CN116139252A - Polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof - Google Patents
Polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof. The polypeptide with the functions is selected from the group consisting of: (a) A polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD; or (b) a polypeptide having the amino acid sequence TIVRAVHKGDSVTITGFGVFEQRRRAARVA; or (c) a polypeptide having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or (f) a polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or (g) a polypeptide having the amino acid sequence TIVRAVGKGDSGITIGFGVFERQRRAARVA. The polypeptide for improving immunity, resisting tumor and prolonging life can improve the immunity of organisms, enhance disease resistance, reduce the incidence of tumor and prolong the life.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof.
Background
In classical immunological theory, T cells develop into CD4 in the thymus + Helper T cells and CD8 + Two major subsets of cytotoxic T cells. Recent studies have found that part of CD4 + T cells can differentiate further outside the thymus to have a CD 8-like profile + Subsets of direct cell killing functions of CTLs and NK cells, namely CD4 + CTLs. Currently, CD4 + CTLs have also been further shown to play an important role in infection, neoplasms, autoimmune diseases, vaccination, and the like. Particularly in infectious diseases, including influenza virus, cytomegalovirus, EBV, human papillomavirus and HIV, etc., have been widely reported. There is literature showing that researchers believe CD4 + The increased number of CTLs and the increased virulence probably represent the overall immunity enhancement of the organism, the change condition of the cell subpopulation can be used as an index for evaluating the immunity function intensity, and the cell subpopulation proportion in the very long life population is found to be obviously higher than that in the normal population, so that the increase of the cell proportion of the subpopulation is related to longevity. Monocytes, on the basis of previous studies, were also shown to be able to be divided into three subgroups, i.e. intermediate (CD 14 ++ CD16 + ) Classical (CD 14) ++ CD16 - ) And non-classical (CD 14) + CD16 ++ ). Wherein warp yarn is typically (CD 14 ++ CD16 - ) In the peripheral blood of healthy people, the total number of monocytes is about 80% -90%, inPlays an important role in the intrinsic immune defense mechanism; intermediate type (CD 14) ++ CD16 + ) About 5% of the total number of monocytes, up to 10% -50% in severe infectious and inflammatory states, with antigen presentation, inflammation and monocyte activation as the primary functions, also known as inflammatory monocyte subpopulations; non-classical (CD 14) + CD16 ++ ) About 5% -10% of the total number of monocytes, can migrate and remove foreign matter from the surface of vascular endothelium, has an immune monitoring function, and plays an important role in early inflammatory response and tissue repair, and is also called as a migrating monocyte subpopulation. The proportion of different monocyte sub-groups changes, and the corresponding cell sub-group functions are also enhanced, which plays a role in prompting the change of the immune function of the organism.
In recent years, the incidence of malignant tumors characterized by abnormal proliferation of cells has been remarkably increased. According to world health organization statistics, 1/5 of men and 1/6 of women worldwide suffer from malignant tumors, wherein almost half of new malignant tumor cases and more than half of malignant tumor death cases worldwide occur in asia, especially china. Malignant tumors are also classified into solid tumors and non-solid tumors, wherein the common solid tumors comprise lung cancer, gastric cancer, breast cancer and the like, and the common non-solid tumors comprise leukemia, lymphoma and the like. At present, no effective radical treatment scheme exists for malignant tumors, and treatment mainly depends on operation, radiotherapy and chemotherapy, but the damage to patients caused by the treatment methods cannot be ignored. The anti-tumor effect of the drug on the solid tumor can be evaluated by calculating the tumor inhibition rate according to the volume of the tumor, and for the non-solid tumor, the inhibition rate of the drug on the proliferation of tumor cells can be verified by in vitro experiments. How to enhance the curative effect, reduce the side effect and improve the life quality of patients by matching different treatment schemes is an important part in the malignant tumor treatment process.
Aging is a biological process that necessarily occurs in all organisms, with progressive, deleterious, and generalized characteristics. With the progress of society, the demands of people for anti-aging and life prolonging are continuously increasing. The anti-aging medicine is a medicine which can improve the life efficiency, improve the physique of human beings, prevent and treat senile diseases and prolong the life of organisms within the limit determined by genetic characteristics; is a medicine for preventing and delaying the aging process of the organism, improving pathological disorder of tissue cells, adjusting important organ functions, regulating the environmental balance in the organism and promoting the whole health. It features that the multi-layer, multi-aspect and longer-time functions are used to regulate the substance, metabolism and function states of tissue and organ of organism, so delaying senility and increasing life.
At present, the demands of residents in China on prolonging the service life and improving the life quality are continuously improved, and according to the past research results, a polypeptide is found to be capable of stimulating the proliferation of organism cell subsets so as to enhance the immunity of a human body, has obvious inhibition effect on tumor cells and can prolong the service life of an individual. The polypeptide can be made into vaccine, and can produce multiple functions of enhancing immunity, resisting tumor and prolonging service life after being inoculated into human body.
Disclosure of Invention
A first object of the present invention is to provide a polypeptide having an improved immunity and an antitumor activity and an improved life; a second object is to provide the use of the polypeptide having immunity-enhancing and antitumor properties and life-prolonging properties.
The first object of the present invention is achieved in that the polypeptide having immunity-enhancing and antitumor properties and life-prolonging properties is selected from the group consisting of:
(a) A polypeptide having an amino acid sequence of MNKAELIDVLTQKLGSDRRQATAAVENVVD (polypeptide 1); or (b)
(b) A polypeptide having an amino acid sequence of TIVRAVHKGDSVTITGFGVFEQRRRAARVA (polypeptide 2); or (b)
(c) A polypeptide having an amino acid sequence of RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA (polypeptide 3); or (b)
(d) A polypeptide having an amino acid sequence of VNKAELIDVLTGGLGSKRRQATAAVEGGVD (polypeptide 4); or (b)
(e) A polypeptide having an amino acid sequence of MGVAGLIDVLTQKLGSGGRQATAAVENDDD (polypeptide 5); or (b)
(f) A polypeptide having an amino acid sequence of TGVRAGHNGDSVTITGFVGFEGRRRAARVA (polypeptide 6); or (b)
(g) A polypeptide having an amino acid sequence of TIVRAVGKGDSGITIGFGVFERQRRAARVA (polypeptide 7); or (b)
(h) A polypeptide having an amino acid sequence of RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA (polypeptide 8); or (b)
(i) A polypeptide having an amino acid sequence of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA (polypeptide 9).
The polypeptide with the functions of improving immunity, resisting tumors and prolonging the service life can improve the immunity of organisms, strengthen the disease resistance, reduce the occurrence rate of tumors and prolong the service life.
The polypeptide component of the invention can stimulate the immune cells of the organism to lead part of immune cell subsets (such as CD4 + CTLs、CD14 + CD16 ++ ) The proportion is increased, and the immune cell subsets have been proved in the past researches to enhance the immune function of organisms, enhance the resistance to various diseases and reduce the prevalence, thereby achieving the aim of enhancing the disease resistance of the organisms. The polypeptide can also enhance the antitumor capability of organism and prolong the service life. Through verification, the polypeptides 1, 2 and 3 with different structures have the functions of enhancing immunity, enhancing the anti-tumor capability of organisms and prolonging the service life, wherein the effect of the polypeptide 1 is most obvious. The amino acid sequences of the three groups of polypeptides are partially changed to obtain polypeptides 4, 5, 6, 7 and 8, 9 respectively, and the polypeptides after the sequence change still have the functions of enhancing immunity, strengthening body anti-tumor capability and prolonging service life through verification, so that the polypeptides which have the same amino acid sequence as the polypeptides 1, 2 and 3 with the structure of 80 percent or more have similar functions.
Drawings
FIG. 1 is a schematic representation of the change in monocyte subpopulations before and after stimulation with polypeptide 1 of the present invention;
FIG. 2 shows CD4 before and after stimulation of polypeptide 1 of the present invention + CTLs ratio change profile;
FIG. 3 is a schematic diagram showing analysis of Kaplan-Meier survival curves of mice before and after intervention with polypeptide 1 of the present invention;
FIG. 4 is a schematic representation of the change in monocyte subpopulations before and after stimulation with polypeptide 6 of the present invention;
FIG. 5 shows CD4 before and after stimulation with polypeptide 6 of the present invention + CellsMiddle CD4 + CTLs ratio change profile;
FIG. 6 is a schematic representation of the change in monocyte subpopulations before and after stimulation with polypeptide 7 of the present invention;
FIG. 7 shows CD4 before and after stimulation of polypeptide 7 of the present invention + CD4 in cells + CTLs ratio change profile;
FIG. 8 is a schematic representation of a Kaplan-Meier survival curve analysis of mice before and after intervention with polypeptide 6 of the present invention;
FIG. 9 is a schematic representation of Kaplan-Meier survival curve analysis of mice before and after intervention with polypeptide 7 of the present invention.
Detailed Description
The invention is further described below with reference to examples and figures, but is not limited in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The polypeptide with the functions of improving immunity, resisting tumors and prolonging life is selected from the following components:
(a) A polypeptide having the amino acid sequence MNKAELIDVLTQKLGSDRRQATAAVENVVD; or (b)
(b) A polypeptide having the amino acid sequence TIVRAVHKGDSVTITGFGVFEQRRRAARVA; or (b)
(c) A polypeptide having the amino acid sequence RNPRTGETVKVKPTSVPAFRPGAQFKAVVAGA; or (b)
(d) A polypeptide having the amino acid sequence VNKAELIDVLTGGLGSKRRQATAAVEGGVD; or (b)
(e) A polypeptide having the amino acid sequence MGVAGLIDVLTQKLGSGGRQATAAVENDDD; or (b)
(f) A polypeptide having the amino acid sequence TGVRAGHNGDSVTITGFVGFEGRRRAARVA; or (b)
(g) A polypeptide having the amino acid sequence TIVRAVGKGDSGITIGFGVFERQRRAARVA; or (b)
(h) A polypeptide having the amino acid sequence RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA; or (b)
(i) A polypeptide having the amino acid sequence of KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA.
The nucleic acid with the functions of improving immunity, resisting tumors and prolonging service life is selected from the following components:
(a) A nucleic acid encoding the polypeptide of claim 1; or (b)
(b) Nucleic acids complementary to the nucleic acid sequences (a), (b) and (c).
The carrier provided by the invention contains nucleic acid with the functions of improving immunity, resisting tumors and prolonging service life.
The host cell of the present invention is characterized by comprising a vector having a nucleic acid having an immunity-enhancing, antitumor and life-prolonging function, or a nucleic acid having an immunity-enhancing, antitumor and life-prolonging function integrated in its genome.
The medicine is a polypeptide vaccine containing polypeptide sequences with the functions of improving immunity, resisting tumors and prolonging service life.
The application of the polypeptide with the functions of improving immunity, resisting tumors and prolonging service life is the application of the polypeptide with the functions of improving immunity in medicines for improving immunity.
The application of the polypeptide with the functions of improving immunity, resisting tumors and prolonging service life is the application of the polypeptide with the functions of improving immunity in preparing medicines with long service life.
The application of the polypeptide with the functions of improving immunity, resisting tumors and prolonging service life is the application of the polypeptide with the functions of improving immunity in preparing antitumor 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 invention is further illustrated by the following examples of specific embodiments:
1. the experiment for improving the immunity of the human body by the polypeptide 1 comprises the following specific operations:
1. inclusion criteria: (1) between 18 and 60 years of age; (2) Normal populations (normal liver and kidney function examination, excluding those infected with tuberculosis, HBV, HCV, tumors, and autoimmune disease).
2. Experimental materials: peripheral venous blood of human; human peripheral blood mononuclear cell separation liquid; PBS buffer; fetal bovine serum; penicillin-streptomycin solution (diabody); PE-CD14 antibody, FITC-CD16 antibody, perpcy-5.5-CD45 antibody, alexaFlour647-GB11 antibody, FITC-CD3 antibody, PE-CD4 antibody; erythrocyte lysate; the fluid rupture of the membrane fixes a body fluid.
3. The experimental procedure is as follows: (1) synthesizing the polypeptide 1 in vitro according to its sequence; (2) peripheral blood PBMC extraction: a) 6 healthy human elbow peripheral blood 5ml was drawn into EDTA anticoagulation tubes, 3 to detect monocyte subpopulations, 3 to detect CD4 + A subset of CTLs; b) 5ml of blood was diluted to 10ml using PBS; c) Taking peripheral blood mononuclear cell separating liquid in a centrifuge tube, 5ml of each tube, and adding 5ml of diluted blood into the upper layer of the separating liquid; d) 1000g, acceleration 4, deceleration 4, centrifugation 25 minutes; e) Carefully sucking out PBMC layer cells by using a suction tube, diluting with a split red liquid, centrifuging, removing supernatant, and repeating for 2 times; f) The cells were resuspended using the prepared cell culture medium (10 ul of diabody+90 ul of fetal bovine serum+900 ul 1640); g) Dividing each sample cell into an experimental group and a control group at average, wherein 500ul of each sample cell is respectively obtained; (3) dissolving the polypeptide 1 powder to a concentration of 1mg/ml using PBS; (4) adding 100ul of polypeptide 1 liquid into the experimental group, and standing for 12h; (5) stream detection: a) Re-suspending cells with 1ml of lyzed red liquid, centrifuging, and removing supernatant; b) 100ul of split red liquid is used for resuspension of cells, and the mononuclear cell group is incubated for 30min in dark after the antibodies of CD14, CD16 and CD45 are added; CD4 + Adding CD3 and CD4 antibodies into the CTLs cell group, and incubating for 30min in a dark place; c) CD4 + Adding a membrane rupture and fixation integrated liquid into the CTLs cell group, and standing for 30min in a dark place; d) Adding 1ml of split red liquid into mononuclear cell group sample, centrifuging, removing supernatant, and CD4 + CTLs cell line were centrifuged directly to remove supernatant; e) Adding 200ul of split red liquid into monocyte group to re-suspend cells, and performing on-machine detection to obtain CD4 + Adding 100ul of membrane rupture fixing solution into CTLs cell group, adding GB11 antibody, and incubating for 30min in dark place; f) CD4 + Adding 1ml of membrane rupture fixing solution into CTLs cell group, centrifuging and removing supernatant; g) CD4 + The CTLs cell group is added with 100ul of membrane rupture fixing solution to resuspend the cells, and the cells are detected by an upper machine.
4. Experimental results: atypical (CD 14) in monocytes after stimulation with polypeptide 1 + CD16 ++ ) CD4 in T cells + The proportion of CTLs subpopulations increases significantly. Suggesting an enhancement of cellular immune function.
5. Statistical analysis was performed on the cell subpopulations after stimulation of polypeptide 1, and the data of the metering data were expressed as mean ± standard error (s.e) and were analyzed using GraphPad Prism8 and SPSS Statistics23 statistical software. If the data accords with the normal distribution, selecting the paired T test, and if the data does not accord with the normal distribution, adopting the Wilcoxon test. The difference of P < 0.05 is significant.
(1) Analysis of monocyte subpopulations changes following stimulation with polypeptide 1
The mononuclear cell ratio of the polypeptide 1 stimulated group is (19.25+/-8.18)%, the control group is (15.79+/-9.08)%, and the difference among the groups has no statistical significance (P is more than 0.05); the proportion of the intermediate monocytes in the polypeptide 1 stimulated group in the monocytes is (3.85+/-2.82)%, the proportion of the intermediate monocytes in the control group is (5.61+/-3.88)%, and the difference between the groups is not statistically significant (P is less than 0.05); the proportion of the classical monocytes in the polypeptide 1 stimulated group in the monocytes is (93.42+/-0.69)%, the proportion of the classical monocytes in the control group is (85.27 +/-7.42)%, and the difference between the groups is not statistically significant (P is less than 0.05); the non-classical type mononuclear cells in the stimulated group of the polypeptide 1 account for 22.77+/-5.82 percent of the mononuclear cells, the control group accounts for 18.85+/-4.82 percent, the difference among the groups is statistically significant (P is less than 0.05), and the non-classical type mononuclear cells after the stimulation of the polypeptide 1 account for the obvious rise of the control group (figure 1).
(2) CD4 after stimulation with polypeptide 1 + Analysis of changes in CTLs cell subsets
2. Experimental study of polypeptide 1 to extend the longevity of C57BL/6J mice:
1. inclusion criteria: c57BL/6J mice (the healthy mice of the strain have the service life of 18-24 months and the longest time of 3 years) which are 8 weeks old and have no Specific Pathogen (SPF) are 40, the lighting and darkness of the raising environment are alternated for 12 hours, the environmental temperature is controlled at 22-25 ℃, the humidity is controlled at 50-60 percent, 1 animal/cage is observed after reaching the experimental environment, and the animals start to take medicine at 1 month old.
2. The experimental procedure is as follows: (1) synthesizing the polypeptide 1 in vitro according to its sequence; (2) Dissolving polypeptide 1 powder into 0.4mg/ml with sterile physiological saline solution, ultrafiltering to obtain sterile solution, aseptically preserving at-20deg.C, and restoring to room temperature before use; (3) Mice were randomly divided into a polypeptide administration group and a vehicle control group, each of 20; (4) The polypeptide administration group is injected with the polypeptide 1, the dosage is 6mg/kg,1 time/3 days, the rat tail vein administration is selected at the same time (9:00-11:00), and the corresponding injection amount of physiological saline is given to the solvent control group; (5) Administration to mice naturally dies, and recording the time of death and most likely cause of death; (6) Mice that died due to accidents or external causes were removed, survival analysis was performed using Kaplan-Meier statistical methods and survival curves were drawn.
3. Experimental results: the change of the service life of the mice after the stimulation of the polypeptide 1 is statistically analyzed, and the data of the measurement data are expressed as mean ± standard error (s.e), and are analyzed by using GraphPad Prism8 and SPSS Statistics23 statistical software. If the data accords with the normal distribution, selecting the paired T test, and if the data does not accord with the normal distribution, adopting the Wilcoxon test. The difference of P < 0.05 is significant. Survival curves were obtained using the Kaplan-Meier statistical method (FIG. 3).
The mice life observation test shows that the average life of the mice of the polypeptide 1 injection group is (26.64 +/-1.72) month, and the median life is (21.00+/-7.83) month; the average life of the mice in the control group is (16.90+/-1.01) months, and the median life is (17+/-0.89) months; the average life of the mice in the polypeptide 1 injection group is longer than that in the control group, and the differences are statistically significant (P is less than 0.05). This demonstrates that the polypeptide 1 intervention can prolong the life of mice from both average life and median life, and has the effect of delaying body aging (table 1).
TABLE 1 mean and median time to survival of mice before and after polypeptide 1 intervention
4. Conclusion: the polypeptide 1 intervenes to delay the aging of organism and prolong the service life of mice.
3. Experimental study of the efficacy of polypeptide 1 against solid tumors:
1. experimental materials: 18-22g male nude mice 40; the tumor cell strain is gastric cancer MGC-803; dissolving polypeptide 1 powder in physiological saline to obtain final concentration of 1mg/ml, filtering with 0.22 μm filter membrane, aseptically packaging, and preserving at 4deg.C; trastuzumab and docetaxel injection.
2. The experimental method comprises the following steps: MGC-803 cell line was incubated at 37℃with 5% CO 2 Culturing in incubator to density above 80%, collecting cells by digestion with 0.25% trypsin digestion solution, centrifuging at 1000rpm, removing supernatant, washing with physiological saline for three times, and re-suspending to 5X10 7 /ml, stored at 4℃for further use.
3. The experimental procedure is as follows: (1) Randomly dividing mice into 4 groups, namely a blank control group, a polypeptide group, a combined drug group and a positive control group, wherein each group comprises 10 cancer cell strain suspensions of 0.2 ml/mouse are inoculated under the right armpit of the mice, the tumor emergence condition is observed every three days after inoculation, and the tumor volume reaches 0.1mm 3 Dosing was started at that time. (2) The positive control group was intravenous trastuzumab 15ml/kg,1 time/3 day; docetaxel 5mg/kg,1 time/7 day; the blank group is injected with 10ml/kg of physiological saline intravenously for 1 time/3 days; 1mg/kg of polypeptide 1 solution prepared by the polypeptide group, 1 time/3 days, and intravenous injection; the combination administration group is intravenous injected with trastuzumab 15ml/kg for 1 time/3 days; docetaxel 5mg/kg,1 time/7 day; 1mg/kg of the prepared polypeptide 1 liquid, 1 time/3 days. (3) Tumor volume was measured 21 days after administration, and tumor inhibition rate (%) = (tumor volume of placebo-administered group)/tumor volume of placebo group according to the formula.
4. Experimental results: statistical analysis was performed on the tumor suppression rate change after the injection of polypeptide 1, and the data of the metering data were expressed as mean ± standard error (s.e.), and were analyzed using GraphPad Prism8 and SPSS Statistics23 statistical software. If the data accords with the normal distribution, selecting the pairing T test, and if the data does not accord with the normal distribution, adopting the Wilcxon test. The difference of P < 0.05 is significant.
The average tumor inhibition rate of the positive control group is (61.66 +/-1.38)%, the average tumor inhibition rate of the polypeptide 1 injection group is (67.62+/-0.90)%, and the average tumor inhibition rate of the combined administration group is (74.06 +/-1.38)%; the average tumor inhibition rate of the combined administration group is higher than that of the positive control group and the polypeptide 1 injection group, the difference has statistical significance (P is less than 0.05), the average tumor inhibition rate of the polypeptide 1 injection group is higher than that of the positive control group, and the difference has statistical significance (P is less than 0.05) (Table 2).
5. Conclusion: the injection of the polypeptide 1 can inhibit the growth of tumors, and can enhance the anti-tumor effect of the drugs when being used in combination with the existing anti-tumor drugs.
Table 2 comparison of tumor inhibition rates of different pharmaceutical intervention solid tumors
4. Experimental study of the efficacy of polypeptide 1 against non-solid tumors
1. Experimental materials: acute lymphoblastic leukemia Jurkat cells; fetal bovine serum; RP-MI1640 medium; 5-fluorouracil (5-FU); the polypeptide 1 powder is prepared into polypeptide liquid of 10ug/ml, 20ug/ml and 30ug/ml by taking DMSO as a solvent.
2. The experimental method comprises the following steps: (1) cell culture: jurkat cells were incubated with RP-MI1640 medium containing 10% fetal bovine serum and 1% diabody at 37℃with a volume fraction of 5% CO 2 Culturing in a saturated humidity incubator for 1 time every 2 days; (2) MTT assay to determine cell proliferation Activity: the Jurkat cells were trypsinized, resuspended in DMSO to form a cell suspension, seeded in 96-well plates with 2X10 cells per well 5 Individual cells, 200ul,37 ℃,5% CO 2 Culturing under the condition for 24 hr, removing supernatant, setting up experimental group A, B, C, adding 200ul of complete culture medium containing 10ug/ml, 20ug/ml, 30ug/ml polypeptide 1 solution, and setting up blank (without adding polypeptide 1) group and cell-free control group, respectively, using commercially available anticancer agentThe medicine 5-FU (5-fluorouracil) is used as a positive control group with anticancer activity, 3 auxiliary holes are respectively arranged in each group, 5mg/ml MTT reagent is added into each hole after 4 hours of culture (the same condition is adopted), 20ul of MTT reagent is added into each hole, the supernatant is discarded after 4 hours of culture again, 150ul of DMSO is added, shaking dissolution is carried out, and an enzyme-labeled instrument is used for measuring OD570 (absorbance value) at 570nm wavelength; according to the formula: inhibition (%) = (control OD 570-experimental OD 570) x 100%/control OD570.
3. Experimental results: the MTT method proves that the polypeptide 1 with different concentrations has the inhibition effect on the proliferation of tumor cells, wherein the inhibition effect on the proliferation of tumor cells is strongest at 30ug/ml, and the inhibition rate of the polypeptide 1 with the concentration is stronger than that of the standard therapeutic drug 5-FU (Table 3).
TABLE 3 comparison of inhibition of in vitro culture of polypeptide 1 at different concentrations with Jurkat cells
5. We speculate that polypeptide 6 and polypeptide 7, which have similar sequences to polypeptide 2, have similar effects, and have carried out relevant experiments to verify
1. Experiments for improving immunity of human body (step and experiment)
(1) Polypeptide 6
(a) Analysis of monocyte subpopulations changes following stimulation with polypeptide 6
The mononuclear cell ratio of the polypeptide 6 stimulation group is (25.60 +/-4.52)%, the control group is (24.20+/-2.41)%, and the difference between the groups has no statistical significance (P is more than 0.05); the proportion of the intermediate monocytes in the polypeptide 6 stimulated group in the monocytes is (10.15+/-0.80)%, the proportion of the intermediate monocytes in the control group is (9.72+/-0.49)%, and the difference between the groups is not statistically significant (P is more than 0.05); the proportion of the polypeptide 6 stimulated group classical monocytes in the monocytes is (88.79 +/-3.52)%, the contrast group is (89.78 +/-1.12)%, and the difference between the groups is not statistically significant (P is more than 0.05); the non-classical type mononuclear cells in the stimulated group of the polypeptide 4 account for 24.00+/-2.41 percent of the mononuclear cells, the control group accounts for 21.75+/-1.86 percent, the difference among the groups is statistically significant (P is less than 0.05), and the non-classical type mononuclear cells after the stimulation of the polypeptide 6 account for the obvious rise of the control group (figure 4).
(b) CD4 after stimulation with polypeptide 6 + Analysis of changes in CTLs cell subsets
Polypeptide 6 stimulating group CD4 + CTLs at CD4 + The cell ratio is (3.11+ -0.41)%, the control group is (1.87+ -0.43)%, and the polypeptide 6 stimulates CD4 + CTLs at CD4 + The ratio of cells was higher than that of the control group, and the difference was statistically significant (P < 0.05) (FIG. 5).
(2) Polypeptide 7
(a) Analysis of monocyte subpopulations changes following stimulation with polypeptide 7
The mononuclear cell ratio of the polypeptide 7 stimulated group is (22.87+/-1.71)%, the control group is (20.71+/-6.70)%, and the difference between the groups has no statistical significance (P is more than 0.05); the proportion of the intermediate monocytes in the polypeptide 7 stimulated group in the monocytes is (10.64+/-3.10)%, the proportion of the intermediate monocytes in the control group is (9.93+/-0.51)%, and the difference between the groups is not statistically significant (P is more than 0.05); the proportion of the polypeptide 7 stimulated group classical monocytes in the monocytes is (89.94 +/-3.57)%, the contrast group is (91.23 +/-3.67)%, and the difference between the groups is not statistically significant (P is more than 0.05); the non-classical type mononuclear cells in the stimulated group of the polypeptide 7 account for 26.24+/-2.89% of the mononuclear cells, the control group accounts for 21.47+/-2.00%, the difference among the groups is statistically significant (P is less than 0.05), and the non-classical type mononuclear cells after the stimulation of the polypeptide 5 account for the obvious rise of the control group (figure 6).
(b) CD4 after stimulation with polypeptide 7 + Analysis of changes in CTLs cell subsets
Polypeptide 7 stimulating group CD4 + CTLs at CD4 + The cell ratio is (2.35+ -0.53)%, the control group is (1.18+ -0.56)%, and the polypeptide 7 stimulates CD4 + CTLs at CD4 + The ratio of cells was higher than that of the control group, and the difference was statistically significant (P < 0.05) (FIG. 7).
2. Experimental study to extend the life of C57BL/6J mice (step same experiment two):
(1) Polypeptide 6
(a) Experimental results: the average life of the mice in the polypeptide injection group is (26.13+/-1.39) months, and the median life is (23+/-2.65) months; the average life of the mice in the control group is (22.85+/-1.15) months, and the median life is (21+/-0.36) months; the average life of the mice in the polypeptide injection group was longer than that in the control group, and the differences were statistically significant (P < 0.05) (Table 4). This shows that the polypeptide intervention can prolong the life of mice from both the average life and the median life, and has the effect of delaying the aging of organisms.
(b) Conclusion: polypeptide 6 has similar functions to polypeptide 2, and can prolong the service life of mice and delay the aging of organisms.
TABLE 4 mean and median time to survival of mice before and after polypeptide 6 intervention
(2) Polypeptide 7
(a) Experimental results: the average life of the mice in the polypeptide injection group is (28+/-5.16) months, and the median life is (22.00+/-2.34) months; the average life of the mice in the control group is (21.00+/-1.19) months, and the median life is (18.00+/-0.56) months; the average life of the mice in the polypeptide injection group was longer than that in the control group, and the differences were statistically significant (P < 0.05) (Table 5). This shows that the polypeptide intervention can prolong the life of mice from both the average life and the median life, and has the effect of delaying the aging of organisms.
(b) Conclusion: polypeptide 7 has similar functions to polypeptide 2, and can prolong the service life of mice and delay the aging of organisms.
TABLE 5 mean, median time to survival of mice before and after polypeptide 7 intervention
3. Experimental study of anti-solid tumor efficacy (step same as experiment three):
(a) Experimental results: the average tumor inhibition rate of the positive control group is (61.66 +/-1.38)%, the average tumor inhibition rate of the polypeptide 6 injection group is (67.12+/-0.69)%, and the average tumor inhibition rate of the combined use 6 group is (68.12+/-1.32)%; the average tumor inhibition rate of the combined administration 6 groups is higher than that of the positive control group and the polypeptide 6 injection group, the difference has statistical significance (P is less than 0.05), the average tumor inhibition rate of the polypeptide 6 injection group is higher than that of the positive control group, the difference has statistical significance (P is less than 0.05), the average tumor inhibition rate of the polypeptide 7 injection group is (69.11 +/-2.13)%, and the average tumor inhibition rate of the combined administration 7 group is (70.35+/-0.89)%; the average tumor inhibition rate of the combined administration 7 groups is higher than that of the positive control group and the polypeptide 7 injection group, the difference has statistical significance (P is less than 0.05), the average tumor inhibition rate of the polypeptide 7 injection group is higher than that of the positive control group, and the difference has statistical significance (P is less than 0.05) (Table 6).
(b) Conclusion: the injection of the polypeptide 6 and the polypeptide 7 can inhibit the growth of tumors, and the combination of the polypeptide and the existing anti-tumor drugs can enhance the anti-tumor effect of the drugs.
Table 6 comparison of tumor inhibition rates of different pharmaceutical intervention solid tumors
4. Experimental study of efficacy against non-solid tumors:
(1) Polypeptide 6 (step and experiment four)
(a) Experimental results: the MTT method proves that the polypeptide 6 solutions with different concentrations have the inhibition effect on the proliferation of tumor cells, wherein the inhibition effect on the proliferation of tumor cells is strongest at 30ug/ml, and the inhibition rate of the polypeptide with the concentration on the proliferation of tumor cells is stronger than that of the standard therapeutic drug 5-FU (Table 7).
TABLE 7 Co-culture inhibition ratio comparison of polypeptide 6 with Jurkat cells at different concentrations
(2) Polypeptide 7 (step and experiment four)
(a) Experimental results: the MTT method proves that the polypeptide 7 with different concentrations has the inhibition effect on the proliferation of tumor cells, wherein the inhibition effect on the proliferation of tumor cells is strongest at 30ug/ml, and the inhibition rate of the polypeptide 7 with the concentration is stronger than that of the standard therapeutic drug 5-FU (Table 8).
TABLE 8 Co-culture inhibition ratio comparison of polypeptide 7 and Jurkat cells at different concentrations
By injecting the above polypeptide, the polypeptide component can stimulate immune cells of human body to make part of immune cell subpopulation (such as CD4 + CTLs、CD14 + CD16 ++ ) The proportion is increased, and the immune cell subsets have been proved in the past researches to enhance the immune function of organisms, enhance the resistance to various diseases and reduce the prevalence, thereby achieving the aim of enhancing the disease resistance of the organisms. The polypeptide can also enhance the antitumor capability of organism and prolong the service life. Through verification, the polypeptides 1, 2 and 3 with different structures have the functions of enhancing immunity, enhancing the anti-tumor capability of organisms and prolonging the service life, wherein the effect of the polypeptide 1 is most obvious. The amino acid sequences of the three groups of polypeptides are partially changed to obtain polypeptides 4, 5, 6, 7 and 8, 9 respectively, and the polypeptides after the sequence change still have the functions of enhancing immunity, strengthening body anti-tumor capability and prolonging service life through verification, so that the polypeptides which have the same amino acid sequence as the polypeptides 1, 2 and 3 with the structure of 80 percent or more have similar functions.
6. Technical key of the protection
The innovation points are as follows: the polypeptide is made into vaccine, which can improve immunity and disease resistance.
Polypeptide 5 sequence: MGVAGLIDVLTQKLGSGGRQATAAVENDDD (the polypeptide is 80% identical to polypeptide 1)
Polypeptide 6 sequence: TGVRAGHNGDSVTITGFVGFEGRRRAARVA (the polypeptide is 80% identical to polypeptide 2)
Polypeptide 7 sequence: TIVRAVGKGDSGITIGFGVFERQRRAARVA (the polypeptide is 80% identical to polypeptide 2)
Polypeptide 8 sequence: RGPGTGETVKVKPTSVPAFRPGAQGKAVVAGA (the polypeptide is 80% identical to polypeptide 3)
Polypeptide 9 sequence: KKGRTGETVKVKPTSVPAFRPGAQGKAGGAGA (the polypeptide is 80% identical to polypeptide 3)
A vaccine: characterized in that the active ingredient is synthesized in vitro according to the polypeptide sequence
The vaccine is administered to the human body by injection, oral administration or other clinical routes
Aims at enhancing immunity, improving disease resistance, resisting tumor and prolonging life
It is believed that polypeptides having more than 80% similarity to the above sequences all have similar functions.
Claims (2)
1. The application of the polypeptide with the amino acid sequence of TGVRAGHNGDSVTITGFVGFEGRRRAARVA is characterized in that the application of the polypeptide in preparing a medicament for prolonging the service life of mice.
2. The application of the polypeptide with the amino acid sequence of TGVRAGHNGDSVTITGFVGFEGRRRAARVA is characterized in that the application of the polypeptide in preparing a medicament 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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