Multifunctional polypeptide and application thereof

Abstract

The invention discloses a multifunctional polypeptide and application thereof, the polypeptide is a polypeptide I or a polypeptide II, and the amino acid sequence of the polypeptide I is shown as VLPF; the amino acid sequence of the polypeptide II is shown as HYF. According to the invention, two high-activity polypeptides are screened from the milk casein of the meadow dzo, have anti-inflammatory and antioxidant activity, and also can promote cell proliferation at the same time.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to a multifunctional polypeptide and application thereof. Background Art

[0002] Chronic inflammation and oxidative stress are the core drivers of many pathological processes, including neurodegenerative diseases (such as Alzheimer's disease), metabolic syndrome, cardiovascular disease and skin aging. Although traditional anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs and glucocorticoids) can effectively inhibit inflammatory responses, long-term use can easily cause gastrointestinal damage, immunosuppression and other side effects; while commonly used antioxidants in clinical practice (such as vitamin C and N-acetylcysteine) have problems such as low bioavailability and single target, making it difficult to achieve precise regulation.

[0003] In recent years, bioactive peptides have become a research hotspot due to their high selectivity, low toxicity and easy modification. Studies have shown that some natural peptides (such as lactoferrin peptides and marine collagen peptides) can function by inhibiting the NF-κB / NLRP3 inflammasome pathway or activating the Nrf2 / ARE antioxidant pathway, but their intensity and stability are limited by the inherent defects of the natural sequence. Although existing synthetic peptide strategies (such as amino acid substitutions and cyclization modifications) can partially improve activity, they mostly focus on a single function (anti-inflammatory or antioxidant), ignoring the synergistic amplification effect of inflammation and oxidative stress, resulting in limited practical application effects. Therefore, the development of peptide molecules that have both efficient anti-inflammatory and antioxidant synergistic effects, stable structures and can be prepared on a large scale is still a technical problem that needs to be overcome in this field. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a multifunctional polypeptide and its application. The present invention screens and obtains two highly active polypeptides, which have anti-inflammatory and antioxidant activities and can also promote cell proliferation.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows: a multifunctional polypeptide, the polypeptide is polypeptide I or polypeptide II, the amino acid sequence of polypeptide I is shown in VLPF; the amino acid sequence of polypeptide II is shown in HYF.

[0006] As a further description of the above scheme: the polypeptide screening process is as follows:

[0007] The Zhongdian yak casein was subjected to simulated gastrointestinal digestion, and a crude extract rich in active peptides was obtained after simulated gastrointestinal digestion. The crude extract of Zhongdian yak casein was then separated by ultrafiltration using an ultrafiltration membrane, and the polypeptide filtrate <3kDa was collected and vacuum-freeze-dried into powder for later use. The peptides were identified by LC-MS / MS, and then scored using the Peptide Ranker database. It was believed that the peptides with a score of >0.8 had potential activity.

[0008] Screen target peptides from multiple peptides with a score greater than 0.8, predict the anti-inflammatory and antioxidant activities of antimicrobial peptides using the BIOPEP-UWM database, and identify multifunctional active peptides through in vitro antioxidant and anti-inflammatory activity assays.

[0009] The present invention also provides the application of the multifunctional polypeptide in the preparation of antioxidant and / or anti-inflammatory products.

[0010] The present invention also provides the application of the multifunctional polypeptide in promoting cell proliferation.

[0011] The present invention also provides a cell culture medium, wherein the polypeptide is added to the medium to promote the proliferation of microorganisms or cells.

[0012] Compared with the prior art, the present invention has the following beneficial effects: Two highly active polypeptides are screened from Zhongdian cattle-cattle hybrid milk casein, which have anti-inflammatory and antioxidant activities and can also promote cell proliferation. Description of the Drawings

[0013] Figure 1 Peptide identification with a molecular weight <3 kDa in Zhongdian cattle-cattle hybrid milk casein; A is the ion chromatogram identified by LC-MS / MS; B is the molecular weight distribution of the polypeptide; C is the number of peptide segments with a score >0.8;

[0014] Figure 2 Effect of 7 polypeptides on the viability of RAW264.7 cells;

[0015] Figure 3 Effect of 7 polypeptides on the viability of Caco2 cells;

[0016] Figure 4 Effect of 7 polypeptides on the viability of HepG2 cells;

[0017] Figure 5 DPPH scavenging activity of 7 polypeptides;

[0018] Figure 6 ABTS scavenging activity of 7 polypeptides;

[0019] Figure 7 Effect of 7 polypeptides on the secretion of NO by RAW264.7 cells;

[0020] Figure 8 Effect of 2 synthetic peptides on the mRNA expression of the inflammation-related gene (iNOS);

[0021] Figure 9 Effect of 2 synthetic peptides on cell proliferation. Detailed Embodiments

[0022] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0023] 1. Preparation of Bifunctional Active Peptides from Milk Source

[0024] The milk of Zhongdian cattle in Shangri-La City, Diqing Prefecture, Yunnan Province was collected. The pH was adjusted to 4.6 - 4.8 by adding acid to precipitate casein and obtain Zhongdian cattle milk casein. The Zhongdian cattle milk casein was subjected to simulated gastrointestinal digestion. After simulated gastrointestinal digestion, a crude extract rich in active peptides was obtained. Then, the crude extract of Zhongdian cattle milk casein was ultrafiltered and separated using a 3 kDa ultrafiltration membrane. The filtrate (<3 kDa) was collected and freeze-dried under vacuum for later use. The peptide segments were identified by LC-MS / MS ( Figure 1 A).

[0025] As Figure 1 shown in Figure 1 B, a total of 386 peptide segments were identified. Among them, the peptides with a molecular weight <0.5 kDa had the highest proportion, accounting for 35.23% of the total peptides. Followed by the peptide segments with a molecular weight in the range of 1.0 kDa - 1.5 kDa, accounting for 21.50% of the total peptides. This indicates that after simulated gastrointestinal digestion, the casein of cattle milk was enzymatically hydrolyzed into more small molecule peptides. As

[0026] shown in

[0027] C, the peptide segments were scored through the Peptide Ranker database. Generally, it is considered that the peptide segments with a score value >0.8 have potential activity. A total of 204 peptide segments had a score value >0.8, among which 136 were <0.5 kDa and 60 were in the range of 0.5 kDa - 1 kDa. This shows that small molecule polypeptides may have stronger activity, and there are a large number of novel active short peptides in the casein of cattle milk.

[0026] 2. Screening of Bifunctional Active Peptides from Milk Source Based on Database

[0027] Target anti-inflammatory peptides were screened from 204 peptide segments with a score greater than 0.8. The anti-inflammatory activity of antibacterial peptides was predicted using the BIOPEP-UWM database. A total of 7 peptide segments were predicted to have anti-inflammatory and antioxidant activities (as shown in Table 1). Then, the toxicity of the peptide segments was predicted through the online database "ToxinPred", and it was found that all 7 potential active peptides were non-toxic. Immediately, the 7 peptides were chemically synthesized, and the bifunctional active peptides were identified through in vitro antioxidant and anti-inflammatory activity assays.

[0028] Table 1 Basic Information of 7 Potential Active Peptides

[0029] Serial number Sequence Molecular weight (Da) Scoring value Toxicity 1 FLGY 498.24783 0.91 Non-Toxin 2 LGLF 448.26857 0.92 Non-Toxin 3 LGYF 498.24783 0.93 Non-Toxin 4 VLPF 474.28422 0.84 Non-Toxin 5 LPFP 472.26857 0.96 Non-Toxin 6 YGF 385.16377 0.97 Non-Toxin 7 HYF 465.20122 0.88 Non-Toxin

[0030] 3. Effects of Polypeptides on the Proliferation Activity of Different Cells

[0031] 3.1 Proliferation Activity of Polypeptides on RAW264.7 Cells

[0032] After treating RAW264.7 cells with synthetic peptides at different concentrations (400 - 25 μg / mL) for 24 h using the MTT method, the effect of the synthetic peptides on the survival rate was observed. The results are shown as Figure 2 follows. Synthetic peptide FLGY could significantly inhibit cell growth within the concentration range of 400 μg / mL, with a cell proliferation rate lower than 85%. The cell proliferation rates of the other six polypeptides were all greater than 85%, indicating that they had no cytotoxicity.

[0033] 3.2 Proliferative activity of polypeptides on Caco2 cells

[0034] After treating Caco2 cells with synthetic peptides at different concentrations (400 - 25 μg / mL) for 24 h using the MTT method, the effect of the synthetic peptides on the survival rate was observed. The results are shown as Figure 3 follows. The cell proliferation rates of the seven polypeptides were all greater than 85%, indicating that they had no cytotoxicity.

[0035] 3.3 Proliferative activity of polypeptides on HepG2 cells

[0036] After treating Caco2 cells with synthetic peptides at different concentrations (400 - 25 μg / mL) for 24 h using the MTT method, the effect of the synthetic peptides on the survival rate was observed. The results are shown as Figure 4 follows. The cell proliferation rates of the seven polypeptides were all greater than 85%, indicating that they had no cytotoxicity and could promote the proliferation of Caco2 cells.

[0037] 4. Screening of milk-derived bifunctional bioactive peptides based on in vitro experiments

[0038] 4.1 Determination of antioxidant activity

[0039] As Figure 5 shown, when the concentrations of the seven polypeptides were in the range of 0.125 - 2 mg / mL, the scavenging rates of DPPH free radicals were all above 20%, indicating that the seven polypeptides could all scavenge DPPH free radicals. Among them, when the concentration of polypeptide HYF was 2 mg / mL, the scavenging rate of DPPH free radicals reached 80%.

[0040] As Figure 6 shown, when the concentrations of the seven polypeptides were in the range of 0.125 - 2 mg / mL, the scavenging rates of ABTS free radicals were all above 20%, indicating that the seven polypeptides could all scavenge ABTS free radicals. Among them, when the concentration was 2 mg / mL, the scavenging rates of ABTS free radicals of polypeptides FLGY, LGYF, and HYF reached above 60%, and the scavenging rate of ABTS free radicals of polypeptide HYF could reach above 80%. The above experimental results show that the seven polypeptides all have antioxidant activity, and the antioxidant activities of polypeptides FLGY, LGYF, and HYF are better than those of the other four polypeptides.

[0041] 4.2 Anti-inflammatory Activity Assay

[0042] To clarify the anti-inflammatory activities of the seven polypeptides, the Griess method was used to detect the effect of the synthetic peptides on NO production in LPS-stimulated RAW264.7 cells at concentrations ranging from 400 to 25 μg / mL. The results are as Figure 7 shown. Compared with the Control (3.87 μM), the NO level in the cell culture medium of the LPS model group increased significantly (22.54 μM) (P < 0.001); compared with the LPS model group, two synthetic peptides (VLPF, HYF) at different concentrations could reduce the NO level in the cells (P < 0.01). When cells produce inflammation, they secrete a large amount of NO. The synthetic peptides VLPF and HYF can significantly reduce the NO produced in the LPS-induced inflammation model. When the concentration is 400 μg / mL, the NO inhibition rate exceeds 50%, indicating that they have good anti-inflammatory activities.

[0043] 4.3 Anti-inflammatory Mechanism Assay

[0044] In NO synthesis, iNOS is the most important enzyme that causes excessive production of NO, which in turn leads to inflammatory responses. In addition, iNOS expressed intracellularly can regulate the secretion of inflammatory cytokines, including IL-6, IL-1β, and TNF-α. The expression level of iNOS was determined by qPCR. From Figure 8 it can be seen that compared with the LPS group, after treatment with the synthetic peptides VLPF and HYF, the mRNA expression level of the iNOS gene decreased significantly (P < 0.05), suggesting that the synthetic peptides VLPF and HYF regulate the secretion of NO at the transcriptional level.

[0045] 5. Polypeptides VLPF and HYF can promote cell proliferation as nutrients

[0046] Adding fetal bovine serum (FBS) to the cell basal medium (DMEM) can provide hormones for maintaining exponential cell growth, nutrients that are absent or in small amounts in the basal medium, and the main low-molecular-weight nutrients. From Figure 9 it can be known that the group with DMEM + FBS is the basal medium supplemented with serum, and the others are groups supplemented with different concentrations of polypeptides in the basal medium. There was no significant difference in the growth of cells in the polypeptide-added groups compared with those in the fetal bovine serum-added group (P > 0.05), and there was a significant difference in cell proliferation between the polypeptide-added groups and the basal medium (DMEM) (P < 0.05), indicating that polypeptides VLPF and HYF can provide nutrients for cell growth, and the nutrients provided can be similar to those of fetal bovine serum and can be added to cell culture media as nutrients to promote the growth of cells or microorganisms.

[0047] It should be understood that the above specific embodiments of the present invention are only for illustrative explanation or interpretation of the principles of the present invention, and do not constitute a limitation to the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention shall be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all variations and modifications that fall within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A multifunctional polypeptide, characterized in that: The polypeptide is polypeptide I or polypeptide II, the amino acid sequence of polypeptide I is shown in VLPF; the amino acid sequence of polypeptide II is shown in HYF.

2. The multifunctional polypeptide according to claim 1, characterized in that The polypeptide screening process is as follows: The Zhongdian yak casein was subjected to simulated gastrointestinal digestion, and a crude extract rich in active peptides was obtained after simulated gastrointestinal digestion. The crude extract of Zhongdian yak casein was then separated by ultrafiltration using an ultrafiltration membrane, and the polypeptide filtrate <3kDa was collected and vacuum-freeze-dried into powder for later use. The peptides were identified by LC-MS / MS, and then scored using the Peptide Ranker database. It was believed that the peptides with a score of >0.8 had potential activity. Target peptides were screened from multiple peptides with scores greater than 0.8, and the anti-inflammatory and antioxidant activities of antimicrobial peptides were predicted using the BIOPEP-UWM database. Multifunctional active peptides were identified through in vitro antioxidant and anti-inflammatory activity assays.

3. Use of the multifunctional polypeptide according to claim 1 in the preparation of antioxidant and / or anti-inflammatory products.

4. Use of the multifunctional polypeptide according to claim 1 in promoting cell proliferation.

5. A cell culture medium, characterized in that The polypeptide is added to the culture medium.

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