A specific sequence nucleic acid molecule and its preparation method and application

Abstract

The application discloses a specific sequence nucleic acid molecule and a preparation method and application thereof, and obtains a new specific sequence nucleic acid molecule, the sequence has certainty, biological activity and stable product quality. The specific sequence PDRN does not have cytotoxicity, is high in safety, and has wide application prospects. Furthermore, the specific sequence PDRN discovered for the first time in the application has a remarkable effect of promoting VEGF expression relative to extracted PDRN, can effectively repair cell damage, and provides a new skin care and repair efficacy component for the cosmetic, pharmaceutical and reproductive medicine industries. The application further provides a preparation method of the specific sequence PDRN, and the preparation method is simple, suitable for popularization and application and industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a specific sequence nucleic acid molecule, its preparation method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are a mixture of deoxyribonucleotides with a molecular weight between 50 and 1500 kDa, derived from the sperm DNA of rainbow trout (Oncorhynchus mykiss) or salmon (Oncorhynchus keta), and prepared through a controlled purification and sterilization process. This process ensures that the product does not contain any active proteins or peptides that may trigger an immune response. PDRNs possess multiple biological activities, including promoting angiogenesis, anti-ischemia, and anti-inflammation, and have shown clear application value in regenerative medicine and the treatment of diabetic foot ulcers.

[0003] In reproductive medicine, PDRN can effectively alleviate chemotherapy-induced premature ovarian failure (POI) by repairing the ovarian microenvironment, restoring AMH and E2 hormone levels, and promoting follicle generation. At the same time, it can reduce testicular damage and improve spermatogenesis, showing unique clinical application prospects in gonadal tissue regeneration and functional reconstruction.

[0004] In vitro and in vivo experiments have shown that the core mechanism of action of PDRN lies in its specific activation of the adenosine A2A receptor. This characteristic is closely related to its DNA source, molecular weight, and manufacturing process, and is a key attribute that distinguishes it from other adenosine receptor-related drugs. By activating the A2A receptor, PDRN exerts its pro-angiogenic and tissue repair effects. Simultaneously, PDRN can provide nucleoside and nucleotide raw materials for the salvage pathway, synergistically supporting DNA repair and survival of damaged cells. Compared to existing adenosine receptor modulators—such as the non-selective activators adenosine and dipyridamole (which are prone to adverse reactions), radondoxin (approved by the FDA for myocardial perfusion imaging, but with different indications), and defibrotide, which has different molecular weights and DNA sources and lacks wound-healing efficacy—PDRN has unique pharmacological advantages in tissue repair applications.

[0005] However, current technologies are limited to extracting PDRN from sperm cells of fish such as rainbow trout, salmon, and chum salmon. These fish resources are limited and subject to supply chain stability and environmental sustainability factors. Therefore, there is an urgent need to develop a method for efficiently obtaining PDRN, the key of which lies in obtaining the relevant functional sequence and establishing a synthetic biology preparation pathway. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a specific sequence nucleic acid molecule, its preparation method, and its application. It has a significant effect on promoting VEGF expression and can effectively repair cell damage, providing a new skin care and repair ingredient for the cosmetics, pharmaceutical, and reproductive medicine industries.

[0007] The technical solution provided by this invention is as follows:

[0008] This invention provides a specific sequence nucleic acid molecule, wherein the nucleic acid molecule comprises any one or more of the following sequences A1) to A3): A1) The nucleic acid molecule contains a nucleic acid sequence shown in any one of SEQ ID No. 1 to SEQ ID No. 15, or a nucleic acid sequence having 98% or more identity with such sequence; preferably, a nucleic acid sequence having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with any one of SEQ ID No. 1 to SEQ ID No. 15; A2) Nucleic acid molecules that hybridize with the sequence described in A1) under stringent conditions; A3) is a nucleic acid molecule whose sequence is complementary to that described in A1).

[0009] 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.

[0010] Preferably, the nucleic acid molecule is a nucleic acid molecule that has the functions of promoting cell proliferation, promoting skin repair, promoting skin wound healing, improving skin inflammation, promoting angiogenesis and / or increasing the content of vascular endothelial growth factor.

[0011] More preferably, the nucleic acid molecule comprises a DNA molecule, specifically at least a single-stranded DNA (ssDNA) or a double-stranded DNA (dsDNA).

[0012] The present invention also provides a method for preparing the above-described nucleic acid molecule, comprising: Using salmon genomic DNA as a template, PCR amplification was performed using primers capable of amplifying the nucleic acid molecules to obtain the amplification product. And the amplification product is purified; The primers used for PCR amplification are nucleic acid sequences shown in SEQ ID No. 16 to SEQ ID No. 45.

[0013] Furthermore, SEQ ID No. 16 and SEQ ID No. 17 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 1; SEQ ID No. 18 and SEQ ID No. 19 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 2; SEQ ID No. 20 and SEQ ID No. 21 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 3; SEQ ID No. 22 and SEQ ID No. 23 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 4; SEQ ID No. 24 and SEQ ID No. 25 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 5; SEQ ID No. 26 and SEQ ID No. 27 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 6; SEQ ID No. 28 and SEQ ID No. 29 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 7; SEQ ID No. 30 and SEQ ID No. 31 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 8; SEQ ID No. 32 and SEQ ID No. 33 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 9; SEQ ID No. 34 and SEQ ID No. 35 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 10; SEQ ID No. 36 and SEQ ID No. 37 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 11; SEQ ID No. 38 and SEQ ID No. 39 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 12; SEQ ID No. 40 and SEQ ID No. 41 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 13; SEQ ID No. 42 and SEQ ID No. 43 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 14; SEQ ID No. 44 and SEQ ID No. 45 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 15.

[0014] The present invention also provides a sodium salt of a nucleic acid molecule, wherein the nucleic acid molecule comprises at least one or more of the following sequences B1) to B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions to any of the specified nucleic acid sequences in B1)-B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0015] Those skilled in the art can prepare the sodium salt using conventional methods, and no further limitations are made herein.

[0016] This invention also provides the application of nucleic acid molecules or their sodium salts in the preparation of PDRN-related products, wherein the nucleic acid molecules contain at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0017] The present invention also provides PDRN, wherein the PDRN comprises the nucleic acid molecule or the sodium salt.

[0018] Preferably, the PDRN is a PDRN that has the functions of promoting cell proliferation, promoting skin repair, promoting skin wound healing, improving skin inflammation, promoting angiogenesis and / or increasing the content of vascular endothelial growth factor.

[0019] This invention also provides the use of nucleic acid molecules or their sodium salts in the preparation of products for skin repair and / or improvement of skin inflammation and / or skin wound healing, wherein the nucleic acid molecules comprise at least one or more of the following sequences B1) to B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0020] Furthermore, the applications include promoting cell proliferation and / or preparing products that promote cell proliferation.

[0021] Furthermore, the applications include promoting angiogenesis and / or preparing products that promote angiogenesis.

[0022] Furthermore, the application includes increasing vascular endothelial growth factor (VEGF) content and / or preparing products that increase VEGF content.

[0023] Furthermore, the products include cosmetics and pharmaceutical / medical devices.

[0024] The applications include the preparation of products that promote cell proliferation, skin repair, skin wound healing, improve skin inflammation, promote angiogenesis, and / or increase vascular endothelial growth factor.

[0025] Among these benefits, promoting skin repair, wound healing, improving skin inflammation, and / or promoting angiogenesis are achieved by increasing the content of vascular endothelial growth factor.

[0026] Preferably, increasing the vascular endothelial growth factor content includes increasing the expression level of vascular endothelial growth factor.

[0027] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in promoting cell proliferation and / or preparing products that promote cell proliferation, wherein the nucleic acid molecule comprises at least one or more of the following sequences B1) to B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0028] This invention demonstrates that the nucleic acid molecule is non-toxic, highly safe, and has a relative cell proliferation rate of over 90%.

[0029] In a preferred embodiment, the cells are human immortalized keratinocytes (HaCat cells).

[0030] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in skin repair and / or the preparation of skin repair products, wherein the nucleic acid molecule comprises at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0031] Preferably, the skin repair includes: increasing cytokine levels.

[0032] More preferably, the cytokines include vascular endothelial growth factor (VEGF).

[0033] In a preferred embodiment, the cells are human immortalized keratinocytes (HaCat cells).

[0034] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in the preparation of skin wound healing products, wherein the nucleic acid molecule comprises at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0035] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in the preparation of products for improving skin inflammation, wherein the nucleic acid molecule comprises at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0036] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in the preparation of products that promote angiogenesis, wherein the nucleic acid molecule comprises at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0037] This invention also provides the use of the nucleic acid molecule, the sodium salt, or the PDRN in increasing vascular endothelial growth factor (VEGF) levels and / or in preparing products that increase VEGF levels, wherein the nucleic acid molecule comprises at least one or more of the following sequences (B1) to (B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence having 98% or more identity with the sequence described in B1); preferably, having 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity. B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

[0038] Preferably, the increase in vascular endothelial growth factor (VEGF) content is achieved by promoting VEGF expression.

[0039] In a preferred embodiment, the cells are human immortalized keratinocytes (HaCat cells).

[0040] The present invention also provides a product containing the above-described nucleic acid molecules as active ingredients; and excipients acceptable for cosmetic or pharmaceutical products.

[0041] Specifically, the excipients may be appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0042] The product of the present invention can be prepared by a general method, wherein one or more diluents or carriers may be added.

[0043] The products of this invention can be prepared into any formulation commonly prepared in this field. For example, they can be formulated into creams, lotions, masks, foundations, medical devices, hair cosmetics, and other formulations. Specifically, they can be skin lotions, skin softeners, hyaluronic acid injections, skin toners, astringents, lotions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, serums, nourishing serums, masks, soaps, cleansing foams, cleansing milks, cleansing creams, lotions, or shower gels.

[0044] In the cosmetics of this invention, other ingredients commonly formulated in cosmetics may also be mixed in as needed. These may include, for example, oils, moisturizers, surfactants, organic pigments, inorganic pigments, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, or purified water.

[0045] On the other hand, the present invention also provides the application of the cosmetic in promoting cell proliferation, promoting skin repair, promoting skin wound healing, improving skin inflammation, promoting angiogenesis and / or increasing the content of vascular endothelial growth factor.

[0046] Those skilled in the art can select the concentration of the nucleic acid molecule, the sodium salt, the PDRN, or the product according to the actual situation, and no specific limitation is made thereto.

[0047] In a preferred embodiment, the concentration of the nucleic acid molecule, the sodium salt, the PDRN, or the product may be 0.0001% to 20%, preferably 0.0001% to 5%.

[0048] The concentration of the nucleic acid molecule, the sodium salt, the PDRN, or the product may be 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01%. 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, and any of these values.

[0049] Beneficial effects

[0050] This invention yields novel, specific PDRN sequences with deterministic characteristics, stable biological activity, and consistent product quality. These specific PDRN sequences are non-cytotoxic, exhibit high safety, and have broad application prospects. Furthermore, this invention is the first to discover that these specific PDRN sequences promote VEGF expression, effectively repairing cell damage and providing a novel skincare and repair ingredient for the cosmetics, pharmaceutical, and reproductive medicine industries.

[0051] This invention also provides a method for preparing specific sequence PDRN, which is simple and suitable for widespread application and industrial production. Attached Figure Description

[0052] Figure 1 Electrophoresis diagram of PDRN for a specific sequence;

[0053] Figure 2 Comparison of the effects of different PDRN sequences on promoting VEGF expression;

[0054] Figure 3 Different concentrations of PDRN promote VEGF expression; where A is a schematic diagram of immunofluorescence and B is a schematic diagram of immunohistochemistry. Detailed Implementation

[0055] To more clearly illustrate the overall concept of the invention, 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.

[0056] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. 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 invention pertains.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] The term "water" as used in this invention includes any feasible type of 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.

[0062] The term "and / or" as used in this invention refers to and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in an alternative manner ("or").

[0063] 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.

[0064] Polydeoxyribonucleotides are single or double-stranded polymers consisting of phosphate, deoxyribose, and four bases (adenine, guanine, thymine, and cytosine).

[0065] Polyribonucleotides are single-chain polymers consisting of phosphate, ribose, and four bases (including adenine, guanine, cytosine, and uracil).

[0066] In this article, PDRN contains polydeoxyribonucleotides and / or polyribonucleotides and their associated salts.

[0067] Furthermore, in this paper, PDRN contains polydeoxyribonucleotides and / or polyribonucleotides and their associated sodium salts with a molecular weight of less than or equal to 1500 kDa.

[0068] 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.

[0069] As is known in the art, the term "identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, "identity" also refers to the degree of sequence correlation between polypeptide or polynucleotide sequences, as determined by matching strings of such sequences. "Identity" can be readily calculated using known methods, including but not limited to those described in: *Computational Molecular Biology* (edited by Lesk, AM), Oxford University Press, New York (1988); *Biocomputing: Informatics and Genome Projects* (edited by Smith, DW), Academic Press, New York (1993); *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); *Sequence Analysis in Molecular Biology* (edited by von Heinje, G.), Academic Press (1987); and *Sequence Analysis Primer* (edited by Gribskov, M. and Devereux, J.), Stockton Press, New York (1991). Preferred methods for determining identity are designed to yield the best match between the tested sequences. Methods for determining identity have been incorporated into publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using sequence analysis software such as the Megalign program of the LASERGENE Bioinformatics Computing Suite (DNASTAR, Madison, WI), the GCG program suite (Wisconsin Package version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, 1228 S. Park St. Madison, WI 53715 USA). In the context of this invention, it will be understood that, when using sequence analysis software for analysis, unless otherwise stated, the results of the analysis will be based on the “default values” of the cited program. As used herein, “default values” refers to any set of values ​​or parameters initially loaded with the software upon initial initialization.

[0070] As used herein, the term "physiologically acceptable" refers to molecular entities and compositions that are physiologically tolerable and, when administered to humans, generally do not produce toxicity or allergic reactions or similar adverse reactions (such as stomach upset, dizziness, etc.).

[0071] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0072] Example 1: Sequence Screening

[0073] Search for assembly data number GCA_023373465.1 in the public database NCBI and download the following files: Oncorhynchus keta genome sequence, annotation features, sequence and annotation, transcription, gene coding sequence, protein sequence and sequence report.

[0074] The total number of genes is 63,346, and the screening process is carried out in multiple steps based on the gene annotation file information.

[0075] (1) Remove negative strand genes, filtering out 31,300 genes. Screening logic: Genes encoded by the positive strand are more direct and clear in transcriptional regulation and functional expression, which is convenient for subsequent sequence design and functional verification; negative strand genes may involve complex regulatory mechanisms such as antisense RNA, which is not conducive to constructing a stable PDRN synthesis template.

[0076] (2) Sequences with a length of 50-1500 bp were screened, and 24,924 genes were filtered out. Screening logic: The molecular weight of natural PDRN is between 50 and 1500 kDa, and the corresponding DNA sequence length is about 50-1500 bp; retaining sequences within this length range can ensure that the molecular weight characteristics of the obtained PDRN are similar to those of the reported biologically active natural PDRN, thereby maintaining its ability to activate adenosine A2A receptor and participate in salvage pathways.

[0077] (3) Filter out poly-CG dinucleotides (CpG) and remove 6102 genes. Screening logic: CpG dinucleotides are known ligands of Toll-like receptor 9 (TLR9), which can activate the innate immune response and trigger the release of inflammatory factors; in order to reduce the potential immunogenicity of synthetic PDRN and avoid unnecessary inflammatory side effects, sequences containing poly-CpG motifs need to be removed.

[0078] (4) Select out Poly-G-rich genes (GGGGG, CGGG, CCCG, etc.), removing 213 genes. Selection logic: Sequences rich in continuous guanine (G) are prone to forming atypical G-quadruplex secondary structures; this higher-order structure may hinder the effective binding of PDRN to adenosine A2A receptor, affecting its biological activity; therefore, sequences containing Poly-G motifs need to be removed.

[0079] (5) Genes with adenine and thymine (A+T) content not in the range of 60%~80% were filtered out, removing 635 genes. Screening logic: The natural ligand of the adenosine A2A receptor is adenosine, which is rich in adenine in its structure; a higher A+T content (especially the adenine ratio) in PDRN may enhance the affinity or activation efficiency of the A2A receptor by providing more adenosine-like structural units; through experimental optimization, this invention has determined that sequences with A+T content between 60% and 80% have better biological activity.

[0080] (6) Remove genes with guanine and cytosine (GC > 60%) content exceeding 60%, as well as highly immunogenic gene sequences. Screening logic: High GC content sequences are too stable, which may make it difficult for PDRN to be effectively taken up or metabolized under physiological conditions; at the same time, it is necessary to further screen and remove known highly immunogenic motifs (such as specific palindromic sequences, TLR recognition motifs, etc.) to ensure the biosafety of synthesized PDRN to the greatest extent. This step screened out 0 sequences, indicating that after the above filtering steps, the GC content and immunogenic motif distribution of the remaining sequences meet the safety requirements.

[0081] (7) Genes with highly repetitive sequences of more than three consecutive identical nucleotides were filtered out, resulting in the rejection of 157 genes. Screening logic: Continuously repetitive nucleotide sequences (such as AAAA, TTTT) are prone to slippage during synthesis or storage, leading to sequence heterogeneity; at the same time, such repetitive sequences may cause non-specific protein or cell membrane binding, interfering with the specific interaction between PDRN and adenosine A2A receptor, thus reducing its therapeutic effect.

[0082] After the above multi-step screening, 15 sequences that meet the characteristics of specific length, base composition and low immunogenicity were finally obtained. The sequence information is shown as SEQ ID No.1 to SEQ ID No.15.

[0083] Example 2: Preparation of Specific Sequences

[0084] PCR amplification primers were designed based on SEQ ID No. 1 to SEQ ID No. 15 obtained in Example 1, and the sequence listing is shown in Table 1.

[0085] Using the salmon genome (derived from laboratory extraction) as a template, 15 selected sequences were amplified using PrimeSTAR® Max DNAPolymerase (from Takara, batch number R047A). The amplification system and amplification program are shown in Tables 2 and 3, respectively.

[0086] Table 1 Amplification Primer Table

[0087]

[0088]

[0089] Table 2 Amplification System

[0090]

[0091] Table 3 Amplification Procedure

[0092]

[0093] Based on the above amplification system and procedure, the specific sequence PDRN was successfully obtained, as shown in the following figures. Figure 1 As shown. Figure 1 The numbers used from left to right are: molecular weight standard (Marker), SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, and SEQ ID No.15.

[0094] After purification using a kit (from Tiangen Biotech Co., Ltd., catalog number DP213), the PCR product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for Sanger sequencing. The results showed that the amplified sequence was consistent with the theoretical sequence.

[0095] Example 3 Cytotoxicity Evaluation

[0096] The specific PDRN sequences SEQ ID No. 1 to SEQ ID No. 15 obtained in Example 2, along with salmon PDRN, were subjected to cytotoxicity safety testing at a sample concentration of 0.01%.

[0097] Specifically, HaCat cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum) at 37°C with 5% CO2. After 24 hours of cell culture, the original medium was discarded, and serum-free (DMEM) medium containing the sample was added, along with 0.1% bovine serum albumin. The cells were then cultured for another 24 hours. A blank control was prepared by replacing the sample with an equal volume of serum-free medium. The relative cell proliferation rate was determined using the CCK-8 assay, and the data are shown in Table 4.

[0098] Table 4 Cytotoxicity Safety Results

[0099]

[0100] Table 4 demonstrates that among the 15 PDRN sequences prepared, 9 sequences showed a relative cell proliferation rate of over 90%, indicating that SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15 are non-cytotoxic.

[0101] Example 4: Efficacy Evaluation

[0102] The efficacy was tested using SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 and salmon PDRN samples at a concentration of 0.01%.

[0103] Specifically, HaCat cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum) at 37°C with 5% CO2. After 24 hours of cell culture, the original medium was discarded, and serum-free (DMEM) medium containing the sample was added, along with 0.1% bovine serum albumin. The cells were cultured for another 24 hours. A blank control was prepared by replacing the sample with an equal volume of serum-free medium. After incubation, the cell supernatant was collected, and the VEGF content was determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. The test data are shown in Table 5. A graph was plotted from the data in Table 5. Figure 2 .

[0104] Table 5 Results of the test on the efficacy of promoting VEGF repair

[0105]

[0106] Note: * indicates p < 0.05 compared with the blank control group; ** indicates p < 0.01 compared with the blank control group; # indicates p < 0.05 compared with the salmon PDNR group; ## indicates p < 0.01 compared with the salmon PDRN group; ### indicates p < 0.001 compared with the salmon PDRN group.

[0107] 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.

[0108] The relative VEGF content indicated that the nine PDRN samples screened in Example 3 promoted VEGF expression. Sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15 showed significant promoting effects, demonstrating that the prepared specific sequence samples possess excellent efficacy in skin repair, wound healing, improvement of skin inflammation, and promotion of angiogenesis. The highly efficient VEGF-inducing effect of the PDRN sequences of this invention holds promise for expanding into the fields of functional recovery and fertility preservation after reproductive tissue injury.

[0109] Example 5: Determination of the optimal concentration of PDRN to promote VEGF expression

[0110] 1. In vitro experiments

[0111] Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were seeded in confocal culture dishes and cultured overnight at 37°C in a 5% CO2 incubator. After cell attachment, the original culture medium was discarded and replaced with fresh culture medium containing different concentrations of PDRN (SEQ ID No. 1). The experiment included a control group (containing only an equal volume of physiological saline without PDRN) and PDRN-treated groups with PDRN concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Each group was divided into three replicates, and the cells were cultured for another 24 hours at 37°C in a 5% CO2 incubator.

[0112] After culture, discard the culture medium and wash the cells three times with PBS for 5 minutes each time. Fix with 4% paraformaldehyde at room temperature for 15 minutes, then wash three times with PBS. Permeabilize with PBS containing 0.1% Triton X-100 at room temperature for 10 minutes, then wash three times with PBS. Block with 5% BSA at room temperature for 1 hour. Discard the blocking solution and add rabbit anti-human VEGF primary antibody (dilution 1:200), incubate overnight at 4°C. The next day, wash three times with PBS, add goat anti-rabbit secondary antibody labeled with Alexa Fluor 488 (dilution 1:500), and incubate at room temperature in the dark for 1 hour. After washing three times with PBS, stain the nuclei with DAPI staining solution at room temperature in the dark for 5 minutes. After washing with PBS, observe and acquire images under a confocal fluorescence microscope.

[0113] 2. In vivo experiments

[0114] Twenty C57BL / 6 mice (n=5 per group) were randomly divided into four groups: a control group (administered with an equal volume of saline), a low-dose PDRN group (10 mg / kg), a medium-dose PDRN group (25 mg / kg), and a high-dose PDRN group (50 mg / kg), using PDRN (SEQ ID No. 1). After establishing a full-thickness skin defect (6 mm in diameter) model on the back, the animals in each group were administered the corresponding dose of PDRN or saline via intraperitoneal injection once daily for 7 consecutive days.

[0115] Twenty-four hours after the last administration, animals were sacrificed, and wound and surrounding skin tissue were harvested. The tissues were fixed in 4% paraformaldehyde, routinely dehydrated, and embedded in paraffin to prepare 4 μm thick sections. After dewaxing, hydration, and antigen retrieval, the sections were subjected to immunofluorescence staining. The staining procedure was the same as in in vitro experiments: after blocking with 5% BSA, rabbit anti-mouse VEGF primary antibody (dilution 1:200) was added and incubated overnight at 4°C; the next day, after washing, fluorescently labeled secondary antibody was added and incubated at room temperature in the dark; DAPI was used to counterstain cell nuclei. After mounting, the slides were observed and images were acquired under a fluorescence microscope.

[0116] 3. Experimental Results

[0117] Immunofluorescence staining results showed ( Figure 3 In the control group (A), VEGF (green fluorescence) expression was weak. After PDRN treatment, the VEGF fluorescence intensity increased significantly with the increase of PDRN concentration. The PDRN 100 μg / mL treatment group showed a strong VEGF positive green fluorescence signal and good co-localization with DAPI nuclear staining (blue fluorescence).

[0118] Immunohistochemical results showed ( Figure 3In the control group (B), there were fewer VEGF-positive cells. VEGF expression was enhanced in all PDRN dose groups compared to the control group, showing a certain dose-dependent effect. The 50 mg / kg PDRN dose group showed the strongest VEGF fluorescence signal, indicating that this dose of PDRN had the most significant effect on promoting VEGF expression.

[0119] In summary, the optimal concentrations of PDRN for promoting VEGF protein expression are 100 μg / mL (in vitro) and 50 mg / kg (in vivo). At these concentrations / dosages, the VEGF fluorescence signal intensity reached its peak, suggesting that PDRN exerts its biological function of promoting angiogenesis and tissue repair by upregulating VEGF expression.

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule with a specific sequence, characterized in that, The nucleic acid molecule comprises any one or more of the following sequences A1) to A3): A1) The nucleic acid molecule contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15, or a nucleic acid sequence that has 98% or more identity with that sequence; A2) Nucleic acid molecules that hybridize with the sequence described in A1) under stringent conditions; A3) is a nucleic acid molecule whose sequence is complementary to that described in A1).

2. The method for preparing nucleic acid molecules according to claim 1, characterized in that, include: Using salmon genomic DNA as a template, PCR amplification was performed using primers capable of amplifying the nucleic acid molecules to obtain the amplification product. The amplification product is then purified. The primers used for PCR amplification are nucleic acid sequences shown in SEQ ID No. 16 to SEQ ID No.

45.

3. The preparation method according to claim 2, characterized in that, SEQ ID No. 16 and SEQ ID No. 17 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 1; SEQ ID No. 18 and SEQ ID No. 19 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 2; SEQ ID No. 20 and SEQ ID No. 21 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 3; SEQ ID No. 22 and SEQ ID No. 23 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 4; SEQ ID No. 24 and SEQ ID No. 25 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 5; SEQ ID No. 26 and SEQ ID No. 27 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 6; SEQ ID No. 28 and SEQ ID No. 29 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 7; SEQ ID No. 30 and SEQ ID No. 31 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 8; SEQ ID No. 32 and SEQ ID No. 33 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 9; SEQ ID No. 34 and SEQ ID No. 35 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 10; SEQ ID No. 36 and SEQ ID No. 37 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 11; SEQ ID No. 38 and SEQ ID No. 39 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 12; SEQ ID No. 40 and SEQ ID No. 41 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 13; SEQ ID No. 42 and SEQ ID No. 43 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No. 14; SEQ ID No. 44 and SEQ ID No. 45 are used to amplify nucleic acid molecules with nucleic acid sequences as shown in SEQ ID No.

15.

4. The application of nucleic acid molecules or their sodium salts in the preparation of PDRN-related products, characterized in that, The nucleic acid molecule described herein contains at least one or more of the following sequences: B1) to B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence that has 98% or more identity with the sequence described in B1); B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

5. The use of nucleic acid molecules or their sodium salts in the preparation of products for skin repair and / or improvement of skin inflammation and / or skin wound healing, characterized in that, The nucleic acid molecule described herein contains at least one or more of the following sequences: B1) to B4): B1) Contains any one of the nucleic acid sequences shown in SEQ ID No. 1 to SEQ ID No. 15; B2) A nucleic acid sequence that has 98% or more identity with the sequence described in B1); B3) Nucleic acid molecules that hybridize under stringent conditions with any of the specified nucleic acid sequences from B1) to B2); Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in B4 and B1-B2).

6. The application according to claim 5, characterized in that, The applications also include the preparation of products that promote cell proliferation.

7. The application according to claim 5, characterized in that, The applications also include the preparation of products that promote angiogenesis.

8. The application according to claim 5, characterized in that, The application also includes the preparation of products that increase the content of vascular endothelial growth factor.

9. The application according to claim 5, characterized in that, The products include cosmetics and pharmaceutical / medical devices.

10. A product characterized in that, It contains the nucleic acid molecule as described in claim 1 as an active ingredient; and excipients acceptable for cosmetic or pharmaceutical products.

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