Pharmaceutical grade pdrn and a method of preparation thereof

Abstract

The application discloses a pharmaceutical-grade PDRN and a preparation method thereof, and belongs to the technical field of biotechnology. The method comprises the following steps: (1) grinding salmon testis tissue in a pre-cooled grinding buffer to obtain a homogenate; (2) using Forskolin to induce the grinding homogenate; the induction conditions are as follows: first, adding Forskolin to 8-12 muM, and standing for 1-3 hours at 4 DEG C; then, increasing the temperature to 10-20 DEG C, adding Forskolin to 18-22 muM and 2 mM glutathione, and treating for 2 hours; and (3) extracting the PDRN through SDS-sodium chloride.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pharmaceutical-grade PDRN and its preparation method. Background Technology

[0002] Polydeoxyribonucleotides (PDRN) are bioactive substances composed of specific deoxyribonucleotide fragments. They have the effects of promoting tissue repair, anti-inflammation, regenerative medicine and skin barrier repair, and are widely used in medical aesthetics, wound healing and chronic disease treatment.

[0003] Current mainstream technologies using human chorionic gonadotropin (hCG) to induce sperm maturation in salmon testes carry significant risks: 1. Biosafety risks: hCG must be obtained from pregnant women's urine or through gene recombination expression, posing a risk of human viral contamination (such as HIV and HBV). This necessitates additional virus inactivation steps (S / D treatment or nanofiltration), resulting in a 15%–20% reduction in PDRN yield. The EU EMA guidelines (CPMP / BWP / 268 / 95) require TSE / BSE risk assessments for animal-derived products, placing additional regulatory scrutiny on the hCG method. 2. Hormone residue side effects: hCG possesses luteinizing hormone (LH) activity; a residual level ≥0.1 IU / mg in the final product can trigger endocrine disorders in humans. The FDA's limits on residual hormones in pharmaceutical excipients are becoming increasingly stringent (21 CFR 610.9), and traditional processes require additional immunoaffinity chromatography, increasing costs. 3. Ethical and compliance obstacles.

[0004] While forskolin, as a cAMP pathway activator, is theoretically feasible, its industrial application in fish sperm induction faces three major technical barriers: 1. Species-specificity barrier: effective concentrations (10-50 μM) in mammals are significantly toxic to salmon testicular cells (apoptosis rate >40%); 2. Poor process compatibility: traditional grinding causes cell membrane damage, preventing forskolin from effectively penetrating to the target site; 3. Increased oxidative stress: mechanical processing generates reactive oxygen species (ROS), which counteract the effects of cAMP signal transduction.

[0005] Therefore, it is crucial to develop a hormone-free, highly safe, and low-cost PDRN large-scale preparation process that breaks through the technical and ethical bottlenecks of traditional hCG methods through a triple technological innovation of low-temperature grinding protection, sequential Forskolin induction, and antioxidant synergy. Summary of the Invention

[0006] The purpose of this invention is to provide a pharmaceutical-grade polydeoxyribonucleotide (PDRN) and its preparation method to solve the problems existing in the prior art. This invention provides a method for preparing ultra-low-risk pharmaceutical-grade PDRN by inducing in vitro maturation of salmon testicular cells using the plant-derived adenylate cyclase activator Forskolin, and by optimizing mechanical grinding and purification processes. This method eliminates hormonal substances, meeting the stringent safety requirements for raw materials in regenerative medicine products.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] One of the technical solutions of this invention is a method for preparing pharmaceutical-grade PDRN, comprising the following steps:

[0009] (1) The salmon testis tissue was ground in pre-cooled grinding buffer to obtain a homogenate;

[0010] (2) Induction of the grinding homogenate with Forskolin; the induction conditions are as follows: first add Forskolin to 8-12 μM, let stand at 4°C for 1-3 hours; then raise the temperature to 10-20°C, add Forskolin to 18-22 μM and 2 mM glutathione, and treat for 2 hours.

[0011] (3) PDRN was obtained by extraction with SDS-sodium chloride.

[0012] The second technical solution of the present invention is a pharmaceutical-grade PDRN prepared by the preparation method.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] (1) Safety breakthrough: Zero hormone residue: Forskolin not detected by HPLC (<0.1ppm), hCG residue <0.01IU / ml (ELISA); No pathogen risk: Plant-derived inducers avoid the risk of hormone residue.

[0015] (2) The production cycle of pharmaceutical-grade PDRN prepared by the present invention is shortened. Forskolin induction requires hours, while hCG method requires 24 hours. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1The HPLC chromatogram shows the content of Forskolin (standard: Forskolin standard, sample: Forskolin residue in PDRN obtained in Example 1, negative control: solvent control). Detailed Implementation

[0018] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0024] This invention provides a method for preparing pharmaceutical-grade PDRN, comprising the following steps:

[0025] (1) The salmon testis tissue was ground in pre-cooled grinding buffer to obtain a homogenate;

[0026] (2) Induction of the grinding homogenate with Forskolin; the induction conditions are as follows: first add Forskolin to 8-12 μM, let stand at 4°C for 1-3 hours; then raise the temperature to 10-20°C, add Forskolin to 18-22 μM and 2 mM glutathione, and treat for 2 hours.

[0027] (3) PDRN was obtained by extraction with SDS-sodium chloride.

[0028] In some specific embodiments, the pre-cooled grinding buffer consists of 5%–10% BSA, 0.5–2 mM EDTA, and 10 mM ascorbic acid, at a temperature of 4°C.

[0029] In some specific implementations, the grinding conditions are: 5s on / 10s off, grinding time is 30min, and the cell disruption rate is controlled to be ≤30%.

[0030] In some specific implementations, the method for SDS-sodium chloride extraction is as follows:

[0031] (1) Add TE buffer containing 5% SDS to 300g of cell homogenate after induction, bring the volume to 1800ml, heat at 55℃ and stir magnetically for 1 hour to obtain lysis buffer;

[0032] (2) Take 1800ml of lysis buffer, add 50ml of 4M NaCl, and stir magnetically at 1000rpm for 20 minutes to dissolve the DNA;

[0033] (3) Add 1 / 10 volume of 5% SDS solution to the solution, stir until white protein precipitates out, centrifuge at 4500 rpm for 30 min, and collect the filtrate.

[0034] (4) Add 1 / 10 volume of 5% SDS solution to the filtrate, mix well and let stand for 10 min, centrifuge at 4500 rpm for 30 min, and collect the filtrate.

[0035] (5) Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir and mix well, let stand at -20℃ for 30 minutes, and then collect the DNA.

[0036] (6) Add 75% ethanol by volume, shake vigorously, wash thoroughly to remove ethanol, and squeeze out the liquid from the white precipitate;

[0037] (7) Repeat step (5) to obtain a white precipitate.

[0038] This invention also provides pharmaceutical-grade PDRN prepared by the aforementioned preparation method.

[0039] Example 1

[0040] 1. Raw material pretreatment:

[0041] Take 100 kg of immature testes from farmed Atlantic salmon, chop them up, and soak them in a pre-cooled grinding buffer (containing 5% BSA + 1 mM EDTA + 10 mM ascorbic acid, 4℃).

[0042] Intermittent grinding (5s on / 10s off) for 30 minutes using an industrial-grade high-speed homogenizer (12,000 rpm), and microscopic examination confirmed a cell disruption rate of 80%.

[0043] 2. Forskolin induction:

[0044] Stage I: Add Forskolin to 10 μM and let stand at 4°C for 2 hours;

[0045] Phase II: Heat to 15°C, add Forskolin to 20 μM and 2 mM glutathione, and treat for 2 hours.

[0046] 3PDRN extraction:

[0047] 3.1 Pour 300g of induced cell homogenate into a beaker, add TE buffer containing 5% SDS to bring the volume to 1800ml, place on a magnetic stirrer, heat and stir at 55℃ for 1 hour to obtain lysis buffer.

[0048] 3.2 Add 50 ml of 4M NaCl to 1800 ml of the lysis buffer from 3.1, and stir at 1000 rpm for 20 minutes using a magnetic stirrer. At this point, the DNA is in a dissolved state.

[0049] 3.3 Add 1 / 10 volume of 5% SDS solution to the solution and stir for 10 min. White protein will precipitate out. Aliquot the liquid into centrifuge tubes or centrifuge cups and centrifuge at 4500 rpm for 30 min. Pour the supernatant into a sieve to filter out some large tissue fragments and collect the filtrate.

[0050] 3.4 Add 1 / 10 volume of 5% SDS solution to the filtrate, mix with a glass rod, and then let stand for 10 minutes.

[0051] 3.5 Dispense the liquid into centrifuge tubes or centrifuge cups, centrifuge at 4500 rpm for 30 min, pour the supernatant onto a sieve to filter out some large tissue fragments, and collect the filtrate.

[0052] 3.6 Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir well, and let stand in a -20°C refrigerator for 30 minutes. Use a glass rod to scoop the DNA into a 50ml centrifuge tube. Discard the liquid.

[0053] 3.7 Add 10ml of 75% ethanol, then cover the container and shake vigorously up and down to wash thoroughly. Then pour off the ethanol. You can use a glass rod to help squeeze out the liquid from the white precipitate.

[0054] 3.8 Repeat step 3.6, squeeze the liquid out of the white precipitate with a glass rod, then pour it onto absorbent paper to wash away the excess liquid, and obtain a white powder, which is the prepared PDRN.

[0055] 3.9 Place the white precipitate in a beaker, add 100 ml of TE buffer, stir at 50°C and 1000 rpm until the precipitate dissolves.

[0056] 3.10 The concentration of DNA, A260 / A280 and A260 / A230 values ​​were detected and recorded using Nano Drop. The results are shown in Table 1.

[0057] Comparative Example 1

[0058] 1. Raw material pretreatment:

[0059] Take 100 kg of immature testes from farmed Atlantic salmon, chop them up, and soak them in a pre-cooled grinding buffer (containing 5% BSA + 1 mM EDTA + 10 mM ascorbic acid, 4℃).

[0060] Intermittent grinding (5s on / 10s off) for 30 minutes using an industrial-grade high-speed homogenizer (12,000 rpm), and microscopic examination confirmed a cell disruption rate of 80%.

[0061] 2hCG induction:

[0062] Add hCG to a final concentration of 1 ng / ml and let stand at 37°C for 24 hours;

[0063] 3PDRN extraction:

[0064] 3.1 Pour 300g of induced cell homogenate into a beaker, add TE buffer containing 5% SDS to bring the volume to 1800ml, place on a magnetic stirrer, heat and stir at 55℃ for 1 hour to obtain lysis buffer.

[0065] 3.2 Add 50 ml of 4M NaCl to 1800 ml of the lysis buffer from 3.1, and stir at 1000 rpm for 20 minutes using a magnetic stirrer. At this point, the DNA is in a dissolved state.

[0066] 3.3 Add 1 / 10 volume of 5% SDS solution to the solution and stir for 10 min. White protein will precipitate out. Aliquot the liquid into centrifuge tubes or centrifuge cups and centrifuge at 4500 rpm for 30 min. Pour the supernatant into a sieve to filter out some large tissue fragments and collect the filtrate.

[0067] 3.4 Add 1 / 10 volume of 5% SDS solution to the filtrate, mix with a glass rod, and then let stand for 10 minutes.

[0068] 3.5 Dispense the liquid into centrifuge tubes or centrifuge cups, centrifuge at 4500 rpm for 30 min, pour the supernatant onto a sieve to filter out some large tissue fragments, and collect the filtrate.

[0069] 3.6 Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir well, and let stand in a -20°C refrigerator for 30 minutes. Use a glass rod to scoop the DNA into a 50ml centrifuge tube. Discard the liquid.

[0070] 3.7 Add 10ml of 75% ethanol, then cover the container and shake vigorously up and down to wash thoroughly. Then pour off the ethanol. You can use a glass rod to help squeeze out the liquid from the white precipitate.

[0071] 3.8 Repeat step 3.6, squeeze the liquid out of the white precipitate with a glass rod, then pour it onto absorbent paper to wash away the excess liquid, and obtain a white powder, which is the prepared PDRN.

[0072] 3.9 Place the white precipitate in a beaker, add 100 ml of TE buffer, stir at 50°C and 1000 rpm until the precipitate dissolves.

[0073] 3.10 The concentration of DNA, A260 / A280 and A260 / A230 values ​​were detected and recorded using Nano Drop. The results are shown in Table 1.

[0074] Example of effect

[0075] The concentrations of PDRN prepared in Example 1 and Comparative Example 1 are shown in Tables 1 and 2.

[0076] Table 1. Concentration of PDRN extracted after Forskolin induction.

[0077] Concentration (ug / ml) A260 / A280 A260 / A230 Dissolution volume (ml) Total (mg) 2295.08 1.82 1.93 2830 6495.08

[0078] Table 2. Concentrations of PDRN extracted after hCG induction.

[0079] Concentration (ug / ml) A260 / A280 A260 / A230 Dissolution volume (ml) Total (mg) 1908 1.82 1.93 2830 5399

[0080] As shown in Tables 1 and 2, the yield of PDRN induced by Forskolin was higher than that induced by hCG.

[0081] 2. The safety of PDRN prepared in Example 1 and Comparative Example 1 was tested.

[0082] 2.1 Acute oral toxicity test

[0083] SPF-grade Sprague-Dawley (SD) rats, half male and half female, weighing 180-220g, were provided by Vital River Pharmaceuticals Beijing. The experiment consisted of three groups: Example 1 group (5000mg / kg test substance administered by gavage), Comparative Example 1 group (5000mg / kg control substance administered by gavage), and a blank control group (equal volume of physiological saline), with 10 animals in each group (5 males and 5 females). After fasting for 12 hours, the animals were administered the test substance via gavage with physiological saline using a stainless steel gavage needle (16G), with a volume not exceeding 20mL / kg. Mortality, weight change (accuracy ±2g), and signs of neurotoxicity (such as loss of righting reflex, ataxia, and abnormal breathing) were closely monitored at 0.5h, 4h, 24h, 48h, 72h, and day 14. If no deaths and no symptoms of poisoning appeared within 14 days, the LD50 was determined. 50 >5000 mg / kg. Data analysis used Fisher's exact test to assess mortality, and t-tests to analyze differences in body weight change.

[0084] 2.2 Subchronic toxicity test (90-day feeding trial)

[0085] SD rats (40 males and 40 females, weighing 60-80g) were selected and housed in separate cages (5 rats / cage) at an ambient temperature of 22±2℃ and humidity of 50±10%, with a 12-hour light-dark cycle. Example 1 and Comparative Example 1 were administered three doses: low (300mg / kg), medium (1000mg / kg), and high (3000mg / kg), respectively, via gavage daily. The dosage was adjusted weekly based on body weight. Body weight, food intake, and water intake were recorded weekly during the experiment. On days 30, 60, and 90, blood samples were collected from the orbital rim to measure ALT, AST, creatinine, and blood urea nitrogen (using an automated biochemical analyzer). Simultaneously, 24-hour urine samples were collected for protein, glucose, and occult blood testing using test strips. After 90 days, the animals were euthanized under ether anesthesia. The liver, kidneys, spleen, and ovaries / testes were dissected, weighed, and their organ coefficients calculated. The sections were fixed in 10% formalin, embedded in paraffin (4μm), and stained with hematoxylin and eosin (HE) for histopathological evaluation. Ovarian / testicular hyperplasia was semi-quantitatively scored from grade 0 (normal) to grade 2 (structural abnormality), and liver / kidney lesions were recorded with indicators such as fatty degeneration and inflammatory cell infiltration. Data were compared between groups using one-way ANOVA, with a significance threshold set at p < 0.05.

[0086] 2.3 Sensitization test (Guinea pig maximum test, GPMT)

[0087] White guinea pigs (half male and half female, weighing 300-500g), 10 in each group, were used. During the induction phase, after hair removal on the nape of the neck, 0.1 mL of a mixture of the test substance (Example 1 or Comparative Example 1, 10% w / v) and Freund's complete adjuvant (FCA) was injected intradermally. Forty-eight hours later, the area was covered with gauze to prevent licking. Seven days later, 0.05 mL of the test substance was applied to the same area and the area was sealed for 24 hours. Erythema and edema reactions were observed at 24h, 48h, and 72h after challenge, and scored from grade 0 (no reaction) to grade 3 (severe ulceration). The sensitization rate was calculated as (number of positive reactions / total number of cases) × 100%. A threshold of ≥30% was considered strong sensitization, and <30% was considered weak sensitization. The entire experiment was evaluated blinded by two lab technicians, and a Kappa value >0.8 was considered acceptable for consistency.

[0088] The experimental results are shown in Table 3.

[0089] Table 3 Comparison of PDRN Detection Items

[0090]

[0091]

[0092] As shown in Table 3, the results indicate that Example 1 has no obvious toxicity or sensitization, while the comparative example shows certain subchronic toxicity and sensitization.

[0093] 2.4 Determination of Forskolin Content

[0094] Sample preparation

[0095] Positive control: Prepare a Forskolin standard solution (e.g., dissolved in 1 mg / mL methanol), filter it, and inject it to ensure that the peak elution occurs under the set conditions. Record the peak elution time of the standard.

[0096] Sample preparation: Take 1g of extracted PDRN sample, add 20mL of methanol, sonicate for 30 minutes, centrifuge (10,000rpm, 10 minutes), filter the supernatant through a 0.22μm filter membrane, and inject the sample after filtration. Record the peak elution time.

[0097] Negative control: A known aqueous solution that does not contain Forskolin was filtered, injected, and the elution time was recorded.

[0098] The chromatographic parameters are shown in Table 4.

[0099] Table 4

[0100] parameter Setting value chromatographic column Reverse-phase C18 column (e.g., Agilent ZORBAX SB-C18, 250 × 4.6 mm, 5 μm) mobile phase Acetonitrile:water = 50:50 (isocratic elution) or gradient optimization Flow rate 1.0 mL / min Column temperature 30℃ Detection wavelength 220nm (near the maximum absorption wavelength of Forskolin) Injection volume 20μL Runtime 15 minutes (to ensure Forskolin peak time is clearly defined)

[0101] HPLC results showed that the Forskolin content in the PDRN of the example was 0.05 ppm.

[0102] Determination of 2.5hCG content

[0103] ① Before using the Human Chorionic Gonadotropin (hCG) Enzyme-Linked Immunosorbent Assay Kit (Certain Biotechnology Co., Ltd., ELK1364), all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, ensure thorough mixing, avoiding foaming as much as possible. If the sample concentration is too high, dilute with sample diluent to bring the sample within the detection range of the kit.

[0104] ② Add 100 μL of standard or test sample, being careful to avoid air bubbles. When adding the sample, place it at the bottom of the well of the microplate, trying not to touch the well wall. Gently shake to mix. Cover the microplate with a cap or membrane and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, please use a fresh standard solution for each experiment.

[0105] ③ Discard the liquid in the wells, shake off excess water, and wash the plate three times. Wash each well with 200ml of washing buffer, soak for 1-2 minutes, and then shake off the liquid in the microplate (or use a plate washer). After the final wash, pat the plate dry on absorbent paper.

[0106] ④ Add 100 μL of biotin antibody working solution to each well (it can be prepared 15 minutes in advance), cover the plate with a membrane, and incubate at 37°C for 50 minutes.

[0107] ⑤ Discard the liquid in the wells and wash the plate three times. Wash each well with 200u of washing buffer and soak for 1-2 minutes to discard the liquid in the plate (or use a plate washer). After the last wash, pat the plate dry on absorbent paper.

[0108] ⑥ Add 100 μL of enzyme conjugate working solution to each well (it can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes.

[0109] ⑦ Discard the liquid in the wells and wash the plate 5 times. Wash each well with 200 μL of washing buffer, soak for 1-2 minutes, and shake off the liquid in the microplate (or use a plate washer). After the last wash, pat the microplate dry on absorbent paper.

[0110] ⑧ Add 90 mL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (adjust the time according to the actual color development, but do not exceed 30 minutes).

[0111] ⑨ Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be as similar as possible to the order of adding the colorimetric reagent. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time has elapsed.

[0112] ⑩ Immediately measure the optical density (OD) of each well using an ELISA reader at a wavelength of 450 nm. The instrument should be warmed up and the detection program set before use.

[0113] A standard curve was plotted, and the hCG concentration of the resulting example was 0.001 IU / mL, while that of the control example was 0.5 IU / mL.

[0114] The comparison table of concentration and OD value is shown in Table 5.

[0115] Table 5

[0116]

[0117]

[0118] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing pharmaceutical-grade PDRN, characterized in that, Includes the following steps: (1) The salmon testis tissue was ground in pre-cooled grinding buffer to obtain a homogenate; (2) Induction of the grinding homogenate with Forskolin; the induction conditions are: first add Forskolin to 8~12 μM, let stand at 4℃ for 1~3 hours; then raise the temperature to 10~20℃, add Forskolin to 18~22 μM and 2 mM glutathione, and treat for 2 hours; (3) PDRN was obtained by extraction with SDS-sodium chloride; The method for SDS-sodium chloride extraction is as follows: ① Add TE buffer containing 5% SDS to 300g of cell homogenate after induction, bring the volume to 1800ml, heat at 55℃ and stir magnetically for 1 hour to obtain lysis buffer; ② Take 1800ml of lysis buffer, add 50ml of 4M NaCl, and stir magnetically at 1000rpm for 20 minutes to dissolve the DNA; ③ Add 1 / 10 volume of 5% SDS solution to the solution, stir until white protein precipitates out, centrifuge at 4500 rpm for 30 min, and collect the filtrate; ④ Add 1 / 10 volume of 5% SDS solution to the filtrate, mix well, let stand for 10 min, centrifuge at 4500 rpm for 30 min, and collect the filtrate; ⑤ Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir and mix well, let stand at -20℃ for 30 minutes, and then collect the DNA. ⑥ Add 75% ethanol by volume, shake vigorously, wash thoroughly to remove ethanol, and squeeze out the liquid from the white precipitate; ⑦ Repeat step ⑤ to obtain a white precipitate.

2. The preparation method according to claim 1, characterized in that, The pre-cooled grinding buffer consists of 5%–10% BSA, 0.5–2 mM EDTA, and 10 mM ascorbic acid, and is kept at a temperature of 4°C.

3. The preparation method according to claim 1, characterized in that, The grinding conditions are: 5s on / 10s off, grinding time is 30min, and the cell breakage rate is controlled to be ≤30%.

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