Polypeptide with improved immunity, anti-tumor and prolonged life and application thereof

Abstract

The application discloses a polypeptide with improved immunity, anti-tumor and prolonged life and application thereof. The polypeptide with the above functions is selected from the following: (a) a polypeptide with an amino acid sequence of MNKAELIDVLTQKLGSDRRQATAAVENVVD; (b) a polypeptide with an amino acid sequence of TIVRAVHKGDSVTITGFGVFEQRRRAARVA; (c) a polypeptide with an amino acid sequence of RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; (g) a polypeptide with an amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA; and (h) a polypeptide with an amino acid sequence of RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA. The polypeptide with improved immunity, anti-tumor and prolonged life can improve the immunity of the organism, enhance the disease resistance, reduce the tumor incidence and prolong the life.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a polypeptide that enhances immunity, has anti-tumor effects, and prolongs lifespan, and its applications. Background Technology

[0002] In classical immunological theory, T cells develop into CD4+ cells in the thymus. + Helper T cells and CD8 + Two major subsets of cytotoxic T cells. Recent studies have found that some CD4+ cells... + T cells can further differentiate into CD8-like cells outside the thymus. + CTLs and a subset of NK cells that perform direct cytotoxic functions, namely CD4 + CTLs. Currently, CD4 + CTLs have been further confirmed to play important roles in infection, tumors, autoimmune diseases, and vaccination. They have been widely reported, especially in infectious diseases including influenza virus, cytomegalovirus, EBV, human papillomavirus, and HIV. Some literature suggests that researchers believe CD4... + An increase in the number and virulence of CTLs may indicate enhanced overall immunity. Changes in this cell subset can serve as an indicator of immune function strength. It has been found that the proportion of this cell subset is significantly higher in extremely long-lived individuals than in normal individuals, suggesting that an increase in the proportion of this cell subset is associated with longevity. Monocytes, according to previous studies, can also be divided into three subsets: intermediate type (CD14...). ++ CD16 + Classic (CD14) ++ CD16 - ) and non-classical (CD14) + CD16 ++ Among them, the classic type (CD14) ++ CD16 - ) accounts for approximately 80%-90% of the total number of monocytes in the peripheral blood of healthy individuals and plays an important role in the innate immune defense mechanism; intermediate type (CD14) ++ CD16 + They account for approximately 5% of the total number of monocytes, but can reach 10%-50% in severe infections and inflammatory states. Their main functions include antigen presentation, inflammation, and monocyte activation; they are also known as the inflammatory monocyte subset. The non-classical type (CD14) + CD16 ++Migrant monocytes, comprising approximately 5%-10% of the total monocyte population, can migrate across the vascular endothelial surface to clear foreign substances. They possess immune surveillance functions and play a crucial role in early inflammatory responses and tissue repair; they are also known as migratory monocyte subsets. Changes in the proportion of different monocyte subsets indicate enhanced function of the corresponding subset, providing clues to changes in the body's immune function.

[0003] In recent years, the incidence of malignant tumors, characterized by abnormal cell proliferation, has increased significantly. According to the World Health Organization, one in five men and one in six women worldwide will develop malignant tumors. Nearly half of all new malignant tumor cases and more than half of all malignant tumor deaths globally occur in Asia, particularly China. Malignant tumors are further divided into solid tumors and non-solid tumors. Common solid tumors include lung cancer, stomach cancer, and breast cancer, while common non-solid tumors include leukemia and lymphoma. Currently, there is no effective cure for malignant tumors; treatment mainly relies on surgery, radiotherapy, and chemotherapy. However, the harm caused to patients by these treatments cannot be ignored. The anti-tumor effect of drugs on solid tumors can be assessed by calculating the tumor inhibition rate based on tumor size. For non-solid tumors, the inhibition rate of drugs on tumor cell proliferation can be assessed through in vitro experiments. How to enhance efficacy, reduce side effects, and improve patients' quality of life through the combination of different treatment regimens is a crucial aspect of the treatment of malignant tumors.

[0004] Aging is an inevitable biological process occurring in all organisms, characterized by its gradual, harmful, and universal nature. With societal progress, the demand for anti-aging and lifespan extension is constantly increasing. Anti-aging drugs refer to a class of medications that improve life efficiency, enhance physical condition, prevent and treat age-related diseases, and extend lifespan within the limits determined by genetic characteristics. These drugs prevent and delay the aging process, improve pathological disorders of tissue cells, regulate the function of vital organs, and balance the internal environment, thus promoting overall health. Their characteristic is that they work through multi-level, multi-faceted, and relatively long-term effects to adjust the body's material, metabolic, and organ functional states, thereby achieving the goal of delaying aging and increasing lifespan.

[0005] Currently, the demand for extended lifespan and improved quality of life among Chinese residents is constantly increasing. Based on our previous research, we have discovered a polypeptide that can stimulate the proliferation of subpopulations of cells, thereby enhancing human immunity, significantly inhibiting tumor cells, and extending individual lifespan. When this polypeptide is formulated into a vaccine and administered to humans, it can produce multiple functions including enhanced immunity, anti-tumor activity, and lifespan extension. Summary of the Invention

[0006] The first objective of this invention is to provide a polypeptide that enhances immunity, has anti-tumor effects, and prolongs lifespan; the second objective is to provide applications of the aforementioned polypeptide that enhances immunity, has anti-tumor effects, and prolongs lifespan.

[0007] The first objective of this invention is achieved by selecting the polypeptides that enhance immunity, have anti-tumor effects, and prolong lifespan from:

[0008] (a) A polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD (polypeptide 1); or

[0009] (b) A polypeptide having the amino acid sequence TIVRAVHKGDSVTITGFGVFEQRRRAARVA (polypeptide 2); or

[0010] (c) A polypeptide (polypeptide 3) having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or

[0011] (d) A polypeptide (peptide 4) having the amino acid sequence VNKAELIDVLTGGLGSKRRQATAAVEGGVD; or

[0012] (e) A polypeptide having the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD (polypeptide 5); or

[0013] (f) A polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or

[0014] (g) A polypeptide having the amino acid sequence TIVRAVGKGDSGITIGFGVFERQRRAARVA; or

[0015] (h) A polypeptide having the amino acid sequence RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA; or

[0016] (i) A polypeptide having the amino acid sequence KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.

[0017] The polypeptides described in this invention, which enhance immunity, fight tumors, and prolong life, can improve the body's immunity, strengthen disease resistance, reduce the incidence of tumors, and prolong life.

[0018] The polypeptide component described in this invention can stimulate the body's immune cells, thereby affecting certain immune cell subsets (such as CD4+). + CTLs, CD14 + CD16 ++The increased proportion of these immune cell subsets has been shown in previous studies to enhance the body's immune function, increase resistance to various diseases, and reduce morbidity, thereby achieving the goal of enhancing the body's disease resistance. This polypeptide can also enhance the body's anti-tumor ability and prolong lifespan. Verification showed that polypeptides 1, 2, and 3, with different structures, all have the functions of enhancing immunity, enhancing the body's anti-tumor ability, and prolonging lifespan, with polypeptide 1 showing the most significant effect. Partial alterations to the amino acid sequences of the three groups of polypeptides yielded polypeptides 4 and 5, polypeptides 6 and 7, and polypeptides 8 and 9, respectively. Verification showed that the sequence-altered polypeptides still possessed the functions of enhancing immunity, strengthening the body's anti-tumor ability, and prolonging lifespan. Therefore, we speculate that polypeptides with 80% or more of the same amino acid sequence as polypeptides 1, 2, and 3 have similar functions. Attached Figure Description

[0019] Figure 1 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 1 of the present invention;

[0020] Figure 2 CD4 before and after stimulation with peptide 1 of the present invention + A diagram illustrating the changes in the proportion of CTLs;

[0021] Figure 3 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 1 of the present invention;

[0022] Figure 4 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 2 of the present invention;

[0023] Figure 5 CD4 before and after stimulation with peptide 2 of the present invention + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0024] Figure 6 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 3 of the present invention;

[0025] Figure 7 The present invention relates to CD4 before and after stimulation by peptide 3. + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0026] Figure 8 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after peptide 2 intervention according to the present invention;

[0027] Figure 9 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after peptide 3 intervention according to the present invention;

[0028] Figure 10 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 4 of the present invention;

[0029] Figure 11 CD4 before and after stimulation with peptide 4 of the present invention + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0030] Figure 12 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with peptide 5 of the present invention;

[0031] Figure 13 The present invention relates to CD4 before and after stimulation by peptide 5. + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0032] Figure 14 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 4 of the present invention;

[0033] Figure 15 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 5 of the present invention;

[0034] Figure 16 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 6 of the present invention;

[0035] Figure 17 The present invention relates to CD4 before and after stimulation by peptide 6. + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0036] Figure 18 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 7 of the present invention.

[0037] Figure 19 CD4 before and after stimulation with peptide 7 of the present invention + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0038] Figure 20 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after peptide 6 intervention according to the present invention;

[0039] Figure 21 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 7 of the present invention;

[0040] Figure 22 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 8 of the present invention.

[0041] Figure 23 The present invention relates to CD4 before and after stimulation by peptide 8. + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0042] Figure 24 This is a schematic diagram showing the changes in monocyte subsets before and after stimulation with polypeptide 9 of the present invention;

[0043] Figure 25 The present invention relates to CD4 before and after stimulation by peptide 9. + CD4 in cells + A diagram illustrating the changes in the proportion of CTLs;

[0044] Figure 26 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 8 of the present invention;

[0045] Figure 27 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after intervention with peptide 9 of the present invention. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0047] The polypeptides described in this invention that enhance immunity, have anti-tumor effects, and prolong lifespan are selected from:

[0048] (a) A polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD; or

[0049] (b) A polypeptide having the amino acid sequence TIVRAVHKGDSVTITGFGVFEQRRRAARVA; or

[0050] (c) A polypeptide having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or

[0051] (d) A polypeptide having the amino acid sequence VNKAELIDVLTGGLGSKRRQATAAVEGGVD; or

[0052] (e) A polypeptide having the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD; or

[0053] (f) A polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or

[0054] (g) A polypeptide having the amino acid sequence TIVRAVGKGDSGITIGFGVFERQRRAARVA; or

[0055] (h) A polypeptide having the amino acid sequence RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA; or

[0056] (i) A polypeptide having the amino acid sequence KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.

[0057] The nucleic acids described in this invention that have the functions of enhancing immunity, fighting tumors, and prolonging lifespan are selected from:

[0058] (a) A nucleic acid encoding the polypeptide as described in claim 1; or

[0059] (b) Nucleic acids complementary to nucleic acid sequences (a), (b), and (c).

[0060] The carrier described in this invention contains nucleic acids that have functions such as enhancing immunity, fighting tumors, and prolonging life.

[0061] The host cell of the present invention is characterized in that it contains a vector containing nucleic acids that have functions of enhancing immunity, fighting tumors, and prolonging lifespan, or its genome integrates nucleic acids that have functions of enhancing immunity, fighting tumors, and prolonging lifespan.

[0062] The drug is a polypeptide vaccine containing polypeptide sequences that have functions such as enhancing immunity, fighting tumors, and prolonging life.

[0063] The application of the polypeptide with functions of enhancing immunity, anti-tumor, and prolonging life, as described in this invention, is the application of the polypeptide with the function of enhancing immunity in drugs that enhance immunity.

[0064] The application of the polypeptide with functions of enhancing immunity, anti-tumor, and prolonging life, as described in this invention, is the application of the polypeptide with the function of enhancing immunity in the preparation of drugs that prolong life.

[0065] The application of the polypeptide with functions of enhancing immunity, anti-tumor activity, and prolonging lifespan described in this invention is the application of the polypeptide with enhanced immunity in the preparation of anti-tumor drugs.

[0066] The tumor in question can be a solid tumor or a non-solid tumor.

[0067] The solid tumor is gastric cancer; the non-solid tumor is acute lymphoblastic leukemia.

[0068] The invention will be further illustrated below with specific implementation experimental examples:

[0069] I. The specific experimental procedures for polypeptide 1 to enhance human immunity are as follows:

[0070] 1. Inclusion criteria: (1) Age between 18 and 60; (2) Normal population (normal liver and kidney function tests, excluding: tuberculosis, HBV, HCV, tumors and autoimmune diseases).

[0071] 2. Experimental materials: Human peripheral venous blood; human peripheral blood mononuclear cell separation medium; PBS buffer; fetal bovine serum; penicillin-streptomycin solution (double antibody); PE-CD14 antibody, FITC-CD16 antibody, Perpcy-5.5-CD45 antibody, AlexaFlour647-GB11 antibody, FITC-CD3 antibody, PE-CD4 antibody; erythrocyte lysis buffer; flow cytometry membrane permeation and fixation solution.

[0072] 3. Experimental procedure: (1) Synthesize polypeptide 1 in vitro according to its sequence; (2) Extract peripheral blood PBMCs: a) Draw 5ml of peripheral blood from the elbow of 6 healthy individuals into EDTA anticoagulant tubes, detect monocyte subsets in 3 cases, and detect CD4 in 3 cases. + CTL subsets; b) Dilute 5 ml of blood to 10 ml with PBS; c) Take 5 ml of peripheral blood mononuclear cell separation medium into centrifuge tubes, add 5 ml of diluted blood to the top of the separation medium; d) 1000g, acceleration 4, deceleration 4, centrifugation for 25 minutes; e) carefully aspirate PBMC layer cells with a pipette, dilute with erythropoietin solution, centrifuge, remove supernatant, repeat twice; f) resuspend cells in prepared cell culture medium (10ul double antibody + 90ul fetal bovine serum + 900ul 1640); g) divide each sample of cells into experimental group and control group, 500ul each; (3) dissolve peptide 1 powder in PBS to a concentration of 1mg / ml; (4) add 100ul peptide 1 solution to the experimental group and let stand for 12h; (5) flow cytometry detection: a) resuspend cells in 1ml erythropoietin solution, centrifuge, and remove supernatant; b) resuspend cells in 100ul erythropoietin solution, add CD14, CD16, and CD45 antibodies to the monocyte group and incubate in the dark for 30min; CD4 + CTLs cells were incubated with CD3 and CD4 antibodies in the dark for 30 minutes; c) CD4 + d) For CTLs, add membrane permeabilization and fixation solution and incubate in the dark for 30 min; d) For monocytes, add 1 ml of erythropoietin solution to samples, centrifuge, discard supernatant, and CD4 + e) For the CTLs cell group, the cells were directly centrifuged and the supernatant was discarded; e) For the mononuclear cell group, the cells were resuspended in 200 μL of lysinic globulin and analyzed by instrumentation, CD4 +CTLs cells were incubated with 100 μL of cell permeabilization and fixation solution and GB11 antibody for 30 min in the dark; f) CD4 + Add 1 ml of cell permeabilization and fixation buffer to the CTLs cell group, centrifuge, and discard the supernatant; g) CD4 + CTLs cells were resuspended in 100 μL of cell permeabilization and fixation solution and then analyzed.

[0073] 4. Experimental Results: After stimulation with peptide 1, non-classical CD14+ β-cell progeny in monocytes... + CD16 ++ CD4 in T cells + The proportion of CTL subsets increased significantly, suggesting enhanced cellular immune function.

[0074] 5. Statistical analysis was performed on the changes in cell subsets after stimulation with peptide 1. All quantitative data are expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 software. Paired t-tests were used for data that followed a normal distribution; Wilcoxon tests were used for data that did not follow a normal distribution. A p-value < 0.05 was considered statistically significant.

[0075] (1) Analysis of changes in monocyte subsets after stimulation with polypeptide 1

[0076] The percentage of monocytes in the peptide 1-stimulated group was (19.25±8.18)%, while that in the control group was (15.79±9.08)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 1-stimulated group was (3.85±2.82)%, while that in the control group was (5.61±3.88)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 1-stimulated group was (93.42±0.69)%, while that in the control group was (85.27±7.42)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 1-stimulated group was (22.77±5.82)%, while that in the control group was (18.85±4.82)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 1 stimulation compared to the control group. Figure 1 ).

[0077] (2) CD4 after stimulation by peptide 1 + Analysis of CTL cell subset changes

[0078] Peptide 1 stimulated group CD4 + CTLs in CD4 +The percentage of CD4 in the cells was (3.43±0.21)%, compared to (0.97±0.04)% in the control group. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 2 ).

[0079] II. Experimental study on the extension of lifespan of C57BL / 6J mice by peptide 1:

[0080] 1. Inclusion criteria: 40 specific pathogen-free (SPF) grade 8-week-old C57BL / 6J mice (the lifespan of healthy mice of this strain can reach 18-24 months, and up to 3 years). The housing environment is 12h light / 12h dark, the ambient temperature is controlled at 22-25℃, and the humidity is controlled at 50-60%. 1 mouse / cage. After the animals arrive at the experimental environment, their growth and activity are observed. Drug administration begins at 1 month of age.

[0081] 2. Experimental procedure: (1) Synthesize polypeptide 1 in vitro according to its sequence; (2) Dissolve polypeptide 1 powder in sterile saline solution to a concentration of 0.4 mg / ml, ultrafilter to a sterile solution and store at -20℃ for sterile preservation, and restore to room temperature before use; (3) Mice are randomly divided into polypeptide administration group and solvent control group, 20 mice in each group; (4) The polypeptide administration group is injected with polypeptide 1 at a dose of 6 mg / kg, once every 3 days, and administered via tail vein at the same time (9:00-11:00), while the solvent control group is given the corresponding amount of saline; (5) The mice are administered the drug until they die naturally, and the time of death and the most likely cause of death are recorded; (6) Remove mice that die due to accidental operation or external causes, and use Kaplan-Meier statistical method to perform survival analysis and draw survival curves.

[0082] 3. Experimental Results: Statistical analysis was performed on the changes in mouse lifespan after peptide 1 stimulation. Quantitative data are expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 software. Paired t-tests were used for data that followed a normal distribution; Wilcoxon tests were used for those that did not. A p-value < 0.05 was considered statistically significant. Survival curves were analyzed using the Kaplan-Meier statistical method (…). Figure 3 ).

[0083] The mouse lifespan observation experiment showed that the average lifespan of mice in the polypeptide 1 injection group was (26.64±1.72) months, and the median lifespan was (21.00±7.83) months; the average lifespan of mice in the control group was (16.90±1.01) months, and the median lifespan was (17±0.89) months. The average and median lifespans of mice in the polypeptide 1 injection group were significantly higher than those in the control group (P<0.05). This indicates that polypeptide 1 intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging (Table 1).

[0084] Table 1. Mean and median survival time of mice before and after peptide 1 intervention

[0085] .

[0086] 4. Conclusion: Intervention with this polypeptide 1 can delay aging and prolong the lifespan of mice.

[0087] III. Experimental study on the anti-solid tumor efficacy of peptide 1:

[0088] 1. Experimental materials: 40 male nude mice weighing 18-22g; tumor cell line: gastric cancer MGC-803; peptide 1 powder was dissolved in physiological saline to a final concentration of 1mg / ml, filtered through a 0.22-micron filter membrane and aseptically dispensed, stored at 4℃ for later use; trastuzumab and docetaxel injection.

[0089] 2. Experimental Methods: MGC-803 cell lines were cultured at 37℃ in a 5% CO2 incubator until the cell density reached over 80%. The culture medium was DMEM high-glucose medium containing 10% FBS. Cells were digested with 0.25% trypsin and collected. After centrifugation at 1000 rpm, the supernatant was discarded, and the cells were washed three times with physiological saline and resuspended to count up to 5 x 10⁻⁶ cells. 7 / ml, store at 4℃ for later use.

[0090] 3. Experimental procedure: (1) Mice were randomly divided into 4 groups: blank control group, polypeptide group, combined drug group, and positive control group, with 10 mice in each group. 0.2 ml of the prepared cancer cell line suspension was injected subcutaneously into the right axilla of each mouse. The tumor growth was observed every three days after inoculation. The tumor volume reached 0.1 mm. 3(2) The positive control group was given intravenous injection of trastuzumab 15ml / kg once every 3 days; docetaxel 5mg / kg once every 7 days; the blank control group was given intravenous injection of normal saline 10ml / kg once every 3 days; the polypeptide group was given intravenous injection of prepared polypeptide 1 solution 1mg / kg once every 3 days; the combined drug group was given intravenous injection of trastuzumab 15ml / kg once every 3 days; docetaxel 5mg / kg once every 7 days; prepared polypeptide 1 solution 1mg / kg once every 3 days. (3) The tumor volume was measured 21 days after administration. The tumor inhibition rate (%) was calculated according to the formula: (tumor volume of blank control group - tumor volume of drug group) / tumor volume of blank control group.

[0091] 4. Experimental Results: Statistical analysis was performed on the changes in tumor inhibition rate after peptide 1 injection. Measurement data are expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 software. Paired t-tests were used for data that followed a normal distribution; Wilcxon tests were used for data that did not follow a normal distribution. A p-value < 0.05 was considered statistically significant.

[0092] The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 1 injection group was (67.62±0.90)%, and the average tumor inhibition rate of the combined drug group was (74.06±1.38)%. The average tumor inhibition rate of the combined drug group was higher than that of the positive control group and the polypeptide 1 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 1 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05) (Table 2).

[0093] 5. Conclusion: Injection of polypeptide 1 can inhibit tumor growth, and its combined use with existing antitumor drugs can enhance their antitumor effects.

[0094] Table 2. Comparison of tumor inhibition rates of different drug interventions for solid tumors

[0095] .

[0096] IV. Experimental Study on the Efficacy of Peptide 1 against Non-Solid Tumors

[0097] 1. Experimental materials: Jurkat cells for acute lymphoblastic leukemia; fetal bovine serum; RP-MI1640 culture medium; 5-fluorouracil (5-FU); using DMSO as solvent, polypeptide 1 powder was prepared into polypeptide solutions of 10ug / ml, 20ug / ml, and 30ug / ml.

[0098] 2. Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator with saturated humidity. The medium was changed every 2 days. (2) MTT assay to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and seeded in 96-well plates, 2 x 10 cells per well. 5 Cells were cultured at 200 μL at 37°C and 5% CO2 for 24 h. After removing the supernatant, experimental groups A, B, and C were established, with 200 μL of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml peptide 1 solution added, respectively. A blank control group (without peptide 1) and a cell-free control group were also established. The commercially available anticancer drug 5-FU (5-fluorouracil) was used as a positive control for anticancer activity. Three accessory wells were set up for each group. After culturing for 4 h (under the same conditions), 20 μL of 5 mg / ml MTT reagent was added to each well. After culturing for another 4 h, the supernatant was discarded, and 150 μL of DMSO was added. The cells were shaken to dissolve, and the OD570 (absorbance value) at 570 nm was measured using a microplate reader. The inhibition rate (%) was calculated as follows: Inhibition rate (%) = (OD570 of control group - OD570 of experimental group) x 100% / OD570 of control group.

[0099] 3. Experimental results: The MTT assay showed that different concentrations of peptide 1 inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 1 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 3).

[0100] Table 3. Comparison of inhibition rates of different concentrations of peptide 1 with Jurkat cells in vitro.

[0101] .

[0102] V. We believe that polypeptides 2 and 3, with different structures, also have similar functions. The results of the verification according to the above experimental protocol are as follows:

[0103] 1. Experiment on how peptides enhance human immunity (steps are the same as in Experiment 1)

[0104] (1) Polypeptide 2

[0105] (a) Analysis of changes in monocyte subsets after stimulation with polypeptide 2

[0106] The percentage of monocytes in the peptide 2-stimulated group was (18.51±8.60)%, while that in the control group was (15.80±9.08)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 2-stimulated group was (4.75±2.93)%, while that in the control group was (5.61±3.65)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 2-stimulated group was (87.95±5.10)%, while that in the control group was (81.23±10.00)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 2-stimulated group was (25.03±2.36)%, while that in the control group was (15.80±9.08)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 2 stimulation compared to the control group. Figure 4 ).

[0107] (b) CD4 after stimulation with peptide 2 + Analysis of CTL cell subset changes

[0108] Peptide 2 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (3.42±0.38)%, while in the control group it was (0.97±0.06)%. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 5 ).

[0109] (2) Polypeptide 3

[0110] (a) Analysis of changes in monocyte subsets after stimulation with polypeptide 3

[0111] The percentage of monocytes in the peptide 3-stimulated group was (24.32±9.47)%, while that in the control group was (22.33±4.55)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 3-stimulated group was (8.85±0.81)%, while that in the control group was (9.63±0.70)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 3-stimulated group was (90.29±1.78)%, while that in the control group was (87.71±6.31)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 3-stimulated group was (27.61±2.63)%, while that in the control group was (23.94±2.81)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 3 stimulation compared to the control group. Figure 6 ).

[0112] (b) CD4 after stimulation by peptide 3 + Analysis of CTL cell subset changes

[0113] Peptide 3 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (3.28±0.41)%, compared to (0.96±0.06)% in the control group. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 7 ).

[0114] 2. Experimental study on prolonging the lifespan of C57BL / 6J mice (steps same as Experiment 2):

[0115] (1) Polypeptide 2

[0116] (a) Experimental results: The average lifespan of mice in the polypeptide 2 injection group was (25.23±0.95) months, and the median lifespan was (22.00±3.91) months; the average lifespan of mice in the control group was (19.80±0.99) months, and the median lifespan was (19.00±1.12) months. The average and median lifespans of mice in the polypeptide 2 injection group were significantly higher than those in the control group (P<0.05) (Table 4). This indicates that polypeptide 2 intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0117] (b) Conclusion: Peptide 2 has similar functions to peptide 1, and both can prolong the lifespan of mice and delay the aging of the body.

[0118] Table 4. Mean and median survival time of mice before and after peptide 2 intervention

[0119] .

[0120] (2) Polypeptide 3

[0121] (a) Experimental results: The average lifespan of mice in the polypeptide 3 injection group was (25.41±1.45) months, and the median lifespan was (21.00±2.17) months; the average lifespan of mice in the control group was (18.55±0.87) months, and the median lifespan was (18±0.74) months. The average and median lifespans of mice in the polypeptide 3 injection group were significantly higher than those in the control group (P<0.05) (Table 5). This indicates that polypeptide 3 intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0122] (b) Conclusion: Peptide 3 has similar functions to peptide 1, and both can prolong the lifespan of mice and delay the aging of the body.

[0123] Table 5. Mean and median survival time of mice before and after peptide 3 intervention

[0124] .

[0125] 3. Experimental study on anti-solid tumor efficacy (steps same as Experiment 3):

[0126] (a) Experimental results: The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 2 injection group was (65.12±0.81)%, and the average tumor inhibition rate of the combined drug 2 group was (68.07±1.02)%. The average tumor inhibition rate of the combined drug 2 group was higher than that of the positive control group and the polypeptide 2 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 2 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 3 injection group was (66.68±1.01)%, and the average tumor inhibition rate of the combined drug 3 group was (70.22±0.88)%. The average tumor inhibition rate of the combined drug 3 group was higher than that of the positive control group and the polypeptide 3 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 3 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05) (Table 6).

[0127] (b) Conclusion: Both peptide 2 and peptide 3 injections can inhibit tumor growth, and their combined use with existing antitumor drugs can enhance the antitumor effect of the drugs.

[0128] Table 6. Comparison of tumor inhibition rates of different drug interventions for solid tumors

[0129] .

[0130] 4. Experimental studies on efficacy against non-solid tumors:

[0131] (1) Polypeptide 2

[0132] (a) Experimental materials: acute lymphoblastic leukemia Jurkat cells; fetal bovine serum; RP-MI1640 culture medium; 5-fluorouracil; using DMSO as solvent, polypeptide 2 powder was prepared into polypeptide solutions of 10ug / ml, 20ug / ml and 30ug / ml.

[0133] (b) Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator with saturated humidity. The medium was changed every 2 days. (2) MTT assay to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and seeded in 96-well plates, 2 x 10 cells per well. 5 Cells were cultured at 200 μL at 37°C and 5% CO2 for 24 h. After removing the supernatant, experimental groups A, B, and C were established, with 200 μL of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml peptide 2 solution added, respectively. A blank control group (without peptide 2) and a cell-free control group were also established. The commercially available anticancer drug 5-FU (5-fluorouracil) was used as the control group for anticancer activity. Three accessory wells were set up for each group. After culturing for 4 h (under the same conditions as before), 20 μL of 5 mg / ml MTT reagent was added to each well. After culturing for another 4 h, the supernatant was discarded, and 150 μL of DMSO was added. The mixture was shaken to dissolve the cells, and the OD570 (absorbance value) at 570 nm was measured using a microplate reader. The inhibition rate (%) was calculated according to the formula: "Inhibition rate (%) = (OD570 of control group - OD570 of experimental group) x 100% / OD570 of control group".

[0134] (c) Experimental results: The MTT assay showed that different concentrations of peptide 2 solution inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide inhibited the proliferation of tumor cells more effectively than the standard therapeutic drug 5-FU (Table 7).

[0135] Table 7. Comparison of inhibition rates of different concentrations of peptide 2 co-cultured with Jurkat cells

[0136] .

[0137] (2) Polypeptide 3

[0138] (a) Experimental materials: acute lymphoblastic leukemia Jurkat cells; fetal bovine serum; RP-MI1640 culture medium; 5-fluorouracil (5-FU); using DMSO as solvent, polypeptide 3 powder was prepared into polypeptide solutions of 10ug / ml, 20ug / ml, and 30ug / ml.

[0139] (b) Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator with saturated humidity. The medium was changed every 2 days. (2) MTT assay to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and seeded in 96-well plates, 2 x 10 cells per well. 5 Cells were cultured at 200 μL at 37°C and 5% CO2 for 24 h. After removing the supernatant, experimental groups A, B, and C were established, with 200 μL of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml peptide 3 solution added, respectively. A blank control group (without peptide 3) and a cell-free control group were also established. The commercially available anticancer drug 5-FU (5-fluorouracil) was used as a control group for anticancer activity. Three auxiliary wells were set up for each group. After culturing for 4 h (under the same conditions), 20 μL of 5 mg / ml MTT reagent was added to each well. After culturing for another 4 h, the supernatant was discarded, and 150 μL of DMSO was added. The cells were shaken to dissolve, and the OD570 (absorbance value) at 570 nm was measured using a microplate reader. The inhibition rate (%) was calculated according to the formula: "Inhibition rate (%) = (OD570 of control group - OD570 of experimental group) x 100% / OD570 of control group".

[0140] (c) Experimental results: The MTT assay showed that different concentrations of peptide 3 inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 3 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 8).

[0141] Table 8. Comparison of inhibition rates of different concentrations of peptide 3 co-cultured with Jurkat cells

[0142] .

[0143] VI. We believe that peptides 4 and 5, whose sequences are similar to peptide 1, also have similar effects, and we have conducted relevant experiments to verify this.

[0144] 1. Experiment to enhance human immunity (steps are the same as Experiment 1)

[0145] (1) Polypeptide 4

[0146] (a) Analysis of changes in monocyte subsets after stimulation with peptide 4

[0147] The percentage of monocytes in the peptide 4-stimulated group was (23.60±9.97)%, while that in the control group was (22.39±0.34)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 4-stimulated group was (8.65±0.49)%, while that in the control group was (9.32±0.39)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 4-stimulated group was (89.28±4.59)%, while that in the control group was (84.42±8.49)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 4-stimulated group was (29.02±2.56)%, while that in the control group was (23.35±1.37)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 4 stimulation compared to the control group. Figure 10 ).

[0148] (b) CD4 after stimulation by peptide 4 + Analysis of CTL cell subset changes

[0149] Peptide 4 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (3.37±0.42)%, while in the control group it was (1.08±0.34)%. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 11 ).

[0150] (2) Polypeptide 5

[0151] (a) Analysis of changes in monocyte subsets after stimulation with peptide 5

[0152] The percentage of monocytes in the peptide 5 stimulation group was (23.60±6.76)%, while that in the control group was (21.69±4.58)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 5 stimulation group was (10.15±0.66)%, while that in the control group was (9.95±0.26)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 5 stimulation group was (89.04±5.41)%, while that in the control group was (89.55±3.03)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 3 stimulation group was (29.01±2.56)%, while that in the control group was (23.35±2.81)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 5 stimulation compared to the control group. Figure 12 ).

[0153] (b) CD4 after stimulation by peptide 5 + Analysis of CTL cell subset changes

[0154] Peptide 5 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (3.28±0.41)%, compared to (0.96±0.06)% in the control group. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 13 ).

[0155] 2. Experimental study on prolonging the lifespan of C57BL / 6J mice (steps same as Experiment 2):

[0156] (1) Polypeptide 4

[0157] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (26.05±1.51) months, and the median lifespan was (27.00±5.56) months; the average lifespan of mice in the control group was (20.56±1.51) months, and the median lifespan was (19.00±0.96) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 9). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0158] (b) Conclusion: Peptide 4 has similar functions to peptide 1, and both can prolong the lifespan of mice and delay the aging of the body.

[0159] Table 9. Mean and median survival time of mice before and after peptide 4 intervention

[0160] .

[0161] (2) Polypeptide 5

[0162] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (27.05±1.53) months, and the median lifespan was (28.00±0.73) months; the average lifespan of mice in the control group was (18.85±1.48) months, and the median lifespan was (18.00±1.11) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 10). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0163] (b) Conclusion: Peptide 5 and peptide 1 have similar functions and can prolong the lifespan of mice and delay the aging of the body.

[0164] Table 10. Mean and median survival time of mice before and after peptide 5 intervention

[0165] .

[0166] 3. Experimental study on anti-solid tumor efficacy (steps same as Experiment 3):

[0167] (a) Experimental results: The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 4 injection group was (66.22±0.79)%, and the average tumor inhibition rate of the combined drug 4 group was (69.12±1.22)%. The average tumor inhibition rate of the combined drug 4 group was higher than that of the positive control group and the polypeptide 4 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 4 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 5 injection group was (64.11±1.01)%, and the average tumor inhibition rate of the combined drug 5 group was (68.35±0.79)%. The average tumor inhibition rate of the combined drug 5 group was higher than that of the positive control group and the polypeptide 5 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 5 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05) (Table 11).

[0168] (b) Conclusion: Both peptide 4 and peptide 5 injections can inhibit tumor growth, and their combined use with existing antitumor drugs can enhance the antitumor effect of the drugs.

[0169] Table 11. Comparison of tumor inhibition rates of different drug interventions for solid tumors

[0170] .

[0171] 4. Experimental studies on efficacy against non-solid tumors:

[0172] (1) Polypeptide 4 (steps are the same as in Experiment 4)

[0173] Experimental results: MTT assay showed that different concentrations of peptide 4 solution inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 12).

[0174] Table 12. Comparison of inhibition rates of different concentrations of peptide 4 co-cultured with Jurkat cells

[0175] .

[0176] (2) Polypeptide 5 (steps are the same as in Experiment 4)

[0177] Experimental results: MTT assay showed that different concentrations of peptide 5 inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 5 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 13).

[0178] Table 13. Comparison of inhibition rates of different concentrations of peptide 5 co-cultured with Jurkat cells

[0179] .

[0180] VII. We hypothesized that peptides 6 and 7, whose sequences are similar to peptide 2, also have similar effects, and we conducted relevant experiments to verify this.

[0181] 1. Experiment to enhance human immunity (steps are the same as Experiment 1)

[0182] (1) Polypeptide 6

[0183] (a) Analysis of changes in monocyte subsets after stimulation with peptide 6

[0184] The percentage of monocytes in the peptide 6 stimulation group was (25.60±4.52)%, while that in the control group was (24.20±2.41)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 6 stimulation group was (10.15±0.80)%, while that in the control group was (9.72±0.49)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 6 stimulation group was (88.79±3.52)%, while that in the control group was (89.78±1.12)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 6 stimulation group was (24.00±2.41)%, while that in the control group was (21.75±1.86)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 6 stimulation compared to the control group. Figure 16 ).

[0185] (b) CD4 after stimulation with peptide 6 + Analysis of CTL cell subset changes

[0186] Peptide 6 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (3.11±0.41)%, compared to (1.87±0.43)% in the control group. + CTLs in CD4 +The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 17 ).

[0187] (2) Polypeptide 7

[0188] (a) Analysis of changes in monocyte subsets after stimulation with peptide 7

[0189] The percentage of monocytes in the peptide 7 stimulation group was (22.87±1.71)%, while that in the control group was (20.71±6.70)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 7 stimulation group was (10.64±3.10)%, while that in the control group was (9.93±0.51)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 7 stimulation group was (89.94±3.57)%, while that in the control group was (91.23±3.67)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 7 stimulation group was (26.24±2.89)%, while that in the control group was (21.47±2.00)%, with a statistically significant difference between the groups (P<0.05). After peptide 5 stimulation, the percentage of non-classical monocytes significantly increased compared to the control group. Figure 18 ).

[0190] (b) CD4 after stimulation with peptide 7 + Analysis of CTL cell subset changes

[0191] Peptide 7 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (2.35±0.53)%, while in the control group it was (1.18±0.56)%. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 19 ).

[0192] 2. Experimental study on prolonging the lifespan of C57BL / 6J mice (steps same as Experiment 2):

[0193] (1) Polypeptide 6

[0194] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (26.13±1.39) months, and the median lifespan was (23±2.65) months; the average lifespan of mice in the control group was (22.85±1.15) months, and the median lifespan was (21±0.36) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 14). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0195] (b) Conclusion: Peptide 6 and peptide 2 have similar functions and can prolong the lifespan of mice and delay the aging of the body.

[0196] Table 14. Mean and median survival time of mice before and after peptide 6 intervention

[0197] .

[0198] (2) Polypeptide 7

[0199] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (28±5.16) months, and the median lifespan was (22.00±2.34) months; the average lifespan of mice in the control group was (21.00±1.19) months, and the median lifespan was (18.00±0.56) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 15). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0200] (b) Conclusion: Peptide 7 and Peptide 2 have similar functions and can prolong the lifespan of mice and delay the aging of the body.

[0201] Table 15. Mean and median survival time of mice before and after peptide 7 intervention.

[0202] .

[0203] 3. Experimental study on anti-solid tumor efficacy (steps same as Experiment 3):

[0204] (a) Experimental results: The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 6 injection group was (67.12±0.69)%, and the average tumor inhibition rate of the combined drug 6 group was (68.12±1.32)%. The average tumor inhibition rate of the combined drug 6 group was higher than that of the positive control group and the polypeptide 6 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 6 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 7 injection group was (69.11±2.13)%, and the average tumor inhibition rate of the combined drug 7 group was (70.35±0.89)%. The average tumor inhibition rate of the combined drug 7 group was higher than that of the positive control group and the polypeptide 7 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 7 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05) (Table 16).

[0205] (b) Conclusion: Both peptides 6 and 7 can inhibit tumor growth, and their combined use with existing antitumor drugs can enhance the antitumor effect of the drugs.

[0206] Table 16. Comparison of tumor inhibition rates of different drug interventions in solid tumors

[0207] .

[0208] 4. Experimental studies on efficacy against non-solid tumors:

[0209] (1) Polypeptide 6 (steps are the same as in Experiment 4)

[0210] (a) Experimental results: The MTT assay showed that different concentrations of peptide 6 solution inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide inhibited the proliferation of tumor cells more effectively than the standard therapeutic drug 5-FU (Table 17).

[0211] Table 17. Comparison of inhibition rates of different concentrations of peptide 6 co-cultured with Jurkat cells

[0212] .

[0213] (2) Polypeptide 7 (steps are the same as in Experiment 4)

[0214] (a) Experimental results: The MTT assay showed that different concentrations of peptide 7 inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 7 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 18).

[0215] Table 18. Comparison of inhibition rates of different concentrations of peptide 7 co-cultured with Jurkat cells

[0216] .

[0217] 8. We hypothesized that peptides 8 and 9, whose sequences are similar to peptide 3, also have similar effects, and we conducted relevant experiments to verify this.

[0218] 1. Experiment to enhance human immunity (steps are the same as Experiment 1)

[0219] (1) Polypeptide 8

[0220] (a) Analysis of changes in monocyte subsets after stimulation with peptide 8

[0221] The percentage of monocytes in the peptide 8 stimulation group was (18.67±2.52)%, while that in the control group was (18.77±2.25)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 8 stimulation group was (9.30±1.00)%, while that in the control group was (8.97±0.75)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 8 stimulation group was (93.27±1.04)%, while that in the control group was (93.07±1.01)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 8 stimulation group was (27.77±3.22)%, while that in the control group was (23.03±1.05)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 8 stimulation compared to the control group. Figure 22 ).

[0222] (b) CD4 after stimulation with peptide 8 + Analysis of CTL cell subset changes

[0223] Peptide 8 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (2.13±1.00)%, while in the control group it was (0.53±0.35)%. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 23 ).

[0224] (2) Polypeptide 9

[0225] (a) Analysis of changes in monocyte subsets after stimulation with polypeptide 9

[0226] The percentage of monocytes in the peptide 9 stimulation group was (18.67±2.52)%, while that in the control group was (18.90±2.14)%, with no statistically significant difference between the groups (P>0.05). The percentage of intermediate monocytes in the peptide 9 stimulation group was (9.07±0.87)%, while that in the control group was (9.47±0.21)%, with no statistically significant difference between the groups (P>0.05). The percentage of classical monocytes in the peptide 9 stimulation group was (93.27±1.04)%, while that in the control group was (94.03±2.55)%, with no statistically significant difference between the groups (P>0.05). The percentage of non-classical monocytes in the peptide 9 stimulation group was (27.53±3.66)%, while that in the control group was (23.37±2.57)%, with a statistically significant difference between the groups (P<0.05). The percentage of non-classical monocytes significantly increased after peptide 9 stimulation compared to the control group. Figure 24 ).

[0227] (b) CD4 after stimulation with peptide 9 + Analysis of CTL cell subset changes

[0228] Peptide 9 stimulated group CD4 + CTLs in CD4 + The percentage of CD4 in the cells was (2.23±1.05)%, while that in the control group was (0.58±0.30)%. + CTLs in CD4 + The proportion of cells in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Figure 25 ).

[0229] 2. Experimental study on prolonging the lifespan of C57BL / 6J mice (steps same as Experiment 2):

[0230] (1) Polypeptide 8

[0231] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (26.35±1.61) months, and the median lifespan was (23.00±6.71) months; the average lifespan of mice in the control group was (18.15±1.14) months, and the median lifespan was (18.00±0.74) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 19). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0232] (b) Conclusion: Peptide 8 and Peptide 3 have similar functions and can prolong the lifespan of mice and delay the aging of the body.

[0233] Table 19. Mean and median survival time of mice before and after peptide 8 intervention.

[0234] .

[0235] (2) Polypeptide 9

[0236] (a) Experimental results: The average lifespan of mice in the polypeptide injection group was (25.30±1.11) months, and the median lifespan was (24.00±1.11) months; the average lifespan of mice in the control group was (21.10±1.11) months, and the median lifespan was (19.00±0.56) months. The average and median lifespans of mice in the polypeptide injection group were significantly higher than those in the control group (P<0.05) (Table 20). This indicates that the polypeptide intervention can extend the lifespan of mice in terms of both average and median lifespan, thus playing a role in delaying aging.

[0237] (b) Conclusion: Peptide 9 and Peptide 3 have similar functions and can prolong the lifespan of mice and delay the aging of the body.

[0238] Table 20. Mean and median survival time of mice before and after peptide 9 intervention.

[0239] .

[0240] 3. Experimental study on anti-solid tumor efficacy (steps same as Experiment 3):

[0241] (a) Experimental results: The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 8 injection group was (65.21±0.78)%, and the average tumor inhibition rate of the combined drug 8 group was (70.32±1.44)%. The average tumor inhibition rate of the combined drug 8 group was higher than that of the positive control group and the polypeptide 8 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 8 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 9 injection group was (62.12±0.12)%, and the average tumor inhibition rate of the combined drug 9 group was (67.35±0.79)%. The average tumor inhibition rate of the combined drug 9 group was higher than that of the positive control group and the polypeptide 9 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the polypeptide 9 injection group was higher than that of the positive control group, and the difference was statistically significant (P<0.05) (Table 21).

[0242] (b) Conclusion: Both peptide 8 and 9 injections can inhibit tumor growth, and their combined use with existing antitumor drugs can enhance the antitumor effect of the drugs.

[0243] Table 21. Comparison of tumor inhibition rates of different drug interventions for solid tumors

[0244] .

[0245] 4. Experimental studies on efficacy against non-solid tumors:

[0246] (1) Peptide 8 (Steps are the same as in Experiment 4)

[0247] (a) Experimental results: The MTT assay showed that different concentrations of peptide 8 solution inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 8 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 22).

[0248] Table 22. Comparison of inhibition rates of different concentrations of peptide 8 co-cultured with Jurkat cells

[0249] .

[0250] (2) Polypeptide 9 (steps are the same as in Experiment 4)

[0251] (a) Experimental results: The MTT assay showed that different concentrations of peptide 9 inhibited the proliferation of tumor cells, with 30 ug / ml showing the strongest inhibitory effect. This concentration of peptide 9 had a stronger inhibitory rate on tumor cell proliferation than the standard therapeutic drug 5-FU (Table 23).

[0252] Table 23. Comparison of inhibition rates of different concentrations of peptide 9 co-cultured with Jurkat cells

[0253] .

[0254] After injection of the aforementioned polypeptides, the polypeptide components can stimulate the body's immune cells, thereby affecting certain immune cell subsets (such as CD4+). + CTLs, CD14 + CD16 ++ The increased proportion of these immune cell subsets has been shown in previous studies to enhance the body's immune function, increase resistance to various diseases, and reduce morbidity, thereby achieving the goal of enhancing the body's disease resistance. This polypeptide can also enhance the body's anti-tumor ability and prolong lifespan. Verification showed that polypeptides 1, 2, and 3, with different structures, all have the functions of enhancing immunity, enhancing the body's anti-tumor ability, and prolonging lifespan, with polypeptide 1 showing the most significant effect. Partial alterations to the amino acid sequences of the three groups of polypeptides yielded polypeptides 4 and 5, polypeptides 6 and 7, and polypeptides 8 and 9, respectively. Verification showed that the sequence-altered polypeptides still possessed the functions of enhancing immunity, strengthening the body's anti-tumor ability, and prolonging lifespan. Therefore, we speculate that polypeptides with 80% or more of the same amino acid sequence as polypeptides 1, 2, and 3 have similar functions.

[0255] VII. Key Technologies for Protection

[0256] Innovation: Using peptides to make vaccines can improve the body's immunity and enhance its disease resistance.

[0257] Peptide 1 sequence: MNKAELIDVLTQKLGSDRRQATAAVENVVD

[0258] Peptide 2 sequence: TIVRAVHKGDSVTITGFGVFEQRRRAARVA

[0259] Peptide 3 sequence: RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA

[0260] Peptide 4 sequence: VNKAELIDVLTGGLGSKRRQATAAVEGGVD (This peptide is 80% identical to peptide 1)

[0261] Peptide 5 sequence: MGVAGLIDVLTQKLGSGGRQATAAVENDDD (This peptide is 80% identical to peptide 1)

[0262] Peptide 6 sequence: TGVRAGHNGDSVTITGFVGFEGRRRAARVA (This peptide is 80% identical to peptide 2)

[0263] Peptide 7 sequence: TIVRAVGKGDSGITIGFGVFERQRRAARVA (This peptide is 80% identical to peptide 2)

[0264] Peptide 8 sequence: RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA (This peptide is 80% identical to peptide 3)

[0265] Peptide 9 sequence: KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA (This peptide is 80% identical to peptide 3)

[0266] A vaccine characterized in that its active ingredient is synthesized in vitro based on a polypeptide sequence;

[0267] The vaccine is administered to humans via injection, oral administration, or other clinical routes.

[0268] The aim is to enhance the body's immunity, improve disease resistance, fight tumors, and prolong life.

[0269] We believe that peptides with a sequence similarity of more than 80% to the above sequence have similar functions.

Claims

1. The application of a polypeptide with the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA, characterized in that... The application of the described polypeptide in the preparation of drugs that prolong the lifespan of mice.

2. The application of a polypeptide with the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA, characterized in that... The application of the polypeptide in the preparation of drugs that inhibit tumor cell growth; the tumor cells are gastric cancer MGC-803 or acute lymphoblastic leukemia Jurkat cells.

Images