CN106729681A - A kind of nanosizing carrier protein platform for improving antigen immunogenicity - Google Patents
A kind of nanosizing carrier protein platform for improving antigen immunogenicity Download PDFInfo
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- CN106729681A CN106729681A CN201611193135.XA CN201611193135A CN106729681A CN 106729681 A CN106729681 A CN 106729681A CN 201611193135 A CN201611193135 A CN 201611193135A CN 106729681 A CN106729681 A CN 106729681A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a kind of nanosizing carrier protein platform for improving antigen immunogenicity, it is characterised in that：By by target in succession in improving antigen immunogenicity on the surface of nano-carrier albumen；The nano-carrier albumen is the nano particle being fabricated by by carrier protein.Capsid surface by the way that polypeptide epitope to be given in nanometer of the invention, protein body is not only for it provides t cell epitope but also as nano-carrier, makes it have the suitably sized identification for being conducive to antigen presenting cell and swallows；Meanwhile, by repeating displaying B cell epitope, the identification of B cell can be conducive to, and then immune effect can be strengthened.
Description
Technical field
The present invention relates to nano vaccine field, in particular it relates to a kind of nanosizing carrier egg for improving antigen immunogenicity
Bai Pingtai.
Background technology
Now there are some researches show the factor that LDL-C (LDL-C) is promotion atherosclerosis, high density
Lipoprotein cholesterol (HDL-C) is to resist the atherosis factor, and existing atherosclerosis correlation prophylactico-therapeutic measures and research are caused mostly
Power is in reduction LDL-C, high density lipoprotein increasing cholesterol.
CETP (CETP, cholesteryl ester transfer protein) be regulation HDL with
The key protein of cholesterol transport between LDL, VLDL, zooscopy show with the polypeptide of CETP build vaccine can reduce LDL-C,
HDL-C is raised, while there is obvious prevention of arterial atherosis, forefathers' research confirms 16 amino acid of the protein carboxyl groups end
Sequence C ETP471-476 (FGFPEHLLVDFLQSLS) can be used for the development of the vaccine, but, it is currently based on CETP vaccines
Research is all the vaccine of molecule or the related gene vaccine of minority, is never successfully entered clinical because can not produce enough
Antibody suppress CETP activity, reduction serum LDL-C.
PCSK9 is proprotein convertase subtilisin kexin9 types (proprotein convertase
Subtilisin-like/kexin type 9, PCSK9) be discovered in recent years regulation blood plasma in LDL-C
(LDL-C) one of key protein matter of content, and be considered as coronary heart disease (CHD) potential therapy target.PCSK9
LDLR on liver plasma membrane can be promoted to degrade, so that reduce LDL-C in serum remove, so suppress PCSK9 can significantly drop
The level of low serum LDL-C.Test result indicate that PCSK9 vaccines can reduce LDL-C, drop is especially shared with lipid-lowering statins
Fat effect is substantially better than alone lipid-lowering statins, and wherein PCSK9207-223 (NVPEEDGTRFHRQASKC) is that relatively have at present
The epitope candidate of potentiality.But the direct relation between PCSK9 vaccines and atherosclerosis is still directly studied without document, due to
APOE can be combined with LDLR, increase the degraded of HDL-C.
Traditional vaccine is that B cell epitope is combined indirectly with carrier protein molecule directly or by thing is linked, for it provides T
Cell epitope.Not yet there is the research of the nm regime of the vaccine at present.
The content of the invention
It is contemplated that overcoming drawbacks described above, there is provided a kind of nanosizing carrier protein that can improve antigen immunogenicity is put down
Platform, and using the nano vaccine with antigen immunogenicity high of platform manufacture.
The invention provides a kind of nanosizing carrier protein platform for improving antigen immunogenicity, it is characterised in that：Pass through
Target is improved into antigen immunogenicity in the surface of nano-carrier albumen in succession；
Above-mentioned nano-carrier albumen is the nano particle being fabricated by by carrier protein.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：It is above-mentioned to be in the specific method on nano-carrier protein surface in succession by target：Nano particle is passed through successively, and contains double bond
Compound A there is addition reaction and crosslinking agent and substitution reaction occur and after target occurs addition reaction, obtain nano vaccine.
This contain double bond compound A can for olefin and its derivatives, cyclenes hydrocarbons and their derivates, heterocyclic alkene and its
Derivative；
Specifically such as：Carbon number is olefin and its derivatives, cyclopentene and its derivative, cyclohexene and its derivative of 1-10
Thing, pyrroles and its derivative are (such as：NEM), Furan and its derivatives etc. can occur the compound of addition reaction with-SH；
The crosslinking agent can be with-NH selected from any one end2There is substitution reaction in group, the other end can be sent out with sulfydryl
The compound of raw substitution reaction, commonly uses such as：sulfo-smcc.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is that concrete technology step is as follows：
Step one, nano particle is dissolved in cushioning liquid and forms nano-solution；Concentration carries out bar by the condition of reaction
Part, preferably：0.2-10mg/ml；
Step 2, compound A is dissolved in reaction solution one is formed in cushioning liquid；Concentration carries out bar by the condition of reaction
Part, preferably：0.2-2mg/ml；
Step 3, crosslinking agent is dissolved in reaction solution two is formed in solvent；Concentration carries out condition by the condition of reaction, excellent
Elect as：0.2-2mg/ml；
Step 4, target is dissolved in target solution is formed in solvent；Concentration carries out condition by the condition of reaction, preferably
For：10-50mg/ml；
Step 5, by nano-solution and reaction solution one, at a temperature of 10-55 DEG C, react 0.5-5 hours；
Step 6, addition reaction solution two, at a temperature of 10-55 DEG C, react 0.5-5 hours；
Step 7, ultrafiltration；
Step 8, addition target solution, at a temperature of 10-55 DEG C, react 0.5-5 hours；
After step 9, ultrafiltration, freeze and obtain target product.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：Above-mentioned nano particle is 5-20 with the mass ratio of compound A:1；The numerical value is general according to the-SH contained on nano particle
Quantity carry out the calculating of the injected volume of compound A；Preferably：The usage amount of compound A is to have reacted all of sulfydryl
It is preferred.
Above-claimed cpd A is 5-15 with the mass ratio of crosslinking agent:1；The numerical value it is general according to contain on nano particle-
NH2Quantity carry out the calculating of the injected volume of crosslinking agent；Preferably：The usage amount of crosslinking agent is by the reaction of all of amino
It is complete to be preferred.
Above-mentioned nano particle is 1-9 with the mass ratio of target:1, the numerical value is general according to the friendship being connected with nano particle
Join the quantity of agent to carry out the calculating of the injected volume of target；Preferably：The usage amount of target is to have reacted all of crosslinking agent
It is preferred.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：Above-mentioned cushioning liquid is selected from TAE, TBE, MES, protein electrophoresis liquid, SSC, SSPE, WB transferring film liquid, TBS, PBS, DPBS, lemon
Lemon acid buffer；
The pH of above-mentioned cushioning liquid is preferably 6-9；
Above-mentioned solvent is selected from ddH2One or more in O, DMF, DMSO, alcohols, ethers, ketone, esters.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：Above-mentioned nano-carrier albumen platform is the OVA nano particles manufactured by thermal polymerization method；
Above-mentioned OVA nano particles are that particle diameter is the nano particle of 30-100nm, the preferably nano particle of 40-60nm.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：The manufacture method of above-mentioned OVA nano particles is as follows：
Step one, OVA molecules are dissolved in solvent；
Step 2, addition cushioning liquid, are mixed；Can also be herein to be added after taking the solution of appropriate step one
Cushioning liquid mixes；The need for product characters, the addition of cushioning liquid can be 0.1-10 times of OVA solution.
Step 3, it is warming up to 50-80 DEG C, stirring reaction 1-5 minutes；
After step 4, the product of step 3 are through dialysis at least one times, prepared OVA nano particles are freezed.
Above-mentioned cushioning liquid be selected from TAE, TBE, MES, protein electrophoresis liquid, SSC, SSPE, WB transferring film liquid, TBS, PBS,
DPBS, citrate buffer solution；
The pH of above-mentioned cushioning liquid is preferably 6-9；
Above-mentioned solvent is selected from ddH2One or more in O, DMF, DMSO, alcohols, ethers, ketone, esters.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：Above-mentioned target is selected from for one or more in the target for adjusting blood fat.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：Above-mentioned target has the CETP of cysteine and/or the aminoterminal increase to have the PCSK9 of cysteine selected from aminoterminal increase.
Further, a kind of nanosizing carrier protein platform of raising antigen immunogenicity that the present invention is provided, its feature
It is：When target has the CETP of cysteine and aminoterminal increase to have the PCSK9 of cysteine selected from aminoterminal increase；
The mass ratio of above-mentioned CETP and PCSK9 is 1:0.01-100.
The concentration of HDL-C can be increased due to CETP, and PCSK9 can degrade to HDL-C, so, the CETP and PCSK
Mass ratio the selection of ratio is preferably carried out according to the balance of HDL-C concentration.
Effect of the invention and effect：
Surface by the way that polypeptide epitope to be given in nanoprotein particle of the invention, protein body is both for it provides T cell
Epitope and as nano-carrier platform.The characteristics of having grain size controllable due to nano-carrier, so, conjunction is had based on it
Suitable size, is more beneficial for antigen presenting cell identification phagocytosis.
Meanwhile, target is modified in the surface of nano-carrier platform, the result for repeating to show B cell epitope can be realized, this
Sample is more beneficial for the identification of B cell, and then can strengthen immune effect.
Specifically such as：In the present invention, by certain processing method, by traditional carrier protein OVA (Ovalbumin) points
Sub- self assembly is the particle of 30-100nm or so, then by crosslinking agent (such as：Sulfo-smcc etc.) will be from target (such as：PCSK9
And CETP) B cell epitope be given in particle surface, found through experiment, the vaccine of the nano-carrier albumen platform of use manufacture
Nano-carrier of its antibody level far above conventional molecular form.
Additionally, in the present invention, for this function of regulation blood fat, it is also proposed that while by CETP471-476、PCSK9207-223
The target of two regulation blood fat is connected on same carrier.On the one hand, the polypeptide of CETP builds vaccine and can reduce LDL-C, rise
HDL-C high, while have the atherosis effect of obvious prevention of arterial, and the polypeptide of PCSK9 builds vaccine and is reducing the same of LDL-C
When can cause the reduction of HDL-C, the concentration that therefore, it can increase HDL-C by CETP is made up caused by PCSK9 to HDL-C's
Degraded, while the two cooperates with reduction LDL-C again, then be just expected to obtain the effect of preferably adjustment blood fat, so as to more preferable
The effect that prevention of arterial is atherosis.
Brief description of the drawings
The DLS figures of Fig. 1, OVA-NP-CETP nano particle；
The TEM figures of Fig. 2, OVA-NP-CETP nano particle；
The SDS-PAGE of Fig. 3, OVA-NP-CETP nano particle；
Wherein, 1 is mark, and 2 is OVA molecules, and 3 is OVA-NP, and 4 is OVA-NEM-SMCC, and 5 is OVA-NEM-SMCC-
CETP, 6 is OVA-NP-NEM-SMCC-CETP；
The anti-CETP antibody levels comparative result figure of Fig. 4, new zealand white rabbit serum.
Specific embodiment
The preparation of embodiment one, OVA nano materials
Specific method is as follows：
Step one, OVA molecules are dissolved in ddH2After O, the mass concentration of solution is 10mg/ml；
Step 2, take the above-mentioned OVA solution of 2.5ml in the reaction bulb (being contained within supporting magneton) of 5ml, add
The buffer solution mixing of the Mes (PH=6.0) of 2.5ml；
Step 3, water-bath is placed in magnetic stirring apparatus, is heated to 70 DEG C, rotating speed is adjusted to 750r, by above-mentioned reaction
Bottle is placed in water-bath, allows it fully to heat 2min10s；
Step 4, by product dialyse (ddH2O 5L, change a water in every 2 hours, totally 5 times), the lyophilized average grain diameter that obtains is
The OVA nano materials (OVA-NP) of 50nm.
In the preparation process of the OVA nano materials, according to the difference of target grain size, to react time, reaction it is dense
Dialysis number of times of degree, rotating speed and post processing etc. is adjusted, and can obtain what average grain diameter respectively 30nm-100nm was not waited
Nano material.
Conventional scheme is such as：(1) particle size, is controlled by control time, the time is more long, and particle diameter is bigger, general 3min is left
The right side can just reach more than 100nm；(2) different particles, can also be obtained by changing concentration, for example, needing to obtain
The nano particle of same particle size, concentration is higher, and the time is shorter.
Concrete scheme has：Scheme one,
Step one, OVA molecules are dissolved in ddH2After O, the mass concentration of solution is 15mg/ml；
Step 2, take the above-mentioned OVA solution of 2.5ml in the reaction bulb (being contained within supporting magneton) of 5ml, add 5ml
PBS (PH=7.2) buffer solution mixing；
Step 3, water-bath is placed in magnetic stirring apparatus, is heated to 80 DEG C, rotating speed is adjusted to 1500r, by above-mentioned reaction
Bottle is placed in water-bath, allows it fully to heat 2min；
Step 4, by product dialyse (ddH2O 5L, change a water in every 2 hours, totally 5 times), the lyophilized average grain diameter that obtains is
The OVA nano materials of 100nm.
Also such as：Scheme two,
Step one, OVA molecules are dissolved in ddH2After O, the mass concentration of solution is 10mg/ml；
Step 2, take the above-mentioned OVA solution of 2.5ml in the reaction bulb (being contained within supporting magneton) of 5ml, add 5ml
PBS (PH=7.2) buffer solution mixing；
Step 3, water-bath is placed in magnetic stirring apparatus, is heated to 80 DEG C, rotating speed is adjusted to 1500r, by above-mentioned reaction
Bottle is placed in water-bath, allows it fully to heat 3min；
Step 4, by product dialyse (ddH2O 5L, change a water in every 2 hours, totally 5 times), the lyophilized average grain diameter that obtains is
The OVA nano materials of 80nm.
Also such as：Scheme three,
Step one, OVA molecules are dissolved in ddH2After O, the mass concentration of solution is 5mg/ml；
Step 2, take the above-mentioned OVA solution of 2.5ml in the reaction bulb (being contained within supporting magneton) of 5ml, add 5ml
TEA (PH=8.0) buffer solution mixing；
Step 3, water-bath is placed in magnetic stirring apparatus, is heated to 50 DEG C, rotating speed is adjusted to 200r, by above-mentioned reaction
Bottle is placed in water-bath, allows it fully to heat 2min；
Step 4, by product dialyse (ddH2O 5L, change a water in every 2 hours, totally 5 times), the lyophilized average grain diameter that obtains is
The OVA nano materials of 30nm.
The preparation of embodiment two, nano vaccine
Nano vaccine specifically to prepare equation as follows：
Option A .peptide is CETP471-476Nano vaccine specific preparation technology it is as follows：
Embodiment A-1
The configuration of step one, reaction dissolvent：
Reaction solution A:OVA-NP is dissolved in PBS (pH=7.2,0.01M), configuration quality concentration is the solution of 2mg/ml；
Reaction solution B：NEM is dissolved in PBS (pH=7.2,0.01M), configuration quality concentration is the solution of 0.6256mg/ml；
Reaction liquid C：Sulfo-smcc is dissolved in ddH2O, configuration quality concentration is the solution of 4.8mg/ml；
Reaction solution D：By CETP471-476(gill biochemical corp customization synthesis, aminoterminal increase has a cysteine) is molten
In DMF or DMSO, configuration quality concentration is the solution of 25mg/ml；
The solution B of step 2, the solution A for taking 2ml and 1.078ml, under room temperature condition, under the rotating speed of 400r/min, instead
Answer 2 hours；
Step 3, the solution C for adding 0.164ml, under room temperature condition, under the rotating speed of 400r/min, react 1 hour；
Step 4, the super filter tube using 3K, secondary water, 4500r*20min*3 times, ultrafiltration；
Step 5, PBS (PH=7.2,0.01M) is used to recover reaction system volume to 4ml；
Step 6,35 μm of solution Ds of l of addition, under room temperature condition, under the rotating speed of 400r/min, react 2 hours；
Step 7, the super filter tube using 10K, secondary water, 4500r*12min*3 times, ultrafiltration；
Step 8, by reacting final product, -80 ° overnight, lyophilized obtain OVA-NP-CETP nano particles.
Embodiment A-2
The configuration of step one, reaction dissolvent：
Reaction solution A:OVA-NP is dissolved in TEA (pH=8,0.02M), configuration quality concentration is the solution of 1mg/ml；
Reaction solution B：NEM is dissolved in TEA (pH=8,0.02M), configuration quality concentration is the solution of 0.2mg/ml；
Reaction liquid C：Sulfo-smcc is dissolved in ddH2O, configuration quality concentration is the solution of 0.2mg/ml；
Reaction solution D：By CETP471-476(gill biochemical corp customization synthesis, aminoterminal increase has a cysteine) is molten
In DMF or DMSO, configuration quality concentration is the solution of 10mg/ml；
Step 2, the solution A for taking 5ml and 1 solution B, under room temperature condition, under the rotating speed of 200r/min, reaction 3 is small
When；
Step 3, the solution C for adding 0.5ml, under room temperature condition, under the rotating speed of 200r/min, react 2 hours；
Step 4, the super filter tube using 3K, secondary water, 4500r*20min*3 times, ultrafiltration；
Step 5, TEA (pH=8,0.02M) is used to recover reaction system volume to 4ml；
Step 6,65 μm of solution Ds of l of addition, under room temperature condition, under the rotating speed of 200r/min, react 5 hours；
Step 7, the super filter tube using 10K, secondary water, 4500r*12min*3 times, ultrafiltration；
Step 8, by reacting final product, -80 ° overnight, lyophilized obtain nano particle.
Embodiment A-3
The configuration of step one, reaction dissolvent：
Reaction solution A:OVA-NP is dissolved in Mes (pH=6,0.01M), configuration quality concentration is the solution of 10mg/ml；
Reaction solution B：NEM is dissolved in Mes (pH=6,0.01M), configuration quality concentration is the solution of 2mg/ml；
Reaction liquid C：Sulfo-smcc is dissolved in ddH2O, configuration quality concentration is the solution of 2mg/ml；
Reaction solution D：By CETP471-476(gill biochemical corp customization synthesis, aminoterminal increase has a cysteine) is molten
In DMSO, configuration quality concentration is the solution of 10mg/ml；
The solution B of step 2, the solution A for taking 5.1ml and 5ml, under room temperature condition, under the rotating speed of 800r/min, reacts
0.5 hour；
Step 3, the solution C for adding 0.5ml, under room temperature condition, under the rotating speed of 800r/min, react 0.5 hour；
Step 4, the super filter tube using 3K, secondary water, 4500r*20min*5 times, ultrafiltration；
Step 5, Mes (pH=6,0.01M) is used to recover reaction system volume to 6ml；
Step 6,60 μm of solution Ds of l of addition, under room temperature condition, under the rotating speed of 800r/min, react 1 hour；
Step 7, the super filter tube using 10K, secondary water, 4500r*12min*6 times, ultrafiltration；
Step 8, by reacting final product, -80 ° overnight, lyophilized obtain nano particle.
Option b .peptide is CETP471-476And PCSK9207-223Nano vaccine specific preparation technology it is as follows：
The configuration of step one, reaction dissolvent：
Reaction solution A:OVA-NP is dissolved in PBS (pH=7.2,0.01M), configuration quality concentration is the solution of 2mg/ml；
Reaction solution B：NEM is dissolved in PBS (pH=7.2,0.01M), configuration quality concentration is the solution of 0.6256mg/ml；
Reaction liquid C：Sulfo-smcc is dissolved in ddH2O, configuration quality concentration is the solution of 0.6256mg/ml；
Reaction solution D：By CETP471-476、PCSK9207-223(the customization synthesis of gill biochemical corp, aminoterminal increases one
Cysteine) with 1:1 ratio is dissolved in DMF, and mass concentration is the solution of 25mg/ml in configuration；
The solution B of step 2, the solution A for taking 3.2ml and 1.072ml, under room temperature condition, under the rotating speed of 400r/min,
Reaction 2 hours；
Step 3, the solution C for adding 0.164ml, under room temperature condition, under the rotating speed of 400r/min, react 1 hour；
Step 4, the super filter tube using 3K, secondary water, 4500r*20min*3 times, ultrafiltration；
Step 5, PBS (PH=7.2,0.01M) is used to recover reaction system volume to 4ml；
Step 6,35 μm of solution Ds of l of addition, under room temperature condition, under the rotating speed of 400r/min, react 2 hours；
Step 7, the super filter tube using 10K, secondary water, 4500r*12min*3 times, ultrafiltration；
Step 8, by reacting final product, -80 ° overnight, it is lyophilized.
Embodiment three, product characteristics and effect experiment
The result of embodiment A-1 products：
As illustrated in fig. 1 and 2, it can be seen that the presence of OVA-NP-CETP nano particles from the grain-size graph of DLS and TEM.
As shown in figure 3, from the figure of SDS-PAGE, the qualitative polypeptide that demonstrates is linked into work(with OVA particles.As shown in figure 4,
The anti-CETP antibody levels of new zealand white rabbit serum (immune rear twice), all serum samples dilute 16000 times, by ELISA
Method detects, each group as shown in Figure 4 OD values 450nm at, hence it is evident that OVA nano-carriers (the i.e., OVA-NP of visible use of the invention
Group) antibody level be far above group of molecules.Herein, group of molecules refers to traditional OVA nano materials.
The product that other embodiment is obtained has similar conclusion to the product of A-1.
Claims (10)
1. it is a kind of improve antigen immunogenicity nanosizing carrier protein platform, it is characterised in that：By by target in succession in receiving
Antigen immunogenicity is improved on the surface of rice carrier protein；
The nano-carrier albumen is the nano particle being fabricated by by carrier protein.
2. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 1, it is characterised in that：
It is described to be in the specific method on nano-carrier protein surface in succession by target：Nano particle is passed through successively, and contains double bond
Compound A there is addition reaction and crosslinking agent and substitution reaction occur and after target occurs addition reaction, obtain nano vaccine.
3. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 2, it is characterised in that tool
Body technology step is as follows：
Step one, nano particle is dissolved in cushioning liquid and forms nano-solution；
Step 2, compound A is dissolved in reaction solution one is formed in cushioning liquid；
Step 3, crosslinking agent is dissolved in reaction solution two is formed in solvent；
Step 4, target is dissolved in target solution is formed in solvent；
Step 5, by nano-solution and reaction solution one, at a temperature of 10-55 DEG C, react 0.5-5 hours；
Step 6, addition reaction solution two, at a temperature of 10-55 DEG C, react 0.5-5 hours；
Step 7, ultrafiltration；
Step 8, addition target solution, at a temperature of 10-55 DEG C, react 0.5-5 hours；
After step 9, ultrafiltration, freeze and obtain target product.
4. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 3, it is characterised in that：
The nano particle is 5-20 with the mass ratio of compound A:1；
The compound A is 5-15 with the mass ratio of crosslinking agent:1；
The nano particle is 1-9 with the mass ratio of target:1.
5. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 3, it is characterised in that：
The cushioning liquid is selected from TAE, TBE, protein electrophoresis liquid, MES, SSC, SSPE, WB transferring film liquid, TBS, PBS, DPBS, lemon
Lemon acid buffer；
The pH of the cushioning liquid is preferably 6-9；
The solvent is selected from ddH2One or more in O, DMF, DMSO, alcohols, ethers, ketone, esters.
6. a kind of nanosizing carrier protein platform of the raising antigen immunogenicity as described in claim 1-5 is any, its feature
It is：
The nano-carrier albumen platform is the OVA nano particles manufactured by thermal polymerization method；
The OVA nano particles are that particle diameter is the nano particle of 30-100nm.
7. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 6, it is characterised in that：
The manufacture method of the OVA nano particles is as follows：
Step one, OVA molecules are dissolved in solvent；
Step 2, addition cushioning liquid, are mixed；
Step 3, it is warming up to 50-80 DEG C, stirring reaction 1-5 minutes；
After step 4, the product of step 3 are through dialysis at least one times, prepared OVA nano particles are freezed.
8. a kind of nanosizing carrier protein platform of the raising antigen immunogenicity as described in claim 1-5 is any, its feature
It is：
The target is selected from for one or more in the target for adjusting blood fat.
9. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 8, it is characterised in that：
The target has the CETP of cysteine and/or the aminoterminal increase to have the PCSK9 of cysteine selected from aminoterminal increase.
10. a kind of nanosizing carrier protein platform for improving antigen immunogenicity as claimed in claim 9, it is characterised in that：
When target has the CETP of cysteine and aminoterminal increase to have the PCSK9 of cysteine selected from aminoterminal increase；
The mass ratio of the CETP and PCSK9 is 1:0.01-100.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611193135.XA CN106729681B (en) | 2016-12-21 | 2016-12-21 | Nanocarrier protein platform for improving antigen immunogenicity |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611193135.XA CN106729681B (en) | 2016-12-21 | 2016-12-21 | Nanocarrier protein platform for improving antigen immunogenicity |
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| CN106729681A true CN106729681A (en) | 2017-05-31 |
| CN106729681B CN106729681B (en) | 2020-02-21 |
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| CN107456575A (en) * | 2017-09-06 | 2017-12-12 | 苏州大学 | A kind of manganese dioxide nano adjuvant and preparation method thereof, application |
| CN109010842A (en) * | 2018-08-13 | 2018-12-18 | 上海交通大学医学院附属新华医院 | A kind of nano platform for the carrier epitope depression effect overcoming vaccine |
| CN109553684A (en) * | 2017-09-25 | 2019-04-02 | 中国科学院过程工程研究所 | A kind of nano-carrier albumen and its preparation method and application |
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| EP2868327A1 (en) * | 2012-06-29 | 2015-05-06 | Universidad Nacional Autónoma De México | Nasal vaccine against the development of atherosclerosis disease and fatty liver |
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| CN107456575A (en) * | 2017-09-06 | 2017-12-12 | 苏州大学 | A kind of manganese dioxide nano adjuvant and preparation method thereof, application |
| CN109553684A (en) * | 2017-09-25 | 2019-04-02 | 中国科学院过程工程研究所 | A kind of nano-carrier albumen and its preparation method and application |
| CN109010842A (en) * | 2018-08-13 | 2018-12-18 | 上海交通大学医学院附属新华医院 | A kind of nano platform for the carrier epitope depression effect overcoming vaccine |
| CN109010842B (en) * | 2018-08-13 | 2021-08-24 | 上海交通大学医学院附属新华医院 | Nanometer platform for overcoming carrier epitope inhibition effect of vaccine |
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| CN106729681B (en) | 2020-02-21 |
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