Hybrid peptide with immunoregulation and anti-oxidation functions as well as preparation method and application of hybrid peptide

Abstract

The invention relates to the technical field of genetic engineering and biological agents, in particular to a hybrid peptide with immunoregulation and anti-oxidation functions and a preparation method and application of the hybrid peptide. The polypeptide provided by the invention has immunoregulation and antioxidation functions at the same time, and in a normal or immunosuppression state, the polypeptide can significantly improve the immunologic function of a body and reduce the damage of immunosuppression to the body; the oxidation resistance of cells and organisms can be enhanced; meanwhile, the polypeptide has the advantages of low cytotoxicity, high safety, simplicity and convenience in preparation and low cost, can be used as an ideal immunomodulator and an antioxidant, is widely applied to the fields of medicines, foods, health care, feeds, nutrition and the like of human and animals, and has very good application potential and value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and biological preparations, and in particular to a hybrid peptide having both immunomodulatory and antioxidant functions, and a preparation method and application thereof. Background Art

[0002] The immune system is a key defense mechanism against infection, inflammation, and autoimmune diseases, primarily protecting the body by identifying and eliminating pathogens. It consists of two main components: the innate immune system and the acquired immune system. The innate immune response plays a vital role as the body's first line of defense, capable of nonspecifically identifying and responding to a variety of pathogens. Immunosuppression, a temporary or permanent immune dysfunction, weakens the immune system, making tissues and organs more susceptible to infection and damage. Therefore, enhancing immunoregulatory functions has long been a research focus within the immune system.

[0003] Balanced redox reactions are crucial for the body to maintain a normal physiological state. Once this balance is broken, it will lead to the accumulation of electrophilic substances, thus triggering oxidative stress in the body. The main cause of oxidative stress is the accumulation of excessive ROS, including O2• - , H₂O₂, and OH₂. Excessive accumulation of these free radicals can damage cellular DNA, proteins, and lipids, leading to cellular dysfunction and a range of diseases. Therefore, the development of highly effective antioxidants, such as antioxidant peptides, to mitigate these harmful effects is of great significance.

[0004] Based on the important role of immunoregulation and anti-oxidation, the development of a new, safe, side-effect-free, environmentally friendly active peptide with immunoregulatory and antioxidant functions has important practical significance and application value for humans or animal husbandry, and is a new breakthrough and new concept in anti-infection and antioxidant strategies. Summary of the Invention

[0005] The present invention provides a hybrid peptide with both immunomodulatory and antioxidant functions, and a preparation method and application thereof.

[0006] To achieve the above objectives, the present invention, based on extensive research into the sequence, structure, and sequence-structure-function relationship of the VLP (amino acid sequence shown in SEQ ID NO. 3) and Aβ (amino acid sequence shown in SEQ ID NO. 4), utilized protein molecular design techniques to optimize the hybridization of the VLP and Aβ peptides, followed by in vitro and in vivo screening. Ultimately, a novel immunomodulatory and antioxidant hybrid peptide, designated VLP-Aβ (VA), was obtained, whose amino acid sequence is shown in SEQ ID NO. 1. The hybrid peptide VA possesses the functions of both parent peptides, namely, immunomodulatory properties: it can enhance the body's immune function under normal or immunosuppressive conditions; and it can enhance the antioxidant function of cells and animals.

[0007] In a first aspect, the present invention provides a polypeptide having any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) The amino acid sequence of a polypeptide with the same function as the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, deleting or inserting one or more amino acids; (3) An amino acid sequence that has at least 85% homology to the amino acid sequence shown in SEQ ID NO. 1 and has the same functional polypeptide.

[0008] The amino acid sequence shown in SEQ ID NO.1 is as follows: VLPVPQKDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG.

[0009] In (2) and (3) above, the derivative polypeptides obtained by modification based on the amino acid sequence shown in SEQ ID NO.1 and having the same function as the hybrid peptide VA include but are not limited to the following polypeptides: (1) A polypeptide obtained by adding a protein tag sequence to the C-terminus and / or N-terminus of the amino acid sequence shown in SEQ ID NO.1, for example, a polypeptide obtained by adding a His tag containing 6 His residues to the C-terminus and / or N-terminus of the amino acid sequence shown in SEQ ID NO.1; or a polypeptide obtained by adding a GST or C-Myc tag to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO.1; Those skilled in the art should understand that adding protein tag sequences to both ends of a polypeptide for purposes such as easy purification and polypeptide labeling is a conventional technical means in the art and does not affect the inherent functions and activities of the polypeptide itself. Therefore, the VA derivatives obtained by adding protein tag sequences to both ends of the hybrid peptide VA as shown in SEQ ID NO.1 are also within the scope of protection of the present invention.

[0010] (2) A polypeptide obtained by making one or more conservative amino acid substitutions in the amino acid sequence shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the polypeptide.

[0012] When the amino acid sequence of the polypeptide is known, those skilled in the art can design coding genes for the polypeptide with different nucleotide sequences according to the needs of polypeptide expression, based on the principle of codon degeneracy and the codon usage preferences of different species.

[0013] In some embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2, or a nucleotide sequence encoding a polypeptide with the same function that is at least 80%, 85%, 90%, 95%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO.2.

[0014] The nucleotide sequence shown in SEQ ID NO. 2 is a gene encoding a hybrid peptide VA designed based on the codon preference of Escherichia coli. Nucleotide sequences encoding polypeptides with the same functional identity and at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the nucleotide sequence shown in SEQ ID NO. 2 also fall within the scope of protection of the present invention.

[0015] In a third aspect, the present invention provides a biological material containing a nucleic acid molecule encoding the polypeptide, wherein the biological material comprises recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or a host cell.

[0016] The host cell includes a microbial cell, an animal or plant cell, or a cell line that cannot develop into an animal or plant individual.

[0017] In a fourth aspect, the present invention provides a method for preparing the above-mentioned polypeptide, which comprises: introducing the above-mentioned nucleic acid molecule into a host cell to express the polypeptide.

[0018] Preferably, the preparation method comprises: connecting the polypeptide encoding gene to an expression vector to construct a recombinant expression vector, and introducing the recombinant expression vector into a host cell by a transgenic method to obtain a host cell into which the polypeptide encoding gene is introduced.

[0019] Among them, the transgenic methods include heat stress transformation, electroporation, transfection, etc.

[0020] The host cells include but are not limited to microbial cells, animal and plant cells.

[0021] In some embodiments of the present invention, the host cell is Escherichia coli.

[0022] When E. coli is used as the host cell, the polypeptide encoding gene sequence obtained by optimizing the codon preference of E. coli, that is, the sequence shown in SEQ ID NO. 2, is used to express the hybrid peptide, which has a better expression amount.

[0023] In some embodiments of the present invention, the preparation of the polypeptide uses Escherichia coli as a host, pCOLD as an expression vector, and is expressed by IPTG induction, specifically comprising the following steps: (1) Connecting the polypeptide encoding gene to the expression vector pCOLD to construct a recombinant expression vector; (2) Transforming the above-mentioned recombinant expression vector into Escherichia coli BL21 (DE3) to construct a recombinant engineered bacterium into which the polypeptide encoding gene is introduced; (3) Cultivating the above-mentioned recombinant engineered bacteria and adding IPTG to induce the expression of the polypeptide; (4) Collecting the culture supernatant and purifying the polypeptide.

[0024] The present invention demonstrates through in vivo and in vitro experiments that the polypeptide can not only enhance the immune activity of the body in normal and immunodeficient states, increase the expression of cytokines, promote the growth of mice, and alleviate the damage to the spleen and thymus of mice caused by immunodeficiency; but also can reduce the level of oxidative damage to HepG2 cells, increase the survival rate of cells, and alleviate liver damage in mice. It also protects the liver function of mice by increasing the activity level of the body's antioxidant enzymes, and has good immune and antioxidant bidirectional regulatory effects.

[0025] Based on the above functions, the fifth aspect of the present invention provides the use of the above-mentioned polypeptide, the nucleic acid molecule, the biological material, or the polypeptide prepared by the preparation method in the preparation of an immunomodulatory preparation.

[0026] Preferably, the immunomodulatory preparation is an immunopotentiator.

[0027] The above-mentioned immunopotentiator can be used for the prevention and treatment of various types of immunosuppression including the body's immunosuppressive reaction caused by cyclophosphamide (CY).

[0028] In a sixth aspect, the present invention provides use of the above-mentioned polypeptide, nucleic acid molecule, biological material, or polypeptide prepared by the preparation method in the preparation of an antioxidant preparation.

[0029] The antioxidant preparation can be used for the prevention and treatment of oxidative damage including hydrogen peroxide-induced oxidative damage or carbon tetrachloride-induced liver damage.

[0030] The preparation of the present invention includes medicine, health care product, food, feed or feed additive.

[0031] In a seventh aspect, the present invention provides the use of the above-mentioned polypeptide or the polypeptide prepared by the method in immunomodulation or anti-oxidation.

[0032] Preferably, the immunomodulation is immunoenhancement, which is to improve the immune function of the body under normal or immunosuppressive conditions.

[0033] Preferably, the immune regulation and anti-oxidation are non-disease treatment purposes.

[0034] In an eighth aspect, the present invention provides a product, wherein the product is the polypeptide described above or comprises a polypeptide prepared by the preparation method.

[0035] Preferably, the product comprises a medicine, a health product, a food, a feed or a feed additive.

[0036] Preferably, in the product, the polypeptide is used as an active ingredient or the polypeptide is compounded with other active ingredients to form an active ingredient of a medicine, health product, food, feed or feed additive.

[0037] Preferably, the product further comprises a carrier or excipient component. For example, the drug may further comprise a carrier or excipient acceptable in the pharmaceutical field.

[0038] The beneficial effects of the present invention include at least: for the first time, the present invention hybridizes VLP and Aβ, and obtains a hybrid peptide through optimization and screening. The hybrid peptide has the functions of two parent peptides, that is, it has both immunoregulatory and antioxidant functions, and its immunoregulatory activity and antioxidant activity are stronger than the corresponding activities of the parent peptides; under normal or immunosuppressive conditions, the hybrid peptide can significantly improve the body's immune function and reduce the damage to the body caused by immunosuppression; and can enhance the antioxidant capacity of cells and the body; at the same time, the hybrid peptide has the advantages of low cytotoxicity, high safety, simple preparation and low cost, and can be used as an ideal immunomodulator and antioxidant, and is widely used in the fields of medicine, food, health care, feed, nutrition, etc. for humans and animals, and has great application potential and value.

[0039] The preparation method of the hybrid peptide provided by the present invention can realize the large-scale and efficient preparation of the hybrid peptide. The prepared hybrid peptide has both immunomodulatory and antioxidant activities and has no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 This is the process of constructing the recombinant expression vector in Example 1 of the present invention.

[0042] Figure 2 This is the fermentation curve of the Escherichia coli engineered bacteria fermenter in Example 1 of the present invention.

[0043] Figure 3 This is the purification result of the recombinant hybrid peptide VA in Example 1 of the present invention.

[0044] Figure 4 This is the effect of the hybrid peptide VA and its parent peptide Aβ (A) and VLP (V) in Example 2 of the present invention on the cell survival rate of mouse macrophages.

[0045] Figure 5 This is the effect of the hybrid peptide VA in Example 3 of the present invention on the cytokine expression of mouse macrophages RAW 264.7; wherein, a is the expression level of TNF-α; b is the expression level of IL-1β, and c is the expression level of IL-6; CON represents the normal group, LPS represents the model group of LPS-induced inflammation, A represents experimental group 1 (treated with Aβ under normal conditions), V represents experimental group 2 (treated with VLP under normal conditions), and VA represents experimental group 3 (treated with VA under normal conditions); * represents a significant difference between the two (p<0.5), ** represents a very significant difference between the two (p<0.01), *** represents a very significant difference between the two (p<0.001), and **** represents a very significant difference between the two (p<0.0001).

[0046] Figure 6 The immunomodulatory effect of the hybrid peptide VA in Example 4 of the present invention on cyclophosphamide-induced immunosuppressive mice; wherein, a is the effect of VA on the body weight of immunosuppressed mice; b is the effect of VA on the thymus of immunosuppressed mice; c is the effect of VA on the spleen of immunosuppressed mice; * represents a significant difference between the two (p<0.5), ** represents a very significant difference between the two (p<0.01), *** represents a very significant difference between the two (p<0.001), and **** represents a very significant difference between the two (p<0.0001).

[0047] Figure 7The regulatory effect of the hybrid peptide VA in Example 4 of the present invention on the spleen lymphocytes of cyclophosphamide-induced immunosuppressive mice; wherein, ah is the effect of VA on the CD4+ / CD8+ ratio of lymphocytes, i is the effect of VA on the proliferation activity of lymphocytes, and j is the effect of VA on the killing activity of natural killer cells; the horizontal axis of ag is titled CD4+, and the scales marked on the horizontal axis are from left to right: 0, 10 3 , 10 4 The vertical axis title is CD8+, and the scales marked on the vertical axis are: 0, 10 from bottom to top. 3 , 10 4 , 10 5 ; * represents a significant difference between the two groups (p<0.5), ** represents a very significant difference between the two groups (p<0.01), *** represents a very significant difference between the two groups (p<0.001), and **** represents a very significant difference between the two groups (p<0.0001).

[0048] Figure 8 The regulatory effect of the hybrid peptide VA in Example 4 of the present invention on immunoglobulins and cytokines in cyclophosphamide-induced immunosuppressive mice; wherein ac is the effect of VA on immunoglobulins IgA, IgG, and IgM, and df is the effect of VA on TNF-α, IL-6, and IL-1β; * represents a significant difference between the two (p<0.5), and ** represents a very significant difference between the two (p<0.01).

[0049] Figure 9 Figure 5 shows the alleviating effect of the hybrid peptide VA in Example 5 of the present invention on hydrogen peroxide-induced oxidative damage in HepG2 cells; wherein a represents the effect of VA on cell survival rate, bd represent the effect of VA on intracellular antioxidant enzyme activities (SOD, CAT, GSH-Px), e represents the effect of VA on intracellular malondialdehyde content, f and h represent the effect of VA on intracellular ROS production, and g and i represent the effect of VA on intracellular mitochondrial damage; * represents a significant difference between the two (p<0.5), and **** represents an extremely significant difference between the two (p<0.0001).

[0050] Figure 10The protective effect of the hybrid peptide VA in Example 6 of the present invention on carbon tetrachloride-induced liver damage in mice; wherein a and b are the H&E staining results of VA on the alleviating effect of mouse liver fibrosis, c is the Masson staining result of VA on the alleviating effect of mouse liver fibrosis, d is the fluorescence staining result of VA on the alleviating effect of mouse liver cell apoptosis, e and f are the effects of VA on mouse serum liver function indicators AST and ALT; * represents a significant difference between the two (p < 0.5), ** represents a very significant difference between the two (p < 0.01), *** represents a very significant difference between the two (p < 0.001), and **** represents a very significant difference between the two (p < 0.0001).

[0051] Figure 11 The hybrid peptide VA in Example 6 of the present invention enhances the antioxidant enzyme activity of mice with carbon tetrachloride-induced liver damage; wherein ac is the enhancing effect of VA on the antioxidant enzyme activity (SOD, CAT, GSH-Px) in mouse serum, d is the effect of VA on the malondialdehyde content in mouse serum, eg is the enhancing effect of VA on the antioxidant enzyme activity (SOD, CAT, GSH-Px) in mouse liver, and h is the effect of VA on the malondialdehyde content in mouse liver; * represents a significant difference between the two (p<0.5), ** represents a very significant difference between the two (p<0.01), *** represents a very significant difference between the two (p<0.001), and **** represents a very significant difference between the two (p<0.0001). DETAILED DESCRIPTION

[0052] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. E. coli competent cells DH5α and BL21 (DE3) were purchased from Invitrogen; pCOLD-TF-SUMO was purchased from Takara.

[0055] Example 1 Preparation of immune antioxidant hybrid peptide VA 1. Construction of recombinant expression vector Based on the amino acid sequence of the hybrid peptide VA (sequence shown in SEQ ID NO.1) and the codon preference of E. coli, the coding gene of the hybrid peptide VA (sequence shown in SEQ ID NO.2) was designed and synthesized, ligated with the expression vector pCOLD-TF-SUMO, and transformed into E. coli DH5α competent cells to construct the recombinant expression vector pCOLD-SUMO-VA. The vector construction process is as follows Figure 1 shown.

[0056] 2. Construction of Escherichia coli engineered bacteria expressing hybrid peptide VA The recombinant expression vector pCOLD-SUMO-VA constructed in step 1 above was transformed into the Escherichia coli BL21 (DE3) strain as follows: the competent Escherichia coli BL21 (DE3) cells were placed on ice and 100 ng of pCOLD-SUMO-VA plasmid was added and mixed evenly. The plasmid was transformed according to the Escherichia coli heat shock transformation method and plated. Positive transformants were verified and identified by PCR to obtain Escherichia coli engineered bacteria expressing the hybrid peptide VA.

[0057] 3. Induced expression and purification of hybrid peptide VA The engineered E. coli bacteria constructed in step 2 were inoculated into a 500 mL conical flask containing 50 mL LB liquid culture medium and cultured at 37°C and 180 rpm for 12 h as the seed liquid for inoculation of the fermenter. The liquid volume in the 2 L fermenter was 500 mL, the inoculation volume was 1%, the fermentation temperature was 37°C, air was introduced into the fermenter, the ventilation volume was 1 vvm, and the dissolved oxygen in the fermentation liquid was maintained at not less than 10% by adjusting the stirring speed. The pH of the fermentation medium was controlled at 7.0 ± 0.2 by adding 50% ammonia water. No feeding was performed in the first 4-5 h after the start of fermentation, and the medium was added after the glucose in the culture medium was consumed. The results are shown in Figure 2 First, the exponential feeding method was used for feeding. After the E. coli grew to 10 hours, IPTG was added to start induction and the pH-stat feeding method was switched to. Induction expression continued for 24 hours. After that, the pH-stat feeding method was used. After the induction expression was completed, the fermentation broth was collected and purified to obtain the hybrid peptide VA. The expression level of VA was determined to be 375 mg / L. The purification results of the recombinant hybrid peptide VA are shown in Figure 3 .

[0058] Example 2 Effect of Hybrid Peptide VA on Mouse Macrophage Cell Survival Rate Macrophage RAW264.7 cells in the logarithmic growth phase were inoculated into 96-well plates, with an initial cell culture density of 1×10 4After overnight incubation at 37°C and 5% CO2, a series of concentration gradients of hybrid peptide VA, Aβ, and VLP (0-100 μg / mL) were added. After 24 hours of incubation, the effect of peptide VA on the survival rate of mouse macrophages was detected using the CCK8 assay. Figure 4 As shown, the cytotoxicity of the hybrid peptide VA is lower than that of its parent peptide Aβ and VLP, and the survival rate of macrophages is greater than 80% in the concentration range of 0-100 μg / mL, indicating that the hybrid peptide VA has low cytotoxicity and high safety.

[0059] Example 3 Immunomodulatory activity of hybrid peptide VA in mouse macrophages The hybrid peptide VA and its parent peptide VLP and Aβ were diluted with DMEM culture medium to prepare a peptide solution with a concentration of 100 μg / mL. The effects of the hybrid peptide VA and its parent peptide VLP and Aβ on the secretion of cytokines such as TNF-α and IL-6 by mouse macrophage RAW264.7 under normal conditions were detected. The normal group (Control), model group (LPS), experimental group 1 (Aβ), experimental group 2 (VLP), and experimental group 3 (VA) were set up respectively. Among them, the normal group did not receive any treatment, and the model group was given LPS to induce inflammation; after overnight culture of the cells, experimental groups 1, 2, and 3 were added with Aβ, VLP or VA solution at a final concentration of 100 μg / mL. The cytokines TNF-α, IL-6 and IL-1β were detected by ELISA, and the results are shown as follows: Figure 5 As shown, the hybrid peptide VA can significantly increase the expression of cytokines TNF-α, IL-6, and IL-1β in mouse macrophages under normal conditions, and the expression of cytokines TNF-α, IL-6, and IL-1β in macrophages in the VA group is significantly higher than that in the Aβ group and the VLP group. This shows that the hybrid peptide VA has an immunomodulatory effect and can enhance the immune activity of cells under normal conditions, and its immune-enhancing effect is better than that of its parent peptide Aβ and VLP.

[0060] Example 4 Immunomodulatory Effects of Hybrid Peptide VA on Immunosuppressed Mice In this example, BALB / c male mice (weighing 20-22 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used for the animal experiments. All experimental procedures followed the guidelines of the European Committee for the Ethics of Laboratory Animals (86 / 609 / EEC) and were approved by the Laboratory Animal Ethics Committee of China Agricultural University. The animals were housed in a clean-grade environment with an ambient temperature of 22 ± 2°C, a humidity of 50% to 55%, and a light on period of 8:00 AM to 8:00 PM. Mice were housed six per cage and had free access to food and water.

[0061] Mice were randomly divided into seven groups (six mice per group): control (CON), CTX (50 mg / kg / day), CTX + LMS (levamisole hydrochloride) (20 mg / kg / day), CTX + Aβ (motherwise derived Aβ peptide, 10 mg / kg / day), CTX + VLP (motherwise derived VLP peptide, 10 mg / kg / day), CTX + VAL (hybrid VA peptide, low dose, 5 mg / kg / day), and CTX + VAH (hybrid VA peptide, high dose, 10 mg / kg / day). Mice were acclimated for 5 days before the start of the formal experiment. Except for the control group, mice in all groups received an intraperitoneal injection of CTX at a dose of 50 mg / kg / day from days 1 to 3 of the formal experiment. The control group received an equivalent volume of saline. From day 4 to day 10, mice were treated daily with the designated drug or peptide (control and CTX groups were again injected with saline). Sixteen hours after the last treatment, mice were euthanized and blood, spleen, and thymus tissues were collected.

[0062] 1. Effects of hybrid peptide VA on body weight and immune organ weights in immunosuppressed mice The spleen and thymus tissues were carefully removed of fat and connective tissue and then weighed. The organ index was calculated as follows: The results are as follows Figure 6 As shown, the results showed that the body weight, spleen index and thymus index of the mice in the model group were significantly lower than those in the control group, indicating that cyclophosphamide can inhibit the growth of mice, reduce the immune organ index and suppress immunity; while the body weight, spleen index and thymus index of the mice in the VAL and VAH experimental groups returned to normal levels and were higher than those in the control group, indicating that the hybrid peptide VA can not only improve the immunity level of mice, but also promote the growth of mice.

[0063] 2. Effects of hybrid peptide VA on spleen immune cells in immunosuppressed mice The results are as follows Figure 7 As shown, splenic T cell subsets were analyzed by flow cytometry, with CD3+ marking all T cells, and CD4+ and CD8+ marking two subsets. Splenic lymphocyte proliferation activity was measured using LPS and ConA. Natural killer cell cytotoxicity was measured using K562 cells as target cells. The results showed that the hybrid peptide VA could enhance the CTX-induced decrease in the CD4+ / CD8+ ratio of mouse splenic lymphocytes and increase lymphocyte proliferation. Furthermore, the hybrid peptide VA also enhanced the cytotoxicity of natural killer cells. These results indicate that the hybrid peptide VA can enhance the immune activity of immune cells in the mouse spleen.

[0064] 3. Effect of hybrid peptide VA on cytokine release in immunosuppressed mice After collecting mouse blood, let it rest at room temperature for 2 hours, then centrifuge it at 5000 rpm at 4°C for 20 minutes. The supernatant was collected as the serum sample. Serum cytokines (TNF-α, IL-6, IL-1β) and immunoglobulins (IgG, IgA, IgM) were measured using ELISA kits. Figure 8 As shown, the results showed that compared with the control group, the immunoglobulin and cytokine levels of mice in the CTX group were significantly reduced; while the administration of hybrid peptide VA could significantly increase the levels of cytokines and immunoglobulins in the serum of immunosuppressed mice, thereby improving the immune activity of immunocompromised mice.

[0065] Example 5 Alleviating effect of hybrid peptide VA on hydrogen peroxide-induced oxidative damage in HepG2 cells HepG2 cell oxidative damage model induced by hydrogen peroxide: HepG2 cells were plated at 2 × 10 5 Cells were plated into 6-well plates and incubated for 12 hours. The medium from each well was aspirated and replaced with 1 mL of VLPs, Aβ, and VA (100 ng / mL) diluted in basal DMEM for an additional 2 hours. Hydrogen peroxide (650 μM) diluted in DMEM was then added to each well and incubated for 6 hours.

[0066] The results are as follows Figure 9 As shown, the hybrid peptide VA can enhance cell survival induced by hydrogen peroxide. It also increases the activity of intracellular antioxidant enzymes (SOD, CAT, GSH-Px) and reduces intracellular malondialdehyde levels. Furthermore, VA can reduce the increase in intracellular ROS levels induced by hydrogen peroxide and protect mitochondria from damage. These results demonstrate that the hybrid peptide VA can alleviate oxidative stress damage at the cellular level.

[0067] Example 6 Protective effect of hybrid peptide VA on carbon tetrachloride (CCl4)-induced liver damage in mice In this example, C57BL / 6 male mice (weighing 20-22 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used for the animal experiments. All experimental procedures followed the guidelines of the European Committee for the Ethics of Laboratory Animals (86 / 609 / EEC) and were approved by the Laboratory Animal Ethics Committee of China Agricultural University. The animals were housed in a clean-grade environment with an ambient temperature of 22 ± 2°C, a humidity of 50% to 55%, and a light cycle of 8:00 AM to 8:00 PM. Mice were housed six per cage and had free access to food and water.

[0068] Mice were randomly divided into seven treatment groups (six mice per group): control, CCl4 (2% CCl4 in olive oil, 5 mL / kg), CCl4 + silymarin (20 mg / kg), CCl4 + Aβ (motherwise derived Aβ peptide, 10 mg / kg), CCl4 + VLP (motherwise derived VLP, 10 mg / kg), CCl4 + VAL (hybrid peptide VA, low dose, 5 mg / kg), and CCl4 + VAH (hybrid peptide VA, high dose, 10 mg / kg). Mice were acclimated for 5 days before the experiment. From day 1 to day 7, mice in each group received an intraperitoneal injection of the corresponding drug or peptide, while the control and CCl4 groups received an equal volume of saline. Two hours after the corresponding drug and peptide injection on day 7, all groups except the control group received an intraperitoneal injection of CCl4, while the control group received an equal volume of olive oil. Sixteen hours after CCl4 treatment, mice were euthanized, and blood and liver tissue samples were collected.

[0069] The results are as follows Figure 10 and Figure 11 As shown, carbon tetrachloride treatment of mice resulted in liver fibrosis and elevated levels of apoptosis in hepatocytes. However, treatment with the hybrid peptide VA reduced liver fibrosis and apoptosis. Furthermore, VA treatment restored serum levels of liver function markers such as ALT and AST, protecting normal liver function. Furthermore, VA increased antioxidant enzyme activities (SOD, CAT, GSH-Px) in the liver and serum of mice and reduced malondialdehyde levels. These results indicate that the hybrid peptide VA can alleviate carbon tetrachloride-induced oxidative damage in the mouse liver.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide has any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) The amino acid sequence of a polypeptide with the same function as the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, deleting or inserting one or more amino acids; (3) An amino acid sequence that has at least 85% homology to the amino acid sequence shown in SEQ ID NO. 1 and has the same functional polypeptide.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2, or a nucleotide sequence encoding a polypeptide with the same function that is at least 80%, 85%, 90%, 95%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO.

2.

4. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule according to claim 2 or 3; the biological material comprises recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or a host cell.

5. The method for preparing the polypeptide according to claim 1, characterized in that: The method comprises: introducing the nucleic acid molecule according to claim 2 or 3 into a host cell to express the polypeptide.

6. The method according to claim 5, characterized in that The host cell is Escherichia coli.

7. Use of the polypeptide according to claim 1, the nucleic acid molecule according to claim 2 or 3, the biomaterial according to claim 4, or the polypeptide prepared by the method according to claim 5 or 6 in the preparation of an immunomodulatory preparation.

8. Use of the polypeptide according to claim 1, the nucleic acid molecule according to claim 2 or 3, the biomaterial according to claim 4, or the polypeptide prepared by the method according to claim 5 or 6 in the preparation of an antioxidant preparation.

9. Use of the polypeptide according to claim 1 or the polypeptide prepared by the method according to claim 5 or 6 in immunomodulation or anti-oxidation.

10. A product, characterized in that The product comprises the polypeptide according to claim 1 or comprises the polypeptide prepared by the method according to claim 5 or 6; Preferably, the product comprises a medicine, a health product, a food, a feed or a feed additive.

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