Histone extraction method, kit and application

Abstract

The invention provides a histone extraction method, a kit and application, and relates to the technical field of biology. The extraction method comprises the following steps: 1) splitting a cell nucleus; (2) adding polyethylene glycol and a glucan solution, and uniformly stirring and mixing; and 3) purifying and separating to obtain the histone. According to the extraction method, the extraction purity and yield of the histone are improved under the condition that the structure of the histone is not damaged; the extraction steps of the histone are simplified, the operation complexity and the time cost of the extraction method are reduced, and the sample treatment efficiency is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a histone extraction method, a kit and an application. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] In the field of biotechnology, protein extraction and purification is a basic and key technology, which is of great significance for subsequent protein function research, structural analysis, and application development.

[0004] Histones are located in the nucleus of eukaryotic organisms, forming a complex chromatin with tightly bound DNA, various non-histones and RNA. Histones usually contain five subunits, including H1, H2A, H2B, H3, and H4, with a molecular weight between 14 and 35 kDa. Among them, two groups of H2A-H2B dimers and two groups of H3-H4 dimers form an octamer core, and DNA is wrapped around the outside of the core, together forming the topological structure of the nucleosome. H1 acts as a linker and binds to the DNA linker region between nucleosomes. Epigenetic studies have found that histone amino acid sequences are modified by methylation, citrullination, acetylation, phosphorylation, ubiquitination, etc., and these modifications are closely related to protein structure and function.

[0005] Each of the five subunits of histones has its own corresponding autoantibody. The autoimmune reaction against DNA is linked to the autoimmune reaction against histones. Patients with positive anti-DNA antibodies often have anti-histone autoantibodies (AHAs) at the same time, but positive AHAs are not necessarily accompanied by anti-DNA antibodies. Due to the complex composition of AHAs, natural histones extracted by different chromatography techniques are used in clinical diagnostic reagents, which may result in weak signal strength or even missed detection. Therefore, different vendors' antigens are mixed to improve the positive detection rate in clinical practice.

[0006] The purity of the extracted histones is not high due to their high affinity for DNA, their tendency to aggregate in the absence of DNA or strong denaturants, proteolytic degradation during long separations, or the presence of non-histone impurities in the extracted histones.

[0007] Traditional histone extraction methods mainly include acid extraction and salt extraction, both of which have their own advantages and disadvantages. Acid extraction is a commonly used histone extraction method, and its main principle is to release histones from chromatin through strong acid treatment (HCl or H2SO4). Although acid extraction can obtain a higher yield of histones, the disadvantage of this method is that strong acid treatment will destroy the structure of histones and affect their biological activity.

[0008] The main principle of the second salt extraction method is to use NaCl (≥2M) and urea (≥5M), and then remove DNA and undissociated chromatin by long-term ultra-high-speed centrifugation (>40000rpm), and then reduce the NaCl concentration step by step by dialysis and centrifuge to remove the precipitated histone and DNA complex, or use molecular sieve chromatography to separate histones. This method takes a long time for dialysis, and molecular sieve takes a long time and is not easy to scale up. The obtained histone product contains non-histone and nucleic acid impurities. There are also salt treatments and centrifugation to separate histones through hydroxyapatite, but high salt elution and urea elution are required to completely elute histones, and the use of urea protein denaturants may destroy the protein structure. It can be seen that although the salt extraction method can keep the structure of histones intact, the extraction steps are cumbersome, laborious and time-consuming, resulting in protein hydrolysis in the separation step. Secondly, many steps involved in the salt extraction method are not easy to carry out on a preparation scale. In addition, the recovery rate is usually low, and the selective loss of some histones cannot be ruled out.

[0009] Therefore, how to improve the extraction yield and purity while maintaining the integrity of histone structure is an important issue facing current histone extraction technology.

[0010] In view of this, the present invention is proposed. Summary of the invention

[0011] The purpose of the present invention is to provide a histone extraction method to alleviate the following prior art problems: to improve the extraction purity and yield of histones without destroying the histone structure; and to simplify the histone extraction steps, reduce the operation complexity and time cost, and improve the sample processing efficiency.

[0012] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0013] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0014] Throughout this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or activity from another entity or activity without necessarily requiring or implying any actual such relationship, order, or importance between such entities or activities, such as numbering (A), (B), and (C), or (I), (II), and (III).

[0015] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0016] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0017] In this document, unless otherwise specified, the numerical range "a-b" represents an abbreviation of any real number combination between a and b, where a and b are real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" have been listed in this document, and "6-22" is just an abbreviation of these numerical combinations. In this document, the "range" disclosed in the form of lower limit and upper limit can be one or more lower limits, and one or more upper limits, respectively.

[0018] Herein, unless otherwise specified, each reaction or operation step may be performed sequentially or non-sequentially. Preferably, the reaction method herein is performed sequentially.

[0019] Herein, "%" refers to mass percentage unless otherwise specified.

[0020] In this article, the unit "mol / L" is also expressed as "M", and the two are used interchangeably; the unit "mmol / L" is also expressed as "mM", and the two are used interchangeably.

[0021] In a first aspect, a histone extraction method is provided, the histone extraction method comprising the following steps:

[0022] 1) Lyse the cell nucleus;

[0023] 2) adding polyethylene glycol and the dextran solution and stirring to mix; and,

[0024] 3) Purify and separate the histones.

[0025] In an optional embodiment, the cell nucleus lysis in step 1) can be performed by conventional lysis methods known in the art, such as but not limited to using a saline solution or a urea solution to lyse the cell nucleus. After the cell nucleus is lysed, histones and nucleic acids are released.

[0026] In an optional embodiment, the step 1) lyses the cell nuclei by a salt lysis method.

[0027] In an optional embodiment, the salt includes at least one of NaCl and KCl, and the working concentration is 1.5 to 2 mol / L, for example, but not limited to 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mol / L.

[0028] In an alternative embodiment, the cell nucleus is derived from thymus tissue.

[0029] In an alternative embodiment, the cell nucleus is derived from bovine thymus tissue.

[0030] Adding the dextran solution in step 2) can increase the solubility of histones in the whole system. Polyethylene glycol is a nucleic acid precipitant, which can combine with nucleic acids to form a precipitate, so that the nucleic acids are separated from the histones dissolved in the dextran solution. The histones fully dissolved in the dextran solution are advantageously separated from the nucleic acids precipitated by polyethylene glycol, so as to improve the yield and purity of the histones.

[0031] In an optional embodiment, the molecular weight of the dextran in step 2) is 10000-500000, for example, it can be but not limited to 10000, 20000, 50000, 80000, 100000, 150000, 200000, 300000, 400000 or 500000; the working concentration is 2-7%, for example, it can be but not limited to 2, 3, 4, 5, 6 or 7%. The molecular weight of the polyethylene glycol in step 2) is 4000-6000, for example, it can be but not limited to 4000, 4500, 5000, 5500 or 6000; the working concentration of the polyethylene glycol is 5-10%, for example, it can be but not limited to 5, 6, 7, 8, 9 or 10%.

[0032] In an optional embodiment, the molecular weight of the dextran in step 2) is 10,000 to 40,000, and the working concentration is 3 to 5%.

[0033] If the molecular weight of dextran is too large, the viscosity of the dextran solution will increase, and it will even condense into a gel, especially under low temperature conditions, making the dextran solution unsuitable for low temperature operation. However, low temperature conditions can reduce the activity of nuclear proteases and reduce the degradation of histones, thereby maintaining the intact structure of histones and improving the yield. Therefore, dextran with a molecular weight of 10,000 to 40,000 is preferred, which can be dissolved at 4°C to avoid increasing the viscosity of the dextran solution under low temperature conditions.

[0034] In an optional embodiment, the stirring and mixing in step 2) is at least 30 minutes, for example, but not limited to, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes.

[0035] In an optional embodiment, the stirring temperature in step 2) is 4°C.

[0036] In an optional embodiment, the purification and separation in step 3) comprises using cation exchange chromatography.

[0037] In an optional embodiment, the chromatography buffer in the cation exchange chromatography contains 0.5-0.6 mol / L of salt, for example, it may be but not limited to 0.5, 0.55 or 0.6 mol / L of salt; the elution buffer contains 0.8-1 mol / L of salt, for example, it may be but not limited to 0.8, 0.9 or 1.0 mol / L of salt.

[0038] In an optional embodiment, the chromatography buffer contains the same type of salt as that in the elution buffer.

[0039] In an optional embodiment, the salt of the elution buffer and the chromatography buffer is selected from NaCl.

[0040] In an optional embodiment, the buffer component in the elution buffer is selected from 10-20mM HEPES, Tris or PB buffer, the pH is 7.4-7, the concentration of the buffer component can be, for example, but not limited to, 10, 15 or 20mmol / L, and the pH can be, for example, but not limited to, 7.4, 7.5 or 7.6.

[0041] In an optional embodiment, the buffer component in the chromatography buffer is selected from 10-20mM HEPES, Tris or PB buffer, the pH is 7.4-7, the concentration of the buffer component can be, for example, but not limited to, 10, 15 or 20mmol / L, and the pH can be, for example, but not limited to, 7.4, 7.5 or 7.6.

[0042] In an optional embodiment, the step 3) further comprises a centrifugal separation step; the solution of step 2) is centrifuged and the supernatant is taken for chromatographic separation.

[0043] In an optional embodiment, the centrifugal speed is 8000-10000 rpm / min, and the centrifugation is for 10-15 min.

[0044] In an optional embodiment, the centrifugal speed is 10000 rpm / min, and the centrifugation is 10 min.

[0045] In a specific embodiment, the histone extraction method comprises:

[0046] (1) Resuspend the cell nuclei in a resuspension buffer and add a salt solution so that the final salt concentration in the lysis system is 1.5 to 2 mol / L;

[0047] (2) adding dextran and polyethylene glycol and mixing them evenly, so that the final concentration of polyethylene glycol in the system is 5-10%, and the final concentration of dextran is 3-5%;

[0048] (3) The supernatant of the lysis system in step (2) is centrifuged and subjected to cation exchange chromatography, followed by one-step elution of histones using an elution buffer.

[0049] In an optional embodiment, the resuspension buffer includes 10-20 mmol / L Tris, HEPES, PB or MOPS buffer, the buffer concentration may be, for example, but not limited to, 10, 15 or 20 mmol / L, and the pH is 7.0-7.6, for example, but not limited to, 7.0, 7.2, 7.5 or 7.6.

[0050] In an optional embodiment, at least 2.5 mL of the resuspension buffer is required for every 1 g of the cell nuclei.

[0051] This embodiment lyses the cell nucleus and dissociates chromatin at a relatively low salt concentration (1.5-2M), releases histones and nucleic acids, and precipitates nucleic acids with polyethylene glycol. However, because chromatin is not completely dissociated at a salt concentration of 1.5-2M, most of the histones will precipitate at the same time, reducing the histone yield. Adding polyethylene glycol and dextran at the same time can promote the dissolution of histones in the solution at a low salt concentration, while nucleic acids bind to polyethylene glycol and precipitate and centrifuge to remove, thereby fully dissociating histones and nucleic acids in chromatin and improving the histone yield.

[0052] In a second aspect, a histone prepared by the histone extraction method described in the first aspect is also provided.

[0053] In the third aspect, the use of the histone described in the second aspect in any one of (I) to (III) is also provided:

[0054] (Ⅰ) Detection of anti-histone antibodies;

[0055] (II) Preparation of products for detecting anti-histone antibodies;

[0056] (III) Preparation of products for diagnosing anti-histone antibody positive diseases.

[0057] In an optional embodiment, the application in any one of items (I) to (III) includes coating the product obtained by the histone extraction method described in the first aspect solely on a solid phase carrier, and the prepared detection product can meet the performance requirements without the need to mix antigens from different merchants to improve the positive detection rate.

[0058] In an optional embodiment, the solid phase carrier comprises magnetic beads, preferably barcoded magnetic beads.

[0059] In an optional embodiment, the anti-histone antibody-positive disease includes, but is not limited to, one or more of systemic lupus erythematosus, drug-induced lupus erythematosus, rheumatoid arthritis, primary biliary cirrhosis and dermatomyositis.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] (1) Improving purity and yield: The present invention provides an improved extraction method, which can significantly improve the extraction purity and yield of nuclear histones by adding glucan with a suitable concentration of polymerization degree during the extraction process. Compared with the traditional acid extraction method and salt extraction method, the extraction method of the present invention can greatly improve the extraction yield while ensuring the integrity of the nuclear histone structure.

[0062] (2) Simplified operation: The extraction method provided by the present invention is simple to operate, short in time and low in cost, which significantly simplifies the operation steps, reduces the operation difficulty and cost, and is more conducive to large-scale production.

[0063] (3) Protecting the nuclear histone structure: The extraction method provided by the present invention can maintain the structural integrity of the nuclear histone, avoiding the damage to the nuclear histone structure that may be caused by the traditional acid extraction method, thereby ensuring the biological activity of the nuclear histone, and the effect of anti-histone antibody detection is significantly better. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0065] Figure 1 This is the electrophoresis diagram of the product obtained by the histone extraction method provided in Example 1. DETAILED DESCRIPTION

[0066] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0067] Example 1

[0068] A method for extracting histones is provided. In this example, 5 g (wet weight) of cell nuclei extracted from 10 g of bovine thymus tissue by conventional methods are subjected to lysis, nucleic acid precipitation and cationic chromatography to finally obtain 60 mg of high-purity histones. The specific implementation steps are as follows (all steps are carried out at 4° C.):

[0069] (1) Weigh 5 g of wet cell nuclei and add 12.5 mL of 20 mM Tirs, pH 7.5, to resuspend the cell nuclei.

[0070] (2) Add an equal volume of 4 M NaCl lysis buffer and stir for 10 min. The final concentration of NaCl in the lysis buffer is 2 M.

[0071] (3) Add 50% polyethylene glycol 6000 to a final concentration of 5%, and add 50% dextran 10000 to a final concentration of 5%, and stir at 4°C for 30 min.

[0072] (4) Centrifuge at 10,000 rpm for 10 min at 4°C and collect the supernatant.

[0073] (5) The supernatant was diluted to 84 mL with 20 mM HEPES, pH 7.5, and the solution was loaded onto a cation exchange chromatography column equilibrated with a chromatography buffer of 20 mM HEPES, pH 7.5, and 0.6 M NaCl solution. The column was rinsed with the chromatography buffer and the protein was eluted with a one-step elution buffer of 20 mM HEPES, pH 7.5, and 1 M NaCl solution. The eluted protein obtained was histone. The yield was calculated and the purity was determined.

[0074] The final yield of the purified product histone in this example was 12.00 mg / 1 g of cell nucleus, OD260 / OD280=0.650,

[0075] The protein purity of the product was determined by 4-20% SDS-PAGE protein electrophoresis. The protein electrophoresis diagram is shown in Figure 1 As shown, non-histone proteins were largely removed.

[0076] Example 2

[0077] A method for extracting histones is provided, referring to Example 1, the only difference from Example 1 is that 3M NaCl lysis solution is added, and the final concentration of NaCl in the lysis buffer is 1.5M.

[0078] Example 3

[0079] A histone extraction method is provided, which differs from that of Example 1 only in that the resuspension buffer is 15 mM HEPES, pH 7.3.

[0080] Example 4

[0081] A histone extraction method is provided, which differs from Example 1 only in that the resuspension buffer is 10 mM PB, pH 7.6.

[0082] Example 5

[0083] A histone extraction method is provided, which differs from that of Example 1 only in that the resuspension buffer is 13 mM MOPS, pH 7.0.

[0084] Example 6

[0085] A histone extraction method is provided. The only difference between the extraction method and Example 1 is that the lysis solution is 3M KCl and the final concentration is 1.5M.

[0086] Example 7

[0087] A histone extraction method is provided. The only difference between the extraction method and Example 1 is that in step (3), 50% polyethylene glycol 6000 is added to a final concentration of 10%.

[0088] Example 8

[0089] A method for extracting histones is provided. The difference between the method and Example 1 is that in step (3), the molecular weight of the polyethylene glycol is 4000.

[0090] Example 9

[0091] A histone extraction method is provided. The only difference between the extraction method and Example 1 is that in step (5), the chromatography buffer is 10 mM Tris, pH 7.5, 0.6 M NaCl solution; and the elution buffer is 10 mM Tris, pH 7.5, 0.8 M NaCl solution.

[0092] Example 10

[0093] A histone extraction method is provided. The difference between the extraction method and Example 1 is that dextran 10000 is added in step (3) with a final concentration of 2%.

[0094] Embodiment 11

[0095] A method for extracting histones is provided. The difference between the extraction method and Example 1 is that dextran 10000 is added in step (3) with a final concentration of 7%.

[0096] Example 12

[0097] A histone extraction method is provided. The difference between the extraction method and Example 1 is that dextran 10000 is added in step (3) with a final concentration of 3%.

[0098] Example 13

[0099] A histone extraction method is provided. The only difference between the extraction method and Example 1 is that dextran 40000 is added in step (3) with a final concentration of 3%.

[0100] Embodiment 14

[0101] A histone extraction method is provided. The only difference between the extraction method and Example 1 is that 100,000 glucan is added in step (3) to a final concentration of 5%.

[0102] Embodiment 15

[0103] A method for extracting histones is provided. The only difference between the extraction method and Example 1 is that the one-step elution buffer used in step (5) is 20 mM HEPES, pH 7.5, 2 M NaCl.

[0104] Comparative Example 1

[0105] A method for extracting histones is provided. The only difference between the method and Example 1 is that dextran 10000 is not added for stirring and mixing.

[0106] Effect Example 1

[0107] The yields of protein extraction methods of Examples 1 to 2, Examples 10 to 14 and Comparative Example 1 were compared and the purity was determined.

[0108] Yield = product mass mg / nucleus mass g; product mass was used to determine protein concentration using the BCA method;

[0109] The product solution was tested for absorbance at 260 nm and 280 nm using a spectrophotometer. The residual nucleic acid in the histone in the product was calculated according to the following formula to determine the protein purity:

[0110] When OD260 / OD280 is less than 0.7, there is basically no nucleic acid residue in the protein; when OD260 / OD280 is greater than 0.7, there is nucleic acid residue. The higher the value, the higher the nucleic acid residue.

[0111] The yield and purity of the extraction methods of Examples 1-2, Examples 10-14 and Comparative Example 1 are shown in Table 1.

[0112] Table 1 Yield and purity test results of each embodiment

[0113]

[0114]

[0115] As can be seen from the above table, when dextran 10000 is not added (Comparative Example 1), histones and nucleic acids are not fully separated, and histones and nucleic acids are precipitated at the same time, resulting in a decrease in histone yield and purity; when the final concentration of dextran 10000 is increased to 7% (Example 11), the solution viscosity increases, which also affects the separation effect, resulting in a decrease in histone yield. When dextran molecular weight ≥ 100000 is added, because the solution is more viscous and even partially forms a gel under low temperature conditions, the solid-liquid separation interface is not obvious after centrifugation, resulting in a decrease in supernatant recovery rate, ultimately resulting in a decrease in histone yield and purity. However, as long as dextran is added in step (3) (Examples 1, 2 and 10-15), the effect of extracting histones is better than that of Comparative Example 1 without adding dextran. And the synergistic effect of the addition of dextran on the histone extraction effect is effective in the range of 1.5-2M salt final concentration in the lysate.

[0116] Effect Example 2

[0117] The sensitivity of the natural histone antigens extracted in Example 1 and Example 15 was verified. The purified protein solutions were concentrated to 2 mg / mL, respectively, and coated with magnetic barcodes (at a ratio of 20 μg / 50K) in the same manner. 16 samples (4 normal serum samples + 12 positive serum samples) were tested by magnetic barcode immunofluorescence method (the detection reagent was alkaline phosphatase-labeled IgG antibody). The results are shown in Table 2 below.

[0118] Table 2 Comparative test results of product sensitivity under different elution buffer conditions in cationic chromatography

[0119]

[0120]

[0121] As can be seen from the above table, although the histone yield of Example 15 increased by 19%, the sensitivity of the eluted histone product of Example 15 decreased sharply by 60%, resulting in a decrease in the detection rate of positive samples, with 5 cases less detected out of 12 positive samples (probably due to the presence of more other impurities in the eluent during high salt elution, which affected the performance). The above results show that the histone product purified by the elution buffer NaCl concentration of 1M in Example 1 is more reactive than the histone product purified by the elution buffer NaCl concentration of 2M in Example 15.

[0122] Effect Example 3

[0123] This effect example verifies the sensitivity of the natural histone antigens extracted from Examples 1 to 14, and uses the natural histone antigens from AROTEC Diagnostics of New Zealand for parallel comparison. This commercial antigen is prepared by salt extraction and extracted and purified from calf thymus tissue without using irritating denaturing agents. The verification method uses magnetic barcode immunofluorescence method, and 32 samples (6 normal serum samples + 26 positive serum samples) are examined. The test results are shown in Table 3:

[0124] Table 3 Comparative test results of Examples 1 and 2 and commercial antigen sensitivity

[0125]

[0126]

[0127] It can be seen from the above table that the natural histones extracted and purified in Examples 1 to 14 have higher sensitivity than commercial antigens without changing the specificity.

[0128] In terms of comprehensive expression efficiency and detection performance, the solution in Example 1 is better.

[0129] 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A histone extraction method, characterized in that: The steps include: 1) Lyse the cell nucleus; 2) adding polyethylene glycol and the dextran solution and stirring to mix; and, 3) Purify and separate the histones.

2. The histone purification method according to claim 1, characterized in that The molecular weight of the dextran in step 2) is 10,000 to 100,000, and the working concentration is 2 to 7%. The molecular weight of the polyethylene glycol in step 2) is 4,000 to 6,000, and the working concentration is 5 to 10%. Optionally, the molecular weight of the dextran is 10,000 to 40,000, and the working concentration is 3 to 5%.

3. The histone purification method according to claim 1, characterized in that The stirring and mixing in step 2) is performed for at least 30 minutes; Optionally, the stirring temperature is 4°C.

4. The histone extraction method according to claim 1, characterized in that The step 1) lyses the cell nuclei by salt lysis method; Optionally, the salt includes at least one of NaCl and KCl, and the working concentration is 1.5-2 mol / L.

5. The histone extraction method according to claim 1, characterized in that The cell nucleus is derived from thymus tissue; Optionally, the cell nucleus is derived from bovine thymus tissue.

6. The histone extraction method according to claim 1, characterized in that: The purification and separation in step 3) comprises using cation exchange chromatography; Optionally, the chromatography buffer in the cation exchange chromatography contains 0.5 to 0.6 mol / L of salt, and the elution buffer contains 0.8 to 1 mol / L of salt; Optionally, the chromatography buffer contains the same type of salt as that in the elution buffer; Optionally, the salt of the elution buffer and the chromatography buffer is selected from NaCl; Optionally, the buffer components in the elution buffer and the chromatography buffer are independently selected from 10-20 mM HEPES, Tris or PB buffer, and the pH is 7.4-7.

6.

7. The histone extraction method according to claim 6, characterized in that: The step 3) further comprises a centrifugal separation step; the solution of step 2) is centrifuged and the supernatant is taken for chromatographic separation; Optionally, the centrifugal speed is 8000-10000 rpm / min, and the centrifugation is 10-15 min; Optionally, the centrifugal speed is 10000 rpm / min, and the centrifugation is performed for 10 min.

8. The histone extraction method according to any one of claims 1 to 7, characterized in that: The steps include: (A) Resuspend the nuclei in resuspension buffer and add salt solution to make the final salt concentration in the lysis system 1.5-2 mol / L; (B) adding dextran and polyethylene glycol and mixing them evenly, so that the final concentration of polyethylene glycol in the system is 5-10%, and the final concentration of dextran is 3-5%; (C) The supernatant was centrifuged and subjected to cation exchange chromatography, and histones were eluted in one step using elution buffer. Optionally, the resuspension buffer comprises 10-20 mmol / L Tris, HEPES, PB or MOPS buffer, with a pH of 7.0-7.6; Optionally, at least 2.5 mL of the resuspension buffer is required per 1 g of the cell nuclei.

9. Histone prepared according to the method according to any one of claims 1 to 8.

10. Use of the histone according to claim 9 in any one of (I) to (III): (I) Detection of anti-histone antibodies for non-therapeutic or diagnostic purposes; (II) Preparation of products for detecting anti-histone antibodies; (III) Preparation of products for diagnosing anti-histone antibody positive diseases.

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