Sheep stem ubiquitin protein as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of sheep stem ubiquitin protein, and belongs to the technical field of bioengineering. The method specifically comprises the following steps: S1, determining a target protein: determining a target molecule of an active component in the sheep stem element; s2, gene synthesis and cloning: synthesizing a target gene sequence by using a DNA synthesis technology, cloning the synthesized gene into an expression vector, and introducing the constructed expression vector into a proper host cell. The sheep stem ubiquitin protein can be applied to the fields of cosmetics for skin care and medical treatment. The preparation method of the sheep stem ubiquitin protein replaces a traditional extraction method through gene synthesis, active ingredients can be prevented from being extracted from human placenta amniotic membranes, and ethical and legal problems are avoided. Efficient production can be achieved, a large amount of target protein can be efficiently produced through gene synthesis and cell expression, and the production cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a sheep stem cell ubiquitin protein and a preparation method and application thereof. Background Art

[0002] Skin aging is a phenomenon of skin aging caused by natural or unnatural factors. After a person is born, the skin tissue develops day by day and its functions become increasingly active. The growth period of skin tissue generally ends at around 25 years old. Since then, growth and aging proceed simultaneously. The elastic fibers of the skin gradually become thicker. In the early aging period of 40 to 50 years old, the aging of the skin gradually becomes obvious. Skin aging is a natural law, and anti-aging is an eternal beauty topic. Although we cannot prevent the natural aging of the skin, we can change the aging state of the skin through scientific and effective measures.

[0003] Ubiquitin is a small molecule protein that is widely present in eukaryotic cells. Its main function in the body is to target target proteins to the proteasome for degradation, thereby participating in various physiological processes of the cell. Ubiquitination is an important way of post-translational modification of proteins. The regulation of proteins is achieved through the covalent connection of ubiquitin to proteins. The ubiquitin-proteasome pathway plays a key role in the degradation of intracellular proteins and is involved in various physiological processes such as cell cycle regulation, apoptosis, and DNA repair. Ubiquitination is a dynamic and multifaceted post-translational modification of proteins involving multiple cellular processes. In this process, ubiquitin (a protein composed of 76 amino acids) is activated and attached to the target protein by specific enzymes (E1, E2, and E3) to form a ubiquitin-protein complex. This complex is then recognized and degraded by the proteasome, thereby regulating the stability and function of the protein.

[0004] As a post-translational modification, ubiquitination has an important influence on protein degradation, stability, activity and localization. Ubiquitination is not only involved in basic life activities such as cell cycle, proliferation, apoptosis, differentiation, but also closely related to signal transduction, damage repair, inflammation and immunity. Abnormal ubiquitination is related to the occurrence and development of many diseases, such as tumors, cardiovascular diseases, neurodegenerative diseases, etc. Therefore, in-depth research on the mechanism of action of ubiquitination in diseases is of great significance for the diagnosis and treatment of diseases.

[0005] Human amniotic mesenchymal stem cells (hA-MSC) are stem cells isolated from the amniotic tissue on the side of the placenta close to the fetus. The amnion is the innermost tissue of the placenta, located on the surface of the fetal chorion, and is an early product of embryonic development that is closely connected to the developing fetus. The amnion is a translucent film with toughness, no nerves, blood vessels, or lymphatic vessels, and wraps the amniotic fluid and the fetus, providing an environment for the embryo to grow and develop. It is only 0.02 to 0.5 mm thick and consists of an epithelial layer, a basal layer, and a matrix layer (connective tissue layer). hAMSCs are derived from the fibroblast layer, which is composed of loose fibroblasts and reticular fibers. Amniotic mesenchymal stem cells have the potential to differentiate into multiple different tissue types. When stimulated by exogenous growth factors or chemicals, hAMSCs can differentiate into cells of different tissue sources in the three germ layers (ectoderm, mesoderm, and endoderm). For example, under specific culture conditions, hAMSCs can differentiate into skeletal muscle cells, pancreatic secretory cells, cardiomyocytes, endothelial cells, etc. Under in vitro culture conditions, hAMSCs can continuously divide for multiple generations, and their proliferation ability will not decrease significantly. This proliferation ability allows hAMSCs to be expanded in large quantities, providing a sufficient source of cells for cell therapy, and can secrete a variety of anti-inflammatory factors and growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), hepatocyte growth factor (HGF), etc. These factors can inhibit inflammatory responses and promote tissue repair and regeneration.

[0006] Amniotic mesenchymal stem cells can be directed to differentiate into a variety of cell types to repair damaged tissues and organs, such as heart disease, liver disease, and nervous system diseases. Amniotic mesenchymal stem cells have immunomodulatory effects, which can inhibit immune responses and reduce inflammation levels, providing new treatments for autoimmune diseases and inflammatory diseases. Amniotic mesenchymal stem cells are rich in antioxidants, such as superoxide dismutase (SOD) and glutathione (GSH), which can remove free radicals in the body and slow down the aging process. At the same time, they can also promote cell proliferation and improve the function of tissues and organs, thereby achieving anti-aging effects. Amniotic mesenchymal stem cells can differentiate into hematopoietic stem cells, providing a steady supply of blood cells for the blood system, and play an important role in the treatment of blood diseases, leukemia, and other diseases. Studies have found that amniotic mesenchymal stem cells can inhibit the proliferation and invasion of tumor cells, and improve the body's ability to clear tumor cells by regulating immune responses.

[0007] A large number of preclinical safety experiments have been completed in the research and application of amniotic mesenchymal stem cells, including acute toxicity, long-term toxicity, tumorigenicity, teratogenicity, local irritation, fever and immunotoxicity tests. The results show that amniotic mesenchymal stem cell treatment is safe and non-toxic, and will not produce immune rejection and allergic reactions. However, the research and application of amniotic mesenchymal stem cells are still in their early stages, and their safety and effectiveness need to be further explored in order to provide more powerful theoretical support for clinical practice. However, the traditional method of extracting active ingredients from human placental amniotic membrane inevitably involves ethical and legal issues. Summary of the invention

[0008] Purpose of the invention: The purpose of the present invention is to provide an amniotic fluid ubiquitin protein and a preparation method and application thereof; it can solve the problem that the traditional method of extracting active ingredients from human placental amniotic membrane inevitably involves ethical and legal issues.

[0009] Technical solution: To solve the above technical problems, according to one aspect of the present invention, more specifically, a sheep stem cell ubiquitin protein comprises the following components: ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water.

[0010] Furthermore, the sheep stem cell ubiquitin protein specifically includes the following components by weight: 0.5-4 parts of ubiquitin protein, 0.3-7 parts of macromolecular sodium hyaluronate, 0.3-7 parts of small molecule sodium hyaluronate, 0.1-1 parts of acetyl hexapeptide-8, 0.5-4 parts of carnosine and 77-98 parts of water.

[0011] According to another aspect of the present invention, a method for preparing a sheep stem cell ubiquitin protein is provided. The method is implemented based on the components in the sheep stem cell ubiquitin protein described above, and specifically comprises the following steps:

[0012] S1. Determination of target protein: Determine the target molecule of the active ingredient in sheep dried extract;

[0013] S2. Gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence, then clone the synthesized gene into an expression vector, and then introduce the constructed expression vector into a suitable host cell;

[0014] S3. Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities, and then the target protein is purified;

[0015] S4. Ubiquitination of target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1. The activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated.

[0016] S5. Mixing the components: ubiquitin protein, large molecule sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are mixed according to the component ratio to obtain a sheep stem cell ubiquitin protein mixture.

[0017] Furthermore, in step S1, the target molecule is one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0018] Furthermore, in step S2, the expression vector is a plasmid or a viral vector; and the host cell is Escherichia coli, yeast, insect cell or mammalian cell.

[0019] Furthermore, in step S4, the optimization of culture conditions includes but is not limited to adjusting the temperature and the concentration of the inducer; the method for purifying the target protein includes but is not limited to affinity chromatography, ion exchange chromatography or gel filtration.

[0020] Furthermore, in step S4, the concentration of ubiquitin activating enzyme E1 is 0.25-10 μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.25-10 μmoL / L, and the concentration of ubiquitin ligase E3 is 0.25-10 μmoL / L.

[0021] According to another aspect of the present invention, an application of a sheep stem cell ubiquitin protein is provided. The sheep stem cell ubiquitin protein described in any one of the above items can be applied to skin care cosmetics and the medical field.

[0022] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM.

[0023] Furthermore, the amount of the ovine stem cell ubiquitin protein in the skin care cosmetic is 2-15% (w / w).

[0024] Beneficial effects: A kind of sheep stem element ubiquitin protein of the present invention has a scientific ratio and a significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve a very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, it is not necessary to inject such skin-beautifying nutrients into the dermis, thereby improving the safety of users during use, and a kind of sheep stem element ubiquitin protein of the present invention can be applied to skin care cosmetics, so that mild, stable and effective anti-aging skin care cosmetics for skin can be prepared. At the same time, a preparation method of a kind of sheep stem element ubiquitin protein of the present invention replaces the traditional extraction method through gene synthesis, which can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be efficiently produced, gene synthesis and cell expression can efficiently produce a large amount of target protein, reducing production costs. And it can ensure the consistency of the protein, the protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch differences that may occur in the natural extraction process, thereby improving the quality of the sheep stem element ubiquitin protein and then improving the use effect of the sheep stem element ubiquitin protein. In cancer treatment, NUB1 can inhibit tumor growth, enhance cancer cell apoptosis, increase sensitivity to chemotherapy, and regulate the cell cycle of cancer cells. In neurodegenerative disease management, it can reduce protein aggregation, enhance the clearance of toxic proteins, protect nerves from degenerative damage, and improve protein quality control. In cancer treatment, EPB41 can regulate cell adhesion, control cell migration, regulate tumor metastasis, and enhance membrane stability. In blood disease management, it can treat hereditary elliptocytosis, improve red blood cell membrane integrity, enhance red blood cell stability, and regulate erythropoiesis. In the treatment of microvascular diseases, ADCY4 can regulate heart function, control blood pressure, regulate vascular smooth muscle, and enhance endothelial cell function. In response to metabolic disorders, it can regulate glucose metabolism, homeostatic energy, control lipid metabolism, and regulate insulin sensitivity. In reproductive medicine, it can be used for infertility treatment, sperm function enhancement, ovarian function regulation, and reproductive hormone signaling. APOA2 can regulate HDL metabolism, enhance cholesterol efflux, control triglyceride metabolism, and prevent atherosclerosis in response to lipid metabolism disorders. It can regulate insulin sensitivity, regulate glucose homeostasis, respond to obesity-related complications, and control energy metabolism in the management of metabolic syndrome. It can treat atherosclerosis, improve lipid profiles, protect blood vessels, and fight inflammation in the treatment of cardiovascular diseases. GDF11 can regenerate skeletal muscle, improve heart function, enhance cognitive function, and prevent age-related tissue decline in response to age-related diseases. It can reduce cardiac hypertrophy, reshape blood vessels, treat heart failure, and prevent heart aging in response to cardiovascular diseases. Its applications in neurology include regulating neural stem cells, preventing brain aging, enhancing neurogenesis, and treating cognitive decline. It can regulate glucose metabolism, stabilize energy, manage obesity, and improve metabolic health in metabolic disorders.Epidermal growth factor can promote cell proliferation, regulate cell differentiation, accelerate cell migration, maintain tissue renewal in terms of cell growth and differentiation regulation. In terms of tissue repair and regeneration, it can promote wound healing, accelerate epidermal repair, enhance angiogenesis, and improve tissue regeneration. At the same time, its anti-aging mechanism can promote collagen synthesis, enhance skin elasticity, improve cell vitality, and delay the aging process. Fibroblast growth factor can maintain stem cell function, promote angiogenesis, improve metabolic function, and protect the nervous system. Insulin-like growth factor can regulate cell growth and development, improve metabolic health, maintain muscle function, and promote tissue repair. Vascular endothelial growth factor can promote angiogenesis, improve circulatory function, accelerate tissue repair, and maintain organ health. Nerve growth factor is an important neurotrophic factor. Its main function is to promote the growth, differentiation, survival, and repair of neurons. Nerve growth factor can interact with the ubiquitin protein system to regulate protein homeostasis, thereby playing a potential role in anti-aging and prevention of multiple diseases. At the same time, it can maintain protein homeostasis, protect and repair neurons, anti-inflammatory and immune regulation, delay cell aging, protect the nervous system, prolong life, and prevent diseases including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, and cancer. CXCL12 can regulate protein homeostasis. In terms of anti-aging, it can delay tissue aging, maintain stem cell homeostasis and promote stem cell regeneration, protect mitochondrial function, and prevent multiple diseases including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, cancer, and immune-related diseases in disease prevention. The application of Complement Component 8Beta in anti-aging is to reduce chronic inflammation, protect cell integrity, and regulate metabolic homeostasis. Its application in disease prevention includes immunity to autoimmune diseases, neurodegenerative diseases, cardiovascular diseases, infectious diseases, and tumors. RASGRF1 can regulate cell proliferation and apoptosis, regulate aging-related signaling pathways, and regulate neural function. Its application in anti-aging can delay cell aging, improve nervous system aging, and regulate metabolic homeostasis. Its application in disease prevention includes tumor prevention and treatment, prevention of neurodegenerative diseases, metabolic diseases, immune-related diseases, fibrotic diseases, cardiovascular diseases, and immune regulation. LUM has anti-inflammatory, immunomodulatory, anti-fibrotic and anti-tumor effects. Its application in anti-aging can delay tissue aging, promote tissue repair and regeneration, and improve metabolic homeostasis. In terms of disease prevention, it can prevent fibrotic diseases, neurodegenerative diseases, cardiovascular diseases, tumors and immune-related diseases. Large molecule sodium hyaluronate has moisturizing, lubricating and buffering, tissue repair and regeneration effects. Small molecule sodium hyaluronate has deep moisturizing, promoting skin nutrition supply and waste excretion, repairing skin damage, anti-oxidation and anti-aging, improving skin quality, and lubricating and protecting. Acetyl hexapeptide-8 has anti-wrinkle and anti-aging effects, promotes collagen synthesis, and has anti-oxidation and moisturizing effects.Carnosine has the effects of anti-oxidation and anti-aging, protecting cell function, regulating immunity, nourishing nerves, improving myocardial metabolism, promoting muscle health and improving cognitive function, reducing pigmentation and promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the method flow chart. DETAILED DESCRIPTION

[0026] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0029] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0030] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0031] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 0.25μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.25μmoL / L, and the concentration of ubiquitin ligase E3 is 0.25μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0032] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 0.5 parts of ubiquitin protein, 0.3 parts of macromolecular sodium hyaluronate, 0.3 parts of small molecule sodium hyaluronate, 0.1 parts of acetyl hexapeptide-8, 0.5 parts of carnosine and 98 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0033] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 2% (w / w).

[0034] Example 2

[0035] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0036] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0037] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0038] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 0.3μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.3μmoL / L, and the concentration of ubiquitin ligase E3 is 0.3μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0039] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 0.6 parts of ubiquitin protein, 0.4 parts of macromolecular sodium hyaluronate, 0.4 parts of small molecule sodium hyaluronate, 0.15 parts of acetyl hexapeptide-8, 0.6 parts of carnosine and 96 parts of water to obtain sheep stem ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0040] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 3% (w / w).

[0041] Example 3

[0042] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0043] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0044] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0045] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 0.35μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.35μmoL / L, and the concentration of ubiquitin ligase E3 is 0.35μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur in the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0046] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 0.7 parts of ubiquitin protein, 0.7 parts of macromolecular sodium hyaluronate, 0.8 parts of small molecule sodium hyaluronate, 0.2 parts of acetyl hexapeptide-8, 0.8 parts of carnosine and 93 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0047] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 4% (w / w).

[0048] Example 4

[0049] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0050] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0051] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0052] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 0.4μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.4μmoL / L, and the concentration of ubiquitin ligase E3 is 0.4μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0053] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 1 part of ubiquitin protein, 1.5 parts of macromolecular sodium hyaluronate, 1.5 parts of small molecule sodium hyaluronate, 0.3 parts of acetyl hexapeptide-8, 1.1 parts of carnosine and 91 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0054] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 5% (w / w).

[0055] Example 5

[0056] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0057] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0058] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0059] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 1.5μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 1.5μmoL / L, and the concentration of ubiquitin ligase E3 is 1.5μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0060] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 1.5 parts of ubiquitin protein, 2 parts of macromolecular sodium hyaluronate, 2 parts of small molecule sodium hyaluronate, 0.4 parts of acetyl hexapeptide-8, 1.7 parts of carnosine and 89 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0061] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 6% (w / w).

[0062] Example 6

[0063] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0064] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0065] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0066] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 3μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 3μmoL / L, and the concentration of ubiquitin ligase E3 is 3μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0067] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 1.9 parts of ubiquitin protein, 2.5 parts of macromolecular sodium hyaluronate, 2.5 parts of small molecule sodium hyaluronate, 0.5 parts of acetyl hexapeptide-8, 1.9 parts of carnosine and 88 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0068] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 7% (w / w).

[0069] Example 7

[0070] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0071] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0072] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0073] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 3.5μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 3.5μmoL / L, and the concentration of ubiquitin ligase E3 is 3.5μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0074] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 2.1 parts of ubiquitin protein, 3 parts of macromolecular sodium hyaluronate, 3 parts of small molecule sodium hyaluronate, 0.6 parts of acetyl hexapeptide-8, 2.1 parts of carnosine and 85 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0075] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 8% (w / w).

[0076] Example 8

[0077] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0078] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0079] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0080] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 4μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 4μmoL / L, and the concentration of ubiquitin ligase E3 is 4μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0081] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 2.7 parts of ubiquitin protein, 3.5 parts of macromolecular sodium hyaluronate, 3.5 parts of small molecule sodium hyaluronate, 0.6 parts of acetyl hexapeptide-8, 2.3 parts of carnosine and 83 parts of water to obtain sheep stem ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0082] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 9% (w / w).

[0083] Example 9

[0084] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0085] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0086] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0087] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 4.5μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 4.5μmoL / L, and the concentration of ubiquitin ligase E3 is 4.5μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0088] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 2.9 parts of ubiquitin protein, 4 parts of macromolecular sodium hyaluronate, 4 parts of small molecule sodium hyaluronate, 0.7 parts of acetyl hexapeptide-8, 2.5 parts of carnosine and 81 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0089] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 10% (w / w).

[0090] Example 10

[0091] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0092] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0093] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0094] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 6μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 6μmoL / L, and the concentration of ubiquitin ligase E3 is 6μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0095] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 3.1 parts of ubiquitin protein, 4.5 parts of macromolecular sodium hyaluronate, 4.5 parts of small molecule sodium hyaluronate, 0.8 parts of acetyl hexapeptide-8, 2.9 parts of carnosine and 80 parts of water to obtain sheep stem ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0096] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 11% (w / w).

[0097] Embodiment 11

[0098] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0099] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0100] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0101] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 7μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 7μmoL / L, and the concentration of ubiquitin ligase E3 is 7μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0102] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 3.4 parts of ubiquitin protein, 6 parts of macromolecular sodium hyaluronate, 6 parts of small molecule sodium hyaluronate, 0.9 parts of acetyl hexapeptide-8, 3.5 parts of carnosine and 89 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect, the skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0103] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 12% (w / w).

[0104] Example 12

[0105] Step 1, determination of target protein: determine the target molecules of active ingredients in sheep stem extract as one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

[0106] Step 2, gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence. Today's technology can efficiently synthesize DNA containing specific sequences, avoiding the complexity and ethical issues of extracting from natural sources. Then clone the synthesized gene into an expression vector. Common vectors include plasmids or viral vectors. This vector needs to contain all the necessary elements for host cell expression, such as promoters, terminators, etc. Then introduce the constructed expression vector into a suitable host cell. Common host cells include Escherichia coli, yeast, insect cells or mammalian cells.

[0107] Step 3: Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities. Common optimization methods include adjusting the temperature, the concentration of the inducer, etc., and then purifying the target protein through affinity chromatography, ion exchange chromatography or gel filtration. The purified protein can be further used for product development and testing.

[0108] Step 4, ubiquitination of the target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1, and the activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. Among them, the concentration of ubiquitin activating enzyme E1 is 10μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 10μmoL / L, and the concentration of ubiquitin ligase E3 is 10μmoL / L. Replacing the traditional extraction method with gene synthesis can avoid extracting active ingredients from human placental amniotic membrane, thereby avoiding ethical and legal issues. And it can be produced efficiently. Gene synthesis and cell expression can efficiently produce a large number of target proteins and reduce production costs. And it can ensure the consistency of the protein. The protein obtained by gene synthesis has a high degree of purity and consistency, avoiding the batch-to-batch differences that may occur during the natural extraction process, thereby improving the quality of sheep stem ubiquitin protein and then improving the use effect of sheep stem ubiquitin protein.

[0109] Step 5, mixing the components: the determined target molecule ubiquitin protein, macromolecular sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are uniformly mixed according to 4 parts of ubiquitin protein, 7 parts of macromolecular sodium hyaluronate, 7 parts of small molecule sodium hyaluronate, 1 part of acetyl hexapeptide-8, 4 parts of carnosine and 77 parts of water to obtain sheep stem cell ubiquitin protein. The ratio is scientific and has significant synergistic effect. Compared with a single anti-aging ingredient, the effect is obvious. Continuous use can achieve very obvious anti-aging effect. The skin becomes firm and full, and restores a youthful state. At the same time, there is no need to inject such skin-beautifying nutrients into the dermis, which improves the safety of users during use. In addition, the sheep stem cell ubiquitin protein in the present invention can be applied to skin care cosmetics, so that a mild, stable and effective anti-aging skin care cosmetic can be prepared.

[0110] When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, ComplementComponent 8Beta, RASGRF1, and LUM, and the dosage of the sheep stem ubiquitin protein is 15% (w / w).

[0111] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A sheep stem cell ubiquitin protein, characterized in that It includes the following components: ubiquitin protein, large molecule sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water.

2. The sheep stem cell ubiquitin protein according to claim 1, characterized in that: The sheep stem cell ubiquitin protein specifically includes the following components by weight: 0.5-4 parts of ubiquitin protein, 0.3-7 parts of macromolecular sodium hyaluronate, 0.3-7 parts of small molecule sodium hyaluronate, 0.1-1 parts of acetyl hexapeptide-8, 0.5-4 parts of carnosine and 77-98 parts of water.

3. A method for preparing a sheep stem cell ubiquitin protein, characterized in that: The method is implemented based on the components in the ovine stem cell ubiquitin protein described in any one of claims 1-2, and specifically comprises the following steps: S1. Determination of target protein: Determine the target molecule of the active ingredient in sheep dried extract; S2. Gene synthesis and cloning: Use DNA synthesis technology to synthesize the target gene sequence, then clone the synthesized gene into an expression vector, and then introduce the constructed expression vector into a suitable host cell; S3. Expression and purification of target protein: By optimizing the culture conditions, the host cells can express the target protein in large quantities, and then the target protein is purified; S4. Ubiquitination of target protein: Ubiquitin molecules first bind to ubiquitin activating enzyme E1. The activated ubiquitin is transferred from E1 to ubiquitin conjugating enzyme E2 through the transesterification process. Ubiquitin ligase E3 recognizes and binds to the specific site of the target protein. E3 enzyme catalyzes the transfer of ubiquitin from E2 to the lysine residue of the target protein, and the target protein is ubiquitinated. S5. Mixing the components: ubiquitin protein, large molecule sodium hyaluronate, small molecule sodium hyaluronate, acetyl hexapeptide-8, carnosine and water are mixed according to the component ratio to obtain a sheep stem cell ubiquitin protein mixture.

4. The method for preparing a sheep stem cell ubiquitin protein according to claim 3, characterized in that: In step S1, the target molecule is one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

5. The method for preparing a sheep stem cell ubiquitin protein according to claim 3, characterized in that: In step S2, the expression vector is a plasmid or a viral vector; the host cell is Escherichia coli, yeast, insect cell or mammalian cell.

6. The method for preparing a sheep stem cell ubiquitin protein according to claim 3, characterized in that: In step S4, the optimization of culture conditions includes but is not limited to adjusting the temperature and the concentration of the inducer; the method for purifying the target protein includes but is not limited to affinity chromatography, ion exchange chromatography or gel filtration.

7. The method for preparing a sheep stem cell ubiquitin protein according to claim 3, characterized in that: In step S4, the concentration of ubiquitin activating enzyme E1 is 0.25-10 μmoL / L, the concentration of ubiquitin conjugating enzyme E2 is 0.25-10 μmoL / L, and the concentration of ubiquitin ligase E3 is 0.25-10 μmoL / L.

8. An application of sheep stem cell ubiquitin protein, characterized in that: The ovine stem cell ubiquitin protein according to any one of claims 1 to 7 can be applied to skin care cosmetics and the medical field.

9. The use of a sheep stem cell ubiquitin protein according to claim 8, characterized in that: When the sheep stem ubiquitin protein is applied to skin care cosmetics, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, and GDF11; when the sheep stem ubiquitin protein is applied to the medical field, the target molecules are: one or more of NUB1, EPB41, ADCY4, APOA2, GDF11, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), CXCL12, Complement Component 8Beta, RASGRF1, and LUM.

10. The use of the sheep stem cell ubiquitin protein according to claim 8, characterized in that: The amount of the ovine stem cell ubiquitin protein in the skin care cosmetics is 2-15% (w / w).

Images