CN115804834A - Polypeptide with functions of improving immunity, resisting tumors and prolonging life and application thereof - Google Patents

Abstract

The invention discloses a polypeptide with the functions of improving immunity, resisting tumor and prolonging life and application thereof. The polypeptide with the 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 the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD; or (f) a polypeptide having an amino acid sequence of TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or (g) a polypeptide having an amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA; or (h) a polypeptide having an amino acid sequence of RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA; or (i) a polypeptide having the amino acid sequence KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA. The polypeptide for improving immunity, resisting tumor and prolonging life can improve the immunity of organisms, enhance the disease resistance, reduce the tumor incidence and prolong the life.

Description

A kind of polypeptide with the functions of improving immunity, anti-tumor and prolonging lifespan and its application

technical field

The invention belongs to the technical field of medicine, and in particular relates to a polypeptide with the functions of improving immunity, anti-tumor and prolonging lifespan and its application.

Background technique

In the classical immunological theory, T cells develop into two main subpopulations in the thymus, CD4 ⁺ helper T cells and CD8 ⁺ cytotoxic T cells. In recent years, studies have found that some CD4 ⁺ T cells can be further differentiated outside the thymus into a subset with direct cell killing functions similar to CD8 ⁺ CTLs and NK cells, that is, CD4 ⁺ CTLs. At present, CD4 ⁺ CTLs have been further confirmed to play an important role in infection, tumor, autoimmune disease, vaccination and other processes. Especially in infectious diseases, including influenza virus, cytomegalovirus, EBV, human papillomavirus and HIV have been widely reported. According to literature, researchers believe that the increase in the number of CD4 ⁺ 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 the immune function. The proportion of the subgroup was significantly higher than that of the normal population, which suggested that the increase of the proportion of the subgroup of cells was related to longevity. According to previous studies, monocytes can also be divided into three subgroups, namely intermediate type (CD14 ⁺⁺ CD16 ⁺ ), classic type (CD14 ⁺⁺ CD16 ^- ) and non-classical type (CD14 ⁺ CD16 ⁺⁺ ). Among them, the classic type (CD14 ⁺⁺ CD16 ^- ) accounts for about 80%-90% of the total monocytes in the peripheral blood of healthy people, and plays an important role in the innate immune defense mechanism; the intermediate type (CD14 ⁺⁺ CD16 ⁺ ) accounts for about The total number of nuclear cells is 5%, and it can reach 10%-50% under severe infection and inflammation. The main functions are antigen presentation, inflammation and monocyte activation, also known as inflammatory monocyte subsets; non-classical (CD14 ⁺ CD16 ⁺⁺ ) accounts for about 5%-10% of the total number of monocytes, can swim on the surface of vascular endothelium and remove foreign matter, has immune surveillance function, and plays an important role in early response to inflammation and tissue repair, also known as Migratory monocyte subsets. When the ratio of different monocyte subgroups changes, the function of the corresponding cell subgroups will also be enhanced, which plays a role in suggesting the changes in the immune function of the body.

In recent years, the incidence of malignant tumors characterized by abnormal cell proliferation has increased significantly. According to the statistics of the World Health Organization, 1/5 of men and 1/6 of women in the world will suffer from malignant tumors, and almost half of the new cases of malignant tumors and more than half of the deaths of malignant tumors in the world occur in Asia, especially in Asia. is China. Malignant tumors are divided into solid tumors and non-solid tumors, common solid tumors such as lung cancer, gastric cancer, breast cancer, etc., common non-solid tumors such as leukemia, lymphoma, etc. At present, there is no effective radical cure for malignant tumors, and the treatment mainly relies on surgery, radiotherapy, and chemotherapy, but the harm caused by these treatments to patients cannot be ignored. The antitumor effect of drugs on solid tumors can be evaluated by calculating the tumor inhibition rate based on the tumor volume. For non-solid tumors, the inhibitory rate of drugs on tumor cell proliferation can be evaluated through in vitro experiments. How to enhance the efficacy, reduce side effects, and improve the quality of life of patients through the cooperation of different treatment options is an important part of the treatment of malignant tumors.

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 prolonging life continues to increase. 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 limit determined by genetic characteristics; , adjust the functions of important organs, regulate the balance of the body's internal environment, and promote overall health. Its characteristic is to adjust the material, metabolism and functional state of tissues and organs of the body through multi-level, multi-faceted and long-term effects, so as to achieve the purpose of delaying aging and increasing life expectancy.

At present, the needs of Chinese residents to prolong life and improve the quality of life are constantly increasing. According to our previous research results, we found that a polypeptide can stimulate the proliferation of subpopulations of body cells to enhance human immunity, and has a significant inhibitory effect on tumor cells. And can prolong the life of the individual. The polypeptide is made into a vaccine, and after being inoculated in the human body, it can produce multiple functions of enhancing immunity, anti-tumor, and prolonging life.

Contents of the invention

The first purpose of the present invention is to provide a polypeptide that can improve immunity, anti-tumor and prolong life; the second purpose is to provide the application of the polypeptide that can improve immunity, anti-tumor and prolong life.

The first object of the present invention is achieved in this way, and the described polypeptide having the functions of improving immunity, anti-tumor and prolonging lifespan is selected from:

(a) a polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD (polypeptide 1); or

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

(c) a polypeptide having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA (polypeptide 3); or

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

(e) a polypeptide having the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD (polypeptide 5); or

(f) a polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA (polypeptide 6); or

(g) a polypeptide having the amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA (polypeptide 7); or

(h) a polypeptide having the amino acid sequence RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA (polypeptide 8); or

(i) A polypeptide having the amino acid sequence KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA (polypeptide 9).

The polypeptide with the functions of improving immunity, anti-tumor and prolonging lifespan according to the present invention can improve the body's immunity, enhance disease resistance, reduce the incidence of tumors and prolong lifespan.

The polypeptide components described in the present invention can stimulate the body's immune cells and increase the proportion of some immune cell subsets (such as CD4 ⁺ CTLs, CD14 ⁺ CD16 ⁺⁺ ). These immune cell subsets have been proven to enhance the body's immunity in previous studies. Function, enhance the resistance to various diseases, reduce the prevalence, so as to achieve the purpose of enhancing the body's disease resistance. The polypeptide can also enhance the anti-tumor ability of the body 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. Partial changes were made to the amino acid sequences of the three groups of polypeptides to obtain polypeptides 4 and 5, polypeptides 6 and 7, and polypeptides 8 and 9 respectively. It has been verified that the sequence-changed polypeptides still have the ability to enhance immunity, strengthen the body's anti-tumor ability, and prolong life Therefore, we speculate that peptides with 80% or more identical amino acid sequences to peptides 1, 2, and 3 have similar functions.

Description of drawings

Fig. 1 is a schematic diagram of changes in monocyte subgroups before and after stimulation by polypeptide 1 of the present invention;

Fig. 2 is a schematic diagram of the changes in the proportion of CD4 ⁺ CTLs before and after stimulation by polypeptide 1 of the present invention;

Fig. 3 is a schematic diagram of Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 1 of the present invention;

Fig. 4 is a schematic diagram of changes in monocyte subgroups before and after stimulation by polypeptide 6 of the present invention;

Figure 5 is a schematic diagram of the change in the proportion of CD4 ⁺ CTLs in CD4 ⁺ cells before and after stimulation by polypeptide 6 of the present invention;

Fig. 6 is a schematic diagram of changes in monocyte subgroups before and after stimulation by polypeptide 7 of the present invention;

Figure 7 is a schematic diagram of the change in the proportion of CD4 ⁺ CTLs in CD4 ⁺ cells before and after stimulation by polypeptide 7 of the present invention;

Fig. 8 is a schematic diagram of Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 6 of the present invention;

Fig. 9 is a schematic diagram of Kaplan-Meier survival curve analysis of mice before and after the intervention of polypeptide 7 of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited in any way. Any transformation or replacement based on the teaching of the present invention belongs to the protection scope of the present invention.

The polypeptides with improving immunity, anti-tumor and prolonging lifespan described in the present invention are selected from:

(a) a polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD; or

(b) a polypeptide having the amino acid sequence of TIVRAVHKGDSVTITGFGVFEQRRRAARVA; or

(c) a polypeptide having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or

(d) a polypeptide having the amino acid sequence VNKAELIDVLTGGLGSKRRQATAAVEGGVD; or

(e) a polypeptide having the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD; or

(f) a polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or

(g) a polypeptide having the amino acid sequence TIVRAVGKGDSGITIGFGVFERQRRAARVA; or

(h) a polypeptide having the amino acid sequence RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA; or

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

The nucleic acid with functions of improving immunity, anti-tumor, and prolonging life according to the present invention is selected from:

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

(b) Nucleic acid complementary to the nucleic acid sequences (a), (b), (c).

The carrier of the present invention contains nucleic acid with the functions of improving immunity, anti-tumor and prolonging life.

The host cell of the present invention is characterized in that it contains the carrier of nucleic acid with functions of improving immunity, anti-tumor and life extension, or the nucleic acid with functions of improving immunity, anti-tumor and life extension is integrated in its genome.

The medicine is a polypeptide vaccine containing a polypeptide sequence with the functions of improving immunity, anti-tumor and prolonging life.

The application of the polypeptide with the functions of improving immunity, anti-tumor and life extension in the present invention is the application of the polypeptide with improving immunity in medicines for enhancing immunity.

The application of the polypeptide with the functions of improving immunity, anti-tumor and life extension in the present invention is the application of the polypeptide with improving immunity in the preparation of medicines for extending life.

The application of the polypeptide with the functions of improving immunity, anti-tumor and life extension in the present invention is the application of the polypeptide with improving immunity in the preparation of anti-tumor drugs.

The tumor is a solid tumor or a non-solid tumor.

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

The present invention will be further described below with concrete implementation experimental case:

1. The specific operation of the experiment of polypeptide 1 improving human immunity is as follows:

1. Inclusion criteria: (1) Age between 18-60; (2) Normal population (normal liver and kidney function tests, exclusion: people infected with tuberculosis, HBV, HCV, tumors and autoimmune diseases).

2. Experimental materials: human peripheral venous blood; human peripheral blood mononuclear cell separation fluid; PBS buffer; fetal bovine serum; penicillin-streptomycin solution (double antibody); PE-CD14 antibody, FITC-CD16 antibody, Perpcy- 5.5-CD45 antibody, AlexaFlour647-GB11 antibody, FITC-CD3 antibody, PE-CD4 antibody; erythrocyte lysate; flow cytometry fixation fluid.

3. Experimental procedure: (1) According to the peptide 1 sequence, synthesize it in vitro; (2) Extract peripheral blood PBMC: a) Take 5ml of peripheral blood from the elbow of 6 healthy people in EDTA anticoagulation tubes, and detect mononuclear cells in 3 cases. Cell subgroups, CD4 ⁺ CTLs subgroups were detected in 3 cases; b) 5ml of blood was diluted to 10ml with PBS; c) Peripheral blood mononuclear cell separation liquid was taken in centrifuge tubes, 5ml per tube, and 5ml of diluted blood was taken Add to the upper layer of the separation medium; d) 1000g, acceleration 4, deceleration 4, centrifuge for 25 minutes; e) Use a pipette to carefully suck out the PBMC layer cells, dilute with lysing red solution and centrifuge, remove the supernatant, repeat 2 times; 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 the experimental group and the control group, each with 500ul; (3) Use PBS to dissolve the peptide 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 cytometric detection: a) Use 1ml of lysing red solution to resuspend the cells, centrifuge and remove the supernatant; b ) ^cells were resuspended in 100ul lysed red solution, CD14, CD16, CD45 antibodies were added to the mononuclear cell group and then incubated in the ^dark for 30min; Membrane fixation solution was kept in the dark for 30 minutes; d) Add 1ml Cleaving Red Solution to the sample of monocyte group and centrifuge to remove the supernatant; CD4 ⁺ CTLs cell group was directly centrifuged and remove the supernatant; e) Add 200ul Cleaving Red to the mononuclear cell group Cells were resuspended in liquid solution and tested on the machine. Add 100ul membrane-permeating fixative to the CD4 ⁺ CTLs cell group, add GB11 antibody and incubate for 30min in the dark; f) add 1ml permeabilization-permeabilizing fixative to the CD4 ⁺ CTLs cell group, centrifuge and remove the supernatant; g ) CD4 ⁺ CTLs cell group, add 100ul permeabilization fixative to resuspend the cells, and test on the machine.

4. Experimental results: After polypeptide 1 stimulation, the proportion of non-classical (CD14 ⁺ CD16 ⁺⁺ ) monocytes and CD4 ⁺ CTLs subsets in T cells increased significantly. It suggested that the cellular immune function was enhanced.

5. Statistical analysis was carried out on the changes of cell subpopulations after polypeptide 1 stimulation, and the data of measurement data were expressed as mean ± standard error (stand error, S.E). GraphPad Prism 8 and SPSS Statistics23 statistical software were used for analysis. If the data conformed to the normal distribution, the paired t-test was selected, and if the data did not conform to the normal distribution, the Wilcoxon test was used. P<0.05 was regarded as a significant difference.

(1) Analysis of changes in monocyte subsets after polypeptide 1 stimulation

The proportion of monocytes in the polypeptide 1 stimulation group was (19.25±8.18)%, and that in the control group was (15.79±9.08)%. There was no significant difference between the groups (P>0.05); The percentage of mononuclear cells was (3.85±2.82)%, and that of the control group was (5.61±3.88)%. There was no significant difference between the groups (P<0.05); The proportion of the middle group was (93.42±0.69)%, and that of the control group was (85.27±7.42)%. The difference between the groups was not statistically significant (P<0.05); Compared with the control group (18.85±4.82)%, the difference between the groups was statistically significant (P<0.05). After polypeptide 1 stimulation, the proportion of non-classical monocytes increased significantly compared with the control group (Fig. 1).

(2) Analysis of changes in CD4 ⁺ CTLs cell subsets after polypeptide 1 stimulation

CD4 ⁺ CTLs accounted for (3.43±0.21)% of CD4 ⁺ cells in the polypeptide 1 stimulation group, and (0.97±0.04)% in the control group, and the proportion of CD4 ⁺ CTLs in the polypeptide ¹ stimulation group was higher than that of the control group, the difference was statistically significant (P<0.05) (Figure 2).

2. Experimental study of polypeptide 1 prolonging the lifespan of C57BL/6J mice:

1. Inclusion criteria: Specific pathogen-free (SPF) 8-week-old C57BL/6J mice (healthy mice of this strain can live up to 18-24 months, up to 3 years) 40, feeding environment 12h light/12h The darkness is alternated, the ambient temperature is controlled at 22-25°C, and the humidity is controlled at 50-60%, 1 animal/cage. After the animals arrive in the experimental environment, the growth and activity are observed, and the drug is started at the age of 1 month.

2. Experimental procedure: (1) According to the peptide 1 sequence, it was synthesized in vitro; (2) The peptide 1 powder was dissolved in sterile saline solution to 0.4mg/ml, ultrafiltered into a sterile solution, and stored at -20°C Store aseptically and return to room temperature before use; (3) The mice were randomly divided into a polypeptide-administered group and a vehicle control group, 20 each; (4) The polypeptide-administered group was injected with polypeptide 1 at a dose of 6 mg/kg, 1 Once/3 days, choose the same time (9:00-11:00) to administer via the tail vein of the rats, and the vehicle control group was given the corresponding injection volume of normal saline; (5) The mice were administered until the mice died naturally, and the time of death was recorded and the most probable cause of death; (6) Remove the mice that died due to operation accidents or external causes, and use the Kaplan-Meier statistical method to perform survival analysis and draw the survival curve.

3. Experimental results: Statistical analysis was carried out on the changes in the lifespan of mice after stimulation with polypeptide 1. The data of the measurement data were expressed as mean ± standard error (stand error, S.E), and were analyzed using GraphPad Prism 8 and SPSS Statistics23 statistical software. If the data conformed to the normal distribution, the paired t-test was selected, and if the data did not conform to the normal distribution, the Wilcoxon test was used. P<0.05 was regarded as a significant difference. Survival curve using Kaplan-Meier statistical method (Figure 3).

The lifespan observation test of mice showed that the average lifespan of mice in the polypeptide 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 The lifespan was (17±0.89) months; the average lifespan and median lifespan of the mice injected with polypeptide 1 were higher than those of the control group, and the differences were statistically significant (P<0.05). This shows that the intervention of peptide 1 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).

Table 1 Average and median survival time of mice before and after polypeptide 1 intervention

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4. Conclusion: The peptide 1 intervention can delay the aging of the body and prolong the life span of mice.

3. Experimental research on the anti-solid tumor efficacy of polypeptide 1:

1. Experimental materials: 40 male nude mice of 18-22g; the tumor cell line is gastric cancer MGC-803; the polypeptide 1 powder was dissolved in normal saline to make the final concentration 1mg/ml, and aseptically separated after filtration with a 0.22 micron filter membrane. Packed and stored at 4°C for later use; trastuzumab and docetaxel injection.

2. Experimental method: culture MGC-803 cell line at 37°C, 5% CO ₂ incubator to a density above 80%, the medium is DMEM high-glucose medium containing 10% FBS, digested with 0.25% trypsin The cells were collected by digestion with liquid solution, the supernatant was discarded by centrifugation at 1000 rpm, washed three times with normal saline, resuspended and counted to 5X10 ⁷ /ml, and stored at 4°C for future use.

3. Experimental procedure: (1) Divide the mice into 4 groups at random, namely the blank control group, the polypeptide group, the combination drug group, and the positive control group, with 10 mice in each group, and prepare 0.2ml of the prepared cancer cell suspension /Only inoculated subcutaneously in the right armpit of mice, observed the tumor appearance every three days after inoculation, and started administration when the tumor volume reached 0.1mm ³ . (2) The positive control group received intravenous injection of trastuzumab 15ml/kg, once every 3 days; docetaxel 5mg/kg, once every 7 days; the blank group received intravenous injection of normal saline 10ml/kg, once every 3 days; times/3 days; the peptide group received intravenous injection of 1 mg/kg prepared peptide solution 1, once/3 days; the combined drug group received intravenous injection of trastuzumab 15ml/kg, once/3 days; Dorsey Hexamate 5mg/kg, 1 time/7 days; the prepared polypeptide 1 solution 1mg/kg, 1 time/3 days. (3) The tumor volume was measured 21 days after the administration, and the tumor inhibition rate (%)=(tumor volume of the blank control group-tumor volume of the administration group)/tumor volume of the blank control group was measured according to the formula.

4. Experimental results: Statistical analysis was carried out on the change of tumor inhibition rate after polypeptide 1 injection, and the data of measurement data were expressed as mean ± standard error (stand erro, S.E). GraphPad Prism8 and SPSS Statistics23 statistical software were used for analysis. If the data conformed to the normal distribution, the paired t-test was selected, and if the data did not conform to the normal distribution, the Wilcxon test was used. P<0.05 was regarded as a significant difference.

The average tumor inhibition rate of the positive control group was (61.66±1.38)%, the average tumor inhibition rate of the polypeptide 1 injection group was (67.62±0.90)%, and the average tumor inhibition rate of the combined drug group was (74.06±1.38)%. The tumor inhibition rate was higher than that of the positive control group and the 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).

5. Conclusion: Polypeptide 1 injection can inhibit tumor growth, and combined use with existing anti-tumor drugs can enhance the anti-tumor effect of drugs.

Table 2 Comparison of tumor inhibition rates of different drugs intervening in solid tumors

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4. Experimental study on the anti-non-solid tumor efficacy of polypeptide 1

1. Experimental materials: acute lymphoblastic leukemia Jurkat cells; fetal bovine serum; RP-MI1640 culture medium; 5-fluorouracil (5-FU); using DMSO as a solvent, make polypeptide 1 powder into 10ug/ml, 20ug/ml, 30ug/ml polypeptide solution.

2. Experimental methods: (1) Cell culture: Jurkat cells were placed in an incubator at 37°C with a volume fraction of 5% CO ₂ and saturated humidity with RP-MI 1640 culture medium containing 10% fetal bovine serum and 1% double antibody In conventional culture, the culture medium was replaced every 2 days; (2) MTT assay was used to measure cell proliferation activity: Jurkat cells in the logarithmic growth phase were digested with trypsin, resuspended in DMSO to make a cell suspension, and inoculated in a 96-well plate In each well, 2x10 ⁵ cells, 200ul, cultured at 37℃, 5% CO ₂ for 24h, removed the supernatant, set up experimental groups A, B, C, added 10ug/ml, 20ug/ml, 30ug/ml 200ul of the complete culture medium of polypeptide 1 solution, and set up a blank control group (without adding polypeptide 1) and a cell-free control group, using commercial anticancer drug 5-FU (5-fluorouracil) as the positive control group for anticancer activity, Set up 3 auxiliary wells for each group, add 20ul of 5mg/ml MTT reagent to each well after culturing for 4 hours (the same conditions as before), discard the supernatant after culturing for another 4 hours, add 150ul of DMSO, shake to dissolve, and use a microplate reader to measure 570nm OD570 (absorbance value) at the wavelength; according to the formula: inhibition rate (%) = (OD570 of the control group-OD570 of the experimental group) x 100%/OD570 of the control group.

3. Experimental results: As verified by the MTT method, different concentrations of polypeptide 1 have inhibitory effects on tumor cell proliferation, among which 30ug/ml has the strongest inhibitory effect, and the inhibitory rate of this concentration of polypeptide 1 on tumor cell proliferation is stronger than Standard treatment drug 5-FU (Table 3).

Table 3 Comparison of inhibition rate between different concentrations of polypeptide 1 and Jurkat cells cultured in vitro

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5. We speculate that peptide 6 and peptide 7, which are similar in sequence to peptide 2, also have similar effects, and relevant experiments have been carried out to verify this

1. Experiment to improve human immunity (steps are the same as experiment 1)

(1) Polypeptide 6

(a) Analysis of changes in monocyte subsets after polypeptide 6 stimulation

The proportion of monocytes in the polypeptide 6 stimulation group was (25.60±4.52)%, and that in the control group was (24.20±2.41)%. There was no significant difference between the groups (P>0.05); The proportion of monocytes was (10.15±0.80)%, and that of the control group was (9.72±0.49)%, and the difference between the groups was not statistically significant (P>0.05); The proportion of the middle group was (88.79±3.52)%, and that of the control group was (89.78±1.12)%. The difference between the groups was not statistically significant (P>0.05); Compared with the control group (21.75±1.86)%, the difference between the groups was statistically significant (P<0.05). After polypeptide 6 stimulation, the proportion of non-classical monocytes increased significantly compared with the control group (Fig. 4).

(b) Analysis of changes in CD4 ⁺ CTLs cell subsets after polypeptide 6 stimulation

CD4 ⁺ CTLs accounted for (3.11±0.41)% of CD4 ⁺ cells in the polypeptide 6 stimulation group, and (1.87±0.43)% in ^the control group, and the proportion of CD4 ⁺ CTLs in the polypeptide 6 stimulation group was higher than that of the control group, the difference was statistically significant (P<0.05) (Figure 5).

(2) Peptide 7

(a) Analysis of changes in monocyte subsets after polypeptide 7 stimulation

The proportion of monocytes in the polypeptide 7 stimulation group was (22.87±1.71)%, and that in the control group was (20.71±6.70)%. There was no significant difference between the groups (P>0.05); The proportion of monocytes was (10.64±3.10)%, and that of the control group was (9.93±0.51)%, and the difference between the groups was not statistically significant (P>0.05); The percentage of non-classical monocytes in the polypeptide 7 stimulation group was (89.94±3.57)%, and that of the control group was (91.23±3.67)%. Compared with the control group (21.47±2.00)%, the difference between the groups was statistically significant (P<0.05). After polypeptide 5 stimulation, the proportion of non-classical monocytes increased significantly compared with the control group (Fig. 6).

(b) Analysis of changes in CD4 ⁺ CTLs cell subsets after polypeptide 7 stimulation

CD4 ⁺ CTLs accounted for (2.35±0.53)% of CD4 ⁺ cells in the polypeptide 7 stimulation group, and (1.18±0.56)% in the control group, and the proportion of CD4 ⁺ CTLs in the polypeptide ⁷ stimulation group was higher than that of the control group, the difference was statistically significant (P<0.05) (Figure 7).

2. Experimental research on prolonging the lifespan of C57BL/6J mice (the steps are the same as experiment 2):

(1) Polypeptide 6

(a) Experimental results: the average lifespan of mice in the polypeptide injection group was (26.13±1.39) months, with a median lifespan of (23±2.65) months; the average lifespan of mice in the control group was (22.85±1.15) months, with a median lifespan of (21±0.36) months; the average lifespan and median lifespan of the mice in the polypeptide 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 polypeptide 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.

(b) Conclusion: Peptide 6 and Peptide 2 have similar functions, both of which can prolong the lifespan of mice and delay the aging of the body.

Table 4 Average and median survival time of mice before and after polypeptide 6 intervention

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(2) Peptide 7

(a) Experimental results: the average lifespan of mice in the polypeptide injection group was (28±5.16) months, with a median lifespan of (22.00±2.34) months; the average lifespan of mice in the control group was (21.00±1.19) months, with a median lifespan of (18.00±0.56) months; the average lifespan and median lifespan of the mice in the polypeptide 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 polypeptide 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.

(b) Conclusion: Peptide 7 has similar functions to Peptide 2, both of which can prolong the lifespan of mice and delay the aging of the body.

Table 5 Average and median survival time of mice before and after polypeptide 7 intervention

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3. Experimental research on anti-solid tumor efficacy (steps are the same as experiment three):

(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 6 combined drug groups was (68.12±1.32) )%; the average tumor inhibition rate of the combination drug group 6 was higher than that of the positive control group and the polypeptide 6 injection group, and the difference was 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, with a difference 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 7 combined drug groups was (70.35±0.89)%; the average tumor inhibitory rate of the 7 combined drug groups was high In the positive control group and the 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 6) .

(b) Conclusion: Both polypeptide 6 and 7 injections can inhibit tumor growth, and combined use with existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.

Table 6 Comparison of tumor inhibition rate of solid tumors intervened by different drugs

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4. Experimental research on anti-non-solid tumor efficacy:

(1) Peptide 6 (the steps are the same as experiment 4)

(a) Experimental results: As verified by the MTT method, different concentrations of polypeptide 6 solutions have inhibitory effects on the proliferation of tumor cells, among which 30ug/ml has the strongest inhibitory effect, and the concentration of polypeptides has a strong inhibitory rate on tumor cell proliferation than the standard treatment drug 5-FU (Table 7).

Table 7 Comparison of inhibition rate of co-culture of different concentrations of polypeptide 6 and Jurkat cells

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(2) Peptide 7 (the steps are the same as experiment 4)

(a) Experimental results: As verified by the MTT method, different concentrations of polypeptide 7 have inhibitory effects on tumor cell proliferation, among which 30ug/ml has the strongest inhibitory effect, and this concentration of polypeptide 7 has a strong inhibitory rate on tumor cell proliferation than the standard treatment drug 5-FU (Table 8).

Table 8 Comparison of inhibition rate of co-culture of different concentrations of polypeptide 7 and Jurkat cells

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After injection of the above polypeptides, the polypeptide components can stimulate the body's immune cells, increasing the proportion of some immune cell subsets (such as CD4 ⁺ CTLs, CD14 ⁺ CD16 ⁺⁺ ), which have been proven in previous studies to enhance the body's immune system. Function, enhance the resistance to various diseases, reduce the prevalence, so as to achieve the purpose of enhancing the body's disease resistance. The polypeptide can also enhance the anti-tumor ability of the body 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. Partial changes were made to the amino acid sequences of the three groups of polypeptides to obtain polypeptides 4 and 5, polypeptides 6 and 7, and polypeptides 8 and 9 respectively. It has been verified that the sequence-changed polypeptides still have the ability to enhance immunity, strengthen the body's anti-tumor ability, and prolong life Therefore, we speculate that the peptides with 80% or more identical amino acid sequences to the structures of peptides 1, 2, and 3 have similar functions.

6. The technical key to claim protection

Innovation point: The polypeptide is made into a vaccine to improve the body's immunity and enhance disease resistance.

Peptide 1 sequence: MNKAELIDVLTQKLGSDRRQATAAVENVVD

Peptide 2 sequence: TIVRAVHKGDSVTITGFGVFEQRRRAARVA

Peptide 3 sequence: RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA

Peptide 4 sequence: VNKAELIDVLTGGLGSKRRQATAAVEGGVD (the peptide is 80% identical to polypeptide 1)

Peptide 5 sequence: MGVAGLIDVLTQKLGSGGRQATAAVENDDD (the peptide is 80% identical to polypeptide 1)

Peptide 6 sequence: TGVRAGHNGDSVTITGFVGFEGRRRAARVA (the polypeptide is 80% identical to polypeptide 2)

Peptide 7 sequence: TIVRAVGKGDSGITIGFGVFERQRRAARVA (the polypeptide is 80% identical to polypeptide 2)

Peptide 8 sequence: RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA (the peptide is 80% identical to peptide 3)

Peptide 9 sequence: KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA (the peptide is 80% identical to peptide 3)

A vaccine: characterized in that the active ingredient is synthesized in vitro according to the polypeptide sequence

The vaccine is administered to humans by injection, oral or other clinical routes

The purpose is to enhance human immunity, improve disease resistance, anti-tumor, and prolong life

We believe that all polypeptides with a similarity of more than 80% to the above sequences have similar functions.

Claims (2)

1. An application of a polypeptide whose amino acid sequence is TIVRAVGKGDSGITIGFGVFERQRRAARVA, characterized in that the application of the polypeptide in the preparation of a drug for prolonging the lifespan of mice. 2. An application of a polypeptide whose amino acid sequence is TIVRAVGKGDSGITIGFGVFERQRRAARVA, characterized in that the polypeptide is used in the preparation of a drug for inhibiting the growth of tumor cells; the tumor cells are gastric cancer MGC-803 or acute lymphoblastic leukemia Jurkat cells.

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