CN106729681B - Nanocarrier protein platform for improving antigen immunogenicity - Google Patents
Nanocarrier protein platform for improving antigen immunogenicity Download PDFInfo
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Abstract
The invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: increasing antigen immunogenicity by attaching a target to the surface of a nanocarrier protein; the nano carrier protein is a nano particle made of carrier protein. According to the invention, the polypeptide epitope is displayed on the surface of the nano protein particle, and the protein particle not only provides T cell epitope for the polypeptide epitope, but also serves as a nano carrier, so that the polypeptide epitope has a proper size and is beneficial to recognition and phagocytosis of antigen presenting cells; meanwhile, the repeated display of the B cell epitope can be beneficial to the recognition of B cells, so that the immune effect can be enhanced.
Description
Technical Field
The invention relates to the field of nano vaccines, in particular to a nano carrier protein platform for improving antigen immunogenicity.
Background
The existing research shows that low-density lipoprotein cholesterol (LDL-C) is a factor for promoting atherosclerosis, high-density lipoprotein cholesterol (HDL-C) is an anti-atherosclerosis factor, and most of the existing prevention and treatment measures and researches related to atherosclerosis aim at reducing the low-density lipoprotein cholesterol and increasing the density lipoprotein cholesterol.
Cholesterol Ester Transfer Protein (CETP) is a key protein for regulating cholesterol transport between HDL (high density lipoprotein) and LDL (low density lipoprotein) and VLDL (very low density lipoprotein), animal studies show that a vaccine constructed by CETP polypeptide can reduce LDL-C and raise HDL-C, and has obvious effect of preventing atherosclerosis, previous studies prove that 16 amino acid sequences CETP471-476(FGFPEHLLVDFLQSLS) at the carboxyl terminal of the protein can be used for developing the vaccine, but the current CETP vaccine-based research is a molecular vaccine or a few related gene vaccines, and has not been successfully introduced into clinic because enough antibodies can not be generated to inhibit the activity of CETP and reduce the LDL-C of serum.
PCSK9 is proprotein convertase subtilisin kexin type 9 (protein convertase subtilisin-like/kexin type 9, PCSK9) is one of the key proteins discovered in recent years to regulate low-density lipoprotein cholesterol (LDL-C) content in plasma, and is considered as a potential therapeutic target for Coronary Heart Disease (CHD). PCSK9 can promote the degradation of LDLR on liver cell membrane, thereby reducing the clearance of LDL-C in serum, so that the inhibition of PCSK9 can significantly reduce the level of LDL-C in serum. Experimental results show that the PCSK9 vaccine can reduce LDL-C, and particularly, the combined lipid-lowering effect of the PCSK9 vaccine and statin lipid-lowering drugs is obviously better than that of the statin lipid-lowering drugs alone, wherein the PCSK9207-223(NVPEEDGTRFHRQASKC) is a potential epitope candidate at present. However, there is no literature on direct studies on the direct relationship between the PCSK9 vaccine and atherosclerosis, since APOE binds to LDLR and increases HDL-C degradation.
Traditional vaccines provide T cell epitopes to B cell epitopes by binding them to carrier protein molecules either directly or indirectly through linkers. At present, the nano-field research of the vaccine is not available.
Disclosure of Invention
The invention aims to overcome the defects and provide a nano carrier protein platform capable of improving the antigen immunogenicity and a nano vaccine with high antigen immunogenicity manufactured by using the platform.
The invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: increasing antigen immunogenicity by attaching a target to the surface of a nanocarrier protein;
the nano carrier protein is a nano particle made of carrier protein.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the specific method for attaching the target to the surface of the nano carrier protein comprises the following steps: the nano-particles are subjected to an addition reaction with a compound A containing double bonds, a substitution reaction with a cross-linking agent and an addition reaction with a target in sequence to obtain the nano-vaccine.
The compound A containing double bonds can be alkene and derivatives thereof, cycloolefin and derivatives thereof, heterocyclic alkene and derivatives thereof;
the method specifically comprises the following steps: compounds capable of undergoing addition reaction with-SH, such as olefins having 1-10 carbon atoms and derivatives thereof, cyclopentene and derivatives thereof, cyclohexene and derivatives thereof, pyrrole and derivatives thereof (e.g., NEM), furan and derivatives thereof, etc.;
the cross-linking agent may be selected from any group of compounds which may be reacted with-NH at either end2The compound in which the group is substituted and the other end can be substituted with a mercapto group is commonly used as follows: sulfo-smcc.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized by comprising the following specific process steps:
dissolving nanoparticles in a buffer solution to form a nano solution; the concentration is carried out according to the reaction conditions, and preferably: 0.2-10 mg/ml;
dissolving a compound A in a buffer solution to form a reaction solution I; the concentration is carried out according to the reaction conditions, and preferably: 0.2-2 mg/ml;
dissolving a cross-linking agent in a solvent to form a reaction solution II; the concentration is carried out according to the reaction conditions, and preferably: 0.2-2 mg/ml;
dissolving the target in a solvent to form a target solution; the concentration is carried out according to the reaction conditions, and preferably: 10-50 mg/ml;
reacting the nano solution and the reaction solution I at the temperature of 10-55 ℃ for 0.5-5 hours;
step six, adding the reaction solution II, and reacting for 0.5-5 hours at the temperature of 10-55 ℃;
step seven, ultrafiltration;
step eight, adding a target solution, and reacting for 0.5-5 hours at the temperature of 10-55 ℃;
and step nine, after ultrafiltration, freeze-drying to obtain a target product.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the mass ratio of the nano particles to the compound A is 5-20: 1; this value is generally calculated from the amount of-SH contained on the nanoparticles; preferably: the amount of compound A used is preferably such that all mercapto groups are reacted.
The mass ratio of the compound A to the cross-linking agent is 5-15: 1; the value is generally based on the-NH groups contained on the nanoparticles2The amount of the crosslinking agent is calculated; preferably: the amount of the crosslinking agent used is preferably such that all amino groups are reacted completely.
The mass ratio of the nanoparticles to the target is 1-9:1, and the target adding amount is generally calculated according to the quantity of the cross-linking agent connected to the nanoparticles; preferably: the amount of target used is preferably such that all the crosslinking agent is reacted.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the buffer solution is selected from TAE, TBE, MES, protein electrophoresis solution, SSC, SSPE, WB membrane transfer solution, TBS, PBS, DPBS and citric acid buffer solution;
the pH of the buffer solution is preferably 6-9;
the above solvent is selected from ddH2O, DMF, DMSO, alcohols, ethers, ketones and esters.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the nano carrier protein platform is OVA nano particles manufactured by a thermal polymerization method;
the OVA nanoparticles are nanoparticles with particle diameter of 30-100nm, preferably 40-60 nm.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the method for producing the above-mentioned OVA nanoparticles is as follows:
dissolving OVA molecules in a solvent;
step two, adding a buffer solution and mixing; taking a proper amount of the solution obtained in the step one, adding a buffer solution, and mixing; the addition amount of the buffer solution can be 0.1-10 times of that of the OVA solution according to the needs of the product properties.
Step three, heating to 50-80 ℃, and stirring for reaction for 1-5 minutes;
and step four, dialyzing the product obtained in the step three at least once, and freeze-drying to obtain the OVA nano-particles.
The buffer solution is selected from TAE, TBE, MES, protein electrophoresis solution, SSC, SSPE, WB membrane transfer solution, TBS, PBS, DPBS and citric acid buffer solution;
the pH of the buffer solution is preferably 6-9;
the above solvent is selected from ddH2O, DMF, DMSO, alcohols, ethers, ketones and esters.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the targets are selected from one or more targets for regulating blood fat.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: the target is selected from CETP with cysteine added at the amino terminal and/or PCSK9 with cysteine added at the amino terminal.
Further, the invention provides a nano carrier protein platform for improving antigen immunogenicity, which is characterized in that: when the target is selected from CETP with an added cysteine at the amino terminus and PCSK9 with an added cysteine at the amino terminus;
the mass ratio of the CETP to the PCSK9 is 1: 0.01-100.
Since CETP increases HDL-C concentration and PCSK9 degrades HDL-C, the mass ratio of CETP to PCSK is preferably selected in proportion to the balance of HDL-C concentration.
The invention has the following functions and effects:
the polypeptide epitope is displayed on the surface of the nano protein particle, and the protein particle not only provides the T cell epitope for the polypeptide epitope, but also serves as a nano carrier platform. Because the nano-carrier has the characteristic of controllable particle size, the nano-carrier is more beneficial to recognition and phagocytosis of antigen presenting cells based on the proper size.
Meanwhile, the target is modified on the surface of the nano carrier platform, so that the result of repeatedly displaying the B cell epitope can be realized, the B cell can be identified more favorably, and the immune effect can be further enhanced.
The method specifically comprises the following steps: in the invention, through a certain treatment method, traditional carrier protein OVA (Ovalbumin) molecules are self-assembled into particles with the size of about 30-100nm, B cell epitopes from targets (such as PCSK9 and CETP) are displayed on the surfaces of the particles through a cross-linking agent (such as sulfo-smcc and the like), and experiments show that the antibody level of vaccines manufactured by the adopted nano carrier protein platform is far higher than that of nano carriers in the traditional molecular form.
In addition, in the invention, the CETP is also proposed to be simultaneously used for the function of regulating blood fat471-476、PCSK9207-223Two blood fat regulating targets are connected on the same carrier. On one hand, the polypeptide construction vaccine of CETP can reduce LDL-C and raise HDL-C, and at the same time has obvious effect of preventing atherosclerosis, while the polypeptide construction vaccine of PCSK9 can reduce LDL-C and at the same time can reduce HDL-C, so that it can utilize CETP to increase HDL-C concentration and compensate HDL-C degradation caused by PCSK9, and at the same time, they can synergistically reduce LDL-C, so that it is hopeful to obtain better effect of regulating blood lipid, and can better prevent atherosclerosis.
Drawings
FIG. 1, DLS plot of OVA-NP-CETP nanoparticles;
FIG. 2, TEM image of OVA-NP-CETP nanoparticles;
FIG. 3 is an SDS-PAGE electrophoresis of OVA-NP-CETP nanoparticles;
wherein, 1 is mark, 2 is OVA molecule, 3 is OVA-NP, 4 is OVA-NEM-SMCC, 5 is OVA-NEM-SMCC-CETP, and 6 is OVA-NP-NEM-SMCC-CETP;
FIG. 4 is a graph showing a comparison of the results of serum anti-CETP antibody levels in New Zealand white rabbits.
Detailed Description
EXAMPLE I preparation of OVA nanomaterials
The specific method comprises the following steps:
step one, dissolving OVA molecules in ddH2After O, the mass concentration of the solution is 10 mg/ml;
step two, taking 2.5ml of the OVA solution, adding 2.5ml of Mes (PH is 6.0) buffer solution into a 5ml reaction bottle (containing matched magnetons), and mixing;
step three, placing the water bath kettle in a magnetic stirrer, heating to 70 ℃, adjusting the rotating speed to 750r, placing the reaction bottle in the water bath kettle, and fully heating for 2min10 s;
step four, dialyzing the product (ddH)2O5L, changing water every 2 hours for 5 times), and freeze-drying to obtain OVA nano material (OVA-NP) with the average particle size of 50 nm.
In the preparation process of the OVA nano material, the reaction time, the reaction concentration, the rotating speed, the dialysis times of post-treatment and the like are adjusted according to the difference of target particle sizes, so that the nano material with the average particle size of 30nm-100nm can be obtained.
The commonly used schemes are as follows: (1) the particle size is controlled by controlling the time, the longer the time, the larger the particle size is, and the particle size can reach more than 100nm generally within about 3 min; (2) it is also possible to obtain different particle sizes by varying the concentration, for example, to obtain nanoparticles of the same size, the higher the concentration the shorter the time.
The specific scheme comprises the following steps: scheme I,
Step one, dissolving OVA molecules in ddH2After O, the mass concentration of the solution is 15 mg/ml;
step two, taking 2.5ml of the OVA solution, adding 5ml of PBS (PH 7.2) buffer solution into a 5ml reaction bottle (containing matched magnetons), and mixing;
step three, placing the water bath kettle in a magnetic stirrer, heating to 80 ℃, adjusting the rotating speed to 1500r, placing the reaction bottle in the water bath kettle, and fully heating for 2 min;
step four, dialyzing the product (ddH)2O5L, changing water every 2 hours for 5 times), and freeze-drying to obtain OVA nano-material with the average particle size of 100 nm.
The method also comprises the following steps: scheme II,
Step one, dissolving OVA molecules in ddH2After O, the mass concentration of the solution is 10 mg/ml;
step two, taking 2.5ml of the OVA solution, adding 5ml of PBS (PH 7.2) buffer solution into a 5ml reaction bottle (containing matched magnetons), and mixing;
step three, placing the water bath kettle in a magnetic stirrer, heating to 80 ℃, adjusting the rotating speed to 1500r, placing the reaction bottle in the water bath kettle, and fully heating for 3 min;
step four, dialyzing the product (ddH)2O5L, changing water every 2 hours for 5 times), and freeze-drying to obtain OVA nano-material with the average particle size of 80 nm.
The method also comprises the following steps: scheme III,
Step one, dissolving OVA molecules in ddH2After O, the mass concentration of the solution is 5 mg/ml;
step two, taking 2.5ml of the OVA solution, adding 5ml of TEA (PH 8.0) buffer solution into a 5ml reaction bottle (containing matched magnetons), and mixing;
step three, placing the water bath kettle in a magnetic stirrer, heating to 50 ℃, adjusting the rotating speed to 200r, placing the reaction bottle in the water bath kettle, and fully heating for 2 min;
step four, dialyzing the product (ddH)2O5L, changing water every 2 hours for 5 times), and freeze-drying to obtain OVA nano-material with the average particle size of 30 nm.
EXAMPLE two, preparation of Nanoprotein
The specific preparation equation of the nano vaccine is as follows:
471-476the specific preparation process of the nano vaccine with peptide as CETP is as follows:
example A-1
Step one, preparation of a reaction solvent:
reaction solution A, OVA-NP was dissolved in PBS (pH 7.2, 0.01M) to prepare a solution having a mass concentration of 2 mg/ml;
reaction solution B: dissolving NEM in PBS (pH 7.2, 0.01M) to prepare 0.6256mg/ml solution;
reaction solution C: dissolving sulfo-smcc in ddH2O, preparing a solution with the mass concentration of 4.8 mg/ml;
reaction solution D: subjecting CETP to471-476(custom-made by Gill Biochemical company, and added with a cysteine at the amino terminal) is dissolved in DMF or DMSO to prepare a solution with the mass concentration of 25 mg/ml;
step two, taking 2ml of solution A and 1.078ml of solution B, and reacting for 2 hours at the rotating speed of 400r/min under the condition of room temperature;
step three, adding 0.164ml of solution C, and reacting for 1 hour at the rotating speed of 400r/min under the condition of room temperature;
step four, using a 3K ultrafiltration tube, secondary water, 4500r 20min 3 times, and performing ultrafiltration;
step five, using PBS (PH 7.2, 0.01M) to restore the volume of the reaction system to 4 ml;
sixthly, adding 35 mu ml of solution D, and reacting for 2 hours at the rotating speed of 400r/min under the condition of room temperature;
step seven, using a 10K ultrafiltration tube, secondary water, 4500r 12min 3 times, and performing ultrafiltration;
and step eight, carrying out overnight freezing at-80 ℃ on the reaction final product to obtain OVA-NP-CETP nanoparticles.
Example A-2
Step one, preparation of a reaction solvent:
reaction solution A, dissolving OVA-NP in TEA (pH 8, 0.02M) to prepare a solution with a mass concentration of 1 mg/ml;
reaction solution B: dissolving NEM in TEA (pH 8, 0.02M) to prepare a solution with the mass concentration of 0.2 mg/ml;
reaction solution C: dissolving sulfo-smcc in ddH2O, preparing a solution with the mass concentration of 0.2 mg/ml;
reaction solution D: subjecting CETP to471-476(custom-made by Gill Biochemical company, with a cysteine added to the amino terminus) dissolved in DMF or DMSO, prepared in concentrated qualityA solution with a degree of 10 mg/ml;
step two, taking 5ml of solution A and 1ml of solution B, and reacting for 3 hours at room temperature and at the rotating speed of 200 r/min;
adding 0.5ml of solution C, and reacting for 2 hours at the room temperature and the rotating speed of 200 r/min;
step four, using a 3K ultrafiltration tube, secondary water, 4500r 20min 3 times, and performing ultrafiltration;
step five, using TEA (pH 8, 0.02M) to restore the volume of the reaction system to 4 ml;
sixthly, adding 65 mu ml of solution D, and reacting for 5 hours at the room temperature and the rotating speed of 200 r/min;
step seven, using a 10K ultrafiltration tube, secondary water, 4500r 12min 3 times, and performing ultrafiltration;
and step eight, carrying out-80 ℃ overnight, and freeze-drying to obtain the nanoparticles.
Examples A to 3
Step one, preparation of a reaction solvent:
reaction solution A, dissolving OVA-NP in Mes (pH 6, 0.01M) to prepare a solution with the mass concentration of 10 mg/ml;
reaction solution B: dissolving NEM in Mes (pH 6, 0.01M) to prepare a solution with the mass concentration of 2 mg/ml;
reaction solution C: dissolving sulfo-smcc in ddH2O, preparing a solution with the mass concentration of 2 mg/ml;
reaction solution D: subjecting CETP to471-476(custom-made and synthesized by Gill Biochemical company, and a cysteine is added at the amino terminal) is dissolved in DMSO to prepare a solution with the mass concentration of 10 mg/ml;
step two, taking 5.1ml of solution A and 5ml of solution B, and reacting for 0.5 hour at the room temperature and the rotating speed of 800 r/min;
step three, adding 0.5ml of solution C, and reacting for 0.5 hour at the room temperature and the rotating speed of 800 r/min;
step four, using a 3K ultrafiltration tube, secondary water, 4500r 20min 5 times, and performing ultrafiltration;
step five, using Mes (pH is 6, 0.01M) to restore the volume of the reaction system to 6 ml;
step six, adding 60 mu ml of solution D, and reacting for 1 hour at the room temperature and the rotating speed of 800 r/min;
step seven, using a 10K ultrafiltration tube, secondary water, 4500r 12min 6 times, and performing ultrafiltration;
and step eight, carrying out-80 ℃ overnight, and freeze-drying to obtain the nanoparticles.
471-476 207-223Scheme b. the specific preparation process of the nano vaccine with peptide CETP and PCSK9 is as follows:
step one, preparation of a reaction solvent:
reaction solution A, OVA-NP was dissolved in PBS (pH 7.2, 0.01M) to prepare a solution having a mass concentration of 2 mg/ml;
reaction solution B: dissolving NEM in PBS (pH 7.2, 0.01M) to prepare 0.6256mg/ml solution;
reaction solution C: dissolving sulfo-smcc in ddH2O, preparing a solution with the mass concentration of 0.6256 mg/ml;
reaction solution D: subjecting CETP to471-476、PCSK9207-223(custom-made by Gill Biochemical company, and added with a cysteine at the amino terminal) is dissolved in DMF according to the proportion of 1:1 to prepare a solution with the medium mass concentration of 25 mg/ml;
step two, taking 3.2ml of solution A and 1.072ml of solution B, and reacting for 2 hours at the room temperature and the rotating speed of 400 r/min;
step three, adding 0.164ml of solution C, and reacting for 1 hour at the rotating speed of 400r/min under the condition of room temperature;
step four, using a 3K ultrafiltration tube, secondary water, 4500r 20min 3 times, and performing ultrafiltration;
step five, using PBS (PH 7.2, 0.01M) to restore the volume of the reaction system to 4 ml;
sixthly, adding 35 mu ml of solution D, and reacting for 2 hours at the rotating speed of 400r/min under the condition of room temperature;
step seven, using a 10K ultrafiltration tube, secondary water, 4500r 12min 3 times, and performing ultrafiltration;
step eight, carrying out-80 ℃ overnight and freeze-drying on the reaction final product.
EXAMPLE III product Property and Effect test
Results for the product of example A-1:
as shown in fig. 1 and 2, the presence of OVA-NP-CETP nanoparticles can be seen from both DLS and TEM particle size diagrams.
As shown in FIG. 3, the successful linkage of the polypeptide to OVA particles was qualitatively demonstrated from the SDS-PAGE pattern. As shown in FIG. 4, the anti-CETP antibody level of the serum of white rabbits in New Zealand (after two immunizations), all serum samples were diluted 16000 times, and the OD value at 450nm of each group shown in FIG. 4 was determined by ELISA, which clearly shows that the antibody level of OVA nano-carriers (i.e., OVA-NP group) adopted in the present invention is much higher than that of the molecular group. Here, the molecular group refers to conventional OVA nanomaterials.
The products obtained in the other examples have similar conclusions as the product of A-1.
Claims (5)
1. A nano carrier protein platform for improving antigen immunogenicity is characterized in that: increasing antigen immunogenicity by attaching a target to the surface of a nanocarrier protein;
the nano carrier protein is nano particles made of carrier protein;
the target is selected from CETP with cysteine added at the amino terminal;
the nano carrier protein is OVA nano particles manufactured by a thermal polymerization method;
the OVA nano-particles are nano-particles with the particle size of 30-100 nm;
the specific method for attaching the target to the surface of the nano carrier protein is as follows: the nanoparticles are subjected to addition reaction with a compound A containing double bonds, substitution reaction with a cross-linking agent and addition reaction with a target in sequence to obtain a nano vaccine;
the compound A containing double bonds is a compound which can perform addition reaction with-SH and is selected from olefins with 2-10 carbon atoms, cyclopentene, cyclohexene, pyrrole and furan;
the cross-linking agent is selected from any one end of the group which can be reacted with-NH2A compound which has a substituent reaction on the group and a substituent reaction on the other end with a mercapto group.
2. The nano-carrier protein platform for improving the immunogenicity of antigens according to claim 1, wherein the specific process steps are as follows:
dissolving nanoparticles in a buffer solution to form a nano solution;
dissolving a compound A in a buffer solution to form a reaction solution I;
dissolving a cross-linking agent in a solvent to form a reaction solution II;
dissolving the target in a solvent to form a target solution;
reacting the nano solution and the reaction solution I at the temperature of 10-55 ℃ for 0.5-5 hours;
step six, adding the reaction solution II, and reacting for 0.5-5 hours at the temperature of 10-55 ℃;
step seven, ultrafiltration;
step eight, adding a target solution, and reacting for 0.5-5 hours at the temperature of 10-55 ℃;
and step nine, after ultrafiltration, freeze-drying to obtain a target product.
3. The Nanocarrier protein platform for enhancing the immunogenicity of an antigen according to claim 2, wherein:
the mass ratio of the nano particles to the compound A is 5-20: 1;
the mass ratio of the compound A to the cross-linking agent is 5-15: 1;
the mass ratio of the nanoparticles to the target is 1-9: 1.
4. The Nanocarrier protein platform for enhancing the immunogenicity of an antigen according to claim 2, wherein:
the buffer solution is selected from TAE, TBE, protein electrophoresis solution, MES, SSC, SSPE, WB membrane transfer solution, TBS, PBS, DPBS and citric acid buffer solution;
the pH of the buffer solution is preferably 6-9;
the solvent is selected from ddH2O, DMF, DMSO, alcohols, ethers, ketones and esters.
5. The Nanocarrier protein platform for enhancing the immunogenicity of an antigen according to claim 1, wherein:
the method for producing the OVA nanoparticles is as follows:
dissolving OVA molecules in a solvent;
step two, adding a buffer solution and mixing;
step three, heating to 50-80 ℃, and stirring for reaction for 1-5 minutes;
and step four, dialyzing the product obtained in the step three at least once, and freeze-drying to obtain the OVA nano-particles.
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| CN1679966A (en) * | 2005-01-19 | 2005-10-12 | 中国药科大学 | Nanometer grainy and mucosal immunity of antiatherosclerosis nucleic vaccine |
| CN103372217A (en) * | 2012-04-28 | 2013-10-30 | 中国科学院深圳先进技术研究院 | Polymer nano carrier preparation as well as preparation method and application thereof |
| EP2868327A1 (en) * | 2012-06-29 | 2015-05-06 | Universidad Nacional Autónoma De México | Nasal vaccine against the development of atherosclerosis disease and fatty liver |
| CN105085684A (en) * | 2014-05-14 | 2015-11-25 | 上海亨臻实业有限公司 | Design and application of PCSK9 targeting recombinant vaccine |
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| CN1679966A (en) * | 2005-01-19 | 2005-10-12 | 中国药科大学 | Nanometer grainy and mucosal immunity of antiatherosclerosis nucleic vaccine |
| CN103372217A (en) * | 2012-04-28 | 2013-10-30 | 中国科学院深圳先进技术研究院 | Polymer nano carrier preparation as well as preparation method and application thereof |
| EP2868327A1 (en) * | 2012-06-29 | 2015-05-06 | Universidad Nacional Autónoma De México | Nasal vaccine against the development of atherosclerosis disease and fatty liver |
| CN105085684A (en) * | 2014-05-14 | 2015-11-25 | 上海亨臻实业有限公司 | Design and application of PCSK9 targeting recombinant vaccine |
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