Preparation method of targeted antigen display MS2 phage VLPs nanoparticle vaccine

Abstract

The invention belongs to the technical field of biological vaccine preparation, and particularly relates to a preparation method of a targeted antigen displayed MS2 phage VLPs nanoparticle vaccine. The preparation method sequentially comprises the following steps: preparation and activation of a targeting ligand, fixed-point transformation and expression of MS2 bacteriophage capsid protein, preparation of targeting antigen-aptamer fusion protein, folding optimization and activity pre-verification of the antigen-aptamer fusion protein, self-assembly and antigen display of targeting MS2-VLPs, purification and characterization of targeting VLPs nanoparticles, and preparation of the vaccine preparation. On the basis, by introducing the targeting ligand aiming at the specific receptor on the surface of the antigen presenting cell, accurate recognition and combination of the VLPs vaccine on the target cell are realized, the problem that the antigen display lacks targeting is fundamentally solved, the antigen delivery efficiency is remarkably improved, the off-target effect is effectively reduced, and the antigen waste is avoided.

Description

Technical Field

[0001] This invention relates to the field of biological vaccine preparation technology, specifically to a method for preparing MS2 phage VLPs nanoparticle vaccines that target antigen display. Background Technology

[0002] Virus-like particles (VLPs) are nanoscale particles formed by the self-assembly of viral structural proteins. They do not contain viral genetic material, possess a spatial conformation similar to that of natural viruses, and can efficiently stimulate the body to produce an immune response without the risk of infection, making them an ideal vaccine vector platform. MS2 bacteriophage, as a single-stranded RNA bacteriophage, has capsid proteins (CP) that can spontaneously assemble into icosahedral VLPs. These VLPs have advantages such as structural stability, strong genetic manipulability, and ease of large-scale preparation. Furthermore, their N-terminal AB loop can tolerate the insertion of exogenous peptides without disrupting particle assembly, and they are widely used in vaccine vector development.

[0003] Current vaccine preparation technologies based on MS2 phage VLPs mainly involve displaying antigens on the surface of VLPs through gene fusion or chemical conjugation. However, several key issues exist: First, antigen display lacks targeting, failing to precisely deliver antigens to specific receptors on the surface of antigen-presenting cells (such as dendritic cells), resulting in low antigen utilization and insufficient immune response strength. Second, traditional fusion or conjugation methods easily disrupt the native conformation of antigens, reducing antigen immunogenicity or causing decreased VLP assembly stability. Third, existing technologies exhibit low antigen-VLP binding efficiency and are prone to generating non-specific binding products, increasing the difficulty of subsequent purification and production costs. Fourth, some chemical conjugation methods require the use of toxic catalysts (such as copper ions), posing biosafety risks and limiting the clinical application prospects of vaccines. Summary of the Invention

[0004] To address the technical problems mentioned above, such as the lack of targeting in antigen display, the tendency of traditional fusion or conjugation methods to disrupt the native conformation of antigens, the low efficiency of antigen-VLP conjugation in existing technologies, and the requirement of toxic catalysts in some chemical conjugation methods, this invention provides the following technical solutions: A method for preparing an antigen-targeting MS2 phage VLP nanoparticle vaccine includes the following specific steps: S1, Preparation and activation of the targeted ligand: S11 targets specific receptors on the surface of antigen-presenting cells (such as the CD11c receptor), screens and synthesizes corresponding targeting ligands (such as the anti-CD11c monoclonal antibody fragment scFv), and introduces an azide group through N-terminal amino modification. ); S12, unreacted modifying reagents are removed by dialysis to obtain activated targeting ligands; the dialysis is performed using phosphate-buffered saline (PBS, pH 7.4) for 8-12 hours to ensure the purity of the activated ligands is ≥95%; Site-specific modification and expression of S2 and MS2 phage capsid proteins: S21, based on the amino acid sequence of MS2 phage capsid protein (CP), introduces a short peptide tag containing an alkyne group (-C≡CH) (such as an alkyne-modified glycine tag) at its C-terminus to construct a modified MS2 capsid protein recombinant expression vector (such as pET-28a-CP-alkynyl tag). S22, the recombinant vector was transformed into E. coli BL21(DE3) competent cells, and after IPTG induction, the modified MS2 capsid protein was purified by nickel ion affinity chromatography. The induction conditions were 37℃, IPTG concentration of 0.5mM, induction time of 4-6h, and the purity of the purified capsid protein was ≥90%. S3, Preparation of target antigen-aptamer fusion protein: S31. Based on the amino acid sequence of the target antigen (such as tumor antigen Her2, viral antigen S protein RBD domain), design and synthesize the corresponding antigen-encoding gene, and simultaneously fuse an MS2 capsid protein AB ring-specific binding aptamer (such as an MS2 aptamer RNA-binding domain mimic peptide) to the N-terminus of the antigen; the binding affinity constant (Kd) of the aptamer to the MS2 capsid protein AB ring is ≤ This ensures efficient integration in the future; S32, the fusion gene was inserted into a prokaryotic expression vector (such as pGEX-6P-1), transformed into Escherichia coli Rosetta (DE3) competent cells, and expressed after low temperature induction (16℃). The protein was then purified by glutathione agarose gel chromatography to obtain the target antigen-aptamer fusion protein. S4, folding optimization and activity pre-validation of antigen-aptamer fusion proteins: S41, the antigen-aptamer fusion protein obtained in S3 was placed in folding buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8.0), and GroEL / GroES molecular chaperone (molar ratio with fusion protein 1:50) was added. The mixture was incubated at 30-35℃ for 4-5 h to assist folding. S42, after folding, the molecular chaperone was removed by ultrafiltration (ultrafiltration membrane with a molecular weight cutoff of 30kDa) to obtain the folded optimized fusion protein; S43, the secondary structure of the fusion protein (α-helix content ≥35%, β-sheet content ≥25%) was detected by circular dichroism (CD) to verify the integrity of its native conformation; S44. The binding activity of the optimized fusion protein to the AB ring of the MS2 capsid protein was detected by ELISA, and the binding activity Kd value ≤5× was screened. The fusion protein is used for subsequent VLP assembly; S5, Self-assembly and antigen display targeting MS2-VLPs: S51, the modified MS2 capsid protein obtained in S2 and the folded optimized antigen-aptamer fusion protein obtained in S4 are mixed at a molar ratio of 180:(30-60) and placed in assembly buffer (containing... In PBS (pH 7.2), incubate at 37-39°C for 2-3 hours to allow the antigen to specifically bind to the capsid protein AB ring via aptamers, thus initially forming antigen-loaded MS2-VLPs. S52, followed by the addition of the activated targeting ligand obtained in S1, and incubation at 25-27℃ for 4-5 h based on the copper-free click chemistry reaction of azide-alkynyl (DBCO mediated), to covalently link the targeting ligand to the alkynyl tag at the C-terminus of the capsid protein, thus completing the self-assembly of the targeting MS2-VLPs; the molar ratio of the targeting ligand to the modified capsid protein is (10-20):180, ensuring that 5-10 targeting ligands are attached to the surface of each VLP; S6, Purification and characterization of VLP-targeted nanoparticles: S61, the assembly product obtained in S5 was purified by gel filtration chromatography (Sepharose 4B chromatography column) and density gradient centrifugation (sucrose density gradient of 10%-40%) to collect the targeted VLPs components with high purity. S62, firstly, the morphology and assembly integrity of VLPs were observed using transmission electron microscopy, then their particle size (target range 20-30 nm) was determined by dynamic light scattering, then Western blotting was used to verify the display effect of antigen and target ligand, and finally ELISA was used to determine the antigen loading and target binding activity. S7, Preparation of vaccine formulation: S71, the targeted VLP nanoparticles purified from S6 are mixed with an adjuvant (such as CpGODN1826) at a mass ratio of 100:(5-10); S72, add an osmotic pressure regulator (such as sodium chloride) and a stabilizer (such as sucrose), and stir evenly at 4-6℃ to obtain an MS2 phage VLP nanoparticle vaccine with targeted antigen display; the pH of the vaccine is adjusted to 6.8-7.8, and the osmotic pressure is 280-320 mOsm / kg.

[0005] Compared with existing technologies: 1. By introducing targeted ligands that target specific receptors on the surface of antigen-presenting cells, VLPs vaccines can accurately recognize and bind to target cells, fundamentally solving the problem of lack of targeting in antigen display, significantly improving antigen delivery efficiency, effectively reducing off-target effects, and avoiding antigen waste; 2. By adopting antigen-aptamer specific binding design combined with molecular chaperone-assisted folding strategy, instead of traditional fusion or conjugation methods, it can maximize the preservation of the antigen's natural conformation, avoid damage to immunogenicity, and ensure the assembly stability of VLPs. 3. By leveraging the high affinity binding of the aptamer to the AB ring of the MS2 capsid protein and combining it with a copper-free click chemically modified targeting ligand, a dual high-efficiency linkage mechanism is formed, which solves the problem of low antigen-VLP linkage efficiency, reduces non-specific binding products, and lowers the difficulty of subsequent purification. 4. Targeted modification is achieved through a copper-free click chemistry reaction mediated by DBCO, which avoids the use of toxic catalysts such as copper ions throughout the process, thus avoiding potential biosafety risks in the preparation stage. Combined with a prokaryotic expression system, it balances safety and the needs of large-scale production. Detailed Implementation

[0006] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. Example

[0007] This invention provides a method for preparing MS2 phage VLPs nanoparticle vaccines with targeted antigen display, comprising the following specific steps: S1, Preparation and activation of the targeted ligand: S11 targets specific receptors on the surface of antigen-presenting cells (such as the CD11c receptor), screens and synthesizes corresponding targeting ligands (such as the anti-CD11c monoclonal antibody fragment scFv), and introduces an azide group through N-terminal amino modification. ); S12, unreacted modifying reagents were removed by dialysis to obtain activated targeting ligands; the dialysis was performed using phosphate-buffered saline (PBS, pH 7.4) for 8 hours to ensure the purity of the activated ligands was ≥95%; Site-specific modification and expression of S2 and MS2 phage capsid proteins: S21, based on the amino acid sequence of MS2 phage capsid protein (CP), introduces a short peptide tag containing an alkyne group (-C≡CH) (such as an alkyne-modified glycine tag) at its C-terminus to construct a modified MS2 capsid protein recombinant expression vector (such as pET-28a-CP-alkynyl tag). S22, the recombinant vector was transformed into E. coli BL21(DE3) competent cells, and after IPTG induction, the modified MS2 capsid protein was purified by nickel ion affinity chromatography. The induction conditions were 37℃, IPTG concentration of 0.5mM, induction time of 4h, and the purity of the purified capsid protein was ≥90%. S3, Preparation of target antigen-aptamer fusion protein: S31. Based on the amino acid sequence of the target antigen (such as tumor antigen Her2, viral antigen S protein RBD domain), design and synthesize the corresponding antigen-encoding gene, and simultaneously fuse an MS2 capsid protein AB ring-specific binding aptamer (such as an MS2 aptamer RNA-binding domain mimic peptide) to the N-terminus of the antigen; the binding affinity constant (Kd) of the aptamer to the MS2 capsid protein AB ring is ≤ This ensures efficient integration in the future; S32, the fusion gene was inserted into a prokaryotic expression vector (such as pGEX-6P-1), transformed into Escherichia coli Rosetta (DE3) competent cells, and expressed after low temperature induction (16℃). The protein was then purified by glutathione agarose gel chromatography to obtain the target antigen-aptamer fusion protein. S4, folding optimization and activity pre-validation of antigen-aptamer fusion proteins: S41, the antigen-aptamer fusion protein obtained in S3 was placed in folding buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8.0), and GroEL / GroES molecular chaperone (molar ratio with fusion protein 1:50) was added. The mixture was incubated at 30°C for 4 h to assist folding. S42, after folding, the molecular chaperone was removed by ultrafiltration (ultrafiltration membrane with a molecular weight cutoff of 30kDa) to obtain the folded optimized fusion protein; S43, the secondary structure of the fusion protein (α-helix content ≥35%, β-sheet content ≥25%) was detected by circular dichroism (CD) to verify the integrity of its native conformation; S44. The binding activity of the optimized fusion protein to the AB ring of the MS2 capsid protein was detected by ELISA, and the binding activity Kd value ≤5× was screened. The fusion protein is used for subsequent VLP assembly; S5, Self-assembly and antigen display targeting MS2-VLPs: S51, the modified MS2 capsid protein obtained in S2 and the folded optimized antigen-aptamer fusion protein obtained in S4 are mixed at a molar ratio of 180:30 and placed in assembly buffer (containing... In PBS (pH 7.2), the antigen was incubated at 37°C for 2 hours to allow the antigen to specifically bind to the capsid protein AB ring via aptamers, thus initially forming antigen-loaded MS2-VLPs. S52, followed by the addition of the activated targeting ligand obtained in S1, and incubation at 25°C for 4 h based on the copper-free click chemistry reaction of azide-alkynyl (DBCO mediated), allows the targeting ligand to be covalently linked to the alkynyl tag at the C-terminus of the capsid protein, completing the self-assembly of the targeting MS2-VLPs; the molar ratio of the targeting ligand to the modified capsid protein is 10:180, ensuring that 5 targeting ligands are attached to the surface of each VLP; S6, Purification and characterization of VLP-targeted nanoparticles: S61, the assembly product obtained in S5 was purified by gel filtration chromatography (Sepharose 4B chromatography column) and density gradient centrifugation (sucrose density gradient of 10%) to collect the targeted VLPs components with high purity. S62, firstly, the morphology and assembly integrity of VLPs were observed using transmission electron microscopy, then their particle size (target range 20 nm) was determined by dynamic light scattering, then Western blotting was used to verify the display effect of antigen and target ligand, and finally ELISA was used to determine the antigen loading and target binding activity. S7, Preparation of vaccine formulation: S71, the targeted VLP nanoparticles purified from S6 are mixed with an adjuvant (such as CpGODN1826) at a mass ratio of 100:5; S72, add an osmotic pressure regulator (such as sodium chloride) and a stabilizer (such as sucrose), and stir evenly at 4°C to obtain an MS2 phage VLP nanoparticle vaccine with targeted antigen display; the pH of the vaccine is adjusted to 6.8 and the osmotic pressure is 280 mOsm / kg. Example

[0008] This invention provides a method for preparing MS2 phage VLPs nanoparticle vaccines with targeted antigen display, comprising the following specific steps: S1, Preparation and activation of the targeted ligand: S11 targets specific receptors on the surface of antigen-presenting cells (such as the CD11c receptor), screens and synthesizes corresponding targeting ligands (such as the anti-CD11c monoclonal antibody fragment scFv), and introduces an azide group through N-terminal amino modification. ); S12, unreacted modifying reagents were removed by dialysis to obtain activated targeting ligands; the dialysis was performed using phosphate-buffered saline (PBS, pH 7.4) for 10 hours to ensure the purity of the activated ligands was ≥95%; Site-specific modification and expression of S2 and MS2 phage capsid proteins: S21, based on the amino acid sequence of MS2 phage capsid protein (CP), introduces a short peptide tag containing an alkyne group (-C≡CH) (such as an alkyne-modified glycine tag) at its C-terminus to construct a modified MS2 capsid protein recombinant expression vector (such as pET-28a-CP-alkynyl tag). S22, the recombinant vector was transformed into E. coli BL21(DE3) competent cells, and after IPTG induction, the modified MS2 capsid protein was purified by nickel ion affinity chromatography. The induction conditions were 37℃, IPTG concentration of 0.5mM, induction time of 5h, and the purity of the purified capsid protein was ≥90%. S3, Preparation of target antigen-aptamer fusion protein: S31. Based on the amino acid sequence of the target antigen (such as tumor antigen Her2, viral antigen S protein RBD domain), design and synthesize the corresponding antigen-encoding gene, and simultaneously fuse an MS2 capsid protein AB ring-specific binding aptamer (such as an MS2 aptamer RNA-binding domain mimic peptide) to the N-terminus of the antigen; the binding affinity constant (Kd) of the aptamer to the MS2 capsid protein AB ring is ≤ This ensures efficient integration in the future; S32, the fusion gene was inserted into a prokaryotic expression vector (such as pGEX-6P-1), transformed into Escherichia coli Rosetta (DE3) competent cells, and expressed after low temperature induction (16℃). The protein was then purified by glutathione agarose gel chromatography to obtain the target antigen-aptamer fusion protein. S4, folding optimization and activity pre-validation of antigen-aptamer fusion proteins: S41, the antigen-aptamer fusion protein obtained in S3 was placed in folding buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8.0), and GroEL / GroES molecular chaperone (molar ratio with fusion protein 1:50) was added. The mixture was incubated at 32.5℃ for 4.5 h to assist folding. S42, after folding, the molecular chaperone was removed by ultrafiltration (ultrafiltration membrane with a molecular weight cutoff of 30kDa) to obtain the folded optimized fusion protein; S43, the secondary structure of the fusion protein (α-helix content ≥35%, β-sheet content ≥25%) was detected by circular dichroism (CD) to verify the integrity of its native conformation; S44. The binding activity of the optimized fusion protein to the AB ring of the MS2 capsid protein was detected by ELISA, and the binding activity Kd value ≤5× was screened. The fusion protein is used for subsequent VLP assembly; S5, Self-assembly and antigen display targeting MS2-VLPs: S51, the modified MS2 capsid protein obtained in S2 and the folded optimized antigen-aptamer fusion protein obtained in S4 are mixed at a molar ratio of 180:45 and placed in assembly buffer (containing... The antigen was incubated in PBS (pH 7.2) at 38°C for 2.5 h to allow the antigen to specifically bind to the capsid protein AB ring via aptamers, thus initially forming antigen-loaded MS2-VLPs. S52, followed by the addition of the activated targeting ligand obtained in S1, and incubation at 26°C for 4.5 h based on the copper-free click chemistry reaction of azide-alkynyl (DBCO mediated), to covalently link the targeting ligand to the alkynyl tag at the C-terminus of the capsid protein, completing the self-assembly of the targeting MS2-VLPs; the molar ratio of the targeting ligand to the modified capsid protein is 15:180, ensuring that 7 targeting ligands are attached to the surface of each VLP; S6, Purification and characterization of VLP-targeted nanoparticles: S61, the assembly product obtained in S5 was purified sequentially by gel filtration chromatography (Sepharose 4B chromatography column) and density gradient centrifugation (sucrose density gradient of 25%) to collect the targeted VLPs components with high purity. S62, firstly, the morphology and assembly integrity of VLPs were observed using transmission electron microscopy, then their particle size (target range 25 nm) was determined by dynamic light scattering, then Western blotting was used to verify the display effect of antigen and target ligand, and finally ELISA was used to determine the antigen loading and target binding activity. S7, Preparation of vaccine formulation: S71, the targeted VLP nanoparticles purified from S6 are mixed with an adjuvant (such as CpGODN1826) at a mass ratio of 100:7.5; S72, add an osmotic pressure regulator (such as sodium chloride) and a stabilizer (such as sucrose), and stir evenly at 5°C to obtain an MS2 phage VLP nanoparticle vaccine with targeted antigen display; the pH of the vaccine is adjusted to 7.3 and the osmotic pressure is 300 mOsm / kg. Example

[0009] This invention provides a method for preparing MS2 phage VLPs nanoparticle vaccines with targeted antigen display, comprising the following specific steps: S1, Preparation and activation of the targeted ligand: S11 targets specific receptors on the surface of antigen-presenting cells (such as the CD11c receptor), screens and synthesizes corresponding targeting ligands (such as the anti-CD11c monoclonal antibody fragment scFv), and introduces an azide group through N-terminal amino modification. ); S12, unreacted modifying reagents were removed by dialysis to obtain activated targeting ligands; the dialysis was performed using phosphate-buffered saline (PBS, pH 7.4) for 12 hours to ensure the purity of the activated ligands was ≥95%; Site-specific modification and expression of S2 and MS2 phage capsid proteins: S21, based on the amino acid sequence of MS2 phage capsid protein (CP), introduces a short peptide tag containing an alkyne group (-C≡CH) (such as an alkyne-modified glycine tag) at its C-terminus to construct a modified MS2 capsid protein recombinant expression vector (such as pET-28a-CP-alkynyl tag). S22, the recombinant vector was transformed into E. coli BL21(DE3) competent cells, and after IPTG induction, the modified MS2 capsid protein was purified by nickel ion affinity chromatography. The induction conditions were 37℃, IPTG concentration of 0.5mM, induction time of 6h, and the purity of the purified capsid protein was ≥90%. S3, Preparation of target antigen-aptamer fusion protein: S31. Based on the amino acid sequence of the target antigen (such as tumor antigen Her2, viral antigen S protein RBD domain), design and synthesize the corresponding antigen-encoding gene, and simultaneously fuse an MS2 capsid protein AB ring-specific binding aptamer (such as an MS2 aptamer RNA-binding domain mimic peptide) to the N-terminus of the antigen; the binding affinity constant (Kd) of the aptamer to the MS2 capsid protein AB ring is ≤ This ensures efficient integration in the future; S32, the fusion gene was inserted into a prokaryotic expression vector (such as pGEX-6P-1), transformed into Escherichia coli Rosetta (DE3) competent cells, and expressed after low temperature induction (16℃). The protein was then purified by glutathione agarose gel chromatography to obtain the target antigen-aptamer fusion protein. S4, folding optimization and activity pre-validation of antigen-aptamer fusion proteins: S41, the antigen-aptamer fusion protein obtained in S3 was placed in folding buffer (containing 50 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8.0), and GroEL / GroES molecular chaperone (molar ratio with fusion protein 1:50) was added. The mixture was incubated at 35°C for 5 h to assist folding. S42, after folding, the molecular chaperone was removed by ultrafiltration (ultrafiltration membrane with a molecular weight cutoff of 30kDa) to obtain the folded optimized fusion protein; S43, the secondary structure of the fusion protein (α-helix content ≥35%, β-sheet content ≥25%) was detected by circular dichroism (CD) to verify the integrity of its native conformation; S44. The binding activity of the optimized fusion protein to the AB ring of the MS2 capsid protein was detected by ELISA, and the binding activity Kd value ≤5× was screened. The fusion protein is used for subsequent VLP assembly; S5, Self-assembly and antigen display targeting MS2-VLPs: S51, the modified MS2 capsid protein obtained in S2 and the folded optimized antigen-aptamer fusion protein obtained in S4 are mixed at a molar ratio of 180:60 and placed in assembly buffer (containing... In PBS (pH 7.2), the antigen was incubated at 39°C for 3 hours to allow the antigen to specifically bind to the capsid protein AB ring via aptamers, thus initially forming antigen-loaded MS2-VLPs. S52, followed by the addition of the activated targeting ligand obtained in S1, and incubation at 27°C for 5 h based on the copper-free click chemistry reaction of azide-alkynyl (DBCO mediated), allows the targeting ligand to be covalently linked to the alkynyl tag at the C-terminus of the capsid protein, completing the self-assembly of the targeting MS2-VLPs; the molar ratio of the targeting ligand to the modified capsid protein is 20:180, ensuring that 10 targeting ligands are attached to the surface of each VLP; S6, Purification and characterization of VLP-targeted nanoparticles: S61, the assembly product obtained in S5 was purified sequentially by gel filtration chromatography (Sepharose 4B chromatography column) and density gradient centrifugation (sucrose density gradient of 40%) to collect the targeted VLPs components with high purity. S62, firstly, the morphology and assembly integrity of VLPs were observed using transmission electron microscopy, then their particle size (target range 30 nm) was determined by dynamic light scattering, then Western blotting was used to verify the display effect of antigen and target ligand, and finally ELISA was used to determine the antigen loading and target binding activity. S7, Preparation of vaccine formulation: S71, the targeted VLP nanoparticles purified from S6 are mixed with an adjuvant (such as CpGODN1826) at a mass ratio of 100:10; S72, add an osmotic pressure regulator (such as sodium chloride) and a stabilizer (such as sucrose), and stir evenly at 6°C to obtain an MS2 phage VLP nanoparticle vaccine with targeted antigen display; the pH of the vaccine is adjusted to 7.8 and the osmotic pressure is 320 mOsm / kg.

[0010] The following data were obtained by comparing the MS2 phage VLPs nanoparticle vaccine with targeted antigen display prepared in Examples 1-3 above:

[0011] As shown in the table above, the MS2 phage VLP nanoparticle vaccines with targeted antigen display prepared in Examples 1-3 all showed good performance in terms of target binding rate, antigen loading, and VLP assembly integrity. After use, Example 2 showed the best results.

[0012] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for preparing an antigen-targeted MS2 phage VLP nanoparticle vaccine, characterized in that, The specific steps are as follows: S1, Preparation and activation of the targeted ligand: S11 targets specific receptors on the surface of antigen-presenting cells, screens and synthesizes corresponding targeting ligands, and introduces azide groups through N-terminal amino modification. S12, unreacted modifying agents are removed by dialysis to obtain activated targeting ligands; Site-specific modification and expression of S2 and MS2 phage capsid proteins: S21, based on the amino acid sequence of MS2 phage capsid protein, introduces a short peptide tag containing an alkyne group at its C-terminus to construct a modified MS2 capsid protein recombinant expression vector; S22, the recombinant vector was transformed into E. coli BL21 competent cells, and after expression was induced by IPTG, the modified MS2 capsid protein was obtained by purification using nickel ion affinity chromatography. S3, Preparation of target antigen-aptamer fusion protein: S31. Based on the amino acid sequence of the target antigen, the corresponding antigen-encoding gene is designed and synthesized, and the MS2 capsid protein AB ring-specific binding aptamer is fused to the N-terminus of the antigen. S32, the fusion gene was inserted into a prokaryotic expression vector, transformed into E. coli Rosetta competent cells, and after low-temperature induction of expression, the target antigen-aptamer fusion protein was obtained by glutathione agarose gel chromatography purification. S4, folding optimization and activity pre-validation of antigen-aptamer fusion proteins: S41, Place the antigen-aptamer fusion protein obtained in S3 in folding buffer, add GroEL / GroES molecular chaperone, and incubate at 30-35℃ for 4-5h to assist folding; S42, after folding, was removed by ultrafiltration to obtain the folded optimized fusion protein; S43, the secondary structure of the fusion protein was detected by circular dichroism spectroscopy to verify the integrity of its native conformation; S44. The binding activity of the optimized fusion protein to the AB ring of the MS2 capsid protein was detected by ELISA, and the binding activity Kd value ≤5× was screened. Fusion protein; S5, Self-assembly and antigen display targeting MS2-VLPs: S51, the modified MS2 capsid protein obtained in S2 and the folded optimized antigen-aptamer fusion protein obtained in S4 are mixed at a molar ratio of 180:(30-60) and placed in assembly buffer. The mixture is incubated at 37-39℃ for 2-3 hours to allow the antigen to specifically bind to the AB ring of the capsid protein through the aptamer, thus initially forming antigen-loaded MS2-VLPs. S52, followed by the addition of the activated targeting ligand obtained in S1, and based on the copper-free click chemistry of azido-alkynyl, incubated at 25-27℃ for 4-5h, so that the targeting ligand is covalently linked to the alkynyl tag at the C-terminus of the capsid protein, thus completing the self-assembly of targeting MS2-VLPs. S6, Purification and characterization of VLP-targeted nanoparticles: S61, the assembly product obtained in S5 was purified sequentially by gel filtration chromatography and density gradient centrifugation to collect the targeted VLPs components with high purity; S62, firstly, the morphology and assembly integrity of VLPs were observed using transmission electron microscopy, then their particle size was determined by dynamic light scattering, followed by Western blotting to verify the display effect of antigen and target ligand, and finally, ELISA was used to determine the antigen loading and target binding activity. S7, Preparation of vaccine formulation: S71, the targeted VLP nanoparticles purified from S6 are mixed with adjuvants at a mass ratio of 100:(5-10); S72, add osmotic pressure regulator and stabilizer, stir evenly at 4-6℃ to obtain MS2 phage VLP nanoparticle vaccine with targeted antigen display.

2. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In step S12, phosphate buffer is used during dialysis, and the dialysis time is 8-12 hours.

3. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In step S22, the induction conditions are 37°C, IPTG concentration is 0.5mM, and induction time is 4-6h.

4. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In S31, the binding affinity constant between the aptamer and the MS2 capsid protein AB ring is ≤ .

5. The method for preparing an MS2 phage VLPs nanoparticle vaccine with targeted antigen display according to claim 1, characterized in that, In S32, the induction temperature is 16°C.

6. The method for preparing an MS2 phage VLPs nanoparticle vaccine with targeted antigen display according to claim 1, characterized in that, In S41, the molar ratio of GroEL / GroES molecular chaperone to antigen-aptamer fusion protein is 1:

50.

7. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In step S51, the assembly buffer solution contains... The phosphate buffer solution has a pH of 7.

2.

8. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In S52, the molar ratio of the targeting ligand to the modified MS2 capsid protein is (10-20):

180.

9. The method for preparing a targeted antigen-displayed MS2 phage VLPs nanoparticle vaccine according to claim 1, characterized in that, In S62, the target range for measuring particle size during dynamic light scattering is 20-30 nm.

10. The method for preparing an MS2 phage VLPs nanoparticle vaccine with targeted antigen display according to claim 1, characterized in that, In S72, the pH of the vaccine is adjusted to 6.8-7.8, and the osmotic pressure is 280-320 mOsm / kg.

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