A hybrid peptide with immunoregulatory and antioxidant functions and a preparation method and application thereof

Abstract

The present application relates to the technical fields of genetic engineering and biological agents, and particularly relates to a hybrid peptide with immunoregulation and antioxidant functions, and a preparation method and application thereof.The polypeptide provided by the present application has both immunoregulation and antioxidant functions, and can significantly improve the immune function of the body and reduce the damage caused by immunosuppression to the body in normal or immunosuppressed states; and can enhance the antioxidant capacity of cells and the body; meanwhile, the polypeptide has the advantages of low cytotoxicity, high safety, simple preparation, low cost, and can be used as an ideal immunoregulator and antioxidant, and can be widely used in the fields of medicine, food, health care, feed, nutrition and the like of humans and animals, and has good application potential and value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biopharmaceutical technology, and in particular to a hybrid peptide with both immunomodulatory and antioxidant functions, its preparation method, and its application. Background Technology

[0002] The immune system is a key defense mechanism against infection, inflammation, and autoimmune diseases, primarily protecting the body by recognizing and eliminating pathogens. It consists of two main components: the innate immune system and the adaptive immune system. The innate immune response plays a crucial role as the body's first line of defense, capable of non-specifically recognizing and responding to various pathogens. Immunosuppression, as a temporary or permanent immune dysfunction, weakens the immune system, making tissues and organs more susceptible to infection and damage. Therefore, enhancing immune regulation has always been a key focus of research in the immune system.

[0003] A balanced redox response is crucial for maintaining normal physiological function. Disruption of this balance leads to the accumulation of electrophilic substances, triggering oxidative stress. The primary cause of oxidative stress is the excessive accumulation of reactive oxygen species (ROS), including O2• - Free radicals such as H2O2 and OH• can accumulate excessively and damage cellular DNA, proteins, and lipids, leading to cellular dysfunction and a range of diseases. Therefore, developing highly effective antioxidants, such as antioxidant peptides, to mitigate these harmful effects is of great importance.

[0004] Based on the important roles of immune regulation and antioxidation, the development of a novel, safe, side-effect-free, environmentally friendly bioactive peptide that simultaneously possesses immune regulation and antioxidation functions has significant practical implications and application value for human or animal husbandry, representing a new breakthrough and concept in anti-infection and antioxidation strategies. Summary of the Invention

[0005] This invention provides a hybrid peptide with both immunomodulatory and antioxidant functions, its preparation method, and its application.

[0006] To achieve the above objectives, this invention, based on extensive research into the sequences, structures, and sequence-structure-function relationship of peptides VLP (amino acid sequence as shown in SEQ ID NO. 3) and Aβ (amino acid sequence as shown in SEQ ID NO. 4), employed protein molecular design techniques to optimize the hybridization of peptides VLP and Aβ, and conducted in vitro and in vivo screening. Ultimately, a novel immunomodulatory and antioxidant hybrid peptide, named VLP-Aβ (VA), with its amino acid sequence shown in SEQ ID NO. 1, was obtained. The hybrid peptide VA possesses the functions of two parent peptides: immunomodulatory function (enhancing the body's immune function under normal or immunosuppressive conditions) and antioxidant function (enhancing the antioxidant function of cells and animal organisms).

[0007] In a first aspect, the present invention provides a polypeptide having any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO.1; (2) An amino acid sequence of a polypeptide with the same function obtained by substituting, deleting or inserting one or more amino acids as shown in SEQ ID NO.1; (3) An amino acid sequence that has at least 85% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function as the 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 modifying the amino acid sequence shown in SEQ ID NO.1 and having the same function as the heteropeptide 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 peptide for purposes such as easy purification and peptide labeling is a conventional technique in the field and will not affect the inherent function and activity of the peptide itself. Therefore, the VA derivative obtained by adding protein tag sequences to both ends of the heteropeptide VA as shown in SEQ ID NO.1 is also within the scope of protection of this invention.

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

[0011] Secondly, the present invention provides a nucleic acid molecule encoding the polypeptide.

[0012] Knowing the amino acid sequence of the polypeptide, 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 has a nucleotide sequence encoding a polypeptide with at least 80%, 85%, 90%, 95%, 98%, or 99% homology to the nucleotide sequence as shown in SEQ ID NO.2.

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

[0015] Thirdly, the present invention provides biological materials containing nucleic acid molecules encoding the polypeptide, said biological materials including recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, or host cells.

[0016] The host cell includes microbial cells, animal or plant cells, or cell lines. The animal or plant cells or cell lines cannot develop into individual animal or plant organisms.

[0017] Fourthly, the present invention provides a method for preparing the above-described polypeptide, the method comprising: introducing the above-described nucleic acid molecule into a host cell to express the polypeptide.

[0018] Preferably, the preparation method includes: linking the coding gene of the polypeptide 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 in which the coding gene of the polypeptide has been introduced.

[0019] The transgenic methods include heat stress transformation, electroconversion, and transfection.

[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 using Escherichia coli as the host cell, the heterozygous peptide is expressed using the polypeptide coding gene sequence optimized by E. coli codon preference, i.e., the sequence shown in SEQ ID NO.2, which has a better expression level.

[0023] In some embodiments of the present invention, the preparation of the polypeptide uses *Escherichia coli* as the host and pCOLD as the expression vector, and is induced by IPTG expression, specifically including the following steps: (1) The coding gene of the polypeptide is ligated into the expression vector pCOLD to construct a recombinant expression vector; (2) The above recombinant expression vector was transformed into Escherichia coli BL21(DE3) to construct a recombinant engineered bacterium with a polypeptide encoding gene introduced; (3) Cultivate the above recombinant engineered bacteria and add IPTG to induce the expression of polypeptides; (4) Collect the culture supernatant and purify it to obtain the polypeptide.

[0024] This invention demonstrates through in vivo and in vitro experiments that the aforementioned polypeptides can not only enhance immune activity in both normal and immunodeficient states, increase cytokine expression, promote mouse growth, and alleviate damage to the spleen and thymus of mice caused by immunodeficiency; but also reduce oxidative damage levels in HepG2 cells, improve cell survival, and alleviate liver damage in mice. Furthermore, by increasing the activity of antioxidant enzymes in the body, it protects liver function in mice, exhibiting a good bidirectional regulatory effect on both immunity and antioxidation.

[0025] Based on the above-described functions, the fifth aspect of the present invention provides the application of the above-described polypeptide or the nucleic acid molecule or the biomaterial or the polypeptide prepared by the preparation method in the preparation of immunomodulatory agents.

[0026] Preferably, the immunomodulatory agent is an immune enhancer.

[0027] The aforementioned immune enhancers can be used for the prevention and treatment of various types of immunosuppression, including immunosuppressive responses caused by cyclophosphamide (CY).

[0028] Sixthly, the present invention provides the application of the above-described polypeptide, the nucleic acid molecule, the biological material, or the polypeptide prepared by the preparation method in the preparation of antioxidant agents.

[0029] The aforementioned antioxidants 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 formulations described in this invention include pharmaceuticals, health products, food, feed, or feed additives.

[0031] In a seventh aspect, the present invention provides the application of the polypeptides described above or polypeptides prepared by the method described above in immune regulation or antioxidation.

[0032] Preferably, the immune regulation is immune enhancement. Immune enhancement refers to improving the body's immune function under normal or immunosuppressive conditions.

[0033] Preferably, the immune regulation and antioxidant effects are for non-disease treatment purposes.

[0034] Eighthly, the present invention provides a product comprising the polypeptide described above or a polypeptide prepared using the preparation method described above.

[0035] Preferably, the product includes pharmaceuticals, health products, food, feed, or feed additives.

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

[0037] Preferably, the product further includes a carrier or excipient component; for example, the drug may also include a pharmaceutically acceptable carrier or excipient.

[0038] The beneficial effects of this invention include at least the following: For the first time, this invention obtains a hybrid peptide by hybridizing VLP and Aβ, followed by optimization and screening. This hybrid peptide simultaneously possesses the functions of two parent peptides, namely, both immunomodulatory and antioxidant functions. Furthermore, compared with the corresponding activities of the parent peptides, its immunomodulatory and antioxidant activities are stronger. Under normal or immunosuppressive conditions, this hybrid peptide can significantly improve the body's immune function and reduce the damage caused by immunosuppression. It can also enhance the antioxidant capacity of cells and the body. At the same time, this hybrid peptide has the advantages of low cytotoxicity, high safety, simple preparation, and low cost. It can be used as an ideal immunomodulator and antioxidant, and can be widely used in the fields of medicine, food, health care, feed, and nutrition for humans and animals, showing great application potential and value.

[0039] The method for preparing heterozygous peptides provided by this invention can achieve the large-scale and efficient preparation of heterozygous peptides. The prepared heterozygous peptides have both immunomodulatory and antioxidant activities, and have no obvious toxic side effects. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

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

[0042] Figure 2 The fermentation curve of the engineered Escherichia coli in the fermenter in Example 1 of this invention is shown.

[0043] Figure 3 The purification results of the recombinant heterozygous peptide VA in Example 1 of this invention are shown.

[0044] Figure 4 This invention relates to the effects of the heterozygous peptide VA and its parent peptides Aβ (A) and VLP (V) on the cell survival rate of mouse macrophages in Example 2 of this invention.

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

[0046] Figure 6 This invention describes the immunomodulatory effect of the heterozygous peptide VA on cyclophosphamide-induced immunosuppressed mice in Example 4 of this invention; where a represents the effect of VA on the body weight of immunosuppressed mice; b represents the effect of VA on the thymus of immunosuppressed mice; c represents the effect of VA on the spleen of immunosuppressed mice; * indicates a significant difference between the two (p<0.5), ** indicates a highly significant difference between the two (p<0.01), *** indicates a highly significant difference between the two (p<0.001), and **** indicates a highly significant difference between the two (p<0.0001).

[0047] Figure 7This invention presents the regulatory effect of the heterozygous peptide VA on splenic lymphocytes in cyclophosphamide-induced immunosuppressed mice in Example 4 of this invention; where ah represents the effect of VA on the CD4+ / CD8+ ratio of lymphocytes, i represents the effect of VA on lymphocyte proliferation activity, and j represents the effect of VA on natural killer cell killing activity; the horizontal axis of ag is labeled CD4+, and the scales on the horizontal axis from left to right are: 0, 10. 3 10 4 The vertical axis title is CD8+, and the vertical axis scales from bottom to top are: 0, 10. 3 10 4 10 5 * indicates a significant difference between the two (p<0.5), ** indicates a highly significant difference between the two (p<0.01), *** indicates a highly significant difference between the two (p<0.001), and **** indicates a highly significant difference between the two (p<0.0001).

[0048] Figure 8 This describes the regulatory effect of the heterozygous peptide VA on immunoglobulins and cytokines in cyclophosphamide-induced immunosuppressed mice in Example 4 of this invention; where ac represents the effect of VA on immunoglobulins IgA, IgG, and IgM, and df represents the effect of VA on TNF-α, IL-6, and IL-1β; * indicates a significant difference between the two (p<0.5), and ** indicates an extremely significant difference between the two (p<0.01).

[0049] Figure 9 Figure 5 illustrates the alleviating effect of the heterozygous peptide VA on hydrogen peroxide-induced oxidative damage in HepG2 cells. Figure a shows the effect of VA on cell viability, bd shows the effect of VA on intracellular antioxidant enzyme activity (SOD, CAT, GSH-Px), e shows the effect of VA on intracellular malondialdehyde content, f and h show the effect of VA on intracellular ROS production, and g and i show the effect of VA on intracellular mitochondrial damage. * indicates a significant difference (p<0.5), and **** indicates an extremely significant difference (p<0.0001).

[0050] Figure 10This is the protective effect of the heterozygous peptide VA against carbon tetrachloride-induced liver injury in mice in Example 6 of the present invention; where a and b are the H&E staining results of VA's effect on alleviating liver fibrosis in mice, c is the Masson staining result of VA's effect on alleviating liver fibrosis in mice, d is the fluorescence staining result of VA's effect on alleviating apoptosis in mouse liver cells, and e and f are the effects of VA on the serum liver function indicators AST and ALT in mice; * indicates a significant difference between the two (p<0.5), ** indicates a highly significant difference between the two (p<0.01), *** indicates a highly significant difference between the two (p<0.001), and **** indicates a highly significant difference between the two (p<0.0001).

[0051] Figure 11 This refers to the effect of the heterozygous peptide VA in Example 6 of the present invention on the enhancement of antioxidant enzyme activity in carbon tetrachloride-induced liver injury in mice; where ac represents the effect of VA on the enhancement of antioxidant enzyme activity (SOD, CAT, GSH-Px) in mouse serum, d represents the effect of VA on malondialdehyde content in mouse serum, eg represents the effect of VA on the enhancement of antioxidant enzyme activity (SOD, CAT, GSH-Px) in mouse liver, and h represents the effect of VA on malondialdehyde content in mouse liver; * indicates a significant difference between the two (p<0.5), ** indicates a highly significant difference between the two (p<0.01), *** indicates a highly significant difference between the two (p<0.001), and **** indicates a highly significant difference between the two (p<0.0001). Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

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

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Among them, 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 heteropeptide VA 1. Construction of recombinant expression vectors Based on the amino acid sequence of the heterozygous peptide VA (as shown in SEQ ID NO.1) and the codon preference of *E. coli*, the encoding gene of the heterozygous peptide VA (as shown in SEQ ID NO.2) was designed and synthesized. This gene was then ligated into 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 As shown.

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

[0057] 3. Induction and purification of heterozygous peptide VA The engineered *E. coli* strain constructed in step 2 was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of LB liquid medium and cultured at 37°C and 180 rpm for 12 h as the seed culture for the fermenter. The 2 L fermenter contained 500 mL of liquid, with an inoculation rate of 1%. The fermentation temperature was 37°C, and air was introduced into the fermenter at a rate of 1 vvm. The dissolved oxygen level in the fermentation broth was maintained at no 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 solution. No feeding was performed for the first 4-5 h after fermentation began, and feeding was only started after the glucose in the medium was depleted. Results are shown below. Figure 2 Initially, an exponential fed-batch method was used for feeding. After 10 hours of E. coli growth, IPTG was added to begin induction, and the feeding method was switched to pH-stat. Induction continued for 24 hours, and thereafter, the pH-stat method was used for all feeding. After induction, the fermentation broth cells were collected and purified to obtain the heterozygous peptide VA. The expression level of VA reached 375 mg / L. The purification results of the recombinant heterozygous peptide VA are shown in [Figure number missing]. Figure 3 .

[0058] Example 2: Effect of heterozygous peptide VA on mouse macrophage cell survival RAW264.7 macrophages in logarithmic growth phase were seeded into 96-well plates at an initial cell density of 1 × 10⁻⁶ cells / well. 4Cells / mL, 100 μL per well, cultured overnight at 37℃ and 5% CO2. Then, a series of concentration gradients of heterozygous peptides VA, Aβ, and VLP (0-100 μg / mL) were added. After 24 h of culture, the effect of peptide VA on mouse macrophage survival was detected using the CCK8 assay. Results are as follows: Figure 4 As shown, the cytotoxicity of the heterozygous peptide VA was lower than that of its parent peptides Aβ and VLP, and the survival rate of macrophages was greater than 80% in the concentration range of 0-100 μg / mL, indicating that the heterozygous peptide VA has low cytotoxicity and high safety.

[0059] Example 3 Immunomodulatory activity of heterozygous peptide VA in mouse macrophages The hybrid peptide VA and its parent peptides VLP and Aβ were diluted with DMEM medium to prepare peptide solutions with a final concentration of 100 μg / mL. The effects of the hybrid peptide VA and its parent peptides VLP and Aβ on the secretion of cytokines such as TNF-α and IL-6 in mouse macrophages RAW264.7 under normal conditions were detected. Control, model (LPS), experimental group 1 (Aβ), experimental group 2 (VLP), and experimental group 3 (VA) were set up. The control group received no treatment, the model group received LPS to induce inflammation, and experimental groups 1, 2, and 3 were treated with Aβ, VLP, or VA solutions at a final concentration of 100 μg / mL after overnight cell culture. The levels of cytokines TNF-α, IL-6, and IL-1β were detected using ELISA. The results are shown below. Figure 5 As shown, the heterozygous peptide VA significantly increased the expression levels of cytokines TNF-α, IL-6, and IL-1β in mouse macrophages under normal conditions, and the expression levels of cytokines TNF-α, IL-6, and IL-1β in macrophages of the VA group were significantly higher than those in the Aβ and VLP groups. This indicates that the heterozygous peptide VA has immunomodulatory effects, can enhance the immune activity of cells under normal conditions, and its immune-enhancing effect is superior to that of its parent peptides Aβ and VLP.

[0060] Example 4: Immunomodulatory effect of heterozygous peptide VA on immunosuppressed mice This embodiment uses male BALB / c mice (weighing 20-22g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) for animal experiments. The entire experimental process follows the guidelines of the European Committee on Laboratory Animal Ethics (86 / 609 / EEC) and is authorized by the Laboratory Animal Ethics Committee of China Agricultural University. The animal housing environment is clean-grade, with an ambient temperature of 22±2℃, humidity of 50%~55%, and light from 8:00 to 20:00. Six mice are housed per cage, with free access to food and water.

[0061] Mice were randomly assigned to seven groups (n=6 per group): control (CON), CTX group (50 mg / kg / day), CTX + LMS (levamisole hydrochloride) group (20 mg / kg / day), CTX + Aβ group (parental peptide Aβ, 10 mg / kg / day), CTX + VLP group (parental peptide VLP, 10 mg / kg / day), CTX + VAL group (hybrid peptide VA, low dose, 5 mg / kg / day), and CTX + VAH group (hybrid peptide VA, high dose, 10 mg / kg / day). Mice were acclimatized for 5 days before the start of the formal experiment. Except for the control group, all mice in all groups received intraperitoneal injections of CTX at a dose of 50 mg / kg / day from day 1 to day 3 of the formal experiment. The control group received an equal volume of saline. From day 4 to day 10, mice were treated daily with the designated drug or peptide (the control and CTX groups were again injected with saline). Sixteen hours after the final treatment, the mice were euthanized and their blood, spleen, and thymus tissue were collected.

[0062] 1. Effects of heterozygous peptide VA on body weight and immune organ weight in immunosuppressed mice After carefully removing fat and connective tissue, the spleen and thymus tissue were weighed. The formula for calculating the organ index is: The results are as follows Figure 6 As shown, the results indicated that the body weight, spleen index, and thymus index of the model group mice were significantly lower than those of the control group, indicating that cyclophosphamide could inhibit mouse growth, reduce immune organ indices, and suppress immunity. In contrast, the body weight, spleen index, and thymus index of the VAL and VAH experimental groups returned to normal levels and were even higher than those of the control group, indicating that the heterozygous peptide VA could not only improve the immunity level of mice but also promote their growth.

[0063] 2. Effects of heterozygous peptide VA on spleen immune cells in immunosuppressed mice The results are as follows Figure 7 As shown, flow cytometry was used to analyze splenic T cell subsets, with CD3+ labeling of all T cells and CD4+ and CD8+ labeling of two subsets. The proliferative activity of splenic lymphocytes was measured using LPS and ConA. The cytotoxic activity of natural killer cells was measured using K562 cells as target cells. The results showed that the heterozygous peptide VA could reduce the CTX-induced decrease in the CD4+ / CD8+ ratio of mouse splenic lymphocytes, while also increasing lymphocyte proliferative activity. Furthermore, the heterozygous peptide VA could also enhance the cytotoxic activity of natural killer cells. These results indicate that the heterozygous peptide VA can enhance the immune activity of immune cells in the mouse spleen.

[0064] 3. Effects of heterozygous peptide VA on cytokine release in immunosuppressed mice Mouse blood was collected and allowed to stand at room temperature for 2 hours, then centrifuged at 5000 rpm for 20 minutes at 4°C. The supernatant was collected as the serum sample. Cytokines (TNF-α, IL-6, IL-1β) and immunoglobulins (IgG, IgA, IgM) in the serum were measured using an ELISA kit. The experimental results are as follows: Figure 8 As shown, the results indicated that, compared with the control group, the levels of immunoglobulins and cytokines in the CTX group mice were significantly reduced; while the administration of the heterozygous peptide VA significantly increased the levels of cytokines and immunoglobulins in the serum of immunosuppressed mice, thereby enhancing the immune activity of immunocompromised mice.

[0065] Example 5: Alleviating effect of heterozygous peptide VA on hydrogen peroxide-induced oxidative damage in HepG2 cells Modeling of hydrogen peroxide-induced oxidative damage in HepG2 cells: HepG2 cells were distributed at a density of 2 × 10⁶ cells per well. 5 Cells were seeded into 6-well plates and incubated for 12 h. The culture medium in each well was aspirated and replaced with 1 mL of VLP, Aβ and VA (100 ng / mL) diluted with basal DMEM and incubated for another 2 h. Then, hydrogen peroxide (650 μM) diluted with DMEM was added to each well and incubated for 6 h.

[0066] The results are as follows Figure 9 As shown, the heterozygous peptide VA can improve cell survival rate induced by hydrogen peroxide, while also increasing the activity levels of intracellular antioxidant enzymes (SOD, CAT, GSH-Px) and reducing intracellular malondialdehyde (MDA) levels. Furthermore, VA can reduce the increase in intracellular ROS levels induced by hydrogen peroxide and protect mitochondria from damage. These results indicate that the heterozygous peptide VA can alleviate oxidative stress damage in cells at the cellular level.

[0067] Example 6: Protective effect of heterozygous peptide VA against carbon tetrachloride (CCl4)-induced liver injury in mice. This embodiment uses male C57BL / 6 mice (weighing 20-22g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) for animal experiments. The entire experimental process follows the guidelines of the European Committee on Laboratory Animal Ethics (86 / 609 / EEC) and is authorized by the Laboratory Animal Ethics Committee of China Agricultural University. The animal housing environment is clean-grade, with an ambient temperature of 22±2℃, humidity of 50%-55%, and light from 8:00 to 20:00. Six mice are housed per cage, with free access to food and water.

[0068] Mice were randomly assigned to seven treatment groups (n=6 per group): control group, CCl4 group (2% CCl4 prepared with olive oil, 5 mL / kg), CCl4 + silymarin group (Silybum, 20 mg / kg), CCl4 + Aβ group (parent peptide Aβ, 10 mg / kg), CCl4 + VLP group (parent peptide VLP, 10 mg / kg), CCl4 + VAL group (hybrid peptide VA, low dose, 5 mg / kg), and CCl4 + VAH group (hybrid peptide VA, high dose, 10 mg / kg). Mice were acclimatized for 5 days prior to the experiment. From day 1 to day 7, each group of mice received an intraperitoneal injection of the corresponding drug or peptide, while the control group and CCl4 group received an equal volume of saline. Two hours after the completion of the drug and peptide injections 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, the 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 in mice induced liver fibrosis and increased hepatocyte apoptosis, while treatment with the heterozygous peptide VA reduced liver fibrosis and decreased hepatocyte apoptosis. Furthermore, VA treatment restored serum levels of liver function indicators ALT and AST, protecting normal liver function. In addition, VA increased the activity of antioxidant enzymes (SOD, CAT, GSH-Px) in the liver and serum of mice and decreased malondialdehyde levels. These results indicate that the heterozygous peptide VA can alleviate carbon tetrachloride-induced oxidative damage to the liver in mice.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a polypeptide or a nucleic acid molecule encoding said polypeptide or a biological material comprising a nucleic acid molecule encoding said polypeptide for the manufacture of an antioxidant preparation, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1; the biological material includes recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector or host cell; The antioxidant is used to prevent and treat carbon tetrachloride-induced liver damage.

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