Immunomodulatory nonapeptide and its application

Abstract

The present invention belongs to the technical field of bioactive peptides, and discloses immunomodulatory nonapeptides and applications. The immunomodulatory peptide provided by the present invention is extracted from white mullet, and the amino acid sequence is shown in SEQ ID NO.1. It is safe and has no toxic side effects, can significantly enhance the phagocytic ability of RAW264.7 cells, and has a strong immunomodulatory function.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides and relates to immunomodulatory nonapeptides and applications. Background Art

[0002] The immune system is an important system for the body to perform immune responses and immune functions. It is composed of immune organs, immune cells and immune molecules. The immune system has the function of identifying and eliminating antigenic foreign bodies, coordinating with other systems of the body, and jointly maintaining the stability of the body's internal environment and physiological balance. At present, there are many drugs on the market that can be used to regulate immunity, but most of them have side effects.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide an immunomodulatory nonapeptide and its application. The immunomodulatory nonapeptide provided by the present invention has the function of regulating the immune system and improving immunity.

[0005] To achieve the above object, the first technical solution adopted by the present invention is:

[0006] The immunomodulatory nonapeptide has an amino acid sequence of KSTHPHFVR, as shown in SEQ ID NO.1.

[0007] Preferably, the immunomodulatory nonapeptide is extracted from white mullet.

[0008] Preferably, the extraction method is: homogenizing the white mullet, and enzymolyzing it with flavor protease and neutral protease to obtain an enzymolysis solution; using ultrafiltration to separate the enzymolysis solution to retain ultrafiltration components with a molecular weight cutoff of less than 3000Da; using gel chromatography to chromatograph the ultrafiltration components; and separating and purifying the components obtained after chromatography.

[0009] The second technical solution adopted in the present invention is:

[0010] Application of immunomodulatory nonapeptides in the preparation of immunity-enhancing products.

[0011] The third technical solution adopted in the present invention is:

[0012] An immunomodulator, comprising the immunomodulatory nonapeptide of the first technical solution.

[0013] The above-mentioned immunomodulatory nonapeptide can also be synthesized using amino acids as raw materials by chemical methods, which is well known in the art.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The immunomodulatory nonapeptide provided by the present invention can significantly increase cell proliferation activity and enhance the phagocytic ability of cells, and has good application prospects in the field of immunity enhancement drugs and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the chromatogram of the ultrafiltration component with a molecular weight below 3000Da after being separated by Sephadex G-15 gel chromatography;

[0017] Figure 2-4 Results of cell proliferation activity, cell phagocytosis rate and cell NO content of different polypeptide components in chromatography groups F1, F2 and F3;

[0018] Figure 5 is the HPLC separation spectrum of the chromatographic component F3;

[0019] Figure 6 is the secondary mass spectrum of the sequence KSTHPHFVR;

[0020] Figure 7 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION PROFILE FOR PURITY CONFIRMATION OF THE SEQUENCE KSTHPHFVR;

[0021] Figure 8 The results of cell proliferation activity of the synthetic sequence KSTHPHFVR;

[0022] Fig. 9 The results of cell phagocytosis rate of the synthetic sequence KSTHPHFVR;

[0023] Fig.10 The results of cellular NO content of the sequence KSTHPHFVR are shown. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. In the embodiment, those who do not indicate specific conditions are carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not indicate manufacturers for reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0025] The first embodiment of the present invention provides an immunomodulatory nonapeptide, the amino acid sequence of which is KSTHPHFVR, as shown in SEQ ID NO.1.

[0026] In the embodiment of the present invention, the immunomodulatory nonapeptide is extracted from white mullet. In fact, amino acids can also be used as raw materials to synthesize a peptide with an amino acid sequence of SEQ ID NO.1 by chemical methods, which is well known in the art.

[0027] In a specific embodiment, the specific method of extracting the immunomodulatory nine peptides is as follows: remove the byproducts of the white mullet, add ultrapure water 2-6 times the weight of the fish meat for homogenization to obtain a homogenous material; heat in a water bath to 50°C, adjust the pH to 7.0, add 4% enzyme according to the weight of the homogenous material, wherein the enzyme is a flavor protease and a neutral protease compounded at a mass ratio of 3:2, enzymolysis for 4 hours, and cool to room temperature to obtain an enzymolysis solution; centrifuge the above enzymolysis solution to obtain the supernatant and vacuum freeze-dry to obtain a white mullet crude peptide powder; ultrafiltration separation of the above crude peptide powder, ultrafiltration components with a molecular weight cutoff of less than 3000Da, freeze-dried and stored at -18°C; the above ultrafiltration components are prepared into a solution with a concentration of 10-50 mg / mL, and chromatographed by Sephadex G-15 dextran gel chromatography, and the obtained components are freeze-dried and stored at -18°C. The components with immunomodulatory activity after chromatography are selected by measuring cell proliferation activity, cell phagocytosis rate, and cell NO content activity, and the selected components are separated and purified by RP-HPLC.

[0028] In a further embodiment, the centrifugation conditions are 4°C, 8000xg, and time 20 min.

[0029] In a further embodiment, the Sephadex G-15 dextran gel chromatography method has a sample loading amount of (2-4) mL, an elution rate of (1-2) mL / min, and a detection wavelength of 220 nm.

[0030] Furthermore, the sample loading volume is 4 mL.

[0031] In the following specific implementation of the present invention, the ultrafiltration fractions are chromatographed by Sephadex G-15 dextran gel chromatography, and the fractions having immunomodulatory effects after chromatography are selected and further separated by RP-HPLC.

[0032] Among them, the immunomodulation assay uses the determination of cell viability and the effect of polypeptide components on cell proliferation activity, cell phagocytosis rate, and cell NO content. The specific determination method is as follows:

[0033] (1) Cell culture

[0034] RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2 for 24-48 hours. When the cell growth density in the culture bottle reached about 80-90%, the cells were dispersed with an elbow pipette to make a suspension, and fresh culture medium was added at a volume ratio of 1:3-1:5, and then the cells were returned to the incubator for further culture.

[0035] (2) Cell proliferation activity assay

[0036] Take cells in the logarithmic growth phase and place them in a 96-well plate, 180 μL per well, and adjust the RAW264.7 cell density to 1×10 4 / mL, after complete attachment, discard the upper culture medium, add different concentrations (0, 1, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μg / mL) of polypeptide solution prepared with DMEM complete culture medium, culture in an incubator at 37°C and 5% CO2 for 24 hours, add CCK-8 dye solution and culture in dark for 2 hours, and use a microplate reader to set the wavelength of 450nm to measure the absorbance. The three concentrations with good effects on the proliferation of RAW264.7 cells and significant differences between groups were set as low, medium and high drug dose groups for subsequent in vitro experiments.

[0037] Take cells in the logarithmic growth phase and adjust the RAW264.7 cell density to 3×10 4 / mL, inoculated in a 96-well plate for culture, and after complete attachment, the upper culture medium was discarded, the blank control group Control was added with DMEM complete culture medium, the positive control group was added with culture medium containing 1μg / mL lipopolysaccharide (LPS), and the drug-treated groups were added with culture medium containing 50, 100 and 200μg / mL polypeptides. After 24h of cell culture, the culture medium was discarded and washed twice with PBS. 220μL of diluted culture medium was added to each well (culture medium: neutral red dye solution = 1:10). After incubation for 2h, the culture medium was discarded, washed twice with PBS, and neutral red detection lysis solution (200μL) was added to each well, and shaken for 10min. Finally, the absorbance was detected at 540nm and the phagocytic rate (%) was calculated.

[0038] (3) Determination of cellular NO secretion

[0039] After 24 hours of drug administration and culture, the cell culture fluid of each group was collected, and 50 μL of the cell supernatant of each group after centrifugation was added to each well of a 96-well plate, and 50 μL of Griess Reagent I and Griess Reagent II were added in sequence, and the absorbance was measured at 490 nm. The NO content was calculated according to the standard curve.

[0040] Example 1

[0041] The specific steps of the method for extracting, separating and purifying the immunomodulatory nonapeptide are as follows:

[0042] Remove byproducts from the white mullet, add ultrapure water 2-6 times the weight of the fish meat, and homogenize to obtain a homogenous material;

[0043] The mixture was heated in a water bath to 50°C, the pH was adjusted to 7.0, 4% enzyme was added according to the mass of the homogenized material, wherein the enzyme was a composite of flavor protease and neutral protease in a mass ratio of 3:2, the mixture was enzymolyzed for 4 hours, and the mixture was cooled to room temperature to obtain an enzymolysis solution; the enzymolysis solution was centrifuged and the supernatant was vacuum freeze-dried to obtain a crude peptide powder of white mullet; the crude peptide powder was separated by ultrafiltration, and the ultrafiltration component with a molecular weight of less than 3000Da was retained, and the mixture was freeze-dried and stored at -18°C.

[0044] The above ultrafiltration components were prepared into a solution with a concentration of 25 mg / mL and filtered through a 0.22 um aqueous microporous filter membrane. Sephadex G-15 dextran gel chromatography was used for chromatography. Double distilled water was used as the eluent. The chromatography method was as follows: the elution flow rate was 1 mL / min, the detection wavelength was 220 nm, the sample volume was 4 mL, and the chromatographic spectrum results were as follows: Figure 1 As shown, the horizontal axis represents the elution time (min) and the vertical axis represents the absorbance A.

[0045] The three separated components obtained in the chromatography collection and elution process are labeled F1, F2, and F3 in sequence, and freeze-dried and stored at -18°C. The obtained components are freeze-dried and stored at -18°C.

[0046] The F1, F2, and F3 groups obtained after chromatography were selected and the effects of peptide sequences at different concentrations (50, 100, and 200 μg / mL) on the proliferation rate of RAW264.7 cells were determined using CCK-8 reagent. The proliferation activity of cells in different groups and their phagocytic rate of neutrophils were also determined. Figure 2-3 As shown, Figure 2 The horizontal axis represents the concentration of each component. Figure 2-3 It can be seen that components F1, F2, and F3 all have good immunomodulatory activity.

[0047] The effects of the three peptide components at different concentrations (50, 100, 200 μg / mL) on the relative cell proliferation rate of RAW264.7 were determined, and then the effects of different peptide sequences on the phagocytic rate and NO synthesis of RAW264.7 were determined at a concentration of 100 μg / mL. Figure 4 It can be seen that the NO content of the F3 component is the lowest, indicating that this component has a good immunomodulatory effect, so F3 was selected for subsequent experiments. Figure 5 The mass spectrum of F3 is shown in Figure 2. The F3 component was selected for the next step of RP-HPLC separation and purification.

[0048] The F3 component was separated and purified by RP-HPLC. The separation conditions of RP-HPLC were as follows: mobile phase A was water containing 0.1% TFA; mobile phase B was acetonitrile containing 0.1% TFA; gradient elution, i.e. 0 min 2% B, 0-18 min 2%-10% B, 18-36 min 10%-20 B, 36-55 min 20%-30 B, 55-70 min 30%-40% B, the analytical column was C18 (3 μm, 100A), the injection volume was 10 μL; the flow rate was 1 mL / min.

[0049] The RP-HPLC separation spectrum of the chromatographic fraction F3 is as follows: Figure 5 In the figure, the horizontal axis represents the elution time (min), and the vertical axis represents the response value (AU).

[0050] The purified components, namely Figure 5 The component F4 at 45-55min was identified. The liquid phase conditions were: mobile phase A was water containing 0.1% formic acid; mobile phase B was water containing 0.1% formic acid and 80% CAN; gradient elution, i.e. 0min 4% B, 0-2min 4%-8% B, 2-45min 8%-28% B, 45-55min 28%-40% B, 55-56min 40%-95% B, 56-66min 95% B, analytical column was packed with Acclaim PepMap RPLC C18 (3μm, 150μm i.d.×150mm); injection volume was 10μL; flow rate was 600nL / min. Mass spectrometry conditions: primary mass spectrometry parameters were Resolution 70000, AGCtarget 3e6, MaximumIT 100 ms, Scanrange 100 to 1500 m / z; secondary mass spectrometry parameters were Resolution 17500, AGCtarget 1e5 MaximumIT 50 ms, TopN20, nce / steppedNCE28.

[0051] The amino acid sequence of the identified immunomodulatory nonapeptide is: Lys-Ser-Thr-His-Pro-His-Phe-Val-Arg (KSTHPHFVR).

[0052] Table 1 shows the information of the immunomodulatory nonapeptide.

[0053] Table 1 Information on immunomodulatory nonapeptides

[0054] .

[0055] Example 2

[0056] To further verify the immunomodulatory activity of the above immunomodulatory nonapeptide, the sequence was synthesized using solid phase synthesis to obtain a synthetic compound of sequence SEQ ID NO.1. Figure 6 The secondary mass spectrum of the synthetic compound of sequence SEQ ID NO.1 is shown in Figure 1. The purity of the synthetic compound of sequence SEQ ID NO.1 is ≥98%. Figure 7 KSTHPHFVR is a synthetic sample of SEQ ID NO.1.

[0057] (1) Cell culture

[0058] RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2 for 24-48 hours. When the cell growth density in the culture bottle reached about 80-90%, the cells were dispersed with an elbow pipette to make a suspension, and fresh culture medium was added at a volume ratio of 1:3-1:5, and then the cells were returned to the incubator for further culture.

[0059] (2) Cell activity assay

[0060] Take cells in the logarithmic growth phase and place them in a 96-well plate, 180 μL per well, and adjust the RAW264.7 cell density to 1×10 4 / mL, after complete attachment, discard the upper culture medium, add different concentrations (0, 1, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400μg / mL) of KSTHPHFVR solution prepared with DMEM complete culture medium, culture in an incubator at 37°C and 5% CO2 for 24h, add CCK-8 dye solution and culture for 2h in dark, and use a microplate reader to set the wavelength of 450nm to measure the absorbance. The three concentrations with good effects on the proliferation of RAW264.7 cells and significant differences between groups were set as low, medium and high drug dose groups for subsequent in vitro experiments.

[0061] Effect of KSTHPHFVR on the proliferation activity of RAW264.7 cells Figure 8As shown. When the concentration of KSTHPHFVR increased from 1μg / mL to 200μg / mL, the viability of RAW264.7 cells did not decrease, but instead effectively promoted the proliferation of RAW264.7 cells in a dose-dependent manner. When the concentration of FRF was 1μg / mL, there was no significant effect on the cell proliferation rate (P>0.05). When the concentration of FRF reached 200μg / mL, the relative proliferation rate was the highest. According to the results of the CCK-8 experiment, FRF had no obvious cytotoxicity within the experimental concentration range and had a certain proliferation effect. Subsequent experiments will select three KSTHPHFVR concentrations (50, 100 and 200μg / mL) with significant differences between the groups (P<0.05) for research. Take cells in the logarithmic growth phase and adjust the RAW264.7 cell density to 3×10 4 / mL, inoculated in a 96-well plate for culture, after complete attachment, discard the upper culture medium, add DMEM complete culture medium to the blank control group, add culture medium containing 1μg / mL LPS to the positive control group, and add culture medium containing 50, 100 and 200μg / mL KSTHPHFVR to the drug-treated group. After 24h of cell culture, discard the culture medium and wash twice with PBS. Add 220μL of diluted culture medium to each well (culture medium: neutral red dye solution = 1:10). After incubation for 2h, discard the culture medium, wash twice with PBS, and add neutral red detection lysis solution (200μL) to each well, and shake for 10min. Finally, detect the absorbance at 540nm and calculate the phagocytic rate (%).

[0062] Effects of KSTHPHFVR on the phagocytic ability of RAW264.7 cells Fig. 9 As shown. The results showed that compared with the normal group cells, the phagocytic rate of neutral red in the LPS group cells was significantly enhanced (P<0.01). After FRF treatment, the phagocytic rate of neutral red dye by cells increased in a dose-dependent manner. And when the concentration of KSTHPHFVR was 200μg / mL, the phagocytic ability of the cells was the highest. The results showed that KSTHPHFVR can significantly enhance the phagocytic ability of RAW 264.7 cells.

[0063] (3) Determination of cellular NO secretion

[0064] After 24 hours of drug administration, the cell culture fluid of each group was collected, and 50 μL of the supernatant of each group of cells after centrifugation was added to each well of a 96-well plate. 50 μL of Griess Reagent I and Griess Reagent II were added in sequence, and the absorbance was measured at 490 nm. The NO content was calculated according to the standard curve. Fig.10As shown. The ability of cells in the normal group to release NO was the lowest, but after KSTHPHFVR and LPS stimulation of cells, the NO content increased significantly compared with the normal group (P<0.01). The results showed that KSTHPHFVR can enhance the NO content in RAW264.7 cells.

[0065] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content of the present invention by a person skilled in the art should be considered as the protection scope of the claims of the present invention.

Claims

1. An immunomodulatory nonapeptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The immunomodulatory nonapeptide according to claim 1, characterized in that The immunomodulatory nonapeptide is extracted from white mullet.

3. The immunomodulatory nonapeptide according to claim 2, characterized in that The extraction method comprises the following steps: homogenizing the white mullet, performing enzymatic hydrolysis with flavor protease and neutral protease to obtain an enzymatic hydrolyzate; using ultrafiltration to separate the enzymatic hydrolyzate to obtain an ultrafiltration component with a molecular weight cutoff less than 3000Da; performing chromatography on the ultrafiltration component by gel chromatography; and separating and purifying the components obtained after chromatography.

4. Use of the immunomodulatory nonapeptide according to any one of claims 1 to 3 in the preparation of a product for improving immunity.

5. An immunomodulator, characterized in that Contains the immunomodulatory nonapeptide according to any one of claims 1-3.

Images