Bovine bone marrow peptide and application thereof in improving immunity

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of bovine bone marrow peptide. The bovine bone marrow peptide is prepared by performing enzymolysis on bovine bone marrow protein. The bovine bone marrow peptide can effectively enhance the activity of immune cells and / or promote the proliferation of the immune cells. The polypeptide can improve the immunity of the organism, has the advantages of being simple to prepare, easy to absorb and the like, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bovine bone marrow peptide, its application in improving immune diseases, and its preparation method. Background Art

[0002] In recent years, the preparation, functional development, and industrial application of bone-derived bioactive peptides have become a hot topic in the field of animal-derived functional foods. Skeleton, composed of periosteum, bone tissue, and bone marrow, is a network of proteins and calcium, with tubes filled with marrow containing a variety of nutrients. Bone marrow contains not only abundant trace elements such as protein, fat, calcium, phosphorus, iron, zinc, copper, and strontium, but also health-promoting factors such as phosphoproteins, phospholipids, chondroitin, mucopolysaccharides, growth hormones, various amino acids, and multiple vitamins, making it a valuable natural medicinal and edible resource. Bone has a high protein content, comparable to that of fresh meat. Furthermore, bone protein is a highly soluble protein with high biological value, making it a high-quality protein source. Currently, collagen and bone peptide injections for promoting fracture healing, prepared from livestock and poultry bones and fish bones, are being marketed both domestically and internationally. Compound bone peptides and bone peptide tablets are also being marketed for the prevention and treatment of rheumatism, fractures, arthritis, osteoporosis, and lumbar disc herniation. With increasing research into their nutritional composition and efficacy, bone proteins are being discovered to possess a variety of potential functions. Studies have shown that bone extracts play an important role in lowering blood pressure, immune regulation, bone disease treatment, antibacterial, and antioxidant effects. With the advancement of health food processing technology, more and more scholars are focusing on the research of the bioactivity and application of animal bone protein peptides and related products.

[0003] A healthy immune system effectively protects against pathogen invasion, maintaining normal functioning and physiological balance. However, with the advancement of technology and fierce competition, people's lifestyles have become irregular, leading to a decline in their immune system. However, because targeted treatments are difficult to obtain, many people turn to functional foods to boost their immunity. Currently, there is a lack of immune-boosting functional foods that utilize traditional foods and herbal remedies. Therefore, developing a health product with immunity-boosting properties is of great significance and value. Summary of the Invention

[0004] In view of the state of the art, the present invention aims to provide a bovine bone marrow peptide with the ability to enhance immunity and its use. Specifically, the bovine bone marrow peptide provided by the present invention can increase the activity of immune cells, enhance the proliferation of immune cells, and thus improve the body's immunity. Furthermore, the bovine bone marrow peptide provided by the present invention is non-toxic, has no side effects, is simple to prepare, and is easily absorbed, and can be used to prepare health products or medicines.

[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 myeloid peptide of the present invention.

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

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

[0010] Another aspect of the present invention also provides use of the bovine bone marrow peptide in preparing health products or medicines for improving 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, wherein the amino acid sequence of the bovine bone marrow peptide is shown in SEQ ID NO: 1.

[0012] The bovine bone marrow peptide of the present invention or the pharmaceutical composition comprising the bovine bone marrow peptide of the present invention is administered in the form of lyophilized powder.

[0013] In some embodiments, the bovine bone marrow peptide of the present invention or the pharmaceutical composition comprising the bovine bone marrow peptide of the present invention can be further used in combination with other immunopotentiators.

[0014] In some embodiments, the immune enhancer can be selected from thymosin, transfer factor, human immunoglobulin, Lentinan, Ganoderma lucidum polysaccharide, Tremella polysaccharide, Versicolor polysaccharide, levamisole, isopropyl creatinine, polyinosinic acid, polyinosinic acid, etc.

[0015] Beneficial effects

[0016] The present invention extracts and produces an active polypeptide from bovine bone marrow, which effectively enhances immune cell activity and / or promotes immune cell proliferation. The polypeptide of the present invention can enhance the body's immunity and has certain clinical application value. Furthermore, the polypeptide 10B3 provided by the present invention is easy to synthesize, low in cost, and readily applicable. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0019] Figure 3 The results shown are the NK cell activities of mice in each group.

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

[0021] Figure 5 The results shown are the serum hemolysin production of mice in each group. DETAILED DESCRIPTION

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

[0023] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” can mean ±1%.

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

[0025] As used herein, "treatment," "treating," or "amelioration" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefits and / or prophylactic benefits.

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

[0027] The term "therapeutically effective amount" refers to an amount that produces the desired effect of its administration. In some embodiments, the term refers to an amount sufficient to treat the disease, disorder, and / or condition when applied to a population suffering from or susceptible to a disease, disorder, and / or condition according to a therapeutic dosing regimen. 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, disorder, and / or condition, and / or delays its onset. Those of ordinary skill in the art will understand that a therapeutically effective amount does not necessarily achieve successful treatment in each specific treatment individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when applied to a patient in need of such treatment. In some embodiments, reference to a therapeutically effective amount can be a reference to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will understand that in some embodiments, a specific agent or therapy that can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective 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 composition of the present invention can be in oral dosage form. 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 ingredients: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, such as paraffin; (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 lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active polypeptide or polypeptides in such compositions can be delayed in a specific region within the digestive tract. Examples of useful encapsulating components include polymeric substances and waxes. If desired, the active polypeptide can also be microencapsulated with one or more of the above-mentioned excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active polypeptide, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof.

[0030] The provided formulations can include a lyoprotectant, for example, which can be selected from sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, 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.

[0031] Alternatively or additionally, in some embodiments, provided formulations may comprise a permeation enhancer, for example, which may be selected from bile salts, such as trihydroxy salts sodium cholate, sodium glycocholate, sodium taurocholate and dihydroxy salts, sodium deoxycholate, sodium glycodeoxycholate, sodium taurodeoxycholate; fatty acids, their salts and esters, for example, oleic acid, lauric acid, cod liver oil extract, sodium laurate, sodium caprate, 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, for example, sodium lauryl sulfate, polysorbate (polysorbate 80), lauryl ether, brijs and benzalkonium chloride; complexing agents, for example, cyclodextrin, dextran sulfate, dextran sulfate, sodium edetate; complexing agents, for example, cyclodextrin, dextran sulfate, sulfate Dextran, sodium edetate, 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, a combination of sodium caprate and alcohol or propylene glycol, a combination of 10% lauric acid in propylene glycol, polyoxyethylene, 2,3-lauryl ether, menthol, sodium caprate, sodium caprylate, sodium glycodeoxycholate, ethylene glycol; polysaccharides such as chitosan and chitosan glutamate; and others It includes, for example, aprotinin, benzalkonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, sodium salicylate, lysophosphatidylcholine, methoxysalicylate, hydroxymethyloleate, 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] Alternatively or additionally, in some embodiments, provided formulations can include an absorption enhancer, for example, which can be selected from surfactants, cholesterol, glycerides, salicylates, bile salts, chelating agents, sodium caprate, salts of capric acid and others including N-(5-chlorosalicylic alcohol)-8-aminocaprylic acid (5-CNAC), 4-((4-chloro-2-hydroxybenzoyl))-amino)butyric acid (4-CNAB) and N-(8-(2-hydroxybenzoyl))-amino)caprylic acid, also known as sodium salicylate (SNAC, caprylic acid, C8, castor oil, medium chain, acylcarnitine, EDTA, monolaurin, bovine beta-casein, tocopherol succinate glycol chitosan conjugate, lecithin, glyceryl monostearate (GMS), chitosan and alginate , PLGA, silicon dioxide, stearic acid, oleic acid, hydrogenated castor oil and trimyristin, etoposide phosphate sulindac enalapril maleate, ramipril, olmesartan medoxomil, valacyclovir, midodrine, gabapentin enacarbil, sulfasalazine, or alternatively or additionally, in some embodiments, provided formulations may comprise a mucosal bioadhesive, for example, which may be selected from sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, 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, carboxymethylcellulose and combinations thereof, mucoadhesive systems, for example, those of natural origin, such as gelatin, agarose, chitosan, hyaluronic acid and synthetic polymers, such as polyvinyl pyrrolidone (PVP), polyacrylates, polyvinyl alcohol, sodium carboxymethylcellulose (SCMC) and pectin, all anionic polymers, chitosan (cationic) and hydroxypropyl methylcellulose (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), eudragitl NE40D is a neutral poly(ethyl methacrylate), hydrophilic polymers, for example, methocel K4M, methocel K15M, SCMC 400, Cekol 700, Cekol10000, CP934P, CP971P and CP974P, carboxyvinyl polymer and triethanolamine, HPC (hydroxypropyl cellulose), CP (Carbopol 934P), Carbopol (CP) Ex-55CMC (sodium carboxymethyl cellulose), HPMC (hydroxypropyl methylcellulose), HEC (hydroxyethyl cellulose), PIP [poly (isoprene)], PIB [poly (isobutylene)], xanthan gum, locust bean gum, pectin, polycarbophil, benzyl esters, hydroxyethyl cellulose Cellulose, poly(acrylic acid), poly(acrylic acid-co-acrylamide), poly(acrylic acid-co-methyl methacrylate), poly(acrylic acid-co-butyl acrylate), (a bioadhesive polymer blend of CP and PIB), a formulation consisting of PVP, cetylpyridinium chloride (as a stabilizer), chitosan chloride, polyethylene oxide, polymethyl vinyl ether / maleic anhydride (PME / MA) and tragacanth gum, polyethylene glycol monomethyl ether monomethacrylate, drum-dried waxy corn starch (DDWM), carbopol 974P and sodium stearyl fumarate, and a cellulose derivative; hydrogels - acrylic acid (polar) and butyl acrylate (non-polar), and combinations thereof.

[0033] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0034] In addition to the active polypeptide, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

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

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

[0037] The pharmaceutical composition of the present invention is 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 buffer components, non-limiting examples of which are tromethamine, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and sodium hydrogen phosphate.

[0038] The composition of the present invention can be administered daily or intermittently, and the frequency of administration can be once a day or 2 to 3 times a day. If each of the two active ingredients is a single formulation, the frequency of administration can be the same as or different from each other. In addition, the composition of the present invention can be used alone or in combination with other antihypertensive drugs. Taking all of the above factors into account, it is important to administer the drug at the lowest dose without side effects to achieve the best therapeutic effect, which can be easily 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, for the remaining life cycle of an individual in need thereof.

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0040] Example 1 Screening of Bovine Bone Marrow Peptides

[0041] 1.1 Extraction of bovine bone marrow protein

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

[0043] 1.2 Enzymatic hydrolysis of bovine bone marrow

[0044] Take 200 mg of bovine bone marrow protein powder, add 100 mL of water, adjust the solution to a pH of 9, add 2% (m / v) alkaline protease, and perform enzymatic hydrolysis at 55°C using a magnetic stirrer for 2 hours. After the hydrolysis is complete, inactivate the enzyme in boiling water for 15 minutes, cool, adjust the pH to 7, and centrifuge at 4000 rpm for 15 minutes. After appropriate concentration, dialysis, and freeze-drying, the bovine bone marrow enzymatic hydrolysate is obtained.

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

[0046] Bovine bone marrow protein hydrolysate was purified. The protein hydrolysate was first filtered through a 0.22 μm microfiltration membrane and then separated using a Superdux peptide 10 / 300GL gel column (10 mm × 300 mm) in an AKTA avant 25 system. Water was used as the mobile phase at a flow rate of 0.4 mL / min. Peak fractions were collected at 280 nm. The collected peak fractions were diluted to the same concentration (0.1 mg / mL) and their effects on immune cell proliferation were compared. The peak fraction with the highest immune cell proliferation activity was concentrated and injected onto an X Bridge Prep C18 RP-HPLC column (10 mm × 100 mm). Gradient elution was performed using water containing 0.1% TFA (phase A) and acetonitrile containing 0.1% TFA (phase B) as the mobile phases at a flow rate of 2 mL / min. The eluate was collected at a wavelength of 220 nm, with a timed collection interval of 6 minutes. The collected fractions were diluted to the same concentration (0.1 mg / mL) and the effects of the collected fractions from different peaks on the proliferation of immune cells were compared.

[0047] Identification of bovine bone marrow peptides

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

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

[0050] Cell viability (%) = OD450nm of experimental group / OD450nm of blank group × 100%.

[0051] A total of 1,387 polypeptides were detected by HPLC-MS / MS, and they were screened and ranked using computer analysis. 23 polypeptides with immune cell proliferation enhancing activity were screened out, and finally the polypeptide 10B3 with the best immune cell proliferation enhancing activity was selected, and its amino acid sequence is shown in SEQ ID NO: 1.

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

[0053] BALB / c mice were randomly divided into three groups, 10 mice each: a blank control group, a model control group, and an experimental group. The experimental group was continuously gavaged with peptide 10B3 (100 mg / kg) for 30 days, while the blank and model control groups received an equal volume of normal saline. Starting after the third week, mice in the model and experimental groups were intraperitoneally injected with cyclophosphamide at a dose of 50 mg / kg every other day for a total of five injections to establish an immunocompromised mouse model. After 30 days of gavage, the following experiments were performed:

[0054] 2.1 Determination of spleen and thymus index

[0055] After 30 days of the experiment, the mice were weighed and sacrificed by cervical dislocation. The spleen and thymus were quickly separated and weighed, and 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. Compared with the blank control group, the experimental group had no significant weight loss. Compared with the blank control group, the spleen and thymus indexes of the model control group were significantly decreased; compared with the model control group, the immune organ indexes of the experimental group were significantly increased.

[0058]

[0059]

[0060] 2.2 Carbon clearance experiment in mice

[0061] The mice were weighed and Indian ink diluted 4 times with physiological saline was injected into the tail vein of the mice at an injection volume of 0.1 mL / 10 g. After the ink was injected, the time was immediately started. 20 μL of blood was collected from the epicanthal venous plexus at 2 minutes and 10 minutes respectively, and immediately added to 2 mL of Na2CO3 solution. The absorbance was measured at a wavelength of 600 nm on an enzyme reader, and the Na2CO3 solution was used as a blank control. The mice were killed by cervical dislocation, and the liver, spleen and thymus were removed. The blood stains on the surface of the organs were blotted with filter paper and weighed. The corrected phagocytic index a is generally used to represent the carbon clearance capacity of mice. The a value is calculated according to the following formula:

[0062]

[0063] Where: a, phagocytic 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 in the figure, compared with the blank control group, the carbon clearance phagocytosis index of mice in the model control group was significantly decreased; while the carbon clearance phagocytosis index of mice in the experimental group was significantly increased compared with the model control group. This indicates that peptide 10B3 has a significant effect on the carbon clearance function of mice, effectively improving the phagocytosis index a, and can enhance nonspecific immunity in the blood.

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

[0066] Each mouse was sacrificed by cervical dislocation, and the spleen was removed aseptically and placed in a small 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 minutes, and the supernatant was discarded. The cell pellet was added with red blood cell lysis buffer, Hank's solution, and centrifuged at 1000 rpm for 10 minutes. The cell pellet was resuspended in 1 mL of RPMI1640 complete culture medium containing 10% calf serum, and the cell concentration was adjusted to 2 × 10 cells / mL with RPMI1640 complete culture medium. 7 pieces / mL.

[0067] (1) Spleen cell proliferation test: The cell suspension was inoculated into a 24-well culture plate, 1 mL per well, and 75 μL / well of concanavalin A (ConA) solution was added. A blank control was set up and the cells were cultured in a 37°C, 5% CO2 incubator for 72 h. 4 h before the end of the culture, the culture medium of each well was discarded, and RPMI1640 culture medium without calf serum and 50 μL of MTT at a concentration of 5 mg / mL were added to each well, and the culture was continued for 4 h. After the culture was completed, the culture medium in the well was gently aspirated, 1 mL of DMSO was added to each well, and the mixture was pipetted to completely dissolve the purple crystals. The cells were divided into 96-well culture plates and the optical density was measured at a wavelength of 570 nm using an enzyme-labeled instrument. The blank well without cells was set to zero, and the spleen cell proliferation capacity was calculated using the following formula:

[0068] Cell proliferation ability = OD with ConA - OD without ConA

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

[0070] (2) NK cell activity assay: 24 h before the experiment, fresh target cells (YAC-1 cells) with a survival rate greater than 95% were subcultured. Before use, the cells were washed three times with Hank's solution and the cell concentration was adjusted to 4×10 5 In the spleen cell proliferation test, when the cells were cultured for 72 hours, the concentration of 2×10 7A spleen cell suspension of 100 μL / mL was used as effector cells. 100 μL each of target cells and effector cells (effector:target ratio 50:1) was taken and added to a U-shaped 96-well culture plate. 100 μL each of target cells and culture medium was added to the wells for natural target cell release, and 100 μL each of target cells and 2.5% Triton was added to the wells for maximum target cell release. The cells were cultured in a 37°C, 5% CO2 incubator for 4 h. The 96-well culture plate was then centrifuged at 1500 rpm for 5 min. 100 μL of the supernatant was aspirated from each well and placed in a flat-bottomed 96-well culture plate. 100 μL of LDH matrix solution was added simultaneously and reacted 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 according to the following formula:

[0071] NK cell activity = (OD reaction well - OD natural release well) / (OD maximum release well - OD natural release well)

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

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

[0074] The abdominal skin of each mouse was depilated to an area of ​​approximately 1 cm × 1 cm using depilatory cream, and 50 μL of DNFB solution was evenly applied to induce sensitization. Five days later, 10 μL of DNFB solution was evenly applied to the right ear (both sides) of the mouse for challenge. The mouse was returned to the cage and killed 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 in the figure, compared with the blank control group, the DTH level in the model control group was significantly reduced; and compared with the model control group, the DTH level in the experimental group was significantly increased. The results show that peptide 10B3 can improve the cellular immunity of mice.

[0076] 2.5 Determination of serum hemolysin

[0077] This study used a hemagglutination assay to measure hemolysin levels. On day 26, 4% (volume fraction) SRBC was diluted with sterile saline to a 2% cell suspension. 0.2 mL of this solution was injected intraperitoneally into each group of mice for immunization. Four days later, the mice's eyes were enucleated and blood was collected in 1.5 mL centrifuge tubes. The tubes were left at room temperature for approximately 1 hour to allow the clotted blood to separate from the tube walls, allowing the serum to fully separate. The serum was then centrifuged at 2000 rpm for 10 minutes and collected. Serum was diluted 1, 2, 4, and 8 times with saline. Each dilution was then placed in a microhemagglutination assay plate, with 100 μL per well. 100 μL of the 0.5% (volume fraction) SRBC suspension was then added. The plates were mixed thoroughly, covered with a moistened plate, 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 model control group mice produced significantly less hemolysin; while the experimental group mice produced significantly more hemolysin than the model control group. These results indicate that peptide 10B3 can effectively increase serum hemolysin levels in mice. SRBCs stimulate the activation and proliferation of T cells, which, with the assistance of T cells, activate B cells to secrete antibodies. Peptide 10B3 acts on T cells through antigens, indirectly stimulating B cells to produce antibodies, thereby enhancing the humoral immunity of 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 the activity of immune cells and / or promote the proliferation of immune cells.

3. A nucleic acid molecule encoding the bovine myeloid peptide according to claim 1 or 2. A vector comprising the nucleic acid molecule according to claim 3 .

5. An isolated host cell comprising the bovine myeloid peptide according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the vector according to claim 4.

6. Use of the bovine bone marrow peptide according to claim 1 or 2 in preparing health products or medicines for improving immunity.

7. A pharmaceutical composition comprising an effective amount of the bovine bone marrow peptide according to claim 1 or 2.

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

9. The pharmaceutical composition according to claim 7 or 8, which can be further used in combination with other immunopotentiators.

10. The pharmaceutical composition according to claim 9, wherein the antihypertensive drug can be thymosin, transfer factor, human immunoglobulin, lentinan, ganoderma lucidum polysaccharide, tremella polysaccharide, versicolor polysaccharide, levamisole, isopropyl creatinine, polyinosinic acid (PCP) or polyinosinic acid (PUP).

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