An immunologically active sea cucumber-derived active peptide, and a preparation method and application thereof

Abstract

The application discloses a sea cucumber-derived active peptide with immunocompetence and a preparation method and application thereof, and belongs to the technical field of biology. An amino acid sequence of the sea cucumber-derived active peptide is shown as SEQ ID NO. 1. The amino acid sequence of the sea cucumber-derived active peptide is obtained by means of a sea cucumber protein database with the aid of separation and purification technology and mass spectrometry identification technology; the interaction between the immunocompetent peptide and a membrane recognition receptor (TLR-2) is explored through multiple rounds of screening and molecular docking technology; and the in-vitro immunocompetence of the polypeptide is verified through solid-phase synthesis and a RAW264.7 cell model. The results show that the novel immunocompetent peptide screened in the application has in-vitro immunocompetence, no biological toxicity, can activate the immune response of macrophages in the body, but will not excessively activate, so that the body is in a healthy and stable state, and the novel immunocompetent peptide has important significance for deep processing of marine protein resources.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an immunomodulatory sea cucumber-derived bioactive peptide, its preparation method, and its application. Background Technology

[0002] In modern society, the fast pace of life and increasing work pressure keep the body in a constant state of stress, leading to weakened immunity, a root cause of many diseases. Research shows that immunologically active peptides can enhance immunity and protect the body from pathogens by inhibiting oxidative stress. Extensive research has been conducted on immunologically active peptides derived from food-based animal and plant proteins. Marine protein-derived immunologically active peptides have attracted widespread attention due to their high safety and structural stability. Therefore, marine biologically derived immunologically active peptides have become a hot topic of research.

[0003] Sea cucumbers belong to the invertebrate phylum Echinodermata, class Holothuroidea, and have been commercially harvested for over 1000 years. Modern research has shown that sea cucumbers contain more than 50 kinds of nutrients beneficial to the body, rich in protein, essential amino acids, various trace elements and vitamins. They also contain many bioactive substances with antioxidant, anti-tumor, anti-fatigue, blood pressure lowering, blood lipid lowering, and immune-regulating effects. Currently, research on sea cucumber bioactive peptides mostly focuses on antioxidant and anti-fatigue aspects, with less research and development on immune-active peptides. Screening and developing novel immune-active peptides from sea cucumbers is of great significance for enriching the variety of immune-active peptides from marine protein sources, preventing diseases, enhancing the body's immunity, and further facilitating the deep processing of marine protein resources. Summary of the Invention

[0004] The purpose of this invention is to provide an immunologically active sea cucumber-derived peptide, its preparation method, and its applications, thereby addressing the problems existing in the prior art. The immunologically active peptide provided by this invention exhibits good immunological activity, is non-toxic, can activate macrophages in the body to induce an immune response and has anti-inflammatory effects, and can remain stable in the intestine, which is of great significance for the intensive processing of marine protein resources.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an immunomodulatory active peptide derived from sea cucumber, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides an immunomodulatory peptide preparation, comprising the aforementioned sea cucumber-derived active peptide.

[0008] The present invention also provides a method for preparing the sea cucumber-derived active peptides, which uses sea cucumber enzymatic hydrolysate as raw material, and separates and purifies it by ultrafiltration and gel chromatography, screens out the component with the strongest immunogenic activity, and then identifies it by secondary mass spectrometry and compares it with a sea cucumber protein database to screen out the sea cucumber-derived active peptides.

[0009] Furthermore, the preparation method of the sea cucumber enzymatic hydrolysate is as follows: sea cucumber is mixed with water, neutral protease is added to the mixture for enzymatic hydrolysis, after enzymatic hydrolysis, the enzyme is inactivated by water bath heating, the supernatant is collected by centrifugation twice to obtain the sea cucumber enzymatic hydrolysate.

[0010] Furthermore, the ratio of sea cucumber to water is 1g:12mL.

[0011] Furthermore, the amount of neutral protease added is 0.6% of the mass of the mixture.

[0012] Furthermore, the enzymatic hydrolysis time is 300 min, the temperature is 50 °C, and the pH is 7.0.

[0013] Furthermore, the water bath heating temperature is 100°C, and the time is 10 minutes.

[0014] The present invention also provides the application of the sea cucumber-derived active peptide or the immune-active peptide preparation in the preparation of products that enhance immunity.

[0015] Furthermore, the product includes pharmaceuticals.

[0016] The present invention discloses the following technical effects:

[0017] This invention utilizes separation and purification techniques and mass spectrometry identification techniques, along with a sea cucumber protein database, to obtain the amino acid sequences of sea cucumber-derived immunoactive peptides. Multiple rounds of screening were conducted by measuring the effects of the active peptides on the survival rate, phagocytic activity, NO release, and cytokine (IL-1β, TNF-α) secretion of RAW264.7 macrophages, as well as the peptides' amino acid composition and biotoxicity. Molecular docking technology was used to explore the interaction between the immunoactive peptides and membrane recognition receptor-2 (TLR-2), and the in vitro immunomodulatory activity of the peptides was verified through solid-phase synthesis and a RAW264.7 cell model. The results show that the novel immunoactive peptides obtained in this invention possess in vitro immunomodulatory activity, are non-toxic, can activate macrophages to induce an immune response and have anti-inflammatory effects without over-activation, and can remain stable in the intestine, maintaining a healthy and stable state in the body. The new active peptide sequences obtained in this invention provide new resources for the development of functional foods. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The diagram shows the interaction between synthetic peptides and TLR-2, where (A)-(C) are molecular docking diagrams of IIENAVQ, FAGDDAPRA, and KSYELP with TLR-2, respectively.

[0020] Figure 2 The effect of synthetic peptides on the survival rate of RAW264.7 macrophages;

[0021] Figure 3 The effect of synthetic peptides on NO release from RAW264.7 macrophages;

[0022] Figure 4 The effects of IIENAVQ activation and LPS induction on the secretion of cytokines IL-1β (A) and TNF-α (B) in RAW264.7 macrophages. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The instruments and chemicals used in the embodiments of this invention are as follows:

[0029] AKTA Purifier 100 protein purification system, EASY-nanoLC 1200 Q Exactive TM Mass spectrometry (ThermoFisher Scientific, MA, USA) combined with electrospray ionization (ESI); RAW264.7 mouse mononuclear macrophages were purchased from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; LPS lipopolysaccharide was purchased from Merck Biotechnology (China) Co., Ltd.; agarose G-10 and neutral red were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; BCA protein concentration assay kit, neutral protease, trypsin, CCK-8, IL-1β and TNF-α detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0030] To obtain high-purity, highly active components, this invention further separates and purifies the sea cucumber enzymatic hydrolysate. Ultrafiltration and gel chromatography are commonly used separation and purification methods. Mass spectrometry identification technology is characterized by its simple operation, high sensitivity, and accurate results. By comparing the mass spectrometry data with a target protein database, the amino acid sequence of the active peptide can be obtained. Molecular docking is an electronic simulation process used to screen and design target, highly efficient bioactive compounds. It is now widely used in high-throughput and virtual enzymatic hydrolysis screening, and for predicting the binding sites, energies, and interaction forces between ligands and receptors. It has the advantages of short processing time and high efficiency. TLR-2 (toll-like receptor 2) is located on the cell surface and is a cytokine with relatively broad ligand specificity. TLR-2 can recognize pathogen-associated molecular patterns to defend against invading organisms and has become an effective therapeutic target.

[0031] Example 1: Isolation and purification of active peptides

[0032] This invention uses sea cucumber enzymatic hydrolysate as raw material, and separates and purifies it by ultrafiltration and gel chromatography. By measuring the effects of each component on the cell survival rate, phagocytic capacity, NO release and cytokine (IL-1β, TNF-α) secretion of macrophages RAW264.7, components with strong immune activity are screened.

[0033] Enzymatic hydrolysis process: The ratio of sea cucumber to water was 1:12 (m / v, g / mL), the amount of neutral protease added was 0.6%, the hydrolysis time was 300 min, the temperature was 50℃, and the pH was 7.0. After the hydrolysis was completed, the enzyme was inactivated by heating in a 100℃ water bath for 10 min, and the supernatant was collected twice by centrifugation at 8000×g and 4℃ to obtain the sea cucumber enzymatic hydrolysate.

[0034] Separation and purification methods:

[0035] (1) Ultrafiltration: Studies have shown that low molecular weight peptides have stronger activity than high molecular weight peptides, and low molecular weight peptides can be completely absorbed in the intestinal environment and retain their original physiological activity. Therefore, ultrafiltration centrifuge tubes with a molecular weight of <3kDa were used to perform ultrafiltration centrifugation on the enzyme hydrolysate to obtain the <3kDa fraction for subsequent activity studies.

[0036] (2) Gel chromatography: A Sephadex G-15 dextran gel resin column was used; the sample concentration was 20 mg / mL; the injection volume was 2 mL; the flow rate was 1 mL / min; and the UV detection wavelength was 214 nm. The collected solutions of the same peak were mixed, concentrated, and freeze-dried. The fraction with the strongest immunogenicity was screened using the following assay indicators.

[0037] Measurement indicators:

[0038] (1) Effect of enzymatic hydrolysate on the survival rate of RAW264.7 macrophages

[0039] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0040] Sample group: RAW264 macrophages were treated with different concentrations (50-800 μg / mL) of enzyme digest for 24 hours.

[0041] The absorbance of different components was determined by the CCK-8 method.

[0042] (2) Effect of enzymatic hydrolysate on the phagocytic capacity of RAW264.7 macrophages

[0043] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0044] Sample group: RAW264 cultured normally was treated with enzyme digests of different concentrations (50-800 μg / mL) for 724 h.

[0045] The absorbance of different components was measured at 550 nm.

[0046] (3) Effects of enzymatic hydrolysate on NO and cytokine (IL-1β, TNF-α) release from RAW264.7 macrophages activated by enzyme hydrolysate

[0047] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0048] Sample group: Normally cultured cells were treated with enzyme digests of different concentrations (50-800 μg / mL) for 24 h.

[0049] Positive control group: Normally cultured cells were treated with 100 ng / mL LPS for 24 h.

[0050] Different components were determined using NO and ELISA kits.

[0051] The results showed that the enzymatic hydrolysate was separated into two fractions, SCH-N-Ⅰ-1 and SCH-N-Ⅰ-2, at 214 nm using a Sephadex G-15 chromatography column. When the SCH-N-Ⅰ-2 fraction was added at 200 μg / mL, the cell viability and phagocytic activity (based on absorbance) were the highest, at 133.95 ± 1.56% and 0.1117 ± 0.0009, respectively. Furthermore, it exhibited high release levels of NO and cytokines (IL-1β, TNF-α) within the specified concentration range. The most potent immunomodulatory fraction, SCH-N-Ⅰ-2, was selected for further studies.

[0052] Example 2: Identification of the amino acid sequence of the active peptide

[0053] The immunocomponent SCH-N-Ⅰ-2 obtained by gel separation and purification was analyzed by secondary mass spectrometry using LC-ESI-MS / MS. By comparing with the sea cucumber protein database, the amino acid sequences of 13 novel immunogenic peptides with high hydrophobicity and immunogenicity were screened out (see Table 1).

[0054] Example 3 Screening of bioactive peptides

[0055] The toxicity of the 13 novel immunomodulatory peptides obtained in Example 2 was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php), and the results are shown in Table 1.

[0056] Table 1. Predicted biotoxicity results of sea cucumber-derived immunomodulatory peptides and Libdock scoring table

[0057]

[0058]

[0059] Example 4: Molecular docking analysis of active peptides with TLR-2

[0060] Using Discovery Studio 2017 R2 software, under the CHARMm force field, 13 immunologically active peptides were molecularly docked with TLR-2 (PDB ID 1FYW). By comparing the Lib Dock Score, the immunologically active peptides IIENAVQ, FAGDDAPRA, and KSYELP with relatively tight docking were screened out. The results are shown in Table 1. The interactions between IIENAVQ (SEQ ID NO.1), FAGDDAPRA, and KSYELP and TLR-2 are shown in Table 1. Figure 1 .

[0061] The main interactions between the active peptide and TLR-2 include van der Waals forces, hydrogen bonds, CH bonds, electrostatic interactions, and alkyl / pialkyl interactions. The amino acid residues that bind to the catalytic site of the active peptide mainly include LEU734, ASP726, LYS751, TRP712, ALA731, ILE755, LYS759, and TYR761.

[0062] Example 5: Verification of the Immunological Activity of Active Peptides

[0063] The three selected immunomodulatory peptides were synthesized in a solid-phase manner, with a purity greater than 98%. The in vitro immunomodulatory activity of the three peptides was then verified.

[0064] (1) Effect of synthetic peptides on the cell survival rate of RAW264.7 macrophages:

[0065] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0066] Sample group: treated with synthetic peptides of different concentrations (50-200 μg / mL) for 24 h.

[0067] The absorbance values ​​of different components were determined using the CCK-8 assay, and the survival rate of RAW264.7 macrophages was calculated based on the absorbance values. The calculation formula is as follows:

[0068]

[0069] The survival rates of RAW264.7 macrophages after treatment with different concentrations of synthetic peptides for 24 hours are as follows: Figure 2 .

[0070] (2) Effect of synthetic peptides on NO release from LPS-induced RAW264.7 macrophages:

[0071] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0072] Induced damage group: After cell culture, cells in the blank group were treated with 100 ng / mL LPS for 24 h, and then treated with synthetic peptides of different concentrations (50-200 μg / mL) for 24 h.

[0073] Negative control group: After cell culture, the blank group cells were treated with 100 ng / mL LPS for 24 h.

[0074] The NO release levels of different components were measured using a NO kit. After treatment with different concentrations of synthetic peptides for 24 hours, the NO release levels of LPS-induced RAW264.7 macrophages were as follows: Figure 3 .

[0075] The results showed that, within the concentration range of 50–200 μg / mL, IIENAVQ had the most significant effect on promoting cell growth, with a cell viability rate of 116.88%. Furthermore, compared with the LPS-negative control group, IIENAVQ significantly reduced the release of RAW264.7NO from LPS-induced macrophages. The peptide with the strongest activity, IIENAVQ, was selected for further research.

[0076] Example 6: Validation of the effect of IIENAVQ on cytokine secretion

[0077] Effects of IIENAVQ activation and LPS-induced secretion of cytokines (IL-1β, TNF-α) in RAW264.7 macrophages

[0078] (1) Effect of IIENAVQ on the secretion of cytokines (IL-1β, TNF-α) in RAW264.7 macrophages

[0079] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0080] Sample group: After cell culture, the blank group cells were treated with different concentrations (50-200 μg / mL) of IIENAVQ for 24 h.

[0081] Positive control group: After cell culture, the blank group cells were treated with 100 ng / mL LPS for 24 h.

[0082] (2) Effects of IIENAVQ on the secretion of cytokines (IL-1β, TNF-α) in LPS-induced RAW264.7 macrophages

[0083] Control group: RAW264.7 macrophages were seeded in 96-well plates and cultured for 24 h.

[0084] Induced damage group: After being cultured, the blank group cells were treated with 100 ng / mL LPS for 24 h, and then treated with different concentrations (50-200 μg / mL) of IIENAVQ for 24 h.

[0085] Negative control group: After cell culture, the blank group cells were treated with 100 ng / mL LPS for 24 h.

[0086] The cytokines of different components were measured using an ELISA kit.

[0087] The final detection results of (1) and (2) are as follows Figure 4 As shown.

[0088] The results show that IIENAVQ maintained a 116.88% survival rate of RAW264.7 cells within a concentration range of 50–200 μg / mL. At a concentration of 200 μg / mL, it significantly reduced NO release, resulting in a final NO concentration of 27.99 ± 0.33 μmol / L. Simultaneously, the secretion of cytokines IL-1β and TNF-α was also significantly reduced to 28.73 ± 1.12 pg / mL and 70.78 ± 5.55 pg / mL, respectively, with inhibition rates of 51.54% and 60.24%. In conclusion, IIENAVQ stimulation can induce an immune response without over-activation, maintaining the body in a healthy and stable state.

[0089] In actual production, immune-active peptides can be produced by solid-phase chemical synthesis and added to pharmaceuticals and health products in powder or granule form.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A sea cucumber-derived active peptide with immunocompetence, characterized in that, The amino acid sequence of the sea cucumber-derived active peptide is shown as SEQ ID NO.

1.

2. An immunologically active peptide preparation, characterized in that, The sea cucumber-derived active peptide of claim 1.

3. Use of the sea cucumber-derived active peptide of claim 1 or the immune active peptide preparation of claim 2 in the preparation of health care products for helping to enhance immunity.

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