Bovine bone marrow peptides and their application in enhancing immunity

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing bovine bone marrow peptides and their applications. This invention prepares bovine bone marrow peptides by proteolytic hydrolysis of bovine bone marrow. The bovine bone marrow peptides can effectively enhance the activity of immune cells and / or promote their proliferation. The polypeptides of this invention can improve the body's immunity and also have the advantages of simple preparation, easy absorption, and ease of widespread application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bovine bone marrow peptide, its application in improving immune diseases, and its preparation method. Background Technology

[0002] In recent years, the preparation, functional development, and industrial application of bone-derived bioactive peptides have become a hot topic in the research of animal-derived functional foods. Bones, composed of periosteum, bone tissue, and bone marrow, are a network structure of proteins and calcium, filled with bone marrow containing various nutrients. Bone marrow not only contains abundant proteins, fats, calcium, phosphorus, iron, zinc, copper, strontium, and other trace elements, but also phosphoproteins, phospholipids, chondroitin, mucopolysaccharides, growth hormones, various amino acids, and multiple vitamins—all beneficial health factors, making it a valuable natural food and medicinal resource. The protein content in bones is very high, comparable to fresh meat, and bone protein is a relatively soluble protein with high biological value, making it a high-quality protein source. Currently, domestic and international manufacturers are producing collagen from livestock and fish bones, or bone peptide injections to promote fracture healing, as well as compound bone peptides and bone peptide tablets for the prevention and treatment of rheumatism, fractures, arthritis, osteoporosis, and lumbar disc herniation. With increasingly in-depth research into their nutritional components and efficacy, it has been discovered that bone proteins possess a variety of potential functions. Studies have shown that bone extracts play an important role in lowering blood pressure, regulating immunity, treating bone diseases, and possessing antibacterial and antioxidant properties. With advancements in health food processing technology, more and more scholars are focusing on the bioactivity and applications of animal bone protein peptides and related products.

[0003] A healthy immune system effectively prevents pathogens from invading and maintains the normal functioning and physiological balance of the body. However, with the development of science and technology and intense competition, irregular lifestyles have led to a decline in immunity. Since targeted treatments are difficult to implement, consuming functional foods to boost immunity has become a popular choice. Currently, there is a lack of functional foods that enhance immunity using traditional foods and herbs. Therefore, developing a health product with immune-boosting effects is of great significance and value. Summary of the Invention

[0004] In view of the current state of the technology, the purpose of this invention is to provide a bovine bone marrow peptide that enhances immunity and its applications. Specifically, the bovine bone marrow peptide provided by this invention can improve the activity of immune cells, enhance their proliferative capacity, and thus improve the body's immunity. Furthermore, the bovine bone marrow peptide provided by this invention is non-toxic, has no side effects, and has the advantages of simple preparation and easy absorption, making it suitable for the preparation of health products or pharmaceuticals.

[0005] The present invention first provides a bovine bone marrow peptide, characterized in that its amino acid sequence is shown in SEQ ID NO:1.

[0006] In some embodiments, the bovine bone marrow peptide can enhance immune cell activity and / or promote immune cell proliferation.

[0007] Another aspect of the present invention provides a nucleic acid molecule encoding the bovine bone marrow peptide described herein.

[0008] Another aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0009] Another aspect of the present invention provides an isolated host cell comprising the bovine bone marrow peptide, the nucleic acid molecule, or the vector described herein.

[0010] Another aspect of the present invention provides the use of the bovine bone marrow peptide in the preparation of health products or medicines that enhance immunity.

[0011] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the bovine bone marrow peptide of the present invention, the amino acid sequence of which is shown in SEQ ID NO:1.

[0012] The bovine bone marrow peptide of the present invention, or a pharmaceutical composition containing the bovine bone marrow peptide of the present invention, is applied in the form of lyophilized powder.

[0013] In some embodiments, the bovine bone marrow peptide of the present invention or a pharmaceutical composition containing the bovine bone marrow peptide of the present invention may be used in combination with other immune enhancers.

[0014] In some embodiments, the immune enhancer may be selected from thymosin, transfer factor, human immunoglobulin, lentinan, Ganoderma lucidum polysaccharide, Tremella fuciformis polysaccharide, Trametes versicolor polysaccharide, levamisole, isoproterenol, polyinosinic-cytosine acid, polyinosinic-uridine acid, etc.

[0015] Beneficial effects

[0016] This invention extracts and prepares an active polypeptide from bovine bone marrow. This active polypeptide effectively enhances the activity of immune cells and / or promotes their proliferation. The polypeptide of this invention can improve the body's immunity and has certain clinical application value. Furthermore, the polypeptide 10B3 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Attached Figure Description

[0017] Figure 1 The results shown are the phagocytic index of carbon clearance in each group of mice.

[0018] Figure 2The results shown represent the spleen cell proliferation capacity of mice in each group.

[0019] Figure 3 The results shown represent the NK cell activity of each group of mice.

[0020] Figure 4 The results shown are the delayed-type hypersensitivity values ​​of mice in each group.

[0021] Figure 5 The results shown represent the serum hemolysin production levels in each group of mice. Detailed Implementation

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Except as provided in the operational examples or otherwise indicated, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0024] In some embodiments, the pharmaceutical compositions provided herein comprise about 1 pg to about 2000 mg of the active polypeptide described herein (e.g., a polypeptide component, which may be a single polypeptide in some embodiments), optionally wherein the pharmaceutical composition comprises about 1 pg to about 1000 mg, about 1 pg to about 500 mg, about 1 pg to about 400 mg, about 1 pg to about 300 mg, about 1 pg to about 200 mg, about 1 pg to about 100 mg, about 1 pg to about 50 mg, about 1 pg to about 25 mg, about 1 pg to about 20 mg, about 1 pg to about 15 mg, about 1 pg to about 10 mg, about 1 pg to about The active polypeptides described herein (e.g., polypeptide components, which in some embodiments may be a single polypeptide) of 5 mg, about 1 pg to about 1 mg, about 1 pg to about 500 pg, about 1 pg to about 250 pg, about 1 pg to about 200 pg, about 1 pg to about 150 pg, about 1 pg to about 100 pg, about 1 pg to about 50 pg, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 400 mg, about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg.

[0025] As used herein, the terms “treatment” or “improvement” are used interchangeably. These terms refer to the means of achieving a beneficial or desired outcome (including, but not limited to, therapeutic and / or preventative benefits).

[0026] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Additionally, the use of the term “part” can include a portion of a part or an entire portion. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0027] The term "therapeuticly effective amount" refers to the amount that produces the desired effect of its administration. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, and / or ailment when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such a disease, condition, and / or ailment. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition, and / or ailment, and / or delays its onset. Those skilled in the art will understand that a therapeutically effective amount does not necessarily achieve successful treatment in every particular individual. Rather, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to patients who require such treatment. In some embodiments, references to a therapeutically effective amount can be to an amount measured, such as in one or more specific tissues (e.g., tissues affected by a disease, condition, or ailment) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a specific agent or therapy can be formulated and / or administered in a single dose. In some implementations, the therapeutic agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0028] The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.

[0029] Furthermore, the pharmaceutical compositions of the present invention can be oral dosage forms. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active polypeptide is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active peptide or peptide in such compositions may be delayed at a site in the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active peptide may also be formed into microcapsules with one or more of the excipients described above. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active peptide, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0030] The provided formulation may include lyophilization protectants, such as those selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucol, maltitol, lactitol, isomaltulose, and mannitol; amino acids, such as arginine, histidine, proline, or glycine; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethyl cellulose, and combinations thereof.

[0031] Alternatively or additionally, in some embodiments, the provided formulation may comprise a penetration enhancer, such as one selected from bile salts, such as sodium trihydroxycholate, sodium glycocholate, sodium taurocholate and dihydroxycholate, sodium deoxycholate, sodium glycodeoxycholate, sodium taurodeoxycholate; fatty acids, their salts and esters, such as oleic acid, lauric acid, cod liver oil extract, sodium lauryl laurate, sodium decanoate, glyceryl monostearate, diethylene glycol monoethyl ether and various sucrose fatty acid esters, medium-chain fatty acid glycerides, polycaprolactone eoma-3 fatty acids, lecithin (phosphatidylcholine), lysophosphatidylcholine; surfactants, such as sodium lauryl sulfate, polysorbate (polysorbate 80), lauryl ether, Brijs and benzalkonium chloride; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; Dextran, sodium EDTA, cosolvents such as ethanol and propylene glycol, a combination of 1% oleic acid and 5% / 10% polyethylene glycol 200, a combination of 2% glyceryl monolaurate and 40% alcohol, sodium decanoate and alcohol or propylene glycol, a combination of 10% lauric acid in propylene glycol, polyoxyethylene, 2,3-lauryl ether, menthol, sodium decanoate, sodium octanoate, sodium glycinate, ethylene glycol; polysaccharides such as chitosan and chitosan glutamate; and others It includes, for example, aprotinin, benzalkonium chloride, hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, sodium salicylate, lysophosphatidylcholine, methoxysalicylate, hydroxymethyl oleate, sodium EDTA, sulfoxide, various alkyl glycosides, ethylenediaminetetraacetic acid (EDTA), tartaric acid; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof.

[0032] Optionally or additionally, in some embodiments, the provided formulation may include an absorption enhancer, such as one selected from surfactants, cholesterol, glycerides, salicylates, bile salts, chelating agents, sodium decanoate, salts of decanoic acid, and others including N-(5-chlorosalicylic acid)-8-aminooctanoic acid (5-CNAC), 4-((4-chloro-2-hydroxybenzoyl))-amino)butyric acid (4-CNAB), and N-(8-(2-hydroxybenzoyl))-amino)octanoic acid, also known as sodium salicylate (SNAC, octanoic acid, C8, castor oil, medium chain, acylcarnitine, EDTA, glyceryl monolaurate, bovine P-casein, tocopheryl succinate glycol chitosan conjugate, lecithin, glyceryl monostearate (GMS), chitosan, and alginate. PLGA, silica, stearic acid, oleic acid, hydrogenated castor oil and trimyridine glyceryl, etoposide phosphate, enalapril maleate, ramipril, olmesartan medoxomil, valacyclovir, midodrine, gabapentin enalacarbide, sulfasalazine, or alternatively or additionally, in some embodiments, the provided formulation may contain a mucosal bioadhesive, such as selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucosyl alcohol, maltitol, lactitol, isomaltulose and mannitol; amino acids, such as arginine or histidine or proline or glycine; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol);Gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof; mucosal adhesion systems, such as those derived from natural sources, such as gelatin, agarose, chitosan, hyaluronic acid, and synthetic polymers, such as polyvinylpyrrolidone (PVP), polyacrylates, polyvinyl alcohol, sodium carboxymethyl cellulose (SCMC), and pectin; all anionic polymers, chitosan (cationic), and hydroxypropyl methyl cellulose (HPMC) as a nonionic polymer; polyacrylic acid (PAA) derivatives (CP934, CP940, PCP), 15% CMC and 35% CP; copolymers of acrylic acid and poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMM); eudragitlNE40D is a neutral poly(ethyl acrylate methacrylate); hydrophilic polymers, such as methocel K4M, methocel K15M, SCMC 400, Cekol 700, Cekol 10000, CP934P, CP971P and CP974P, carboxyvinyl polymers and triethanolamine, HPC (hydroxypropyl cellulose), CP (Carbopol 934P), Carbopol (CP) Ex-55CMC (sodium carboxymethyl cellulose), HPMC (hydroxypropyl methyl cellulose), HEC (hydroxyethyl cellulose), PIP [poly(isoprene)], PIB [poly(isobutylene)], xanthan gum, locust bean gum, pectin, polycarbofil, benzyl ester, hydroxyethyl cellulose Formulations comprising: poly(acrylic acid), poly(acrylic acid-co-acrylamide), poly(acrylic acid-co-methyl methacrylate), poly(acrylic acid-co-butyl acrylate), (bioadhesive polymer blends of CP and PIB), composed of PVP, hexadecylpyridinium chloride (as stabilizer), chlorinated chitosan, polyethylene oxide, polymethyl vinyl ether / maleic anhydride (PME / MA) and tragacanth gum, polyethylene glycol monomethyl ether monomethyl acrylate, drum-dried waxy corn starch (DDWM), carbopol 974P and stearyl fumarate sodium, and cellulose derivatives; hydrogels of acrylic acid (polar) and butyl acrylate (non-polar) and combinations thereof.

[0033] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0034] In addition to active peptides, suspensions may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0035] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0036] Dosage forms of the polypeptides of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

[0037] The pharmaceutical compositions of the present invention are formulated to a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by low concentrations of suitable biocompatible buffering agents, non-limiting examples of which are glycerol, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0038] The compositions of the present invention can be administered daily or intermittently, with a frequency of once or two to three times daily. If each of the two active ingredients is a single formulation, their administration frequencies can be the same or different. Furthermore, the compositions of the present invention can be used alone or in combination with other antihypertensive drugs. Considering all the foregoing factors, it is important to administer the lowest possible dose to achieve optimal efficacy without side effects, which can be readily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times, or more; for example, repeated over the remaining lifespan of the individual in need.

[0039] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0040] Example 1: Screening of bovine bone marrow peptides

[0041] 1.1 Bovine bone marrow protein extraction

[0042] Fresh fetal bovine bones were collected, and the bone marrow was separated from the bone tissue. The bones were initially washed to remove residual fragments and excess meat residue, and excess blood was washed away. The resulting bovine bone marrow was frozen to -20°C to harden, then placed in a mortar and pulverized into powder using liquid nitrogen at a ratio of approximately 1:6. The powder was stored at -40°C for later use. 100g of bovine bone marrow powder was taken, and bovine bone marrow protein was extracted using distilled water as a solvent. The extraction was performed three times under reflux at 45°C using a magnetic stirrer, with extract ratios of 1:10, 1:7, and 1:5 g / mL, respectively. The extraction times were 2, 1, and 0.5 h, respectively. The extracts were combined. The oil and water layers were separated using a separatory funnel, and the aqueous layer was collected. The aqueous layer was defatted three times with petroleum ether. The aqueous solution was then appropriately concentrated, dialyzed (3500 Da, 48 h), and freeze-dried under vacuum to obtain bovine bone marrow protein powder.

[0043] 1.2 Bovine bone marrow protein hydrolysis

[0044] Take 200 mg of bovine bone marrow protein powder, add 100 mL of water, adjust the pH of the solution to 9, add 2% (m / V) alkaline protease, and enzymatically hydrolyze at 55℃ using a magnetic stirrer for 2 h. After enzymatic hydrolysis, inactivate the enzyme with boiling water for 15 min, cool, adjust the pH to 7, and centrifuge at 4000 r / min for 15 min. After appropriate concentration, dialyze, and freeze-dry to obtain bovine bone marrow protease hydrolysate.

[0045] 1.3 Isolation and purification of bovine bone marrow peptides

[0046] Bovine bone marrow protein hydrolysate was purified. The hydrolysate was first filtered through a 0.22 μm microfiltration membrane, then separated using an AKTA Avant 25 system Superdux peptide 10 / 300GL gel column (10 mm × 300 mm) with water as the mobile phase at a flow rate of 0.4 mL / min. Peaks were collected at 280 nm, and the collected peak fractions were diluted to the same concentration (0.1 mg / mL). The effects of different peak fractions on immune cell proliferation were compared. The peak with the best immune cell proliferation activity was concentrated and injected into an X Bridge Prep C18 RP-HPLC column (10 mm × 100 mm). Gradient elution was performed using water (phase A) containing 0.1% TFA and acetonitrile (phase B) containing 0.1% TFA as the mobile phase at a flow rate of 2 mL / min. Eluent was collected over time at 220 nm, with one tube of eluent collected every 6 min. The collected components were diluted to the same concentration (0.1 mg / mL), and the effects of different peak components on immune cell proliferation were compared.

[0047] Identification of bovine bone marrow peptides

[0048] After purification, the fraction enhancing immune cell proliferation activity was desalted and lyophilized using a Pierce C18 Spin Tips column. The peptide sample was redissolved in 0.1% formic acid-water (solvent A), and its concentration was analyzed using a Q-Exactive Plus and ThermoFisher Scientific EASY-nanoLC 1200 system. In a 60-min gradient, 3 μL of peptide sample (1.5 mg / mL) was loaded onto a PepMap C18 column (75 μm × 250 mm), starting with 2% buffer B (80% acetonitrile plus 0.1% formic acid), gradually increasing to 35% buffer B, and then increasing to 100% buffer B within 1 min, maintaining this level for 12 min. The flow rate was maintained at 300 nL / min, and the column temperature at 40 °C. The electrospray voltage was set to 2 kV. Full-scan MS spectra (m / z 200–1800) were obtained in Orbitrap at a resolution of 70,000.

[0049] The effect of the peptide on immune cell proliferation was detected using the CCK-8 assay. Specifically, RAW264.7 cells in logarithmic growth phase were selected, and the cell concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated for 24 h. After changing the medium, bovine bone marrow peptide samples were added, and the plates were incubated for another 24 h. A blank control group was also included. After discarding the culture medium, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 1.5 h. The OD value was measured at 450 nm, and cell viability was calculated using the following formula:

[0050] Cell viability (%) = Experimental group OD450nm / Blank group OD450nm × 100%.

[0051] A total of 1387 peptides were detected by HPLC-MS / MS. They were screened and ranked using computer analysis, and 23 peptides with the ability to enhance immune cell proliferation were selected. Finally, peptide 10B3, which has the best immune cell proliferation activity, was selected. Its amino acid sequence is shown in SEQ ID NO:1.

[0052] Example 2. Effects of polypeptide 10B3 on immune function

[0053] BALB / c mice were randomly divided into three groups of 10 mice each: a blank control group, a model control group, and an experimental group. The experimental group received peptide 10B3 (100 mg / kg) via gavage for 30 consecutive days, while the blank control and model control groups received an equal volume of physiological saline. Starting from week 3, mice in both the model control and experimental groups received cyclophosphamide via intraperitoneal injection at a dose of 50 mg / kg every other day for a total of 5 injections to establish a mouse model of immunodeficiency. After 30 days of gavage, the following experiments were conducted:

[0054] 2.1 Measurement of spleen and thymus index

[0055] After 30 days of experimentation, the mice were weighed, euthanized by cervical dislocation, and their spleen and thymus were quickly separated and weighed. The spleen and thymus indices were calculated as follows:

[0056] Spleen and thymus index = Spleen or thymus (mg) / Body weight (g)

[0057] The results are shown in the table below. The experimental group showed no significant decrease in body weight compared to the blank control group. Compared to the blank control group, the spleen and thymus indices of the model control group were significantly decreased; compared to the model control group, the immune organ indices of the experimental group were significantly increased.

[0058]

[0059]

[0060] 2.2 Mouse carbon clearance test

[0061] Mice were weighed, and Indian ink diluted 4 times with physiological saline was injected into their tail veins at a dosage of 0.1 mL / 10 g. Immediately after injection, timing was initiated, and 20 μL of blood was drawn from the internal canthal venous plexus at 2 min and 10 min, respectively. This blood was immediately added to 2 mL of Na₂CO₃ solution, and the absorbance was measured at 600 nm using a microplate reader, with the Na₂CO₃ solution serving as a blank control. Mice were euthanized by cervical dislocation, and their liver, spleen, and thymus were harvested. Blood stains on the surface of the organs were blotted dry with filter paper, and the organs were weighed. The carbon clearance capacity of mice is generally represented by the corrected phagocytic index α, calculated using the following formula:

[0062]

[0063] Where: a, phagocytosis index; K, clearance index; A1, absorbance at 2 min; A2, absorbance at 10 min; t1 = 2 min; t2 = 10 min.

[0064] The results are as follows Figure 1 As shown, compared with the blank control group, the phagocytic index of carbon clearance in the model control group mice was significantly decreased; compared with the model control group, the phagocytic index of carbon clearance in the experimental group mice was significantly increased. This indicates that peptide 10B3 has a significant effect on carbon clearance function in mice, effectively increasing the phagocytic index α, and can enhance non-specific immunity in the blood.

[0065] 2.3 Spleen cell proliferation assay and NK cell activity assay

[0066] Each mouse was euthanized by cervical dislocation, and the spleen was aseptically removed and placed in a small petri dish containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with forceps to prepare a single-cell suspension. The cells were washed twice with Hank's solution and centrifuged at 1000 rpm for 10 min, discarding the supernatant. Red blood cell lysis buffer was added to the cell pellet, followed by Hank's solution. The mixture was centrifuged at 1000 rpm for 10 min, resuspended in 1 mL of RPMI 1640 complete culture medium containing 10% fetal bovine serum, and finally adjusted to a cell concentration of 2 × 10⁶ cells / mL with RPMI 1640 complete culture medium. 7 per mL.

[0067] (1) Spleen cell proliferation assay: Cell suspension was seeded into 24-well plates at 1 mL per well, with 75 μL of concanavalin A (ConA) solution added per well. A blank control was also included. The plates were incubated at 37°C with 5% CO2 for 72 h. Four h before the end of the incubation period, the culture medium in each well was discarded, and RPMI 1640 culture medium without fetal bovine serum and 50 μL of MTT solution (5 mg / mL) were added to each well. The plates were then incubated for another 4 h. After the incubation period, the culture medium was gently aspirated from each well, and 1 mL of DMSO was added to each well. The mixture was then pipetted to dissolve the purple crystals completely. The cells were aliquoted into 96-well plates, and the optical density was measured at 570 nm using a microplate reader. The plate was zeroed using a cell-free blank well, and the spleen cell proliferation capacity was calculated using the following formula.

[0068] Cell proliferation capacity = OD plus Con - OD without ConA

[0069] The results are as follows Figure 2 As shown, compared with the blank control group, the proliferative capacity of spleen cells in the model control group was significantly decreased; compared with the model control group, the proliferative capacity of spleen cells in the experimental group was significantly increased. The results indicate that peptide 10B3 can significantly enhance the transformation capacity of ConA-induced mouse spleen lymphocytes and improve mouse cellular immunity.

[0070] (2) NK cell activity assay: Fresh target cells (YAC-1 cells) with a viability greater than 95% were passaged 24 hours before the experiment. Before use, the cells were washed three times with Hank's solution and the cell concentration was adjusted to 4 × 10⁻⁶ cells / mL with RPMI 1640 complete culture medium. 5 Cells / mL. In the spleen cell proliferation assay, after 72 hours of cell culture, a concentration of 2×10⁻⁶ was used. 7Spleen cell suspension at 100 cells / mL was used as effector cells. 100 μL each of target cells and effector cells (effector-target ratio 50:1) were added to a U-shaped 96-well plate. 100 μL each of target cells and culture medium were added to the target cell spontaneous release wells, and 100 μL each of target cells and 2.5% Triton were added to the target cell maximum release wells. The plates were incubated at 37℃ in a 5% CO2 incubator for 4 h. Then, the 96-well plate was centrifuged at 1500 rpm for 5 min. 100 μL of supernatant was collected from each well and placed in a flat-bottomed 96-well plate. 100 μL of LDH matrix solution was added simultaneously. The reaction was allowed to proceed for 3 min. 30 μL of 1 mol / L HCl was added to each well. The absorbance was measured at 490 nm using a microplate reader. NK cell activity was calculated using the following formula:

[0071] NK cell activity = (OD reaction wells - OD spontaneous release wells) / (OD maximum release wells - OD spontaneous release wells)

[0072] The results are as follows Figure 3 As shown, compared with the blank control group, the NK cell activity in the model control group was significantly decreased; compared with the model control group, the NK cell activity in the experimental group was significantly increased. The results indicate that peptide 10B3 can significantly enhance the activity of mouse NK cells.

[0073] 2.4 Delayed-type hypersensitivity (DTH) in mice

[0074] Hair removal cream was used to remove approximately 1cm x 1cm of hair from the abdominal skin of each mouse. 50μL of DNFB solution was applied evenly to induce sensitization. Five days later, 10μL of DNFB solution was applied evenly to the right ear (both sides) of the mouse to induce a sensitization reaction. The mice were then returned to their cages and euthanized by cervical dislocation 24 hours later. The difference in weight between the left and right ears was used as the delayed-type hypersensitivity reaction value.

[0075] The results are as follows Figure 4 As shown, compared with the blank control group, the DTH level in the model control group was significantly decreased; compared with the model control group, the DTH level in the experimental group was significantly increased. The results indicate that peptide 10B3 can enhance cellular immunity in mice.

[0076] 2.5 Determination of serum hemolysin

[0077] This study used a hemagglutination assay to detect hemolysin levels. On day 26, 4% (v / v) SRBC was diluted with sterile physiological saline to a 2% cell suspension. 0.2 mL of this suspension was injected intraperitoneally into mice in each group for immunization. Four days later, blood was collected from the mice after enucleation and placed in 1.5 mL centrifuge tubes. The tubes were left at room temperature for approximately 1 hour to allow the coagulated blood to fully separate from the tube wall, thus allowing for complete serum separation. The tubes were centrifuged at 2000 rpm for 10 minutes, and the serum was collected. The serum was serially diluted 1, 2, 4, and 8 times with physiological saline. 100 μL of each dilution was added to a micro-volume hemagglutination plate, and 100 μL of 0.5% (v / v) SRBC suspension was added to each well. The plate was mixed, covered, and incubated at 37°C for 3 hours. The degree of hemagglutination was then observed.

[0078] The results are as follows Figure 5 As shown, compared with the blank control group, the hemolysin production of mice in the model control group was significantly decreased; compared with the model control group, the hemolysin production of mice in the experimental group was significantly increased. These results indicate that peptide 10B3 can effectively increase serum hemolysin levels in mice. SRBCs stimulate the activation and proliferation of T cells, and with the assistance of T cells, B cells are activated and secrete antibodies. Peptide 10B3, through antigen action on T cells, stimulates B cells to indirectly produce antibodies, thereby enhancing humoral immunity in mice.

Claims

1. A bovine bone marrow peptide, characterized in that, The amino acid sequence of the bovine bone marrow peptide is shown in SEQ ID NO:

1.

2. The bovine bone marrow peptide according to claim 1, wherein the bovine bone marrow peptide can enhance immune cell activity and / or promote immune cell proliferation.

3. A nucleic acid molecule encoding the bovine bone marrow peptide as described in claim 1 or 2.

4. A vector comprising the nucleic acid molecule as described in claim 3.

5. An isolated host cell comprising the bovine bone marrow peptide as claimed in claim 1 or 2, the nucleic acid molecule as claimed in claim 3, or the vector as claimed in claim 4.

6. The use of the bovine bone marrow peptide according to claim 1 or 2 in the preparation of health products or medicines that enhance immunity.

7. A pharmaceutical composition comprising an effective amount of the bovine bone marrow peptide as described in claim 1 or 2.

8. The pharmaceutical composition according to claim 7, wherein it is administered in the form of a lyophilized powder.

9. The pharmaceutical composition according to claim 7 or 8 may further be used in combination with other immune enhancers.

10. The pharmaceutical composition according to claim 9, wherein the other immune enhancer is thymosin, transfer factor, human immunoglobulin, lentinan, Ganoderma lucidum polysaccharide, Tremella fuciformis polysaccharide, Trametes versicolor polysaccharide, levamisole, isoproterenol, polyinosinic-cytosine monophosphate, or polyinosinic-uridine monophosphate.

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