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] The present invention belongs to the field of medical technology, and specifically relates to a polypeptide having the functions of improving immunity, resisting tumors and prolonging life, and its application. Background Art

[0002] In classical immunology, T cells develop into CD4 + Helper T cells and CD8 + There are two main subpopulations of cytotoxic T cells. Recent studies have found that some CD4 + T cells can further differentiate into CD8 T cells outside the thymus + The direct cell killing function of CTLs and NK cells is a subset of CD4 + CTLs. Currently, CD4 + CTLs have also been further confirmed to play an important role in infection, tumors, autoimmune diseases, vaccination, etc. Especially in infectious diseases, including influenza virus, cytomegalovirus, EBV, human papillomavirus and HIV, there have been extensive reports. Some literature shows that researchers believe that CD4 + The increase in the number of CTLs and the enhancement of virulence may indicate the enhancement of the body's overall immunity. The changes in this cell subset can be used as an indicator to evaluate the strength of immune function. It was found that the proportion of this cell subset in super-long-lived people is significantly higher than that in normal people, which suggests that the increase in the proportion of this cell subset is related to longevity. According to previous studies, monocytes can also be divided into three subsets, namely intermediate (CD14 ++ CD16 + ), classic type (CD14 ++ CD16 - ) and nonclassical (CD14 + CD16 ++ Among them, the classic type (CD14 ++ CD16 - ) accounts for about 80%-90% of the total number of monocytes in the peripheral blood of healthy people and plays an important role in the innate immune defense mechanism; intermediate (CD14 ++ CD16 + ) account for about 5% of the total number of monocytes, and can reach 10%-50% in severe infection and inflammatory conditions. Their main functions include antigen presentation, inflammation, and monocyte activation. They are also called inflammatory monocyte subsets; non-classical (CD14 + CD16 ++These cells, also known as migratory monocyte subsets, account for approximately 5%-10% of the total monocyte population. They migrate along the vascular endothelium, clearing foreign matter and performing immune surveillance. They play a crucial role in early inflammatory responses and tissue repair. Changes in the proportions of different monocyte subsets correlate with enhanced function of the corresponding subset, providing insights into 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 a malignant tumor. Almost half of new malignant tumor cases and over half of deaths from malignant tumors occur in Asia, particularly China. Malignant tumors are categorized as solid tumors and non-solid tumors. Common solid tumors include lung cancer, gastric cancer, and breast cancer, while common non-solid tumors include leukemia and lymphoma. Currently, there is no effective cure for malignant tumors, and treatment primarily relies on surgery, radiotherapy, and chemotherapy. However, the harm these treatments cause to patients cannot be ignored. The anti-tumor effect of drugs on solid tumors can be evaluated by calculating the tumor inhibition rate based on tumor size. For non-solid tumors, the rate of inhibition of tumor cell proliferation can be verified through in vitro experiments. Coordinating different treatment options to enhance efficacy, minimize side effects, and improve patient quality of life is a crucial component of malignant tumor treatment.

[0004] Aging is a biological process that inevitably occurs in all organisms, characterized by its gradual, harmful, and universal nature. As society progresses, the demand for anti-aging and life extension continues to grow. Anti-aging drugs refer to a class of medications designed to enhance life efficiency, improve physical fitness, prevent and treat age-related diseases, and, within the limits determined by genetic characteristics, extend lifespan. They prevent and delay the aging process, improve pathological disorders of tissues and cells, regulate the functions of vital organs, regulate the body's internal environment, and promote overall health. These drugs are characterized by their multi-level, multifaceted, and long-term effects, adjusting the body's substances, metabolism, and tissue and organ functions, thereby delaying aging and extending lifespan.

[0005] Currently, Chinese residents are increasingly seeking to extend their lifespans and improve their quality of life. Our previous research has revealed a peptide that can stimulate the proliferation of cell subsets, thereby enhancing immunity, significantly inhibiting tumor cells, and potentially extending lifespan. This peptide, when formulated into a vaccine and administered to the human body, has multiple benefits: enhancing immunity, fighting tumors, and extending lifespan. Summary of the Invention

[0006] The first purpose of the present invention is to provide a polypeptide having the effects of improving immunity, resisting tumors and prolonging life; the second purpose is to provide the application of the polypeptide having the effects of improving immunity, resisting tumors and prolonging life.

[0007] The first object of the present invention is achieved in that the polypeptide having the functions of improving immunity, resisting tumors and prolonging life is selected from the group consisting of:

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

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

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

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

[0012] (e) a polypeptide having the amino acid sequence of 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 of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.

[0017] The polypeptides of the present invention that have the functions of improving immunity, resisting tumors and prolonging life can improve the body's immunity, enhance disease resistance, reduce the incidence of tumors and prolong life.

[0018] The polypeptide component of the present invention can stimulate the body's immune cells, so that some immune cell subpopulations (such as CD4 + CTLs, CD14 + CD16 ++) proportion increases. These immune cell subsets have been shown in previous studies to enhance the body's immune function, strengthen resistance to various diseases, and reduce the incidence of disease, thereby achieving the goal of enhancing the body's disease resistance. This polypeptide can also enhance the body's anti-tumor ability and prolong life. It has been verified 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 life, among which polypeptide 1 has the most obvious effect. The amino acid sequences of the three groups of polypeptides were partially changed to obtain polypeptides 4 and 5, polypeptides 6 and 7, and polypeptides 8 and 9, respectively. It has been verified that the polypeptides with sequence changes still have the functions of enhancing immunity, strengthening the body's anti-tumor ability, and prolonging life. Therefore, we speculate that polypeptides with 80% or more identical amino acid sequences to polypeptides 1, 2, and 3 have similar functions. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 CD4 before and after stimulation by polypeptide 1 of the present invention + Schematic diagram of 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 the intervention of the polypeptide 1 of the present invention;

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

[0023] Figure 5 CD4 before and after stimulation by polypeptide 2 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

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

[0025] Figure 7 CD4 before and after stimulation by polypeptide 3 of the present invention + CD4 + Schematic diagram of 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 the intervention of the polypeptide 2 of the present invention;

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

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

[0029] Figure 11 CD4 before and after stimulation by polypeptide 4 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

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

[0031] Figure 13 CD4 before and after stimulation by polypeptide 5 of the present invention + CD4 + Schematic diagram of 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 the intervention of the polypeptide 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 the intervention of polypeptide 5 of the present invention;

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

[0035] Figure 17 CD4 before and after stimulation by polypeptide 6 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

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

[0037] Figure 19 CD4 before and after stimulation by polypeptide 7 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

[0038] Figure 20 Schematic diagram of Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 6 of the present invention;

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

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

[0041] Figure 23 CD4 before and after stimulation by polypeptide 8 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

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

[0043] Figure 25 CD4 before and after stimulation by polypeptide 9 of the present invention + CD4 + Schematic diagram of the changes in the proportion of CTLs;

[0044] Figure 26 Schematic diagram of Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 8 of the present invention;

[0045] Figure 27 Schematic diagram of Kaplan-Meier survival curve analysis of mice before and after intervention with polypeptide 9 of the present invention. DETAILED DESCRIPTION

[0046] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0047] The polypeptides of the present invention that have the functions of improving immunity, resisting tumors and prolonging life are selected from the group consisting of:

[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 of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.

[0057] The nucleic acid having the functions of improving immunity, anti-tumor and prolonging life of the present invention is selected from:

[0058] (a) a nucleic acid encoding the polypeptide of claim 1; or

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

[0060] The vector of the present invention contains nucleic acid with the functions of improving immunity, resisting tumors and prolonging life.

[0061] The host cell of the present invention is characterized in that it contains a vector of a nucleic acid having the functions of improving immunity, anti-tumor, and prolonging life, or a nucleic acid having the functions of improving immunity, anti-tumor, and prolonging life is integrated into its genome.

[0062] The medicine is a polypeptide vaccine containing a polypeptide sequence with the functions of improving immunity, resisting tumors and prolonging life.

[0063] The application of the polypeptide with immunity-enhancing, anti-tumor and life-extending functions of the present invention is the application of the polypeptide with immunity-enhancing in medicines for enhancing immunity.

[0064] The application of the polypeptide with immunity-enhancing, anti-tumor and life-extending functions of the present invention is the application of the polypeptide with immunity-enhancing in the preparation of medicines for prolonging life.

[0065] The application of the polypeptide with the functions of improving immunity, resisting tumors and prolonging life described in the present invention is the application of the polypeptide with the functions of improving immunity in the preparation of anti-tumor drugs.

[0066] The tumor is 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 present invention is further described below with reference to specific experimental cases:

[0069] 1. The specific operation of the experiment on peptide 1 to improve human immunity is as follows:

[0070] 1. Inclusion criteria: (1) Aged 18-60 years; (2) Normal population (normal liver and kidney function tests, excluding those infected with tuberculosis, HBV, HCV, tumors, and autoimmune system 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; red blood cell lysis buffer; flow cytometry permeabilization and fixation integrated solution.

[0072] 3. Experimental procedures: (1) Based on the sequence of peptide 1, it was synthesized in vitro; (2) Peripheral blood PBMCs were extracted: a) 5 ml of peripheral blood was drawn from the elbows of 6 healthy subjects and placed in EDTA anticoagulant tubes, 3 of which were used to detect monocyte subsets, and 3 of which were used to detect CD4 + CTL subsets; b) Dilute 5 ml of blood to 10 ml with PBS; c) Place 5 ml of peripheral blood mononuclear cell separation solution in centrifuge tubes, and add 5 ml of the diluted blood to the top layer of the separation solution; d) 1000g, acceleration 4, deceleration 4, centrifuge for 25 minutes; e) Use a pipette to carefully aspirate the PBMC layer cells, dilute with lysate and centrifuge, remove the supernatant, and repeat twice; f) Use the prepared cell culture medium (10ul double antibody + 90ul fetal bovine serum + 900ul1640) to resuspend the cells; g) Divide each sample cell into experimental group and control group, 500ul each; (3) Use PBS to dissolve polypeptide 1 powder to a concentration of 1mg / ml; (4) Add 100ul of polypeptide 1 solution to the experimental group and let it stand for 12h; (5) Flow cytometry: a) Use 1ml of lysate to resuspend the cells and centrifuge, remove the supernatant; b) Use 100ul of lysate to resuspend the cells, add CD14, CD16, and CD45 antibodies to the monocyte group and incubate in the dark for 30min; CD4 + CTLs cell group was added with CD3 and CD4 antibodies and incubated in the dark for 30 minutes; c) CD4 + CTLs cell group was added with membrane rupture fixation solution and kept in the dark for 30 minutes; d) Monocyte group was added with 1 ml of lysate solution and centrifuged, and the supernatant was removed. + CTLs cell group was directly centrifuged and the supernatant was removed; e) monocyte group was added with 200ul lysate and resuspended, and the cells were tested by flow cytometry. +100 μl of permeabilization fixative was added to the CTLs cell group, followed by the addition of GB11 antibody and incubation in the dark for 30 min; f) CD4 + Add 1 ml of permeabilization fixative to the CTLs cell group, centrifuge, and remove the supernatant; g) CD4 + In the CTLs cell group, 100 μl of membrane permeabilization fixative was added to resuspend the cells and tested on the microscope.

[0073] 4. Experimental results: After stimulation with peptide 1, non-classical monocytes (CD14 + CD16 ++ ), CD4 + The proportion of CTLs subsets increased significantly, indicating enhanced cellular immune function.

[0074] 5. Statistical analysis of changes in cell subpopulations after peptide 1 stimulation was performed. Quantitative data were expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 statistical software. Paired t-tests were used if the data conformed to a normal distribution; Wilcoxon tests were used if not. P < 0.05 was considered significant.

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

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

[0077] (2) CD4 after peptide 1 stimulation + Analysis of changes in CTLs cell subsets

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

[0079] 2. Experimental study on the effect of peptide 1 on the lifespan extension of C57BL / 6J mice:

[0080] 1. Inclusion Criteria: 40 8-week-old specific pathogen-free (SPF) C57BL / 6J mice (healthy mice of this strain can live up to 18-24 months, and up to 3 years), housed in a 12-hour light / 12-hour dark cycle, with a temperature of 22-25°C and a humidity of 50-60%, one mouse per cage. After arrival at the experimental environment, the growth and activity of the animals were observed, and drug administration began at 1 month of age.

[0081] 2. Experimental procedure: (1) According to the sequence of polypeptide 1, it was synthesized in vitro; (2) Peptide 1 powder was dissolved in sterile saline solution to 0.4 mg / ml, ultrafiltered to a sterile solution, and stored aseptically at -20°C. It was returned to room temperature before use; (3) Mice were randomly divided into a polypeptide administration group and a vehicle control group, with 20 mice in each group; (4) The polypeptide administration group was injected with polypeptide 1 at a dose of 6 mg / kg, once every 3 days, at the same time (9:00-11:00) through the tail vein of mice, and the vehicle control group was injected with the corresponding amount of saline; (5) The mice were given the drug until natural death, and the time of death and the most likely cause of death were recorded; (6) Mice that died due to operational accidents or external reasons were removed, and the Kaplan-Meier statistical method was used for survival analysis and survival curves were drawn.

[0082] 3. Experimental Results: Statistical analysis was performed on the changes in mouse lifespan after peptide 1 stimulation. Quantitative data were expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 statistical software. Paired T-tests were used if the data conformed to a normal distribution; Wilcoxon tests were used if the data did not conform to a normal distribution. P < 0.05 was considered significant. Survival curves were calculated using the Kaplan-Meier method ( Figure 3 ).

[0083] Mouse lifespan observation experiments showed that the average lifespan of mice in the peptide 1 injection group was (26.64±1.72) months, with a median lifespan of (21.00±7.83) months; the average lifespan of mice in the control group was (16.90±1.01) months, with a median lifespan of (17±0.89) months. The average and median lifespans of mice in the peptide 1 injection group were significantly higher than those in the control group (P<0.05). This suggests that peptide 1 intervention can extend the lifespan of mice in terms of both average and median lifespan, 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: The intervention of peptide 1 can delay the aging of the body and prolong the life span of mice.

[0087] 3. Experimental study on the efficacy of peptide 1 against solid tumors:

[0088] 1. Experimental Materials: 40 male nude mice weighing 18-22 g; gastric cancer MGC-803 cell line; Peptide 1 powder was dissolved in normal saline to a final concentration of 1 mg / ml, filtered through a 0.22 μm filter membrane, and sterile packaged and stored at 4°C until use; Trastuzumab and docetaxel injections.

[0089] 2. Experimental method: MGC-803 cells were cultured in a 37°C, 5% CO2 incubator to a density of more than 80%. The culture medium was DMEM high-glucose medium containing 10% FBS. The cells were digested and collected with 0.25% trypsin solution. The supernatant was discarded after centrifugation at 1000 rpm. The cells were washed three times with physiological saline and then resuspended and counted to 5×10 7 / ml, store at 4℃ for future use.

[0090] 3. Experimental process: (1) The mice were randomly divided into 4 groups, namely blank control group, peptide group, combined drug group and positive control group, with 10 mice in each group. 0.2 ml of the prepared cancer cell suspension was inoculated subcutaneously under the right armpit of the mouse. The tumor was observed every three days after inoculation. When the tumor volume reached 0.1 mm 3The drug was started at 1:00 pm. (2) The positive control group was given intravenous injection of trastuzumab 15 ml / kg, once every 3 days; docetaxel 5 mg / kg, once every 7 days; the blank group was given intravenous injection of normal saline 10 ml / kg, once every 3 days; the peptide group was given intravenous injection of prepared peptide 1 solution 1 mg / kg, once every 3 days; the combined drug group was given intravenous injection of trastuzumab 15 ml / kg, once every 3 days; docetaxel 5 mg / kg, once every 7 days; prepared peptide 1 solution 1 mg / kg, once every 3 days. (3) The tumor volume was measured 21 days after drug administration. The tumor inhibition rate (%) was calculated according to the formula = (tumor volume of blank control group - tumor volume of drug administration group) / tumor volume of blank control group.

[0091] 4. Experimental Results: Statistical analysis of changes in tumor inhibition rates after peptide 1 injection was performed. Quantitative data were expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 statistical software. Paired t-tests were used if the data conformed to a normal distribution; Wilcxon tests were used if not. P < 0.05 was considered significant.

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

[0093] 5. Conclusion: Injection of peptide 1 can inhibit tumor growth, and its combination with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

[0094] Table 2. Comparison of tumor inhibition rates of solid tumors treated with different drugs

[0095] .

[0096] IV. Experimental study on the efficacy of peptide 1 against non-solid tumors

[0097] 1. Experimental materials: Jurkat cells, fetal bovine serum, RP-MI1640 culture medium, 5-fluorouracil (5-FU). Peptide 1 powder was prepared into peptide solutions at 10 μg / ml, 20 μg / ml, and 30 μg / ml using DMSO as the solvent.

[0098] 2. Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37°C, 5% CO2, and saturated humidity. The culture medium was replaced every 2 days. (2) MTT assay for cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and inoculated into a 96-well plate at 2×10 cells per well. 5 10 cells, 200 μl of complete culture medium were added and cultured at 37°C, 5% CO2 for 24 h. The supernatant was removed and experimental groups A, B, and C were established. 200 μl of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml of peptide 1 were added, respectively. A blank control group (no peptide 1) and a cell-free control group were also established. The commercial anticancer drug 5-FU (5-fluorouracil) was used as a positive control for anticancer activity. Three replica wells were set up for each group. After 4 h of culture (same conditions as before), 20 μl of 5 mg / ml MTT reagent was added to each well. After another 4 h of culture, the supernatant was discarded and 150 μl of DMSO was added. The cells were dissolved by shaking and the OD570 (absorbance) at a wavelength of 570 nm was measured using a microplate reader. The formula: 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 all inhibited the proliferation of tumor cells, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of peptide 1 at this concentration on tumor cell proliferation was stronger than that of the standard therapeutic drug 5-FU (Table 3).

[0100] Table 3. Comparison of the inhibitory rate of different concentrations of peptide 1 on Jurkat cells in vitro

[0101] .

[0102] 5. We believe that peptides 2 and 3 with different structures also have similar functions. The results of the above experimental scheme are as follows:

[0103] 1. Experiment on how peptides can improve human immunity (same steps as Experiment 1)

[0104] (1) Peptide 2

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

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

[0107] (b) CD4 after peptide 2 stimulation + Analysis of changes in CTLs cell subsets

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

[0109] (2) Peptide 3

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

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

[0112] (b) CD4 after peptide 3 stimulation + Analysis of changes in CTLs cell subsets

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

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

[0115] (1) Peptide 2

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

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

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

[0119] .

[0120] (2) Peptide 3

[0121] (a) Experimental results: The mean lifespan of mice in the peptide 3 injection group was (25.41±1.45) months, and the median lifespan was (21.00±2.17) months; the mean lifespan of mice in the control group was (18.55±0.87) months, and the median lifespan was (18±0.74) months. The mean and median lifespans of mice in the peptide 3 injection group were significantly higher than those in the control group (P<0.05) (Table 5). This indicates that peptide 3 intervention can extend the lifespan of mice in terms of both mean and median lifespan, 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 (same steps 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 peptide 2 injection group was (65.12±0.81)%, and the average tumor inhibition rate of the combination drug group was (68.07±1.02)%. The average tumor inhibition rate of the combination drug group was higher than that of the positive control group and the peptide 2 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 2 injection group was higher than that of the positive control group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 3 injection group was (66.68±1.01)%, and the average tumor inhibition rate of the combination drug group was (70.22±0.88)%. The average tumor inhibition rate of the combination drug group was higher than that of the positive control group and the peptide 3 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 3 injection group was higher than that of the positive control group, and the differences were statistically significant (P<0.05) (Table 6).

[0127] (b) Conclusion: Injection of peptides 2 and 3 can inhibit tumor growth. Their combined use with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

[0128] Table 6. Comparison of tumor inhibition rates of different drugs in solid tumors

[0129] .

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

[0131] (1) Peptide 2

[0132] (a) Experimental materials: Jurkat cells, fetal bovine serum, RP-MI1640 culture medium, 5-fluorouracil. Peptide 2 powder was prepared into peptide solutions at 10 μg / ml, 20 μg / ml, and 30 μg / ml using DMSO as the solvent.

[0133] (b) Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37°C, 5% CO2, and saturated humidity. The culture medium was replaced every 2 days. (2) MTT assay for cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and inoculated into 96-well plates at 2 × 10 cells per well. 5 Each well was cultured for 24 hours at 37°C and 5% CO2. The supernatant was removed and experimental groups A, B, and C were established. 200 μl of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml of Peptide 2 were added, respectively. A blank control group (no Peptide 2) and a cell-free control group were also established. The commercial anticancer drug 5-FU (5-fluorouracil) was used as a control group for anticancer activity. Three replica wells were set up for each group. After 4 hours of culture (same conditions as before), 20 μl of 5 mg / ml MTT reagent was added to each well. After another 4 hours of culture, the supernatant was discarded and 150 μl of DMSO was added. The cells were dissolved by shaking and the OD570 (absorbance) at a wavelength of 570 nm was measured using a microplate reader. The inhibition rate (%) was determined according to the formula "OD570 of the control group - OD570 of the experimental group" x 100% / OD570 of the control group.

[0134] (c) Experimental results: The MTT assay confirmed that peptide 2 solutions at different concentrations all had inhibitory effects on tumor cell proliferation, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of this concentration of peptide on tumor cell proliferation was higher than that of the standard therapeutic drug 5-FU (Table 7).

[0135] Table 7. Comparison of the inhibitory rate of co-culture of different concentrations of peptide 2 with Jurkat cells

[0136] .

[0137] (2) Peptide 3

[0138] (a) Experimental materials: Jurkat cells, fetal bovine serum, RP-MI1640 culture medium, and 5-fluorouracil (5-FU). Peptide 3 powder was prepared into peptide solutions at 10 μg / ml, 20 μg / ml, and 30 μg / ml using DMSO as the solvent.

[0139] (b) Experimental methods: (1) Cell culture: Jurkat cells were cultured in RP-MI 1640 medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37°C, 5% CO2, and saturated humidity. The culture medium was replaced every 2 days. (2) MTT assay for cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to prepare a cell suspension, and inoculated into 96-well plates at 2 × 10 cells per well. 5 Each well was cultured for 4 h at 37°C with 5% CO2. The supernatant was discarded and experimental groups A, B, and C were established. 200 μl of complete culture medium containing 10 μg / ml, 20 μg / ml, and 30 μg / ml of peptide 3 solution was added, respectively. A blank control group (no peptide 3) and a cell-free control group were also established. The commercial anticancer drug 5-FU (5-fluorouracil) was used as a control group for anticancer activity. Three replica wells were set up for each group. After 4 h of culture (same conditions as before), 20 μl of 5 mg / ml MTT reagent was added to each well. After another 4 h of culture, the supernatant was discarded and 150 μl of DMSO was added. The cells were dissolved by shaking and the OD570 (absorbance) at a wavelength of 570 nm was measured using a microplate reader. The formula "inhibition rate (%) = (control group OD570 - experimental group OD570) x 100% / control group OD570" was used.

[0140] (c) Experimental results: The MTT assay confirmed that different concentrations of peptide 3 all inhibited the proliferation of tumor cells, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of peptide 3 at this concentration on tumor cell proliferation was stronger than that of the standard therapeutic drug 5-FU (Table 8).

[0141] Table 8. Comparison of the inhibitory rate of co-culture of different concentrations of peptide 3 with Jurkat cells

[0142] .

[0143] 6. We believe that peptides 4 and 5, which have similar sequences to peptide 1, also have similar effects, and we have conducted relevant experiments to verify this.

[0144] 1. Experiment on improving human immunity (same steps as Experiment 1)

[0145] (1) Peptide 4

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

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

[0148] (b) CD4 after peptide 4 stimulation + Analysis of changes in CTLs cell subsets

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

[0150] (2) Peptide 5

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

[0152] The proportion of monocytes in the peptide 5 stimulation group was (23.60±6.76)%, while that in the control group was (21.69±4.58)%. There was no significant difference between the two groups (P>0.05). The proportion 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)%. There was no significant difference between the two groups (P>0.05). The proportion 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). There was no significant difference between the two groups (P>0.05). The proportion 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). There was significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 5 stimulation was significantly increased compared with the control group ( Figure 12 ).

[0153] (b) CD4 after stimulation with peptide 5 + Analysis of changes in CTLs cell subsets

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

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

[0156] (1) Peptide 4

[0157] (a) Experimental results: The mean lifespan of mice in the peptide injection group was (26.05±1.51) months, with a median lifespan of (27.00±5.56) months; the mean lifespan of mice in the control group was (20.56±1.51) months, with a median lifespan of (19.00±0.96) months. The mean and median lifespans of mice in the peptide injection group were significantly higher than those in the control group (P<0.05) (Table 9). This suggests that peptide intervention can extend the lifespan of mice in terms of both mean and median lifespan, thereby 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) Peptide 5

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

[0163] (b) Conclusion: Peptide 5 has similar functions to peptide 1, and both 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 (same steps 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 peptide 4 injection group was (66.22±0.79)%, and the average tumor inhibition rate of the combination drug group was (69.12±1.22)%. The average tumor inhibition rate of the combination drug group was higher than that of the positive control group and the peptide 4 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 4 injection group was higher than that of the positive control group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 5 injection group was (64.11±1.01)%, and the average tumor inhibition rate of the combination drug group was (68.35±0.79)%. The average tumor inhibition rate of the combination drug group was higher than that of the positive control group and the peptide 5 injection group, and the differences were statistically significant (P<0.05). The average tumor inhibition rate of the peptide 5 injection group was higher than that of the positive control group, and the differences were statistically significant (P<0.05) (Table 11).

[0168] (b) Conclusion: Injection of peptides 4 and 5 can inhibit tumor growth. Their combined use with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

[0169] Table 11. Comparison of tumor inhibition rates of solid tumors treated with different drugs

[0170] .

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

[0172] (1) Peptide 4 (same steps as Experiment 4)

[0173] Experimental results: The MTT assay showed that peptide 4 solutions at different concentrations all had inhibitory effects on tumor cell proliferation, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of this concentration of peptide on tumor cell proliferation was higher than that of the standard therapeutic drug 5-FU (Table 12).

[0174] Table 12. Comparison of the inhibitory rate of co-culture of different concentrations of peptide 4 with Jurkat cells

[0175] .

[0176] (2) Peptide 5 (same steps as Experiment 4)

[0177] Experimental results: The MTT assay showed that different concentrations of peptide 5 all had an inhibitory effect on the proliferation of tumor cells, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of peptide 5 on tumor cell proliferation at this concentration was stronger than that of the standard therapeutic drug 5-FU (Table 13).

[0178] Table 13. Comparison of the inhibitory rate of co-culture of different concentrations of peptide 5 with Jurkat cells

[0179] .

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

[0181] 1. Experiment on improving human immunity (same steps as Experiment 1)

[0182] (1) Peptide 6

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

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

[0185] (b) CD4 after stimulation with peptide 6 + Analysis of changes in CTLs cell subsets

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

[0187] (2) Peptide 7

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

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

[0190] (b) CD4 after stimulation with peptide 7 + Analysis of changes in CTLs cell subsets

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

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

[0193] (1) Peptide 6

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

[0195] (b) Conclusion: Peptide 6 has similar functions to peptide 2, and both 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) Peptide 7

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

[0200] (b) Conclusion: Peptide 7 has similar functions to peptide 2, and both 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 (same steps 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 combination drug group was (68.12±1.32)%. The average tumor inhibition rate of the combination drug 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 differences were 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 combination drug group was (70.35±0.89)%. The average tumor inhibition rate of the combination drug 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 differences were statistically significant (P<0.05) (Table 16).

[0205] (b) Conclusion: Injection of peptides 6 and 7 can inhibit tumor growth. Their combined use with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

[0206] Table 16. Comparison of tumor inhibition rates of solid tumors treated with different drugs

[0207] .

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

[0209] (1) Peptide 6 (same steps as Experiment 4)

[0210] (a) Experimental results: The MTT assay confirmed that peptide 6 solutions at different concentrations all had inhibitory effects on tumor cell proliferation, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of peptide 6 at this concentration on tumor cell proliferation was higher than that of the standard therapeutic drug 5-FU (Table 17).

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

[0212] .

[0213] (2) Peptide 7 (same steps as Experiment 4)

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

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

[0216] .

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

[0218] 1. Experiment on improving human immunity (same steps as Experiment 1)

[0219] (1) Peptide 8

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

[0221] The proportion of monocytes in the peptide 8 stimulation group was (18.67±2.52)%, while that in the control group was (18.77±2.25)%. There was no significant difference between the two groups (P>0.05). The proportion 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)%. There was no significant difference between the two groups (P>0.05). The proportion 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)%. There was no significant difference between the two groups (P>0.05). The proportion 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). There was significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 8 stimulation was significantly increased compared with the control group ( Figure 22 ).

[0222] (b) CD4 after stimulation with peptide 8 + Analysis of changes in CTLs cell subsets

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

[0224] (2) Peptide 9

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

[0226] The proportion of monocytes in the peptide 9 stimulation group was (18.67±2.52)%, while that in the control group was (18.90±2.14)%. There was no significant difference between the two groups (P>0.05). The proportion 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)%. There was no significant difference between the two groups (P>0.05). The proportion 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)%. There was no significant difference between the two groups (P>0.05). The proportion 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). There was significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 9 stimulation was significantly increased compared with the control group ( Figure 24 ).

[0227] (b) CD4 after stimulation with peptide 9 + Analysis of changes in CTLs cell subsets

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

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

[0230] (1) Peptide 8

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

[0232] (b) Conclusion: Peptide 8 has similar functions to peptide 3, and both 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) Peptide 9

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

[0237] (b) Conclusion: Peptide 9 has similar functions to peptide 3, and both 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 (same steps 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 combination drug group was (70.32±1.44)%. The average tumor inhibition rate of the combination drug 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 differences were 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 combination drug group was (67.35±0.79)%. The average tumor inhibition rate of the combination drug 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 differences were statistically significant (P<0.05) (Table 21).

[0242] (b) Conclusion: Injection of peptides 8 and 9 can inhibit tumor growth. Their combined use with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

[0243] Table 21. Comparison of tumor inhibition rates of solid tumors treated with different drugs

[0244] .

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

[0246] (1) Peptide 8 (same steps as Experiment 4)

[0247] (a) Experimental results: The MTT assay confirmed that different concentrations of Peptide 8 solutions all had an inhibitory effect on tumor cell proliferation, with 30 μg / ml having the strongest inhibitory effect. The inhibitory rate of Peptide 8 at this concentration on tumor cell proliferation was stronger than that of the standard therapeutic drug 5-FU (Table 22).

[0248] Table 22. Comparison of the inhibitory rate of different concentrations of peptide 8 co-cultured with Jurkat cells

[0249] .

[0250] (2) Peptide 9 (same steps as Experiment 4)

[0251] (a) Experimental results: The MTT assay confirmed that different concentrations of peptide 9 all inhibited the proliferation of tumor cells, with 30 μg / ml showing the strongest inhibitory effect. The inhibitory rate of peptide 9 at this concentration on tumor cell proliferation was higher than that of the standard therapeutic drug 5-FU (Table 23).

[0252] Table 23. Comparison of the inhibitory rate of different concentrations of peptide 9 co-cultured with Jurkat cells

[0253] .

[0254] After injection of the above polypeptides, the polypeptide components can stimulate the body's immune cells, making some immune cell subsets (such as CD4 + CTLs, CD14 + CD16 ++ ) proportion increases. These immune cell subsets have been shown in previous studies to enhance the body's immune function, strengthen resistance to various diseases, and reduce the incidence of disease, thereby achieving the goal of enhancing the body's disease resistance. This polypeptide can also enhance the body's anti-tumor ability and prolong life. It has been verified that peptides 1, 2, and 3 with different structures all have the functions of enhancing immunity, enhancing the body's anti-tumor ability, and prolonging life, among which peptide 1 has the most obvious effect. The amino acid sequences of the three groups of peptides were partially altered to obtain peptides 4 and 5, peptides 6 and 7, and peptides 8 and 9, respectively. It has been verified that the peptides with altered sequences still have the functions of enhancing immunity, strengthening the body's anti-tumor ability, and prolonging life. Therefore, we speculate that peptides with 80% or more identical amino acid sequences to peptides 1, 2, and 3 have similar functions.

[0255] VII. Key Technologies Requested for Protection

[0256] Innovation: Making peptides into vaccines to improve the body's immunity and enhance 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 the 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 purpose is to enhance human immunity, improve disease resistance, fight tumors, and prolong life;

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

Claims

1. An application of a polypeptide having an amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA, characterized in that The polypeptide is used in preparing a drug for prolonging the lifespan of mice.

2. Use of a polypeptide having an amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA, characterized in that The polypeptide is used in the preparation of a drug for inhibiting tumor cell growth; the tumor cells are gastric cancer MGC-803 cells or acute lymphoblastic leukemia Jurkat cells.

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