A high-purity small molecule fish sperm DNA-Na raw material and preparation method

Abstract

The present invention belongs to the field of nucleotide and biochemical technology, and specifically relates to a high-purity small molecule fish sperm DNA-Na raw material and a preparation method. The protein content in the fish sperm DNA-Na raw material is less than 0.05%, and the content of deoxyribonucleotides below 20kDa is not less than 80%. The fish sperm DNA-Na raw material is prepared by fish sperm through fish sperm pretreatment, DNA fragmentation, deproteinization and freeze-drying steps, wherein the deproteinization does not use SDS-NaCl treatment and ethanol precipitation, thereby improving the level of green industrialization. The fish sperm DNA-Na raw material obtained by the preparation process has a low protein content, which can reduce the immunogenicity caused by heterologous proteins; the proportion of low molecular weight nucleic acids is high, which is conducive to further improving skin permeability. The product has good application value in medical beauty fields such as skin health maintenance and damage repair.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleotides and biochemistry, and particularly relates to a high-purity small-molecule fish sperm DNA-Na raw material and a preparation method thereof. Background Art

[0002] DNA-Na, also known as polydeoxyribonu-cleo-tide (PDRN), is a low molecular weight DNA derivative of natural origin with a molecular weight ranging from 50 to 1500 kDa. The most representative molecular weight of DNA-Na is 80 to 200 kDa, and its base length is 50 to 2000 bp. It is a linear polymer of deoxyribonucleotides with phosphodiester bonds. The DNA-Na structure contains double-stranded deoxyribonucleotides, and the two polydeoxyribonucleotides are connected by hydrogen bonds between base pairs (adenine (A) = thymine (T); guanine (G) = cytosine (C)), eventually forming a double helix structure.

[0003] DNA-Na is mainly extracted and purified from the sperm cells of salmon (Oncorhynchus keta) or rainbow trout (Oncorhynchus mykiss). The base composition of DNA-Na extracted from the seminal vesicles of salmon or rainbow trout is as similar as 98% to human DNA, which obviously can explore its potential medical use. Based on this factor, its clinical application scope has gradually expanded, and it has shown obvious effects in tissue repair, wound healing, anti-ischemia, anti-inflammation and other aspects. In vitro and in vivo experiments have shown that the most relevant mechanism of action of DNA-Na is as an agonist of adenosine A2A receptors, activating A2A receptors and producing a variety of physiological effects. In addition to activating adenosine A2A receptors, DNA-Na also provides nucleosides and nucleotides for the so-called "salvage pathway".

[0004] Commonly used DNA-Na is mainly the product of small fragments after the degradation of macromolecular DNA. DNA-Na of different molecular weights has different application fields. Generally, DNA-Na of 700KDa or above is used for skin regeneration, joint cartilage regeneration and bone formation promotion, 350~500kDa is used in pharmaceutical treatment to achieve tissue regeneration, corneal regeneration and treatment of musculoskeletal diseases, and 40~60kDa (<60kDa) is used in cosmetics to achieve the effects of repairing barriers, improving wrinkles and skin beauty, etc. DNA-NA of smaller molecular weight has better skin permeability and is more easily absorbed by the epidermis when applied, but there are few reports on the extraction and purification technology of small molecular weight DNA-NA.

[0005] CN201911191251.1 provides a method for preparing small molecule DNA-Na (PDRN), which uses a DNA shearing instrument and requires repeated low-temperature treatment such as 0°C ethanol precipitation, which is not suitable for industrial production; the molecular weight of its DNA-Na (PDRN) is about 50~500bp (calculated based on the average molecular weight of DNA base pairs of 650Da, the molecular weight range of its DNA-Na is about 32.5kDa~325kDa), and the molecular weight distribution is large. CN202011188974.9 provides a method for preparing small molecule DNA-Na (PDRN), which uses endonucleases to control the molecular weight of the product. The molecular weight range of DNA-Na (PDRN) can be concentrated in 50~200bp (32.5kDa~130kDa), but the cost of the enzyme digestion method using endonucleases is too high and is not suitable for large-scale production. CN201610497384.1 uses saturated sodium chloride combined with ultrasonic treatment and ethanol precipitation to prepare DNA-Na(PDRN), which is not suitable for large-scale production; the molecular weight of the obtained DNA-Na(PDRN) is about 100~300 bp (65kDa~195kDa).

[0006] Generally speaking, the enzymatic digestion and high temperature methods used in the current traditional processes for extracting small molecule DNA-Na have high industrial costs, or are only suitable for small-batch production in the laboratory and are not suitable for large-scale industrial production. The deproteinization step is also relatively complicated, involving the use of a large amount of organic reagents such as sodium dodecyl sulfate (SDS) and ethanol, which can easily lead to industrial pollution and does not meet the requirements of green production. The traditional method of extracting and purifying DNA-Na generally controls the purity of the prepared DNA-Na active ingredient to no less than 90%, and often ignores the control of the content of impurities, because when precipitation technology is generally used to separate DNA and large proteins, the curling of the higher-level structure of the DNA fragment often forms a package for water-soluble polypeptides and small proteins. When this kind of product containing more impurities is used in the field of damaged skin repair and injection beauty, the immunogenic reaction caused by the impurities is very harmful, ranging from skin redness, swelling and inflammation to severe allergic reactions, endangering the life of the user. Therefore, it is urgent to improve the safety of the process and the extraction efficiency, increase the control of impurity content, remove pharmacologically active proteins and peptides as much as possible, ensure the safety of the product, and reduce the possibility of immune side effects. At the same time, for skin beauty cosmetics and food DNA-Na that repair the barrier and improve wrinkles, effectively obtaining DNA-Na less than 60kDa is also a basic requirement for its product function. Summary of the invention

[0007] In view of the above problems in the prior art, the object of the present invention is to provide a high-purity small molecule fish sperm DNA-Na raw material and a preparation method.

[0008] The technical solution of the present invention is as follows:

[0009] A high-purity small molecule fish sperm DNA-Na raw material, wherein the fish sperm DNA-Na raw material is prepared from fish testes, wherein the protein content of the fish sperm DNA-Na raw material is less than 0.05% by mass as a freeze-dried product; and the content of deoxyribonucleotides below 20 kDa in the fish sperm DNA-Na raw material is not less than 80%.

[0010] A method for preparing the above-mentioned high-purity small molecule fish sperm DNA-Na raw material comprises the following steps:

[0011] S1 fish spermatozoa pretreatment: The fish spermatozoa were frozen and crushed, and cleaved and enzymatically hydrolyzed using Tris-water solution, sodium hydroxide solution, and trypsin to obtain a pretreatment mixture;

[0012] S2 DNA fragmentation: Add Tris-water solution and sodium hydroxide solution to the pre-treatment mixture, control the temperature at 4°C to 30°C, and perform ultrasound at 20KHz for 30 to 60 minutes to obtain a DNA fragmentation treatment mixture;

[0013] S3 deproteinization treatment: the DNA fragmentation treatment mixture is centrifuged to remove insoluble matter, the pH is adjusted to 7.5-8.0, and then centrifuged again to remove precipitation, and the supernatant is taken as the pretreatment solution; the pretreatment solution is filtered through a 1 μm filter membrane, added to a nanofiltration treatment system, and passed through a 5 kDa ultrafiltration membrane to prepare a DNA-Na raw material crude solution; the DNA-Na raw material crude solution is taken and treated with hydrophobic chromatography to obtain a high-purity small molecule fish sperm DNA-Na raw material solution;

[0014] S4 freeze-drying: Take the high-purity small molecule fish sperm DNA-Na raw material liquid and freeze-dry it to collect the dry powder as the high-purity small molecule fish sperm DNA-Na raw material.

[0015] Preferably, in step S1, the pH of lysis and enzymatic hydrolysis is 7.5-8.0.

[0016] Preferably, in step S1, the amount of trypsin used is not less than 250 KU / L, and the enzymatic hydrolysis conditions are stirring and digesting at 37° C. for 24 hours.

[0017] Preferably, in step S2: ultrasonic treatment is performed for 40 minutes with a 1 second pause after every 5 seconds; and the pH of the pre-treatment mixed solution is adjusted to 7.5-8.0 before the ultrasonic treatment.

[0018] Preferably, in step S3: the DNA fragmentation treatment mixture is first adjusted to pH 8.5-9.0 and centrifuged at a centrifugal speed of 8000 rpm; then the pH is adjusted to pH 7.5-8.0, and then allowed to stand for 8 hours and centrifuged again to remove the precipitate.

[0019] Preferably, in step S3: in the preparation of the crude DNA-Na raw material solution, after filtering with a 1 μm filter membrane, first adjust the pH of the pretreatment liquid to 8.5-9.0, adjust the membrane inlet pressure of the nanofiltration treatment system to <0.2 MPa, control the temperature to 20°C-25°C, and the circulation time to not less than 4 hours to prepare the first filtrate; then prepare a 20 mmol / L sodium dihydrogen phosphate solution with a pH of 7.0 as a top wash liquid, add the nanofiltration system, and circulate the top wash until the volume is less than one-sixth of the original volume to prepare the second filtrate; the first filtrate and the second filtrate are combined and adjusted to pH 7.5-8.0 as the crude DNA-Na raw material solution.

[0020] Preferably, the hydrophobic chromatography separation conditions in step S3 are:

[0021] Stationary phase: butyl hydrophobic chromatographic filler; sample loading: no more than 2BV;

[0022] Loading speed: linear speed 0.5 cm / min, elution speed: linear speed 1 cm / min;

[0023] Mobile phase A: 100 mmol NaCl solution;

[0024] Mobile phase B: purified water;

[0025] Mobile phase C: 20mmol potassium hydrogen phosphate solution;

[0026] Mobile phase D: 0.1 mol / L sodium hydroxide aqueous solution;

[0027] Flow rate: 8BV / h.

[0028] Preferably, the elution procedure of the hydrophobic chromatography separation is: gradient setting: pre-equilibrium (mobile phase A: mobile phase B = 1:1) 2BV→ loading→ post-equilibrium (mobile phase A: mobile phase B = 1:1) 3BV→ elution one (mobile phase A: mobile phase B = 1:2) 2BV→ elution two (mobile phase A: mobile phase B: mobile phase C = 1:2:1) 2BV→ elution three (mobile phase A: mobile phase B: mobile phase C = 1:1:2) 1BV→ elution four (mobile phase B: mobile phase D = 5:1) 2BV; collect the fractions obtained from elution four.

[0029] Preferably, the freeze-drying procedure of step S4 is:

[0030] Pre-freezing temperature: -40℃, pre-freezing time: 2h; collector temperature: -60℃; heating rate: 1℃ / min; heating gradient: -25℃ for 10h, -10℃ for 10h, 15℃ for 10h, 35℃ for no less than 20h.

[0031] Beneficial effects of the present invention:

[0032] The high-purity small molecule fish sperm DNA-Na raw material of the present invention has the advantages of low protein impurity content, high content of deoxyribonucleotides below 20 kDa and concentrated molecular weight distribution. The protein impurity content is less than 0.05%, which can greatly reduce the risk of immunogenicity induced by heterologous proteins. The low molecular weight and concentrated molecular weight distribution of deoxyribonucleotides are conducive to improving percutaneous absorption, and are conducive to playing its role in repairing barriers, improving wrinkles and skin beauty.

[0033] The method for preparing high-purity small molecule fish sperm DNA-Na raw material of the present invention has the following advantages:

[0034] (1) In terms of protein impurity removal, the existing technology overcomes the need to use sodium dodecyl sulfate (SDS)-NaCl and ethanol precipitation or even ethanol precipitation below 0°C, reducing the amount of organic solvents, improving the green level of large-scale industrial preparation, reducing industrial pollution, and the protein / peptide impurity content is less than 0.05%. After enzyme treatment, the centrifuged sample is first filtered with a microporous membrane to remove insoluble matter, and then treated with an ultrafiltration membrane to remove most of the insoluble matter such as large molecular proteins, peptide nucleic acids, polysaccharides, or polysaccharide polymers.

[0035] (2) In terms of DNA-Na molecular weight control, the molecular weight and molecular weight distribution of fish sperm DNA-Na raw materials were effectively controlled by means of multi-parameter control measures such as pH, temperature control, and ultrasonic conditions, and the use of expensive reagents such as endonucleases and non-large-scale industrial equipment such as DNA fragmentation instruments were avoided, making the product molecular weight and molecular weight distribution unexpectedly exceed the level of existing technologies.

[0036] (3) The hydrophobic chromatography system can mount the target DNA-Na on the stationary phase, use a high salt system to elute to remove soluble protein peptides, and then use an alkaline water mobile phase to elute the target. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the protein content detection results of the BCA method in Example 8;

[0038] Figure 2 This is a graph showing the detection results of the nucleic acid molecular weight distribution in Example 9. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific embodiments. The following examples are only exemplary cases of the present invention and are not to be regarded as limiting the scope of protection. In the fragmentation of DNA fragments, the temperature control range during ultrasound is 4°C to 30°C, preferably 10°C to 30°C.

[0040] Example 1 Pretreatment of fish sperm cells

[0041] Step 1: Take frozen and broken rainbow trout (Oncorhynchus mykiss) testis, add 2 times the volume of pre-prepared 40mmol / L Tris-water solution to disperse and mix, add 0.2% (mass volume ratio) of Triton-100, and then add 0.5mol / L sodium hydroxide solution to adjust the pH to 7.5 and stir for 8h.

[0042] Step 2: Use a crusher to grind the material from the previous step into a slurry, adjust the pH to 7.5 with 0.5 mol / L sodium hydroxide solution again, add trypsin solution (the amount added in terms of activity units is not less than 250 KU / L, 250 KU / L here), adjust the temperature to 37°C, and stir to digest for 24 hours.

[0043] Example 2 Fragmentation of DNA fragments

[0044] After digestion was completed as in Example 1, a milky white turbid liquid with a pungent odor was obtained. Without centrifugation, an equal volume of 40mmol / L Tris-water solution was added again for mixing, 0.1% (by mass volume ratio) of Triton-100 was added, and then 0.5mol / L sodium hydroxide solution was added to adjust the pH to 7.5. The above solution was transferred to an ultrasonic crusher, the temperature was controlled at 20°C±5°C (i.e., the temperature control accuracy was ±5°C), the frequency was 20KHz, and the ultrasonic treatment was performed for 5s and 1s, with a total treatment time of 40min.

[0045] Example 3 Separation of DNA and protein

[0046] Step 1. Example 2: After the crushing treatment is completed, add 0.5 mol / mol sodium hydroxide solution to adjust the pH to 8.5, stir thoroughly for 20 min, transfer to disc centrifuge, set the liquid flow rate to 0.1 m³ / h, set the slag discharge cycle to 400 s, the speed to 8000 rpm, centrifuge to remove insoluble matter, take a sample of the supernatant and centrifuge at 12000 rpm for 3 min without obvious precipitation visually, which is qualified.

[0047] Step 2: The pH of the supernatant was adjusted to 7.5, and the supernatant was allowed to stand for 8 hours to precipitate part of the water-soluble protein again, and the precipitate was removed by centrifugation (this intermediate product was named: centrifuge fluid).

[0048] Example 4 Membrane treatment of separation liquid

[0049] Step 1: The centrifugal supernatant obtained in Example 3 was filtered through a 1 μm filter membrane, added to a nanofiltration treatment system, passed through a 5 kDa ultrafiltration membrane, the membrane inlet pressure was <0.2 MPa, condensed water was passed to control the temperature at 20°C~25°C, the pH was adjusted to 8.5, the circulation time was not less than 4 h (here 4 h), and the filtrate was prepared for use.

[0050] Step 2, prepare a 20 mmol / L sodium dihydrogen phosphate solution with a pH of 7.0 as a top wash solution (the amount is one-fourth of the treatment amount in step 1 of this embodiment), add it to the nanofiltration system, circulate the top wash until the volume is less than one-sixth of the original volume, combine the obtained filtrate with the filtrate in step 1 of this embodiment, and adjust the pH to 7.5, at this time, a DNA-Na product solution from which most of the protein has been removed is obtained (this intermediate is named: crude product solution).

[0051] Example 5 Column chromatography treatment of DNA-Na

[0052] Step 1: Add an equal volume of 20 mmol / L sodium chloride solution to the filtrate (crude product solution) obtained in Example 4 to observe whether a precipitate is precipitated. If a precipitate is precipitated, filter it with a 1 μm filter membrane.

[0053] Step 2: Column chromatography separation, the specific parameters are as follows:

[0054] Stationary phase: Butyl hydrophobic chromatography packing, model example: Proteomix®HIC Butyl;

[0055] Column efficiency: Theoretical plate number is not less than 6000 / m;

[0056] Sample volume: no more than 2BV

[0057] Loading speed: linear speed 0.5 cm / min, elution speed: linear speed 1 cm / min;

[0058] Mobile phase A: 100mmol NaCl solution

[0059] Mobile phase B: purified water

[0060] Mobile phase C: 20mmol potassium hydrogen phosphate solution

[0061] Mobile phase D: 0.1 mol / L sodium hydroxide aqueous solution

[0062] Flow rate: 8BV / h

[0063] Gradient setting: pre-equilibrium (mobile phase A: mobile phase B = 1:1) 2BV→ loading → post-equilibrium (mobile phase A: mobile phase B = 1:1) 3BV→ elution one (mobile phase A: mobile phase B = 1:2) 2BV→ elution two (mobile phase A: mobile phase B: mobile phase C = 1:2:1) 2BV→ elution three (mobile phase A: mobile phase B: mobile phase C = 1:1:2) 1BV→ elution four (mobile phase B: mobile phase D = 5:1) 2BV;

[0064] Collect the eluted fractions;

[0065] Step 3: Column chromatography post-treatment

[0066] The chromatographic column treated in the previous step was eluted again with mobile phase D for 2 BV, the obtained fractions were collected, combined with elution phase 4, and the pH was adjusted to 7.5 to obtain a refined solution.

[0067] Note: In this example, BV means times volume.

[0068] Example 6 Lyophilization of fish sperm DNA-Na

[0069] The refined solution obtained in Example 5 was loaded into a flat-plate freeze dryer and the freeze drying process was performed according to the following parameters:

[0070] Pre-freezing temperature: -40℃, pre-freezing time: 2h;

[0071] Trap temperature: -60℃;

[0072] Heating rate: 1℃ / min;

[0073] Temperature gradient: -25℃ for 10h, -10℃ for 10h, 15℃ for 10h, 35℃ for no less than 20h (here 20h);

[0074] Freeze-drying endpoint judgment: pressure rise test result, the pressure rise rate does not exceed 1.0pa / 30S;

[0075] After lyophilization, the dry powder is collected to obtain the DNA-Na product with impurities fully removed.

[0076] Example 7: Protein content detection of products by ninhydrin method

[0077] In order to determine the protein content removal effect of the product of Example 6, the protein content was detected by the ninhydrin method.

[0078] Step 1: Preparation of ninhydrin stock solution: Dissolve 25 g of ninhydrin in 100 ml of anhydrous ethanol;

[0079] Step 2: Preparation of standard solution: quantitatively weigh glycine hydrochloride and dissolve it in water to prepare a 10.0 mg / ml solution;

[0080] Step 3, sample solution preparation: quantitatively weigh the DNA-Na product of Example 6 and prepare a 0.01 mg / ml solution;

[0081] Step 4: Colorimetric method: Use a pipette to take 200 μl of each of the standard solution, crude solution and sample solution, drop them on a silica gel plate, add 100 μl of ninhydrin stock solution to each, place the silica gel plate in an oven at 120°C for 5 min, observe the color development, the sample group should not be darker than the standard solution group, at this time the content of impurity protein / peptide in the sample is less than 0.1%;

[0082] Example 8 Product protein content detection by BCA method

[0083] In order to determine the protein content removal effect of the product, the BCA method protein content test was carried out.

[0084] In addition to the sample prepared in Example 6 (sample batch 1), two more batches of samples were prepared according to the methods of Examples 1-6.

[0085] Sample batch 2: prepared according to the method of Examples 1-6, except that:

[0086] In the pretreatment of fish sperm cells: in step 1, take the frozen and broken fish spermatozoa, add 3 times the volume of the pre-prepared 40mmol / LTris-water solution to disperse and mix; adjust the pH to 8.0 with sodium hydroxide solution; in step 2, adjust the pH to 8.0 with sodium hydroxide solution, and add 450KU / L of trypsin solution in terms of activity units.

[0087] During the fragmentation of DNA fragments: adjust the pH value of the sodium hydroxide solution to 8.0; control the temperature to 25°C ± 5°C (i.e., the temperature control accuracy is ± 5°C).

[0088] In the separation of DNA and protein: in step 1, the pH of the sodium hydroxide solution is adjusted to 9.0, and in step 2, the pH of the supernatant is adjusted to 8.0.

[0089] In the membrane treatment of the separation liquid: in step 1, the pH is adjusted to 9.0, and in step 2, the filtrate is combined and the pH is adjusted to 8.0.

[0090] In the column chromatography treatment of DNA-Na: adjust the pH to 8.0 in step 3.

[0091] Sample batch 3: Prepared according to the method of sample batch 2, except that:

[0092] In the pretreatment of fish sperm cells: the trypsin solution is added in an amount of 650 KU / L in terms of activity units.

[0093] In the column chromatography treatment of DNA-Na: in step 1, 0.45 μm filter membrane is used for filtration.

[0094] Step 1. Take the BCA kit for use. Example model: BlueKit BCA kit, including standard protein, A solution, B solution, and sample lysis solution (the sample lysis solution of the kit is not required for this experiment);

[0095] Step 2: Preparation of BCA working solution: Take out solution A and solution B from the kit, mix them evenly in a ratio of 50:1, and set aside;

[0096] Step 3, sample solution preparation: quantitatively weigh the DNA-Na products of sample batches 1 to 3 and prepare them into 0.01 mg / ml solution; separately take the crude product solution obtained in the preparation of sample batches 1 to 3 (obtained after separation liquid membrane treatment) and prepare the lyophilized product according to the method of Example 6, and prepare it into 0.01 mg / ml solution as a control drug.

[0097] Step 4: Add 1, 2, 5, 10, 50 μl of standard protein, 50 μl of sample solution, 50 μl of crude solution to a 96-well plate, add 200 μl of BCA working solution to each well, and incubate at room temperature for 20 min;

[0098] Step 4. Detection: Set the wavelength of the microplate reader to 562nm, measure the absorbance value of each well, and calculate the protein concentration of the sample solution using the standard protein regression equation curve, which must not exceed 0.1%;

[0099] Measurement results:

[0100] The results are as follows Figure 1 The results show that the protein content of three batches of samples, sample batches 1 to 3, was less than 0.05% (mass percentage). Compared with the samples without hydrophobic chromatography treatment (crude solution freeze-dried product control), the protein content was significantly reduced, achieving the goal of basically completely removing the protein. The protein content of the samples without hydrophobic chromatography treatment (crude solution) was about 4% to 5%, while the protein content of the corresponding samples of sample batches 1 to 3 was less than 0.05%. Figure 1 In order to facilitate display and comparison, the vertical axis uses proportional coordinates rather than equidistant coordinates.

[0101] Example 9 Detection of product nucleic acid molecular weight distribution

[0102] The molecular weight distribution of the sample in Example 6 (i.e., sample batch 1) was determined using size exclusion chromatography, and the chromatographic conditions were as follows:

[0103] Column: Waters Insulin HMWP, 10 μm, 7.8 mm × 300 mm

[0104] Mobile phase: Prepare 500 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 5 mM phosphoric acid, and 50% (v / v) 2-propanol solution. Specifically, dissolve 146.1 g sodium chloride (NaCl) and 6.9 g sodium dihydrogen phosphate hydrate (NaH2PO4, H2O) in about 2000 ml water and 1.7 ml concentrated phosphoric acid (H3PO4). Add 2500 ml 2-propanol. Add water to 5000 ml and mix well.

[0105] Chromatographic conditions: flow rate 0.5 ml / min, detection wavelength 260 nm, isocratic elution;

[0106] Test results: The product of sample batch 1 was prepared into a 0.1 g / ml concentration sample, filtered through a 0.22 μm microporous membrane, and injected together with 5 kDa, 10 kDa, 20 kDa, and 50 kDa molecular weight standards. Figure 2 As shown in the figure, the target chromatographic peak detected in the range above 50kDa is less than 3%, and in the range below 20kDa it reaches 86.9%, of which the DNA content in the range of 5kDa~20kDa reaches 85%. Compared with the centrifuge solution and the crude solution, the nucleic acid molecules with a molecular weight greater than 50kDa are basically completely removed, and the proportion of DNA molecules in the range of 5kDa~20kDa is greatly increased.

[0107] The same method was used to measure sample batches 2 and 3 prepared in Example 8. The DNA content of the two batches of samples was less than 3% in the range above 50 kDa, and 87.2% and 89.4% below 20 kDa, respectively. Since the products prepared by this method have a high DNA content below 20 kDa, especially 5-20 kDa, it is conducive to transdermal effect, and thus more suitable as skin beauty cosmetics for repairing barrier and improving wrinkles.

[0108] Assuming the total nucleic acid content accounts for 1% of the cell dry weight and the feed testis cell dry weight accounts for 10%,

[0109] The theoretical yield calculation formula is as follows: yield = product quality / (testicular cell feed amount*10%*1%)*100%.

[0110] The feed amount of Example 1 is 40Kg, the output of the product of Example 6 is 16.7g, and the yield of this method is calculated to be 41.75%.

Claims

1. A high-purity small molecule fish sperm DNA-Na raw material, characterized in that: The fish sperm DNA-Na raw material is prepared by fish sperm, the protein content of the fish sperm DNA-Na raw material is less than 0.05% by mass percentage in terms of freeze-dried product; the content of deoxyribonucleotides below 20 kDa in the fish sperm DNA-Na raw material is not less than 80%; the preparation method of the fish sperm DNA-Na raw material comprises the following steps: S1 fish testis pretreatment: fish testis is frozen and crushed, and cleaved and enzymolyzed with Tris-water solution, sodium hydroxide solution and trypsin to obtain a pretreatment mixture; S2 DNA fragmentation: Add Tris-water solution and sodium hydroxide solution to the pre-treatment mixture, control the temperature at 4°C to 30°C, and perform ultrasound at 20KHz for 30 to 60 minutes to obtain a DNA fragmentation treatment mixture; S3 deproteinization treatment: the DNA fragmentation treatment mixture is centrifuged to remove insoluble matter, the pH is adjusted to 7.5-8.0, and then centrifuged again to remove precipitation, and the supernatant is taken as the pretreatment solution; the pretreatment solution is filtered through a 1 μm filter membrane, added to a nanofiltration treatment system, and passed through a 5 kDa ultrafiltration membrane to prepare a DNA-Na raw material crude solution; the DNA-Na raw material crude solution is taken and treated with hydrophobic chromatography to obtain a high-purity small molecule fish sperm DNA-Na raw material solution; S4 freeze drying: taking the high-purity small molecule fish sperm DNA-Na raw material liquid and freeze drying it to collect the dry powder as the high-purity small molecule fish sperm DNA-Na raw material; In the step S1: the pH of the lysis and enzymatic hydrolysis is 7.5-8.0; In the step S1: the amount of trypsin used is not less than 250 KU / L, and the enzymatic hydrolysis conditions are stirring and digesting at 37° C. for 24 hours; The hydrophobic chromatography separation conditions in step S3 are: Stationary phase: butyl hydrophobic chromatographic filler; sample loading: no more than 2BV; Loading speed: linear speed 0.5 cm / min, elution speed: linear speed 1 cm / min; Mobile phase A: 100 mmol NaCl solution; Mobile phase B: purified water; Mobile phase C: 20mmol potassium hydrogen phosphate solution; Mobile phase D: 0.1 mol / L sodium hydroxide aqueous solution; Flow rate: 8BV / h; The elution procedure of the hydrophobic chromatography separation is as follows: gradient setting: pre-equilibrium (mobile phase A: mobile phase B = 1:1) 2BV→ loading→ post-equilibrium (mobile phase A: mobile phase B = 1:1) 3BV→ elution one (mobile phase A: mobile phase B = 1:2) 2BV→ elution two (mobile phase A: mobile phase B: mobile phase C = 1:2:1) 2BV→ elution three (mobile phase A: mobile phase B: mobile phase C = 1:1:2) 1BV→ elution four (mobile phase B: mobile phase D = 5:1) 2BV; The eluted fractions were collected.

2. A high-purity small molecule fish sperm DNA-Na raw material according to claim 1, characterized in that: In the step S2, ultrasonic treatment was performed for 40 minutes with a 1 second pause after every 5 seconds, and the pH of the pre-treatment mixed solution was adjusted to 7.5-8.0 before ultrasonic treatment.

3. A high-purity small molecule fish sperm DNA-Na raw material according to claim 1, characterized in that: In the step S3: the DNA fragmentation treatment mixture is first adjusted to pH 8.5-9.0 and centrifuged at a centrifugal speed of 8000 rpm; then the pH is adjusted to pH 7.5-8.0, and then allowed to stand for 8 hours and centrifuged again to remove the precipitate.

4. A high-purity small molecule fish sperm DNA-Na raw material according to claim 1, characterized in that: In the step S3: in the preparation of the crude DNA-Na raw material solution, after filtering with a 1 μm filter membrane, firstly adjust the pH of the pretreatment liquid to 8.5-9.0, adjust the membrane inlet pressure of the nanofiltration treatment system to less than 0.2 MPa, control the temperature to 20° C.-25° C., and the circulation time to not less than 4 hours to prepare the first filtrate; then prepare a 20 mmol / L sodium dihydrogen phosphate solution with a pH of 7.0 as a top wash liquid, add the solution to the nanofiltration system, circulate the top wash until the volume is less than one-sixth of the original volume, and prepare the second filtrate; combine the first filtrate and the second filtrate to adjust the pH to 7.5-8.0 as the crude DNA-Na raw material solution.

5. A high-purity small molecule fish sperm DNA-Na raw material according to claim 1, characterized in that: The freeze-drying procedure of step S4 is: Pre-freezing temperature: -40℃; pre-freezing time: 2h; collector temperature: -60℃; heating rate: 1℃ / min; heating gradient: -25℃ for 10h, -10℃ for 10h, 15℃ for 10h, 35℃ for no less than 20h.

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