An active peptide or active peptide composition and its use in the preparation of products having immunomodulatory effects
By preparing active peptide compositions with specific amino acid sequences, the problem of the lack of natural and safe immunomodulators in the prior art has been solved, and significant immunomodulatory effects have been achieved, especially the enhancement of macrophage proliferation rate by the compositions of SEQ ID NO:1 and SEQ ID NO:3.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-17
- Publication Date
- 2026-03-31
AI Technical Summary
There is a lack of natural, safe and effective immunomodulators in the current technology, especially for people with weakened immune systems due to diseases and medical treatments, and the immunomodulatory activity of pigeon meat active peptides is still unclear.
Three specific amino acid sequences of active peptides (LLPPPPPPA, NFYYSHWK, YDWEWH) and their combinations were provided and prepared by solid-phase peptide synthesis. It was found that they have a synergistic effect in enhancing immune activity, especially the combination of SEQ ID NO:1 and SEQ ID NO:3, which has significantly higher immune activity than the individual active peptides.
It achieves natural and safe immunomodulatory effects, provides an active peptide composition with significant immunomodulatory activity, and can be used to prepare drugs with immunomodulatory effects, significantly improving the proliferation rate of macrophages.
Smart Images
Figure CN118812639B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioactive peptide technology, specifically to an active peptide or active peptide composition and its application in the preparation of products with immunomodulatory effects. Background Technology
[0002] Pigeons belong to the class Aves, order Columbiformes, family Columbidae, and genus Columba. They are popularly known as "sweet-blooded animals," believed to aid in the recovery of health for those with anemia, and are often described as "one pigeon is better than nine chickens." Both the bones and meat of pigeons can be used as functional products to regulate the heart, nourish blood, replenish qi, prevent disease, eliminate fatigue, and improve appetite. Pigeon meat protein, due to its rich and comprehensive nutrition, high protein, high energy, and low fat content, is an excellent protein source for functional foods and the development of bioactive peptides. Currently, the number of people with weakened immune systems due to disease and medical treatment is increasing year by year, but most commonly used immunomodulators in clinical practice have certain toxic side effects. Therefore, researching immunomodulators that are naturally derived, safe, effective, and free of toxic side effects is particularly important. In recent years, food-derived immunomodulatory peptides have become a popular area of research in bioactive peptides due to their natural origin, high safety, strong stability, and easy absorption. Pigeon meat protein, with its excellent amino acid composition, can serve as an important protein source for the preparation of immunomodulatory peptides. Current research on pigeon meat focuses mainly on its nutritional components or flavor compounds, while research on pigeon meat protein peptides is still in its early stages, and it remains unclear whether pigeon meat bioactive peptides have immune activity.
[0003] In their preliminary research, the inventors discovered that the crude peptides from enzymatic hydrolysis of pigeon breast meat possess certain immunomodulatory activity; however, these crude peptides are a mixture of numerous bioactive peptides with an unclear composition. Therefore, developing a bioactive peptide with a defined amino acid composition has significant application value. Summary of the Invention
[0004] In order to solve at least one of the above-mentioned technical problems existing in the prior art, the present invention provides an active peptide or active peptide composition and its application in the preparation of products with immunomodulatory effects.
[0005] The above-mentioned technical problem to be solved by the present invention is achieved through the following technical solution:
[0006] The present invention first provides an active peptide having the amino acid sequence shown in SEQ ID NO:1; the amino acid sequence shown in SEQ ID NO:1 is LLPPPPPPA.
[0007] The present invention also provides an active peptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence shown in SEQ ID NO:2 is NFYYSHWK.
[0008] The present invention also provides an active peptide having the amino acid sequence shown in SEQ ID NO:3; the amino acid sequence shown in SEQ ID NO:3 is YDWEWH.
[0009] The inventors discovered in their research that the active peptides with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 have good immune activity.
[0010] The active peptides with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 can be isolated from pigeon breast meat.
[0011] The active peptides with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 can also be prepared by conventional synthetic methods.
[0012] The present invention provides an active peptide composition comprising any two or more combinations of active peptides having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
[0013] The present invention provides an active peptide composition comprising active peptides with amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3.
[0014] The inventors were surprised to discover that combining the active peptides of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 could synergistically enhance immune activity; its immune activity was significantly higher than that of the active peptides of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 alone. However, the composition obtained by combining the other two amino acids of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 did not have significantly higher immune activity than the active peptides of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 alone.
[0015] Preferably, the weight ratio of the active peptides of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 is 1~3:1~3.
[0016] Most preferably, the weight ratio of the active peptides of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 is 1:1.
[0017] The present invention also provides the application of the above-mentioned active peptide or active peptide composition in the preparation of products with immunomodulatory effects.
[0018] The present invention also provides the use of the above-mentioned active peptide or active peptide composition in the preparation of products with immune activity.
[0019] Preferably, the product is a pharmaceutical product.
[0020] Beneficial effects: This invention provides a novel active peptide with a novel amino acid composition. Studies have shown that the active peptide of this invention has good immunomodulatory activity; therefore, it has important application value in further using it as an active ingredient to prepare drugs with immunomodulatory effects. Attached Figure Description
[0021] Figure 1 The image shows the HPLC purity analysis of the active peptide with the amino acid sequence shown in SEQ ID NO:1, prepared by the method described in Example 1.
[0022] Figure 2 The mass spectrum is of the active peptide with the amino acid sequence shown in SEQ ID NO:1 prepared by the method described in Example 1.
[0023] Figure 3 The image shows the HPLC purity analysis of the active peptide with the amino acid sequence shown in SEQ ID NO:2, prepared by the method described in Example 2.
[0024] Figure 4 The mass spectrum is of the active peptide with the amino acid sequence shown in SEQ ID NO:2 prepared by the method described in Example 2.
[0025] Figure 5 The image shows the HPLC purity analysis of the active peptide with the amino acid sequence shown in SEQ ID NO:3, prepared by the method described in Example 3.
[0026] Figure 6 The mass spectrum is of the active peptide with the amino acid sequence shown in SEQ ID NO:3 prepared by the method described in Example 3. Detailed Implementation
[0027] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.
[0028] Example 1 Synthesis of bioactive peptides
[0029] In this embodiment, the active peptide with the amino acid sequence (LLPPPPPPA) shown in SEQ ID NO:1 was synthesized using a conventional solid-phase peptide synthesis method.
[0030] 1. Solid-phase peptide synthesis procedure:
[0031] I. Resin swelling
[0032] Weigh 1 g of 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.84 mmol / g, place the resin in a reaction tube, add DCM (15 ml / g), and shake for 30 min.
[0033] II. Connect the first amino acid.
[0034] The solvent was removed by filtration through a sand filter. 1.5 molar excess of Fmoc-L-His(Trt)-OH amino acid was added, followed by 10 molar excess of DIEA. Finally, a small amount of DMF was added to dissolve the mixture, and it was shaken for 1 h. The mixture was then washed 6 times alternately with DMF and DCM.
[0035] III. Sealing
[0036] Add a certain amount of methanol to the reaction solution. This will block any excess reaction sites and prevent them from affecting subsequent reactions.
[0037] IV. Deprotection
[0038] Add 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 5 min, remove the solution, then add another 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 15 min.
[0039] V. Testing
[0040] Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5 min, and a deep blue color indicates a positive reaction.
[0041] VI. Washing
[0042] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice
[0043] VII. Condensation
[0044] Protective amino acid (Fmoc-L-Trp(Boc)-OH) in three-fold excess and HBTU in three-fold excess, both dissolved in as little DMF as possible, added to the reaction tube, and immediately added in ten-fold excess NMM. React for 30 min.
[0045] 8. Wash
[0046] DMF (10 ml / g) once, methanol (10 ml / g) twice, DMF (10 ml / g) twice.
[0047] 9. Repeat steps four through eight, connecting the amino acids in the sequence from right to left.
[0048] 10. After the last amino acid is attached, deprotect the resin and wash it according to the following method.
[0049] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, DCM (10 ml / g) twice, then evaporated for 10 min.
[0050] XI. Cutting peptides from resin
[0051] Preparation of cutting fluid (10 ml / g): TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%
[0052] Add the resin to a flask or centrifuge tube at a resin-to-cutting fluid ratio of 10 ml / g, and incubate at a constant temperature with shaking for 120 min.
[0053] 12. Dry and wash
[0054] The lysis buffer was dried as much as possible with nitrogen gas, precipitated by ether chromatography, washed six times with ether, and then evaporated to dryness at room temperature. The crude peptide sequence was obtained.
[0055] 13. Purification of polypeptides by HPLC
[0056] Detailed operation steps:
[0057] (1) Take 200 mg of crude peptide and put it into a container. Dissolve it in 2-5 ml of 50% acetonitrile aqueous solution. You can sonicate it slightly for 2 minutes.
[0058] (2) Filter the solution using a 0.45 μm filter membrane.
[0059] (3) Analysis: Take 3 μL and analyze the crude product using analytical grade HPLC. The mobile phase is water and acetonitrile, the time is 30 min, gradient elution, first equilibrate the HPLC with the initial gradient for 5 min and then inject the sample. The initial gradient is 95% water and 5% acetonitrile, and the final ratio is 5% water and 95% acetonitrile. (Note: Innovative Tongheng LC3000 high performance liquid chromatography)
[0060] (4) Preparation: Prepare the dissolved sample for injection. Equilibrate with preparative HPLC for 10 min, starting with a gradient of 95% water and 5% acetonitrile, and ending with a gradient of 25% water and 75% acetonitrile for 40 min. Collect the sample from the detector. (Note: Beijing Qingbohua P1300 HPLC system)
[0061] (5) Identification: The collected samples will be taken for purity and MS analysis. (Note: watersZQ2000)
[0062] 14. Finally, freeze-dry the purified solution to obtain the final product.
[0063] 15. Analyze the purity of the peptides. (Note: Waters 2695 liquid chromatography analyzer)
[0064] (1) Take 1 Mg of the white powdered polypeptide and dissolve it in an appropriate amount of H2O. If the water solubility is poor, an appropriate amount of organic solvent can be used to help dissolve it.
[0065] (2) Select an appropriate acetonitrile gradient analysis based on the sequence length.
[0066] (3) If the analysis is qualified, seal the package and store at -20 degrees.
[0067] 2. Structural identification methods:
[0068] 2.1 Instrument Parameters
[0069] 1) Instrument: Ultimate U3000 nano-Lumos three-in-one liquid chromatography-mass spectrometry system
[0070] 2) Chromatographic columns: all packing material is 1.9 μm; pre-column: 2 cm x 100 μm; analytical column: 15 cm x 100 μm.
[0071] 3) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid, 80% acetonitrile
[0072] 4) Chromatographic gradient: 0-8 min 2% B; 8-9 min 2-10% B; 9-63 min 10-44% B; 63-68 min 44-99% B. Flow rate: 300 nL / min
[0073] 5) Mass spectrometry parameters:
[0074] Spray voltage: 2.2kV
[0075] Capillary temperature: 320℃
[0076] Level 1 scan: Resolution 60,000, scan range 350-1600 m / z
[0077] Secondary scan: resolution 15000, HCD collision energy: 30%
[0078] 2.2 Data Processing
[0079] The raw data in .raw format is converted to .mgf format using MSConvert software, and then processed using pNovo software. The main parameters are:
[0080] 1) Fragmentation method: HCD
[0081] 2) Enzyme digestion: non-specific
[0082] 3) Quality error: 20 ppm for both primary and secondary spectra.
[0083] 4) Modification: Oxidation (Met) is a variable modification.
[0084] Example 2 Synthesis of bioactive peptides
[0085] In this embodiment, the active peptide with the amino acid sequence (NFYYSHWK) shown in SEQ ID NO:2 was synthesized using a conventional solid-phase peptide synthesis method.
[0086] 1. Solid-phase peptide synthesis procedure:
[0087] I. Resin swelling
[0088] Weigh 1 g of 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.84 mmol / g, place the resin in a reaction tube, add DCM (15 ml / g), and shake for 30 min.
[0089] II. Connect the first amino acid.
[0090] The solvent was removed by filtration through a sand filter. 1.5 molar excess of Fmoc-L-His(Trt)-OH amino acid was added, followed by 10 molar excess of DIEA. Finally, a small amount of DMF was added to dissolve the mixture, and it was shaken for 1 h. The mixture was then washed 6 times alternately with DMF and DCM.
[0091] III. Sealing
[0092] Add a certain amount of methanol to the reaction solution. This will block any excess reaction sites and prevent them from affecting subsequent reactions.
[0093] IV. Deprotection
[0094] Add 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 5 min, remove the solution, then add another 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 15 min.
[0095] V. Testing
[0096] Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5 min, and a deep blue color indicates a positive reaction.
[0097] VI. Washing
[0098] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice
[0099] VII. Condensation
[0100] Protective amino acid (Fmoc-L-Trp(Boc)-OH) in three-fold excess and HBTU in three-fold excess, both dissolved in as little DMF as possible, added to the reaction tube, and immediately added in ten-fold excess NMM. React for 30 min.
[0101] 8. Wash
[0102] DMF (10 ml / g) once, methanol (10 ml / g) twice, DMF (10 ml / g) twice.
[0103] 9. Repeat steps four through eight, connecting the amino acids in the sequence from right to left.
[0104] 10. After the last amino acid is attached, deprotect the resin and wash it according to the following method.
[0105] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, DCM (10 ml / g) twice, then evaporated for 10 min.
[0106] XI. Cutting peptides from resin
[0107] Preparation of cutting fluid (10 ml / g): TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%
[0108] Add the resin to a flask or centrifuge tube at a resin-to-cutting fluid ratio of 10 ml / g, and incubate at a constant temperature with shaking for 120 min.
[0109] 12. Dry and wash
[0110] The lysis buffer was dried as much as possible with nitrogen gas, precipitated by ether chromatography, washed six times with ether, and then evaporated to dryness at room temperature. The crude peptide sequence was obtained.
[0111] 13. Purification of polypeptides by HPLC
[0112] Detailed operation steps:
[0113] (1) Take 200 mg of crude peptide and put it into a container. Dissolve it in 2-5 ml of 50% acetonitrile aqueous solution. You can sonicate it slightly for 2 minutes.
[0114] (2) Filter the solution using a 0.45 μm filter membrane.
[0115] (3) Analysis: Take 3 μL and analyze the crude product using analytical grade HPLC. The mobile phase is water and acetonitrile, the time is 30 min, gradient elution, first equilibrate the HPLC with the initial gradient for 5 min and then inject the sample. The initial gradient is 95% water and 5% acetonitrile, and the final ratio is 5% water and 95% acetonitrile. (Note: Innovative Tongheng LC3000 high performance liquid chromatography)
[0116] (4) Preparation: Prepare the dissolved sample for injection. Equilibrate with preparative HPLC for 10 min, starting with a gradient of 95% water and 5% acetonitrile, and ending with a gradient of 25% water and 75% acetonitrile for 40 min. Collect the sample from the detector. (Note: Beijing Qingbohua P1300 HPLC system)
[0117] (5) Identification: The collected samples will be taken for purity and MS analysis. (Note: watersZQ2000)
[0118] 14. Finally, freeze-dry the purified solution to obtain the final product.
[0119] 15. Analyze the purity of the peptides. (Note: Waters 2695 liquid chromatography analyzer)
[0120] (1) Take 1 Mg of the white powdered polypeptide and dissolve it in an appropriate amount of H2O. If the water solubility is poor, an appropriate amount of organic solvent can be used to help dissolve it.
[0121] (2) Select an appropriate acetonitrile gradient analysis based on the sequence length.
[0122] (3) If the analysis is qualified, seal the package and store at -20 degrees.
[0123] 2. Structural identification methods
[0124] 2.1 Instrument Parameters
[0125] 1) Instrument: Ultimate U3000 nano-Lumos three-in-one liquid chromatography-mass spectrometry system
[0126] 2) Chromatographic columns: all packing material is 1.9 μm; pre-column: 2 cm x 100 μm; analytical column: 15 cm x 100 μm.
[0127] 3) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid, 80% acetonitrile
[0128] 4) Chromatographic gradient: 0-8 min 2% B; 8-9 min 2-10% B; 9-63 min 10-44% B; 63-68 min 44-99% B. Flow rate: 300 nL / min
[0129] 5) Mass spectrometry parameters:
[0130] Spray voltage: 2.2kV
[0131] Capillary temperature: 320℃
[0132] Level 1 scan: Resolution 60,000, scan range 350-1600 m / z
[0133] Secondary scan: resolution 15000, HCD collision energy: 30%
[0134] 2.2 Data Processing
[0135] The raw data in .raw format is converted to .mgf format using MSConvert software, and then processed using pNovo software. The main parameters are:
[0136] 1) Fragmentation method: HCD
[0137] 2) Enzyme digestion: non-specific
[0138] 3) Quality error: 20 ppm for both primary and secondary spectra.
[0139] 4) Modification: Oxidation (Met) is a variable modification.
[0140] Example 3 Synthesis of bioactive peptides
[0141] In this embodiment, the active peptide with the amino acid sequence (YDWEWH) shown in SEQ ID NO:3 was synthesized using a conventional solid-phase peptide synthesis method.
[0142] 1. Solid-phase peptide synthesis procedure:
[0143] I. Resin swelling
[0144] Weigh 1 g of 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.84 mmol / g, place the resin in a reaction tube, add DCM (15 ml / g), and shake for 30 min.
[0145] II. Connect the first amino acid.
[0146] The solvent was removed by filtration through a sand filter. 1.5 molar excess of Fmoc-L-His(Trt)-OH amino acid was added, followed by 10 molar excess of DIEA. Finally, a small amount of DMF was added to dissolve the mixture, and it was shaken for 1 h. The mixture was then washed 6 times alternately with DMF and DCM.
[0147] III. Sealing
[0148] Add a certain amount of methanol to the reaction solution. This will block any excess reaction sites and prevent them from affecting subsequent reactions.
[0149] IV. Deprotection
[0150] Add 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 5 min, remove the solution, then add another 15 ml of 20% piperidine DMF solution (15 ml / g), incubate for 15 min.
[0151] V. Testing
[0152] Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃-110℃ for 5 min, and a deep blue color indicates a positive reaction.
[0153] VI. Washing
[0154] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice
[0155] VII. Condensation
[0156] Protective amino acid (Fmoc-L-Trp(Boc)-OH) in three-fold excess and HBTU in three-fold excess, both dissolved in as little DMF as possible, added to the reaction tube, and immediately added in ten-fold excess NMM. React for 30 min.
[0157] 8. Wash
[0158] DMF (10 ml / g) once, methanol (10 ml / g) twice, DMF (10 ml / g) twice.
[0159] 9. Repeat steps four through eight, connecting the amino acids in the sequence from right to left.
[0160] 10. After the last amino acid is attached, deprotect the resin and wash it according to the following method.
[0161] DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, DCM (10 ml / g) twice, then evaporated for 10 min.
[0162] XI. Cutting peptides from resin
[0163] Preparation of cutting fluid (10 ml / g): TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%
[0164] Add the resin to a flask or centrifuge tube at a resin-to-cutting fluid ratio of 10 ml / g, and incubate at a constant temperature with shaking for 120 min.
[0165] 12. Dry and wash
[0166] The lysis buffer was dried as much as possible with nitrogen gas, precipitated by ether chromatography, washed six times with ether, and then evaporated to dryness at room temperature. The crude peptide sequence was obtained.
[0167] 13. Purification of polypeptides by HPLC
[0168] Detailed operation steps:
[0169] (1) Take 200 mg of crude peptide and put it into a container. Dissolve it in 2-5 ml of 50% acetonitrile aqueous solution. You can sonicate it slightly for 2 minutes.
[0170] (2) Filter the solution using a 0.45 μm filter membrane.
[0171] (3) Analysis: Take 3 μL and analyze the crude product using analytical grade HPLC. The mobile phase is water and acetonitrile, the time is 30 min, gradient elution, first equilibrate the HPLC with the initial gradient for 5 min and then inject the sample. The initial gradient is 95% water and 5% acetonitrile, and the final ratio is 5% water and 95% acetonitrile. (Note: Innovative Tongheng LC3000 high performance liquid chromatography)
[0172] (4) Preparation: Prepare the dissolved sample for injection. Equilibrate with preparative HPLC for 10 min, starting with a gradient of 95% water and 5% acetonitrile, and ending with a gradient of 25% water and 75% acetonitrile for 40 min. Collect the sample from the detector. (Note: Beijing Qingbohua P1300 HPLC system)
[0173] (5) Identification: The collected samples will be taken for purity and MS analysis. (Note: watersZQ2000)
[0174] 14. Finally, freeze-dry the purified solution to obtain the final product.
[0175] 15. Analyze the purity of the peptides. (Note: Waters 2695 liquid chromatography analyzer)
[0176] (1) Take 1 Mg of the white powdered polypeptide and dissolve it in an appropriate amount of H2O. If the water solubility is poor, an appropriate amount of organic solvent can be used to help dissolve it.
[0177] (2) Select an appropriate acetonitrile gradient analysis based on the sequence length.
[0178] (3) If the analysis is qualified, seal the package and store at -20 degrees.
[0179] 2. Structural identification methods
[0180] 2.1 Instrument Parameters
[0181] 1) Instrument: Ultimate U3000 nano-Lumos three-in-one liquid chromatography-mass spectrometry system
[0182] 2) Chromatographic columns: all packing material is 1.9 μm; pre-column: 2 cm x 100 μm; analytical column: 15 cm x 100 μm.
[0183] 3) Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid, 80% acetonitrile
[0184] 4) Chromatographic gradient: 0-8 min 2% B; 8-9 min 2-10% B; 9-63 min 10-44% B; 63-68 min 44-99% B. Flow rate: 300 nL / min
[0185] 5) Mass spectrometry parameters:
[0186] Spray voltage: 2.2kV
[0187] Capillary temperature: 320℃
[0188] Level 1 scan: Resolution 60,000, scan range 350-1600 m / z
[0189] Secondary scan: resolution 15000, HCD collision energy: 30%
[0190] 2.2 Data Processing
[0191] The raw data in .raw format is converted to .mgf format using MSConvert software, and then processed using pNovo software. The main parameters are:
[0192] 1) Fragmentation method: HCD
[0193] 2) Enzyme digestion: non-specific
[0194] 3) Quality error: 20 ppm for both primary and secondary spectra.
[0195] 4) Modification: Oxidation (Met) is a variable modification.
[0196] Example 4 Preparation of active peptide composition
[0197] The active peptide composition is obtained by mixing the active peptides with the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 at a weight ratio of 1:1.
[0198] Example 5 Preparation of active peptide composition
[0199] The active peptide composition is obtained by mixing the active peptides with the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 at a weight ratio of 1:1.
[0200] Example 6 Preparation of active peptide composition
[0201] The active peptide composition is obtained by mixing the active peptides with the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3 at a weight ratio of 1:1.
[0202] Experimental Example: Analysis of Pigeon Meat Active Peptides After In Vitro Immunization
[0203] The phagocytic activity of macrophages makes them crucial as the first line of defense in the immune system; therefore, macrophage proliferation rate is used to evaluate the immunomodulatory activity of pigeon meat bioactive peptides. This invention uses the methylene blue method to determine macrophage proliferation rate.
[0204] (1) Preparation and dispensing of culture medium
[0205] Preparation of complete culture medium: Add DMEM culture medium (90 mL / bottle) to a clean bench, and add 10 mL fetal bovine serum and 1 mL double antibiotics to each bottle and mix well. Store at 4°C for later use.
[0206] Elution buffer (EB): Take 245 mL of PBS, add 4 mL of acetic acid, then add 250 mL of anhydrous ethanol, and store at room temperature.
[0207] Methylene blue staining solution: Take 500 mL of HBSS, add 3.0 g of methylene blue, and finally add 12.5 mL of (50%) glutaraldehyde in a fume hood.
[0208] (2) Cell culture and passage
[0209] RAW264.7 cells were cultured in DMEM medium and placed in a 37°C, 5% CO2 cell culture incubator. Cells were ready for experiments when the cell density reached 80%–90%.
[0210] (3) Assay of cell proliferation activity
[0211] RAW264.7 cells were collected, and when the confluence of RAW264.7 cells reached 80%, they were diluted with culture medium to a cell density of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated at 37°C for 24 h in a 5% CO2 incubator. After 24 h, the supernatant was discarded, and the cells were washed once with PBS. Then, 500 μg / mL of the test sample solution (100 μL / well, diluted with complete culture medium) was added. The blank control group was added with complete culture medium, with 3 replicates per group. PBS was added to the remaining blank wells to prevent evaporation of the cell supernatant. After incubation for another 24 h, the culture medium was aspirated, and the cells were washed once with PBS. Then, methylene blue staining solution (50 μL / well) was added, and the plate was incubated at 37°C for 1 h. After incubation, the 96-well plate was gently soaked six times with ultrapure water (or until the water remained clear), the plate was inverted on a piece of tissue paper and patted dry, and finally 100 μL of EB was added to each well. The plate was placed on a shaker and shaken for 15 min. The absorbance of each well was measured at 595 nm. Cell proliferation rate is calculated based on OD value.
[0212] The calculation formula is as follows:
[0213] Relative proliferation rate (%) =
[0214] The test samples were active peptides with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, as well as the compositions prepared in Examples 4 to 6; the test results are shown in Table 1.
[0215] Table 1. Test results of the relative proliferation rate of macrophages by the pigeon meat bioactive peptides of the present invention.
[0216]
[0217] As can be seen from the experimental results in Table 1, the active peptides with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 all have good relative macrophage proliferation rates; this indicates that the active peptides described in this invention all have good immune activity.
[0218] As can be seen from the experimental results in Table 1, the composition prepared in Example 4 exhibits a significantly higher relative macrophage proliferation rate than the active peptides with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. This indicates that combining the active peptides with amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 can synergistically enhance immune activity, and its immune activity is significantly higher than that of the individual active peptides with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0219] As can be seen from the experimental results in Table 1, the relative macrophage proliferation rate of the compositions prepared in Examples 5 and 6 was not significantly increased compared with the active peptides of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. This indicates that the composition obtained by combining the active peptides of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 has significantly higher immunomodulatory activity than the active peptides of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 alone. However, the composition obtained by combining the other two amino acids of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 does not have significantly higher immunomodulatory activity than the active peptides of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 alone.
Claims
1. An active peptide composition having an immunomodulatory effect, characterized by, an active peptide of the amino acid sequence shown as SEQ ID NO: 1 and the amino acid sequence shown as SEQ ID NO: 3 is composed of 1:1 by weight; the amino acid sequence shown as SEQ ID NO: 1 is LLPPPPPP A; the amino acid sequence shown as SEQ ID NO: 3 is YDWEWH.