Hippocampal glycoprotein with immune regulation function as well as preparation method and application of hippocampal glycoprotein

Abstract

The invention discloses hippocampus glycoprotein with an immune regulation function as well as a preparation method and application of the hippocampus glycoprotein, and belongs to the technical field of biological medicines. The hippocampus glycoprotein is prepared through optimization of a membrane separation process, the hippocampus glycoprotein is an N-glycosylation and chondroitin sulfate co-modified composite glycoprotein, the protein totally contains 65 different types of N-carbohydrate chains, the ratio of sialylated N-carbohydrate chains exceeds 30%, and the carbohydrate chain structure types comprise H5N4S1, H5N4S2, H6N5S1, H6N5S2 and the like; meanwhile, rich glycosaminoglycans (GAGs) are combined on the hippocampus glycoprotein, chondroitin sulfate is taken as a main type, and the total sulfation degree is 0.4-0.6. The hippocampus glycoprotein can effectively activate macrophages and promote secretion of immune factors such as NO, TNF-alpha and IL-6, and has remarkable immunomodulatory activity. According to the hippocampus glycoprotein provided by the invention, the hippocampus glycoprotein co-modified by N-glycosylation and chondroitin sulfate is taken as an active ingredient, and is supplemented with an acceptable carrier to prepare a medicine or a health care product, so that a new direction is provided for later development and application of related functions for immunoregulation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a hippocampus glycoprotein with immune regulation function and a preparation method and application thereof. BACKGROUND

[0002] Immune regulation plays a key role in maintaining body homeostasis, and its core is to balance the intensity of immune response by precisely regulating the activity of immune cells and the cytokine network. When it is out of function, not only will it trigger autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, causing the immune system to attack the body's own tissues, but also will weaken the ability to clear bacteria, viruses and other pathogens, making the body susceptible to infection, and more likely to fail to identify and remove mutant cells in time due to the failure of immune surveillance mechanism, thereby promoting tumor proliferation and metastasis. Macrophages, as the core effector cells of innate immunity, can secrete nitric oxide (NO) after activation, which can destroy the cell membrane structure of pathogens and inhibit their metabolism, and effectively play an anti-infection role; tumor necrosis factor -α (TNF-α) can start an inflammatory cascade reaction, and activate T cells, B cells and other adaptive immune cells; interleukin -6 (IL-6) can maintain the dynamic balance of immune response by regulating the proliferation and differentiation of immune cells and inflammatory signal transduction. The three of them constitute the molecular basis of immune regulation, and their secretion levels and the activation state of macrophages are key indicators for evaluating the activity of immune regulation.

[0003] At present, the immune regulators on the market are mostly chemical synthetic drugs or recombinant protein biological agents, which have the problems of obvious side effects, insufficient biocompatibility, high cost and the like. Therefore, it has become a research focus in the fields of medicine and functional food to mine safe and efficient immune regulation substances from natural products.

[0004] Hippocampus (Hippocampus spp.) is a marine organism of the Hippocampus genus of the Syngnathidae family, and is named after its head resembling a horse and its body side having many stripes. It is a traditional precious Chinese medicinal material. The 2025 edition of Chinese Pharmacopoeia records that hippocampus has the medicinal effects of warming kidney and strengthening yang and resolving masses and detumescence. Modern research shows that hippocampus has certain traditional application basis in anti-tumor, anti-thrombus, anti-fatigue, anti-aging and immune enhancement. However, the research on the correlation between specific components and activities of hippocampus is still insufficient, and it has further mining value. SUMMARY

[0005] Based on the above needs, the purpose of the present application is to provide hippocampus glycoprotein with immune regulation function and its preparation method and application, the present application provides a method for preparing hippocampus glyproteins, the prepared hippocampus glyproteins have sialylation N-glycosylation modification and chondroitin sulfate modification, the characteristic sulfation mode provides a strong clue and theoretical basis for elucidating the mechanism of hippocampus glyproteins in immune function regulation, also expands the use of hippocampus glyproteins in the field of immune regulation technology, and has wide application prospect.

[0006] To achieve the above-mentioned purpose of the application, the technical scheme is adopted as follows:

[0007] The present application provides a hippocampus glycoprotein with immune regulation function, the molecular structure of the hippocampus glycoprotein has sialylation N-glycosylation modification and chondroitin sulfate modification, and the molecular weight of the hippocampus glycoprotein is 20-220kDa.

[0008] Further, the mass ratio of total glycosaminoglycan in the hippocampus glycoprotein to the hippocampus glycoprotein is 0.1%-0.5%, and the sulfation degree of chondroitin sulfate is 0.4-0.6, wherein the mass ratio of 6-sulfated chondroitin sulfate to the hippocampus glycoprotein is 0.1%-0.2%.

[0009] Further, the structure type of the sialylated N-glycan chain includes at least one of H5N4S1, H5N4S2, H6N5S1 and H6N5S2. The N represents N-acetylglucosamine (GlcNAc), the H represents mannose (Man) or galactose (Gal), the S represents sialic acid (NeuNAc), and the numbers represent the number of corresponding sugar units.

[0010] The present application also provides a preparation method of the hippocampus glycoprotein, characterized in that it comprises the following steps:

[0011] S1: cutting hippocampus, soaking in water, homogenizing to obtain hippocampus homogenate;

[0012] S2: extracting the hippocampus homogenate by enzymatic method or water extraction method to obtain supernatant;

[0013] S3: performing membrane separation operation on the supernatant to obtain desalted solution;

[0014] S4: adding ethanol to the desalted solution for alcohol precipitation, and separating the precipitate after centrifugation to obtain alcohol precipitation precipitate;

[0015] S5: drying the alcohol precipitation precipitate to obtain hippocampus glycoprotein.

[0016] Further, the hippocampus is at least one of Hippocampus kelloggi, Hippocampus histrix, Hippocampus kuda, Hippocampus trimaculatus, Hippocampus parviceps and Hippocampus belly.

[0017] Further, the ratio of hippocampus to water in step S1 is 1:5-20; the membrane separation operation in step S3 is desalination using a nanofiltration membrane with a molecular weight cut-off of 150-300 Da.

[0018] Further, the specific operation steps of the enzymatic method in step S2 are: a adjusting the pH of hippocampus homogenate, adding biological protease for extraction to obtain hippocampus extract; b heating the hippocampus extract to inactivate the enzyme, cooling to room temperature, centrifuging to remove the precipitate impurities to obtain the supernatant.

[0019] Further, the pH is 1-9, the biological protease is at least one of pepsin, neutral protease, trypsin and pancreatin, the amount of biological protease is 500-3000 U / g based on the mass of hippocampus, the enzymolysis temperature is 30-45℃, and the enzymolysis time is 0.5-3h.

[0020] Further, the specific operation steps of the water extraction method are: a homogenizing hippocampus at 30-100℃ under 0-1 Mpa for 0.5-5 hours, extracting 1-3 times to obtain hippocampus extract; b centrifuging the hippocampus extract to remove the precipitate impurities to obtain the supernatant.

[0021] Further, the final concentration of ethanol in step S4 is 40-85%.

[0022] Further, the drying method in step S5 is at least one of vacuum drying, freeze-drying and spray drying.

[0023] The application also provides the use of hippocampus glycoprotein in the preparation of preparations or drugs with immunomodulatory function.

[0024] Further, the hippocampus glycoprotein can effectively promote the proliferation of macrophages and the secretion of inflammatory factors.

[0025] Further, the hippocampus glycoprotein can promote the release of NO by macrophage RAW264.7 and regulate the secretion of IL-6 and TNF-α.

[0026] Further, the concentration of the hippocampus glycoprotein is 5-120 μg / mL.

[0027] Compared with the prior art, the application has the following advantages and beneficial effects:

[0028] (1) The application provides a simple, efficient and operable preparation method of hippocampus glycoprotein, and provides two different preparation methods of hippocampus extract, including water extraction method and enzymatic method, which provides data support for later related research and has reference significance.

[0029] (2) The present application first characterizes hippocampus glycoprotein, determines that the hippocampus glyproteins have unique glycosylation characteristics and high glycosaminoglycan (GAGs) content, especially high proportion of sialylated N-glycan (more than 30%) and high sulfated chondroitin sulfate as the main structural characteristics. N-glycan analysis shows that the hippocampus glycoprotein contains a variety of typical high sialylation structures. Such modification can significantly improve the biological activity and stability of the protein molecule, prolong its in vivo half-life, improve its solubility and binding capacity with receptors, and enhance its ability to be recognized by immune cells and regulate immune response.

[0030] (3) The hippocampus glycoprotein provided by the present application is rich in 4-sulfated CS and 6-sulfated CS, wherein the proportion of CS-6S is >30%, and has a high CS sulfation degree (0.4-0.6). The biological activity of glycosaminoglycan is closely related to its sulfation mode (sulfation site affects protein interaction, and the abundance of specific CS chain unit regulates biological function). These characteristics help to improve the molecular activity of glycoprotein, so that it can more effectively participate in immune regulation, cell activation and related biological processes. Thus, a molecular basis is laid for its application in the field of immune regulation.

[0031] (4) The present application first confirms that hippocampus glycoprotein can promote the NO release of mouse macrophage RAW264.7 and regulate the secretion of important inflammatory factors such as IL-6 and TNF-α, confirms its ability to regulate immune cell function, and reveals its molecular mechanism of realizing immune activity through polysaccharide chain modification. The present application provides a hippocampus glycoprotein which is a complex glycoprotein co-modified by N-glycosylation and chondroitin sulfate and has a significant immune-enhancing effect. Its characteristic sulfation mode provides a strong clue and theoretical basis for elucidating the mechanism of hippocampus glycoprotein in immune function regulation.

[0032] (5) The hippocampus glycoprotein prepared by the present application has a sugar type including any one or a combination of H5N4S1, H5N4S2, H6N5S1 and H6N5S2. Such high sialylated N-glycosylation structure can enhance the biological activity and stability of hippocampus glycoprotein, prolong its in vivo half-life, improve its solubility and molecular affinity, and regulate its recognition and binding capacity with cells or receptors, thereby significantly improving its application advantages in the field of immune regulation. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The sialylated N-glycan with a peak area greater than 1% in hippocampus glycoprotein (HG) and its relative content.

[0034] Figure 2 The contents of CS-0S, CS-4S and CS-6S in hippocampus glycoprotein (HG).

[0035] Figure 3 The content of chondroitin sulfate (CS) in hippocampal glycoprotein (HG) at different sulfation sites.

[0036] Figure 4 The effect of hippocampal glycoprotein (HG-D) on the secretion of TNF-α by Raw264.7 cells.

[0037] Figure 5 The effect of hippocampal glycoprotein (HG-D) on the secretion of IL-6 by Raw264.7 cells.

[0038] Figure 6 The effect of hippocampal glycoprotein (HG-D) on the release of NO by Raw264.7 cells. DETAILED DESCRIPTION

[0039] Other advantages and features of the present application will become more apparent from the following detailed description of the application when read in conjunction with the accompanying drawings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The following examples are provided to illustrate certain embodiments of the application and are not intended to limit the scope of the application. The ratio of hippocampus to water in the feed solution of the present application is the mass-volume ratio of hippocampus to water, with the mass unit being g and the volume unit being mL.

[0040] Example 1: Preparation of hippocampal glycoprotein (HG)

[0041] The preparation method of hippocampal glycoprotein in this example includes the following steps:

[0042] S1: Take hippocampus and soak it in water at a ratio of 1:20 for 3 hours, then homogenize it for 5 minutes to obtain hippocampus homogenate.

[0043] S2: Extract the hippocampus homogenate at 100°C and 0.4 MPa for 2 hours, and extract it twice to obtain hippocampus extract.

[0044] S3: Cool the hippocampus extract to room temperature, centrifuge it at 3000 rpm for 10 minutes to remove precipitated impurities, and separate the supernatant.

[0045] S4: Desalt the supernatant from step S3 by passing it through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da.

[0046] The specific process of nanofiltration desalting is as follows: add an equal volume of double distilled water to the solution to be nanofiltered, supplement the solution during the desalting process, and keep the volume constant until the conductivity value of the effluent approaches that of double distilled water, thus obtaining a desalted solution.

[0047] S5: Add ethanol to the desalted solution from step S4 to a final concentration of 80%, let it stand overnight, centrifuge it at 3000 rpm for 20 minutes, and separate the precipitate.

[0048] S6: The precipitate was removed by ethanol, and the precipitate was freeze-dried at 20-40 Pa and -60°C to -40°C for 2-3 days to obtain hippocampal glycoprotein (HG).

[0049] Example 2: Preparation of hippocampal glycoprotein (HG-D)

[0050] This example is the same as example 1 except the following steps.

[0051] S2: The pH of the hippocampal homogenate was adjusted to 2.0, and pepsin was added at 20 U per gram of hippocampus. The extraction was performed at 37°C and a stirring speed of 150 rpm for 2 hours. The pH was adjusted to 7.5, and trypsin was added at 800 U per gram of hippocampus. The extraction was performed at 37°C and a stirring speed of 150 rpm for 3 hours to obtain hippocampal extract.

[0052] S3: The hippocampal extract was heated to 100°C to inactivate the enzyme for 10 minutes, and then cooled to room temperature. The precipitate was removed by centrifugation at 4000 rpm for 10 minutes to separate the supernatant.

[0053] S5: The precipitate was removed by ethanol, and the precipitate was dried under reduced pressure at -0.1 MPa to 0 MPa and 30°C to 60°C for 2-3 days to obtain hippocampal glycoprotein (HG-D).

[0054] Example 3: Determination of the molecular weight of hippocampal glycoprotein and analysis of monosaccharide composition

[0055] The molecular weight of hippocampal glycoprotein was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: chromatographic column: TSK-Gel G3000PW chromatographic column (7.5 x 300 mm, 5 μm); mobile phase: 0.2% trifluoroacetic acid aqueous solution and acetonitrile mixed at 7:3 (v / v,%); flow rate: 0.6 mL / min; column temperature: 30°C; injection volume: 10 μL; detector: diode array detector (equipped with Agilent 1260 Infinity series HPLC instrument); detection wavelength: 220 nm. The data were processed and the molecular weight of the sample was calculated using Astra software. The monosaccharide composition was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization high performance liquid chromatography (HPLC).

[0056] The specific operation process is as follows: taking hippocampus glycoprotein sample, treating with acid hydrolysis to break the sugar chain in it into free monosaccharide, then adding PMP derivatization reagent for derivatization reaction, converting the monosaccharide into PMP derivative with ultraviolet absorption; using a reversed-phase C18 chromatographic column (Agilent ZORBAX SB-C18) to separate the derived monosaccharide, using acetonitrile-phosphate buffer as the mobile phase for gradient elution, and detecting the chromatographic peaks of each monosaccharide derivative by a diode array detector at a wavelength of 254 nm; qualitatively analyzing according to the retention time and standard monosaccharide comparison, and calculating the relative content of each monosaccharide by peak area external standard method, so as to determine the types and proportions of monosaccharides in hippocampus glycoprotein.

[0057] The results show that hippocampus glycoprotein (HG) is composed of 7 kinds of monosaccharides, and the composition and molar mass ratio of each monosaccharide is as follows: mannose: glucosamine: glucuronic acid: galactosamine: glucose: galactose: fucose = 13.07: 34.38: 3.90: 3.90: 21.20: 20.30: 3.25, and the molecular weight distribution range is 20 kDa to 220 kDa.

[0058] Example 4, N-glycan profile analysis by PGC-HESI-MS / MS analysis

[0059] PNGase F enzymatic method was used to release N-glycan from hippocampus glycoprotein samples, and 2-AA was used for labeling. Vanquish Flex ultra-high performance liquid chromatography (UHPLC, Thermo Scientific, USA) equipped with Orbitrap Exploris 240 mass spectrometer was used for analysis. Hypercarb PGC column (150 mm x 2.1 mm i.d., 3 μm, Thermo Scientific, USA) was used for N-glycan separation, and the column temperature was 35°C. The mobile phase was 10 mM ammonium bicarbonate solution (A) and 80% acetonitrile solution (containing 20% 10 mM ammonium bicarbonate, B), and the flow rate was 0.3 mL / min. The gradient elution program was as follows: 0-2 min, 12% B; 2-15 min, 12%-28% B; 15-16 min, 28%-90% B; 16-18 min, 90% B; 18-19 min, 90%-12% B; 19-22 min, 12% B. Data acquisition was performed in negative ion mode (ESI⁻), with a spray voltage of 2500 V. The full scan range was 150-2000 m / z, with a resolution of 120,000 (m / z 200), and the acquisition mode was profile mode. MS / MS analysis used HCD collision mode, with a collision energy of 30 V and a resolution of 30,000. Ion source parameters: sheath gas 35 arb (units), auxiliary gas 10 arb (units), purge gas 0 arb (units); ion transmission tube temperature 320°C, gasification temperature 350°C, radio frequency lens 70%.

[0060] The results are shown in Figure 1 A total of 65 different types of N-glycans were identified in hippocampus glycoprotein (HG), and the relative content of each type of N-glycan was obtained by calculating the peak area. There were 14 N-glycans with a relative content of more than 1%. Sialylated N-glycans accounted for more than 30%. Figure 1 The N-glycans with a relative content of more than 1% in hippocampus glycoprotein and their relative contents are shown. The results show that hippocampus glycoprotein (HG) contains a relatively rich variety of N-glycans, and sialylated N-glycans are the main ones.

[0061] Example 5, determination of glycosaminoglycan disaccharide composition by multiple reaction monitoring (MRM) analysis

[0062] The glycosaminoglycan disaccharides were generated by enzymatic hydrolysis using a mixture of glycosaminoglycan lyase (containing heparin lyase I, II, III and chondroitinase ABC, AC, B, 40 mU of each enzyme), and the obtained disaccharides and standard products were labeled by 2-aminoacridone reduction amination method. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using an UltiMate3000 ultra-high performance liquid chromatography (UHPLC) separation system equipped with a TSQ-Quantiva mass spectrometer (Thermo Scientific, USA), in multiple reaction monitoring (MRM) mode, with a Poroshell 120 C18 chromatographic column (3.0×150 mm i.d, 2.7 µm, Agilent, USA) at a column temperature of 45°C. The mobile phase A was water / methanol (85:15, v / v), and the mobile phase B was water / methanol (35:65, v / v), both containing 50 mM ammonium acetate (pH adjusted to 6.5 with acetic acid) at a flow rate of 0.2 mL / min. The gradient elution program was as follows: 0-2 min, 0% B; 2-8 min, 0%-12% B; 8-15 min, 12%-25% B. Data acquisition was performed in negative ion mode (ESI⁻) with a spray voltage of 3 kV; ion source gas parameters: sheath gas flow rate 35 arb (units), auxiliary gas flow rate 10 arb (units); ion transmission tube temperature and gasification temperature were both 350°C. The peak intensity of MRM transition was used to draw the external standard calibration curve for quantification, and the linear relationship was evaluated by the correlation coefficient (r²).

[0063] The results are shown in Table 1 below. In the glycosaminoglycan (GAGs) analysis, the content of three types of GAGs in hippocampal glycoprotein (HG) was more than 3.7 mg / g, and chondroitin sulfate (CS) was the main component.

[0064] Table 1 GAGs components and contents in hippocampal glycoprotein (HG)

[0065]

[0066] The results are shown in Table 1 below. In the glycosaminoglycan (GAGs) analysis, the content of three types of GAGs in hippocampal glycoprotein (HG) was more than 3.7 mg / g, and chondroitin sulfate (CS) was the main component. Figure 2 As shown in Table 1, in terms of GAG subtype distribution, the content of 4-sulfated chondroitin sulfate in hippocampal glycoprotein (HG) was the highest, followed by 6-sulfated chondroitin sulfate (CS-6S), and the content of non-sulfated chondroitin sulfate (CS-0S) was the lowest. Calculation showed that the CS sulfation degree of hippocampal glycoprotein (HG) was 0.4-0.6.

[0067] As shown in Table 1, in terms of GAG subtype distribution, the content of 4-sulfated chondroitin sulfate in hippocampal glycoprotein (HG) was the highest, followed by 6-sulfated chondroitin sulfate (CS-6S), and the content of non-sulfated chondroitin sulfate (CS-0S) was the lowest. Calculation showed that the CS sulfation degree of hippocampal glycoprotein (HG) was 0.4-0.6. Figure 3 As shown in Table 1, in terms of GAG subtype distribution, the content of 4-sulfated chondroitin sulfate in hippocampal glycoprotein (HG) was the highest, followed by 6-sulfated chondroitin sulfate (CS-6S), and the content of non-sulfated chondroitin sulfate (CS-0S) was the lowest. Calculation showed that the CS sulfation degree of hippocampal glycoprotein (HG) was 0.4-0.6.

[0068] These results show that hippocampal glycoprotein (HG) contains rich glycosaminoglycans, of which chondroitin sulfate (CS) is the main component, accounting for the vast majority of total GAGs. In terms of subtype distribution, hippocampal glycoprotein (HG) is rich in 4-sulfated chondroitin sulfate (CS-4S) and 6-sulfated chondroitin sulfate (CS-6S), and the degree of CS sulfation is 0.4-0.6. Combined with the fact that the biological activity of glycosaminoglycans is closely related to its sulfation pattern (sulfation sites affect protein interactions, and the abundance of specific CS chain units regulates biological functions), and CS-6S has multiple biological significance, the high content of CS and characteristic sulfation pattern (especially the higher proportion of CS-6S) in hippocampal glycoprotein (HG) suggests that it has significant biological activity, providing an important basis for further exploring its role in related physiological function regulation.

[0069] Example 6, Effect of hippocampal glycoprotein (HG-D) on TNF-α secretion of mouse-derived macrophages

[0070] Logarithmic growth phase RAW 264.7 cells were plated in a 96-well plate at a density of 5×10 4 cells / well, 160 μL of medium per well, and cultured in a 37°C, 5% CO2 humidified incubator for a night to stabilize the cells. The next morning, 40 μL of fresh medium containing different concentrations of HG-D was added to make the final concentration of HG-D 5, 25, 50, 100 μg / mL per well, and the system was 200 μL / well. At the same time, a blank control group (complementing complete medium to 200 μL / well) and 1 μg / mL LPS as a positive control were set. The cell culture solution was appropriately diluted and used for TNF-α detection.

[0071] As shown in Figure 4 , hippocampal glycoprotein (HG-D) can promote the secretion of TNF-α by Raw264.7 macrophages, and the effect is more significant at higher concentrations; when the concentration is 100 μg / mL, the secretion of TNF-α induced by hippocampal glycoprotein (HG-D) is the highest (4166.28 pg / mL), which reflects its strong ability to promote TNF-α secretion.

[0072] Example 7, Effect of hippocampal glycoprotein (HG-D) on IL-6 secretion of mouse-derived macrophages

[0073] Logarithmic growth phase RAW 264.7 cells were plated in a 96-well plate at a density of 5×10 4Cells were seeded into 96-well plates with 160 μL of culture medium per well and cultured overnight at 37°C in a 5% CO2 humidifier to stabilize the cells. The next morning, 40 μL of fresh culture medium containing different concentrations of HG-D was added to each well to achieve final HG-D concentrations of 25, 50, and 100 μg / mL, with a total volume of 200 μL / well. A blank control group (with culture medium replenished to 200 μL / well) and a positive control group (1 μg / mL LPS) were also included. The cell culture medium was then appropriately diluted for IL-6 detection.

[0074] The results are as follows Figure 5 As shown, hippocampal glycoprotein (HG-D) can stimulate Raw264.7 macrophages to secrete IL-6, and the effect is the most significant; when the concentration is 100 μg / mL, HG-D induces IL-6 secretion of 2201.28 pg / mL, showing its outstanding activity in promoting IL-6 secretion.

[0075] Example 8: Effect of hippocampal glycoprotein (HG-D) on NO release from mouse-derived macrophages

[0076] RAW 264.7 cells in logarithmic growth phase were divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded into 96-well plates with 160 μL of culture medium per well and cultured overnight at 37°C in a 5% CO2 humidified incubator to stabilize the cells. The next morning, 40 μL of fresh culture medium containing different concentrations of HG-D was added to each well to achieve final HG-D concentrations of 5, 50, and 100 μg / mL, with a total volume of 200 μL / well. A blank control group (with culture medium replenished to 200 μL / well) and a positive control group (1 μg / mL LPS) were also included, with three replicates for each group. After 24 h of further culture, 50 μL of the supernatant was aspirated into a new 96-well plate. 50 μL of Griess I and 50 μL of Griess II were added to each well sequentially, and the mixture was shaken thoroughly. The absorbance was measured at 540 nm. The nitrite (NO3) content in the cell supernatant was calculated using a standard curve equation based on the sample OD value. 2- The concentration of ).

[0077] The results are as follows Figure 6 The results showed that, compared with the NC group, hippocampal glycoprotein (HG-D) could stimulate Raw264.7 macrophages to release NO, and the effect was the most significant. When the concentration was 100 μg / mL, the amount of NO released induced by hippocampal glycoprotein (HG-D) was 16.17 μM. The binding of NO can activate the function of macrophages to kill or inhibit microorganisms, further indicating that hippocampal glycoprotein (HG-D) has significant immunomodulatory potential.

[0078] In summary, the hippocampus glycoprotein has strong immune response potential verified by in vitro experiments, and its unique structure or composition characteristics: sialylated N-glycan and its rich CS-6S structure play an important role, can enhance the interaction with cytokine and NO signal related receptors in macrophages, and can simultaneously up-regulate multiple key immune mediators, showing significant advantages in immune regulation, laying a foundation for its application and mechanism in-depth research in the field of functional food, biomedical and other fields, highlighting its application prospect in immune regulation.

[0079] In summary, the hippocampus glycoprotein becomes an important material basis for enhancing the immune function of hippocampus due to its rich high-sialylated N-glycan and high-sulfated GAGs. The present application not only provides the structure and function of hippocampus glycoprotein and related knowledge, but also provides experimental basis for its application in the fields of immune regulation, functional food and new drug development.

[0080] The present application innovatively proves that the hippocampus glycoprotein has significant immune regulation activity, has many advantages such as clear source, controllable preparation process, unique component characteristics, good biocompatibility and good stability, and can be used for developing health care products or functional foods for immune regulation needs, and has broad development and application prospect in the field of biomedical and health industry.

[0081] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and the protection scope of the present application is not limited thereto; any skilled person in the art can make other different forms of changes or variations without departing from the spirit and technical scope of the present application; here, all the embodiments cannot be exhausted, and any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.

[0082] On the basis of the above embodiments, the technical features involved therein and the functions and effects of the technical features in the present application are described in detail to help the skilled person in the art to fully understand the technical solutions of the present application and to reproduce them.

[0083] Finally, although the present application is described according to the embodiments, each embodiment does not contain only one independent technical solution, and the description manner of the specification is only for the sake of clarity, and the skilled person in the art should consider the specification as a whole, and the technical solutions in each embodiment can be combined to form other embodiments which can be understood by the skilled person.

Claims

1. A hippocampus glycoprotein having an immunomodulatory function, characterized by, The hippocampus glycoprotein has both sialylation N-glycosylation modification and chondroitin sulfate modification, and the molecular weight of the hippocampus glycoprotein is 20-220 kDa.

2. The hippocampus glycoprotein according to claim 1, characterized in that, The total glycosaminoglycan accounts for 0.1%-0.5% of the mass of the hippocampus glycoprotein, the sulfation degree of chondroitin sulfate is 0.4-0.6, and the 6-sulfated chondroitin sulfate accounts for 0.1%-0.2% of the mass of the hippocampus glycoprotein.

3. The method of producing hippocampus glycoprotein according to claim 1 or 2, characterized in that, The method comprises the following steps: S1: cutting hippocampus, soaking in water, and homogenizing to obtain hippocampus homogenate; S2: extracting the hippocampus homogenate by enzymatic method or water extraction method to obtain supernatant; S3: performing membrane separation operation on the supernatant to obtain desalted solution; S4: adding ethanol to the desalted solution for alcohol precipitation, and separating the precipitate after centrifugation to obtain alcohol precipitation precipitate; S5: drying the alcohol precipitation precipitate to obtain hippocampus glycoprotein.

4. The production method according to claim 3, characterized by, The hippocampus is at least one of Hippocampus kelloggi, Hippocampus histrix, Hippocampus kuda, Hippocampus trimaculatus, Hippocampus parvus, and Hippocampus ingens.

5. The preparation method according to claim 3, characterized in that, In the step S1, the ratio of hippocampus to water is 1:5-20, and in the step S3, the membrane separation operation is desalination using a nanofiltration membrane with a molecular weight cut-off of 150-300 Da.

6. The preparation method according to claim 3, characterized in that, In the step S2, the specific operation steps of the enzymatic method are as follows: a, adjusting the pH of the hippocampus homogenate, adding biological protease for extraction to obtain hippocampus extract; b, heating the hippocampus extract to inactivate the enzyme, cooling to room temperature, centrifuging to remove the precipitate impurities, and obtaining the supernatant.

7. The production method according to claim 6, wherein The pH is 1-9, the biological protease is at least one of pepsin, neutral protease, trypsin, and pancreatin, the amount of biological protease is 500-3000 U / g based on the mass of hippocampus, the enzymolysis temperature is 30-45℃, and the enzymolysis time is 0.5-3 h.

8. The preparation method according to claim 3, characterized in that, In the step S2, the specific operation steps of the water extraction method are as follows: a, extracting the hippocampus homogenate at 30-100℃ and 0-1 Mpa for 0.5-5 hours, extracting 1-3 times to obtain hippocampus extract; b, centrifuging the hippocampus extract to remove the precipitate impurities to obtain the supernatant.

9. The hippocampus glycoprotein of claim 1 is used for preparing a preparation or a drug with immunomodulatory function.

10. Use according to claim 9, characterized in that, The hippocampus glycoprotein can effectively promote the proliferation of macrophages and the secretion of inflammatory factors.

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