CN109010842B - Nanometer platform for overcoming carrier epitope inhibition effect of vaccine - Google Patents
Nanometer platform for overcoming carrier epitope inhibition effect of vaccine Download PDFInfo
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Abstract
The invention provides a nano platform for overcoming the carrier epitope suppression effect of a vaccine, which is characterized in that: taking albumin as a raw material, and obtaining a target vaccine carrier through reduction, thermal polymerization, oxidation, concentration and external connection of an epitope; wherein the reduction is to reduce disulfide bonds in albumin to sulfhydryl groups; the heat polymerization is nanoparticles formed based on albumin self-assembly; the oxidation is the oxidation of sulfhydryl groups to disulfide bonds. The invention can completely inhibit the generation of carrier protein antibody, the synthesis process is green and simple, and no toxic chemical or complex flow is involved; low cost, small batch difference and wide clinical transformation prospect.
Description
Technical Field
The invention relates to the field of nano vaccines, in particular to a nano platform for overcoming the carrier epitope suppression effect of vaccines.
Background
In order to solve the problem of the "carrier inhibition effect", there are documents that polylactic-co-glycolic acid (PLGA) and liposome are used as a material for coating carrier protein, and the material has the problems that the generation of carrier protein antibodies is not completely inhibited, the synthetic method is not green (toxic organic solvent is involved in the synthetic process, etc.), the synthetic process is complicated, and the synthetic material is complicated (comprising various lipid components and high molecular materials).
In addition, there is a literature that polyethylene glycol (PEG) is used to modify carrier protein to reduce the "carrier inhibition effect", but the proposal does not completely inhibit the generation of carrier protein antibody, and long-term use of PEG also generates antibody against PEG, which results in accelerated clearance of PEG by the body.
In addition, plasmid transfection and prokaryotic expression are used in the literature to produce recombinant proteins fused with self-antigens and non-self-antigens to reduce the "vector inhibition effect", but the scheme does not completely inhibit the generation of carrier protein antibodies, and the production operation is complex and the cost is high.
Disclosure of Invention
The invention aims to overcome the defects and provides a nano platform for overcoming the carrier epitope suppression effect of the vaccine, in particular to a nano vaccine carrier which is used for overcoming the carrier epitope suppression effect and is formed by pure protein composite based on albumin-carrier protein disulfide bond crosslinking, can completely suppress the generation of carrier protein antibodies, has green and simple synthesis process and does not relate to toxic chemicals or complex processes; low cost, small batch difference and wide clinical transformation prospect.
The invention provides a nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nano particles, which is characterized in that: taking albumin as a raw material, and obtaining a target vaccine carrier through reduction, thermal polymerization, oxidation, concentration and external connection of an epitope;
wherein the reduction is to reduce disulfide bonds in albumin to thiol groups;
the thermal polymerization is nanoparticles formed by albumin self-assembly;
the oxidation is to oxidize the thiol group into a disulfide bond.
The albumin is selected from human albumin (such as human serum albumin), or animal albumin (such as mouse serum albumin or bovine serum albumin).
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: namely, the reduction step is:
s1, preparing a solution A: dissolving albumin and carrier protein in a buffer;
s2, preparing a solution B: dissolving a reducing agent in water;
and S3, mixing the solution A and the solution B, and stirring at room temperature for reaction for 0.5-2 hours.
The buffer solution may preferably be selected from weak alkaline buffer solutions having a pH of 8-10, such as: weak acids and their salts, weak bases and their salts, acid salts of polybasic weak acids and their corresponding secondary salts. The method specifically comprises the following steps: phosphate, borate, citrate, carbonate, acetate, barbiturate, Tris (Tris) system, etc. In this step, a phosphoric acid buffer solution is preferably used.
The concentration of the A solution is preferably 2-10mg/ml in percentage by mass.
In the solution B, the reducing agent can be any reducing agent capable of reducing disulfide bonds into sulfydryl, and the mass percentage concentration of the reducing agent after the reducing agent is dissolved in water is preferably 8-20 mg/ml.
In S3, the volume ratio of the solution A to the solution B is preferably 3: 0.1-1.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: that is, the mass ratio of the albumin to the carrier protein is greater than 4: 1; the present inventors have found that antibodies against KLH can be produced when the mass ratio of albumin to carrier protein is less than or equal to 4:1, possibly due to the excess carrier protein not being coated with albumin.
The mass ratio of the albumin to the reducing agent is 1: 0.1-1.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: that is, the thermal polymerization step is:
s1, reacting buffer solution, anionic surfactant, water and a product of reduction reaction for 5-20 minutes at a water bath temperature of 50-100 ℃;
s2, cooling in a water bath at the temperature of below 10 ℃.
The buffer solution may preferably be selected from weakly acidic buffer solutions having a pH of 5.5 to 7, such as: weak acids and their salts, acid salts of polybasic weak acids and their corresponding secondary salts. The method specifically comprises the following steps: phosphate, borate, citrate, carbonate, acetate, barbiturate, and the like. In this step, a MES buffer solution is preferably used.
The anionic surfactant may be replaced with any long chain alkyl anionic surfactant, such as: sodium tetradecyl sulfate, sodium hexadecane sulfonate, etc., and in the present invention, an anionic surfactant can be used to control the particle size.
In addition, due to the action of thermal polymerization stirring and the like, the protein firstly forms nanoparticles with good uniformity, and in the process, partial sulfydryl forms new disulfide bonds again.
The volume ratio of the buffer solution, the anionic surfactant, the water and the reduction reaction product is 1:0.01-0.3:0.1-0.5: 0.4-1.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: namely, the oxidation step is: reacting the thermal polymerization product with an oxidant at room temperature for 4-10 hours;
the oxidizing agent may be selected from any agent capable of oxidizing a thiol group to a disulfide bond, and is preferably a peroxide, which may be an organic peroxide or an inorganic peroxide.
Generally, the peroxide is an agent soluble in water, and is used by preparing a solution having a concentration of not more than 50%.
The hot polymerization product is generally used after being diluted with water to a concentration of 0.5 to 2 mg/ml.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: that is, the mass ratio of the hot polymerization product to the oxidizing agent is 1: 0.1-1.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: namely, the concentration step is:
s1, dialyzing the oxidation product for 12-48 hours; the dialysis is typically performed every 4 hours.
S2, carrying out ultrafiltration concentration on the product of S1 by using a buffer solution for 5-60 minutes. The ultrafiltration process is generally carried out 3 times or more.
The buffer solution may preferably be selected from weakly alkaline buffer solutions having a pH of 7-8, such as: weak acids and their salts, weak bases and their salts, acid salts of polybasic weak acids and their corresponding secondary salts. The method specifically comprises the following steps: phosphate, borate, citrate, carbonate, acetate, barbiturate, Tris (Tris) system, etc. In this step, a phosphoric acid buffer solution is preferably used.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: namely, the process for externally binding an epitope comprises:
s1, reacting the concentrated product with a cross-linking agent for 0.5-3 hours at room temperature;
s2, carrying out ultrafiltration on the product of the S1 by using a buffer solution to remove unreacted cross-linking agents;
s3, diluting the product of S2 by using a buffer solution, adding an epitope, and reacting for 1-5 hours at room temperature;
s4, ultrafiltering the product of S3 with buffer solution to remove unreacted antigen epitope;
s5, diluting the product of S4 by using a buffer solution, and storing at the temperature below-20 ℃.
The cross-linking agent is generally a protein cross-linking agent which can be dissolved in water and can act on-SH bonds, and during the use process, the cross-linking agent is generally prepared by adopting an aqueous solution of the cross-linking agent, and the mass percentage concentration of the cross-linking agent is 5-50 mg/ml.
The epitope can be any epitope according to the use requirement, and the epitope is generally dissolved in a buffer solution and then is used, wherein the mass percentage concentration of the epitope is 20-50 mg/ml.
The buffer solution may preferably be selected from weakly alkaline buffer solutions having a pH of 7-8, such as: weak acids and their salts, weak bases and their salts, acid salts of polybasic weak acids and their corresponding secondary salts. The method specifically comprises the following steps: phosphate, borate, citrate, carbonate, acetate, barbiturate, Tris (Tris) system, etc. In this step, a phosphoric acid buffer solution is preferably used.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: namely, the mass ratio of the albumin to the cross-linking agent is 1: 0.5-15;
the mass ratio of the albumin to the epitope is 1: 0.5-15.
Further, the nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles provided by the invention also has the characteristics that: i.e. for overcoming the inhibitory effect of the carrier epitope.
The invention has the following functions and effects:
the invention adopts pure protein as raw material to synthesize carrier protein, and the raw material is nontoxic and harmless and is easy to obtain; the synthetic process is green, environment-friendly and simple, and toxic chemicals or complex processes are not involved; and the cost is low, the batch difference is small, and the clinical transformation prospect is realized.
In addition, the antibody titer result of the carrier protein provided by the invention shows that the nano vaccine carrier can completely inhibit the generation of carrier protein antibodies.
Drawings
FIG. 1. scheme for synthesis of the nano-vaccine vectors of example 1 and example 2;
FIG. 2(a) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 2(b) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 2(c) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 2(d) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 2(e) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 2(f) is a diagram showing the physical and chemical characteristics of examples 1 and 2;
FIG. 3, SDS-PAGE gel electrophoresis;
FIG. 4(a) is a graph showing the variation of MKN particle size after different treatments;
FIG. 4(b) is a graph of the change in particle size of HKN after various treatments;
FIG. 5(a) is a graph of the change in particle size of MKPN in PBS;
FIG. 5(b) is a graph of the change in particle size of MKPN in RPMI1640 of 10% FBS;
FIG. 6 is a graph showing biological safety evaluation of MKPN;
FIG. 7 is a graph comparing whether the vaccine completely inhibited the production of carrier protein antibodies;
FIG. 8 is a graph comparing the ability of a vaccine to increase the antibody titer of a polypeptide of interest after the "vector suppression effect" is eliminated.
Detailed Description
A. Feedstock and apparatus
1) Raw materials: human Serum Albumin (HSA), Mouse Serum Albumin (MSA), which can be replaced with any albumin of human or animal origin, as required for use.
Proprotein convertase subtilisin/kexin type 9 short peptide (PCSK9), the epitope of which can be replaced by any epitope according to the needs of use;
dithiothreitol (DTT), which reducing agent can be replaced by any agent capable of effecting-SH reduction; sulfosuccinimidyl 4 (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-smcc), the crosslinker may be replaced by any protein crosslinker capable of acting on-SH;
sodium Dodecyl Sulfate (SDS), the anionic surfactant may be replaced with any long chain alkyl anionic surfactant, such as: sodium tetradecane sulfate, sodium hexadecane sulfonate, etc.;
30% hydrogen peroxide, which can be replaced by any reagent capable of oxidizing mercapto into-SH;
sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), which can be any acid or base agent capable of adjusting the pH of the buffer solution;
2- (N-morpholino) ethanesulfonic acid (MES), disodium hydrogen phosphate dodecahydrate (Na2HPO4 & 12H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4 & 2H2O), such buffer solutions may be composed of aqueous solutions of any weak acid and its salts, weak bases and its salts, acid salts of polybasic weak acids and their corresponding secondary salts. The method specifically comprises the following steps: phosphate, borate, citrate, carbonate, acetate, barbiturate, Tris (Tris) and the like.
2) Equipment: magnetic stirrer, magneton, 2.5ml/5ml/10ml glass reaction bottle, water purifier, electronic analytical balance, measuring cylinder, pipette, ultrafiltration tube (10kD), dialysis bag (MWCO:14000), glass reaction vessel, volumetric flask, 5L glass flask, and a plurality of beakers
3) Preparing buffer solution
a) pH 9.00.01M carbonate buffer: 0.6g of anhydrous sodium carbonate and 3.7g of sodium bicarbonate were weighed and dissolved in 100ml of deionized water (0.5M), and 1ml of the above solution was taken and added with water to 50ml (20-fold dilution) to obtain a carbonate buffer solution having a pH of 9.00.01M
b) pH 8.00.01M phosphate buffer: weighing Na2HPO4·12H2O7.16 g, dissolved in 100ml of deionized water, and NaH was weighed2PO4·2H2Dissolving O3.12 g in 100ml of deionized water, wherein 9.47ml of the former and 0.53ml of the latter are mixed to obtain 0.2M pH 8.0 phosphate buffer, and 2.5ml of the mixture is added with water to 50ml (diluted 20 times) to obtain 0.01M pH 8.0 phosphate buffer
c) pH 7.40.01M phosphate buffer: separately weighing Na2HPO4·12H2O 58.019g， NaH2PO4·2H2O5.928 g, mixed and placed in a 500mL beaker, and 500mL of secondary water was added and dissolved with stirring. The solution in the beaker is poured into a 1000mL volumetric flask, and secondary water is added to reach the constant volume of 1000 mL. 2.5mL of the stock solution was taken, and water was added to 50mL (20-fold dilution) to obtain a phosphate buffer solution having a pH of 7.40.01M
a)0.1M MES buffer: MES1.952g was weighed and dissolved in 100ml of deionized water
b) 6% SDS solution: 0.6g of SDS was weighed and dissolved in 10ml of deionized water
The above reagents are only directed to the following examples one and two, and the concentrations thereof can be adjusted according to the needs of the reaction in the actual reaction process.
B. As shown in figure 1, the preparation of the first and second nanometer vaccine carriers in the following examples uses albumin as raw material, and obtains the target vaccine carrier through reduction, thermal polymerization, oxidation, concentration and external connection of antigen epitope.
The mass ratio of the albumin to the carrier protein is more than 4: 1;
the mass ratio of the albumin to the reducing agent is 1: 0.1-1.
The mass ratio of the thermal polymerization product to the oxidant is 1: 0.1-1.
The mass ratio of the albumin to the cross-linking agent is 1: 0.5-15;
the mass ratio of the albumin to the epitope is 1: 0.5-15.
The first embodiment,
1)Reducing MSA (for reducing albumin disulfide bond to form sulfhydryl group for reassembling crosslinking)
Reduction of MSA/KLH: weighing KLH 1.5mg and MSA 10.5mg, dissolving in 2.4ml carbonic acid buffer solution with pH 9.0 to obtain solution A with total protein concentration of 5 mg/ml; weighing a plurality of mg of DTT, and dissolving the DTT in deionized water to obtain a solution B with the final concentration of 10 mg/ml; the solution 62ulB and the solution A were mixed in a 2.5ml reaction flask, and placed on a flat magnetic stirrer to react at room temperature for 1h at 300r to obtain solution C.
2)Thermal polymerization to form MAS-KLH crosslinked nano-carrier (MKN) (using SDS to control particle size, thermal polymerization stirring and other actions The protein is firstly formed into nano particles with good uniformity, and partial sulfydryl is reformed into new disulfide bond in the process
Thermal polymerization to form MKN: adding water into a reaction vessel, preheating to 73 ℃ before lifting a heating stirrer, adding 1ml of MES buffer solution into a 2.5ml reaction bottle, adding 30ul of SDS solution, adding 0.2ml of deionized water, finally adding 0.8ml of solution C, tightly covering the bottle cap of the reaction bottle, putting the reaction bottle into the water preheated to 73 ℃, adjusting the rotation speed to 750r, heating and stirring for 10min, taking out the reaction bottle, putting the reaction bottle into water (4 ℃) soaked with ice blocks, and cooling to obtain the clear milky MKN solution
3)Oxidizing MKN (to allow more thiol groups to form disulfidesBonds to make the whole particle cross-linked more tightly, and also avoid adding Mercapto-blocking agent)
Respectively diluting the MKN/HKN solution with deionized water to 1mg/ml, adding 2ul of 30% hydrogen peroxide per mg of protein, placing on a flat magnetic stirrer at 300r, and stirring at room temperature for 6 hr
4)MKN after dialysis and concentration oxidation
Adding the oxidized MKN into a dialysis bag (MWCO:14000), dialyzing in a 5L glass bottle for 24h, and changing water every 4h for 4 times; transferring the solution into 15ml ultrafiltration tube (10kD) after dialysis, ultrafiltering and concentrating with the above phosphate buffer solution (pH: 7.40.01M), ultrafiltering at 6500r for 15min for 3 times, and concentrating to obtain MKN solution 2mg/ml
5)MKPN is synthesized by MKN circumscribed PCSK9 short peptide (model antigen short peptide)
Solution preparation: several mg of sulfo-smcc are weighed, dissolved into 5mg/ml by deionized water, dissolved by ultrasonic acceleration, and dozens of mg of PCSK9 short peptide are weighed, and dissolved into 25mg/ml by the phosphate buffer solution with the pH value of 7.40.01M.
The synthesis process comprises the following steps: the MKN solution having a concentration of 2mg/ml obtained by the above-mentioned ultrafiltration concentration was put into a 5ml reaction glass bottle (volume: 2ml), 150. mu.l of the above-mentioned sulfo-smcc solution was added, and the mixture was placed on a flat magnetic stirrer and reacted at 400r at room temperature for 1 hour. Then, the unreacted sulfo-smcc was removed by ultrafiltration using the above-mentioned pH 7.4PBS buffer, 6500r, 15min, and ultrafiltration was performed 3 times, the volume was restored to 2ml using the above-mentioned pH 7.4PBS solution, 150ul of the above-mentioned PCSK9 solution was added, the mixture was placed on a plate magnetic stirrer and reacted at 400r at room temperature for 2h, and the unreacted PCSK9, 6500r, 15min was removed by ultrafiltration using the above-mentioned pH 7.4PBS buffer, and ultrafiltration was performed 3 times. The volume was restored to 2ml with PBS solution at pH 7.4 to obtain MKPN solution, which was stored at-20 ℃.
Example two
(1)Reducing MSA/HAS (to reduce albumin disulfide bonds to form sulfhydryl groups to facilitate reassembling crosslinks)
Reduction of HAS/KLH: weighing a plurality of mg of DTT, and dissolving the DTT in deionized water to obtain a solution B with the final concentration of 10 mg/ml; KLH 1mg and HSA 9mg were weighed out and dissolved in 2ml of phosphate buffer pH 8.0 to give a final total protein concentration of 5mg/ml as solution D; the solution 62ulB and the solution D were mixed in a 2.5ml reaction flask, and placed on a flat magnetic stirrer to react at room temperature for 1h at 300r to obtain a solution E.
(2)Forming HSA-KLH crosslinked nano-carrier (HKN) by thermal polymerization (adjusting particle size by SDS, thermal polymerization with stirring, etc The protein is firstly formed into nano particles with good uniformity, and part of sulfhydryl groups are reformed into new disulfide bonds in the process)
Thermal polymerization formation HKN: adding water into a reaction vessel, preheating to 70 ℃ before lifting a heating stirrer, adding 1ml of MES buffer solution into a 2.5ml reaction bottle, adding 30ul of SDS solution, adding 0.2ml of deionized water, finally adding 0.8ml of solution E, tightly covering the bottle cap of the reaction bottle, putting the reaction bottle into the water preheated to 70 ℃, adjusting the rotation speed to 750r, heating and stirring for 10min, taking out the reaction bottle, putting the reaction bottle into water (4 ℃) soaked with ice blocks, and cooling to obtain clear milky HKN solution
(3)Oxidation HKN (to allow more sulfhydryl groups to form disulfide bonds and to allow more compact cross-linking of the whole particle, and to avoid the need for additional reagents Mercapto-blocking agent)
Diluting the HKN solutions with deionized water to 1mg/ml, adding 2ul 30% hydrogen peroxide per mg protein, stirring at 300r for 6 hr at room temperature
(4)Dialyzing and concentrating the oxidized HKN
Adding the oxidized HKN into a dialysis bag (MWCO:14000), dialyzing in a 5L glass bottle for 24h, and changing water every 4h for 4 times; transferring the solution into 15ml ultrafiltration tube (10kD) after dialysis, ultrafiltering with phosphate buffer solution (pH: 7.40.01M) for 3 times at 6500r for 15min, and concentrating to obtain 2mg/ml HKN solution
(5)HKN Synthesis of HKPN by externally connecting PCSK9 short peptide (model antigen short peptide)
The synthesis process comprises the following steps: the HKN solution having a concentration of 2mg/ml obtained by the above ultrafiltration concentration was put into a 5ml reaction glass bottle (volume: 2ml), 150. mu.l of the above sulfo-smcc solution was added, and the mixture was placed on a flat magnetic stirrer and reacted at 400r at room temperature for 1 hour. Then, the unreacted sulfo-smcc was removed by ultrafiltration using the above-mentioned pH 7.4PBS buffer, 6500r, 15min, and ultrafiltration was performed 3 times, the volume was restored to 2ml using the above-mentioned pH 7.4PBS solution, 150ul of the above-mentioned PCSK9 solution was added, the mixture was placed on a plate magnetic stirrer and reacted at 400r at room temperature for 2h, and the unreacted PCSK9, 6500r, 15min was removed by ultrafiltration using the above-mentioned pH 7.4PBS buffer, and ultrafiltration was performed 3 times. The volume was restored to 2ml with PBS (pH 7.4) to obtain HKPN solution, which was stored at-20 ℃.
C. Physical and chemical characterization
As shown in FIG. 2(a), the DLS particle size of MKN was about 70nm, and that of MKPN was about 80 nm.
As shown in FIG. 2(b), the MKN potential is about-20 mV and the MKPN potential is about-24 mV.
As shown in fig. 2(c) and 2(d), the TEM images of MKN, MKPN show nanoparticles whose morphology is nearly spherical.
As shown in FIG. 2(e), the DLS particle size of HKN was about 70 nm.
FIG. 2(f) shows TEM images of nanoparticles whose morphology is nearly spherical.
D. Verification of success of MKN and PCSK9 short peptide linkage
As shown in FIG. 3, SDS-PAGE gel electrophoresis showed that the MKPN molecular weight was significantly larger.
E. Verification of the principal forces developed by MKN/HKN
As shown in fig. 4(a), the MKN particle size varied after different treatments.
As shown in fig. 4(b), the particle size of HKN changed after different treatments.
F. Evaluation of physiological stability of MKPN
As shown in FIG. 5, it was revealed that the MKPN showed no significant change in particle size in PBS in FIG. 5(a) and RPMI1640 containing 10% FBS in FIG. 5(b), and had good physiological stability.
G. Evaluation of biosafety of MKPN
As shown in FIG. 6, the MKPN and DC2.4 cells with different concentration gradients were co-cultured for 24h, the cell activities were all above 100%, and the MKPN biological safety was very good.
H. Evaluation of whether the vaccine completely inhibited the production of carrier protein antibody
As shown in fig. 7, the MKN and MKPN experimental groups had almost no anti-KLH antibody production, which is on par with the negative control group.
I. Evaluating whether the vaccine can improve the target polypeptide antibody titer after eliminating the carrier inhibition effect
As shown in fig. 8, the antibody titer of the MKPN test group was slightly higher than that of the conjugate vaccine at 40 days after the first immunization, and was significantly higher than that of the conjugate vaccine after 45 days.
Claims (9)
1. A nano vaccine carrier based on albumin-carrier protein disulfide bond crosslinking to form pure protein composite nanoparticles is characterized in that: taking albumin as a raw material, and obtaining a target vaccine carrier through reduction, thermal polymerization, oxidation, concentration and external connection of an epitope;
wherein the reduction is to reduce disulfide bonds in albumin to sulfhydryl groups;
the thermal polymerization is based on the albumin self-assembly mode to form nanoparticles;
the oxidation is oxidation of sulfhydryl groups to disulfide bonds;
the reduction process comprises the following steps:
s1, preparing a solution A: dissolving albumin and carrier protein in a buffer;
s2, preparing a solution B: dissolving a reducing agent in water;
s3, mixing the solution A and the solution B, and stirring at room temperature for reaction for 0.5-2 hours;
the carrier protein is KLH.
2. The nano-vaccine vector of claim 1, based on albumin-carrier protein disulfide cross-linking to form pure protein composite nanoparticles, wherein:
the mass ratio of the albumin to the carrier protein is more than 4: 1;
the mass ratio of the albumin to the reducing agent is 1: 0.1-1.
3. The nano-vaccine vector of claim 1, based on albumin-carrier protein disulfide cross-linking to form pure protein composite nanoparticles, wherein:
the thermal polymerization process comprises the following steps:
s1, reacting buffer solution, anionic surfactant, water and a product of reduction reaction for 5-20 minutes at a water bath temperature of 50-100 ℃;
s2, cooling in a water bath at the temperature of below 10 ℃.
4. The nano-vaccine vector of claim 1, based on albumin-carrier protein disulfide cross-linking to form pure protein composite nanoparticles, wherein:
the oxidation process comprises the following steps: reacting the thermal polymerization product with an oxidant at room temperature for 4-10 hours;
the oxidant is peroxide.
5. The nano-vaccine vector of claim 4, which is based on albumin-carrier protein disulfide bond cross-linking to form pure protein composite nanoparticles, wherein:
the mass ratio of the thermal polymerization product to the oxidant is 1: 0.1-1.
6. The nano-vaccine vector of claim 1, based on albumin-carrier protein disulfide cross-linking to form pure protein composite nanoparticles, wherein:
the concentration process comprises the following steps:
s1, dialyzing the oxidation product for 12-48 hours;
s2, carrying out ultrafiltration concentration on the product of S1 by using a buffer solution for 5-60 minutes.
7. The nano-vaccine vector of claim 1, based on albumin-carrier protein disulfide cross-linking to form pure protein composite nanoparticles, wherein:
the process of externally connecting the epitope comprises the following steps:
s1, reacting the concentrated product with a cross-linking agent for 0.5-3 hours at room temperature;
s2, carrying out ultrafiltration on the product of the S1 by using a buffer solution to remove unreacted cross-linking agents;
s3, diluting the product of S2 by using a buffer solution, adding an epitope, and reacting for 1-5 hours at room temperature;
s4, ultrafiltering the product of S3 with buffer solution to remove unreacted antigen epitope;
s5, diluting the product of S4 by using a buffer solution, and storing at the temperature below-20 ℃.
8. The nano-vaccine vector of claim 7, wherein the nano-vaccine vector is based on albumin-carrier protein disulfide bond cross-linking to form pure protein composite nanoparticles, and is characterized in that:
the mass ratio of the albumin to the cross-linking agent is 1: 0.5-15;
the mass ratio of the albumin to the epitope is 1: 0.5-15.
9. The nano-vaccine vector according to any one of claims 1 to 8, which is based on albumin-carrier protein disulfide bond cross-linking to form pure protein composite nanoparticles, wherein: used for overcoming the inhibitory effect of carrier epitope.
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| CN106729681A (en) * | 2016-12-21 | 2017-05-31 | 上海交通大学医学院附属新华医院 | A kind of nanosizing carrier protein platform for improving antigen immunogenicity |
| CN107157933A (en) * | 2017-05-04 | 2017-09-15 | 同济大学 | A kind of albumen self assembly novel nano vaccine and preparation method thereof |
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| CN106729681A (en) * | 2016-12-21 | 2017-05-31 | 上海交通大学医学院附属新华医院 | A kind of nanosizing carrier protein platform for improving antigen immunogenicity |
| CN107157933A (en) * | 2017-05-04 | 2017-09-15 | 同济大学 | A kind of albumen self assembly novel nano vaccine and preparation method thereof |
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| Title |
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| "Minimalist" Nanovaccine Constituted from Near Whole Antigen for Cancer Immunotherapy;Kun Wang et al.;《ACS Nano》;20180621;第12卷;第6398-6409页 * |
| Self-Templated, Green-Synthetic, Size-Controlled Protein Nanoassembly as a Robust Nanoplatform for Biomedical Application;Ya Wen et al.;《ACS Appl. Mater. Interfaces 》;20180306;第10卷;第11457-11466页 * |
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