DNA isothermal amplification enzyme mutant and application thereof in preparation of PDRN

Abstract

This invention provides a DNA isothermal amplase mutant and its application in the preparation of PDRN, relating to the field of genetic engineering technology. This application, through methods such as predicting the protein structure of phi29 DNA isothermal amplase and screening for enzyme mutations, has for the first time discovered beneficial mutation sites in phi29 DNA isothermal amplase, obtaining a DNA isothermal amplase mutant with high amplification activity using low-concentration templates and common primers. This mutant can also directly generate small-molecule PDRN, simplifying the strand breakage problem in the later stages of long-chain PDRN preparation. The PDRN preparation method provided in this application has the advantages of short cycle time, simple and scalable process, and can directly prepare low-molecular-weight PDRN in vitro using a DNA isothermal amplase mutant. The PDRN product has high purity, no exogenous microbial-related problems, and also has advantages such as high reaction efficiency and controllable product.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a DNA isothermal amplification enzyme mutant and its application in the preparation of PDRN. Background Technology

[0002] PDRN possesses clinical efficacy in treating arthritis, diabetic foot ulcers, and promoting tissue regeneration, and also exhibits significant anti-inflammatory, repairing, anti-aging, and skin-whitening effects. Conventional PDRN extraction requires extraction from salmon testis tissue, a process involving testis crushing, extraction, fragmentation, centrifugation, freeze-drying, quality control, and packaging. This method suffers from low extraction yield, unstable product quality, high production costs, potential biosafety risks, and difficulty in controlling product molecular weight. Furthermore, artificially farmed salmon or trout also face challenges such as high costs, technical difficulties, and long production cycles. Therefore, finding a safe, efficient, and inexpensive source of PDRN is a pressing issue in this field.

[0003] The development of synthetic biology has made it possible to prepare PDRN using biological methods. Synthetic biology-based PDRN preparation ensures the sustainability of raw material sources, is no longer limited to the farming or fishing of specific fish species, and is more eco-friendly. PDRN prepared using biological methods has less protein residue than that prepared using traditional methods, making it less likely to cause allergic reactions. Synthetic biology-based PDRN preparation has high production efficiency and a greater cost advantage. Furthermore, the synthetic biology process does not use animal-derived materials, reducing the risk of pathogenic microorganism transmission; however, mature processes have not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a DNA isothermal amplification enzyme mutant and a new method for preparing PDRN, which solves the problems of high cost, technical difficulty and long cycle caused by existing PDRN preparation methods through in vitro amplification.

[0005] On the one hand, this application provides a DNA isothermal amplification enzyme mutant, said DNA isothermal amplification enzyme mutant comprising at least one of the following A1)-A3): A1) The amino acid sequence contains a sequence obtained by mutating one or both amino acid residues at positions 12 and 66 as shown in SEQ ID NO.1; A2) comprises proteins obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the mutant described in A1), which have 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% identity with and function the same as the protein described in A1. A3) A fusion protein with the same function is obtained by linking a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2).

[0006] Optionally, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 may be as shown in SEQ ID No.4 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No.4.

[0007] Optionally, A3) ligating a tag protein to the N-terminus of the mutant described in A1) and / or A2) yields a fusion protein with the same function.

[0008] Optionally, those skilled in the art will understand that the tagged protein may include "tags" that facilitate purification, such as histidine (HIS) tags, glutathione-S-transferase tags (GST), maltose-binding protein tags (MBP), calmodulin-binding peptide tags (CBP), etc. Such tagged peptides can be easily purified, for example, from conditioned media by chelation chromatography or affinity chromatography.

[0009] Optionally, the tag is a histidine (HIS) tag, the sequence of which is shown in SEQ ID No. 2.

[0010] Optionally, the tag is located at the N end of the sequence; more preferably, the sequence after adding the tag to SEQ ID NO.1 is as shown in SEQ ID NO.3.

[0011] Optionally, the DNA isothermal amplase mutant comprises a sequence obtained by mutating amino acid residues at positions 12 and 66 of the sequence shown in SEQ ID NO.1.

[0012] Furthermore, the amino acid sequence of A1 also includes a sequence obtained by mutating any one or more amino acid residue sites at positions 8, 51, 97, 197, 221, 497, 512, and 526 in the sequence shown in SEQ ID NO.1.

[0013] Optionally, the DNA isothermal amplase mutant comprises a sequence obtained by mutating amino acid residues at positions 8, 12, 51, 66, 97, 197, 221, 497, 512, and 526 in the sequence shown in SEQ ID NO.1.

[0014] Furthermore, the mutation includes at least one or more of the following: B1)-B10): B1) The 8th amino acid residue is mutated from a methionine residue to an arginine residue; B2) The 12th amino acid residue is mutated from an aspartic acid residue to an alanine residue; B3) The 51st amino acid residue is mutated from a valine residue to an alanine residue; B4) The 66th amino acid residue is mutated from an aspartic acid residue to an alanine residue; B5) The 97th amino acid residue is mutated from a methionine residue to a threonine residue; B6) The 197th amino acid residue is mutated from a glycine residue to an aspartic acid residue; B7) The amino acid residue at position 221 is mutated from a glutamic acid residue to a lysine residue; B8) The 497th amino acid residue is mutated from a glutamine residue to a proline residue; B9) The amino acid residue at position 512 is mutated from a lysine residue to a glutamic acid residue; The 526th amino acid residue in B10 is mutated from a phenylalanine residue to a leucine residue.

[0015] Optionally, the DNA isothermal amplase mutant comprises the sequence AA-phi29 obtained by mutating the 12th amino acid residue of the sequence shown in SEQ ID NO.1 from an aspartic acid residue to an alanine residue and the 66th amino acid residue from a valine residue to an alanine residue.

[0016] Alternatively, the DNA isothermal amplase mutant may contain the amino acid sequence shown in SEQ ID No. 5.

[0017] Alternatively, the amino acid sequence of the DNA isothermal amplase mutant further includes a histidine tag located at the N-terminus of the sequence, and the DNA isothermal amplase mutant comprises the amino acid sequence shown in SEQ ID No. 6.

[0018] Alternatively, the nucleotide sequence encoding the DNA isothermal amplase mutant may comprise the nucleotide sequence shown in SEQ ID NO.7.

[0019] Optionally, the DNA isothermal amplification enzyme mutant comprises the sequence shown in SEQ ID NO.1, wherein the 8th amino acid residue is mutated from methionine to arginine, the 12th amino acid residue is mutated from aspartic acid to alanine, the 51st amino acid residue is mutated from valine to alanine, the 66th amino acid residue is mutated from aspartic acid to alanine, the 97th amino acid residue is mutated from methionine to threonine, the 197th amino acid residue is mutated from glycine to aspartic acid, the 221st amino acid residue is mutated from glutamic acid to lysine, the 497th amino acid residue is mutated from glutamine to proline, the 512th amino acid residue is mutated from lysine to glutamic acid, and the 526th amino acid residue is mutated from phenylalanine to leucine, i.e., 8T2A-phi29.

[0020] Alternatively, the DNA isothermal amplase mutant may contain the amino acid sequence shown in SEQ ID No. 8.

[0021] Alternatively, the amino acid sequence of the DNA isothermal amplase mutant further includes a histidine tag located at the N-terminus of the sequence, and the amino acid sequence of the DNA isothermal amplase mutant is shown in SEQ ID No. 9.

[0022] Alternatively, the nucleotide sequence encoding the DNA isothermal amplase mutant comprises the nucleotide sequence shown in SEQ ID NO.10.

[0023] It is understandable that those skilled in the art can select appropriate gene editing systems and gene editing methods to obtain the above-mentioned mutants based on the actual situation.

[0024] On the other hand, this application also provides biological materials, said biological materials comprising at least one or more of the following C1)-C6): C1) A nucleic acid molecule, wherein the nucleic acid molecule contains a nucleic acid molecule encoding the DNA isothermal amplification enzyme mutant; C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2); C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in C1), the expression cassette described in C2), the recombinant vector described in C3), the recombinant microorganism described in C4), and / or the recombinant cell described in C5).

[0025] The expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of C1 nucleic acid molecules can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0026] The recombinant vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed-circular plasmids. Vectors can be self-replicating vectors (i.e., complete structures existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures self-replication. Alternatively, a vector is one that, when introduced into a host cell, integrates into the genome and replicates along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used, as those skilled in the art can choose according to the specific circumstances; no excessive limitations are imposed here.

[0027] In one alternative implementation, the carrier may be pGS-21.

[0028] The whole-cell catalyst may include suspensions, metabolites, extracts, etc. of recombinant microorganisms or recombinant cells.

[0029] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain to construct recombinant microorganisms, as long as they can complete the expression of the mutants described in this application. No specific strains are limited here.

[0030] Furthermore, the recombinant microorganism is one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.

[0031] Optionally, the recombinant microorganism is Escherichia coli.

[0032] In one alternative embodiment, the recombinant microorganism is Escherichia coli BL21(DE3).

[0033] Further, the nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.7, or a nucleotide sequence as shown in SEQ ID NO.10 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.10.

[0034] Optionally, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO. 7 or a nucleotide sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 7, or a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence with at least 95%, 96%, 97%, 98%, 98.1%, 98 ...8.3%, 98.4%, 98.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 7, or a nucleotide sequence as shown in SEQ ID NO. 10. NO.10 has a nucleotide sequence with at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% identity.

[0035] It is understood that those skilled in the art can select appropriate gene editing systems and gene editing methods to obtain the above-mentioned biological materials based on the actual situation.

[0036] On the other hand, this application also provides a method for preparing the DNA isothermal amplification enzyme mutant, the method comprising: fermenting the DNA isothermal amplification enzyme mutant using the recombinant microorganism and / or the whole-cell catalyst.

[0037] The fermentation can be carried out using conventional methods.

[0038] Optionally, the preparation method includes the following steps: Step 1: Construct recombinant microorganisms expressing DNA isothermal amplification enzyme mutants; Step 2: Fermentation culture of the recombinant microorganisms.

[0039] Optionally, the preparation method further includes the steps of cell disruption, centrifugation to collect DNA isothermal amplification enzyme mutants, and purification.

[0040] Since this enzyme is an intracellular enzyme, it needs to be collected by cell disruption, centrifugation, and then purified.

[0041] The cell disruption method described herein can be performed using conventional methods by those skilled in the art, as long as cell disruption can be achieved.

[0042] In one alternative implementation, the method for disrupting cells employs high-pressure homogenization, including 200 bar for one cycle and 750 bar for six cycles.

[0043] The centrifugation can be performed by those skilled in the art under conventional conditions, as long as it can achieve the separation of the DNA isothermal amplase mutant from other substances.

[0044] The purification can be carried out using conventional methods.

[0045] In one optional embodiment, the purification is performed on the DNA isothermal amplase mutant using the AKTA protein purifier nickel column affinity purification method.

[0046] Optionally, the purification may further include dialysis for desalting and ultrafiltration.

[0047] In one optional embodiment, the preparation method includes: Step 1: Construct recombinant microorganisms expressing DNA isothermal amplification enzyme mutants; Step 2: Ferment and culture the recombinant microorganisms at 25℃-40℃ and 100-300 rpm until OD reaches zero. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.1-0.5 mM and incubate at 15℃-24℃ for 12-36 h.

[0048] Optionally, the preparation method includes: Step 1: Construct recombinant microorganisms expressing DNA isothermal amplification enzyme mutants; Step 2: Inoculate the recombinant microbial single colony into seed culture medium and incubate at 20℃-40℃ for 8-48 h to obtain seed liquid; Step 3: Inoculate the seed culture into the fermentation medium at an inoculation rate of 1%-5% and incubate at 25℃-40℃ and 100-300 rpm until OD reaches 100%. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.1-0.5 mM, cool to 15℃-24℃ and incubate for 12-36 h; Step 4: Disrupt cells, centrifuge to collect DNA isothermal amplification enzyme mutants, and purify them.

[0049] The seed culture medium is LB medium.

[0050] The DNA isothermal amplase mutant described in this application has the characteristics of low exonuclease activity, no need for resistance-modified primers, high amplification conversion rate, high polymerase activity, and high amplification efficiency.

[0051] The conversion rate can reach up to 58%, close to the ideal value of 60%.

[0052] On the other hand, this application also provides compositions comprising the DNA isothermal amplification enzyme mutant or the biological material.

[0053] The composition of this application may also contain excipients, which may be suitable solvents, solubilizers, co-solvents, emulsifiers, osmotic pressure regulators, stabilizers, pH regulators, buffers, diluents, protectants, etc.

[0054] The compositions of this application can be prepared by common methods, wherein one or more protective agents or carriers can be added to form dosage forms commonly used in the art.

[0055] In one alternative implementation, a storage solution can be used to preserve the DNA isothermal amplification enzyme mutant in order to ensure its activity.

[0056] The storage solution was formulated with 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% (v / v) NP-40, 0.5% (v / v) Tween 20, 50% (v / v) glycerol, and the balance being water.

[0057] On the other hand, this application also provides a product comprising the DNA isothermal amplification enzyme mutant, the biological material, or the composition.

[0058] On the other hand, this application also provides the application of the DNA isothermal amplification enzyme mutant and / or the biological material and / or the composition, wherein the application is selected from at least one of the following D1)-D4): D1) is used to prepare nucleic acid products; D2) is used to prepare products containing nucleic acids; D3) is a product used to prepare amplified nucleic acid sequences; D4) is used in the preparation of PDRN and related products.

[0059] On the other hand, this application also provides the use of the DNA isothermal amplification enzyme mutant and / or the biomaterial and / or the composition described herein in the preparation of nucleic acid products.

[0060] On the other hand, this application also provides the use of the DNA isothermal amplification enzyme mutant and / or the biomaterial and / or the composition described herein in the preparation of products containing nucleic acids.

[0061] On the other hand, this application also provides the use of the DNA isothermal amplification enzyme mutant and / or the biological material and / or the composition described herein in the preparation of products that amplify nucleic acid sequences.

[0062] On the other hand, this application also provides the use of the DNA isothermal amplification enzyme mutant and / or the biomaterial and / or the composition described herein in the preparation of PDRN and related products.

[0063] On the other hand, this application also provides a method for preparing PDRN, the method comprising performing an amplification reaction using the DNA isothermal amplification enzyme mutant, the biological material, or the composition; optionally, the reaction temperature of the amplification reaction is 30℃-45℃; optionally, the amount of primer added for the amplification reaction is 1-50 μM; optionally, the amount of DNA isothermal amplification enzyme mutant added is 0.5-2.5 U / μL; optionally, the amplification time of the amplification reaction is 4-50 h.

[0064] in, The reaction temperature of the amplification reaction, or the upper or lower limit of the reaction temperature range, can be taken from any value among 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, and 45℃.

[0065] Optional, 37℃-45℃; even more optional, 42℃.

[0066] The primer addition amount or the upper limit or lower limit of the primer addition amount range can be taken from any value among 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM.

[0067] Optionally, the primer addition amount is 1-10 μM; more preferably, the primer addition amount is 5 μM.

[0068] The amount of DNA isothermal amplase mutant added, or the upper limit or lower limit of the range of DNA isothermal amplase mutant added, can be taken from any value among 0.5 U / μL, 0.6 U / μL, 0.7 U / μL, 0.8 U / μL, 0.9 U / μL, 1 U / μL, 1.1 U / μL, 1.2 U / μL, 1.3 U / μL, 1.4 U / μL, 1.5 U / μL, 1.6 U / μL, 1.7 U / μL, 1.8 U / μL, 1.9 U / μL, 2 U / μL, 2.1 U / μL, 2.2 U / μL, 2.3 U / μL, 2.4 U / μL, and 2.5 U / μL.

[0069] Optionally, the amount of the DNA isothermal amplification enzyme mutant added is 0.5-2 U / μL.

[0070] The amplification time or the upper limit or lower limit of the amplification time range can be any value among 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 47 h, 48 h, 49 h, and 50 h.

[0071] Optionally, the amplification time is 6-48 h.

[0072] Optionally, the primers are modified primers and / or unmodified primers.

[0073] Alternatively, the primers may be unmodified 6N random primers.

[0074] The unmodified 6N random primer sequence is NNNNNN, and the modified primers are two thiolated 6N random primers at the 3' end with the sequence NNNN*N*N, where * indicates that the position is modified.

[0075] Optionally, the amplification reaction further includes dNTPs, which include dATP, dTTP, dCTP, and dGTP, with each dNTP having a concentration of 0.1-10 mM; or each dNTP having a concentration of 1 mM.

[0076] Optionally, the amplification reaction further includes a buffer (isothermal amplification enzyme reaction buffer) comprising 33 mM Tris-acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v / v) Tween 20, 1 mM DTT, and the balance being water.

[0077] Optionally, the amplification reaction further includes a PDRN template, the concentration of which is 1-100 ng / μL; optionally, 10 ng / μL.

[0078] The PDRN template can be natural PDRN or artificially synthesized PDRN.

[0079] In one alternative implementation, the PDRN template is salmon PDRN.

[0080] The amplification system includes: 6N random primers, DNA isothermal amplification enzyme mutant, dNTPs, PDRN template, buffer, and solvent.

[0081] Optionally, the solvent is water.

[0082] Furthermore, the method further includes a crushing step; optionally, the crushing method includes ultrasonic crushing.

[0083] Alternatively, the power of the ultrasonic fragmentation is 200-350 W.

[0084] The power of the ultrasonic breaking and the upper or lower limit of the power range can be any value among 200 W, 210 W, 220 W, 230 W, 240 W, 250 W, 260 W, 270 W, 280 W, 290 W, 300 W, 310 W, 320 W, 330 W, 340 W, and 350 W.

[0085] Alternatively, the ultrasonic fragmentation time is 30-90 min.

[0086] The time and the upper or lower limit of the time range can be any value among 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min.

[0087] Alternatively, the duty cycle of the ultrasonic breaker can be 1-5:1-5, specifically 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:2, 2:3, 2:4, 2:5, 3:1, 3:2, 3:3, 3:4, 3:5, 4:1, 4:2, 4:3, 4:4, 4:5, 5:1, 5:2, 5:3, 5:4, 5:5.

[0088] In one optional embodiment, a method for preparing PDRN includes the following steps: Step 1: Amplification reaction is performed using the DNA isothermal amplification enzyme mutant, the biological material, or the composition to obtain crude PDRN. The amplification system includes: 6N random primers, DNA isothermal amplification enzyme mutant, dNTPs, PDRN template, buffer, and solvent. The amplification reaction conditions include amplification at 30℃-45℃ for 6-50 h. Optionally, the amplification system includes 1-50 μM 6N random primers, 0.5-2.5 U / μL DNA isothermal amplification enzyme mutant, dNTPs with concentrations of 0.1-10 mM each of dATP, dTTP, dCTP, and dGTP, and 1-100 ng / μL PDRN template. Step 2: Crush the crude PDRN product; optionally, the crushing method includes ultrasonic crushing, the ultrasonic crushing power is 200-350 W, and the ultrasonic crushing time is 30-90 min.

[0089] Optionally, a method for preparing PDRN includes the following steps: Step 1: Amplification reaction is performed using the DNA isothermal amplification enzyme mutant, the biological material, or the composition to obtain crude PDRN. The amplification system includes: 6N random primers, DNA isothermal amplification enzyme mutant, dNTPs, PDRN template, buffer, and solvent. The amplification reaction includes mixing the 6N random primers, PDRN template, buffer, and solvent, denaturing at 90℃-95℃ for 5-10 min in a PCR instrument, slowly cooling to 25℃, adding dNTPs and the DNA isothermal amplification enzyme mutant, mixing well, and amplifying at 30℃-45℃ for 6-50 h. After amplification, the temperature is raised to inactivate the enzyme, and then the temperature is lowered to complete the amplification reaction. Optionally, the amplification system includes 1-50 μM 6N random primers, 0.5-2.5 U / μL DNA isothermal amplification enzyme mutant, dNTPs with concentrations of 0.1-10 mM each of dATP, dTTP, dCTP, and dGTP, and 1-100 ng / μL PDRN template. Step 2: Crush the crude PDRN product; optionally, the crushing method includes ultrasonic crushing, the ultrasonic crushing power is 200-350 W, and the ultrasonic crushing time is 30-90 min.

[0090] The method further includes a purification step. In an optional embodiment, the purification involves ultrafiltration to remove dNTPs substrates that have not formed a DNA double strand, yielding a pure PDRN double-stranded solution, followed by ethanol precipitation to obtain the PDRN product.

[0091] The above method can be used to obtain PDRN solutions with uniform size (less than 500 bp).

[0092] On the other hand, this application also provides PDRN prepared using the method described above.

[0093] The PDRNs are of uniform size and the fragments are less than or equal to 500 bp.

[0094] On the other hand, this application also provides a product comprising the PDRN.

[0095] On the other hand, this application also provides the application of the PDRN prepared by the method or the PDRN described therein in the preparation of skin repair products and / or skin wound healing products and / or products that promote angiogenesis and / or products that increase the relative cell proliferation rate.

[0096] Optionally, the skin repair, skin wound healing, and angiogenesis promotion are achieved by promoting the increase of vascular endothelial growth factor content or promoting cell proliferation.

[0097] Optionally, the increase in vascular endothelial growth factor (VEGF) content includes promoting VEGF expression.

[0098] Optionally, the cell proliferation promotion includes increasing the relative cell growth rate (RGR).

[0099] The active expression of cytokines in wound repair makes them a new reference parameter for inferring damage levels, serving as a molecular-level "living response" indicator in living wounds. VEGF is a major factor in the late inflammatory and proliferative phases of wound healing, playing a crucial role in granulation tissue formation, wound healing, angiogenesis, collagen fiber synthesis, and the repair of hair follicles and skin appendages.

[0100] In one alternative implementation, the cells are HaCat cells (human immortalized keratinocytes).

[0101] The present invention has the following beneficial effects: Commercially available phi29 DNA isothermal amplases, due to their exonuclease activity, require thiolated primers for amplification and necessitate high concentrations of template and primers, representing a high cost for industrial production. This application, through protein structure prediction and enzyme mutation screening of phi29 DNA isothermal amplases, has for the first time discovered beneficial mutation sites in phi29 DNA isothermal amplases, obtaining a DNA isothermal amplase mutant with high amplification activity using lower concentrations of template and common primers. Compared to commercially available DNA isothermal amplases, the DNA isothermal amplase mutant prepared in this patent not only solves the exonuclease activity problem but also directly generates small-molecule PDRN, simplifying the strand breakage problem in the later stages of long-chain PDRN preparation. Furthermore, it simplifies the process and reduces costs in PDRN preparation.

[0102] The PDRN preparation method provided in this application has the advantages of short cycle, simple process and scalability. It can directly prepare low molecular weight PDRN using DNA isothermal amplification enzyme mutant in vitro enzymatic method. The PDRN product has high purity, no exogenous microbial related problems, and also has the advantages of high reaction efficiency and controllable product. Attached Figure Description

[0103] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 The graph shows the expression of phi29 isothermal amplification enzyme. A: SDS-PAGE protein electrophoresis verification of phi29 isothermal amplification enzyme expression; B: Western blotting verification of protein expression (using histagged antibody). Figure 2 SDS-PAGE protein electrophoresis image of the phi29 isothermal amplification enzyme purification process; Figure 3 Amplification results using resistant primers for commercially available controls and self-prepared phi29 isothermal amplification enzyme; Figure 4 Comparison of amplification effects of Sangon Biotech Super phi29 / Thermo EquiPhi29 / AA-phi29 self-made enzymes / 8T2A-phi29 self-made enzymes using resistant primers (anti-) and non-resistant common primers (common); Figure 5 The graph shows the effect of different denaturation temperatures on enzyme amplification efficiency. Figure 6 Figures showing the ultrasonic disruption of PDRN solution at different powers and times; Figure 7Comparison of ultrasonic disruption of 8T2A-phi29 self-made enzyme amplification sample and commercially available enzyme amplification sample. Detailed Implementation

[0104] Technical terms: Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0105] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0106] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0107] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0108] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0109] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0110] Whole-cell catalysts: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The complete biological organism that participates in this catalytic process is called a whole-cell catalyst.

[0111] The term “and / or” as used in this invention refers to and covers any and all possible combinations of one or more of the related listed items, as well as the absence of a combination when interpreted in an alternative manner (“or”).

[0112] In this invention, the terms "nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any phosphate ester analogue thereof, such as single-stranded or double-stranded helical thiophosphates and thioesters. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The "product" of this disclosure comprises one or more nucleic acids as described herein.

[0113] The PDRN in this invention relates to polydeoxyribonucleotides and / or polyribonucleotides and their associated salts.

[0114] The PDRN in this invention relates to polydeoxyribonucleotides and / or polyribonucleotides and their associated sodium salts with a molecular weight of less than or equal to 1500 kDa.

[0115] The polydeoxyribonucleotides involved in this invention are single or double-stranded polymers composed of phosphate, deoxyribose and four bases (adenine, guanine, thymine and cytosine). The polyribonucleotides involved in this invention are single-chain polymers composed of phosphate, ribose and four bases (including adenine, guanine, cytosine and uracil).

[0116] Furthermore, those skilled in the art will recognize that PDRN often exists as a water-soluble salt formed with sodium ions, and the PDRN prepared by this invention also covers the case of sodium salts of nucleic acid molecules.

[0117] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0118] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0119] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0120] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0121] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0122] In this specification, mutations in amino acids are referred to as "original amino acid, site, substituted amino acid". For example, the mutation of aspartic acid D to alanine A at position 9, starting from the N (nitrogen) end of the sequence and counting sequentially from the C (carbon) end, is represented as D9A.

[0123] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0124] LB medium formula: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L, with the remainder being water; for LB solid (plate) medium, simply add an appropriate amount of agar (15-20 g / L). Fermentation medium formula: glycerol 10 g / L, diammonium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 10.5 g / L, citric acid 1.7 g / L, peptone 10 g / L, anhydrous magnesium sulfate 1.68 g / L, with the remainder being water.

[0125] The 10 kDa ultrafiltration tubes were purchased from Merck Amicon. ® Ultrafiltration tube, part number: UFC9010.

[0126] pGS-21 plasmid was purchased from Nanjing Genscript Biotech Co., Ltd.

[0127] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0128] Example 1: Preparation of phi29 isothermal amplification enzyme 1.1 Construction of expression strains In this embodiment, Nanjing Genscript Biotech Co., Ltd. synthesized the phi29 isothermal amplification enzyme, the amino acid sequence of which is shown in SEQ ID No. 1 and the nucleotide sequence of which is shown in SEQ ID No. 4. To facilitate subsequent purification, a histidine tag (sequence shown in SEQ ID No. 2) was added to the N-terminus of the sequence, and the final amino acid sequence of the phi29 isothermal amplification enzyme is shown in SEQ ID No. 3. This enzyme was then ligated into the plasmid expression vector pGS-21 to obtain the phi29 DNA plasmid.

[0129] Strain construction: The synthesized phi29 DNA plasmid was electroporated into BL21(DE3) competent cells and named BL21(DE3) / pGS-21-N-8his-phi29. After incubation at 37°C for 1 h in a shaker, it was plated on LB agar plates and incubated overnight at 37°C. Single colonies were picked and cultured in LB medium. After 16 h of culture, a portion of the bacterial culture was transferred to glycerol tubes and frozen for preservation with 50% glycerol. The empty pGS-21 vector was electroporated into BL21(DE3) competent cells using the same method to obtain a blank control strain named BL21(DE3) / pGS-21 empty vector.

[0130] Shake-flask fermentation: Blank control bacterial cultures of BL21(DE3) / pGS-21-N-8his-phi29 and BL21(DE3) / pGS-21 empty vectors were respectively taken from glycerol tubes and inoculated into seed culture on LB medium at 37°C overnight. The seed culture was then inoculated into fermentation medium at a 1% inoculum in shake flasks and cultured at 37°C and 220 rpm until OD reached... 600 When the pH was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the mixture was cultured at 20℃ for 24 h to induce enzyme expression. After fermentation, the cells were collected by centrifugation and homogenized under high pressure (200 bar, 1 cycle; 750 bar, 6 cycles) to obtain whole-cell lysates. The cells were then centrifuged (7500 rpm, 30 min) to collect the supernatant and precipitate, respectively. The whole-cell lysates, precipitates, and supernatants were subjected to SDS-PAGE protein electrophoresis to verify expression. The blank control was the BL21(DE3) / pGS-21 empty vector. The results are as follows: Figure 1 As shown in Figure A. The target protein was confirmed using Western blotting antigen-antibody binding in the fragmented supernatant and fragmented whole-cell fluid, and the results are as follows. Figure 1 As shown in B. In summary, as... Figure 1 The results showed that the phi29 isothermal amplification enzyme was successfully expressed, and no inclusion bodies were produced.

[0131] 1.2 Purification and preparation of phi29 isothermal amplification enzyme Following the shake-flask fermentation method described in section 1.1, after low-temperature induction culture, the bacterial sludge was collected by centrifugation. The sludge was rinsed with buffer A (pH = 7.520 mM Tris-HCl, 150 mM NaCl, balance water) to remove residual culture medium, and then resuspended in buffer A to OD. 600 =Approximately 10, the bacterial cells were disrupted by high-pressure homogenization (200 bar, 1 cycle, 750 bar, 6 cycles in sequence), and then the target protein was purified by nickel column affinity purification using an AKTA protein purification instrument.

[0132] The purification steps were as follows: The bacterial culture, after high-pressure homogenization, was centrifuged at 7500 rpm for 30 min. The supernatant was collected and filtered through a 0.22 μm aqueous filter membrane to obtain the disrupted supernatant. The AKTA protein purifier and nickel column were equilibrated with buffer A. Then, the enzyme was loaded onto the column at a low flow rate to allow for sufficient column adhesion. The flow-through was collected. Gradient elution was then performed with buffer B (pH=7.5, 20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, balance water). Elutions at different elution times were collected to obtain Elution 1, Elution 2, and Elution 3. The collected flow-through and elutions were then subjected to SDS-PAGE protein electrophoresis to verify the purification results. Figure 2 The elution enzyme solution was dialyzed to remove salts, and then concentrated by ultrafiltration to prepare the phi29 isothermal amplification enzyme solution, which was then added to the storage solution for preservation. The positive control was a commercially available phi29 protease (Sangon Biotech Super Phi29 DNA isothermal amplification enzyme (B110082)) with a molecular weight consistent with the mutant prepared in this study.

[0133] The storage solution was formulated as follows: 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% (v / v) NP-40, 0.5% (v / v) Tween 20, 50% (v / v) glycerol, with the balance being water.

[0134] 1.3 Validation of the multiple displacement isothermal amplification system Using the phi29 isothermal amplification enzyme prepared in section 1.2 as the sample, and Sangon Biotech Super Phi29 DNA isothermal amplification enzyme (B110082) / Thermo EquiPhi29 (A65394) as the positive control, salmon PDRN samples were prepared (specific preparation method: 0.5 L of disruption buffer (0.9% sodium chloride, 0.9% sodium citrate buffer, pH 8.0) was added to 25 kg of salmon testes, and mechanically disrupted for 3 min. The sample was washed once with disruption buffer and centrifuged to collect the precipitate. The precipitate was dispersed in 15 times its weight of enzymatic digestion buffer (0.15 M NaCl / 1.0 mM EDTA-2Na aqueous solution), and neutral protease and papain (1:4 mass ratio) were added for enzymatic digestion. The enzyme amount was 15000 U / g salmon testes, the pH was 6.6, the temperature was 35℃, and the digestion time was 5 h. The viscosity of the resulting enzymatic digest was 7. After enzymatic hydrolysis at mPa·s, sodium chloride was added to bring the total concentration to 1.5 M, and the mixture was heated to 90℃ and maintained for 10 min to inactivate the enzyme. Solids were removed by centrifugation and filtration. An appropriate amount of ethanol was added to the filtrate to bring the ethanol concentration to 49% (v / v), resulting in a white precipitate. A 70±2% ethanol solution was prepared as a washing buffer and the precipitate was washed three times. The precipitate was then dehydrated with 95% ethanol solution until the concentration of the ethanol solution after dehydration became 87%. The dehydrated precipitate was then vacuum-dried at a temperature gradient of 30℃-40℃ to obtain the product. Using dNTPs as the substrate and multiple displacement amplification PCR as the amplification template, the feasibility of using isothermal amplification enzymes to amplify PDRN was verified.

[0135] The steps are as follows: First, salmon PDRN samples were prepared as templates for the reaction. Suzhou Genewiz Biotechnology Co., Ltd. synthesized two thiolated 6N random primers (NNNN*N*N) at the 3' end. The isothermal amplification enzyme reaction buffer, thiolated 6N random primers, PDRN template, and water were added to the reaction system according to the concentrations described in Table 1. After mixing, the mixture was placed in a PCR instrument at 95°C for 5 min to denature, then slowly cooled to 25°C. dNTPs and phi29 isothermal amplification enzyme (the phi29 isothermal amplification enzyme prepared in 1.2, or Sangon Biotech Super Phi29 (B110082) or Thermo EquiPhi29 (A65394)) were added, mixed, and amplified at 30°C for 4 h. After amplification, the temperature was raised to 65°C and held for 10 min to inactivate the enzyme, then cooled to room temperature to complete the amplification reaction.

[0136] Table 1 Isothermal amplification reaction system

[0137] Weigh out agarose powder to prepare a 1% agarose gel. Detect the amplification quality using DNA electrophoresis. Observe the results using ultraviolet imaging on a gel imaging system. Figure 3 As shown.

[0138] 1.4 Optimization of the target protein expression sequence To further improve the amplification effect, the protein structure of the phi29 isothermal amplification enzyme obtained in 1.2 was predicted in this embodiment, and the phi29 isothermal amplification enzyme was modified by enzyme mutation screening and other methods. The D12A and D66A sites were mutated to obtain the AA-phi29 self-made enzyme (amino acid as shown in SEQ ID No. 5, amino acid sequence with histidine tag as shown in SEQ ID No. 6, nucleotide sequence as shown in SEQ ID No. 7); the combination of M8R, D12A, V51A, D66A, M97T, G197D, E221K, Q497P, K512E, and F526L sites was mutated to construct the 8T2A-phi29 self-made enzyme (amino acid as shown in SEQ ID No. 8, amino acid sequence with histidine tag as shown in SEQ ID No. 9, nucleotide sequence as shown in SEQ ID No. 10). Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the self-made enzymes AA-phi29 and 8T2A-phi29. The expression strain construction method was as described in 1.1, the purification method in 1.2, and the PDRN amplification method in 1.3. During PDRN amplification, non-resistant, unmodified ordinary 6N random primers (NNNNNN, synthesized by Suzhou Genewise Biotechnology Co., Ltd.) at the same concentration were used instead of the thiomodified 6N random primers for amplification. The results are as follows. Figure 4 As shown.

[0139] Compared to commercially available Sangon Biotech Super phi29 and Thermo EquiPhi29 enzymes, this study found that the exonuclease activities of the self-made AA-phi29 and 8T2A-phi29 enzymes were reduced, eliminating the need for resistance-modified primers; high-efficiency amplification could be achieved using ordinary primers. Furthermore, the self-made 8T2A-phi29 enzyme exhibited higher polymerase activity, enabling highly efficient amplification reactions and rapidly linking monomeric dNTPs into DNA double strands, thus achieving high-efficiency PDRN preparation. Figure 4 ).

[0140] Example 2 Optimization of a Multiple Substitution Isothermal Amplification System 2.1 Calculation of amplification conversion rate Under different amplification conditions, the amplification efficiency varies with the same substrate addition amount, and the yield of DNA double strands formed by primers using long-chain PDRN as a template and consuming dNTPs also differs. Simply measuring the amplification concentration using a Nanodrop ultra-micro UV spectrophotometer cannot eliminate the influence of individual dNTPs. To verify the utilization rate of dNTPs, the amplification product needs to be purified to remove unbound substrate dNTPs and excess primers, and the actual PDRN amplification efficiency needs to be calculated.

[0141] The specific method for detecting PDRN amplification efficiency involves first determining the deoxyribonucleotide content (C1) and volume (V1) in the solution using NanoDrop before amplification. After amplification, the reaction solution is passed through a 10KD ultrafiltration tube and washed three times with ultrapure water to remove unchained monomeric dNTPs before determining the deoxyribonucleotide content (C2) and corresponding volume (V2) in the solution. Because each dNTP trisodium salt releases sodium pyrophosphate (Na2PPi) upon addition to the DNA strand, resulting in mass loss, the theoretical conversion rate is 60%.

[0142] In this embodiment, the salmon PDRN sample extracted by method 1.3 in Example 1 was used as the PDRN template. High-purity PDRN sample was prepared by using the isothermal amplification enzyme mutant 8T2A-phi29 (amino acid sequence shown in SEQ ID No. 9) prepared in Example 1 and dNTPs as substrate materials to optimize the amplification conditions of the multiple displacement isothermal amplification system.

[0143] 2.2 Optimization of amplification temperature Add substrates according to the reaction system in Table 2. Under the same amplification conditions, set different amplification temperatures (30℃ / 33℃ / 37℃ / 42℃ / 45℃) to find the optimal amplification temperature for the enzyme. This includes: adding isothermal amplification enzyme reaction buffer, 6N random primers, PDRN template, and water according to the concentrations in the reaction system described in Table 2, mixing well, and then placing it in a PCR instrument for denaturation at 95℃ for 5 min. Slowly cool to 25℃, add dNTPs and the isothermal amplification enzyme mutant 8T2A-phi29, mix well, and amplify at 30℃, 33℃, 37℃, 42℃, or 45℃ for 4 h. After amplification, raise the temperature to 65℃ and hold for 10 min to inactivate the enzyme, then cool to room temperature to complete the amplification reaction. The remaining methods are the same as described in Example 1.1.3. The conversion rates at 30℃, 33℃, 37℃, 42℃, and 45℃ are 15%, 17%, 20%, 23%, and 22%, respectively. The amplification results are as follows: Figure 5 As shown, it can achieve good amplification results at temperatures ranging from 30℃ to 45℃, with the highest amplification efficiency at 42℃.

[0144] Table 2 Isothermal amplification reaction system

[0145] 2.3 Validation of different amplification times, primer concentrations, and enzyme addition amounts The final concentration of dNTPs in the reaction system was fixed at 1 mM. Multiple sets of variables were set, including 6N common primer concentrations of 10 μM, 5 μM, and 2 μM; amplification times of 4 h, 8 h, 16 h, 24 h, and 48 h; and enzyme addition amounts of 0.5 U / μL, 1 U / μL, and 2 U / μL for crossover experiments. The amplification conditions included isothermal amplification of enzyme reaction 10x Buffer (330 mM Tris-acetate, 100 mM magnesium acetate, 660 mM potassium acetate, 1% (v / v) Tween 20, 10 mM DTT, pH=8.8), different concentrations of 6N common random primers, PDRN template, and water. After mixing, the mixture was placed in a PCR instrument at 95°C for denaturation for 5 minutes. The temperature was slowly lowered to 25°C, and dNTPs and different amounts of the isothermal amplification enzyme mutant 8T2A-phi29 were added. After mixing, the mixture was amplified at 42°C for different reaction times. After amplification, the temperature was raised to 65°C and held for 10 min to inactivate the enzyme, and then cooled to room temperature to complete the amplification reaction. After amplification, monomeric dNTPs were removed by ultrafiltration using a 10 kDa ultrafiltration tube (Merck Amicon® Ultra ultrafiltration tube, 10 kDa MWCO, catalog number: UFC9010). The amplification efficiency under different reaction conditions was compared, as shown in Table 3.

[0146] Table 3 Conversion rates under different reaction conditions a

[0147] a Conversion rate = (Concentration of reaction solution after ultrafiltration washing * volume) / (Concentration of reaction solution before amplification * volume) As shown in Table 3, the conversion rate increased with increasing primer and enzyme addition amounts, and also increased with prolonged amplification time. When the primer addition amount was 1-50 μM and the enzyme addition amount was 0.5-2.5 U / μL, the conversion rate was higher than 10% after amplification for 6-50 h. Among them, when the primer addition amount was 5 μM and the enzyme addition amount was 1 U / μL, the conversion rate was 58% after amplification for 48 h, which was close to the theoretical conversion rate.

[0148] Example 3 Preparation of PDRN samples of uniform size 3.1 The PDRN sample prepared in Example 2 was subjected to ultrasonic disruption using an ultrasonic disruptor. 3.1.1 The PDRN sample prepared in Example 2 was subjected to ultrasonic disruption. Add isothermal amplification enzyme reaction buffer, 6N random primers, PDRN template, and water to the reaction system as described in Table 4. After mixing, denature at 95°C for 5 min in a PCR instrument, slowly cool to 25°C, add dNTPs and the isothermal amplification enzyme mutant 8T2A-phi29 obtained in Example 1, mix well, and amplify at 42°C for 48 h. After amplification, raise the temperature to 65°C and hold for 10 min to inactivate the enzyme, then cool to room temperature to complete the amplification reaction. Then, use a 10 kDa filter tube (Merck Amicon® Ultra ultrafiltration tube, 10 kDa MWCO, catalog number: UFC9010) to remove dNTPs substrate that did not form DNA double strands, obtaining a pure PDRN double-stranded solution. Add four volumes of pure ethanol to the solution, allow to stand to precipitate, centrifuge, wash twice with 70% ethanol solution, collect the precipitate, and dry in an oven or freeze-dry to prepare PDRN powder.

[0149] Table 4 Isothermal amplification reaction system

[0150] 3.1.2 The PDRN sample prepared in Example 2 was subjected to ultrasonic disruption using an ultrasonic disruptor. Based on the experimental conditions, solutions of different concentrations were prepared for fragmentation treatment: a 1000 ng / μL PDRN solution was prepared, and the ultrasonic fragmenter duty cycle was set to 3:3. The degree of fragmentation of the amplified samples was compared under different power conditions (150 W, 200 W, 250 W, 300 W, 350 W) and different ultrasonic times (10 min, 30 min, 60 min). The solution sample before fragmentation was used as a control. Agarose gel electrophoresis was used to verify the fragmentation. The results are shown in [Figure number missing]. Figure 6.like Figure 6 As shown, a uniform PDRN solution (below 500 bp) can be obtained with a power of 200 W or more and a crushing time of about 30 min or more.

[0151] 3.2 PDRN samples prepared with different enzymes under the same conditions were subjected to ultrasonic disruption. PDRN samples were prepared using the same method as described in 3.1.1, except that Thermo EquiPhi29 enzyme was used instead of the isothermal amplification enzyme mutant 8T2A-phi29 to obtain samples amplified by Thermo EquiPhi29 enzyme. Samples amplified by the phi29 isothermal amplification enzyme mutant were prepared as described in 3.1.1. PDRN was dissolved to 1000 ng / μL, and samples amplified by Thermo EquiPhi29 enzyme were prepared at the same concentration as a control. The ultrasonic homogenizer was set to a duty cycle of 3:3, and homogenized at 250 W for 30 min and 60 min. Thermo EquiPhi29 enzyme amplification samples before ultrasonic homogenization and samples amplified by the phi29 isothermal amplification enzyme mutant 8T2A-phi29 were used as control groups. Agarose gel electrophoresis was used to verify the homogenization status. Figure 7 As shown, the phi29 isothermal amplification enzyme mutant (self-made phi29 amplification product) prepared in Example 2 reduces the difficulty of fragmentation in amplified samples and produces more uniform products (below 500 bp).

[0152] Example 4: Validation of the efficacy of amplified samples VEGF is a key pro-angiogenic factor, and its increased secretion is crucial for improving tissue microcirculation, promoting wound healing, and skin repair. Relative cell proliferation rate (RGR) reflects the effects of substances on cell proliferation and repair. Four samples were prepared under the optimized amplification conditions of Example 2 (as described in 3.1.1 of Example 3). After ultrafiltration, they were homogenized by sonication at 250 W for 30 min, and the ethanol precipitate was redissolved to obtain four fragmented PDRN products with consistent concentrations.

[0153] The efficacy of the fragmented short cells was verified as follows: HaCat cells were used as the experimental subject, and the culture system was DMEM medium (with 10% fetal bovine serum added) under the conditions of 37°C and 5% CO2 in a carbon dioxide incubator. After 24 h of cell culture, the original culture medium was discarded, and serum-free (DMEM) medium mixed with the sample (or positive control PC) was added for another 24 h of culture. An equal volume of serum-free medium was added to the blank control. After the incubation period, the cell supernatant was collected, and the VEGF content was determined using a human VEGF ELISA (enzyme-linked immunosorbent assay) kit (catalog number KE00216) according to the manufacturer's instructions.

[0154] HaCat cells were used as the experimental subject. The culture system was serum-free DMEM medium, and the culture conditions were a CO2 incubator at 37°C and 5% CO2. After 24 h of cell culture, the original culture medium was discarded, and serum-free medium containing the sample (or positive control PC) was added for another 24 h of culture. The control group consisted of water. CCK was used. The relative cell proliferation rate was detected by method 8 (catalog number PF00004), and the results are shown in Table 5.

[0155] The results are shown in Table 5. NC is the blank control, 1-4 are the four prepared fragmented PDRN products, and the comparative example (PC) is a commercially available PDRN sample (Haiya Tou Ning). In the PC positive control, the amount of TGF-β1 added was 100 ng / μL, the amount added to samples 1-4 was 10 ng / μL, and the amount added to the comparative example was 10 ng / μL.

[0156] Table 5. Efficacy Measurement Results Data

[0157] The results indicate that the PDRN prepared by this method has a good promoting effect on the secretion of endothelial vascular endothelial growth factor and the activity of cell RGR, indicating that it can effectively promote cell proliferation and repair capacity, especially in skin tissue, where it has significant anti-aging and wound healing potential.

[0158] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A DNA isothermal amplification enzyme mutant, characterized in that, The DNA isothermal amplase mutant contains at least one of the following A1)-A3): A1) The amino acid sequence contains a sequence obtained by mutating one or both amino acid residues at positions 12 and 66 as shown in SEQ ID NO.1; A2) A protein having 98% or more identity with and functioning the DNA isothermal amplase mutant of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of the mutant described in A1). A3) A fusion protein with the same function is obtained by linking a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2).

2. The DNA isothermal amplification enzyme mutant according to claim 1, characterized in that, The amino acid sequence of A1) further includes sequences obtained by mutating any one or more amino acid residue sites at positions 8, 51, 97, 197, 221, 497, 512, and 526 in the sequence shown in SEQ ID NO.

1.

3. The DNA isothermal amplification enzyme mutant according to any one of claims 1 or 2, characterized in that, The mutation includes at least one or more of the following B1)-B10): B1) The 8th amino acid residue is mutated from a methionine residue to an arginine residue; B2) The 12th amino acid residue is mutated from an aspartic acid residue to an alanine residue; B3) The 51st amino acid residue is mutated from a valine residue to an alanine residue; B4) The 66th amino acid residue is mutated from an aspartic acid residue to an alanine residue; B5) The 97th amino acid residue is mutated from a methionine residue to a threonine residue; B6) The 197th amino acid residue is mutated from a glycine residue to an aspartic acid residue; B7) The amino acid residue at position 221 is mutated from a glutamic acid residue to a lysine residue; B8) The 497th amino acid residue is mutated from a glutamine residue to a proline residue; B9) The amino acid residue at position 512 is mutated from a lysine residue to a glutamic acid residue; The 526th amino acid residue in B10 is mutated from a phenylalanine residue to a leucine residue.

4. A biomaterial, characterized in that, The biomaterial comprises at least one or more of the following C1)-C6): C1) A nucleic acid molecule, wherein the nucleic acid molecule contains a nucleic acid molecule encoding a DNA isothermal amplification enzyme mutant according to any one of claims 1-3; C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2); C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C6) A whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in C1), the expression cassette described in C2), the recombinant vector described in C3), the recombinant microorganism described in C4), and / or the recombinant cell described in C5).

5. A composition, characterized in that, The composition comprises the DNA isothermal amplase mutant as described in any one of claims 1-3 or the biological material as described in claim 4.

6. The use of the DNA isothermal amplification enzyme mutant according to any one of claims 1-3 or / and the biomaterial according to claim 4 or / and the composition according to claim 5, characterized in that, The application is selected from at least one of the following D1)-D4): D1) is used to prepare nucleic acid products; D2) is used to prepare products containing nucleic acids; D3) is a product used to prepare amplified nucleic acid sequences; D4) is used in the preparation of PDRN and related products.

7. A method for preparing PDRN, characterized in that, The method includes performing an amplification reaction using the DNA isothermal amplification enzyme mutant according to any one of claims 1-3, the biological material according to claim 4, or the composition according to claim 5; optionally, the reaction temperature of the amplification reaction is 30℃-45℃; optionally, the amount of primer added for the amplification reaction is 1-50 μM; optionally, the amount of DNA isothermal amplification enzyme mutant added is 0.5-2.5 U / μL; optionally, the amplification time of the amplification reaction is 6-50 h.

8. The method according to claim 7, characterized in that, The method further includes a crushing step; optionally, the crushing method includes ultrasonic crushing.

9. PDRN prepared using the method described in any one of claims 7-8.

10. The use of the PDRN prepared by the method of any one of claims 7-8 or the PDRN as described in claim 9 in the preparation of skin repair products and / or skin wound healing products and / or products that promote angiogenesis and / or products that increase the relative cell proliferation rate.

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