A polypeptide capable of improving immunity, resisting tumors and prolonging life and its application

Abstract

The present invention discloses a polypeptide having the functions of improving immunity, resisting tumors and prolonging life and its application. The polypeptide having the above functions is selected from: (a) a polypeptide having an amino acid sequence of MNKAELIDVLTQKLGSDRRQATAAVENVVD; or (b) a polypeptide having an amino acid sequence of TIVRAVHKGDSVTITGFGVFEQRRRAARVA; or (c) a polypeptide having an amino acid sequence of RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or (d) a polypeptide having an amino acid sequence of VNKAELIDVLTGGLGSKRRQATAAVEGGVD; or (e) a polypeptide having an amino acid sequence of MGVAGLIDVL TQKLGSGGRQATAAVENDDD amino acid sequence; or (f) a polypeptide having a TGVRAGHNGDSVTITGFVGFEGRRRAARVA amino acid sequence; or (g) a polypeptide having a TIVRAVGKGDSGITIGFGVFERQRRAARVA amino acid sequence; or (h) a polypeptide having a RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA amino acid sequence; or (i) a polypeptide having a KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA amino acid sequence. The polypeptides for improving immunity, resisting tumors and prolonging life of the present invention can improve the immunity of the body, enhance disease resistance, reduce tumor incidence and prolong 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 span and its application. Background Art

[0002] In classical immunology, T cells develop into CD4 + Helper T cells and CD8 + There are two main subgroups of cytotoxic T cells. Recent studies have found that some CD4 + T cells can further differentiate into CD8 + 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, etc. have been widely reported. Some literature shows that researchers believe that CD4 + The increase in the number of CTLs and the enhancement of virulence may represent 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 the super-long-lived population is significantly higher than that in the normal population, suggesting 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 - ) account for about 80%-90% of the total number of monocytes in the peripheral blood of healthy people and play 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, and they are also called inflammatory monocyte subsets; non-classical (CD14 + CD16 ++) account for about 5%-10% of the total number of monocytes. They can wander on the surface of vascular endothelium and remove foreign bodies. They have immune surveillance functions and play an important role in early inflammatory response and tissue repair. They are also called migratory monocyte subsets. When the proportion of different monocyte subsets changes, the function of the corresponding cell subset will also be enhanced, which plays a role in indicating the 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 statistics from the World Health Organization, 1 / 5 of men and 1 / 6 of women worldwide will suffer from malignant tumors, of which almost half of the new cases of malignant tumors and more than half of the deaths from malignant tumors occur in Asia, especially China. Malignant tumors are divided into solid tumors and non-solid tumors. Common solid tumors include lung cancer, gastric cancer, breast cancer, etc., and common non-solid tumors include leukemia, lymphoma, etc. At present, there is no effective cure for malignant tumors. Treatment mainly relies on surgery, radiotherapy, and chemotherapy, but the harm caused to patients by these treatments cannot be ignored. The anti-tumor effect of drugs on solid tumors can be evaluated by calculating the tumor inhibition rate based on the size of the tumor. For non-solid tumors, the inhibition rate of drugs on tumor cell proliferation can be evaluated by in vitro experiments. How to enhance the efficacy, reduce side effects, and improve the quality of life of patients through the coordination of different treatment plans is an important part of the treatment of malignant tumors.

[0004] Aging is a biological process that inevitably occurs in all organisms, and is characterized by gradualness, harmfulness, and universality. With the progress of society, people's demand for anti-aging and life extension is constantly increasing. Anti-aging drugs refer to a class of drugs that can improve life efficiency, improve human physique, prevent and treat senile diseases, and prolong the life of the body within the limits determined by genetic characteristics; they are drugs that can prevent and delay the aging process of the body, improve tissue and cell pathological disorders, adjust the functions of important organs, regulate the balance of the body's internal environment, and promote overall health. Its characteristics are to adjust the body's substances, metabolism, and tissue and organ functional states through multi-level, multi-faceted and long-term effects, thereby achieving the purpose of delaying aging and increasing life.

[0005] At present, the demand of Chinese residents for extending life span and improving quality of life is increasing. According to our previous research results, we have found a peptide that can stimulate the proliferation of cell subpopulations in the body to enhance human immunity, has a significant inhibitory effect on tumor cells, and can extend individual life span. The peptide is made into a vaccine and inoculated into the human body to produce multiple functions such as enhancing immunity, anti-tumor, and extending life span. 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:

[0008] (a) a polypeptide having an 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 an amino acid sequence of RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA (polypeptide 3); or

[0011] (d) a polypeptide having an amino acid sequence of 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 of 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 an amino acid sequence of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.

[0017] The polypeptide having the functions of improving immunity, resisting tumors and prolonging lifespan of the present invention can improve the immunity of the body, enhance the disease resistance, reduce the incidence of tumors and prolong lifespan.

[0018] The polypeptide component of the present invention can stimulate the body's immune cells, making some immune cell subpopulations (such as CD4 + CTLs, CD14 + CD16 ++) proportion increased. These immune cell subsets have been shown in previous studies to enhance the body's immune function, enhance resistance to a variety of diseases, and reduce the prevalence of disease, thereby achieving the purpose of enhancing the body's disease resistance. The 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 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, 5, polypeptides 6, 7, and polypeptides 8, 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 with 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 by 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 by 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 by 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] Fig. 9 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after the intervention of the polypeptide 3 of the present invention;

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

[0029] Fig.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] Fig.12 This is a schematic diagram of the changes in monocyte subpopulations before and after stimulation by polypeptide 5 of the present invention;

[0031] Fig.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] Fig.14 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 4 of the present invention;

[0033] Fig.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] Fig.16 This is a schematic diagram of the changes in monocyte subpopulations before and after stimulation by the polypeptide 6 of the present invention;

[0035] Fig.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] Fig.18 This is a schematic diagram of the changes in monocyte subpopulations before and after stimulation by the polypeptide 7 of the present invention;

[0037] Fig.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] Fig. 20 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 6 of the present invention;

[0039] Fig.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] Fig. 22 This is a schematic diagram of the changes in monocyte subpopulations before and after stimulation by the polypeptide 8 of the present invention;

[0041] Fig.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] Fig.24 This is a schematic diagram of the changes in monocyte subpopulations before and after stimulation by the polypeptide 9 of the present invention;

[0043] Fig.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] Fig.26 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after the intervention of the polypeptide 8 of the present invention;

[0045] Fig. 27 This is a schematic diagram of the Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 9 of the present invention. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with 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 belong to the protection scope 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:

[0048] (a) a polypeptide having the amino acid sequence of 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 of 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 an 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 described in the present invention is characterized in that it contains a vector of a nucleic acid with the function of improving immunity, anti-tumor, and prolonging life, or a nucleic acid with the function of improving immunity, anti-tumor, and prolonging life is integrated into its genome.

[0062] The medicine is a polypeptide vaccine containing a polypeptide sequence having 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 described in 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 lymphocytic leukemia.

[0068] The present invention is further described below with 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 between 18 and 60 years old; (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 solution; 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 solution; flow cytometry permeabilization and fixation integrated solution.

[0072] 3. Experimental process: (1) According to the sequence of peptide 1, it was synthesized in vitro; (2) Peripheral blood PBMC was extracted: a) 5 ml of peripheral blood was extracted from the elbows of 6 healthy people and placed in EDTA anticoagulant tubes, 3 of which were tested for monocyte subsets, and 3 were tested for CD4 + CTLs subsets; b) dilute 5 ml of blood to 10 ml with PBS; c) take peripheral blood mononuclear cell separation solution into centrifuge tubes, 5 ml per tube, take 5 ml of the diluted blood and add it to the upper 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 an experimental group and a 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, centrifuge and remove the supernatant; b) Resuspend the cells with 100ul of lysate, 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 dark for 30 minutes; d) Monocyte group was added with 1 ml of lysate solution and centrifuged, the supernatant was removed, and CD4 + CTLs cell group was directly centrifuged and supernatant was removed; e) monocyte group was added with 200ul lysate to resuspend cells and tested on the machine, CD4 +100ul permeabilization fixative was added to the CTLs cell group, followed by GB11 antibody and incubation for 30min in the dark; 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, 100ul of membrane permeabilization fixative was added to resuspend the cells and tested on the instrument.

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

[0074] 5. Statistical analysis was performed on the changes in cell subpopulations after stimulation with peptide 1. The data of quantitative data were expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics 23 statistical software. If the data conformed to the normal distribution, a paired T test was selected. If it did not conform to the normal distribution, a Wilcoxon test was used. P < 0.05 was considered to be significantly different.

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

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

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

[0078] Peptide 1 stimulated CD4 + CTLs in CD4 +The proportion 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 in the control group 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 extending the lifespan of C57BL / 6J mice:

[0080] 1. Inclusion criteria: 40 8-week-old C57BL / 6J mice of specific pathogen-free (SPF) grade (the life span of healthy mice of this strain can reach 18-24 months, and the longest can reach 3 years), raised in an environment with 12h light / 12h dark alternation, ambient temperature controlled at 22-25℃, humidity controlled at 50-60%, 1 mouse / cage, growth and activity of the animals were observed after arriving at the experimental environment, and drug administration was started at 1 month of age.

[0081] 2. Experimental process: (1) According to the sequence of polypeptide 1, it was synthesized in vitro; (2) The polypeptide 1 powder was dissolved in sterile saline solution to 0.4 mg / ml, ultrafiltered into a sterile solution, and then stored aseptically at -20°C. It was restored to room temperature before use; (3) The 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, and the same time (9:00-11:00) was selected to be administered through the tail vein of the 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) The 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: The changes in the life span of mice after stimulation with peptide 1 were statistically analyzed. The data of the quantitative data were expressed as mean ± standard error (SE) and analyzed using GraphPad Prism 8 and SPSS Statistics23 statistical software. If the data conformed to the normal distribution, the paired T test was selected. If it did not conform to the normal distribution, the Wilcoxon test was used. P < 0.05 was considered to be significantly different. The survival curve was calculated using the Kaplan-Meier statistical method ( Figure 3 ).

[0083] The mouse lifespan observation test 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 lifespan and median lifespan of mice in the polypeptide 1 injection group were higher than those in the control group, and the differences were statistically significant (P<0.05). This shows that the polypeptide 1 intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body (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; the tumor cell line is gastric cancer MGC-803; the polypeptide 1 powder is dissolved in physiological saline to a final concentration of 1 mg / ml, filtered through a 0.22 μm filter membrane, and then aseptically packaged and stored at 4°C for later use; trastuzumab and docetaxel injection.

[0089] 2. Experimental method: MGC-803 cell line was 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 digestion solution, centrifuged at 1000 rpm and the supernatant was discarded. After washing three times with physiological saline, the cells were resuspended and counted to 5X10 7 / ml, stored at 4℃ for future use.

[0090] 3. Experimental procedure: (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 mice. 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 liquid 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 liquid 1 mg / kg, once every 3 days. (3) The tumor volume was measured 21 days after drug administration, and 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: The changes in tumor inhibition rate after the injection of peptide 1 were statistically analyzed. The data of quantitative data were expressed as mean ± standard error (SE), and analyzed using GraphPad Prism8 and SPSS Statistics23 statistical software. If the data conformed to the normal distribution, the paired T test was selected, and if it did not conform to the normal distribution, the Wilcxon test was used. P < 0.05 was considered to be significantly different.

[0092] The average tumor inhibition rate of the positive control group was (61.66±1.38)%, and the average tumor inhibition rate of the polypeptide 1 injection group was (67.62±0.90)%, and the average tumor inhibition rate of the combination drug group was (74.06±1.38)%; the average tumor inhibition rate of the combination drug group was higher than that of the positive control group and polypeptide 1 injection group, and the difference was 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 peptide 1 can inhibit tumor growth, and its combined use 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] 4. Experimental study on the efficacy of peptide 1 against non-solid tumors

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

[0098] 2. Experimental methods: (1) Cell culture: Jurkat cells were routinely cultured in RP-MI 1640 culture 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 to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to make a cell suspension, and inoculated into a 96-well plate at 2×10 cells per well. 5 cells, 200ul, cultured at 37℃, 5%CO2 for 24h, discarded the supernatant, set up experimental groups A, B, and C, added 200ul of complete culture medium containing 10ug / ml, 20ug / ml, and 30ug / ml of peptide 1 solution, respectively, and set up a blank control (no peptide 1) group and a cell-free control group. The commercial anticancer drug 5-FU (5-fluorouracil) was used as the positive control group for anticancer activity. Three counter-wells were set up in each group. After culturing for 4h (same conditions as before), 20ul of 5mg / ml MTT reagent was added to each well. After culturing for another 4h, the supernatant was discarded, 150ul DMSO was added, and the mixture was dissolved by shaking. The OD570 (absorbance value) at a wavelength of 570nm was measured using an enzyme reader; according to the formula: inhibition rate (%) = (control group OD570-experimental group OD570) x 100% / control group OD570.

[0099] 3. Experimental results: The MTT method verified that different concentrations of polypeptide 1 had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of polypeptide 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 inhibition 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 stimulation with peptide 2

[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)%, with 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)%, with 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)%, with 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)%, with significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 2 stimulation was significantly increased compared with that in the control group ( Figure 4 ).

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

[0108] Peptide 2 stimulated CD4 + CTLs in CD4 + The proportion 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 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) Peptide 3

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

[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)%, with 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)%, with 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)%, with 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)%, with significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 3 stimulation was significantly increased compared with that in the control group ( Figure 6 ).

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

[0113] Peptide 3 stimulates CD4 + CTLs in CD4 + The proportion 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 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 extending the lifespan of C57BL / 6J mice (same steps as Experiment 2):

[0115] (1) Peptide 2

[0116] (a) Experimental results: The average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide 2 injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 4). This shows that the peptide 2 intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0117] (b) Conclusion: Peptide 2 has similar functions to peptide 1 and can both extend 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) Peptide 3

[0121] (a) Experimental results: The average 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 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 lifespan and median lifespan of mice in the peptide 3 injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 5). This shows that the peptide 3 intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0122] (b) Conclusion: Peptide 3 has similar functions to peptide 1, and both can extend 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 polypeptide 2 injection group was (65.12±0.81)%, and the average tumor inhibition rate of the combination drug group 2 was (68.07±1.02)%; the average tumor inhibition rate of the combination drug group 2 was higher than that of the positive control group and polypeptide 2 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 2 injection group was higher than that of the positive control group, 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 combination drug group 3 was (70.22±0.88)%; the average tumor inhibition rate of the combination drug group 3 was higher than that of the positive control group and polypeptide 3 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 3 injection group was higher than that of the positive control group, the difference was statistically significant (P<0.05) (Table 6).

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

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

[0129] .

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

[0131] (1) Peptide 2

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

[0133] (b) Experimental methods: (1) Cell culture: Jurkat cells were routinely cultured in RP-MI 1640 culture 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 to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to make a cell suspension, and inoculated into a 96-well plate at 2 × 10 cells per well. 5 cells, 200ul, cultured at 37℃, 5%CO2 for 24h, discarded the supernatant, set up experimental groups A, B, C, added 200ul of complete culture medium containing 10ug / ml, 20ug / ml, 30ug / ml peptide 2 solution, respectively, and set up blank control (no peptide 2 added) group and cell-free control group, and used commercial anticancer drug 5-FU (5-fluorouracil) as the control group for anticancer activity; set up 3 counter-wells for each group, cultured for 4h (same conditions as before), added 20ul of 5mg / ml MTT reagent to each well, cultured for another 4h, discarded the supernatant, added 150ul DMSO, vortexed to dissolve, and used a microplate reader to measure OD570 (absorbance value) at a wavelength of 570nm; according to the formula "inhibition rate (%) = (control group OD570-experimental group OD570) x 100% / control group OD570".

[0134] (c) Experimental results: The MTT method verified that different concentrations of peptide 2 solution all had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of this concentration of peptide on tumor cell proliferation was stronger than that of 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) Peptide 3

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

[0139] (b) Experimental methods: (1) Cell culture: Jurkat cells were routinely cultured in RP-MI 1640 culture 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 to determine cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to make a cell suspension, and inoculated into a 96-well plate at 2 × 10 cells per well. 5 cells, 200ul, cultured at 37℃, 5%CO2 for 24h, discarded the supernatant, set up experimental groups A, B, and C, added 200ul of complete culture medium containing 10ug / ml, 20ug / ml, and 30ug / ml peptide 3 solution, respectively, and set up a blank control (no peptide 3 added) group and a cell-free control group. The commercial anticancer drug 5-FU (5-fluorouracil) was used as the control group for anticancer activity. Three counter-wells were set up for each group. After culturing for 4h (same conditions as before), 20ul of 5mg / ml MTT reagent was added to each well. After culturing for another 4h, the supernatant was discarded, 150ul DMSO was added, and the mixture was dissolved by shaking. The OD570 (absorbance value) at a wavelength of 570nm was measured using an enzyme reader; according to the formula "inhibition rate (%) = (control group OD570-experimental group OD570) x 100% / control group OD570".

[0140] (c) Experimental results: The MTT assay showed that different concentrations of peptide 3 all had an inhibitory effect on the proliferation of tumor cells, with 30ug / 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 inhibition rates of different concentrations of peptide 3 co-cultured 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 to improve human immunity (same steps as experiment 1)

[0145] (1) Peptide 4

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

[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)%, with 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)%, with 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)%, with 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)%, with significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 4 stimulation was significantly increased compared with that in the control group ( Fig.10 ).

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

[0149] Peptide 4 stimulated CD4 + CTLs in CD4 + The proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.11 ).

[0150] (2) Peptide 5

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

[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)%, with 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)%, with 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)%, with 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)%, 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 that in the control group ( Fig.12 ).

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

[0154] Peptide 5 stimulates CD4 + CTLs in CD4 + The proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.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 average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 9). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0158] (b) Conclusion: Peptide 4 has similar functions to peptide 1 and can both extend 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 average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 10). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0163] (b) Conclusion: Peptide 5 has similar functions to peptide 1, and both can extend 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 polypeptide 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 polypeptide 4 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 4 injection group was higher than that of the positive control group, 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 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 polypeptide 5 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 5 injection group was higher than that of the positive control group, the difference was statistically significant (P<0.05) (Table 11).

[0168] (b) Conclusion: Injection of peptides 4 and 5 can inhibit tumor growth. Their combination 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 the efficacy against non-solid tumors:

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

[0173] Experimental results: The MTT method verified that different concentrations of peptide 4 solutions all had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of this concentration of peptide on tumor cell proliferation was stronger than that of 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) Peptide 5 (same steps as experiment 4)

[0177] Experimental results: The MTT method verified that different concentrations of polypeptide 5 all had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of polypeptide 5 at this concentration on tumor cell proliferation was stronger than that of the standard therapeutic drug 5-FU (Table 13).

[0178] Table 13. Comparison of the inhibition 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 to improve human immunity (same steps as experiment 1)

[0182] (1) Peptide 6

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

[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 that in the control group ( Fig.16 ).

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

[0186] Peptide 6 stimulates CD4 + CTLs in CD4 + The proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.17 ).

[0187] (2) Peptide 7

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

[0189] The proportion 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 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)%, while 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)%, while 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)%, while 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 that in the control group ( Fig.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 proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.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 average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 14). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0195] (b) Conclusion: Peptide 6 has similar functions to peptide 2, and both can extend 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 average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 15). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0200] (b) Conclusion: Peptide 7 has similar functions to peptide 2, and both can extend 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 polypeptide 6 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 6 injection group was higher than that of the positive control group, 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 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 polypeptide 7 injection group, the difference was statistically significant (P<0.05), and the average tumor inhibition rate of the polypeptide 7 injection group was higher than that of the positive control group, the difference was 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 with different drug interventions

[0207] .

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

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

[0210] (a) Experimental results: The MTT method verified that different concentrations of peptide 6 solution all had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of this concentration of peptide on tumor cell proliferation was stronger than that of the standard therapeutic drug 5-FU (Table 17).

[0211] Table 17. Comparison of inhibition rate of co-culture of different concentrations of peptide 6 and Jurkat cells

[0212] .

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

[0214] (a) Experimental results: The MTT assay showed that different concentrations of peptide 7 all had an inhibitory effect on the proliferation of tumor cells, with 30ug / ml having 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 rate of co-culture of different concentrations of peptide 7 and Jurkat cells

[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 to improve human immunity (same steps as experiment 1)

[0219] (1) Peptide 8

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

[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)%, with 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)%, with 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)%, with 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)%, with significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 8 stimulation was significantly increased compared with that in the control group ( Fig. 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 proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.23 ).

[0224] (2) Peptide 9

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

[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)%, with 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)%, with 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)%, with 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)%, with significant difference between the two groups (P<0.05). The proportion of non-classical monocytes after peptide 9 stimulation was significantly increased compared with that in the control group ( Fig.24 ).

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

[0228] Peptide 9 stimulates CD4 + CTLs in CD4 + The proportion 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 in the control group was higher than that in the control group, and the difference was statistically significant (P < 0.05). Fig.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 average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 19). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

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

[0236] (a) Experimental results: The average lifespan of mice in the peptide 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 lifespan and median lifespan of mice in the peptide injection group were higher than those in the control group, and the differences were statistically significant (P<0.05) (Table 20). This shows that the peptide intervention can prolong the lifespan of mice in terms of average lifespan and median lifespan, and play a role in delaying the aging of the body.

[0237] (b) Conclusion: Peptide 9 has similar functions to peptide 3, and both can extend 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)%, and 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 group was (70.32±1.44)%; the average tumor inhibition rate of the combined drug group was higher than that of the positive control group and the polypeptide 8 injection group, and the difference was statistically significant (P<0.05), and 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 group was (67.35±0.79)%; the average tumor inhibition rate of the combined drug group was higher than that of the positive control group and the polypeptide 9 injection group, and the difference was statistically significant (P<0.05), and 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: 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 the efficacy against non-solid tumors:

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

[0247] (a) Experimental results: The MTT method verified that different concentrations of polypeptide 8 solution all had an inhibitory effect on the proliferation of tumor cells, among which 30ug / ml had the strongest inhibitory effect. The inhibitory rate of polypeptide 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 inhibition rates 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 showed that different concentrations of peptide 9 inhibited the proliferation of tumor cells, with 30ug / ml having the strongest inhibitory effect. The inhibitory rate of peptide 9 at this concentration on tumor cell proliferation was stronger than that of 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 above polypeptides, the polypeptide components can stimulate the body's immune cells, making some immune cell subsets (such as CD4 + CTLs, CD14 + CD16 ++ ) proportion increased. These immune cell subsets have been shown in previous studies to enhance the body's immune function, enhance resistance to a variety of diseases, and reduce the prevalence of disease, thereby achieving the purpose of enhancing the body's disease resistance. The 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 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, 5, polypeptides 6, 7, and polypeptides 8, 9, respectively. It has been verified that the polypeptides after 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 with polypeptides 1, 2, and 3 have similar functions.

[0255] VII. Key technologies for which protection is sought

[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 according to the 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 KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA, characterized in that The polypeptide is used in preparing a drug for prolonging the life span of mice.

2. An application of a polypeptide having an amino acid sequence of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA, characterized in that The polypeptide is used in preparing a drug for inhibiting tumor cell growth; the tumor cell is gastric cancer MGC-803 or acute lymphoblastic leukemia Jurkat cell.

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