A kind of PDRN derived from HEK293 cells and preparation method thereof

Abstract

The present invention relates to a PDRN derived from HEK293 cells and a preparation method thereof. The steps include: (1) centrifuging HEK293 cells, adding a lysate to obtain a lysate solution; (2) adding magnesium sulfate, protease and nuclease to the lysate solution, adding sodium dodecyl sulfate and sodium chloride after incubation, and obtaining a crude PDRN product after enzyme inactivation and alcohol precipitation; (3) dissolving the crude PDRN product in a sodium chloride solution, filtering, alcohol precipitation, washing, dehydration and drying to obtain PDRN. The PDRN preparation method provided by the present invention uses HEK293 cells as a source, uses a nuclease and a protease double enzyme one-pot method to prepare PDRN, has simple process control, does not have special environments such as high temperature, strong acid and alkali, and has relatively mild conditions. It does not need to add special equipment, and can be industrially produced on a large scale. In addition, the base composition ratio of the PDRN derived from HEK293 cells is completely consistent with that of the human body, has anti-inflammatory function and tissue repair activity, and has great application value in the fields of soft tissue repair, medical and aesthetic post-repair.

Description

Technical Field

[0001] The invention relates to a PDRN derived from HEK293 cells and a preparation method thereof, and belongs to the field of biotechnology. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleic acid with a molecular weight of 50-1500KDa. It exists in the human placenta and is the raw material for the production of DNA in cells. PDRN exists in the extracellular matrix (ECM) for cell metabolism. In addition to participating in cell growth, many preclinical and clinical studies have demonstrated that PDRN can efficiently activate A on the cell surface. 2A Adenosine receptors promote the production of vascular endothelial growth factor (VEGF) and collagen, the migration of various cells, significantly enhance the proliferation of fibroblasts, reduce inflammatory responses, and activate aging or damaged cells. In addition, based on its efficacy in promoting human cell regeneration, accelerating wound healing, reducing scar formation, and shortening the skin metabolism cycle, PDRN also has broad application prospects in beauty repair. At present, PDRN-related beauty products mainly include Italy's PDRN salmon water light needle, South Korea's Demena PDRN salmon microneedle introduction repair essence, HPCell Pulilan baby needle, etc., which are deeply loved by women.

[0003] Generally, commercialized PDRN is derived from the gonads of salmon, king salmon or rainbow trout, and is obtained using DNA extraction technology through crude extraction, purification, and refinement. The distribution composition of bases in this type of PDRN is about 98% similar to that of the human body.

[0004] Chinese patent document CN115960153A discloses a method for preparing and applying a low-endotoxin small molecule PDRN. First, the salmonid testicles are pretreated, minced, alkaline lysed, the lysate supernatant is filtered, adsorbed with activated carbon, filtered to obtain the supernatant, precipitated with isopropanol, and the precipitate is freeze-dried to obtain a PDRN product with endotoxin <0.1EU / mg. However, this method uses isopropanol to precipitate the extracted PDRN. The boiling point of isopropanol is relatively high, at 82.5°C. It is difficult to remove isopropanol from the precipitate obtained. At the same time, isopropanol belongs to the list of three types of carcinogens published by the World Health Organization's International Agency for Research on Cancer. Therefore, the PDRN prepared by this patented process has a greater safety hazard.

[0005] Chinese patent document CN114107283A discloses an efficient extraction and shearing technology for preparing polydeoxyribonucleic acid for skin. First, salmon testis is taken out and homogenized by tissue crusher. Then proteinase K is added to hydrolyze the protein and the supernatant is collected by centrifugation. Then saturated sodium chloride solution is added and the supernatant is collected by centrifugation. Then anhydrous ethanol is added and DNA is precipitated at low temperature. Adaptive Focused Acoustic TM , AFA) technology of genomic DNA fragmentation instrument shearing DNA, and finally freeze drying to obtain PDRN, the molecular weight of the obtained PDRN is between 200 and 300bp. However, this method requires the use of high-end instruments for preparation, which is only suitable for the preparation of small batches of products and cannot be carried out for large-scale industrial production.

[0006] It can be seen that the existing technologies all obtain PDRN from fish testes. Although it is easier to extract from fish testes, there are also disadvantages such as insufficient sources. First of all, these fish are not unlimited resources. Overfishing will lead to resource depletion, and artificial breeding also has technical difficulties; secondly, it takes a certain amount of time for fry to grow into adult fish, and fish only spawn and ejaculate during the breeding season, which makes it difficult to obtain fish sperm; thirdly, PDRN is obtained by extracting DNA from fish gonads. Its base composition is up to 98% similar to that of the human body, and it has certain heterology and potential safety hazards. The current problem of insufficient sources for the preparation of PDRN leads to high costs and high prices of related products, which greatly limits the promotion and use of PDRN. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a HEK293 cell-derived PDRN and a preparation method thereof.

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

[0009] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0010] (1) resuspending HEK293 cells in PBS buffer and centrifuging to collect cell pellets; then adding lysis buffer to the cell pellets for high-pressure homogenization to obtain a lysate solution;

[0011] (2) adding magnesium sulfate, protease and nuclease to the lysate solution obtained in step (1), incubating at 45-55° C. for 1-2 h, adding sodium dodecyl sulfate (SDS) and sodium chloride after the incubation, stirring until fully dissolved, inactivating the enzyme and precipitating with alcohol to obtain a crude PDRN;

[0012] (3) Dissolve the crude PDRN obtained in step (2) in a sodium chloride solution, adjust the pH to 6.0-8.0, raise the temperature to 50-60° C., add activated carbon, stir for 1-2 h, filter, precipitate with alcohol, wash, dehydrate and dry to obtain PDRN.

[0013] Preferably according to the present invention, in step (1), the composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4.

[0014] Preferably according to the present invention, in step (1), the volume ratio of the lysate to the PBS buffer is (3-6):(10-15).

[0015] Preferably, according to the present invention, in step (1), the centrifugation parameters are 5000-10000 r / min, 15-30 min; the high-pressure homogenization is carried out in a high-pressure homogenizer, and the parameters are pressure 300-500 bar, and time 10-30 min.

[0016] Preferably according to the present invention, in step (2), the final concentration of magnesium sulfate in the lysate solution is 25-35 mM.

[0017] Preferably, according to the present invention, in step (2), the protease is one or a combination of two or more of the protease protamex 1.6, the protease Alcalase 2.4L FG, the protease Flavourzyme 500MG, and the protease Novozym 2778; and the mass ratio of the protease to the lysate solution is (0.05-0.15):100.

[0018] According to the preferred embodiment of the present invention, in step (2), the nuclease is a nuclease mutant RXG-M, whose amino acid sequence is shown in SEQ ID NO.1, and whose nucleotide sequence is shown in SEQ ID NO.2, and the enzyme has a high degradation activity on the DNA of HEK293 cells. The volume mass ratio of the nuclease to the lysate solution is (0.2-0.6):100, unit: mL / g; the nuclease is obtained by heterologous recombinant expression, fermentation and extraction according to the prior art method.

[0019] Preferably, according to the present invention, in step (2), the mass ratio of the sodium dodecyl sulfate to the lysate solution is (0.2-2):100; the mass ratio of the sodium chloride to the lysate solution is (2-20):100.

[0020] Preferably, in step (2), the enzyme inactivation and alcohol precipitation are as follows: the temperature is raised to 60-100° C. and maintained for 2-4 hours to fully inactivate the protease and nuclease, then the temperature is lowered to 20-30° C., and 1-1.5 times the volume of the lysate solution is added with 95% ethanol for precipitation.

[0021] Preferably, according to the present invention, in step (3), the mass volume ratio of the crude PDRN and the sodium chloride solution is 1:(5-100), unit: g / mL; and the mass concentration of the sodium chloride solution is 4-10%.

[0022] Preferably, according to the present invention, in step (3), the mass volume ratio of the activated carbon and the sodium chloride solution is (1-2):100, unit: g / mL.

[0023] Preferably, in step (3), the filtration, alcohol precipitation, washing, dehydration and drying are as follows: filtering through a 0.45 μm sterile filter plate to remove the activated carbon, then filtering through a 0.22 μm filter to obtain a refined filtrate, cooling the refined filtrate to 20 to 30° C., adding 1 to 1.5 times the volume of a sodium chloride solution and 95% ethanol for precipitation, washing and dehydrating the precipitate with ethanol, and drying at 40 to 60° C. for 3 to 6 hours.

[0024] The present invention also provides PDRN derived from HEK293 cells prepared by the above method. The PDRN extracted from HEK293 is a fully humanized nucleotide, which is completely consistent with the base composition ratio of the human body.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention successfully prepared PDRN with a molecular weight of 200,000 to 1 million Daltons using HEK293 cells as the source. In addition, HEK293 cells have the characteristics of high transfection efficiency and easy culture, which effectively solves the problem of extracting the source of PDRN raw materials, reduces the cost and price of PDRN, and greatly expands the scope of use of PDRN.

[0027] 2. The PDRN preparation method provided by the present invention uses HEK293 cells as the source, and uses a one-pot method of nuclease and protease to prepare PDRN. The process control is simple, there is no special environment such as high temperature, strong acid and alkali, the conditions are relatively mild, and no additional special equipment is required, and large-scale industrial production can be carried out.

[0028] 3. The nuclease used in the present invention is the nuclease mutant RXG-M, which has a wider optimum temperature range and pH range, has a high degradation activity on HEK293 cell DNA, and can obtain PDRN with smaller molecular fragments and higher activity.

[0029] 4. The HEK293 cell-derived PDRN prepared by the present invention has a base composition ratio that is completely consistent with that of the human body, has anti-inflammatory function and tissue repair activity, and has great application value in the fields of soft tissue repair, medical and aesthetic post-operative repair, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Electrophoretograms of HEK293 cell-derived PDRN prepared in Examples 1 to 3 and Comparative Examples 1 to 2;

[0031] In the figure, lane 1 is comparative example 1, lane 2 is comparative example 2, lane 3 is example 3, lane 4 is example 2, and lane 5 is example 1.

[0032] Figure 2 This is a diagram showing the effect of the HEK293 cell-derived PDRN and salmon sperm-derived PDRN prepared by the present invention on inhibiting the inflammatory factor TNF-α.

[0033] Figure 3 This is a diagram showing the effect of the HEK293 cell-derived PDRN and salmon sperm-derived PDRN prepared by the present invention on inhibiting the inflammatory factor IL-1β.

[0034] Figure 4 This is a diagram showing the scratch repair effect of HEK293 cell-derived PDRN prepared in the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0036] The protease protamex 1.6, protease Alcalase 2.4L FG, protease Flavourzyme 500MG, and protease Novozym 2778 described in the examples are available from Novozymes (China) Biotechnology Co., Ltd.

[0037] The nuclease used in the present invention is a nuclease mutant RXG-M, whose amino acid sequence is shown in SEQ ID NO.1, and whose nucleotide sequence is shown in SEQ ID NO.2. The nuclease mutant RXG-M is a mutant RXG-M obtained by rationally designing enzyme molecules and optimizing site-directed mutation screening with nuclease RXG (GenBank: RXG77175.1) as the initial nuclease. The mutant enzyme has mutation sites of V120A, Q135P, R146A, and Q150L. Compared with the wild-type enzyme, it has a wider optimum temperature range and pH range, has a high degradation activity for HEK293 cell DNA, and can be used together with protease to achieve a one-pot extraction of PDRN.

[0038] The nuclease mutant RXG-M can be obtained by heterologous recombination expression, fermentation and extraction according to the prior art method. The specific method is as follows: artificially synthesize the nucleotide sequence of the nuclease mutant RXG-M (SEQ ID NO.2), connect it to the expression vector pET-28(a)+ using BamH I and Hind III restriction sites to obtain a recombinant plasmid pET-28(a)+-RXG-M; heat-shock transform the recombinant plasmid into Escherichia coli BL21 competent cells, and obtain the target strain E. coli after kanamycin resistance screening. BL21 / pET-28(a)+-RXG-M; the recombinant bacteria were inoculated into LB liquid culture medium, and after activation culture, they were inoculated into fresh LB culture medium, and cultured until OD600 = 0.8-1.2, and IPTG with a final concentration of 0.05 mM was added to induce protein expression, the induction temperature was 20°C, and the induction time was controlled to be 12-20 h; after the fermentation was completed, the fermentation liquid was taken out, centrifuged at 10000 r / m for 20 min, the supernatant was discarded, the precipitate was collected, and 100 mM PBS buffer was added according to 10 times the mass of the precipitate to re-suspend the cell precipitate, centrifuged at 10000 r / m for 20 min, the supernatant was discarded, the precipitate was collected, and the same 100 mM The precipitate was suspended in PBS buffer and placed in an ultrasonic crusher with the crushing condition controlled at 400W for 20 minutes until the solution became clear visibly, then centrifuged at 10000r / m for 20 minutes, the precipitate was discarded, and the supernatant was collected as the crude enzyme solution of nuclease RXG-M. After purification by nickel ion affinity chromatography column and desalting by desalting column, the enzyme solution of nuclease mutant RXG-M was obtained.

[0039] The wild-type nuclease RXG (GenBank: RXG77175.1) was obtained by the same method.

[0040] Those skilled in the art can utilize the technical means or technical implications of the present invention to extract PDRN using other DNA donor tissues as raw materials, which also falls within the protection scope of the present invention.

[0041] Example 1

[0042] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0043] (1) 5 L of HEK293 cell culture medium was centrifuged at 8000 r / min for 20 min, the HEK293 cells obtained after centrifugation were resuspended in 1 L of PBS buffer, and centrifuged at 8000 r / min for 20 min to collect the cell precipitate; then 500 mL of lysis solution was added to the cell precipitate and high-pressure homogenization was performed in a high-pressure homogenizer to obtain a lysate solution; the high-pressure homogenization parameters were pressure 400 bar and time 20 min;

[0044] The composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4;

[0045] (2) magnesium sulfate (final concentration of 30 mM), 2 mL of nuclease mutant RXG-M enzyme solution, and 0.5 g of protease protamex 1.6 were added to 500 g of the lysate solution obtained in step (1) in sequence, and incubated at 50° C. for 2 h. After the incubation, 5 g of sodium dodecyl sulfate (SDS) and 50 g of sodium chloride were added, stirred until fully dissolved, heated to 90° C. and maintained for 1 h to fully inactivate the protease and nuclease, then cooled to 25° C., and 1.5 times the volume of the lysate solution was added with 95% ethanol for precipitation to obtain a crude PDRN;

[0046] (3) 5 g of the crude PDRN obtained in step (2) was dissolved in 500 mL of a 10% sodium chloride solution, the pH was adjusted to 7.0, the temperature was raised to 55°C, 7.5 g of activated carbon was added, and the mixture was stirred for 1.5 h. The activated carbon was removed by filtering through a 0.45 μm sterile filter plate, and then filtered through a 0.22 μm filter to obtain a refined filtrate. After the refined filtrate was cooled to 30°C, 1.5 times the volume of 95% ethanol in the sodium chloride solution was added for precipitation. After sufficient precipitation for 3 h, the mixture was washed three times with 80% alcohol, and finally dehydrated with anhydrous ethanol to an alcohol content of more than 95%, allowed to stand for 5 h, the precipitate was collected, and dried at 45°C for 8 h to obtain 4.5 g of powdered PDRN.

[0047] Example 2

[0048] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0049] (1) 5 L of HEK293 cell culture medium was centrifuged at 8000 r / min for 20 min, the HEK293 cells obtained after centrifugation were resuspended in 1 L of PBS buffer, and centrifuged at 8000 r / min for 20 min to collect the cell precipitate; then 500 mL of lysis solution was added to the cell precipitate and high-pressure homogenization was performed in a high-pressure homogenizer to obtain a lysate solution; the high-pressure homogenization parameters were pressure 400 bar and time 20 min;

[0050] The composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4;

[0051] (2) magnesium sulfate (final concentration of 30 mM), 2 mL of nuclease mutant RXG-M enzyme solution, 0.1 g of protease Alcalase 2.4L FG, 0.2 g of protease Flavourzyme 500MG, and 0.2 g of protease Novozym 2778 were added to 500 g of the lysate solution obtained in step (1) in sequence, and the mixture was incubated at 50° C. for 2 h. After the incubation, 5 g of sodium dodecyl sulfate (SDS) and 50 g of sodium chloride were added, and the mixture was stirred until fully dissolved. The mixture was heated to 90° C. and maintained for 1 h to fully inactivate the protease and nuclease, and then cooled to 25° C., and 1.5 times the volume of the lysate solution of 95% ethanol was added for precipitation to obtain a crude PDRN product;

[0052] (3) 4.5 g of the crude PDRN obtained in step (2) was dissolved in 500 mL of a 10% sodium chloride solution, the pH was adjusted to 7.0, the temperature was raised to 55°C, 6.5 g of activated carbon was added, and the mixture was stirred for 1.5 h. The activated carbon was removed by filtering through a 0.45 μm sterile filter plate, and then filtered through a 0.22 μm filter to obtain a refined filtrate. After the refined filtrate was cooled to 30°C, 1.5 times the volume of 95% ethanol in the sodium chloride solution was added for precipitation. After sufficient precipitation for 3 h, the mixture was washed three times with 80% alcohol, and finally dehydrated with anhydrous ethanol to an alcohol content of more than 95%, allowed to stand for 5 h, the precipitate was collected, and dried at 50°C for 6 h to obtain 4.1 g of powdered PDRN.

[0053] Example 3

[0054] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0055] (1) 50 L of HEK293 cell culture medium was taken and centrifuged at 8000 r / min for 20 min. The HEK293 cells obtained after centrifugation were resuspended in 10 L of PBS buffer and centrifuged at 8000 r / min for 20 min to collect the cell precipitate; then 5 L of lysis solution was added to the cell precipitate and high-pressure homogenization was performed in a high-pressure homogenizer to obtain a lysate solution; the high-pressure homogenization parameters were pressure 400 bar and time 20 min;

[0056] The composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4;

[0057] (2) magnesium sulfate (final concentration of 30 mM), 25 mL of crude nuclease RXG-M enzyme solution, 2 g of protease protamex 1.6, 1 g of protease Alcalase 2.4L FG, 2 g of protease Flavourzyme 500MG, and 1 g of protease Novozym 2778 were added to 5000 g of the lysate solution obtained in step (1), and the mixture was incubated at 50° C. for 2 h. After the incubation, 50 g of sodium dodecyl sulfate (SDS) and 500 g of sodium chloride were added, stirred until fully dissolved, heated to 90° C. and maintained for 1 h to fully inactivate the protease and nuclease, then cooled to 25° C., and 1.5 times the volume of the lysate solution of 95% ethanol was added for precipitation to obtain a crude PDRN product;

[0058] (3) 48 g of the crude PDRN obtained in step (2) was dissolved in 6 L of a 10% sodium chloride solution, the pH was adjusted to 7.0, the temperature was raised to 55°C, 60 g of activated carbon was added, and the mixture was stirred for 1.5 h. The activated carbon was removed by filtering through a 0.45 μm sterile filter plate, and then filtered through a 0.22 μm filter to obtain a refined filtrate. After the refined filtrate was cooled to 30°C, 1.5 times the volume of 95% ethanol in the sodium chloride solution was added for precipitation. After sufficient precipitation for 3 h, the mixture was washed three times with 80% alcohol, and finally dehydrated with anhydrous ethanol to an alcohol content of more than 95%, allowed to stand for 5 h, the precipitate was collected, and dried at 50°C for 6 h to obtain 44 g of powdered PDRN.

[0059] Comparative Example 1

[0060] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0061] (1) 5 L of HEK293 cell culture medium was centrifuged at 8000 r / min for 20 min, the HEK293 cells obtained after centrifugation were resuspended in 1 L of PBS buffer, and centrifuged at 8000 r / min for 20 min to collect the cell precipitate; then 500 mL of lysis solution was added to the cell precipitate and high-pressure homogenization was performed in a high-pressure homogenizer to obtain a lysate solution; the high-pressure homogenization parameters were pressure 400 bar and time 20 min;

[0062] The composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4;

[0063] (2) magnesium sulfate (final concentration of 30 mM), 2 mL of nuclease RXG enzyme solution, and 0.5 g of protease protamex 1.6 were added to 500 g of the lysate solution obtained in step (1) in sequence, and incubated at 50° C. for 2 h. After the incubation, 5 g of sodium dodecyl sulfate (SDS) and 50 g of sodium chloride were added, stirred until fully dissolved, heated to 90° C. and maintained for 1 h to fully inactivate the protease and nuclease, then cooled to 25° C., and 1.5 times the volume of the lysate solution was added with 95% ethanol for precipitation to obtain a crude PDRN product;

[0064] (3) 4 g of the crude PDRN obtained in step (2) was dissolved in 500 mL of a 10% sodium chloride solution, the pH was adjusted to 7.0, the temperature was raised to 55°C, 7.5 g of activated carbon was added, and the mixture was stirred for 1.5 h. The activated carbon was removed by filtering through a 0.45 μm sterile filter plate, and then filtered through a 0.22 μm filter to obtain a refined filtrate. After the refined filtrate was cooled to 30°C, 1.5 times the volume of 95% ethanol in the sodium chloride solution was added for precipitation. After sufficient precipitation for 3 h, the mixture was washed three times with 80% alcohol, and finally dehydrated with anhydrous ethanol to an alcohol content of more than 95%, allowed to stand for 5 h, the precipitate was collected, and dried at 45°C for 6 h to obtain 2.1 g of powdered PDRN.

[0065] Comparative Example 2

[0066] A method for preparing PDRN derived from HEK293 cells comprises the following steps:

[0067] (1) 5 L of HEK293 cell culture medium was centrifuged at 8000 r / min for 20 min, the HEK293 cells obtained after centrifugation were resuspended in 1 L of PBS buffer, and centrifuged at 8000 r / min for 20 min to collect the cell precipitate; then 500 mL of lysis solution was added to the cell precipitate and high-pressure homogenization was performed in a high-pressure homogenizer to obtain a lysate solution; the high-pressure homogenization parameters were pressure 400 bar and time 20 min;

[0068] The composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4;

[0069] (2) magnesium sulfate (final concentration of 30 mM), 2 mL of nuclease RXG enzyme solution, 0.1 g of protease Alcalase 2.4L FG, 0.2 g of protease Flavourzyme 500MG, and 0.2 g of protease Novozym 2778 were added to 500 g of the lysate solution obtained in step (1) in sequence, and the mixture was incubated at 50° C. for 2 h. After the incubation, 5 g of sodium dodecyl sulfate (SDS) and 50 g of sodium chloride were added, stirred until fully dissolved, heated to 90° C. and maintained for 1 h to fully inactivate the protease and nuclease, then cooled to 25° C., and 1.5 times the volume of the lysate solution was added with 95% ethanol for precipitation to obtain a crude PDRN product;

[0070] (3) 3.9 g of the crude PDRN obtained in step (2) was dissolved in 550 mL of a 10% sodium chloride solution, the pH was adjusted to 7.0, the temperature was raised to 55°C, 6.5 g of activated carbon was added, and the mixture was stirred for 1.5 h. The activated carbon was removed by filtering through a 0.45 μm sterile filter plate, and then filtered through a 0.22 μm filter to obtain a refined filtrate. After the refined filtrate was cooled to 30°C, 1.5 times the volume of 95% ethanol in the sodium chloride solution was added for precipitation. After sufficient precipitation for 3 h, the mixture was washed three times with 80% alcohol, and finally dehydrated with anhydrous ethanol to an alcohol content of more than 95%, allowed to stand for 5 h, the precipitate was collected, and dried at 50°C for 6 h to obtain 2.3 g of powdered PDRN.

[0071] Experimental Example 1

[0072] Weigh 0.05 g of each of the PDRN powders prepared in Example 1, Example 2, Example 3 and Comparative Example 1 and Comparative Example 2, add 100 mL of purified water, stir to fully dissolve, and then perform 1% agarose gel electrophoresis at a constant voltage of 120 V for 30 min to identify the size of the PDRN fragments. The results are as follows: Figure 1 shown.

[0073] The DNA content in PDRN was determined by ultraviolet spectrophotometry, the protein content in PDRN was determined by Folin phenol method, and the endotoxin content in PDRN was determined by turbidity method. The results are shown in Table 1.

[0074] Table 1. PDRN related indicators extracted

[0075]

[0076] Depend on Figure 1As shown in Table 1, the present invention can extract PDRN with higher purity from HEK293 cells, with DNA content>99%, protein content<0.5%, and extraction rate>0.8‰. The above three indicators are better than those of Comparative Examples 1 and 2, indicating that the nuclease obtained by the present invention has a better application value in extracting and preparing PDRN. At the same time, from the perspective of molecular weight, the molecular weight distribution of Examples 1, 2 and 3 is 200-1000KDa, which is lower than that of Comparative Examples 1 and 2, indicating that the nuclease mutant RXG-M obtained by the present invention has a better enzymolysis effect, and can obtain PDRN with smaller molecular fragments and higher activity.

[0077] Experimental Example 2

[0078] 1. Determine the inhibitory effect of PDRN on the release of inflammatory factors TNF-α and IL-1β in human skin keratinocytes-HaCaT induced by UVA. The specific method is as follows

[0079] HaCaT cells were cultured in MEM medium (containing 1‰ non-essential amino acids, 10% fetal bovine serum, 100U / mL penicillin, and 100mg / mL streptomycin) at 37°C, 5% carbon dioxide, and saturated humidity. When the cells were in a monolayer and sub-confluent state, they were digested with 0.25% trypsin to make a single cell suspension for subculture.

[0080] The subcultured cells were inoculated in a 9.0 cm dish and cultured until the cells were in a sub-confluent state; the samples were divided into four groups (blank group, UV group, PDRN group of the present invention and salmon PDRN group), PDRN (DMSO dissolved) and salmon PDRN (DMSO dissolved) prepared in Example 1 of the present invention were added to the cell culture medium of the PDRN group of the present invention and the salmon PDRN group at a final concentration of 3 μM, an equal amount of DMSO was added to the blank group and the UV group, the culture medium was discarded after 1 hour, the PBS buffer was rinsed 3 times, and PBS buffer was added at 5 mL / dish, the blank group was not irradiated, the UV group, the PDRN group of the present invention and the salmon PDRN group were irradiated at 2.4 J / cm 2 After irradiation for 2 h, the levels of TNF-α and IL-1β in the cell supernatant were detected by ELISA.

[0081] Depend on Figure 2 and Figure 3 It can be seen that compared with the blank group (not receiving UV irradiation), the TNF-α and IL-1β levels in the UV group were significantly increased, indicating that 2.4 J / cm 2Irradiation for 2 hours can induce the production of cellular inflammatory factors TNF-α and IL-1β. The contents of inflammatory factors TNF-α and IL-1β in both the experimental groups, whether the PDRN group of the present invention or the salmon PDRN group, were lower than those in the UV group, indicating that PDRN can effectively reduce the production and release of UV-induced inflammatory factors, and the inhibitory effects of the PDRN of the present invention and the salmon PDRN are almost equivalent.

[0082] Therefore, the HEK293 cell-derived PDRN extracted in the present invention can effectively inhibit the release of inflammatory factors TNF-α and IL-1β, thereby playing an anti-inflammatory and soothing and repairing role, and its anti-inflammatory activity is almost consistent with that of PDRN derived from salmon sperm.

[0083] 2. Determine the cell scratch repair function of PDRN. The specific method is as follows:

[0084] ① Cell culture: First, use a marker pen to mark each well in the back of the 6-well plate in three equal parts horizontally and vertically, and divide each well into approximately (5-15)×10 5 HaCaT cells were inoculated, and three groups were set up: a negative control group, a PDRN group of the present invention, and a salmon sperm PDRN group, with 3 replicate wells in each group.

[0085] ② Scratch test: Use a marker pen to draw 3 horizontal lines at the bottom of the well plate with a ruler. After 24 hours of cell culture, use a 10μL gun tip to align the well plate vertically with a ruler (you can also use a special cell culture dish for scratching), push the longitudinal line downward to form a scratch, rinse the cells 3 times with PBS buffer, remove the scratched cells, add 2mL of serum-free medium to the PDRN group and salmon sperm PDRN group of the present invention, respectively, to prepare PDRN and salmon sperm PDRN in Example 1 of the present invention, with a final concentration of 0.5mg / mL, and only serum-free medium is added to the negative control group. Cultivate in a 37°C, 5% carbon dioxide incubator, take pictures under a 40x microscope at 24h, 48h, and 72h, with the intersection of the horizontal and longitudinal lines as the core, and obtain photos of 9 parts in each well, and 27 data in total for each group.

[0086] ③Data processing: Measure the scratch area, compare it with the initial scratch area, and calculate the percentage of the scratch area.

[0087] Depend on Figure 4 It can be seen that after treating keratinocytes for 0 to 72 hours, the percentage of scratch area in the PDRN group continued to decrease compared with the negative control, and the repair effects of the HEK293 cell-derived PDRN and salmon sperm PDRN extracted by the present invention were almost equivalent, indicating that the PDRN prepared by the present invention has the effect of repairing and activating cells.

Claims

1. A method for preparing PDRN derived from HEK293 cells, characterized in that: The steps include: (1) HEK293 cells were resuspended in PBS buffer and centrifuged to collect the cell pellet; then lysis buffer was added to the cell pellet for high-pressure homogenization to obtain a lysate solution; (2) adding magnesium sulfate, protease and nuclease to the lysate solution obtained in step (1), incubating at 45-55°C for 1-2 hours, adding sodium dodecyl sulfate and sodium chloride after the incubation, stirring until fully dissolved, inactivating the enzyme and precipitating with alcohol to obtain a crude PDRN; Wherein, the nuclease is a nuclease mutant RXG-M, whose amino acid sequence is shown in SEQ ID NO.1, and whose nucleotide sequence is shown in SEQ ID NO.2; (3) The crude PDRN obtained in step (2) is dissolved in a sodium chloride solution, the pH is adjusted to 6.0-8.0, the temperature is raised to 50-60°C, activated carbon is added, and the mixture is stirred for 1-2 hours. After filtering, alcohol precipitation, washing, dehydration and drying, PDRN is obtained.

2. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (1), the composition of the lysate is 50 mM Tris, 250 mM NaCl, 5 mM EDTA, pH 7.4; the volume ratio of the lysate to PBS buffer is (3-6):(10-15); the centrifugation parameters are 5000-10000 r / min, 15-30 min; the high-pressure homogenization is carried out in a high-pressure homogenizer, the parameters are pressure 300-500 bar, time 10-30 min.

3. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (2), the final concentration of magnesium sulfate in the lysate solution is 25-35 mM.

4. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (2), the protease is one or a combination of two or more of the protease protamex 1.6, the protease Alcalase 2.4 L FG, the protease Flavourzyme 500 MG, and the protease Novozym 2778; the mass ratio of the protease to the lysate solution is (0.05-0.15):

100.

5. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (2), the volume mass ratio of the nuclease to the lysate solution is (0.2-0.6):100, unit: mL / g; the mass ratio of the sodium dodecyl sulfate to the lysate solution is (0.2-2):100; the mass ratio of the sodium chloride to the lysate solution is (2-20):

100.

6. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (2), the enzyme inactivation and alcohol precipitation are specifically as follows: heating to 60-100°C and maintaining for 2-4 hours to fully inactivate proteases and nucleases, then cooling to 20-30°C, and adding 1-1.5 times the volume of the lysate solution of 95% ethanol for precipitation.

7. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (3), the mass volume ratio of the crude PDRN to the sodium chloride solution is 1:(5-100), unit: g / mL; the mass concentration of the sodium chloride solution is 4-10%.

8. The preparation method of PDRN derived from HEK293 cells as claimed in claim 1, characterized in that, In step (3), the mass volume ratio of the activated carbon to the sodium chloride solution is (1-2): 100, unit: g / mL; the filtering, alcohol precipitation, washing, dehydration and drying are specifically as follows: filtering through a 0.45 μm sterile filter plate to remove the activated carbon, then filtering through a 0.22 μm filter to obtain a refined filtrate, cooling the refined filtrate to 20-30°C, adding 1-1.5 times the volume of the sodium chloride solution to 95% ethanol for precipitation, washing and dehydrating the precipitate with ethanol, and drying at 40-60°C for 3-6 hours.

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