A culture method of pdrn genetically engineered cells, a product purification method and application thereof

Abstract

The present application belongs to the field of bioengineering technology, and particularly relates to a culture method of PDRN genetically engineered cells, a product purification method and application. The present application solves the problems of low yield, growth-synthesis imbalance and poor purification effect in the production of existing recombinant PDRN. During the culture, the fermentation medium is optimized, the nucleotide metabolic pathway is up-regulated by low-dose PDRN synthesis precursors and metabolic activators in cooperation, and the balance between bacterial growth and product synthesis is maintained by controlling the fermentation pH and dissolved oxygen in stages. During the purification, the fermentation broth is pretreated, then microfiltration, ultrafiltration, Q-agarose gel FF strong anion exchange chromatography and freeze-drying are sequentially performed. The present application improves the yield of PDRN, and the finished product has high purity and yield, which is suitable for industrial production. The obtained PDRN can be used in the fields of biomaterials, medicine, medical aesthetics and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for culturing PDRN gene-engineered cells, a method for purifying the product, and its application. Background Technology

[0002] PDRN (polydeoxyribonucleotide) is a natural biological macromolecule polymerized from deoxyribonucleotides. It has excellent biocompatibility and multiple biological activities, and can play a core biological role in promoting tissue repair, anti-inflammatory and analgesic effects, regulating collagen synthesis and repairing the skin barrier.

[0003] Currently, industrial-scale PDRN production mainly relies on extraction from the gonads of aquatic animals such as salmon. While this process can yield PDRN with a natural structure, it has significant limitations: the salmon extraction process is complex, raw material supply is constrained by factors such as the fish's reproductive and maturation cycle and the fishing season, production costs fluctuate greatly, and production capacity is unstable, making it difficult to support large-scale, continuous supply demands. To overcome these shortcomings, researchers are gradually exploring the use of genetic engineering technology to construct recombinant microorganisms for PDRN production. By introducing exogenous genes related to PDRN synthesis enzyme systems into host bacteria such as Escherichia coli, efficient fermentation production of PDRN can be achieved. Compared with traditional extraction methods, the recombinant fermentation method has significant advantages such as readily available raw materials, short production cycle, good batch stability, and no risk of animal-derived contamination.

[0004] However, existing recombinant PDRN fermentation technologies still face a series of pressing technical challenges. First, the introduction of exogenous genes increases the host's metabolic burden, affecting normal cell growth and leading to low fermentation density; simultaneously, plasmids are easily lost, resulting in poor genetic stability. Second, existing culture media lack precise nutritional design for the PDRN synthesis pathway, and optimizing the dosage of key precursors such as 5-phosphate ribose, histidine, and uridine has become a bottleneck limiting PDRN synthesis. Third, existing technologies lack effective regulation of key metabolic nodes such as the cAMP signaling pathway and tricarboxylic acid cycle intermediates, making it difficult to efficiently guide metabolic flux towards PDRN synthesis. Fourth, fermentation processes often employ simple isothermal and constant-rate cultivation, without staged, precise control of parameters such as pH, dissolved oxygen, and temperature for different stages of PDRN synthesis, and the feeding strategy is simplistic. Fifth, downstream purification processes suffer from low yields and unstable purity.

[0005] In summary, existing recombinant PDRN production technologies suffer from several problems, including limited cell growth, insufficient precursor supply, lack of metabolic regulation, crude process parameters, and low purification yields. These issues result in generally low PDRN fermentation yields, making it difficult to meet the economic requirements of industrial production. Therefore, developing a genetically engineered cell culture method and supporting purification process that can effectively increase PDRN yield, maintain the growth stability of engineered strains, and achieve highly efficient metabolic flux guidance is of significant application value. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a method for culturing PDRN-engineered cells, a method for purifying the product, and its applications. This invention optimizes the fermentation medium formulation, employing a specific combination of low-dose precursors and metabolic activators to synergistically promote the upregulation of nucleotide metabolic pathways in engineered bacteria, significantly increasing PDRN yield. Simultaneously, by combining staged fermentation process control, the balance between cell growth and product synthesis is effectively maintained. Furthermore, a complete purification process has been established, yielding high-purity, high-yield PDRN products.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a method for culturing PDRN genetically engineered cells, wherein the PDRN genetically engineered cells are recombinant Escherichia coli TA4-41, numbered CGMCC No. 33625; the culturing method includes the following steps: (1) Recombinant Escherichia coli TA4-41 was inoculated into seed culture medium for activation culture to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium for fermentation culture. During the fermentation process, feed medium is added and the fermentation conditions are controlled to obtain a fermentation broth containing PDRN.

[0008] Specifically, the fermentation medium comprises the following components: disodium hydrogen phosphate 2.5-3.0 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, ammonium sulfate 0.5-0.8 g / L, magnesium chloride 0.03-0.05 g / L, calcium nitrate tetrahydrate 0.02-0.04 g / L, glycerol 5-8 g / L, L-histidine 0.02-0.05 g / L, uridine 0.01-0.03 g / L, ribose 5-phosphate 0.008-0.015 g / L, cyclic adenosine monophosphate 0.0008-0.0015 g / L, and α-ketoglutarate 0.03-0.08 g / L; The supplemental culture medium contains the following components: dipotassium hydrogen phosphate 30-60 g / L, sodium dihydrogen phosphate 10-25 g / L, ammonium sulfate 5-15 g / L, glycerol 120-180 g / L, uridine 0.02-0.06 g / L, cyclic adenosine monophosphate 0.001-0.003 g / L, and α-ketoglutarate 0.05-0.12 g / L.

[0009] Preferably, the fermentation medium comprises the following components: disodium hydrogen phosphate 2.8 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 0.6 g / L, magnesium chloride 0.04 g / L, calcium nitrate tetrahydrate 0.03 g / L, glycerol 6 g / L, L-histidine 0.03 g / L, uridine 0.02 g / L, ribose 5-phosphate 0.01 g / L, cyclic adenosine monophosphate (cAMP) 0.001 g / L, and α-ketoglutarate 0.05 g / L; The supplemental culture medium contains the following components: 45 g / L dipotassium hydrogen phosphate, 18 g / L sodium dihydrogen phosphate, 10 g / L ammonium sulfate, 150 g / L glycerol, 0.04 g / L uridine, 0.002 g / L cyclic adenosine monophosphate and 0.08 g / L α-ketoglutarate.

[0010] Specifically, the seed activation culture described in step (1) includes primary seed recovery and secondary seed activation; The seed culture medium used for the primary seed regeneration contains 0.01-0.02 g / L L-histidine, 0.005-0.015 g / L uridine, and 0.0003-0.0008 g / L cyclic adenosine monophosphate. The seed culture medium used for the secondary seed activation contains 0.02-0.05 g / L L-histidine, 0.01-0.03 g / L uridine, 0.0008-0.0015 g / L cyclic adenosine monophosphate, and 0.01-0.04 g / L α-ketoglutarate.

[0011] Preferably, the seed culture medium used for the primary seed regeneration contains 0.015 g / L L-histidine, 0.01 g / L uridine, and 0.0005 g / L cyclic adenosine monophosphate. The seed culture medium used for the secondary seed activation contains 0.03 g / L L-histidine, 0.02 g / L uridine, 0.001 g / L cyclic adenosine monophosphate, and 0.025 g / L α-ketoglutarate.

[0012] Specifically, the fermentation conditions described in step (2) are as follows: 0-24h, pH value controlled at 7.0-7.2, temperature 35-39℃, stirring speed 200-280rpm, maintaining dissolved oxygen concentration not less than 45%; 24-48h, pH value controlled at 6.7-6.9, temperature 34-38℃, stirring speed 280-320rpm, maintaining dissolved oxygen concentration not less than 30%. The feeding method for the fed-batch culture medium is as follows: feeding is started when fermentation has progressed to 8-12 hours, and fed at a constant rate of 10-15 mL / h until fermentation ends.

[0013] In another aspect, the present invention provides a method for purifying PDRN, comprising the following steps: (1) The fermentation broth obtained by the above culture method is subjected to solid-liquid separation, the fermentation supernatant is discarded, and the cell precipitate is collected; (2) After washing the bacterial precipitate, resuspend it, add lysozyme for enzymatic hydrolysis and cell disruption, then homogenize under high pressure, centrifuge and collect the supernatant to obtain crude PDRN extract. (3) Filter the crude PDRN extract through a microfiltration membrane and collect the microfiltrate; (4) The microfiltration filtrate was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to obtain PDRN concentrate; (5) Purify the PDRN concentrate by anion exchange chromatography, collect the eluent, and obtain the pure PDRN solution; (6) Freeze-dry the pure PDRN solution to obtain the finished PDRN product.

[0014] Specifically, in step (2), the final concentration of lysozyme added is 1-3 mg / mL, and the enzymatic hydrolysis conditions are incubation at 35-39℃ for 0.5-1.5 h; the pressure of the high-pressure homogenization is 600-1000 bar, and the number of homogenization times is 1-3. The conditions for anion exchange chromatography described in step (5) are as follows: Q-agarose gel FF (Q Sepharose Fast Flow) anion exchange medium is used, the equilibration solution is Tris-HCl buffer, pH 7.8-8.2; after loading the sample, it is washed with equilibration solution containing 0.15-0.25 mol / L NaCl; elution is performed with equilibration solution containing 0.6-1.0 mol / L NaCl, and the eluent corresponding to the 260 nm UV absorption peak is collected; The freeze-drying process described in step (6) is gradient freeze-drying, with the following procedure: pre-freezing at -40℃ to -35℃ for 1-3 hours, freezing at -55℃ to -45℃ for 2-6 hours, and deep freezing at -65℃ to -55℃ for 12-24 hours, with a vacuum degree ≤10Pa.

[0015] Preferably, the concentration of the Tris-HCl buffer solution is 10-50 mM; more preferably, it is 10-30 mM.

[0016] In another aspect, the present invention provides a method for preparing PDRN, wherein recombinant Escherichia coli TA4-41 is cultured using the aforementioned culture method, and the PDRN obtained from the culture is purified using the aforementioned purification method to obtain the PDRN product.

[0017] In another aspect, the present invention provides the application of PDRN obtained by the aforementioned culture method, purification method or preparation method in the preparation of biomaterials, pharmaceuticals or cosmetics.

[0018] Specifically, the biomaterial is a biomaterial used in tissue engineering, wound dressings, or tissue repair scaffolds; The drug is used to promote tissue repair, reduce inflammation, promote wound healing, or treat osteoarthritis; The cosmetics mentioned are cosmetics used for skin repair, anti-aging, or improving skin barrier function.

[0019] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0020] More specifically, the pharmaceutically acceptable excipients are selected from one or more of the following: fillers, binders, disintegrants, lubricants, preservatives, antioxidants, buffers, solubilizers, emulsifiers, stabilizers, suspending agents, wetting agents, flavoring agents, colorants, osmotic pressure regulators, pH regulators, complexing agents, diluents, absorption enhancers, and surfactants.

[0021] Preferably, the filler is lactose, starch, microcrystalline cellulose, mannitol, sorbitol, dextrin, sucrose, glucose, dicalcium phosphate, calcium sulfate, or calcium carbonate. The adhesive is polyvinylpyrrolidone, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, starch paste, gelatin, gum arabic, sodium alginate, or polyvinyl alcohol. The disintegrant is sodium carboxymethyl starch, crospovidone, crospovidone sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, starch or microcrystalline cellulose; The lubricant is magnesium stearate, stearic acid, sodium stearate fumarate, talc, micronized silica gel, polyethylene glycol, or sodium lauryl sulfate. The preservatives are benzyl alcohol, benzalkonium chloride, sodium benzoate, potassium sorbate, methylparaben, propylparaben, chlorobutanol, or thimerosal. The antioxidant is vitamin C, vitamin E, cysteine, sodium thiosulfate, sodium bisulfite, sodium metabisulfite, butylated hydroxyanisole, or butylated hydroxytoluene. The buffer is a phosphate buffer, acetate buffer, citrate buffer, Tris buffer, histidine buffer, or succinate buffer. The solubilizer is polysorbate, poloxamer, polyoxyethylene hydrogenated castor oil, sodium lauryl sulfate, or sucrose fatty acid ester. The emulsifier is lecithin, soybean lecithin, lanolin, beeswax, glyceryl monostearate, stearic acid, cetyl alcohol, or polyoxyethylene fatty acid ester; The stabilizer is sucrose, trehalose, mannitol, sorbitol, glycine, arginine, monosodium glutamate, human serum albumin, polyethylene glycol, or cyclodextrin. The suspending agent is sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, xanthan gum, guar gum, gum arabic, tragacanth gum, or magnesium aluminum silicate; The wetting agent is glycerin, propylene glycol, polyethylene glycol, polysorbate, or poloxamer; The flavoring agent is sucrose, sodium saccharin, aspartame, steviol glycoside, menthol, citric acid, or flavoring. The osmotic pressure regulator is sodium chloride, potassium chloride, glucose, glycerol, or mannitol; The pH adjuster is hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, or disodium hydrogen phosphate. The complexing agent is ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, or calcium sodium ethylenediaminetetraacetic acid.

[0022] Specifically, the dosage form of the drug is an injection, a lyophilized powder for injection, a topical preparation, a spray, a gel, an ointment, a cream, a patch, a solution, a suspension, an emulsion, a tablet, a capsule, a granule, a pill, or a powder.

[0023] Specifically, the cosmetics also include excipients acceptable in the cosmetics field.

[0024] More specifically, the excipients acceptable in the cosmetics field are selected from one or more of the following: moisturizers, emulsifiers, thickeners, preservatives, antioxidants, pH adjusters, chelating agents, transdermal absorption enhancers, surfactants, solvents, fragrances, pigments, cooling agents, and skin conditioning agents.

[0025] Preferably, the moisturizer is glycerin, propylene glycol, butylene glycol, pentanediol, sorbitol, sodium hyaluronate, polyethylene glycol, trehalose, betaine, urea, sodium lactate, or ceramide. The emollient is squalane, squalene, jojoba oil, olive oil, almond oil, silicone oil, lanolin, petrolatum, mineral oil, isopropyl myristate, or isopropyl palmitate. The emulsifier is lecithin, soybean lecithin, stearic acid, cetyl alcohol, cetearyl alcohol, glyceryl monostearate, polyoxyethylene fatty acid ester, polyoxyethylene fatty alcohol ether, sorbitan fatty acid ester, or polysorbate. The thickener is carbomer, xanthan gum, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, acrylic acid copolymer, guar gum, sodium alginate, or polyvinyl alcohol; The preservative is phenoxyethanol, methylparaben, propylparaben, sodium benzoate, potassium sorbate, chlorphenesin, pentylene glycol, or ethylhexylglycerin. The antioxidants are vitamin C, vitamin E, vitamin E acetate, coenzyme Q10, ferulic acid, resveratrol, tea polyphenols, butylated hydroxyanisole, or butylated hydroxytoluene. The pH adjuster is citric acid, sodium citrate, lactic acid, sodium lactate, triethanolamine, sodium hydroxide, or hydrochloric acid; The chelating agent is ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, or tetrasodium ethylenediaminetetraacetic acid; The transdermal absorption enhancer is azone, propylene glycol, oleic acid, linoleic acid, menthol, eucalyptus oil, urea, or dimethyl sulfoxide; The surfactant is polysorbate, poloxamer, sodium lauryl sulfate, fatty acid glycerides, or sucrose esters; The solvent is water, ethanol, propylene glycol, butylene glycol, glycerol, or polyethylene glycol; The cooling agent is menthol, menthol, camphor, or borneol; The skin conditioning agents are allantoin, panthenol, niacinamide, ceramide, hyaluronic acid, collagen, squalane, aloe vera extract, chamomile extract, or centella asiatica extract.

[0026] Specifically, the dosage form of the cosmetic is an aqueous solution, serum, lotion, cream, gel, spray, mask, freeze-dried powder, aerosol, stick, powder, or oil.

[0027] The beneficial effects of this invention are as follows: 1. This invention provides the necessary raw materials for PDRN biosynthesis by scientifically combining the precursor and activator in the culture medium, while avoiding the metabolic burden on the cells caused by excessive precursor. At the same time, it activates the product synthesis-related pathways, achieving a synergistic improvement in efficient cell proliferation and efficient PDRN expression, and significantly improving the fermentation culture efficiency of PDRN.

[0028] 2. The fermentation product obtained by the cultivation process of the present invention, combined with a suitable purification process, can efficiently remove various impurities while ensuring the structural stability of PDRN, thereby achieving high purity and high yield of the product, significantly improving the quality of the finished product, forming a synergistic effect with the efficient cultivation process, and ensuring the overall production effect of PDRN.

[0029] 3. The culture medium components and reagents used in the PDRN gene-engineered cell culture process of this invention are all conventional commercial products in the field of bioengineering. The culture parameters are easy to adjust and the operation process is simple. It can be automated through conventional fermentation equipment without special customization, which reduces the production threshold and cost and is suitable for industrial-scale culture and production. Detailed Implementation

[0030] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0031] Example 1 1.1 Fermentation medium The formula composition is as follows: Disodium hydrogen phosphate 2.8 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 0.6 g / L, magnesium chloride 0.04 g / L, calcium nitrate tetrahydrate 0.03 g / L, glycerol 6 g / L, L-histidine 0.03 g / L, uridine 0.02 g / L, ribose 5-phosphate 0.01 g / L, cyclic adenosine monophosphate (cAMP) 0.001 g / L, α-ketoglutarate 0.05 g / L, in ultrapure water, diluted to 1 L.

[0032] 1.2 Feeding Culture Medium The formula composition is as follows: Dipotassium hydrogen phosphate 45 g / L, sodium dihydrogen phosphate 18 g / L, ammonium sulfate 10 g / L, glycerol 150 g / L, uridine 0.04 g / L, cyclic adenosine monophosphate (cAMP) 0.002 g / L, α-ketoglutarate 0.08 g / L, with ultrapure water as solvent, and brought to a final volume of 1 L.

[0033] 1.3 Other reagents (1) Ampicillin sodium solution (100mg / mL): Weigh 1g of ampicillin sodium, dissolve it in 10mL of ultrapure water, filter it through a 0.22μm filter membrane to remove bacteria, dispense it into aliquots and freeze it at -20℃ for later use; (2) 5mol / L ammonia solution: Measure 33.5mL of concentrated ammonia (25%-28%), make up to 100mL with ultrapure water, filter through a 0.22μm filter membrane for sterilization and use. (3) PBS buffer (0.01mol / L, pH 7.4): Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4, dissolve them in ultrapure water and bring the volume up to 1L. Sterilize at 121℃ for 20min before use. (4) TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0): Weigh 1.211g of Tris and 0.372g of EDTA-Na2, dissolve them in ultrapure water and bring the volume up to 1L, adjust the pH to 8.0 with HCl, and autoclave at 121℃ for 20min before use. (5) Other reagents: PDRN standard (Aladdin, CAS: 100403-24-5); nucleic acid dye SYBR Green I was purchased from Thermo Fisher Scientific; sodium azide was analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] 1.4 Preparation method of culture medium Preparation of fermentation medium: Weigh each component accurately according to the formula, add ultrapure water, stir at room temperature for 30 minutes until completely dissolved, autoclave at 115℃ for 15 minutes, cool to below 60℃ and then use aseptically.

[0035] Preparation of supplemented culture medium: Weigh each component accurately according to the formula, add ultrapure water, stir at room temperature for 60 min until completely dissolved, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use.

[0036] Example 2: Fermentation Production of PDRN This embodiment uses the culture medium and matching reagents prepared in Example 1 to ferment and culture recombinant Escherichia coli TA4-41 (CGMCC No. 33625, disclosed in CN 121320218 A). The specific steps are as follows: 2.1 Preparation of highly active seed liquid (1) Primary seed revival: Take 200 μL of TA4-41 bacterial culture frozen at -80℃ and inoculate it into a 250 mL shake flask containing 40 mL of LB medium. The LB medium contains 0.015 g / L L-histidine, 0.01 g / L uridine, and 0.0005 g / L cAMP, and add 40 μL of 100 mg / mL ampicillin sodium solution (final concentration 1‰ v / v). Incubate at 37℃ and 250 rpm for 3 h with shaking.

[0037] (2) Secondary seed activation: Take 10 mL of the revived bacterial culture and transfer it to a 500 mL shake flask containing 100 mL of fresh LB medium. The LB medium contains 0.03 g / L L-histidine, 0.02 g / L uridine, 0.001 g / L cAMP, and 0.025 g / L α-ketoglutarate. Incubate at 37 °C and 280 rpm for 2 h with shaking until the bacterial culture reaches OD500. 600 A value of 3.2 ± 0.1 indicates a highly active seed solution.

[0038] 2.2 Fermentation culture in fermenters It uses a 5L fully automatic fermenter with a liquid capacity of 4L.

[0039] 4 L of the fermentation medium prepared in Example 1 was poured into a fermenter, and 20 mL of 5 mol / L ammonia solution and 10 mL of defoamer were added. The mixture was autoclaved at 115 °C for 15 min. After cooling to 37 °C, sterile air was introduced at a flow rate of 2.2 L / min, and the stirring speed was set to 200 rpm to pre-equilibrate dissolved oxygen to above 80%.

[0040] Add 4 mL of 100 mg / mL ampicillin sodium solution (final concentration 1‰ v / v) to the container, and then inoculate the highly active seed liquid prepared in step 2.1 at an inoculation rate of 1.2% (v / v).

[0041] During fermentation, the following parameters should be controlled: (1) 0-24h: pH value is controlled at 7.1±0.1 (maintained by automatic addition of 5mol / L ammonia solution), temperature is 37℃, and stirring speed is gradually increased in the range of 200-280rpm to maintain dissolved oxygen concentration not less than 45%; (2) 24-48h: The temperature is reduced to 36℃, the pH value is adjusted to 6.8±0.1, and the stirring speed is increased to 280-320rpm to maintain the dissolved oxygen concentration not less than 30%.

[0042] When fermentation reached 10 hours, the feeding program was started, and the feeding medium prepared in Example 1 was fed at a constant rate of 12 mL / h until the end of fermentation.

[0043] During fermentation, when the pH value deviates from the set range, a 5mol / L ammonia solution is automatically added to correct the pH; when foam is generated, a small amount of defoamer is added to control it.

[0044] Fermentation is terminated after 48 hours.

[0045] 2.3 Fermentation endpoint detection Take 3 mL of the fermentation broth after 48 hours of fermentation and test the following indicators. Each sample is repeated three times: (1) Bacterial OD 600 Value: Using uninoculated fermentation medium as a blank control, the absorbance value was measured at a wavelength of 600 nm using a spectrophotometer.

[0046] (2) PDRN yield: Quantitative fluorescence method was used. The fermentation supernatant was diluted appropriately, stained with SYBR Green I nucleic acid dye, and the fluorescence intensity was detected using a fluorescence spectrophotometer. Simultaneously, standard curves were established using PDRN standard solutions at concentrations of 0, 50, 100, 200, 500, and 1000 mg / L (linear correlation coefficient RL). 2 (≥0.99), the concentration of PDRN in the sample was calculated based on the standard curve.

[0047] The test result was: bacterial OD 600 Peak value = 18.5 ± 0.4, PDRN yield = 980 ± 25 mg / L (based on fermentation broth volume).

[0048] Example 3: Purification of PDRN and Detection of Finished Product 3.1 Fermentation broth pretreatment and combined cell wall disruption Take the fermentation broth after the fermentation in Example 2, centrifuge it at 4℃ and 8000rpm for 10min, discard the fermentation supernatant, and collect the cell precipitate.

[0049] The bacterial cell pellet was washed twice with PBS buffer and then resuspended in PBS buffer at a ratio of 1:4 (w / v). Lysozyme was added to a final concentration of 2 mg / mL, and the pellet was incubated at 37°C for 1 h. The pellet was then homogenized twice using an autoclave at 800 bar to ensure complete cell disruption.

[0050] The cell wall disruption solution was centrifuged at 4℃ and 10000rpm for 15min, and the supernatant was collected to obtain crude PDRN extract.

[0051] 3.2 Microfiltration-Ultrafiltration Combined Filtration for Impurity Removal and Concentration The crude PDRN extract was vacuum filtered through a 0.22 μm microporous membrane, and the microfiltrate was collected to remove large particulate impurities and cell debris.

[0052] The microfiltration filtrate was transferred into a stirred ultrafiltration cup equipped with a 50 kDa molecular weight cutoff ultrafiltration membrane and concentrated by ultrafiltration at 4°C until the volume of the concentrate was 1 / 10 of the original volume, thus obtaining PDRN concentrate.

[0053] 3.3 Anion exchange chromatography purification Purification was performed using anion exchange chromatography.

[0054] (1) Equilibration: Pack the anion exchange medium (Q-agarose gel FF (Q Sepharose Fast Flow), a strong anion exchange medium with quaternary ammonium groups) into the chromatography column, and equilibrate the column with equilibration buffer (20 mM Tris-HCl buffer, pH 8.0) at an appropriate flow rate until the pH and conductivity of the effluent are stable and the baseline is stable (OD). 260 <0.01); (2) Sample loading: Load the PDRN concentrate obtained in step 3.2 at a flow rate of 1 mL / min; (3) Washing: Wash the chromatography column with equilibration buffer containing 0.2 mol / L NaCl at a flow rate of 1 mL / min to remove unbound impurity proteins and nucleic acids until the baseline is stable; (4) Elution: Elute with an equilibrium solution containing 0.6-1.0 mol / L NaCl, and collect the eluent corresponding to the 260 nm UV absorption peak, which is the pure PDRN solution; (5) Regeneration: Clean the chromatography column in place with 1-2 column volumes of 1 mol / L NaOH solution, and then rebalance it with equilibration buffer for the next use.

[0055] 3.4 Gradient freeze drying and finished product preparation The collected PDRN purified solution was diluted or concentrated with TE buffer to a concentration of 10 mg / mL. Sodium azide was added to a final concentration of 0.01% as a preservative.

[0056] The above solution was placed in a low-temperature vacuum gradient freeze dryer and freeze-dried according to the following procedure: pre-freezing at -40℃ for 2 hours → freezing at -50℃ for 4 hours → deep freezing at -60℃ for 18 hours, maintaining a vacuum degree of ≤10Pa throughout the process.

[0057] After freeze-drying, the product is sealed and packaged under aseptic conditions to obtain a white powdered PDRN product.

[0058] 3.5 Finished Product Quality Inspection The PDRN freeze-dried product obtained in step 3.4 was subjected to quality testing. The testing methods and results are as follows: (1) Purity detection Purity was determined by high performance liquid chromatography (HPLC). An appropriate amount of lyophilized PDRN was dissolved and diluted to a suitable concentration with the mobile phase, injected, and analyzed. The chromatographic purity of PDRN was calculated using the peak area normalization method.

[0059] Test results: The HPLC purity of the three batches of samples were 98.5%, 98.1%, and 98.8%, respectively, with an average purity greater than 98%.

[0060] (2) Purification yield Purification yield (%) = (Total PDRN after purification / Total PDRN in bacterial cells before cell disruption) × 100%. The total PDRN in the bacterial cells before cell disruption is calculated by measuring the PDRN concentration and volume of the crude extract after cell disruption.

[0061] Test results: The purification yields of the three batches of samples were 85.2%, 84.8%, and 86.0%, respectively, with an average yield greater than 85%.

[0062] Comparative Example Comparative Example 1: The fermentation medium differed from that of Example 1 in that the low-dose precursor was replaced with a high-concentration precursor (L-histidine 0.15 g / L, uridine 0.1 g / L, 5-phosphoribose 0.08 g / L), and the activators cAMP and α-ketoglutarate were not added. The remaining components were the same as those of the fermentation medium of Example 1.

[0063] Comparative Example 2: The fermentation medium differed from that of Example 1 in that the activator cAMP and α-ketoglutarate were not added, and only the same low-dose precursors (L-histidine 0.03 g / L, uridine 0.02 g / L, 5-phosphoribose 0.01 g / L) at the same concentration as in Example 1 were retained, while the other components were the same as those of the fermentation medium of Example 1.

[0064] Comparative Example 3: The fermentation medium differed from that of Example 1 in that L-histidine, uridine, and ribose 5-phosphate were not added, but the same concentration of activator (cAMP 0.001 g / L, α-ketoglutarate 0.05 g / L) was retained as in Example 1, and the remaining components were the same as those of the fermentation medium of Example 1.

[0065] After each group's fermentation was completed, the cell OD was measured uniformly. 600 Peak values ​​and PDRN yields (both based on fermentation broth) are shown in Table 1.

[0066] Table 1

[0067] The results above show that the bacterial OD in Example 2 is... 600 The peak concentration reached 18.5±0.4 mg / L, and the PDRN yield reached 980±25 mg / L, both significantly higher than Comparative Examples 1-3. Comparative Example 2, with only a low dose of precursor and no activator, showed the best cell growth and product expression among the comparative examples, but was still 11.9% and 29.1% lower than Example 2, respectively. Comparative Example 3, with only an activator and no precursor, lacked synthetic raw materials, so the activator could not function, and its effect was weaker than Comparative Example 2. Comparative Example 1, with a high dose of precursor and no activator, resulted in excessive precursor causing metabolic burden on the cells, inhibiting cell growth and PDRN synthesis, and its indicators were the lowest among all groups.

[0068] The above results confirm that low-dose precursors provide raw materials for PDRN synthesis without metabolic toxicity, and cAMP and α-ketoglutarate activators can regulate metabolic pathways. The two work synergistically to increase bacterial biomass and PDRN production.

[0069] Furthermore, the purification process of this invention yields PDRN products with an average HPLC purity of over 98% and an average purification yield of over 85%. The process is simple to operate, highly controllable, effectively removes impurities, and ensures the stability of the PDRN structure, making it suitable for industrial-scale production.

[0070] In summary, the fermentation and purification technology of this invention solves the problem of balancing cell growth and product expression in traditional PDRN fermentation, and has the advantages of high yield, high purity and low cost. It provides a feasible solution for the large-scale production of PDRN and has good prospects for industrial application.

[0071] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for culturing PDRN genetically engineered cells, characterized in that, The PDRN-engineered cells are recombinant Escherichia coli TA4-41, numbered CGMCC No. 33625; the culture method includes the following steps: (1) Recombinant Escherichia coli TA4-41 was inoculated into seed culture medium for activation culture to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium for fermentation culture. During the fermentation process, the feed medium is added and the fermentation conditions are controlled to obtain a fermentation broth containing PDRN. The fermentation medium comprises the following components: disodium hydrogen phosphate 2.5-3.0 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, ammonium sulfate 0.5-0.8 g / L, magnesium chloride 0.03-0.05 g / L, calcium nitrate tetrahydrate 0.02-0.04 g / L, glycerol 5-8 g / L, L-histidine 0.02-0.05 g / L, uridine 0.01-0.03 g / L, ribose 5-phosphate 0.008-0.015 g / L, cyclic adenosine monophosphate 0.0008-0.0015 g / L, and α-ketoglutarate 0.03-0.08 g / L; The supplemental culture medium contains the following components: dipotassium hydrogen phosphate 30-60 g / L, sodium dihydrogen phosphate 10-25 g / L, ammonium sulfate 5-15 g / L, glycerol 120-180 g / L, uridine 0.02-0.06 g / L, cyclic adenosine monophosphate 0.001-0.003 g / L, and α-ketoglutarate 0.05-0.12 g / L.

2. The cultivation method according to claim 1, characterized in that, The fermentation medium contains the following components: disodium hydrogen phosphate 2.8 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 0.6 g / L, magnesium chloride 0.04 g / L, calcium nitrate tetrahydrate 0.03 g / L, glycerol 6 g / L, L-histidine 0.03 g / L, uridine 0.02 g / L, ribose 5-phosphate 0.01 g / L, cyclic adenosine monophosphate 0.001 g / L, and α-ketoglutarate 0.05 g / L; The supplemental culture medium contains the following components: 45 g / L dipotassium hydrogen phosphate, 18 g / L sodium dihydrogen phosphate, 10 g / L ammonium sulfate, 150 g / L glycerol, 0.04 g / L uridine, 0.002 g / L cyclic adenosine monophosphate and 0.08 g / L α-ketoglutarate.

3. The cultivation method according to claim 1, characterized in that, The activation culture described in step (1) includes primary seed revival and secondary seed activation; The seed culture medium used for the primary seed regeneration contains 0.01-0.02 g / L L-histidine, 0.005-0.015 g / L uridine, and 0.0003-0.0008 g / L cyclic adenosine monophosphate. The seed culture medium used for the secondary seed activation contains 0.02-0.05 g / L L-histidine, 0.01-0.03 g / L uridine, 0.0008-0.0015 g / L cyclic adenosine monophosphate, and 0.01-0.04 g / L α-ketoglutarate.

4. The cultivation method according to claim 3, characterized in that, The seed culture medium used for the primary seed revival contained 0.015 g / L L-histidine, 0.01 g / L uridine, and 0.0005 g / L cyclic adenosine monophosphate. The seed culture medium used for the secondary seed activation contains 0.03 g / L L-histidine, 0.02 g / L uridine, 0.001 g / L cyclic adenosine monophosphate, and 0.025 g / L α-ketoglutarate.

5. The cultivation method according to claim 1, characterized in that, The fermentation conditions described in step (2) are as follows: 0-24h, pH value controlled at 7.0-7.2, temperature 35-39℃, stirring speed 200-280rpm, maintaining dissolved oxygen concentration not less than 45%; 24-48h, pH value controlled at 6.7-6.9, temperature 34-38℃, stirring speed 280-320rpm, maintaining dissolved oxygen concentration not less than 30%. The feeding method for the fed-batch culture medium is as follows: feeding is started when fermentation has progressed to 8-12 hours, and fed at a constant rate of 10-15 mL / h until fermentation ends.

6. A method for purifying PDRN, characterized in that, Includes the following steps: (1) The fermentation broth obtained by the culture method according to any one of claims 1-5 is subjected to solid-liquid separation, the fermentation supernatant is discarded, and the cell precipitate is collected; (2) After washing the bacterial precipitate, resuspend it, add lysozyme for enzymatic hydrolysis and cell disruption, then homogenize under high pressure, centrifuge and collect the supernatant to obtain crude PDRN extract. (3) Filter the crude PDRN extract through a microfiltration membrane and collect the microfiltrate; (4) The microfiltration filtrate was concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to obtain PDRN concentrate; (5) The PDRN concentrate was purified by anion exchange chromatography, and the eluent was collected to obtain pure PDRN solution; (6) Freeze-dry the pure PDRN solution to obtain the finished PDRN product.

7. The purification method according to claim 6, characterized in that, In step (2), the final concentration of lysozyme added is 1-3 mg / mL, and the enzymatic hydrolysis conditions are incubation at 35-39℃ for 0.5-1.5 h; the pressure of the high-pressure homogenization is 600-1000 bar, and the number of homogenization times is 1-3. The conditions for anion exchange chromatography in step (5) are as follows: Q-agarose gel FF anion exchange medium is used, the equilibration solution is Tris-HCl buffer, pH 7.8-8.2; after loading the sample, it is washed with equilibration solution containing 0.15-0.25 mol / L NaCl; elution is performed with equilibration solution containing 0.6-1.0 mol / L NaCl, and the eluent corresponding to the 260 nm UV absorption peak is collected. The freeze-drying process described in step (6) is gradient freeze-drying, with the following procedure: pre-freezing at -40℃ to -35℃ for 1-3 hours, freezing at -55℃ to -45℃ for 2-6 hours, and deep freezing at -65℃ to -55℃ for 12-24 hours, with a vacuum degree ≤10Pa.

8. A method for preparing PDRN, characterized in that, Recombinant Escherichia coli TA4-41 was cultured using the culture method described in any one of claims 1-5, and the PDRN obtained from the culture was purified using the purification method described in any one of claims 6-7 to obtain the PDRN product.

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