparative example provides an endotoxin removal kit, which only contains an endotoxin remover: 2% Triton X-114.

[0104] Comparative Example 5

[0105] This comparative example provides an endotoxin removal kit, which only contains an endotoxin remover: 1% polymyxin B.

[0106] Example 3

[0107] This embodiment provides a method for removing endotoxins from nucleic acids, which is to use the endotoxin removal kit of embodiment 1 or 2 to treat the nucleic acid sample, and the treatment steps are as follows:

[0108] (1) Add a high salt ion solution accounting for 1 / 3 of the volume and an endotoxin scavenger composition accounting for 1 / 2 of the volume to the nucleic acid sample to be processed, place on ice for 10 minutes, centrifuge at 10000g and 4°C for 20 minutes, discard the supernatant, and collect the precipitate;

[0109] (2) After washing the precipitate obtained in step (1) with 70% ethanol, the precipitate is dissolved, and then the nucleic acid is precipitated with a precipitating agent, and then the precipitate is washed, dried and redissolved.

[0110] Example 4

[0111] This embodiment provides a plasmid extraction method, which comprises the following steps:

[0112] (1) Resuspend the cells in 15 mL of PBS buffer, centrifuge at 3500 g, 4°C for 10 min, and discard the supernatant.

[0113] (2) Add 6 mL of suspension to the bacterial pellet from step (1) to resuspend the bacteria.

[0114] (3) Add 6 mL of lysis buffer, gently invert upside down, and leave at room temperature for 5 minutes.

[0115] (4) Add 5 mL of neutralizing solution, gently invert the tube, place on ice for 10 min, centrifuge at 10,000 g, 4°C for 20 min, and discard the precipitate.

[0116] (5) Add 2 / 3 volume of isopropanol to the supernatant solution from the previous step, mix well, place on ice for 10 minutes, centrifuge at 10,000 g, 4°C for 20 minutes, discard the supernatant, and dissolve the precipitate in 1 mL of deionized water.

[0117] (6) Add 1 mL of RNA precipitant, place on ice for 10 min, centrifuge at 10,000 g, 4°C for 20 min, and discard the precipitate.

[0118] (7) Transfer the supernatant solution from the previous step to a new centrifuge tube and perform endotoxin removal treatment using an endotoxin removal kit: add 1 / 3 volume of high salt ion solution and 1 / 2 volume of endotoxin removal agent composition, place on ice for 10 minutes, centrifuge at 10,000 g, 4°C for 20 minutes, and discard the supernatant.

[0119] (8) Add 1 mL of 70% ethanol, suspend the precipitate, centrifuge at 10,000 g, 4°C for 5 min, and discard the supernatant.

[0120] (9) Dissolve the precipitate with 200 μL of deionized water. After the precipitate is completely dissolved, add 1 / 2 volume of DNA precipitant and 2 volumes of anhydrous ethanol. Mix well and place at room temperature for 5 minutes. Centrifuge at 10,000 g at 4°C for 5 minutes.

[0121] (10) Add 1 mL of 70% ethanol, suspend the precipitate, centrifuge at 10,000 g, 4°C for 5 min, and discard the supernatant.

[0122] (11) After the precipitate is dry, add 100 μL of dissolving solution to dissolve it.

[0123] The main reagents used in the above method are as follows:

[0124] Suspension: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0);

[0125] Lysis buffer: 0.2 M NaOH, 1% SDS;

[0126] Neutralizing solution: 3M potassium acetate, 12% acetic acid;

[0127] RNA precipitation agent: 5M LiCl;

[0128] DNA precipitant: 10 M ammonium acetate;

[0129] The endotoxin removal kit is the endotoxin removal kit in Example 1 or 2;

[0130] Dissolving solution: 100mM Tris-HCl, 10mM EDTA, pH 8.0, also known as TE buffer.

[0131] In order to verify the endotoxin removal effect of the endotoxin removal kits of Examples 1-2 and various comparative examples and their application effect in plasmid extraction, the following experimental examples use different plasmids as objects and use the endotoxin removal kits of Examples 1-2 and various comparative examples to perform endotoxin removal in plasmid extraction.

[0132] Experimental example

[0133] 1. Bacterial culture preparation

[0134] Pick a monoclonal DH5α strain containing the PUC18 plasmid (one colony is sufficient), add it to 50 mL of liquid culture medium containing the corresponding antibiotics, and culture it overnight at 37°C. The next day, collect the overnight bacterial culture, centrifuge it at 3500g, 4°C for 10 minutes, discard the supernatant, and keep the bacterial cells.

[0135] Pick the monoclonal DH5α strain containing the pET-32a plasmid, add it to 50 mL of liquid culture medium containing the corresponding antibiotics, and culture it overnight at 37°C. The next day, collect the overnight bacterial culture, centrifuge it at 3500g, 4°C for 10 minutes, discard the supernatant, and keep the bacterial cells.

[0136] Pick the monoclonal DH5α strain containing the pEGFP-N1 plasmid, add it to 50 mL of liquid culture medium containing the corresponding antibiotics, and culture it overnight at 37°C. The next day, collect the overnight bacterial culture, centrifuge it at 3500g, 4°C for 10 minutes, discard the supernatant, and keep the bacterial cells.

[0137] The monoclonal DH5α strain containing the pcDNA3.1-EGFP plasmid was picked and added to 50 mL of liquid culture medium containing the corresponding antibiotics, and cultured overnight at 37°C. The next day, the bacterial culture cultured overnight was collected, centrifuged at 3500g, 4°C for 10 minutes, and the supernatant was discarded, retaining the bacterial cells.

[0138] 2. Plasmid extraction

[0139] For the bacteria collected in step 1, the method of Example 4 was used to extract plasmids.

[0140] The concentration and A260 / 280 of the extracted plasmid were measured using Thermo's NanoDropOne instrument. Usually, the A260 / 280 of the plasmid is between 1.8-2.0, indicating that the purity is relatively high; in addition, 0.5 μL of the plasmid was taken, 6× Loading Buffer was added, and 1% agarose gel electrophoresis was performed, and the results were observed using the Thermo Scientific Image Lab gel imaging instrument.

[0141] Furthermore, the horseshoe crab reagent of Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd. was used to detect endotoxins in the plasmid samples extracted from the above experimental examples, with pyrogen-free water as the negative control, and endotoxin standard samples diluted with pyrogen-free water as the positive control. The detection limit was 0.1EU / µg, and the plasmid samples to be tested were diluted and tested with sterile TE buffer.

[0142] The results of plasmid concentration, purity and endotoxin content are shown in Tables 1 and 2. The results of 1% agarose gel electrophoresis are shown in Figure 1-10 shown.

[0143] Table 1 Concentration and purity of different plasmid DNA and endotoxin content test results (1)

[0144]

[0145] Note: The three values ​​of each index of the same plasmid in Table 1 are the test results of three parallel samples.

[0146] Table 2 Concentration and purity of different plasmid DNA and endotoxin content test results (2)

[0147]

[0148] Note: The three values ​​of each index of the same plasmid in Table 2 are the test results of three parallel samples.

[0149] Result analysis: Endotoxin removal was performed in plasmid extraction using the endotoxin removal kits of Example 1 and Example 2. Four different plasmids were selected for extraction. The results were detected by 1% agarose gel electrophoresis ( Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 ) It can be seen that the four extracted plasmids have no other impurities and the supercoiled structure accounts for more than 90%, the plasmid concentration is >2000ng / µL, and the total amount of plasmid extracted from 50mL of bacterial culture in a single time exceeds 200µg (the current commercially available column-based plasmid extraction kit with better results can obtain up to 270µg of plasmid for 50mL of overnight culture solution), and the A260 / 280 value is between 1.8-2.0, which proves that the purity of the plasmid DNA is high, and the A260 / 230 value is greater than 2.0, which proves that it is not contaminated by other impurities such as salts; endotoxin was detected by Xiamen horseshoe crab reagent, and the results showed that the endotoxin content of the extracted plasmids was lower than 0.1EU / µg, which can meet the cell transfection level requirements.

[0150] The endotoxin removal kit of Comparative Example 1-5 was used to remove endotoxin in plasmid extraction, and the PEGFP-N1 plasmid was selected for extraction. The results of 1% agarose gel electrophoresis ( Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 ) It can be seen that the extracted plasmid has no other impurities and the supercoiled structure accounts for more than 90%, and the plasmid concentration and purity are not significantly different from the embodiment. Among them, Comparative Example 4 belongs to the traditional method, and the recovery rate of the plasmid is low, only about 500ng / µL. Endotoxin was detected by Xiamen horseshoe crab reagent, and the results showed that the endotoxin content of the plasmid extracted in each comparative example was higher than 0.5EU / µg, which could not meet the cell transfection level requirements.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for removing endotoxins from nucleic acids, characterized in that: The method comprises: mixing a nucleic acid sample to be treated with a sodium salt solution and an endotoxin scavenger composition and then incubating the mixture; then separating a supernatant and a precipitate, and collecting the precipitate; The endotoxin scavenger composition uses water as a solvent and is composed of: 15-30 mM magnesium salt, 20%-40% PEG8000, and 0.5%-1% polymyxin B; The amount of sodium salt solution added is such that the working concentration of sodium salt in the endotoxin removal treatment system is 0.5-1M; The sodium salt and the magnesium salt are neutral salts; The nucleic acid is plasmid DNA.

2. The method for removing endotoxins from nucleic acid according to claim 1, characterized in that: The concentration of the sodium salt solution is 3-5M.

3. The method for removing endotoxins from nucleic acid according to claim 1 or 2, characterized in that: In the treatment system, the volume ratio of the endotoxin scavenger composition added to the nucleic acid sample to be treated is (0.4-1):

1.

4. The method for removing endotoxins from nucleic acid according to claim 1 or 2, characterized in that: The incubation is carried out at 0-4°C.

5. The method according to claim 1 or 2, characterized in that: The incubation time is 5-15 min.

6. A method for extracting plasmid DNA, characterized in that: The method comprises: separating plasmid DNA from bacterial lysate to obtain a solution containing plasmid DNA, mixing the solution containing plasmid DNA with a sodium salt solution and an endotoxin scavenger composition to perform endotoxin removal treatment; separating a supernatant and a precipitate after the treatment is completed, and collecting the precipitate; The endotoxin scavenger composition uses water as a solvent and is composed of: 15-30 mM magnesium salt, 20%-40% PEG8000, and 0.5%-1% polymyxin B; The amount of sodium salt solution added is such that the working concentration of sodium salt in the endotoxin removal treatment system is 0.5-1M; The sodium salt and the magnesium salt are neutral salts.

7. The method according to claim 6, characterized in that The concentration of the sodium salt solution is 3-5M.

8. The method according to claim 6 or 7, characterized in that: The volume ratio of the endotoxin scavenger composition added to the solution containing plasmid DNA is (0.4-1):

1.

9. The method according to claim 6 or 7, characterized in that: The endotoxin removal treatment is incubated at 0-4°C.

10. The method according to claim 6 or 7, characterized in that: The treatment time is 5-15 minutes.

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