A complex peptide for enhancing animal immunity and its preparation method

Abstract

This invention belongs to the field of biotechnology, specifically relating to a complex peptide for enhancing animal immunity and its preparation method. The complex peptide is composed of InflamBlock-9 peptide and ImmunoEnhance-7 peptide in a mass ratio of 3:1. InflamBlock-9 peptide reduces the excessive secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α by targeting and inhibiting the key protein IκB kinase (IKK) in the NF-κB signaling pathway. ImmunoEnhance-7 peptide promotes the synthesis of immunoglobulins IgG, IgA, and apolipoprotein A1 (Apo-A1) by activating the JAK-STAT signaling pathway. The combined use of these two peptides significantly reduces the levels of IL-1β, IL-6, and SAA in cat serum under stress (P<0.05) and increases the levels of IL-10, IgG, and Apo-A1 (P<0.05 or P<0.10), effectively alleviating excessive inflammatory response and improving immunosuppression. The preparation method of this invention achieves efficient peptide preparation through a dual prokaryotic and eukaryotic expression system. The process is controllable and low-cost, making it suitable for the development of functional pet foods or health products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a complex peptide that enhances animal immunity and its preparation method. Background Technology

[0002] With the increasing frequency of stressful events such as pet transportation and relocation, immune dysfunction in cats caused by environmental changes and unfamiliar social interactions has become a common ailment in veterinary clinics. Under stress, the hypothalamic-pituitary-adrenal (HPA) axis in cats is overactivated, leading to increased secretion of stress hormones such as cortisol. This, in turn, inhibits immune cell function and induces excessive secretion of pro-inflammatory factors (such as IL-1β, IL-6, and TNF-α), resulting in intestinal mucosal damage, systemic inflammatory responses, and immunosuppression. Simultaneously, impaired B cell differentiation leads to reduced secretion of immunoglobulins (such as IgG and IgA), significantly decreasing disease resistance and making cats more susceptible to secondary infectious diseases (such as feline infectious peritonitis and stomatitis).

[0003] Currently, the main immune regulation methods for stressed cats include: (1) Antibiotic prophylaxis: Although it can suppress secondary infections, long-term use can easily lead to intestinal flora imbalance and drug resistance; (2) Nutritional supplements (such as vitamin E and zinc): They can only help maintain the activity of immune cells and cannot directly regulate the inflammatory response; (3) Probiotic preparations: They can indirectly improve immunity by regulating the intestinal flora, but their effect on improving the level of systemic inflammatory factors and immunoglobulins is limited; (4) Single-function peptides: Such as antimicrobial peptides (which only inhibit pathogens) or immunomodulatory peptides (which only promote the activation of immune cells), which are difficult to meet the dual needs of "inhibiting inflammation" and "enhancing immunity" at the same time.

[0004] Studies have shown that peptides have unique advantages in the field of immune regulation due to their small molecular weight (<50 kDa), easy absorption (no need for complete enzymatic digestion), and high biological activity (they can act directly on target cells). However, the function of a single peptide is limited, and developing complex peptides with synergistic effects of different functional peptides is a key direction for solving immune problems in stressed cats.

[0005] Based on the above background, this invention provides a new solution for immune regulation in cats under stress by screening two functionally complementary peptides (one targeting and inhibiting inflammatory signaling pathways, and the other activating immune response pathways) and optimizing the expression process to achieve efficient preparation. Summary of the Invention

[0006] The first objective of this invention is to provide a complex peptide that enhances animal immunity, the amino acid sequence of which is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0007] A second objective of the present invention is to provide a nucleotide sequence encoding the above-mentioned complex peptide, which is a DNA molecule as shown in any of the following examples.

[0008] A third objective of this invention is to provide a recombinant expression vector containing the above-mentioned nucleotide sequence, wherein the vector is a pET-28a vector.

[0009] A fourth objective of the present invention is to provide a host bacterium containing the above-mentioned recombinant expression vector, wherein the host bacterium is Escherichia coli BL21(DE3).

[0010] The fifth objective of this invention is to provide a method for preparing the above-mentioned complex peptide, comprising the following steps:

[0011] (1) Preparation of InflamBlock-9 peptide: pET-28a (+)-InflamBlock-9 recombinant plasmid was constructed, transformed into Escherichia coli BL21(DE3), and purified by glutathione agarose resin affinity chromatography after IPTG induction expression.

[0012] (2) Preparation of ImmunoEnhance-7 peptide: artificial synthesis;

[0013] (3) Mix InflamBlock-9 peptide and ImmunoEnhance-7 peptide at a mass ratio of 3:1, add 0.1% poloxamer F68 to aid dissolution, and freeze dry to obtain composite peptide powder.

[0014] The sixth objective of this invention is to provide the application of the above-mentioned complex peptide in the preparation of products that enhance animal immunity, wherein enhancing animal immunity includes reducing the levels of IL-1β, IL-6, and SAA, and increasing the content of IL-10, IgG, and Apo-A1 in cat serum.

[0015] A seventh object of the present invention is to provide a composition for improving intestinal health and preventing diarrhea in animals, comprising the above-mentioned complex peptide and a pharmaceutically acceptable carrier; said carrier being starch, microcrystalline cellulose or starch paste.

[0016] In some embodiments, the polypeptide is 0.1%-1.0% by mass.

[0017] An eighth objective of the present invention is to provide the use of the above-mentioned complex peptide in the preparation of pet feed additives or oral liquids for enhancing animal immunity.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] (1) In terms of inflammation suppression: InflamBlock-9 peptide targets and binds to the IκB kinase (IKK) regulatory subunit, blocking the activation of the NF-κB signaling pathway, thereby reducing the secretion of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α (IL-1β and IL-6 levels at T3 were significantly lower than those in the control group, P<0.05).

[0020] (2) Immunoenhance-7 peptide promotes B cell differentiation by activating the JAK-STAT signaling pathway and enhances the synthesis of IgG, IgA and Apo-A1 (IgG level at T4 was significantly higher than that in the control group P<0.05, and Apo-A1 content returned to normal level).

[0021] (3) Overall effect: The experiment showed that at T4, the IL-10 level (anti-inflammatory factor) in the experimental group was significantly higher than that in the blank group (P<0.05), indicating a combined effect of inflammation suppression and immune enhancement. In addition, this invention uses a dual expression system of prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) peptides to optimize peptide stability (e.g., InflamBlock-9 peptide reduces pancreatic enzyme degradation through GST tagging). The preparation process is simple and low-cost, and is suitable for large-scale production of pet food or health products. Attached Figure Description

[0022] Figure 1. Detection results of tumor necrosis factor-α; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0023] Figure 2. Detection results of interferon-γ; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0024] Figure 3. Detection results of interleukin-1-β; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0025] Figure 4. Detection results of interleukin-6; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0026] Figure 5. Detection results of interleukin-10; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0027] Figure 6. Detection results of immunoglobulin A; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0028] Figure 7. Detection results of immunoglobulin M; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10).

[0029] Figure 8The results of immunoglobulin G detection; different letters indicate significant differences (P<0.05), and the symbol (#) indicates a trend of significant difference (P<0.10). Detailed Implementation

[0030] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0031] Example 1: Preparation and Validation of Complex Peptides

[0032] 1. Preparation of InflamBlock-9 peptide

[0033] (1) Construction of recombinant vector: Based on the frequency of E. coli codon usage in the NCBI database, the InflamBlock-9 peptide (H-Glu-Leu-Asp-Ser-Met-Thr-Lys-Pro-Arg-OH, SEQ ID NO:1) encoding gene was optimized and obtained with GC content of 50%. After synthesis, it was cloned into the pET-28a (+) vector with HIS tag (EcoR I / Xho I restriction site) to construct pET-28a (+)-InflamBlock-9 plasmid.

[0034] (2) Host bacterial transformation: The plasmid was transformed into BL21(DE3) competent cells (ice bath for 30 min → heat shock at 42℃ for 90 s → ice bath for 2 min), plated on LB agar containing 100 μg / mL ampicillin, and cultured at 37℃ for 16 h. Single clones were picked and inoculated into 5 mL LB (containing ampicillin), and cultured at 37℃ and 200 rpm until OD600=0.6. The plasmid was extracted and positive clones were identified by PCR (named BL21(DE3)-pET-28a (+)-InflamBlock-9).

[0035] (3) Fermentation and purification: Positive strains were inoculated into 500 mL LB (containing ampicillin) and pre-cultured at 37℃ and 200 rpm until OD600=0.8. 0.1 mM IPTG was added for induction for 4 h. Cells were collected by centrifugation (8000g×10 min, 4℃), resuspended in PBS, and then sonicated (200 W, 3 s working time / 3 s interval, total time 10 min). The supernatant was purified and eluted using a Ni-NTA column. HPLC analysis using a Tricine-SDS-PAGE system (designed specifically for small peptides) showed a main band molecular weight of approximately 1.0 kDa and an HPLC purity of 96.3%.

[0036] 2. Preparation of ImmunoEnhance-7 peptide: ImmunoEnhance-7 peptide (H-Phe-Trp-Ile-His-Gln-Arg-Leu-OH, SEQ ID NO:2) was synthesized by Nanjing Genscript Biotech Co., Ltd. The HPLC purity was 98.7% as reported in the test report.

[0037] 3. Preparation of complex peptides: InflamBlock-9 peptide (30 mg) and ImmunoEnhance-7 peptide (10 mg) were mixed at a mass ratio of 3:1, 0.1% poloxamer F68 was added to aid dissolution, and the mixture was freeze-dried (-50℃, vacuum degree 10 Pa) for 48 h to obtain complex peptide powder.

[0038] Example 2

[0039] The peptide efficacy evaluation experiment was conducted entirely at the Experimental Animal Center of South China Agricultural University, lasting 22 days. The experiment was divided into four phases: a transition period (7 days), a pre-feeding period (7 days), a transportation period (1 day), and a recovery period (7 days). Twelve healthy adult British Shorthair cats were selected as experimental animals and randomly divided into two groups based on sex and weight: a control group and a compound peptide group. All cats were individually housed in cages (108cm*70cm*76cm) within the same temperature-controlled environment, with free access to food and water. After the transition period, the experimental group cats were additionally fed 0.2% (by weight) of cat strips containing the compound peptide prepared in Example 1, while the control group cats were fed blank cat strips until the end of the experiment. Prior to the experiment, all experimental cats underwent necessary immunization and deworming treatments. The litter box was cleaned once daily in the morning, the litter was changed weekly, and the cat enclosure was cleaned and disinfected daily to maintain cleanliness.

[0040] Every morning at 8:30, each cat was fed 60-90g of cat food (free access). The amount fed and the amount left over for each cat were accurately recorded daily. The mental state of the experimental cats was checked and fecal scores were calculated. The cats were weighed on an empty stomach on the first day of food change, at the end of the transportation period, and at the end of the recovery period. Blood samples were collected 1 day before the pre-feeding period, 1 hour before transportation, 1 hour after transportation, and at the end of the recovery period to detect inflammatory factors and immunoglobulins in the serum.

[0041] Example 3: Effects of complex peptides on inflammatory factors and immunoglobulins in cat serum

[0042] At T3, TNF-α levels in both groups were significantly increased (P<0.05), indicating that transport may have triggered an inflammatory response. At T4, these levels significantly decreased (P<0.05), returning to T2 levels, with no significant difference between the two groups (Figure 1). IFN-γ levels increased over time, and at T4, IFN-γ levels significantly decreased (P<0.05), but remained significantly higher than at T2 (P<0.05, Figure 2).

[0043] from Figures 3-4 As shown in Figure d, the trends of IL-1β and IL-6 were relatively consistent. At T3, both levels increased significantly (P<0.05), but overall, the levels in the experimental group were lower than those in the control group. At T3, the IL-1β level in the experimental group was significantly lower than that in the control group, and IL-6 also showed a significant trend of being significantly lower in the experimental group than in the control group (P<0.10). IL-10 levels increased in both groups at T2, with the experimental group showing a significant upward trend (P<0.10). At T3, the IL-10 levels in both groups increased significantly (P<0.05), and at T4, the IL-10 levels decreased significantly (P<0.05, Figure 5).

[0044] As shown in Figure 6, IgA levels in both groups increased to some extent at T2, with the experimental group showing a significantly higher level than one week prior (P<0.05). At T3, IgA levels in both groups decreased significantly (P<0.05), and at T4, the experimental group's level was slightly higher than the control group. IgM levels decreased significantly at T3 (P<0.05), and at T4, the experimental group's IgM level gradually returned to normal, showing a trend of being significantly higher than the control group (P<0.10, Figure 7). In Figure 8, at T2, the experimental group's IgG level was significantly higher than the control group (P<0.05), and at T3, the experimental group's IgG level was significantly higher than the control group (P<0.05). The experimental group's IgG level decreased significantly (P<0.05), while the control group's IgG level showed a significant decreasing trend (P<0.10). At T4, IgG levels in both groups increased, with both the experimental and control groups returning to T2 levels, but the experimental group's IgG level remained significantly higher than the control group (P<0.05).

[0045] In summary, the compound peptide powder of the present invention can reduce the levels of IL-1β, IL-6, and SAA, and increase the content of IL-10, IgG, and Apo-A1 in cat serum, thereby reducing the excessive development of inflammation. At the same time, it can improve the immune status of cats under stress by increasing the content of immunoglobulins.

[0046] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex peptide for enhancing animal immunity, characterized in that, The complex peptide is composed of InflamBlock-9 peptide and ImmunoEnhance-7 peptide; the amino acid sequence of InflamBlock-9 peptide is shown in SEQ ID NO:1, and the amino acid sequence of ImmunoEnhance-7 peptide is shown in SEQ ID NO:2; the animal is a cat.

2. The nucleotide encoding the complex peptide of claim 1.

3. A recombinant expression vector containing the nucleotides of claim 2.

4. A host bacterium containing the recombinant expression vector of claim 3, characterized in that, The host bacteria are Escherichia coli BL21(DE3) or Pichia pastoris GS115.

5. The use of the complex peptide according to claim 1 in the preparation of products that enhance animal immunity, characterized in that, The improvement of animal immunity is achieved by reducing the levels of IL-1β and IL-6 and increasing the levels of IL-10 and IgG in cat serum; the animal is a cat.

6. The application according to claim 5, characterized in that, The product is a feed additive or oral liquid.

7. A composition for improving intestinal health and preventing diarrhea in animals, characterized in that, The compound peptide of claim 1 is contained in the compound peptide, and a pharmaceutically acceptable carrier, wherein the carrier is starch, microcrystalline cellulose or starch paste; and the animal is a cat.

8. The composition according to claim 7, characterized in that, The mass percentage of the complex peptide is 0.1%-1.0%.

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