CN115246871A

CN115246871A - Preparation method of mytilus coruscus immunocompetence hexapeptide

Info

Publication number: CN115246871A
Application number: CN202111608952.8A
Authority: CN
Inventors: 余方苗; 曾瑜; 何康; 唐云平
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-10-28
Anticipated expiration: 2041-12-27
Also published as: CN115246871B

Abstract

The invention provides a preparation method of mytilus coruscus immunocompetence hexapeptide, and belongs to the technical field of preparation of polypeptide. The preparation method of the mytilus coruscus immunological competence hexapeptide comprises the following steps: mincing and degreasing mytilus coruscus meat, carrying out enzymolysis, carrying out ultrafiltration to obtain an enzymolysis solution, carrying out rotary evaporation on the enzymolysis solution, and carrying out freeze drying to obtain mytilus coruscus crude peptide; step (2) separating and purifying by using a gel column and separating and purifying by using RP-HPLC; and (3) detecting the mytilus coruscus active peptide by using LC-MS/MS to determine the sequence of the mytilus coruscus active peptide. The mytilus coruscus immunological activity hexapeptide prepared by the method can be used for preparing medicines and health care products for improving cellular immunity and humoral immunity and improving immune organ functions.

Description

Preparation method of mytilus coruscus immunocompetence hexapeptide

Technical Field

The invention relates to the technical field of polypeptide preparation, in particular to a preparation method of mytilus coruscus immunological activity hexapeptide.

Background

Mussel (Mytilus sp.) Belongs to the phylum of mollusca (Mollusca) Class of lamellibranches (Lanellibranchia) Different from the column order (Anisomyaria) Mytilus galloprovincialis (A)Mytilacea）And Mytilidae (Mytidea) It is a bivalve mollusk widely distributed on the coastline of the world. The activity research of the modern medicine on the mussels as the traditional Chinese medicine shows that the mussels have the functional activities of anti-inflammation, antioxidation, antifatigue, antitumor, antibiosis, immunoregulation and the like, and the activities benefit from various active substances contained in the mussels. For example, isolated antimicrobial peptides extracted from mussels have different antimicrobial spectra and participate in different stages of the immune response of different types in the body of the mussel, thereby forming the natural broad-spectrum antimicrobial capability of the mussel. In addition, chinese patent CN109206483A discloses the use of mussel active peptide in the anti-tumor aspect, and chinese patent CN106676155A discloses the use of mussel polypeptide in the anti-thrombus aspect. Therefore, the mussel active peptide has extremely high research value. However, the effect of mussel active peptide on immunity is rarely reported.

The preparation method of common bioactive peptide comprises chemical synthesis method, direct extraction method, enzyme hydrolysis method, fermentation method, gene recombination synthesis method, etc. of known amino acid sequence. Chemical synthesis involves many organic reagents and many are toxic, which is contrary to the ultimate food-grade localization of bioactive peptides, which is the limitation of chemical synthesis. The gene recombination synthesis method is still in a relatively basic stage at present, has higher operation difficulty and seriously unbalanced input-output ratio, and cannot be developed to the degree of industrial scale production. Therefore, more intensive research on the preparation method of the mussel active peptide is needed. The enzyme hydrolysis method for producing the active peptide has the advantages of high efficiency, little damage to the nutritional value of protein, no peculiar smell, extremely high product safety, mild production conditions, capability of producing specific peptide in a positioning way and low cost, and the enzyme hydrolysis method becomes the most main production method of the active peptide. The zymolyte obtained after proteolysis is further separated and purified, and the separation and purification of the active peptide are more refined and efficient due to the application of a membrane separation technology and a high performance liquid chromatography technology in the aspect of protein purification. However, few systematic research reports are reported for preparing the mytilus coruscus active peptide by the enzyme hydrolysis method at present.

Disclosure of Invention

The invention aims to provide a mytilus coruscus active hexapeptide with immune activity and a preparation method thereof.

In order to achieve the purpose of the invention, the following technical scheme is adopted.

The invention discloses a mytilus coruscus immunological activity hexapeptide prepared by the preparation method, and the sequence of the mytilus coruscus immunological activity hexapeptide is shown as SEQ ID NO. 2.

Preferably, the Mytilus coruscus immunologically active hexapeptide has a molecular weight of 636.40Da.

The invention also discloses a preparation method of the mytilus coruscus immunological activity hexapeptide, which comprises the following steps:

step (1), preparing thick-shell mussel crude peptide;

separating and purifying the mytilus coruscus active peptide;

step (3) detecting the mytilus coruscus active peptide;

wherein the Mytilus coruscus active peptide comprises 6 short peptides with the sequence shown as SEQ ID NO.1-SEQ ID NO. 6.

Preferably, step (1) comprises: mincing mytilus coruscus meat, degreasing, performing enzymolysis, performing ultrafiltration to obtain enzymolysis liquid, and performing rotary evaporation, freeze drying to obtain mytilus coruscus crude peptide.

The invention adopts the enzymolysis method for extraction, and the enzyme has specific enzyme cutting sites, so the reaction has high specificity, and the reaction can be accurately controlled only by selecting proper enzyme and proper reaction conditions, thereby being beneficial to establishing standard production standards in large-scale production.

Preferably, the substances contained in the enzymatic hydrolysate have a molecular weight of less than 1kDa.

Preferably, the degreasing temperature is 50-60 ℃ and the time is 1-2.5h.

Preferably, the enzymatic hydrolysis uses pepsin and/or trypsin; the enzymolysis temperature is 35-40 ℃; the enzymolysis time is 5-8h.

Preferably, ultrafiltration uses the TFF Cogent μ Scale system.

Preferably, step (1) comprises:

removing sufficient filaments from Mytilus coruscus meat, washing with running water, and mincing in tissue homogenizer; mixing the minced meat with isopropanol according to a proportion of 1 g; after degreasing, removing supernatant, washing the residual mytilus coruscus meat with pure water until no alcohol smell exists, and collecting precipitate; adding pure water according to a feed-liquid ratio of 1; after cooling, centrifuging 5000g for 10-12min under the precooling condition of 2-4 ℃ to obtain supernatant; and (3) ultrafiltering the obtained supernatant by a TFF Cogent mu Scale system to finally obtain an enzymolysis solution with the molecular weight of less than 1kDa, and performing rotary evaporation, freeze drying to obtain the crude peptide of the mytilus coruscus.

More preferably, step (1) comprises:

removing byssus from the thick-shell mussel meat, washing with running water, and mincing in a tissue homogenizer. Mixing the minced meat with isopropanol according to a ratio of 1g to 4mL, uniformly stirring, degreasing at 55 ℃ for 2h, and repeating for 2 times; after degreasing, removing supernatant, washing the residual mytilus coruscus meat with pure water until no alcohol smell exists, and collecting precipitate; adding pure water according to a feed-liquid ratio of 1; after cooling, centrifuging for 10min at 5000g under the condition of precooling at 4 ℃ to obtain supernatant; and (3) ultrafiltering the obtained supernatant by a TFF Cogent mu Scale system to finally obtain an enzymolysis solution with the molecular weight of less than 1kDa, and performing rotary evaporation, freeze drying to obtain the mytilus coruscus crude peptide.

More preferably, the defatted in step (1) is carried out by mixing the ground meat with isopropanol, indole-5-carboxylic acid, benzhydrylamine in a proportion of 1g: 0.2-0.5g, 0.5-0.8mL, uniformly stirring, degreasing at 50-60 ℃ for 1-2.5h, and repeating for 2 times.

The degreasing degree can influence the preparation and purification of the subsequent peptide, and the degreasing treatment is carried out by using the mixed solution of isopropanol, indole-5-carboxylic acid and benzhydrylamine, so that the degreasing effect can be further improved, and the negative influence of lipid residue on the subsequent steps can be reduced.

Preferably, step (2) comprises separation and purification using a gel column and separation and purification using RP-HPLC.

Preferably, the gel column separation and purification comprises the following steps:

packing the gel into a column; filtering the prepared thick-shell mussel crude peptide solution, and then loading the sample for elution; eluting and collecting samples corresponding to each elution peak to perform an MTT experiment; and selecting the optimal peptide according to the experimental result.

More preferably, the concentration of the crude mytilus coruscus peptide solution is 0.1-0.5g/mL.

More preferably, the cell used in the MTT assay is RAW 264.7.

More preferably, the gel column separation and purification comprises the following steps:

loading the gel to a column with a volume of 5.0 × 45cm, and balancing 3-5 column volumes by using 1.0-1.2mL/min ultrapure water as a mobile phase; preparing a solution with the concentration of 0.1-0.5g/mL by using the freeze-dried mytilus coruscus crude peptide, wherein the sample loading amount is 1-2mL, filtering the solution by using a filter membrane with the diameter of 0.22 mu m before loading, eluting the solution by using ultrapure water with the concentration of 1.0-1.2mL/min, collecting each tube for 2-3min, and measuring the absorbance of each tube at the position of 280 nm; and drawing a component curve by taking the number of tubes as an abscissa and the absorbance as an ordinate, collecting each peak, freeze-drying, carrying out experiments, and screening out the optimal peak peptide solution with the highest relative proliferation rate to the macrophage RAW 264.7.

Still more preferably, the gel column separation purification comprises the steps of:

loading the pretreated Sephadex G-25 gel into a column with the column volume of 5.0 × 45cm, and balancing 4 column volumes by using ultrapure water of 1.0mL/min as a mobile phase; preparing a solution with the concentration of 0.2g/mL by using the freeze-dried mytilus coruscus crude peptide, wherein the sample loading amount is 2mL, filtering the solution by using a filter membrane with the diameter of 0.22 mu m before loading, eluting the solution by using ultrapure water with the concentration of 1.0mL/min, collecting each tube for 3min, and measuring the absorbance of each tube at the position of 280 nm; and drawing a component curve by taking the number of tubes as an abscissa and the absorbance as an ordinate, collecting each peak, freeze-drying, performing MTT (maximum temperature transfer) experiment, and screening out the optimal peak peptide solution with the highest relative proliferation rate to macrophage RAW 264.7.

Preferably, the separation and purification using RP-HPLC comprises the following steps:

and (3) further separating and purifying the optimal peak peptide solution by using an analytical chromatographic column, wherein the column temperature is 23-26 ℃, the sample injection amount is 10-15 mu L, and ultrapure water is used as a mobile phase A, and acetonitrile is used as a mobile phase B for elution.

More preferably, the separation and purification using RP-HPLC comprises the following steps:

and further separating and purifying the optimal peak peptide solution by using a ZORBAX SB-C18 analytical chromatographic column (5 mu m, 9.4X 250 mm), wherein the column temperature is 25 ℃, the sample injection amount is 10 mu L, ultrapure water is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the elution conditions are as follows: 0-5 min 10% B elution, 5-15 min 10% -50% B elution, 15-25 min 50% B elution, 25-30 min 50% -100% B elution, 30-35 min 100% B elution; the elution flow rate is 0.5 mL/min, and the peak is detected at 280nm to obtain the Mytilus coruscus active peptide.

Preferably, step (3) comprises detecting Mytilus coruscus active peptide by LC-MS/MS to determine Mytilus coruscus active peptide sequence.

The invention also discloses application of the mytilus coruscus immunological activity hexapeptide in preparation of medicines and/or health products for improving spleen tissue injury and/or thymus gland atrophy.

The invention also discloses application of the mytilus coruscus immunological activity hexapeptide in preparation of medicines and/or health care products for improving cellular immunity and/or humoral immunity.

Compared with the prior art, the invention has the beneficial effects that:

the mytilus coruscus active peptide is prepared by an enzymolysis method, the reaction condition is mild, the operation is easy, and the enzymolysis reaction has high specificity, so that the unified standard can be established in large-scale preparation; the mytilus coruscus immunological activity hexapeptide prepared by the method can be used for preparing medicines or health care products for repairing immune organ injury, improving phagocytic capacity of macrophages and improving cellular immunity and humoral immunity functions. In addition, when thick-shell mussel meat is degreased, the mixed liquid of isopropanol, indole-5-carboxylic acid and benzhydrylamine is used for degreasing, so that the degreasing rate is effectively improved and can reach over 45%.

Drawings

FIG. 1 shows Sephadex G-25 elution peak;

FIG. 2 shows the relative proliferation rate of each elution peak;

FIG. 3 shows the elution peaks of RP-HPLC column;

FIG. 4 is a graph showing cell proliferation activity of various Mytilus coruscus active peptides at different concentrations;

FIG. 5 is a graph showing the determination of NO content in various Mytilus coruscus active peptides at different concentrations;

FIG. 6 is a mass spectrum of the Mytilus coruscus immunological activity hexapeptide sequence LVVLGH;

FIG. 7 is a graph of the effect of Mytilus coruscus immunologically active hexapeptides on the immune organ index of mice;

FIG. 8 shows the result of staining spleen and thymus HE of mice; wherein A1-5 is spleen HE staining result, and B1-5 is thymus HE staining result; WP denotes white marrow, RP denotes red marrow;

FIG. 9 is a graph showing the effect of Mytilus coruscus immunoreactive hexapeptide on the levels of TNF- α, IL-1 β, and IL-6 in serum;

FIG. 10 is a graph of the effect of Mytilus coruscus immunologically active hexapeptides on serum IgA, igG, and IgM levels.

Detailed Description

The exemplary embodiments will be described herein in detail, and the implementations described in the exemplary embodiments below do not represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Preparation of mytilus coruscus active peptide

(1) Preparation of mytilus coruscus crude peptide

Removing sufficient filaments from the thick-shell mussel meat, washing with running water, and mincing in a tissue homogenizer. Mixing the minced meat with isopropanol according to the proportion of 1g; after degreasing, discarding the supernatant, washing the residual mytilus coruscus meat with pure water until no alcohol smell exists, and collecting the precipitate. Adding pure water according to a feed-liquid ratio of 1; after cooling, centrifuging for 10min at 5000g under the condition of precooling at 4 ℃ to obtain supernatant; and (3) ultrafiltering the obtained supernatant by a TFF Cogent mu Scale system to finally obtain an enzymolysis solution with the molecular weight of less than 1kDa, and carrying out rotary evaporation, freeze drying to obtain Mytilus coruscus crude peptide (MCP).

(2) Separation and purification of mytilus coruscus active peptide

(2.1) gel column separation and purification

Loading the pretreated Sephadex G-25 gel into a column with the column volume of 5.0 × 45cm, and balancing 4 column volumes by using ultrapure water of 1.0mL/min as a mobile phase; preparing freeze-dried mytilus coruscus crude peptide into a solution with the concentration of 0.2g/mL, loading the solution with the amount of 2mL, filtering the solution with a filter membrane of 0.22 mu m before loading, eluting the solution with ultrapure water of 1.0mL/min, collecting each tube at a time interval of 3min, and measuring the absorbance of each tube at 280 nm; the number of tubes is plotted as abscissa and absorbance is plotted as ordinate to form a composition curve as shown in FIG. 1, each peak is collected and freeze-dried, and MTT test is performed, with the test results shown in FIG. 2.

As can be seen from FIG. 1, after separation by Sephadex G-25 gel column, 2 elution peaks were obtained at 280nm in absorbance value of each tube: MCP-I and MCP-II. Collecting each elution peak, performing MTT experiment after freeze drying, determining the relative proliferation rate of different components, and taking MCP crude peptide as comparison.

As can be seen from FIG. 2, the relative proliferation rate of MCP-II fraction to RAW264.7 cells was higher than that of MCP-I fraction, so that MCP-II fraction was selected as the optimum peak peptide solution for further separation and purification by RP-HPLC.

(2.2) RP-HPLC separation and purification

And (2) further separating and purifying the optimal peak peptide solution MCP-II by using a ZORBAX SB-C18 analytical chromatographic column (5 mu m 9.4 multiplied by 250 mm), wherein the column temperature is 25 ℃, the sample injection amount is 10 mu L, ultrapure water is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the elution conditions are as follows: 0-5 min 10% B elution, 5-15 min 10% -50% B elution, 15-25 min 50% B elution, 25-30 min 50% -100% B elution, 30-35 min 100% B elution; the elution flow rate was 0.5 mL/min, and the peak was detected at 280 nm. The results of the detection are shown in FIG. 3.

As can be seen from FIG. 3, the highest peak appeared around 20min, which is much higher than other peaks; the yield of other peaks is too low, so the highest peak is collected as the mytilus coruscus active peptide, and the sequencing is carried out after freeze-drying.

(3) Detection of Mytilus coruscus active peptide

Performing LC-MS/MS detection on the Mytilus coruscus active peptide:

the mytilus coruscus active peptide is prepared into a 1mg/mL solution, filtered by a 0.22 mu m filter membrane and then subjected to LC-MS/MS analysis, and the chromatographic conditions are shown in Table 1.

Performing De-novo search and database search on the obtained result by using PEAKS software; sample application in NCBIMytilusA protein database of species.

TABLE 1 chromatographic conditions

Assembly	Parameter(s)
		Chromatographic column	C18，3µm，250mm×75µm（Eksigent）
Sample volume	4μL
		Flow rate of flow	300nL/min
Mobile phase	Phase A is acetonitrile, 0.1% formic acid; phase B is water, 0.1% formic acid; 0-1695in 93% -88% A,16-51min 88% -70% A,51-71min 70% -58%,71-72min 58% -5% A,72-78min 5% A

The mass spectrometer is Orbitrap Fusion Lumos (thermoldissher), and the detection conditions are as follows: positive ion detection mode, primary resolution 120000, agc set to 3e6, scan range 300-1400m/z. The 10 ions with the highest intensity were selected from 1 MS spectrum for MS/MS analysis with a secondary resolution of 15000, AGC set to 2e4, and a separation window of 1.6m/z.

After LC-MS/MS detection, the confidence level is passed and the Uniprot protein database is searchedMytilusAnd 6 Mytilus coruscus active peptide sequences are screened out according to the comparison result of the protein data of the species, and are shown in the table 2.

Table 2 physicochemical Properties of 6 peptides selected from MCP

Sequence	ALC（%）	Length	Molecular weight （Da）	SEQIDNO
					FGGGGF	99	6	540.2332	1
LVVLGH	99	6	636.3959	2
					FVLPR	99	5	630.3853	3
LLTDY	99	5	623.3167	4
					LLAL	99	4	428.2999	5
FLFER	99	5	710.3751	6

Example 2

Preparation of mytilus coruscus immunological activity hexapeptide

The 6 Mytilus coruscus active peptides FGGGGF, LVVLGH, FVLPR, LLTDY, LLAL and FLFER are numbered as (1), (2), (3), (4), (5) and (6) respectively, and the influence of peptide sequences with different concentrations (50, 100 and 200 mug/mL) on the relative cell proliferation rate and NO secretion of RAW264.7 is measured.

The relative cell proliferation rate is measured by a CCK-8 method:

RAW264.7 cells in logarithmic growth phase were selected, diluted with medium and transferred to 96-well plates, 200. Mu.L of cell suspension was added to each well, and the cell density was 1X 10 ⁴ Per well; 37 ℃ and 5% CO ₂ Culturing overnight under the condition; removing culture medium after culturing, adding culture medium containing Mytilus coruscus active peptide at different concentrations, 37 deg.C, and 5% CO ₂ Culturing for 24h under the condition; adding 20 mu L of CCK-8 solution into each well after culture, incubating for 1h under the same condition, detecting absorbance at 450nm by using an enzyme-labeling instrument, and calculating the relative proliferation rate of cells:

relative increment rate (%) = (OD experimental wells-OD blank wells)/(OD control wells-OD blank wells);

wherein the experimental holes are holes added with mytilus coruscus bioactive peptide culture media with different concentrations, the control holes are holes added with mytilus coruscus bioactive peptide culture media without mytilus coruscus bioactive peptide, and the blank holes are holes without cells.

The effect of different concentrations (50, 100, 200. Mu.g/mL) of peptide sequences on RAW264.7 NO secretion was determined using the Griess reaction, using the NO kit (Shanghai Biyuntian Biotech Co., ltd.) according to the instructions.

The results are shown in FIGS. 4 and 5.

As can be seen from FIGS. 4 and 5, the LVVLGH (named Mytilus coruscus immunologically active hexapeptide, expressed as LH-6) peptide (No. 2) has the highest relative proliferative effect on RAW264.7 and NO synthesis promoting ability. The Mytilus coruscus immunoreactive hexapeptide was detected by mass spectrometry, and the results are shown in FIG. 6.

Example 3

Preparation of mytilus coruscus active peptide

(1) Preparing thick-shell mussel crude peptide;

removing byssus from the thick-shell mussel meat, washing with running water, and mincing in a tissue homogenizer; mixing the minced meat, isopropanol indole-5-carboxylic acid and benzhydrylamine according to a ratio of 1g; after degreasing, removing supernatant, washing the residual mytilus coruscus meat with pure water until no alcohol smell exists, and collecting precipitate; adding pure water according to a feed-liquid ratio of 1; after cooling, centrifuging for 10min at 5000g under the condition of precooling at 4 ℃ to obtain supernatant; and (3) ultrafiltering the obtained supernatant by a TFF Cogent mu Scale system to finally obtain an enzymolysis solution with the molecular weight of less than 1kDa, and performing rotary evaporation, freeze drying to obtain the mytilus coruscus crude peptide (MCP).

The steps (2) and (3) are the same as the embodiment 1, and because the results in the steps (2) and (3) are almost completely consistent with the embodiment 1, the difference is within the error range, and the result schematic diagram and the description are omitted.

Test example 1

Determination of thick-shell mussel meat defatting rate

The fat content of the mytilus coruscus minced meat before and after the degreasing treatment in example 1 and example 3 is measured according to GB/T5009.6-2016 method for measuring fat in food, and the degreasing rate is calculated after the measurement:

the degreasing rate (%) = (fat content before degreasing-fat content after degreasing)/fat content after degreasing × 100%.

The measurement shows that the defatting rate of the minced mytilus coruscus in example 1 is 33.47%, and the defatting rate in example 3 is 47.16%. The degreasing rate is obviously increased in the example 3, which shows that the mixed solution of isopropanol indole-5-carboxylic acid and benzhydrylamine is better in degreasing effect.

Test example 2

Test for improving mouse immunoregulation function by mytilus coruscus immunocompetence hexapeptide

1. Grouping and modeling of experimental animals

Experimental animals 50 male ICR mice were divided into 5 groups of 10 mice each and were adaptively fed for 7 days. 4 groups of the three were selected to be injected intraperitoneally with 80 mg/kg/d Cyclophosphamide (CTX) for immunosuppression model induction for 5 days. The other group was used as normal group to inject normal saline intraperitoneally. From day 4 onwards, 3 groups of immunosuppressive mice were administered with Mytilus coruscus immunologically active hexapeptide once a day by gavage (200. Mu.L) at concentrations of 50mg/kg,100mg/kg,200mg/kg, and the normal group and the model group were administered with the same amount of physiological saline, respectively, and the procedure was repeated for 15 days.

2. Influence of Mytilus coruscus immunocompetent hexapeptide on immunosuppressed mouse immune organ index

Mice were weighed twice daily, each prior to feeding, for 20 days. The day before sacrifice, the patient is deprived of food for 24 hours, then the eyeball is bled, the thymus and the spleen are rapidly picked up, after blood stains are cleaned in physiological saline and residual water drops on the surface are sucked off by gauze, the weighing is carried out, and the organ index is calculated according to the following formula:

immune organ index = immune organ weight (mg)/mouse weight (g).

The influence of Mytilus coruscus immunocompetent hexapeptide on the immune organ index of a mouse is shown in figure 7, the spleen index and the thymus index of a model group are both obviously lower than those of a normal group, and the successful induction of an immune hypofunction model is shown. The indexes of two immune organs of each mytilus coruscus immune activity hexapeptide group are different from those of the model group, which shows that the mytilus coruscus immune activity hexapeptide can repair the injury of CTX to the immune organs.

3. Histopathological observation

Thymus and spleen were fixed in 4% paraformaldehyde for histopathological examination. Then, thymus and spleen tissues of each mouse were gradually dehydrated in gradient ethanol and embedded in paraffin. The paraffin-embedded sections were cut into sections 4 μm thick using a microtome, stained with hematoxylin and eosin, and then examined and photographed on an optical microscope, and the photograph is shown in FIG. 8, in which A1-5 is the spleen HE staining result and B1-5 is the thymus HE staining result; WP represents white marrow and RP represents red marrow.

Spleen HE staining results showed: compared with the normal group, the spleen has unclear white marrow and red marrow boundaries and mature lymphocytes are reduced in the model group mice; the above-mentioned lesions were reduced in each group after mytilus coruscus immunoreactive hexapeptide treatment and spleen tissue was nearly normal. The mytilus coruscus immunocompetent hexapeptide can improve spleen tissue damage and function degradation caused by CTX.

Thymus HE staining results showed: thymosomes of the model group mice are not obviously formed, cortical medulla is interwoven together, and after administration, the thoracic gland pith is clearly demarcated, and the cortex is thickened. The mytilus coruscus immunological activity hexapeptide can improve spleen tissue damage and thymus atrophy caused by CTX.

4. Effect on immunosuppressed mice delayed allergy

4 days before the last administration, 2% (v/v) Sheep Red Blood Cells (SRBC) were intraperitoneally injected into each group of mice, and 200. Mu.L (about 1X 10) per mouse was injected ⁸ SRBC) to achieve a sensitizing effect. After 3 days of the immunoreaction, the thickness of the left hind paw of each group of mice was measured using a vernier caliper, followed by injection of 20. Mu.L (about 1X 10) of the left hind paw of the mice ⁸ Individual cells) 20% (v/v) SRBC, and the left hind plantar injection site thickness was measured 24h later, 3 times to balance the error. The difference between the two measurements before and after injection of the left hind plantar aspect of the mice of each group was finally used to indicate the degree of delayed allergy. The delayed allergic response results are shown in table 3.

TABLE 3 Effect of Mytilus coruscus Immunoactive hexapeptide on SRBC-induced mouse DTH

Group of	Initial thickness/(cm)	Final thickness/(cm)	Difference/(cm)
				Normal group	2.45±0.07	2.96±0.09	0.51±0.07
Model set	2.06±0.08	2.32±0.06	0.26±0.03**
				Mytilus coruscus immunocompetence hexapeptide-50 mg/kg	2.29±0.05	2.61±0.11	0.32±0.05**
Mytilus coruscus immunocompetence hexapeptide-50 mg/kg	2.31±0.09	2.68±0.07	0.37±0.04*#
				Immunity activity hexapeptide of Mytilus coruscus-50 mg/kg	2.27±0.09	2.70±0.17	0.43±0.11##

As can be seen from Table 3, the thickness difference of the 24h toe of the mice is significantly changed compared with that of the model group, and the high-dose mytilus coruscus immunocompetent hexapeptide can effectively repair the DTH of the mice inhibited by CTX, so that the cellular immune function of the immunosuppressed mice is improved.

5. Determination of mouse serum cytokines

After the last time of feeding the mytilus coruscus with the immune activity hexapeptide for 24 hours, the mice are killed by dislocation of cervical vertebra, blood is taken from eyeballs, 500g of the blood is centrifuged at low temperature for 10min, and serum is collected. A standard curve is drawn according to the instruction of each cytokine kit (Nanjing Senega Biotechnology Co., ltd.), and then the concentrations of TNF-alpha, IL-6 and IL-1 beta in serum are determined.

Cytokines are important immune mediator small molecules in humoral immunity. As shown in FIG. 9, the Mytilus coruscus immunoreactive hexapeptide was able to dose-dependently significantly elevate the levels of the cytokines TNF- α, IL-6, IL-1 β.

6. Determination of immunoglobulins (IgG, igA, igM) in mouse serum

Blood is taken from eyeballs after mice are sacrificed, serum is collected by centrifugation, a standard curve is drawn according to each immunoglobulin reagent, and then the concentration of the immunoglobulin IgG, igA and IgM in the serum is measured.

The measurement results are shown in FIG. 10.

The immune globulin plays an irreplaceable role in the humoral immunity of an organism, and an experimental result shows that the LH-6 with medium and high dose can obviously improve the immune globulin level in an immunosuppressive mouse body, so that the humoral immunity function of the immunosuppressive mouse is repaired.

Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.

The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Zhejiang ocean university

<120> preparation method of mytilus coruscus immunocompetence hexapeptide

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

Phe Gly Gly Gly Gly Phe

1 5

<210> 2

<211> 6

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

Leu Val Val Leu Gly His

1 5

<210> 3

<211> 5

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 3

Phe Val Leu Pro Arg

1 5

<210> 4

<211> 5

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 4

Leu Leu Thr Asp Tyr

1 5

<210> 5

<211> 4

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 5

Leu Leu Ala Leu

1

<210> 6

<211> 5

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 6

Phe Leu Phe Glu Arg

1 5

Claims

1. An immunological active hexapeptide of Mytilus coruscus has a sequence shown in SEQ ID NO. 2.

2. The Mytilus coruscus bioactive hexapeptide of claim 1, wherein the Mytilus coruscus bioactive hexapeptide has a molecular weight of 636.40Da.

3. A method for preparing the mytilus coruscus immunological activity hexapeptide of claim 1, comprising the steps of:

step (1), preparing thick-shell mussel crude peptide;

step (2) separating and purifying the mytilus coruscus active peptide;

step (3) detecting the mytilus coruscus active peptide;

wherein, the Mytilus coruscus active peptide comprises 6 short peptides with the sequence shown as SEQ ID NO.1-SEQ ID NO. 6.

4. The method of claim 3, wherein the step (1) comprises: mincing mytilus coruscus meat, degreasing, performing enzymolysis, performing ultrafiltration to obtain enzymolysis liquid, and performing rotary evaporation, freeze drying to obtain mytilus coruscus crude peptide.

5. The method according to claim 3, wherein a molecular weight of a substance contained in the enzymatic hydrolysate is less than 1kDa.

6. The method according to claim 3, wherein the step (2) comprises separation and purification using a gel column and separation and purification using RP-HPLC.

7. Use of the mytilus coruscus immunologically active hexapeptide of claim 1 in the manufacture of a medicament and/or health product for ameliorating spleen tissue damage and/or thymus atrophy.

8. Use of the mytilus coruscus immunologically active hexapeptide of claim 1 in the manufacture of a medicament and/or health care product for improving cellular and/or humoral immunity.