Preparation method and application of anti-aging vaccine

Abstract

The invention discloses a preparation method and application of an anti-aging vaccine, and relates to the technical field of medical treatment. Comprising the following steps: 1, synthesizing a polypeptide with a specific sequence; 2, coupling the polypeptide with a carrier protein; adding an immunologic adjuvant, and fully and uniformly mixing at room temperature to obtain a polypeptide solution; 3, freeze-drying the polypeptide solution to obtain a freeze-dried pure product; and dissolving the freeze-dried pure product in ultrapure water, filling into a sterile EP tube, and storing at-20 DEG C to obtain the anti-aging polypeptide vaccine. The anti-aging polypeptide vaccine has the beneficial effects that the polypeptide sequences (A: RRGDGRWKD, B: RSQHLRFPDR, and C: TGRRLFCYS) are designed to create the polypeptide vaccine, and the polypeptide vaccine can effectively act on cells with high expression of GPNMB, so that the anti-aging effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing an anti-aging vaccine and application thereof. Background Art

[0002] Anti-aging vaccines are an innovative medical technology that aims to slow down or reverse the aging process. Although anti-aging vaccines are still under research and development and there are no mature products for widespread use, scientists are exploring how to delay aging through the immune system.

[0003] Researchers are trying to deal with these senescent cells. Some research teams are developing new anti-aging cell drugs (senolytics), some are trying to transform immune cells, and others hope to use genetic tools to kill senescent cells. Currently, there are about 20 clinical trials underway. Researchers hope that these methods can remove senescent cells, fight aging, and protect human health.

[0004] At present, traditional vaccines have problems with recurrent virulence or incomplete inactivation, and traditional anti-aging only sees the appearance of aging but is far from seeing the essence of aging. Starting only from the appearance of aging, it can only achieve temporary improvement in appearance and symptoms.

[0005] Therefore, the present invention prepares an anti-aging polypeptide vaccine. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing an anti-aging vaccine and its application to solve the problems raised in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing an anti-aging polypeptide vaccine comprises the following steps:

[0009] Step 1: Synthesize a peptide with a specific sequence;

[0010] Step 2: coupling the polypeptide with the carrier protein; adding an immune adjuvant and mixing thoroughly at room temperature to obtain a polypeptide solution;

[0011] Step 3: Freeze-dry the polypeptide solution to obtain a lyophilized pure product; dissolve the lyophilized pure product in ultrapure water, put it into a sterile EP tube, and store it at -20°C to obtain an anti-aging polypeptide vaccine.

[0012] More optimally: the polypeptides include polypeptide A, polypeptide B, and polypeptide C; the sequence of polypeptide A is RRGDGRWKD, the sequence of polypeptide B is RSQHLRFPDR, and the sequence of polypeptide C is TGRRLFCYS;

[0013] Among them, the molecular weight of polypeptide A is 3832.3Da, the molecular weight of polypeptide B is 5049.9Da, and the molecular weight of polypeptide C is 4161.8Da.

[0014] More optimally: the preparation method of the polypeptide is as follows: (1) using Fmoc solid phase synthesis method, selecting 4-branched MAP resin (multimeric antigen peptide resin), and preparing polypeptide resin in a polypeptide synthesizer; (2) adding the polypeptide resin to the cutting solution under ice bath; after warming to room temperature, stirring the reaction for 1.5-2h, filtering out the resin, evaporating the filtered filtrate to 2-3mL at room temperature and low pressure, and using 60-70mL of pre-cooled ether to precipitate the polypeptide, leaving it at 4-5°C overnight, and then passing it through a G6 glass sand funnel overnight, vacuum drying for 3-4h to obtain a crude polypeptide; dissolving the crude polypeptide to a concentration of 10-12mg / mL, filtering it with a 0.45μm fiber filter membrane, and aspirating 2-3mL to a medium pressure liquid chromatograph for purification, harvesting the polypeptide solution when the main peak appears in the chromatogram to obtain the polypeptide.

[0015] More optimally: the parameters of the medium pressure liquid chromatography are set as follows: the chromatographic column is SOURCE-30-RPC or POROS-50-R1; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is composed of 0.1% trifluoroacetic acid and 10% ethanol, and mobile phase B is composed of 0.1% trifluoroacetic acid and 90% ethanol; the elution gradient is set as follows: in the first 1.5 column volumes, the chromatographic column is completely filled with mobile phase A, and in the next 8 column volumes, the concentration of mobile phase B increases from 0% to 80%, and in the next 0.5 column volumes, the concentration of mobile phase B increases from 80% to 100%, and in the last 1.5 column volumes, the chromatographic column is again completely filled with mobile phase A.

[0016] More optimally: the purity of the polypeptide is identified by reverse phase high pressure liquid chromatography, the analytical column is C18, the mobile phase is acetonitrile solution (containing 0.1% trifluoroacetic acid), and the speed is 1 mL / min; the molecular weight is determined by liquid chromatography / mass spectrometry, where the molecular weight of polypeptide A is 3832.3 Da, the molecular weight of polypeptide B is 5049.9 Da, and the molecular weight of polypeptide C is 4161.8 Da.

[0017] More optimally: the cutting solution includes the following components: 0.25-0.30 mL of deionized water, 0.25-0.30 mL of 1,2-ethanedithiol, and 9.5-10 mL of trifluoroacetic acid. The cutting solution can not only detach the peptide chain from the resin, but also remove a variety of protective groups.

[0018] More optimally: bovine serum albumin and glutaraldehyde coupling agent are mixed, activated at pH = 7.2-7.8 and room temperature for 2-4 hours to obtain activated carrier protein; polypeptide is added, coupled at 37°C for 3-4 hours; immune adjuvant is added, fully mixed and emulsified at room temperature to obtain polypeptide solution.

[0019] More optimally, the ratio of bovine serum albumin to glutaraldehyde coupling agent is 1:30; the ratio of activated carrier protein to polypeptide is 1:1; and the ratio of polypeptide to immune adjuvant is 1:1.

[0020] More optimally, the immune adjuvant includes QS-21.

[0021] More optimally: in the anti-aging polypeptide vaccine, the concentration of the lyophilized pure product is 15-30 mg / L.

[0022] More optimally: the use of the anti-aging polypeptide vaccine in a drug for slowing down, preventing or delaying individual aging.

[0023] The present invention uses the data analysis tool GEO2R and the protein transmembrane analysis software TMHMM2.0, and the expression of GPNMB in aging human umbilical vein endothelial cells (HUVECs) is significantly increased. GPNMB is also upregulated in other tissues of elderly mice. GPNMB is upregulated in the vascular endothelial cells of patients with atherosclerosis, but not in the cells of patients without atherosclerosis. Similarly, compared with the leukocytes of patients without atherosclerosis, the expression of GPNMB in the leukocytes of patients with atherosclerosis is also significantly increased. Therefore, the present invention can effectively act on cells that highly express GPNMB by designing polypeptide sequences (A: RRGDGRWKD, B: RSQHLRFPDR, C: TGRRLFCYS) and creating polypeptide vaccines therefrom, thereby achieving an anti-aging effect.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The polypeptide vaccine obtained by the present invention is a subunit vaccine with certain advantages and application prospects, and is simple to prepare, and can reduce or avoid allergic reactions caused by vaccination. At the same time, the peptide segment used to prepare the polypeptide vaccine is a purified immunogenic molecule, which can be synthesized in large quantities and customized at a relatively low cost, and can be refrigerated and stored;

[0026] (2) In the process of preparing the polypeptide vaccine of the present invention, an immune adjuvant QS-21 is added. QS-21 is a mixture containing triterpenoid saponins. These saponin molecules have surface activity and can bind to antigens to form stable complexes, promote the uptake and processing of antigens by antigen presenting cells (APCs), stimulate APC activation and secretion of cytokines, and enhance the response of T cells and B cells, thereby enhancing the immunogenicity of vaccine antigens and promoting the body to produce a stronger immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart of systemic inflammatory factors in mice in the experimental group and the control group in the detection experiment (II) of the present invention;

[0028] Figure 2 This is a comparison chart of the life span of mice in the experimental group and the control group in the detection experiment (II) of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1: A method for preparing an anti-aging polypeptide vaccine, comprising the following steps:

[0031] Step 1: (1) Use 10 mL of DMF to swell the four-branched MAP resin at room temperature for 30 minutes. After completion, filter out the solvent for later use; (2) Use 3 mL of DMF to dissolve arginine and add 2 mL of condensation reagent (HBTU / HOBT). After the solution is fully dissolved by vortex mixing, add it to the reactor and react at room temperature, wherein the molar ratio of MAP resin, arginine, HBTU and HOBt is set to 1:4:4:4; (2) After the reaction, use 7 mL of DMF to wash 4 times to remove unreacted reagents and by-products in the reaction, and use ninhydrin reagent to detect the completeness of coupling. If the coupling is incomplete, the coupling needs to be repeated once;

[0032] After the coupling is complete, the above (1) and (2) are repeated according to the polypeptide sequence (A: RRGDGRWKD). After the last amino acid condensation is completed, the Fmoc protecting group needs to be removed. After deprotection, 7 mL DMF is used for washing 4 times again; and the resin is blown dry with nitrogen flow for 30 minutes. The synthesis process according to the polypeptide sequence (B: RSQHLRFPDR, C: TGRRLFCYS) is the same as the above method. Finally, a polypeptide resin is obtained;

[0033] (3) Add the polypeptide resin to 10 mL of a cutting solution in an ice bath (wherein the cutting solution is composed of TFA / thioanisole / benzene / EDT in a volume ratio of 90:5:3:2), stir and react for 2 h when the temperature rises to room temperature, filter out the resin, evaporate the filtered filtrate to 3 mL at room temperature and low pressure, and use 70 mL of pre-cooled ether to precipitate the polypeptide, keep it at 5°C overnight, and then use a G6 glass sand funnel overnight, vacuum dry for 4 h, and finally store the obtained crude polypeptide in a -20°C refrigerator to obtain a crude polypeptide;

[0034] (4) The crude polypeptide was dissolved to a concentration of 12 mg / mL, filtered using a 0.45 μm fiber filter membrane, and 3 mL was drawn into a medium pressure liquid chromatograph for purification, wherein the parameters of the medium pressure liquid chromatography were set as follows: the chromatographic column was SOURCE-30-RPC or POROS-50-R1; the mobile phase included mobile phase A and mobile phase B, wherein mobile phase A consisted of 0.1% trifluoroacetic acid and 10% ethanol, and mobile phase B consisted of 0.1% trifluoroacetic acid and 90% ethanol; the elution gradient was set as follows: in the first 1.5 column volumes, the chromatographic column was completely filled with mobile phase A, and in the next 8 column volumes, the concentration of mobile phase B increased from 0% to 80%, and in the next 0.5 column volumes, the concentration of mobile phase B increased from 80% to 100%, and in the last 1.5 column volumes, the chromatographic column was completely filled with mobile phase A again, and the polypeptide solution was harvested when the main peak appeared in the chromatogram to obtain polypeptides (including polypeptide A, polypeptide B, and polypeptide C);

[0035] Step 2: Mix bovine serum albumin and glutaraldehyde coupling agent; activate for 2 hours at pH = 7.5 and room temperature to obtain activated carrier protein; add polypeptide and couple at 37°C for 3 hours; add immune adjuvant QS-21, mix and emulsify at room temperature to obtain polypeptide solution; wherein the ratio of bovine serum albumin to glutaraldehyde coupling agent is 1:30; the ratio of the activated carrier protein to the polypeptide is 1:1; the ratio of the polypeptide to the immune adjuvant is 1:1.

[0036] Step 3: Freeze-dry the polypeptide solution to obtain a lyophilized pure product; dissolve the lyophilized pure product in ultrapure water at a concentration of 20 mg / mL, put it into a sterile EP tube, and store it at -20°C to obtain an anti-aging polypeptide vaccine.

[0037] Detection experiment (I): Using the aging mouse model, apolipoprotein E (APOE) knockout mice, supplemented with a high-fat diet as an atherosclerosis mouse model, 40 male mice of the same age, gender, and weight were randomly divided into two groups. The experimental group had 20 mice injected with 20 mg / mL of vaccine, with an injection volume of 0.5 mL, and the control group had 20 mice injected with normal saline, with an injection volume of 0.5 mL.

[0038] 1. Detection of atherosclerotic plaque area

[0039] (1) Frozen sections of the mouse vascular tissues to be stained in the control group and the experimental group were made. After the sections were thawed, they were gently washed with PBS three times, each time for 5 minutes.

[0040] (2) Circle the tissue with a histochemical pen and add an appropriate volume of staining fixative to fully cover the tissue for 30 min.

[0041] (3) Remove the staining fixative and wash with PBS three times, 8 min each time;

[0042] (4) Remove PBS and add hematoxylin-eosin to each tissue for staining. The sections are immersed in the staining solution and can be covered with plastic wrap to prevent evaporation. Incubate at 37°C overnight.

[0043] (5) On the next day, discard the working solution, seal the slides with sealing tablets, and observe and photograph them under an optical microscope.

[0044] The atherosclerotic plaque area was detected in the pathological sections of the blood vessels of the mice in the experimental group and the control group. The results were as follows: the atherosclerotic plaque area in the experimental group was significantly smaller than that in the control group by 20%.

[0045] 2. Lipofuscin detection

[0046] Blood samples were collected from the experimental group and the control group and tested using an ELISA kit:

[0047] (1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and place them back at 4°C;

[0048] (2) Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well;

[0049] (3) First add 10 μL of the sample to be tested to the sample well, and then add 40 μL of diluent; do not add anything to the blank well;

[0050] (4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard and sample wells, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60 min; then discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 min, discard the washing solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate); then, add 50 μL of substrate A and B (the color developer of the ELISA detection kit) to each well, and incubate at 37°C in the dark for 15 min; finally, add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0051] With the concentration of the standard as the horizontal axis and the OD value as the vertical axis, draw a standard curve on the coordinate paper, find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample. The results are as follows:

[0052] The plasma concentration of lipofuscin in the experimental group was 64.61±3.42uM, and that in the control group was 89.76±5.78uM, and the difference was statistically significant at P<0.01.

[0053] Detection experiment (II): A total of 40 mice with the same age, sex, and weight of the pulmonary fibrosis model were randomly divided into two groups: 20 mice in the experimental group were injected with 20 mg / mL of vaccine, with an injection volume of 0.5 mL; and 20 mice in the control group were injected with normal saline, with an injection volume of 0.5 mL.

[0054] 1. Pulmonary fibrosis degree detection:

[0055] The left lung was fixed with 10% formaldehyde for histological examination, routinely paraffin-embedded, and sliced ​​(5 μm thick). The tissue sections were then stained with HE to determine the degree of inflammation, and the degree of pulmonary fibrosis was scored strictly according to the SZAPIEL scoring standard. The results obtained are as follows:

[0056] Level 0 Level 1 Level 2 Level 3 Level 4 SZAPIE score Control group 0 3 10 5 2 45.76 Experimental Group 0 12 7 1 0 16.45

[0057] Table 1

[0058] 2. The mice in the control group and experimental group in the test experiment (II) were tested for systemic inflammatory factors (TNF-α, IL-6, IL-8), and the data obtained were as follows: Figure 1 shown.

[0059] 3. Record the life span of mice in the control group and experimental group in the test experiment (II). The survival curve was drawn using the Kaplan-Meier method. The data obtained are as follows: Figure 2 shown.

[0060] Conclusion: In the mouse model of the application test experiment (I), the area of ​​atherosclerotic plaques in the experimental group with the vaccine was 20% smaller than that in the control group without the vaccine; the lipofuscin level in the experimental group was lower than that in the control group. In the mouse model of the application test experiment (II), the degree of lung and liver fibrosis in the experimental group was reduced, the liver fibrosis area was improved, and the system score of the experimental group was significantly lower than that of the control group; the serum concentration of inflammatory factors in the experimental group was significantly lower than that of the control group; the average life span of the experimental group was also increased by 30% compared with the control group. Therefore, the vaccine obtained by the present invention can play a good role in anti-aging.

[0061] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing an anti-aging polypeptide vaccine, characterized in that: The following steps are involved: Step 1: Synthesize a peptide with a specific sequence; Step 2: coupling the polypeptide with the carrier protein; adding an immune adjuvant and mixing thoroughly at room temperature to obtain a polypeptide solution; Step 3: Freeze-dry the polypeptide solution to obtain a lyophilized pure product; dissolve the lyophilized pure product in ultrapure water, put it into a sterile EP tube, and store it at -20°C to obtain an anti-aging polypeptide vaccine.

2. The method for preparing an anti-aging polypeptide vaccine according to claim 1, characterized in that: The polypeptides include polypeptide A, polypeptide B and polypeptide C; the sequence of polypeptide A is RRGDGRWKD, the sequence of polypeptide B is RSQHLRFPDR, and the sequence of polypeptide C is TGRRLFCYS.

3. The method for preparing an anti-aging polypeptide vaccine according to claim 1, characterized in that: The preparation method of the polypeptide is as follows: (1) using Fmoc solid phase synthesis method, selecting 4-branched MAP resin, and preparing polypeptide resin in a polypeptide synthesizer; (2) adding the polypeptide resin to a cutting solution under an ice bath; after the solution is heated to room temperature, stirring the reaction for 1.5-2 hours, filtering out the resin, washing, precipitating, and drying to obtain a crude polypeptide; purifying, drying, and freeze-drying to obtain the polypeptide.

4. The method for preparing an anti-aging polypeptide vaccine according to claim 3, characterized in that: The cutting fluid comprises the following components: 0.25-0.30 mL of deionized water, 0.25-0.30 mL of 1,2-ethanedithiol, and 9.5-10 mL of trifluoroacetic acid.

5. The method for preparing an anti-aging polypeptide vaccine according to claim 1, characterized in that: The preparation method of the polypeptide solution is as follows: bovine serum albumin and glutaraldehyde coupling agent are mixed, activated for 2-4 hours at pH = 7.2-7.8 and room temperature to obtain activated carrier protein; polypeptide is added, coupled at 37°C for 3-4 hours; immune adjuvant is added, fully mixed and emulsified at room temperature to obtain polypeptide solution.

6. The method for preparing an anti-aging polypeptide vaccine according to claim 5, characterized in that: The ratio of bovine serum albumin to glutaraldehyde coupling agent is 1:30; the ratio of activated carrier protein to polypeptide is 1:1; and the ratio of polypeptide to immune adjuvant is 1:

1.

7. The method for preparing an anti-aging polypeptide vaccine according to claim 1, characterized in that: The immune adjuvant includes QS-21.

8. The method for preparing an anti-aging polypeptide vaccine according to claim 1, characterized in that: In the anti-aging polypeptide vaccine, the concentration of the lyophilized pure product is 15-30 mg / L.

9. The anti-aging polypeptide vaccine prepared according to the method for preparing an anti-aging polypeptide vaccine according to any one of claims 1 to 8.

10. An application of an anti-aging polypeptide vaccine, characterized in that: The use of the anti-aging polypeptide vaccine in medicines for slowing down, preventing or delaying individual aging.

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