CN115960153A - Preparation method and application of low-endotoxin small-molecule PDRN - Google Patents
Preparation method and application of low-endotoxin small-molecule PDRN Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a preparation method of low-endotoxin micromolecule PDRN, belonging to the technical field of biology. The preparation method comprises pretreating testis of Salmonidae, mincing, cracking with alkali cracking solution, filtering the cracked supernatant, adsorbing with active carbon, filtering, separating to obtain supernatant, precipitating with isopropanol to obtain wet solid of PDRN, and freeze drying to obtain the product PDRN. The invention takes the testis of the salmonidae fish as a raw material to obtain functional small-segment PDRN, the endotoxin content is extremely low and is lower than 0.1EU/mg, the purity of the small-molecule PDRN is 1.8-2.0, and the yield reaches 17-18%. Meanwhile, protease with higher cost is not needed, a DNA breaking instrument is not needed, and the method is simple in process, easy to operate and easy to amplify and produce.
Description
Technical Field
The invention relates to a preparation method and application of low-endotoxin small-molecule PDRN, belonging to the technical field of biology.
Background
Small-molecule Polydeoxyribonucleotides (PDRNs) are a kind of active polynucleotide mixture, the molecular weight range is 50-2000 bp, a small amount of PDRNs can be extracted from human placenta or sperm cells, but the raw materials are limited, so the small-molecule Polydeoxyribonucleotides (PDRNs) are mainly extracted from the spermatic fluid or the spermary tissue of salmonidae fish at present. Researches show that PDRN can start various signal paths, increase anti-inflammatory factors, reduce inflammatory factors and inhibit inflammatory reactions by combining with adenosine A2A receptors; purine or pyrimidine can also be provided through a salvage pathway to accelerate DNA synthesis to repair damaged skin; in addition, it can promote osteoblast activity, collagen synthesis and angiogenesis. At present, PDRN is used as a cosmetic and medicine raw material and is increasingly widely applied to the fields of medical cosmetology, cosmetics, medical instruments, health-care food and medicines.
Endotoxin is a generic term for toxic substances present in gram-negative bacteria. Is a cell wall component of various gram-negative bacteria, and is called as a pyrogen after the bacteria are cracked to release toxin. Bacterial endotoxins are composed primarily of phospholipids, lipoproteins, and lipopolysaccharides, the biologically active portion of the endotoxin complex, the toxic component of which is lipid a. Since lipid a structures of different gram-negative bacteria are basically similar, toxic effects caused by infection with such bacteria are almost the same, and they are mainly manifested as fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation and the like. When bacteria die and lyse or autolyze, endotoxin is released in large quantities. For drugs, medical instruments and the like which are liable to introduce endotoxin, endotoxin must be detected by endotoxin. Endotoxin is highly heat-resistant and chemically stable, so that it is difficult to remove or inactivate endotoxin by a general method.
Because the endotoxin molecules have low isoelectric points and have more negative charges in a common solution, the endotoxin can be removed by selecting a cation or anion exchange medium according to different properties of a target sample to separate the endotoxin from the target sample, or by utilizing a specific affinity chromatography filler, usually a certain polymer or agarose is used as a matrix and is coupled with ligands which have specific interaction with the endotoxin, such as polymyxin B, histamine, histidine, polylysine, polycation ligands and the like. The disadvantage of the chromatography method is that it requires the use of relatively expensive chromatography packing and also requires special chromatography equipment. Another method for removing endotoxin which is more commonly used is phase separation, and the commonly used reagents include phenols, triton X-114, triton X-100 and the like. Triton (Triton) is a nonionic surfactant, has high solubility at low temperature, and endotoxin is dissolved in the Triton, the solubility of Triton is gradually reduced along with the increase of temperature, when the temperature is raised to the half point, triton and endotoxin form precipitates together, the precipitates are separated from water phase, and the precipitates are removed by centrifugation, so that the endotoxin in a supernatant sample is removed. However, triton is irritating and harmful to the human body if inhaled, ingested or absorbed by the skin.
Chinese patent CN201911191251.1 (CN 110747194A) in the prior art discloses a preparation method of salmon small molecular PDRN with molecular weight concentrated in the efficacy interval, which comprises (1) cracking salmon tissues by using an alkali cracking solution, and carrying out thermostatic water bath at 90-100 ℃ for 15-60 min; (2) Adding Tris-HCl into the reaction system obtained in the step (1); (3) Adding HCl into the reaction system obtained in the step (2); (4) Centrifuging the reaction system obtained in the step (3), and collecting a supernatant; (5) Performing molecular disruption on the supernatant obtained in the step (4) by using a DNA disruptor; (6) Adding NH into the reaction system obtained in the step (5) 4 Ac, adding absolute ethyl alcohol, and standing for more than 30 min; (7) And (4) centrifuging the reaction system obtained in the step (6), and reserving the precipitate to obtain the micromolecule polydeoxyribonucleotide. The yield disclosed by the patent is 9-14%; the purity is 1.6-1.9. When the purity is less than 1.8, it indicates that more protein impurities remain. Meanwhile, the patent uses a DNA breaking instrument (the manufacturer is Misonix corporation of America) with the model of Sonicator-4000, the maximum processing capacity of the device is 4L, the working principle is ultrasonic breaking, heat is easily generated in the process of crushing DNA, sample precipitation occurs, and the operation is not easy in large-scale industrial production.
Chinese patent CN202010243275.3 (CN 111214694B) discloses a dressing with hemostatic and wound healing accelerating functions, comprising Small Molecule Polydeoxyribonucleotides (SMPDRN) extracted from salmon. The preparation method of the SMPDRN comprises the following steps: (1) Cracking salmon tissue by using an alkali cracking solution, wherein the alkali cracking solution contains 1-3 mol/LNaOH, 50-200mM NaCl, 5-25 mg/ml proteinase K, and is subjected to constant-temperature water bath at 90-100 ℃ for 20-40 min; (2) Adding Tris-HCl into the reaction system obtained in the step (1); (3) Adding HCl into the reaction system obtained in the step (2); (4) Centrifuging the reaction system obtained in the step (3), and collecting a supernatant; (5) Adding the supernatant obtained in the step (4) into isopropanol with the same volume, and standing for more than 30 min; (6) And (5) centrifuging the reaction system obtained in the step (5), and reserving the precipitate to obtain the micromolecule poly-deoxyribonucleotide. This patent uses a relatively expensive protease.
Chinese patent CN202211007267.4 (CN 115074357A) discloses the following preparation steps: comprises collecting the sperm of male salmon, centrifuging at low speed, adding the precipitate into a lysis solution for lysis (1-20 mM urea, 10-100 mM Tris-HCl, 100-500nM NaCl, 100-500 nM CaCl) 2 ，1～10mM Na 2 EDTA, proteinase K), centrifuging the lysate to obtain the supernatant, adding saturated sodium chloride, centrifuging again to obtain the supernatant, precipitating with ethanol, drying, dissolving in physiological saline, decomposing DNA with ultrasonic waves, filtering, and freeze-drying. Wherein the filtration step uses a 0.2 μm filtration membrane. Yield published in the examples: 14.2-16.6%, and the purity is 2.02-2.09, and when the purity is more than 2.0, the residual RNA impurities are more. The patent is to collect the semen of salmon, and the raw materials are not easy to obtain.
The small-molecule PDRN reported in the prior art at present does not control endotoxin, and has low purity and low yield, so that a small-molecule PDRN process with low endotoxin needs to be developed.
Disclosure of Invention
The invention aims to provide a preparation process of low-endotoxin high-purity micromolecule PDRN, the method is low in cost, high in product purity and high in yield, a DNA breaking instrument which is high in cost and cannot be industrially implemented is not needed, and the endotoxin content is lower than 0.1EU/mg, so that the problems of high endotoxin content, low micromolecule PDRN purity and low yield in micromolecule PDRN in the prior art are solved.
The invention provides a preparation method of low-endotoxin micromolecule PDRN, which comprises the following steps:
(1) Carrying out cracking heating reaction on the testis tissue of the salmonidae fish by using an alkali cracking solution, wherein the alkali cracking solution contains EDTA, naOH and SDS;
(2) Adding Tris-HCl into the reaction system obtained in the step (1);
(3) Adding HCl into the reaction system obtained in the step (2);
(4) Centrifuging the reaction system obtained in the step (3), filtering for the first time, and collecting supernatant;
(5) Adding activated carbon into the reaction system obtained in the step (4);
(6) Centrifuging the reaction system obtained in the step (5), filtering for the second time, collecting supernatant, and adding ammonium acetate;
(7) And (5) adding isopropanol into the reaction system obtained in the step (6), standing at room temperature, centrifuging, filtering, and collecting precipitate to obtain the micromolecule PDRN wet solid.
As an embodiment of the present invention, the Salmonidae fishes include Salmonidae, coregonidae, and anisideidae, such as Pacific salmon (including salmon, rainbow trout, and the like), copelagi, whitefish, hucho, cornus pelagi, corynonella, pakiss, bigler, trout, ottelia, pimenta, northern salmon, anethrus, ekistrotus, and the like. As an embodiment of the invention, the working conditions of the step (1) are as follows: the dosage ratio of the testis tissue of the salmonidae fishes to the alkali lysate is 1g: 3-8 mL, preferably in a ratio of 1g:6mL; the heating reaction temperature is 70-90 ℃, the reaction time is 10-30 min, and the preferable reaction condition is heating at 80 ℃ for 20min.
As an embodiment of the present invention, the operating conditions in step (2) are: and (2) cooling the reaction system obtained in the step (1) to room temperature, adding 2M Tris-HCl according to 1/2-2 of the volume of the lysate, preferably according to 1/2 of the volume of the lysate, and uniformly mixing.
As an embodiment of the present invention, the working conditions in step (3) are: adding 1M HCl into the reaction system obtained in the step (2) according to 1/10-1 of the volume of the reaction solution, preferably according to 1/5 of the volume of the reaction solution, and uniformly mixing.
In one embodiment of the present invention, the operating conditions in step (4) to step (6) are: centrifuging the reaction system obtained in the step (3) at room temperature and 4500rpm for 5-10 min, collecting supernatant, carrying out first filtration, wherein the filter medium comprises filter paper, a 0.22 mu M filter membrane or a 0.45 mu M filter membrane, centrifuging after adsorbing by activated carbon, carrying out second filtration, and adding 10M NH according to 1/50-1/10 of the total volume of the supernatant, preferably according to 1/20 of the total volume of the supernatant 4 Ac, mixed evenly and then precipitated by isopropanol.
As an embodiment of the present invention, the first filtration in step (3) is performed with a 0.45 μm filter membrane, so that incompletely lysed tissue fragments can be removed.
As an embodiment of the present invention, the operating conditions in step (5) are: the dosage of the activated carbon is 0.5-5% of the mass of the reaction system obtained in the step (4), the preferred dosage of the activated carbon is 2%, and the activated carbon has the function of adsorbing protein and endotoxin.
As an embodiment of the present invention, the operating conditions in step (6) are: centrifuging at 4500rpm for 5-10 min at room temperature, collecting supernatant, and performing secondary filtration with 0.45 μm and 0.22 μm filter membranes to remove impurities such as microorganism.
As an embodiment of the present invention, the working conditions in step (7) are: adding 0.5-2 times volume of isopropanol into the filtrate obtained in the step (6), preferably 0.7 times volume of isopropanol, standing at room temperature for 20-60 min, preferably 30min, centrifuging at 4500rpm for 5min, collecting precipitate, discarding supernatant, mashing the precipitate, adding 70% ethanol, washing the precipitate, centrifuging at 3000rpm for 5min, collecting precipitate, repeatedly washing for 2-5 times, preferably washing for 2 times, and collecting precipitate to obtain the PDRN wet solid.
As an embodiment of the invention, the preparation method further comprises the steps of dissolving the PDRN wet solid by using a TE buffer solution and then carrying out vacuum freeze drying.
In one embodiment of the invention, the endotoxin content in the small molecule polydeoxyribonucleotide is less than 0.1EU/mg; the purity of the micromolecule polydeoxyribonucleotide is 1.8-2.0.
As an embodiment of the invention, the average molecular weight of the small molecule polydeoxyribonucleotides is 10 to 2000bp, and may be, for example, 10 to 1000bp,10 to 500bp or 10 to 250bp.
In one embodiment of the present invention, the average molecular weight of the small molecule polydeoxyribonucleotide is 85% or more, and more preferably 95% or more of the total mass of the 10-250 bp fragment.
In another aspect of the invention, the invention provides an application of the preparation method in the preparation of small-molecule polydeoxyribonucleotides in the fields of food, skin care, medical and cosmetic and medicine.
Compared with the prior art, the invention has the following advantages: 1. the invention provides a method for preparing stable small-molecule PDRN with the average molecular weight of 10-250 bp segments accounting for more than 95% of the total mass and the endotoxin content lower than 0.1EU/mg by taking the testis of salmonidae fishes as a raw material, wherein the purity of the small-molecule PDRN is in the range of 1.8-2.0 (the larger the value is, the better the value is), preferably about 1.9, which shows that the residual RNA and protein impurities are greatly reduced; the yield of the micromolecule PDRN reaches 17-18%, and the problems of high endotoxin content, low purity and low yield of the micromolecule PDRN in the prior art are solved.
2. According to the preparation process of the low-endotoxin high-purity micromolecule PDRN, the method does not need to use protease with higher cost, does not need to use a DNA breaking instrument, and is low in overall cost and low in equipment investment; the small molecular PDRN with concentrated molecular weight can be obtained by reaction at 90 ℃ or below, the temperature is lower than that of the prior art, the requirement on the rotating speed of centrifugal equipment is low, and therefore, the energy consumption is reduced. The method of the invention is easy to be produced in an enlarged way, and has wide application prospect in the fields of medical cosmetology, cosmetics, medical instruments, health food and medicine.
Drawings
FIG. 1 is an agarose gel electrophoresis of samples prepared at different reaction temperatures, wherein lane 1 uses DL2000 DNA molecular weight Marker to represent standards of different molecular weight indications.
FIG. 2 is a map obtained by calculating the gray level value lane of a DL2000 DNA standard using Image analysis software.
Fig. 3 is a map obtained by calculating the grayscale value lane of the small molecule PDRN obtained in example 1 using Image analysis software.
FIG. 4 is a map obtained by calculating the grayscale value lane of the small molecule PDRN obtained in example 2 by using Image analysis software.
Fig. 5 is a map obtained by calculating the grayscale value lane of the small molecule PDRN obtained in example 3 using Image analysis software.
Fig. 6 is a graph in which the gray level value lane of the small molecule PDRN obtained in comparative example 1 was calculated using Image analysis software.
FIG. 7 is a graph of gray level lane of the small molecule PDRN obtained in comparative example 2 calculated using Image analysis software.
FIG. 8 is a gel diagram of limulus reagent assay by gel method in example 2 and comparative examples 3 to 5.
Detailed Description
The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.
The technical solutions of the present invention will be further described below with reference to specific examples in order to facilitate the understanding of the present invention by those skilled in the art, but the following should not limit the scope of the present invention as claimed in the claims in any way. The raw materials or reagents used in the examples of the present invention and comparative examples were commercially available without specific indication.
The molecular weight of the small molecule PDRD was determined by agarose gel electrophoresis. The principle is that the agarose gel has a network structure, substance molecules can be subjected to resistance when passing through the agarose gel, and macromolecular substances are subjected to large resistance when surging, so that in gel electrophoresis, the migration speed is low, small molecular substances are migrated quickly, and the molecular weight of sample molecules can be determined by comparing the positions of the small molecular substances with different molecular weights in a standard product.
The yield of small molecule PDRD was calculated as yield = product weight/testis weight.
Purity of small molecule PDRD: after the product is dissolved in TE buffer solution (10 mM Tris-HCl,1mM EDTA, pH 8.0), the absorbance at 260nm and 280nm is respectively detected by using a spectrophotometer, and the purity of the polydeoxyribonucleotide is calculated by calculating the ratio of A260 nm/A280 nm (the PDRN is better purified and has less residual RNA and protein impurities when the ratio of A260 nm/A280 nm is between 1.8 and 2.0).
The endotoxin content is detected by gel method with limulus reagent, and the principle is that under proper conditions (temperature, pH value and non-interference matter), bacterial endotoxin can activate procoagulant enzyme in limulus reagent to make limulus reagent produce agglutination reaction to form gel.
Example 1
A preparation method of small molecule low endotoxin PDRN comprises the following steps:
(1) Accurately weighing 100g of salmon testis, cleaning with pure water, removing blood vessel, mincing into paste, cracking with 600ml of alkaline lysis solution (0.1M EDTA,2M NaOH,0.5% SDS), and heating in 70 deg.C constant temperature water bath for 20min.
(2) Cooling the reaction system obtained in the step (1) to room temperature, and adding 300ml of buffer 2M Tris-HCl (pH8.0);
(3) To the reaction system obtained in step (2), 180ml of a 1M HCl solution was added.
(4) Centrifuging the reaction system obtained in the step (3), centrifuging at 4500rpm for 10min, filtering through a 0.45-micron filter membrane, and collecting a supernatant.
(5) Adding 2% by weight of activated carbon to the reaction system obtained in step (4).
(6) Centrifuging the reaction system obtained in step (5), 4500rpm,30min, filtering with 0.45 μm filter membrane and 0.22 μm filter membrane respectively, collecting supernatant, adding 50ml of 10M NH 4 Ac solution.
(7) And (3) adding 0.7-time volume of isopropanol into the reaction system obtained in the step (6), standing at room temperature for 30min, centrifuging at 4500rpm for 5min, filtering, collecting precipitates, performing stirred washing on the precipitates for 2 times by using 70% ethanol, and centrifuging at 4500rpm for 5min to obtain the micromolecular PDRN wet solid.
(8) Taking a small amount of the precipitate prepared in step (7), dissolving the precipitate in TE buffer, determining the concentration of the precipitate, and performing agarose gel electrophoresis, specifically, referring to lane 4 in FIG. 1 and FIG. 3, the gray value lane% of which is 86.1% calculated by using Image analysis software (calculated from the gray value of the band on the electrophoretogram, it can be understood as the electrophoresis purity), which indicates that the mass of the fragment with the molecular weight of 10-250 bp of the small-molecule polydeoxyribonucleotide accounts for 86.1% of the total mass of the small-molecule polydeoxyribonucleotide.
(9) Dissolving the precipitate obtained in step (7) with TE buffer solution, and freeze-drying in a vacuum freeze-dryer to obtain white PDRN powder. The purity and yield of the product are shown in table 1.
Example 2:
a preparation method of small molecule low endotoxin PDRN comprises the following steps:
(1) 100g of spermary of salmon is accurately weighed, washed with pure water, blood vessel removed, minced into paste, lysed with 600ml of alkaline lysis solution (0.1M EDTA,2M NaOH,0.5% SDS), and heated in 80 deg.C constant temperature water bath for 20min.
(2) Cooling the reaction system obtained in the step (1) to room temperature, and adding 300ml of buffer 2M Tris-HCl (pH8.0);
(3) To the reaction system obtained in step (2), 180ml of a 1M HCl solution was added.
(4) Centrifuging the reaction system obtained in the step (3), centrifuging at 4500rpm for 10min, filtering through a 0.45-micron filter membrane, and collecting a supernatant.
(5) Adding 2% by weight of activated carbon to the reaction system obtained in the step (4).
(6) Centrifuging the reaction system obtained in step (5), 4500rpm,30min, filtering with 0.45 μm filter membrane and 0.22 μm filter membrane respectively, collecting supernatant, adding 50ml of 10M NH 4 Ac solution.
(7) Adding isopropanol with the volume 0.7 times that of the reaction system obtained in the step (6), standing at room temperature for 30min, centrifuging at 4500rpm for 5min, filtering, collecting precipitate, performing stirred washing on the precipitate with 70% ethanol for 2 times, and centrifuging at 4500rpm for 5min to obtain the micromolecular PDRN wet solid.
(8) Taking a small amount of the precipitate prepared in step (7), dissolving the precipitate in TE buffer, determining the concentration of the precipitate, and performing agarose gel electrophoresis, wherein the gray value lane% of the precipitate is 96.9% as shown in lane 5 of FIG. 1 and Image analysis software shown in FIG. 4, which indicates that the mass of the fragment with the average molecular weight of about 10-250 bp of the small-molecule polydeoxyribonucleotide accounts for 96.9% of the total mass of the small-molecule polydeoxyribonucleotide.
(9) Dissolving the precipitate obtained in step (7) with TE buffer solution, and freeze-drying in a vacuum freeze-dryer to obtain white PDRN powder. The purity and yield of the product are shown in table 1.
Example 3:
a preparation method of small molecule low endotoxin PDRN comprises the following steps:
(1) Accurately weighing 100g of salmon testis, cleaning with pure water, removing blood vessel, mincing into paste, cracking with 600ml of alkaline lysis solution (0.1M EDTA,2M NaOH,0.5% SDS), and heating in 90 deg.C constant temperature water bath for 20min.
(2) Cooling the reaction system obtained in the step (1) to room temperature, and adding 300ml of buffer 2M Tris-HCl (pH8.0);
(3) To the reaction system obtained in step (2), 180ml of a 1M HCl solution was added.
(4) Centrifuging the reaction system obtained in the step (3), centrifuging at 4500rpm for 10min, filtering through a 0.45-micron filter membrane, and collecting a supernatant.
(5) Adding 2% by weight of activated carbon to the reaction system obtained in step (4).
(6) Centrifuging the reaction system obtained in step (5), 4500rpm,30min, filtering with 0.45 μm filter membrane and 0.22 μm filter membrane respectively, collecting supernatant, adding 50ml 10M NH 4 Ac solution.
(7) Adding isopropanol with the volume 0.7 times that of the reaction system obtained in the step (6), standing at room temperature for 30min, centrifuging at 4500rpm for 5min, filtering, collecting precipitate, performing stirred washing on the precipitate with 70% ethanol for 2 times, and centrifuging at 4500rpm for 5min to obtain the micromolecular PDRN wet solid.
(8) Taking a small amount of the precipitate prepared in step (7), dissolving the precipitate in TE buffer solution, determining the concentration of the precipitate, and performing agarose gel electrophoresis, specifically, as shown in lane 6 of FIG. 1 and in FIG. 5, the gray value lane% is 95.8% by using Image analysis software, which indicates that the mass of the fragment with the molecular weight of the small-molecule polydeoxyribonucleotide being between 10 and 250bp accounts for 95.8% of the total mass of the small-molecule polydeoxyribonucleotide.
(9) Dissolving the precipitate obtained in step (7) with TE buffer solution, and freeze-drying in a vacuum freeze-dryer to obtain white PDRN powder. The purity and yield of the product are shown in table 1.
Comparative example 1:
a preparation method of small molecule low endotoxin PDRN comprises the following steps:
(1) 100g of spermary of salmon is accurately weighed, washed with pure water, blood vessel removed, minced into paste, lysed with 600ml of alkaline lysis solution (0.1M EDTA,2M NaOH,0.5% SDS), and heated in a constant temperature water bath at 50 ℃ for 20min.
(2) Cooling the reaction system obtained in the step (1) to room temperature, and adding 300ml of buffer 2M Tris-HCl (pH8.0);
(3) To the reaction system obtained in step (2), 180ml of a 1M HCl solution was added.
(4) Centrifuging the reaction system obtained in the step (3), centrifuging at 4500rpm for 10min, filtering through a 0.45-micron filter membrane, and collecting a supernatant.
(5) Adding 2% by weight of activated carbon to the reaction system obtained in the step (4).
(6) Centrifuging the reaction system obtained in step (5), 4500rpm,30min, filtering with 0.45 μm filter membrane and 0.22 μm filter membrane respectively, collecting supernatant, adding 50ml of 10M NH 4 Ac solution.
(7) And (3) adding 0.7-time volume of isopropanol into the reaction system obtained in the step (6), standing at room temperature for 30min, centrifuging at 4500rpm for 5min, filtering, collecting precipitates, performing stirred washing on the precipitates for 2 times by using 70% ethanol, and centrifuging at 4500rpm for 5min to obtain the micromolecular PDRN wet solid.
(8) Taking a small amount of the precipitate prepared in step (7), dissolving the precipitate with TE buffer solution, determining the concentration of the precipitate, and performing agarose gel electrophoresis, wherein the gray value lane% of the precipitate is 69.1% as shown in lane 2 of FIG. 1 and as shown in FIG. 6 by using Image analysis software, which indicates that the mass of the fragment of the small-molecule polydeoxyribonucleotide is 69.1% of the total mass of the fragment with the molecular weight of 10-250 bp.
(9) Dissolving the precipitate obtained in step (7) with TE buffer solution, and freeze-drying in a vacuum freeze-dryer to obtain white PDRN powder. The purity and yield of the product are shown in table 1.
Comparative example 2:
a preparation method of small molecule low endotoxin PDRN comprises the following steps:
(1) Accurately weighing 100g of salmon testis, cleaning with pure water, removing blood vessel, mincing into paste, cracking with 600ml of alkaline lysis solution (0.1M EDTA,2M NaOH,0.5% SDS), and heating in 60 deg.C constant temperature water bath for 20min.
(2) Cooling the reaction system obtained in the step (1) to room temperature, and adding 300ml of buffer solution 2M Tris-HCl (pH8.0);
(3) To the reaction system obtained in step (2), 180ml of 1M HCl solution was added.
(4) And (4) centrifuging the reaction system obtained in the step (3), centrifuging at 4500rpm for 10min, filtering through a 0.45-micron filter membrane, and collecting a supernatant.
(5) Adding 2% by weight of activated carbon to the reaction system obtained in step (4).
(6) Centrifuging the reaction system obtained in step (5), 4500rpm,30min, filtering with 0.45 μm filter membrane and 0.22 μm filter membrane respectively, collecting supernatant, adding 50ml of 10M NH 4 Ac solution.
(7) Adding isopropanol with the volume 0.7 times that of the reaction system obtained in the step (6), standing at room temperature for 30min, centrifuging at 4500rpm for 5min, filtering, collecting precipitate, performing stirred washing on the precipitate with 70% ethanol for 2 times, and centrifuging at 4500rpm for 5min to obtain the micromolecular PDRN wet solid.
(8) Taking a small amount of the precipitate prepared in step (7), dissolving the precipitate with TE buffer solution, determining the concentration of the precipitate, and performing agarose gel electrophoresis, wherein the gray value lane% of the precipitate is 68.1% as shown in lane 3 of FIG. 1 and as shown in FIG. 7 by using Image analysis software, which indicates that the mass of the fragment of the small-molecule polydeoxyribonucleotide is 68.1% of the total mass of the fragment with the molecular weight of 10-250 bp.
(9) Dissolving the precipitate obtained in step (7) with TE buffer solution, and freeze-drying in a vacuum freeze-dryer to obtain white PDRN powder. The purity and yield of the product are shown in table 1.
As can be seen from FIG. 1 and FIGS. 2 to 7, the molecular weights of the products obtained in examples 1 to 3 are very concentrated, and most of the products are concentrated on the molecular weight of 100bp, wherein the same effect can be achieved at a lower temperature level in example 2 compared with example 3, the energy consumption is relatively less, the product yield is highest, and the purity is best, so that example 2 is the best example; the molecular weight of the products obtained in comparative example 1 and comparative example 2 is not concentrated, the tailing is serious, the purity is lower than 1.8, and the yield is lower than 17%.
TABLE 1 comparison of purity and yield
Comparative example 3: comparative example 2, step (4) and step (6) were carried out without filtration through 0.22 μm/0.45 μm filter, and the other conditions were the same as in example 1.
Comparative example 4: the procedure (5) of comparative example 2 was carried out under the same conditions as in example 1, except that activated carbon adsorption was not added.
Comparative example 5: in contrast to example 2, the filtration was not performed under 0.22um/0.45um in step (4) and step (6), and the adsorption was not performed under activated carbon in step (5), and the other conditions were the same as in example 1.
Claims (10)
1. A preparation method of low endotoxin micromolecule PDRN is characterized by comprising the following steps:
(1) Carrying out lysis heating reaction on the testis tissue of the salmonidae fish by using an alkaline lysis solution, wherein the alkaline lysis solution contains EDTA, naOH and SDS;
(2) Adding Tris-HCl into the reaction system obtained in the step (1);
(3) Adding HCl into the reaction system obtained in the step (2);
(4) Centrifuging the reaction system obtained in the step (3), filtering for the first time, and collecting supernatant;
(5) Adding activated carbon into the reaction system obtained in the step (4);
(6) Centrifuging the reaction system obtained in the step (5), filtering for the second time, collecting supernatant, and adding ammonium acetate;
(7) Adding isopropanol into the reaction system obtained in the step (6), standing at room temperature, centrifuging, filtering, collecting precipitate,
obtaining the micromolecular PDRN wet solid.
2. The method according to claim 1, wherein the filtration in the steps (4) and (6) is performed by using filter paper or membrane.
3. The method according to claim 2, wherein the first filtration in the step (4) is performed with a 0.45 μm membrane filter; and the second filtration is sequentially carried out by adopting a 0.45 mu m filter membrane and a 0.22 mu m filter membrane.
4. The process according to any one of claims 1 to 3, wherein the amount of the activated carbon used in step (5) is 0.5 to 5% by mass, preferably 2% by mass, based on the mass of the reaction system obtained in step (4).
5. The method according to any one of claims 1 to 4, wherein the ratio of the amount of the Salmonidae fish testis tissue to the alkali lysate in step (1) is 1g: 3-8 mL, preferably in a ratio of 1g:6mL.
6. The process according to any one of claims 1 to 4, wherein the heating in step (1) is carried out at a reaction temperature of 70 to 90 ℃ for 10 to 30min, preferably at a reaction condition of 80 ℃ for 20min.
7. The method according to any one of claims 1 to 6, wherein the endotoxin content in the small-molecule polydeoxyribonucleotide is less than 0.1EU/mg; the purity of the micromolecule polydeoxyribonucleotide is 1.8-2.0.
8. The method according to any one of claims 1 to 6, wherein the molecular weight of the small-molecule polydeoxyribonucleotide is 50 to 2000bp.
9. The method according to claim 8, wherein the molecular weight of the small polydeoxyribonucleotide is at least 95% of the total mass of the fragment of 10 to 250bp.
10. The use of the process according to any one of claims 1 to 9 for the preparation of small-molecule polydeoxyribonucleotides in the fields of food, skin care, medical and cosmetic applications and medicine.
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