Silk protein-derived immunopeptide, preparation method and application thereof

Abstract

The application discloses a silk protein source immunopeptide and a preparation method and application thereof, and the immunopeptide is a single-chain polypeptide, and the amino acid sequence is SEQ ID NO:1.Compared with the prior art, the immunopeptide has the advantages that (1) the raw material source of the immunopeptide is convenient and easy to obtain, the preparation steps are simple, the production cost is low, and the immunopeptide is beneficial to industrialization; (2) the immunopeptide has good stability and no obvious change in immunological activity after in-vitro digestion by pepsin and trypsin; (3) the immunopeptide can effectively promote the proliferation of mouse B lymphocytes and T lymphocytes, and thus is expected to become a main component of functional food and immunoregulation drugs; and (4) from the economic agricultural perspective, the application of the immunopeptide improves the economic benefit and social benefit of the silkworm and mulberry industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and relates to an effective component of a functional food or a biological medicine, in particular to a silkworm silk protein-derived immunopeptide and a preparation method and application thereof. BACKGROUND

[0002] Silkworm silk is a protein substance and has high nutritional and medicinal values. After degumming (silk gum protein), the silkworm silk fiber obtained from the silkworm cocoon is mainly silk fibroin, which is mainly composed of beta keratin and has a stable molecular spatial structure. The silk fibroin is referred to as silkworm silk protein, which has good biocompatibility, no toxic side effects, no irritation, and can promote cell metabolism. Therefore, in addition to fiber clothing, the silkworm silk protein is also favored in the fields of medical treatment and skin care products, and many literatures have been reported in the prior art.

[0003] It has been reported in the prior art that a variety of animal proteins can be separated into bioactive peptides after enzymolysis, and the enzymolysis products have good antioxidant, antitumor, antibacterial, and immunoregulatory effects. Among them, the reports on silkworm pupa protein active peptides include: “A preparation process of silkworm pupa active protein peptide” (CN106834398A), Zheng Shiyuan (Enzymolysis process of silkworm pupa protein for preparing polypeptides, Chemical Engineer, 2009, 23 (05): 5-12) introduced the enzymolysis process of silkworm pupa protein for preparing polypeptides based on the modification of silkworm pupa protein. Yang Anshu et al. (Study on the process of preparing immunologically active peptides by enzymatic hydrolysis of silkworm pupa protein. Food Industry Science and Technology, 2008, (1): 225-227.) prepared small molecule polypeptides by using complex enzymes to hydrolyze silkworm pupa protein, and the small molecule polypeptides in the silkworm pupa protein enzymolysis solution can enhance the phagocytic function of the reticuloendothelial system of mice. Liu Guofei and Baidao Ming invented “A silkworm pupa bioactive small peptide composition for enhancing immunity” (CN105250987A). “A method for improving the functional characteristics of silkworm pupa protein by using ultrasonic wave and enzymolysis technology, products thereof and applications thereof” (ZL201310113821.1), “A method for continuously preparing silkworm pupa protein antioxidant peptides by using an enzyme membrane reactor” (ZL201310137894.4), “A method for continuously preparing silkworm pupa protein ACE inhibitory peptides by using an enzyme membrane reactor, products thereof and applications thereof” (ZL201310113267.7), and the like.

[0004] However, there is no related report on obtaining immunopeptides from silkworm silk protein. SUMMARY

[0005] The technical problem solved by the present application is to overcome the deficiencies of the prior art, obtain an immunopeptide extracted from silkworm silk protein, and use it in functional foods or biological medicines. The present application provides a silkworm silk protein-derived immunopeptide and a preparation method and application thereof.

[0006] Technical solution: Silk protein-derived immune peptide, which is a single-chain polypeptide and has an amino acid sequence of SEQ ID NO: 1: DHAV.

[0007] Preferably, the molecular weight of the immune peptide is 401.09-556.17 Da.

[0008] The preparation method of the silk protein-derived immune peptide described above uses NaOH-urea solution to dissolve silk fibers to obtain silk fibroin, and then uses ultrasonic crushing-alkaline protease combined treatment to obtain an enzymatic hydrolysis product. The enzymatic hydrolysis product is successively separated by Sephadex-G100 and Sephadex-G15 to obtain an effective component with a molecular weight of less than 1000Da. The effective component is purified by HPLC to obtain a short peptide compound with a molecular weight of 401.09-556.17Da, which is the silk protein-derived immune peptide, and filtered through a ceramic membrane to obtain an immune peptide concentrate.

[0009] Preferably, the method comprises the following specific steps:

[0010] S1. Preparation of silk fibroin

[0011] The silk fibers were dissolved in NaOH-urea solution and stirred until completely dissolved to obtain a silk fibroin solution, which was then centrifuged, precipitated, dialyzed, and freeze-dried;

[0012] S2, ultrasonic grinding

[0013] The product obtained in S1 was crushed using an ultrasonic crusher under the following conditions: ultrasonic power 550-560 W, material-liquid ratio 10.5-11.5%, temperature 37-38 ° C, and time 93-95 min;

[0014] S3, alkaline protease enzymatic hydrolysis

[0015] The crushed product of S2 was enzymatically hydrolyzed with alkaline protease under the following conditions: enzyme dosage 5800 U / g, temperature 56°C, pH 8.0;

[0016] S4. Separation and purification

[0017] The products of S3 enzymatic hydrolysis were separated using Sephadex G-100 and Sephadex G-15 columns, respectively, and the best active fractions were screened and collected using the increase in mouse spleen lymphocyte proliferation rate as an evaluation index. The products were then purified by HPLC to obtain silk protein immune peptide products with a molecular weight range of 401.09-556.17 Da.

[0018] S5. Enrichment of immune peptide activity

[0019] The immune peptide product obtained in S4 is filtered through a ceramic membrane, the working pressure is 0.15-0.25 MPa, and the working time is 1.5-2.5 h, so that a silk protein immune peptide concentrate is obtained, and the immune activity is 8-9 times that before concentration;

[0020] S6, immune peptide drying

[0021] The concentrate obtained in S5 is frozen and dried at-80 DEG C to obtain a silk protein source immune peptide dry powder.

[0022] Preferably, the mass-volume ratio of the silk fiber to the NaOH-urea solution in S1 is g:L = 19:1-22:1.

[0023] Preferably, the NaOH concentration of the NaOH-urea solution in S1 is 1.2-1.5 mol / L, and the urea concentration is 8-10 mol / L.

[0024] Preferably, the high performance liquid chromatography-mass spectrometry technology is used to analyze the molecular weight and structure of the silk protein immune peptide.

[0025] The silk protein source immune peptide described above is used in promoting the proliferation of mouse B lymphocytes and T lymphocytes.

[0026] The silk protein source immune peptide described above is used in preparing a functional food for improving immunity.

[0027] The silk protein source immune peptide described above is used in preparing a drug for improving immunity.

[0028] Beneficial effects: (1) The raw material source of the immune peptide described in the application is convenient and easy to obtain, the preparation steps are simple, the production cost is low, and it is conducive to industrialization; (2) the immune activity of the immune peptide does not change obviously after in vitro digestion by pepsin and trypsin, and has good stability; (3) the immune peptide can effectively promote the proliferation of mouse B lymphocytes and T lymphocytes, and thus is expected to become the main component of functional food and immunoregulatory drugs; (4) from the economic agricultural point of view, the application of the immune peptide improves the economic and social benefits of the silkworm and mulberry industry. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Sephadex-G100 gel chromatogram of ultrasonic-protease enzymolysis silk protein;

[0030] Figure 2 Effect of component one and component two after ultrasonic-protease enzymolysis of silk protein on mouse spleen lymphocyte proliferation;

[0031] Figure 3 Sephadex-G15 gel chromatogram of component two of ultrasonic-protease enzymolysis silk protein;

[0032] Figure 4 Figure 1 is a high performance liquid chromatogram of silk protein component 2 subjected to ultrasonic wave-protease enzymolysis;

[0033] Figure 5 Figure 2 is a secondary mass spectrum of silk protein component 2 subjected to ultrasonic wave-protease enzymolysis. DETAILED DESCRIPTION

[0034] The following examples further illustrate the present application, but should not be construed as limiting the application. Modifications and variations of the method, steps or conditions of the application are possible without departing from the spirit and scope of the application. If not specifically mentioned, the technical means used in the examples are conventional means known to those skilled in the art.

[0035] Example 1 Preparation of silk protein-derived immunopeptide

[0036] (1) Preparation of silk protein: 20 g / L of silk fiber was subjected to stirring hydrolysis at a NaOH concentration of 1.2-1.5 mol / L and a urea concentration of 8-10 mol / L at a temperature of 40°C for 3-4 h, and completely dissolved into silk protein. The precipitate was collected by centrifugation, dialyzed, and freeze-dried.

[0037] (2) Ultrasonic pulverization: the protein obtained in step (1) was subjected to pulverization treatment using an ultrasonic wave disrupter under the following conditions: ultrasonic power 550-560 W, solid-liquid ratio 10.5-11.5%, temperature 37-38°C, and time 93-95 min;

[0038] (3) Alkaline protease enzymolysis: the protein obtained in step (2) was subjected to enzymolysis using alkaline protease. Alcalase 2.4L FG alkaline protease was used in the enzymolysis test, and the enzymolysis conditions were as follows: enzyme amount 5800 U / g, temperature 56°C, and pH 8.0;

[0039] (4) Separation and purification: Sephadex G-100 and Sephadex G-15 dextran gel chromatography columns were used for separation, and the proliferation rate of mouse spleen lymphocytes was used as an evaluation index to screen and collect the best active component. The silk protein immunopeptide product having a molecular weight of 401.09-556.17 Da was obtained by purification using a high performance liquid chromatograph (HPLC).

[0040] (5) Immunopeptide activity enrichment: the silk protein immunopeptide product obtained in step (4) was subjected to ceramic membrane filtration under the following conditions: working pressure 0.15-0.25 MPa and working time 1.5-2.5 h, to obtain a concentrated solution of silk protein immunomodulatory peptide, and the immunomodulatory activity of the concentrated solution was 8.6 times that before concentration.

[0041] (6) Molecular structure identification of silk protein immune peptide: The silk protein immune peptide concentrate obtained in step (5) was subjected to relative molecular mass and amino acid sequence analysis. The relative molecular mass analysis was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (4800MALDI-TOF / TOF) on the sample, and the amino acid sequence was determined using a PPsQ-33A fully automatic protein peptide sequencer (SHIMADZU) on the sample treated by Edman degradation to analyze the amino acid sequence of the N-terminus of the polypeptide protein. The short peptide sequence was detected to be: aspartic acid-histidine-alanine-valine, with a molecular weight of 401.09-556.17Da.

[0042] (7) Drying of silk protein immune peptides: freeze-drying the silk protein immune peptide concentrate obtained in step (5) at ultra-low temperature (-80°C) to obtain immune peptide dry powder;

[0043] like Figure 1 As shown, the silk fibroin hydrolyzate after alkaline protease hydrolysis was separated by Sephadex G-100 column to obtain two components: component one and component two.

[0044] like Figure 2 As shown, the effects of different concentrations of component one and component two on lymphocyte proliferation rate showed that 100 μg / mL had the highest cell proliferation rate, and the cell proliferation rate of component two at each dose (25, 50, 100 μg / mL) was significantly higher than that of component one.

[0045] like Figure 3 As shown, component 2 was subjected to Sephadex-G15 gel chromatography to obtain a single isolate, namely, silk immune peptide.

[0046] like Figure 4 As shown in FIG, the silk immune peptide isolate separated by Sephadex-G15 gel chromatography obtained a main peak after high performance liquid chromatography separation, and the peak elution time was 19 minutes. The isolate is the silk immune peptide.

[0047] like Figure 5 As shown, Figure 4 The isolated isolate was subjected to secondary mass spectrometry analysis, which showed that the immune peptide was composed of aspartic acid (D)-histidine (H)-alanine (A)-valine (V) with a molecular weight of 401.09-556.17 Da.

[0048] Example 2 Application of silk protein-derived immune peptides

[0049] (1) In vitro stability experiment of silk protein immune peptide

[0050] According to the Ministry of Agriculture issued "transgenic organisms and their products food safety testing - simulation of gastric juice exogenous protein digestion stability test method" for the relevant operation. Take 2 mL silk protein immune peptide concentrate in test tube, add 4 mL gastric juice 37 ℃ water bath 1 h, and then inactivate the pepsin in the simulated gastric juice (100 ℃, 5 min). When the temperature is reduced to room temperature, adjust the pH value of the above solution system to neutral (7.0-7.5) with 0.2 mol / L NaHCO3 solution, then add equal volume of intestinal juice simulation body temperature treatment (37 ℃, 1 h), and inactivate the trypsin in the simulated intestinal juice (100 ℃, 5 min) to terminate the reaction. In vitro simulation of gastric juice intestinal juice treatment and then mouse spleen cell experiment, determination of mouse spleen lymphocyte proliferation rate.

[0051] After 4h of in vitro digestion with pepsin and trypsin, its immunological activity did not change significantly, showing good stability, and the results are shown in Table 1.

[0052] Table 1 Stability of silk protein immune peptide in simulated human gastrointestinal environment

[0053]

[0054] Note: Each group is repeated three times, and the results are mean ± standard deviation.

[0055] Preparation of gastric juice: 0.2 g NaCl and 80.2 mg pepsin were added to 70 mL distilled water, and the pH was adjusted to 1.2 with HCL solution, and the volume was made to 100 mL with distilled water.

[0056] Preparation of intestinal juice: 0.7 g KH2PO4 was added to 25 mL distilled water, and 19 mL 0.2 mol / L NaOH solution and 40 mL distilled water were added, respectively. Then add 1.0 g trypsin, adjust the pH to 7.5 with 0.2 mol / L NaOH solution, and make the volume to 100 mL with distilled water.

[0057] (2) Mouse spleen lymphocyte proliferation experiment

[0058] Take 8 weeks ICR mice cervical dislocation, soak in 70% ethanol for 5 min, take the spleen aseptically, grind with a syringe plug, pass through a 200 mesh copper screen, collect the cell suspension 400g centrifugation for 15 min, remove the supernatant, add 2 ml red blood cell lysis solution, resuspend at 37 ℃ for 2 min, then 400g centrifugation for 15 min, add 5 mL phosphate buffer for resuspension, 400g centrifugation for 15 min, remove the supernatant, add 5 ml cell culture medium for resuspension, take 100ul cell counting.

[0059] Take the above spleen lymphocyte suspension 0.1 mL plus 3.9 mL phosphate buffer, mix, take 0.9 mL of the suspension plus 0.1 mL of 0.4% trypan blue solution, mix and count under an inverted microscope. Then, again diluted with RPMI-1640 complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin), the spleen cell concentration is adjusted to 5 x 10 6 per mL.

[0060] In 96-well plates, add 5 x 10 6 per mL of spleen cell suspension, 100 μL per well. Then add 100 μL of silk protein sample solution, 3 concentrations (25, 50 and 100 μg / mL) per sample, 3 wells per concentration. The control group of the spleen lymphocyte proliferation test is added with 100 μL of RPMI-1640 culture medium, and cultured at 37°C in a 5% CO2 incubator for 48 h. Then, 20 μL of 5 mg / mL MTT solution is added per well, and cultured for another 4 h. After removal, centrifuge at 1000 r / min for 5 min, aspirate the supernatant from each well, add 100 μL of DMSO to each well, and gently shake to dissolve the purple crystals. After 15 min of reaction in the dark at room temperature, measure the absorbance value at 570 nm using a microplate reader.

[0061] Lymphocyte proliferation rate (%) = (experimental group OD value - control group OD value) / experimental group OD value

[0062] The mouse spleen lymphocyte induction experiment showed that the B lymphocyte proliferation rate under the action of the immune peptide and lipopolysaccharide (LPS, 10 μg / mL) was 86.04-89.58% (LPS control was 15.59%); the T lymphocyte proliferation rate under the action of the immune peptide and concanavalin A (ConA, 5 μg / mL) was 88.89-90.87% (CoA control was 18.99%). The immune peptide can effectively promote the proliferation of mouse B lymphocytes and T lymphocytes, and the results are shown in Table 2. Among them, LPS and ConA are general inducers for inducing the proliferation of B lymphocytes and T lymphocytes, respectively, and are commonly used as positive controls for studying immune response. The results of the present application show that the proliferation rate of B lymphocytes and T lymphocytes after the action of the silk immune peptide and LPS or ConA is significantly higher than that of the positive control, indicating that the silk immune peptide has a significant immune-promoting effect.

[0063] Table 2 Effect of silk protein immune peptide on the proliferation rate of mouse spleen lymphocytes induced by ConA and LPS (%)

[0064]

[0065] Note: Each group is repeated three times, and the results are the average ± standard deviation.

Claims

1. A method for preparing a silk protein-derived immunopeptide, characterized by, The method adopts NaOH-urea solution to dissolve silk fibers to obtain silk fibroin, and then adopts ultrasonic crushing-alkaline protease combined treatment to obtain enzymatic products, and the enzymatic products are sequentially separated by Sephadex-G100 and Sephadex-G15 to obtain effective components with a molecular weight less than 1000 Da, and the effective components are purified by HPLC to obtain short peptide compounds with a molecular weight of 401.09-556.17 Da, that is, silk protein source immunopeptides, and the immunopeptides are filtered by a ceramic membrane to obtain an immunopeptide concentrate. The immunopeptide is a single-chain polypeptide, and the amino acid sequence is SEQ ID NO:

1.

2. The method for preparing the silk protein-derived immune peptide according to claim 1, characterized in that: The method comprises the following specific steps: S1, preparing silk fibroin The silk fibers are dissolved in NaOH-urea solution, stirred until completely dissolved, and then centrifuged to collect the precipitate, dialyzed, and freeze-dried to obtain a silk fibroin solution; S2, ultrasonic crushing The product obtained in S1 is crushed by an ultrasonic crusher, and the crushing conditions are as follows: ultrasonic power 550-560 W, solid-liquid ratio 10.5%-11.5%, temperature 37-38 ℃, and time 93-95 min; S3, alkaline protease enzymolysis The crushed product in S2 is subjected to alkaline protease enzymolysis, and the enzymolysis conditions are as follows: enzyme amount 5800 U / g, temperature 56 ℃, and pH 8.0; S4, separation and purification The product after enzymolysis in S3 is sequentially separated by a dextran gel chromatography column Sephadex G-100 and Sephadex G-15, and the active component with the best activity is collected by taking the mouse spleen lymphocyte proliferation rate as an evaluation index, and then purified by HPLC to obtain a silk protein immunopeptide product with a molecular weight of 401.09-556.17 Da; S5, immunopeptide activity enrichment The immunopeptide product obtained in S4 is filtered by a ceramic membrane, the working pressure is 0.15-0.25 Mpa, and the working time is 1.5-2.5 h, so as to obtain a silk protein immunopeptide concentrate, and the immunological activity is 8-9 times that before concentration; S6, immunopeptide drying The concentrate obtained in S5 is freeze-dried at-80 °C to obtain a silk protein source immunopeptide dry powder.

3. The method for preparing the silk protein-derived immune peptide according to claim 2, characterized in that: In S1, the mass-volume ratio of the silk fibers to the NaOH-urea solution is g:L=19:1-22:

1.

4. The method for preparing silk protein-derived immune peptide according to claim 2, characterized in that: In S1, the NaOH concentration of the NaOH-urea solution is 1.2-1.5 mol / L, and the urea concentration is 8-10 mol / L.

5. The method for preparing silk protein-derived immune peptide according to claim 2, characterized in that: The molecular weight and structure of the silk protein immunopeptide are analyzed by high performance liquid chromatography-mass spectrometry.

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