Mirror-coupled protein nanoparticle conjugates and uses thereof

Abstract

The invention relates to the technical field of biology, in particular to a mirror image coupled protein nanoparticle conjugate and application thereof. The conjugate comprises: ii) a functional protein labeled by a first peptide tag, and ii) a scaffold protein labeled by a second peptide tag, the first peptide tag and the second peptide tag being composed of D-amino acid residues; the first peptide tag and the second peptide tag form a peptide partner pair, and the first peptide tag and the second peptide tag are coupled and combined through an isopeptide bond. The conjugate provided by the invention is easier to escape from the recognition of protease, can obviously prolong the half-life period in a biological system, is not easy to cause unnecessary immunoreaction, and has good biological stability and safety.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a mirror-coupled protein nanoparticle conjugate and application thereof. Background Art

[0002] Protein-based virus-like particle (VLP) vaccines offer significant advantages over traditional live attenuated and inactivated vaccines. They are typically composed of a central particle scaffold, a coupling component, and various antigen-specific elements, allowing them to mimic the structure of real viruses without being infectious, making them safer for immunocompromised individuals. Artificially assembled vaccines with multiple copies of an antigen can elicit a stronger, more durable, and potentially broader immune response than a single antigen. This effective immunization strategy reduces the need for frequent booster shots and provides stronger protection against the virus.

[0003] However, concerns about the stability and integrity of VLP assembly have always existed, and due to the inherent complexity of antigenic proteins, modular vaccine assembly seems to be more reliable than gene fusion methods in vaccine assembly. It is well known that antigenic proteins usually need to be expressed in eukaryotic systems to maintain their activity. Therefore, genetic fusion between antigenic proteins and the basic units of VLP nanoparticles will conflict with the requirements of protein folding and post-translational modification, which will aggravate the problem of symmetry mismatch and make the assembly of protein nanoparticles with eukaryotic antigenic proteins quite complicated. This requires the development of effective coupling strategies between VLP and antigenic proteins to achieve optimal vaccine assembly while shortening manufacturing time. In the past few decades, click chemistry has shown great prospects in VLP antigenic protein assembly, and some non-covalent and covalent methods have been continuously developed. Covalent modification provides greater stability to VLP vaccine particles, making them less susceptible to environmental changes. Among these methods, Spy Chemistry stands out for its efficient and stable process, which can form genetically encodable and spontaneous isopeptide bonds even under harsh conditions. At present, SpyTag / SpyCatcher and its derivatives have been widely used in VLP vaccine development.

[0004] There are many research works that have successfully used Spy Chemistry as a method to anchor antigens to multimeric protein nanoparticles (such as Dodecin, ferritin, AP205 and Mi3). The general method is to fuse SpyCatcher with the core assembly unit of VLP nanoparticles and to fuse SpyTag with antigen proteins purified from heterologous host systems. When these two labeled protein components are mixed together, they react spontaneously to form VLP vaccine particles with multiple antigen molecules aggregated on the surface. The reaction between SpyTag and SpyCatcher is a very efficient and robust covalent reaction.

[0005] However, due to the large molecular weight of SpyCatcher and SpyTag, the molecular scar left by Spy Chemistry may be severe, and the molecular weight of the formed protein complex is about 14kDa. Due to steric effects, this may hinder the further assembly and antigen anchoring of VLP. In addition, the reaction between SpyTag and SpyCatcher occurs immediately after contact, which makes the reaction process less controllable. From a design perspective, the coupling fragments involved in the assembly of vaccine VLP particles may inadvertently trigger autoimmunity, which is undesirable. In addition, since SpyCatcher is derived from the membrane protein of the pathogenic bacterium Streptococcus pyogenes, there is a risk of inducing unnecessary immune responses, which may limit its future clinical application.

[0006] The protein ligase SpyStapler can mediate the assembly of VLP vaccines. SpyStapler is separated from SpyCatcher, and the remaining fragment is named BDTag. Isopeptide bonds can still be formed between BDTag and SpyTag under the catalysis of SpyStapler. Due to the non-covalent nature of the interaction between SpyStapler and the other two peptide fragments, SpyStapler acts like a ligase and can be removed once the reaction is completed.

[0007] However, the connection efficiency of BDTag and SpyTag needs to be further improved. The VLP vaccine assembled in this way is not stable, and there is a risk that the vaccine connection components will easily produce unnecessary immune responses. Summary of the invention

[0008] In order to solve at least one of the above problems, the present disclosure provides a conjugate and application, wherein the conjugate is more likely to escape recognition by proteases, can significantly prolong the half-life in biological systems, is less likely to induce unnecessary immune responses, and has good biological stability and safety.

[0009] According to one aspect of the present disclosure, a conjugate is provided, wherein the conjugate comprises: i) a functional protein labeled with a first peptide tag, and ii) a scaffold protein labeled with a second peptide tag, wherein the first peptide tag and the second peptide tag are composed of D-amino acid residues; the first peptide tag and the second peptide tag form a peptide partner pair, and the first peptide tag and the second peptide tag are coupled to each other via an isopeptide bond.

[0010] In some embodiments, the first peptide tag and the functional protein and / or the scaffold protein and the second peptide tag contain a peptide such as LPXT (G) w The amino acid sequence is shown.

[0011] In some embodiments, the functional protein labeled with the first peptide tag includes functional protein, LPXT (G) from N-terminus to C-terminus. w and a first peptide tag, or comprising a first peptide tag, LPXT(G) w and a functional protein, optionally with a linker sequence between the functional protein and LPXT(G)w.

[0012] In some embodiments, the scaffold protein labeled with the second peptide tag includes scaffold protein, LPXT (G) from N-terminus to C-terminus. w and a second peptide tag, or including a second peptide tag, LPXT(G) w and a scaffold protein, optionally, between the scaffold protein and LPXT (G) w There is a linker sequence in between.

[0013] In some embodiments, wherein X is any L-amino acid, w is any integer from 1 to 20.

[0014] In some embodiments, X is A, I, L, V, F, W, Y, N, C, Q, M, S, T, R, H, K, D, E, P, or G.

[0015] In some preferred embodiments, X is E.

[0016] In some embodiments, w=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0017] In some embodiments, the linker sequence is selected from a flexible linker, a rigid helical linker, a flexible curled linker and / or a β-linked linker, preferably a flexible curled linker.

[0018] In some embodiments, the flexible linker is selected from (G v S) h or (G) h .

[0019] In some embodiments, the flexible linker comprises an amino acid sequence as shown in GGS or GGSGGS (SEQ ID NO: 21), or an amino acid sequence having at least 65% sequence identity thereto.

[0020] In some embodiments, the rigid helical connector is selected from EA v K、A(EA v K) h A.

[0021] In some embodiments, the rigid helical linker comprises the amino acid sequence shown as EAAAK (SEQ ID NO: 22), or an amino acid sequence having at least 65% sequence identity thereto.

[0022] In some embodiments, the flexible curled linker comprises the amino acid sequence shown in RVNPVELG (SEQ ID NO: 23), or an amino acid sequence having at least 65% sequence identity thereto and having a linker function.

[0023] In some embodiments, the β-linked linker comprises the amino acid sequence shown as VSVATNWG (SEQ ID NO: 24), or an amino acid sequence having at least 65% sequence identity thereto.

[0024] In some embodiments, v is any integer from 1 to 10.

[0025] In some embodiments, v=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0026] In some embodiments, h is any integer from 1 to 30.

[0027] In some embodiments, h=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0028] In some embodiments, the first peptide tag and the second peptide tag are selected from one or more of the following peptide partner pairs: constituting a SpyTag and BDTag pair, a SpyTag and SpyCatcher pair, a SnoopTag and SnoopCatcher pair, a SnoopTagJr and SnoopCatcher pair, a SnoopTag and DogTag pair, a SnoopTagJr and DogTag pair, a DogTag and DogCatcher pair, a Pilin-C and Isopeptag pair, a Isopeptag-N and Pilin-N pair, a SdyCatcher and SdyTag pair, a PsCsCatcher and PsCsTag pair, a Jo and In pair, a RrgATag and RrgACatcher pair, a DogTag and RrgACatcher pair, a kTAG and SpyTAG pair, a RumTrunkTag and Mooncake pair, a RumTrunkTag and Katl pair, or a combination of variants or fragments thereof.

[0029] In some embodiments, the SpyTag comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 65% sequence identity thereto.

[0030] In some embodiments, the BDTag comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 65% sequence identity thereto.

[0031] In some embodiments, the isopeptide bond between the first peptide tag and the second peptide tag is formed by a spontaneous reaction or in the presence of a ligase.

[0032] In some embodiments, the amino acid sequence of the ligase consists of D-amino acid residues.

[0033] In some embodiments, the ligase comprises Spyligase, SnoopLigase and / or SpyStapler.

[0034] In some embodiments, the conjugate further comprises the ligase.

[0035] In some embodiments, the SpyStapler comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 65% sequence identity thereto.

[0036] In some embodiments, the functional protein includes one or more of an antigen, an antibody or antigen-binding fragment, a therapeutic peptide, a detectable tag, a cell-penetrating peptide, a targeting peptide, or a membrane fusion peptide.

[0037] In some embodiments, the SpyStapler is used to catalyze the SpyTag / BDTag to generate an isopeptide bond.

[0038] In some embodiments, the immunogenic protein includes one or more immunogenic proteins or fragments thereof.

[0039] In some embodiments, the functional protein is composed of L-amino acids.

[0040] In some embodiments, the scaffold protein is composed of L-amino acids.

[0041] In some embodiments, the scaffold protein is capable of forming nanoparticles.

[0042] In some embodiments, the nanoparticles formed by the scaffold protein have a regular or irregular three-dimensional structure, such as a cubic, polyhedral, granular, tubular, linear, dendritic, and combinations thereof, more preferably a virus-like particle (VLP) structure.

[0043] In some embodiments, the scaffold protein is capable of self-assembling into nanoparticles.

[0044] In some embodiments, the nanoparticles formed by the scaffold protein comprise a virus-like particle (VLP) structure.

[0045] In some embodiments, the scaffold protein comprises i3-01 protein, ferritin, encapsulin protein, 03-33 protein, SL protein, IMX313 protein, DPSL protein, I53-50 protein, Dodecin protein, AP205 protein, PD protein or mi3 protein, or variants or fragments thereof.

[0046] In some embodiments, the functional protein is displayed on the surface of the nanoparticle.

[0047] In some embodiments, the second peptide tag-labeled scaffold protein comprises Mi3-LPXT(G) w -SpyTag / BDTag, wherein X is any L-amino acid, and w is any integer from 1 to 20.

[0048] In some embodiments, the scaffold protein labeled with the second peptide tag comprises Mi3-RVNPVELG-LPXT(G) w -SpyTag / BDTag, wherein X is any L-amino acid, and w is any integer from 1 to 20. In some embodiments, the scaffold protein labeled with the second peptide tag comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 6, 13, 16 to 20, or an amino acid sequence having at least 65% sequence identity thereto.

[0049] In some embodiments, the functional protein labeled with the first peptide tag comprises functional protein-LPXT(G) w -BDTag / SpyTag, wherein X is any L-amino acid, and w is any integer from 1 to 20. In some embodiments, the functional protein labeled with the first peptide tag comprises an amino acid sequence as shown in SEQ ID NO: 7-12, 14 or an amino acid sequence having at least 65% sequence identity therewith.

[0050] In some embodiments, the SpyTag and the BDTag are coupled to each other via an isopeptide bond to form a peptide-linked pair.

[0051] In some embodiments, the SpyTag and the BDTag are both composed of D-amino acids.

[0052] In some embodiments, the Mi3-RVNPVELG-LPXT(G) w-SpyTag / BDTag or the antigen protein-LPXT(G) w - BDTag / SpyTag is conjugated via an isopeptide bond coupling between the SpyTag and the BDTag.

[0053] In some embodiments, the carboxyl terminus is tagged with LPXT(G) in the presence of a transpeptidase. n The first peptide tag is amino-terminal with (G) m The functional protein is coupled and connected to obtain the functional protein labeled with the first peptide tag.

[0054] In some embodiments, the carboxyl terminus is tagged with LPXT(G) in the presence of a transpeptidase. n The functional protein has a (G) m The first peptide tag is coupled and connected to obtain a peptide-tagged protein.

[0055] In some embodiments, the carboxyl terminus is tagged with LPXT(G) in the presence of a transpeptidase. n The second peptide tag is amino-terminal with (G) m The scaffold protein is coupled and connected to obtain a scaffold protein labeled with a second peptide tag.

[0056] In some embodiments, the carboxyl terminus is tagged with LPXT(G) in the presence of a transpeptidase. n The scaffold protein has a (G) m The second peptide tag is coupled and connected to obtain a scaffold protein labeled with the second peptide tag.

[0057] In an alternative embodiment, the scaffold protein is conjugated to LPXT(G) n There is also the above-mentioned linker sequence between them.

[0058] In some embodiments, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0059] In some embodiments, m=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0060] In some embodiments, the preparation further comprises a purification step.

[0061] In some embodiments, the purification comprises size exclusion chromatography purification, affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size exclusion chromatography (SEC) or gel permeation chromatography (GPC), etc.

[0062] In some embodiments, the transpeptidase comprises a sortase.

[0063] In some embodiments, the transpeptidase comprises one or more of sortaseA, sortase B, sortase C, sortaseD, sortase E, or sortase F.

[0064] In some embodiments, the transpeptidase is sortaseA, and the recognition motif of sortaseA is LPXT(G) n , wherein X is any L-amino acid, and n=1-10.

[0065] In some embodiments, the functional protein / scaffold protein has m glycine residues at the amino terminus, or LPXT (G) residues at the carboxyl terminus. n The functional protein / the scaffold protein is encoded by nucleic acid or synthesized chemically.

[0066] In some embodiments, the carboxyl terminus carries LPXT(G) n The first peptide tag / the second peptide tag, or the first peptide tag / the second peptide tag having m glycines at the amino terminus is chemically synthesized.

[0067] According to yet another aspect of the present disclosure, a nanoparticle is provided, comprising the conjugate.

[0068] In some preferred embodiments, the functional protein is displayed on the surface and / or in the cavity of the nanoparticle.

[0069] In some embodiments, the nanoparticles may include, but are not limited to, one or more of i3-01 protein nanoparticles, ferritin nanoparticles, encapsulin protein nanoparticles, 03-33 protein nanoparticles, SL protein nanoparticles, IMX313 protein nanoparticles, DPSL protein nanoparticles, I53-50 protein nanoparticles, Dodecin protein nanoparticles, AP205 protein nanoparticles, PD protein nanoparticles or mi3 protein nanoparticles.

[0070] In some embodiments, the nanoparticles are further loaded with one or more of additional polypeptides, proteins, drugs or nucleic acid molecules.

[0071] According to one aspect of the present disclosure, a protein labeled with a peptide tag is provided, wherein the peptide tag is composed of D-amino acids.

[0072] In some embodiments, the peptide tag contains a LPXT (G) between the protein. wThe amino acid sequence shown, wherein X is any L-amino acid, and w is any integer from 1 to 20.

[0073] In some embodiments, the peptide tag-labeled protein includes the protein, LPXT (G) from the N-terminus to the C-terminus. w and the peptide tag. In some embodiments, the peptide-tagged protein includes the peptide tag, LPXT(G)w and the protein from the N-terminus to the C-terminus. In an optional embodiment, the protein and LPXT(G) w There is a linker sequence in between, wherein the linker sequence is as defined above.

[0074] In some embodiments, the peptide tag includes any one or more of SpyTag, BDTag, SpyCatcher, SnoopTag, SnoopCatcher, SnoopTagJr, DogTag, DogCatcher, Pilin-C, Isopeptag, Isopeptag-N, Pilin-N, SdyCatcher, SdyTag, PsCsCatcher, PsCsTag, Jo, In, RrgATag, RrgACatcher, kTAG, RumTrunkTag, Mooncake, Katl or a combination of their variants.

[0075] In some embodiments, the protein is composed of L-amino acid residues.

[0076] In some embodiments, the protein includes the functional protein and / or the scaffold protein.

[0077] In some embodiments, the functional protein includes one or more of an antigen, an antibody or antigen-binding fragment, a therapeutic peptide, a detectable tag, a cell-penetrating peptide, a targeting peptide, or a membrane fusion peptide.

[0078] In some embodiments, the scaffold protein is capable of forming nanoparticles.

[0079] In some embodiments, the scaffold protein is capable of self-assembling into nanoparticles.

[0080] In some embodiments, the nanoparticles formed by the scaffold protein have a regular or irregular three-dimensional structure.

[0081] In some embodiments, the nanoparticles formed by the scaffold protein have a structure of cube, polyhedron, granule, tube, wire, dendrite, and combinations thereof.

[0082] In some embodiments, the nanoparticles formed by the scaffold protein have a structure of virus-like particles (VLP).

[0083] In some embodiments, the scaffold protein comprises one or more of i3-01 protein, ferritin, encapsulin protein, 03-33 protein, SL protein, IMX313 protein, DPSL protein, I53-50 protein, Dodecin protein, AP205 protein, PD protein or mi3 protein, or variants or fragments thereof, preferably including mi3 protein or variants or fragments thereof.

[0084] In some embodiments, the protein includes, from N-terminus to C-terminus, a functional protein, LPXT (G) w and peptide tag, or including peptide tag, LPXT(G) w In an optional embodiment, the functional protein and LPXT (G) w In a specific embodiment, the protein has an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 12 and 14, or an amino acid sequence having at least 65% sequence identity therewith.

[0085] In some embodiments, the protein includes, from N-terminus to C-terminus, a scaffold protein, LPXT (G) w and peptide tag, or including peptide tag, LPXT(G) w and a scaffold protein. In an optional embodiment, there is a linker sequence between the scaffold protein and LPXT(G)w. In a specific embodiment, the protein has an amino acid sequence as shown in any one of SEQ ID NOs: 5, 6, 13, 16 to 20 or an amino acid sequence having at least 65% sequence identity therewith.

[0086] According to another aspect of the present disclosure, a method for constructing a protein labeled with the peptide tag is provided, the method comprising the following steps: in the presence of a transpeptidase, the carboxyl end of the protein is labeled with LPXT (G) n The peptide tag is amino-terminal with (G) m Protein coupling connection, or carboxyl-terminal with LPXT (G) n Protein with (G) at the amino terminus m A peptide tag is coupled and connected to obtain a protein labeled with a peptide tag, wherein n and m are each independently selected from integers of 1 to 10.

[0087] In some embodiments, the method further comprises a purification step.

[0088] In some embodiments, the purification comprises size exclusion chromatography purification, affinity chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), charge-based separation, size exclusion chromatography (SEC) or gel permeation chromatography (GPC), etc.

[0089] In some embodiments, the transpeptidase comprises a sortase.

[0090] In some embodiments, the transpeptidase comprises one or more of sortaseA, sortase B, sortase C, sortaseD, sortase E, or sortase F.

[0091] In some embodiments, the transpeptidase is sortaseA, and the recognition motif of sortaseA is LPXT(G)n, wherein X is any L-amino acid, and n=1-10.

[0092] In some embodiments, the peptide tag is chemically synthesized.

[0093] In some embodiments, the protein is encoded by a gene or synthesized chemically.

[0094] According to another aspect of the present disclosure, a pharmaceutical composition is provided, comprising the conjugate and a pharmaceutically acceptable excipient.

[0095] According to yet another aspect of the present disclosure, the pharmaceutical composition comprises a vaccine.

[0096] In some embodiments, the pharmaceutically acceptable excipients include, but are not limited to, one or more of excipients, adjuvants, fillers, diluents, disintegrants, lubricants, glidants, binders, solubilizers, surfactants, emulsifiers, preservatives, antioxidants, flavoring agents, colorants, osmotic pressure regulators, and proppants.

[0097] In some embodiments, the pharmaceutically acceptable excipients include one or more of liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, and mesoporous silica nanoparticles.

[0098] In some embodiments, the pharmaceutical composition is a tablet, capsule, solution, granule, pill, powder, ointment, pill, suspension, powder, injection, suppository, cream, spray or patch.

[0099] According to another aspect of the present disclosure, provided is a use of the conjugate, the nanoparticle or the pharmaceutical composition in preparing a drug for preventing and / or treating a disease.

[0100] According to another aspect of the present disclosure, provided is a use of the conjugate, the nanoparticle or the pharmaceutical composition in preparing a drug for preventing and / or treating a disease.

[0101] In some embodiments, the medicament comprises a vaccine.

[0102] In some embodiments, the pharmaceutical composition comprises a nanoparticle vaccine.

[0103] In some embodiments, the prevention and / or treatment of a disease comprises administering a pharmaceutically effective amount of the pharmaceutical composition to a subject.

[0104] Those skilled in the art will appreciate that the disease may be any disease associated with immunogenic proteins.

[0105] According to yet another aspect of the present disclosure, there is provided a use of the nanoparticles as a delivery vehicle for delivering a drug, wherein the drug is used in an effective amount and is formulated to be encapsulated in the nanoparticles for administration to a subject to deliver the drug to a target site.

[0106] According to yet another aspect of the present disclosure, a method for preventing and / or treating a disease is provided, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a subject, thereby stimulating an immune response in the subject.

[0107] The present invention successfully uses a mirror peptide tag system to connect functional proteins and scaffold proteins. The mirror peptide tag system is composed of D-amino acids. This D-type polypeptide can more easily evade the recognition of proteases, can significantly prolong the half-life in biological systems, is less likely to induce unnecessary immune responses, and becomes "invisible". This "invisible" mirror connection component further improves the stability of the VLP vaccine, can further enhance the stability of the protein and reduce its unnecessary immunogenicity in the body. Furthermore, the use of connectors, especially flexible coil connectors, significantly improves the connection efficiency between the central particle scaffold and the antigen-specific element, making it a safer and more effective choice for VLP development. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 A schematic diagram of the mirror image VLP vaccine assembly is shown. Figure 1 A shows the SortaseA-mediated transpeptidase reaction for D-type peptide labeling on Mi3 and antigenic proteins. In the presence of Sortase, proteins containing the LPETGG motif were conjugated to GGG-D-BDTag or GGG-D-SpyTag. Figure 1 B shows the assembly of antigen-modified Mi3 nanoparticle vaccine by SpyStapler-mediated D-type peptide coupling. Antibody response was detected by ELISA after immunization of mice.

[0109] Figure 2 The effect of protein linker sequence on mirror-image assembly ligation efficiency is shown. Figure 2 A shows the sequences of six protein linkers and the related structures were generated using AlphaFold. Figure 2 B shows the coupling efficiency of Mi3-Linker xD-SpyTag and CyRPA-D-BDTag reaction in the SDS-PAGE diagram; the Mi3 Linker xD-SpyTag control group is shown on the left and the reaction group is shown on the right. Figure 2 C shows the coupling efficiency of Mi3-Linker xD-BDTag and CyRPA-D-SpyTag reaction in the SDS-PAGE diagram; the Mi3 Linkerx-D-BDTag control group is on the left and the reaction product is on the right.

[0110] Figure 3 TEM verifying the integrity of the mirror-connected protein nanoparticles is shown. Figure 3 A shows SDS-PAGE verification of mirror-linked Mi3R nanoparticles modified with various proteins; Figure 3 B shows the TEM analysis results, confirming that Figure 3 Nanoparticle integrity of each Mi3 nanoparticle analyzed in A, including modified and unmodified VLPs. Inset is a size distribution plot of particle diameters.

[0111] Figure 4 Protease resistance testing of mirror-linked VLPs is shown. Figure 4 A shows SDS-PAGE analysis of tryptic digestion products of Mi3-SpyTag+YFP-BDTag and Mi3-D-SpyTag+YFP-PD-BDTag over time. Figure 4 B shows SDS-PAGE analysis of proteinase K digestion products of Mi3-SpyTag+YFP-BDTag and Mi3-D-SpyTag+YFP-PD-BDTag over time. Figure 4 C shows the quantitative analysis of the residual percentage of Mi3-YFP conjugate (L&D) over time during trypsin digestion. Figure 4 D shows the quantitative analysis of the residual percentage (L&D) of Mi3-YFP conjugate during the proteinase K process.

[0112] Figure 5 Mirror-image VLP vaccines using the malarial antigen CyRPA were shown to enhance antibody responses. Figure 5 A shows the immunization schedule. Figure 5B shows the changes in antibody titers after mice were immunized with free CyRPA and CyRPA-modified protein nanoparticles. DETAILED DESCRIPTION

[0113] Mirror-image life refers to artificial biological systems in which all macromolecules exist as enantiomeric mirror images. Unlike natural proteins that are easily degraded by endogenous and environmental proteases, D-amino acids can evade recognition by these proteases, resulting in significantly extended half-lives in biological systems. Therefore, mirror-image proteins and peptides made from D-amino acids have attracted increasing attention due to their significantly enhanced stability and lower immunogenicity compared to L-amino acid enantiomers. In addition, by making these elements "invisible" to the host's immune system, it is possible to minimize the side effects that may be triggered by the introduction of spy chemical components (such as SpyTag / SpyCatcher and SpyTag / SpyStapler / BDTag). However, the technical limitations of synthesizing D-peptides have brought new challenges, especially for peptides longer than 70 amino acids, which can make the production of D-SpyCatcher expensive and time-consuming.

[0114] SortaseA is a transpeptidase widely used in biocoupling reactions of peptides and proteins. It catalyzes the formation of LPXT-GGG bonds through an amide bond exchange reaction between peptides or proteins containing an LPXTG motif (X is any residue except proline) and an NH2-GGG residue. The inventors found that the achiral nature of glycine may allow D-type polypeptides to bind to L-amino acid proteins through the transpeptidase activity of SortaseA, thus making it possible to use D-SpyTag and D-BDTag to label nanoparticle scaffold proteins and antigenic proteins (and vice versa). Based on this, the present disclosure has developed a method in which an isopeptide bond is formed between a nanoparticle scaffold protein labeled with a D-SpyTag or D-BDTag and a functional protein (e.g., an antigenic protein), and a D-SpyStapler is added to obtain nanoparticles decorated with antigenic proteins.

[0115] The present disclosure will LPET (G) n The motifs were genetically fused with nanoparticle assembly units (also known as scaffold proteins) and functional proteins (e.g., antigenic proteins also known as immunogenic proteins). n The nanoparticle assembly unit and antigen protein with motif were labeled with D-SpyTag and D-BDTag respectively using SortaseA-mediated transpeptidase reaction ( Figure 1A). Then, purification is performed to obtain nanoparticle assembly units labeled with D-SpyTag or D-BDTag and functional proteins labeled with D-BDTag or D-SpyTag. Nanoparticles modified with antigens are assembled through peptide-peptide coupling between SpyTag and BDTag mediated by D-SpyStapler. Those skilled in the art will appreciate that the nanoparticle assembly units and functional proteins used in the present disclosure, as well as nanoparticle assembly units and functional proteins labeled with D-SpyTag or D-BDTag, can also be obtained by biosynthesis.

[0116] Those skilled in the art will appreciate that any scaffold protein that can self-assemble into nanoparticles is applicable to the scheme of the present disclosure. In a specific embodiment, the nanoparticle assembly unit used in the present disclosure may be Mi3.

[0117] Those skilled in the art should understand that the functional protein used in the present disclosure can be any protein with biological function.

[0118] Those skilled in the art will appreciate that the antigen protein used in the present disclosure may be any antigen intended for immunization. In an exemplary embodiment, the antigen protein used in the present disclosure is CyRPA.

[0119] The results disclosed herein demonstrate that the mirror image SpyStapler can effectively catalyze the formation of isopeptide bonds between proteins labeled with D-SpyTag and D-BDTag. These well-assembled nanoparticles obtained by the present disclosure display high-copy antigen molecules on their surfaces. In some embodiments, the nanoparticles (VLPs) with high-copy antigen molecules displayed on the surface of the present disclosure are used as immunogens to immunize animals, and the serum collected from the immunized animals is analyzed ( Figure 1 B), the results demonstrate that the VLPs disclosed herein are capable of generating safe and efficient immune responses in animals.

[0120] On the other hand, the fusion protein linker sequence plays a vital role in stabilizing and maintaining the biological activity of the recombinant fusion protein. It should be understood by those skilled in the art that any conventionally used linker sequence can be used between the nanoparticle unit protein and the D-type polypeptide tag. In some embodiments, different linker sequences can be introduced between the protein and the peptide tag to improve the efficiency of the mirror reaction and the efficiency of the functional protein anchoring the central particle scaffold. In some embodiments, the linker sequence may include a flexible linker, a rigid helical linker, a flexible coil linker and / or a β-linked linker. In a preferred embodiment, the linker sequence may include a flexible linker, a flexible coil linker and / or a β-linked linker. In an exemplary embodiment, the linker sequence may have an amino acid sequence such as GGS, GGSGGS (SEQ ID NO: 21), EAAAK (SEQ ID NO: 22), RVNPVELG (SEQ ID NO: 23) or VSVATNWG (SEQ ID NO: 24). In a more preferred embodiment, the linker sequence may include an amino acid sequence such as RVNPVELG (SEQ ID NO: 23).

[0121] After the vaccine assembly method disclosed in the present invention is processed through multiple steps such as gene construction, recombinant protein expression and purification, and SpyStapler-mediated antigen modification on protein nanoparticles, virus-like particles (VLPs) with or without linker sequences can still maintain the polyhedral structure of the central particle scaffold unchanged. Maintaining the polyhedral structure of nanoparticles is crucial for future vaccine applications, ensuring that antigen decoration occurs in a highly concentrated arrangement so as to better mimic natural viruses. The mirror-image plug-and-play vaccine assembly strategy provided by the present disclosure is not only gentle, but also can ensure the stability and integrity of nanoparticles, which is conducive to the application of VLPs in vaccines.

[0122] The disclosed method of displaying immunogenic proteins on nanoparticles using D-type peptide tags can further enhance the stability of proteins and reduce the unnecessary immunogenicity caused by them in vivo, thereby reducing exogenous protein contamination. This feature is crucial in designing VLP-based vaccines because it can "hide" the introduction of unnecessary exogenous proteins or peptide fragments as much as possible during antigen connection. By using D-type peptides, it can be ensured that the immune system does not recognize these components, ultimately developing more effective and safer vaccines.

[0123] definition

[0124] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0125] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0126] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0127] The term "D-amino acid" as used herein is a mirror image of a natural amino acid (i.e., L-amino acid). In this article, the terms "mirror image amino acid" and "D-amino acid" can be used interchangeably. Unless otherwise specified, the amino acid residues in the proteins, tags, peptides, and amino acid sequences involved in the present disclosure are all L-amino acids.

[0128] The term "D-type SpyStapler" used in this article is a mirror image of the natural SpyStapler (i.e., L-type SpyStapler). The terms "mirror image SpyStapler" and "D-SpyStapler" and "D-type SpyStapler" may be used interchangeably. The protein ligase SpyStapler can mediate the assembly of VLP vaccines. SpyStapler is separated from SpyCatcher, and the remaining fragment is named BDTag. Isopeptide bonds can still be formed between BDTag and SpyTag under the catalysis of SpyStapler. Due to the non-covalent nature of the interaction between SpyStapler and the other two peptide fragments, SpyStapler acts like a ligase and can be removed once the reaction is completed.

[0129] The term "enantiomer" is used to describe one of a pair of molecular isomers that are mirror images of each other and are not superimposable. Other terms used to designate or refer to enantiomers include "stereoisomer" (because of the different arrangements or stereochemistry around a chiral center; although all enantiomers are stereoisomers, not all stereoisomers are enantiomers) or "optical isomer" (because of the optical activity of pure enantiomers, which is the ability of different pure enantiomers to rotate plane polarized light in different directions). Enantiomers generally have the same physical properties, such as melting and boiling points, and also have the same spectral characteristics. Enantiomers can differ from each other with respect to their interaction with plane polarized light and with respect to biological activity.

[0130] The term "antibody" as used herein encompasses immunoglobulins (whether naturally occurring or partially or completely synthetically produced) and fragments thereof. The term also covers any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides comprising framework regions that specifically bind and recognize antigens from immunoglobulin genes or fragments thereof. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, their fragments, and also includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments), bifunctional antibodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function. In a specific embodiment, the antibody of the present disclosure can be a monoclonal antibody or an antigen-binding fragment thereof.

[0131] The term "antigen" or "immunogenic protein" as used herein refers to a substance that can stimulate the body to produce an immune response and can bind to the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0132] The term "fusion protein" as used herein refers to a genetically engineered protein encoded by a nucleotide sequence formed by two or more complete or partial genes or a series of nucleic acids combined together. Alternatively, a fusion protein can be made by combining two or more heterologous peptides.

[0133] The term "linker" or "connection sequence" used herein refers to one or more (e.g., about 2-10) amino acid residues: between two adjacent motifs, regions or domains of a polypeptide, such as between antigenic peptides or between antigenic peptides and adjacent peptides encoded by multiple translation leader sequences, or between antigenic peptides and spacers or cleavage sites. The connecting peptide can be derived from the construct design of the fusion protein (e.g., amino acid residues caused by the use of restriction enzyme sites during the construction of nucleic acid molecules encoding fusion proteins).

[0134] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments, and fragments produced by any one or more of connection, cutting, endonuclease action or exonuclease action. In certain embodiments, the nucleic acid of the present invention is produced by PCR. Nucleic acid can be composed of monomers, which are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally occurring nucleotides) or combinations thereof. Modified nucleotides can have modifications in sugar moieties, or pyrimidine or purine base moieties, or in place of sugar moieties, or pyrimidine or purine base moieties.

[0135] The term "expression vector" used herein means a DNA construct containing a nucleic acid molecule operably connected to a suitable control sequence, which can achieve expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter for achieving transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genome insert. Viral vectors may be based on DNA (e.g., adenovirus or vaccinia virus) or RNA, including oncolytic virus vectors (e.g., VSV), which may be replicable or non-replicable. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, may be integrated into the genome itself. In this specification, "plasmid", "expression plasmid", and "vector" are often used interchangeably.

[0136] The term "expression" as used herein refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes transcription and translation. Translation can begin with an unconventional start codon, such as a CUG codon, or translation can begin with several start codons (standard AUG and unconventional) to produce more protein (based on per molar amount) than the mRNA produced.

[0137] As used herein, the term "sequence identity" refers to the "percentage of sequence identity" or "percentage of sequence identity" between two polynucleotide sequences or amino acid sequences, i.e., the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. At least 65% sequence identity includes at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the entire length of the sequence. In some embodiments, the amino acid sequence with sequence identity has the same or similar function as the reference sequence. In some embodiments, the function includes coupling with a polypeptide formed by another amino acid sequence by forming an isopeptide bond. In some embodiments, the function includes exerting enzyme activity to catalyze the formation of an isopeptide bond between polypeptides formed by two amino acid sequences for coupling. In some embodiments, the function includes a connection function. In some embodiments, the connection includes a flexible connection, a rigid connection, etc.

[0138] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0139] The term "VLP" or "virus-like particle" used herein refers to a non-replicating viral coat derived from any of several viruses. VLP can be naturally occurring or synthesized by the expression of viral structural proteins alone, and can then self-assemble into a virus-like structure. VLP is usually composed of one or more viral proteins, such as particle-forming proteins called capsids, coats, shells, surfaces and / or envelope proteins, or particle-forming polypeptides derived from these proteins. In some embodiments, VLPs can be spontaneously formed when recombinantly expressing proteins in a suitable expression system. VLPs can differ in morphology, size, and number of subunits. Methods for producing specific VLPs are known in the art. After recombinant expression of viral proteins, the presence of VLPs can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, etc. (see, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505). For example, VLPs can be separated by density gradient centrifugation and / or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question and images recorded under appropriate exposure conditions.

[0140] The term "SortaseA" as used herein is a transpeptidase widely used in peptide and protein bioconjugation reactions. It catalyzes the formation of LPXT-GGG bonds through an amide bond exchange reaction between peptides or proteins containing the LPXTG motif (X is any residue except proline) and NH2-GGG residues. The achiral nature of glycine enables D-type peptides to be conjugated to L-amino acid proteins through the transpeptidase activity of SortaseA.

[0141] The terms "vaccine" and "vaccine composition" as used herein refer to a pharmaceutical composition containing a corresponding viral antigen, which can induce, stimulate or enhance an immune response of a subject against the corresponding virus. The vaccine of the present invention can be delivered, for example, by intramuscular injection, subcutaneously, intranasally, through mucosal presentation, intravenously, or by intradermal or subcutaneous administration.

[0142] The term "CyRPA (cell surface protein)" used in this article is a protein on the surface of Plasmodium falciparum cells, which forms a multiprotein complex with PfRH5 and PfRipr, which plays a key role in the invasion of red blood cells by Plasmodium. This complex can unravel the red blood cell membrane, allowing Plasmodium to enter the cell and induce infection.

[0143] The term "variant" as used herein refers to a polynucleotide or polypeptide having a sequence that is substantially similar or identical to a reference (e.g., parent) polynucleotide or polypeptide. In the case of a polynucleotide, a variant may have a deletion, substitution, or addition of one or more nucleotides at the 5' end, 3' end, and / or one or more internal sites compared to a reference polynucleotide. The sequence similarity and / or difference between a variant and a reference polynucleotide may be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those produced using, for example, site-directed mutagenesis. Typically, variants of polynucleotides, including but not limited to DNA, may have at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide, as determined by sequence alignment programs known in the art. In the case of a polypeptide, a variant may have one or more amino acid deletions, substitutions, additions compared to a reference polypeptide. Conventional techniques known in the art, such as Western blotting, may be used to detect the similarity and / or differences of the sequence between a variant and a reference polypeptide. As measured by sequence alignment programs known in the art, a variant of a polypeptide may have, for example, at least or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide.

[0144] As used herein, the term "prevention and / or treatment" refers to inhibiting the replication, spread or preventing the corresponding virus from settling in the host, as well as alleviating the symptoms of a disease or condition caused by the virus. If the viral load is reduced, the symptoms are alleviated and / or the food intake and / or growth are increased, then the treatment is considered to have achieved a therapeutic effect.

[0145] As used herein, the term "therapeutically effective amount (or dose)" or "effective amount (or dose)" means an amount of a compound sufficient to cause improvement in one or more symptoms of the disease being treated in a statistically significant manner. The exact amount depends on numerous factors, such as the activity of the composition, the method of delivery employed, the immunostimulatory capacity of the composition, the intended patient and patient considerations, etc., and can be readily determined by one of ordinary skill in the art. The therapeutic effect may directly or indirectly include a reduction in one or more symptoms of the disease, and the therapeutic effect may also directly or indirectly include a reduction in one or more symptoms of the disease.

[0146] The term "pharmaceutically acceptable" as used herein refers to being able to be applied to humans and / or other animals as subjects, and will not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to auxiliary materials that are present in the pharmaceutical preparation at the same time as the active ingredient and will not produce excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceutically acceptable excipient" refers to a high-safety excipient that is suitable for a specific pharmaceutical preparation and is routinely used in pharmacy. It includes, but is not limited to, liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0147] The term "subject" as used herein can be any organism capable of a cellular immune response, such as a human, pet, livestock, exhibit animal, zoo specimen, or other animal. For example, the subject can be a human, non-human primate, dog, cat, rabbit, rat, mouse, guinea pig, horse, cow, sheep, goat, pig, etc.

[0148] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. The actual protection scope of the present invention is set forth in the claims. In the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples are not mentioned in terms of their sources, and are all commercially available on the market or obtained by conventional methods known to those skilled in the art.

[0149] Example

[0150] Materials and Methods

[0151] Gene construction

[0152] Gene constructs containing genes encoding Mi3 (Genebank ID: AXF54357.1), yellow fluorescent protein (YFP, Genebank ID: ATB19151), cyan fluorescent protein (CFP, Genebank ID: AAG34040), and sortaseA (GenBank: HEI7765168) were generated by conventional molecular cloning techniques.

[0153] The recognition motif LPETGG of sortaseA was appended to the C-terminus of Mi3, YFP and CFP, and the linker sequences GGS, GGSGGS, EAAAK, RVNPVELG and VSVATNWG were inserted between the LPETGG motif and the Mi3 sequence. The integrity of all constructs was confirmed by sequencing by Sangon Biotech, Shanghai, China. The pQE801 plasmid was used for Mi3 and YFP, and the pET28a plasmid was used for CFP and SortaseA. The molecular cloning process involved the use of Escherichia coli strain DH5α, which was cultured in LB broth medium.

[0154] Recombinant protein expression and purification

[0155] We used E. coli strain BL21 (DE3) for protein expression. Cells were cultured in Luria broth with 100 μg / mL ampicillin or kanamycin at 37°C and 220 rpm shaking. When the OD value reached 0.6-0.8, IPTG (500 μM) was added and cultured overnight at 16°C (or induced at 37°C for SortaseA expression for 3 hours) to induce protein expression, followed by overnight culture at 4°C and then harvesting the cells by centrifugation at 4,000 g for 20 minutes.

[0156] In order to extract fluorescent proteins CFP and YFP, the cell pellet was resuspended in buffer A (300mM NaCl, 50mM Tris-HCl, pH 8.0) containing phenylmethylsulfonyl fluoride (PMSF, 1mM) at room temperature, and the cell was broken using the filter pressure method. Cell debris was removed by centrifugation at 20,000g and 4°C for 30 minutes, and proteins were separated from cell lysates using HisTrap Ni-NTA columns (GE Healthcare) and eluted with buffer B (300mM NaCl, 50mM Tris-HCl, 500mM imidazole, pH 8.0). Further purification was performed using size exclusion chromatography (SEC) on an AKTAPURE fast protein liquid chromatography (FPLC) system equipped with S200 (GE Healthcare).

[0157] To separate the VLP precursor protein Mi3, the cell pellet was resuspended in buffer C (150 mM NaCl, 50 mM Tris-HCl, pH 8.5) containing PMSF (1 mM) at room temperature. The supernatant was collected by ultrasonic treatment on ice and then centrifuged at 20,000 g and 4 ° C for 30 minutes. Protein was extracted using HisTrap Ni-NTA columns (GE Healthcare) and eluted with buffer D (150 mM NaCl, 50 mM Tris-HCl, 500 mM imidazole, pH 8.5) and then purified by SEC (Superose 6, GE Healthcare).

[0158] For transpeptidase, i.e. Sortase A, cell pellets were resuspended in buffer E (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol) containing PMSF (1 mM) at room temperature. Cells were disrupted by sonication on ice, then centrifuged and proteins were separated using HisTrap Ni-NTA columns (GE Healthcare). Eluted with buffer F (150 mM NaCl, 50 mM Tris-HCl, 10% glycerol, 500 mM imidazole, pH 7.5) and further purified using SEC (Superose 200, GE Healthcare).

[0159] The protein fractions were further verified using SDS-PAGE, and the protein concentration was determined based on the absorbance at 280 nm. The proteins were quickly frozen in liquid nitrogen and stored at -80 °C until use.

[0160] The CyRPA antigen was expressed and obtained by Mingyan Company through the HEK293 cell line.

[0161] In order to purify SpyStapler, cells were resuspended in buffer A (300mM NaCl, 50mM Tris-HCl, pH 8.0) and 8M urea was added for denaturation purification, and cell disruption was performed using the method of filter pressure. Cell debris was removed by centrifugation at 20,000g and 4°C for 30 minutes, and proteins were separated from cell lysates using HisTrap Ni-NTA columns (GE Healthcare), and eluted with buffer B (300mM NaCl, 50mM Tris-HCl, 500mM imidazole, pH 8.0). Further purification was performed using size exclusion chromatography (SEC) on the AKTAPURE fast protein liquid chromatography (FPLC) system equipped with S75 (GE Healthcare).

[0162] Transpeptidation reaction

[0163] Proteins with LEPTGG terminal motifs (such as Mi3, YFP, CFP, CyRPA, etc.) were mixed with GGG-SpyTag / GGG-BDTag and SortaseA at a molar ratio of 1:10:1 and reacted overnight in a buffer containing 50mM Tris HCl buffer, 150mM NaCl (pH 7.5), and 2mM β-ME (Merck).

[0164] SpyStapler-mediated antigen modification on protein nanoparticles

[0165] The target protein with D-BDTag / SpyTag was generated by transpeptidation reaction with LPETGG motif and GGG-D-BDTag / SpyTag mediated by Sortase A. Subsequently, the obtained product was further purified by SEC gel filtration chromatography using Superose 200 / 6 from GE Healthcare.

[0166] The specified mixture of Mi3-VLP, Mi3-D-SpyTag or Mi3-D-BDTag was combined with D-SpyStapler and different amounts of specific proteins (such as YFP-D-SpyTag, CFP-D-SpyTag, CyRPA-D-SpyTag, YFP-D-BDTag, CFP-D-BDTag or CyRPA-D-BDTag, synthesized by Keshengjing peptide), and the sequences involved in the embodiments are shown in Table 1. To optimize the coupling efficiency. All reactions were carried out in PBS buffer to maintain the consistency of the reaction system. The reactions were left overnight at 4°C and then analyzed using SDS-PAGE and BioRad Image Lab analysis tools. The resulting coupling products were centrifuged and resuspended in PBS to remove excess reactants.

[0167] Table 1. Sequence table of the examples

[0168]

[0169]

[0170]

[0171] Note: The underlined parts in the table are D-amino acids.

[0172] Protease degradation resistance test

[0173] Mi3-SpyTag and YFP-BDTag were conjugated to SpyStapler in a 1:1 molar ratio to obtain Mi3-YFP L-conjugate. Using a similar method, Mi3-D-SpyTag and YFP-D-BDTag were conjugated to D-SpyStapler in a 1:1:2 ratio to form Mi3-YFPD-conjugate. After centrifugation of the mixture at 20,000 rpm for 2 minutes, the nanoparticle conjugate was separated to remove any unreacted free protein or peptide. Degradation experiments 0.25 mg / ml trypsin (Sangon Biotechnology) and 0.001 mg / ml proteinase K (Sangon Biotechnology) were added to 20 μl of nanoparticle conjugate, and each band was quantitatively detected using the BioRad Image Lab analysis tool after 0, 1, 5, 10, 15 and 20 minutes of reaction.

[0174] TEM scanning

[0175] The morphology and size of protein nanoparticles were observed by transmission electron microscopy (TEM). After the protein nanoparticle samples were diluted to about 0.1 mg / ml in buffer C (150 mM NaCl, 50 mM Tris-HCl, pH 8.5), these samples were deposited on 200 mesh copper grids for 10 minutes, stained with 2% uranyl acetate for 1 minute, and air-dried. Then, 100 kV, JEM-1200EX transmission electron microscope was used for observation.

[0176] Mouse immunization

[0177] After the VLP / antigen was reacted at 4°C for at least 12 hours using a D-SpyStapler, the resulting product was centrifuged and resuspended in PBS to remove excess reactants before being used for mouse immunization. Each vaccine (excluding blank VLP) was diluted to contain 1 μg of antigen per dose for vaccination. All vaccination samples were prepared the day before immunization.

[0178] All animal procedures were approved by the Animal Use and Care Committee of the Shenzhen Peking University Hong Kong University of Science and Technology Medical Center (SPHMC) with protocol number 2020-812. Specific pathogen-free female C57BL6 mice (Guangdong Yaokang), 6 to 8 weeks old, 6 per group, were injected into their leg muscles with the indicated samples (25 μl) premixed with AddaVax adjuvant (25 μl) from InvivoGen. For CyRPA-related immunization, blood samples were collected through the tail vein 14 days before the booster shot and 28, 42, and 56 days after the booster. The collected whole blood samples were coagulated overnight at 4°C. Serum was separated by centrifugation at 10,000 g for 5 minutes. The clarified serum was heat inactivated at 56°C for 10 minutes and then stored at -20°C.

[0179] ELISA detection of anti-CyRPA specific antibodies

[0180] CyRPA was coated at a concentration of 1 μg / mL in coating solution (pH 9.6, coating solution, Sangon Biotech) overnight at 4°C. Subsequently, the plates were washed six times with PBST buffer (PBS containing 0.05% Tween-20) and then blocked with 200 μL of 10% skim milk in PBST for 1 hour at room temperature. The serum samples were first diluted at a dilution factor of 1 / 160, and then several rounds of 3-fold serial dilutions were performed in PBST. The diluted serum (100 μL) was added to the plate and incubated at room temperature for 1 hour. Goat anti-mouse IgG was combined with alkaline phosphatase (IgGAP, GeneTex, 1 mg / mL) at a dilution factor of 1 / 3000 and incubated at room temperature for 1 hour. Between each step, the plate was washed six times with PBST, and alkaline phosphatase yellow (pNPP, Sigma) was used as the developing matrix. The absorbance at 405 nm was measured using a TECAN Infinite 200PRO microplate reader. The baseline absorbance value (threshold) was set to 0.2.

[0181] Example 1. Effect of different linker sequences on mirror reaction efficiency

[0182] The fusion protein linker sequence plays a vital role in stabilizing and maintaining the biological activity of the recombinant fusion protein. This example tests the effect of introducing different linker sequences between the nanoparticle unit protein and the D-type polypeptide tag on the mirror reaction efficiency. Five types of linker sequences were fused between the Mi3 and LPETGG motifs to generate the Mi3-linker X-LPETGG construct (where X represents a specific type of linker sequence), ( Figure 2 A) The five linker sequences are GGS, GGSGGS, EAAAK, RVNPVELG, and VSVATNWG. Next, the efficiency of the mirror reaction involving the corresponding Mi3 linker sequence proteins was tested. Among them, GGS and GGSGGS are flexible GS linkers, which are known to enhance the folding and stability of fusion proteins; the EAAAK linker is a rigid helical linker widely used to promote correct folding; the RVNPVELG linker is a flexible curly linker; and the VSVATNWG is a β-linked linker. Figure 2 The structure of the linker sequence shown in A was generated using AlphaFold. Five linker sequences were tested on Mi3-D-SpyTag and Mi3-D-BDTag nanoparticles. After inserting the linker sequence, we purified the relevant proteins. SDS-PAGE analysis was then performed to evaluate the reaction efficiency between D-peptide-tagged CyRPA and Mi3 fused with various linker sequences ( Figure 2 B and Figure 2C).

[0183] The coupling reactions between Mi3-D-PyTag (without linker sequence and with different linker sequences) and CyRPA-D-BDTag and between Mi3-D-BDTag (without linker sequence and with different linker sequences) and CyRPA-D-SpyTag were compared. Figure 2As shown in B and 2C, the left side shows the control group of Mi3-linker X without antigen protein, while the right side shows different reaction products and reaction efficiency. The reaction between Mi3-linker XD-SpyTag and CyRPA-D-BDTag showed different degrees of coupling efficiency. CyRPA-D-SpyTag (43.9 kDa) and CyRPA-D-BDTag (44.9 kDa) were purified by size exclusion chromatography and then combined with Mi3-D-BDTag and Mi3-D-SpyTag in the presence of D-SpyStapler. The molecular weight of the resulting coupling product is approximately 69 kDa. By quantifying the results of SDS-PAGE gel, it can be seen that the coupling efficiencies of Mi3-D-SpyTag without a linker, Mi3-GGS-D-SpyTag with a linker, Mi3-GGSGGS-D-SpyTag, Mi3-EAAAK-D-SpyTag, Mi3-RVNPVELG-D-SpyTag and Mi3-VSVATNWG-D-SpyTag with CyRPA-D-BDTag are approximately 5%, 10%, 0%, 2%, 40% and 35%, respectively. The coupling efficiencies of Mi3-D-BDTag, Mi3-GGS-D-BDTag, Mi3-GGSGGS-D-BDTag, Mi3-EAAAK-D-BDTag, Mi3-RVNPVELG-D-BDTag and Mi3-VSVATNWG-D-BDTag without a linker and CyRPA-D-SpyTag were 20%, 5%, 10%, 1%, 40% and 15%, respectively. The results showed that Mi3-D-SpyTag / D-BDTag without a linker could be coupled with CyRPA-D-BDTa / D-SpyTag, and for the coupling of Mi3-D-SpyTag and CyRPA-D-BDTag, both the RVNPVELG linker and the VSVATNWG linker showed higher coupling efficiency than those without a linker; for the coupling of Mi3-D-BDTag and CyRPA-D-SpyTag, the RVNPVELG linker showed higher coupling efficiency than those without a linker. Moreover, based on these results, Mi3-RVNPVELG-LPETGG (hereinafter referred to as Mi3R) was selected for subsequent vaccine assembly. It is speculated that the flexible curled linker is expected to expand the reaction space and increase the contact probability between the D-SpyTag and D-BDTag labeled proteins.

[0184] Example 2.

[0185] In this example, Mi3R nanoparticles (Mi3-RVNPVELG-LPETGG) were used for SDS-PAGE analysis to confirm their binding efficiency with different proteins and peptides. Subsequently, the structure of the mirror-image nanoparticles was verified using transmission electron microscopy (TEM).

[0186] Nanoparticles of Mi3R with peptide tags D-BDTag, D-SpyTag, and connected to different proteins YFP and CyRPA were subjected to SDS-PAGE analysis, in which Mi3R, YFP-D-BDTag, YFP-D-SpyTag, CyRPA-D-BDTag, and CyRPA-D-SpyTag were used as controls. The results showed that coupling reactions occurred between Mi3R-D-SpyTag and YFP-D-BDTag, and between Mi3R-D-BDTag and YFP-D-SpyTag, producing Mi3R-D-SpyTag+YFP-D-BDTag, Mi3R-D-SpyTag+CyRPA-D-BDTag, Mi3R-D-BDTag+YFP-D-SpyTag, and Mi3R-D-BDTag+YFP-D-SpyTag conjugates ( Figure 3 A).

[0187] In order to verify whether the polyhedral structure of Mi3R nanoparticles remains unchanged after multiple steps of treatment, TEM imaging was performed ( Figure 3B). The results of TEM imaging showed that the purified VLP nanoparticles: Mi3R-D-SpyTag (control) and Mi3R-D-BDTag (control) were produced by Mi3R and D-SpyTag / D-BDTag through sortase A enzyme reaction, with diameters of 23.62±1.72nm and 23.71±1.04nm, respectively. In addition, the effects of YFP and CyRPA conjugated to Mi3R-D-SpyTag and Mi3R-D-BDTag were also evaluated. The diameters produced by the combinations of Mi3R-D-SpyTag+YFP-D-BDTag, Mi3R-D-SpyTag+CyRPA-D-BDTag, Mi3RD-BDTag+YFP-D-SpyTag and Mi3R-D-BDTag+CyRPA-D-SpyTag were 23.28±1.52nm, 22.33±1.04nm, 23.03±1.18nm and 22.06±1.14nm, respectively, all maintaining a uniform polyhedral particle shape. Maintaining the polyhedral structure of nanoparticles is crucial for future vaccine applications, ensuring that antigen decoration occurs in a highly concentrated arrangement so that natural viruses can be better mimicked. The complex mechanisms of surface chemistry and structural folding of proteins and peptides on the anchor make the above-measured particle size changes more difficult. However, these results indicate that the mirror-image plug-and-play vaccine assembly strategy disclosed in the present invention is mild and can ensure the stability and integrity of the nanoparticles, which is crucial to their performance in subsequent vaccine applications.

[0188] Example 3

[0189] Mi3-YFP combined with L- and D-SpyTag was digested with trypsin (Sangon Biotechnology) and proteinase K (Sangon Biotechnology), respectively, and the digestion products were analyzed by SDS-PAGE. We used 1 mg / ml of Mi3-SpyTag in combination with YFP-BDTag to obtain L-type Mi3-YFP, and used 1 mg / ml of Mi3-D-SpyTag in combination with YFP-D-BDTag to obtain D-type Mi3-YFP. The two VLPs were digested with 0.001 mg / ml proteinase K and 0.25 mg / ml trypsin for 0, 1, 5, 10, 15 and 20 minutes, respectively. The results are shown in the figure. Figure 4 shown.

[0190] In the trypsin digestion experiment, Figure 4 A and Figure 4As shown in Figure C, we observed that the L-type Mi3-YFP was rapidly digested and the digestion products increased over time. The L-SpyStapler showed a clear degradation process and was almost undetectable after 5 minutes. In contrast, the digestion rate of the Mi3-YFP conjugate of the D-type product was much slower. There were almost no digestion products at the same position. And the D-SpyStapler showed almost no changes throughout the 20-minute digestion process. We quantified the digestion trends of the L-type and D-type Mi3-YFP conjugate bands by measuring the changes in band brightness in the SDS-PAGE images. The L-type Mi3-YFP was rapidly degraded, with about 60% digestion within 1 minute and the conjugate band decreased to 50% after 20 minutes. In contrast, the D-type conjugate retained about 90% after 1 minute and 82% after 20 minutes, verifying the high stability of the D-type product.

[0191] In the proteinase K digestion experiment, the trend was similar to that of trypsin digestion ( Figure 4 B and Figure 4 D), the digestion rate of L-type Mi3-YFP is much faster than that of D-type Mi3-YFP. Over time, digestion degradation products were only detected in the L-type digestion experiment, while no relevant degradation products were detected in the D-type detection area. After 1 minute of digestion, L-SpyStapler was almost undetectable, while D-SpyStapler showed no change. Quantitative detection analysis showed that after 1 minute of proteinase K digestion, the L-type conjugate decreased by about 60%, while the D-type conjugate decreased by only about 20%. These results confirm that SpyStapler-mediated D-type polypeptide connection enhances the stability of VLP protein nanoparticles. This method allows the exogenous connection component (D-type polypeptide connection) to escape host recognition, thereby reducing potential side effects associated with vaccine products.

[0192] Example 4

[0193] When immunized in mice, the mirror-linked VLP vaccine of the present invention triggered a strong immune response after binding to specific antigenic proteins. Using free CyRPA as the control group, we immunized 6-week-old C57BL / 6 mice with Mi3 nanoparticles linked to CyRPA via L- and D-type peptides, respectively. The treatment groups included Mi3SpyTag+CyRPA-BDTag, Mi3BDTag+CyRPA-SpyTag, Mi3-D-SpyTag+CyRPA-D-BDTag, and Mi3-D-BDTag+CyRPA-D-SpyTag. The mice were given a booster shot 14 days later. Serum samples were collected through the tail vein on days 14, 28, and 42, and by cardiac puncture on day 56 ( Figure 5A). All five types of immunogens elicited antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) 14 days after primary immunization. Free CyRPA produced lower levels of immune responses (~10 2 ), while the other four VLP immunogens produced antibody levels that were approximately 50-fold higher. There was no significant difference in antibody levels between the L-peptide and D-peptide-linked VLP vaccines. At day 28 after the booster, antibody responses were significantly enhanced for all immunogen types. Free CyRPA increased only tenfold, to 10 3 The antibody responses of the other four VLP antigens exceeded 10 4 The results are shown in Figure 5 B. These results indicate that mirror-image VLPs activated the same level of antibody response as L-peptide-linked VLPs. The antibody levels produced by L- and D-peptide-linked VLPs were significantly higher than those produced by free antigens, highlighting the advantages of using VLPs as a basis for vaccines. As shown in the results of Example 4, the "invisible" mirror-image linkage further improves the stability of the VLP vaccine and reduces the introduction of exogenous proteins, making it a safer choice for VLP development. We continued to monitor the antibody levels of immunized mice, and on days 42 and 56, we observed that serum antibody titers for all four VLP immunogens decreased slightly over time at a similar rate, which is consistent with the expected decay of antibodies in the host.

[0194] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A conjugate, wherein the conjugate comprises: i) a functional protein labeled with a first peptide tag, and ii) a scaffold protein labeled with a second peptide tag, in, The first peptide tag and the second peptide tag are composed of D-amino acid residues; The first peptide tag and the second peptide tag form a peptide partner pair, and the first peptide tag and the second peptide tag are coupled to each other via an isopeptide bond.

2. The conjugate according to claim 1, characterized in that The first peptide tag and the functional protein and / or the scaffold protein and the second peptide tag contain LPXT (G) w The amino acid sequence shown, wherein X is any L-amino acid, and w is any integer from 1 to 20; Preferably, the functional protein labeled with the first peptide tag includes the functional protein, LPXT (G) from the N-terminus to the C-terminus. w and a first peptide tag, or including a first peptide tag, LPXT(G) w and a functional protein, optionally, between the functional protein and LPXT (G) w There is a linker sequence between them; Preferably, the scaffold protein labeled with the second peptide tag includes scaffold protein, LPXT (G) from N-terminus to C-terminus. w and a second peptide tag, or including a second peptide tag, LPXT(G) w and a scaffold protein, optionally, between the scaffold protein and LPXT (G) w There is a linker sequence between them; Preferably, the linker sequence is selected from a flexible linker, a rigid helical linker, a flexible curled linker and / or a β-linked linker, more preferably a flexible curled linker; Preferably, the flexible linker is selected from (G v S) h or (G) h , v is any integer from 1 to 10, and h is any integer from 1 to 30; Preferably, the rigid helical connector is selected from EA v K、A(EA v K) h A; Preferably, the flexible crimp joint is selected from RVNPVELG; Preferably, the beta-linked linker is selected from VSVATNWG.

3. The composition according to claim 1, characterized in that The first peptide tag and the second peptide tag are selected from one or more peptide partner pairs: SpyTag and BDTag pair, SpyTag and SpyCatcher pair, SnoopTag and SnoopCatcher pair, SnoopTagJr and SnoopCatcher pair, SnoopTag and DogTag pair, SnoopTagJr and DogTag pair, DogTag and DogCatcher pair, Pilin-C and Isopeptag pair, Isopept ag-N and Pilin-N pairs, SdyCatcher and SdyTag pairs, PsCsCatcher and PsCsTag pairs, Jo and In pairs, RrgATag and RrgACatcher pairs, DogTag and RrgACatcher pairs, kTAG and SpyTAG pairs, RumTrunkTag and Mooncake pairs, RumTrunkTag and Katl pairs, or a combination of variants or fragments thereof, preferably selected from SpyTag and BDTag pairs or variants or fragments thereof, Preferably, the isopeptide bond between the first peptide tag and the second peptide tag is formed by a spontaneous reaction or in the presence of a ligase. Preferably, the amino acid sequence of the ligase consists of D-amino acid residues. Preferably, the ligase comprises Spyligase, SnoopLigase and / or SpyStapler, Preferably, the conjugate further comprises the ligase.

4. The conjugate according to claim 1, characterized in that The functional protein includes one or more of an antigen, an antibody or an antigen-binding fragment, a therapeutic peptide, a detectable label, a cell-penetrating peptide, a targeting peptide or a membrane fusion peptide; Preferably, the functional protein consists of L-amino acid residues.

5. The conjugate according to any one of claim 1, characterized in that The scaffold protein is composed of L-amino acid residues; Preferably, the scaffold protein is capable of forming nanoparticles, more preferably, the scaffold protein is capable of self-assembling into nanoparticles; Preferably, the nanoparticles formed by the scaffold protein have a regular or irregular three-dimensional structure; Preferably, the nanoparticles formed by the scaffold protein have a cubic, polyhedral, granular, tubular, linear, dendritic and combinations thereof structure, more preferably a virus-like particle (VLP) structure; Preferably, the scaffold protein comprises one or more of i3-01 protein, ferritin, encapsulin protein, 03-33 protein, SL protein, IMX313 protein, DPSL protein, I53-50 protein, Dodecin protein, AP205 protein, PD protein or mi3 protein, or variants or fragments thereof, preferably including mi3 protein or variants or fragments thereof.

6. A nanoparticle, comprising the conjugate according to any one of claims 1 to 5, Preferably, the functional protein is displayed on the surface and / or in the cavity of the nanoparticle. Preferably, the nanoparticles comprise one or more of i3-01 protein nanoparticles, ferritin nanoparticles, encapsulin protein nanoparticles, 03-33 protein nanoparticles, SL protein nanoparticles, IMX313 protein nanoparticles, DPSL protein nanoparticles, I53-50 protein nanoparticles, Dodecin protein nanoparticles, AP205 protein nanoparticles, PD protein nanoparticles or mi3 protein nanoparticles; Preferably, the nanoparticles are also loaded with one or more of additional polypeptides, proteins, drugs or nucleic acid molecules.

7. A protein labeled with a peptide tag, wherein the peptide tag is composed of D-amino acid residues.

8. The peptide-tagged protein according to claim 7, characterized in that: The peptide tag contains LPXT (G) between the protein w The amino acid sequence shown, wherein X is any L-amino acid, and w is any integer from 1 to 20; Preferably, the protein consists of L-amino acid residues; Preferably, the peptide-tagged protein includes the protein, LPXT (G) from the N-terminus to the C-terminus. w and the peptide tag, or comprising the peptide tag, LPXT (G) w and the protein, optionally, in the protein and LPXT (G) w There is a linker sequence between them; Preferably, the linker sequence is selected from a flexible linker, a rigid helical linker, a flexible curled linker and / or a β-linked linker, more preferably a flexible curled linker; Preferably, the flexible linker is selected from (G v S) h or (G) h , v is any integer from 1 to 10, and h is any integer from 1 to 30; Preferably, the rigid helical connector is selected from EA v K、A(EA v K) h A; Preferably, the flexible curled linker comprises the amino acid sequence shown in RVNPVELG, or an amino acid sequence having at least 65% sequence identity thereto; Preferably, the β-linker comprises the amino acid sequence shown in VSVATNWG, or an amino acid sequence having at least 65% sequence identity thereto; Preferably, the peptide tag comprises any one or more of SpyTag, BDTag, SpyCatcher, SnoopTag, SnoopCatcher, SnoopTagJr, DogTag, DogCatcher, Pilin-C, Isopeptag, Isopeptag-N, Pilin-N, SdyCatcher, SdyTag, PsCsCatcher, PsCsTag, Jo, In, RrgATag, RrgACatcher, kTAG, RumTrunkTag, Mooncake, Katl or variants or fragments thereof; Preferably, the protein comprises the functional protein and / or scaffold protein, Preferably, the functional protein comprises one or more of an antigen, an antibody or an antigen-binding fragment, a therapeutic peptide, a detectable label, a cell-penetrating peptide, a targeting peptide or a membrane fusion peptide. Preferably, the scaffold protein is capable of forming nanoparticles, more preferably, the scaffold protein is capable of self-assembling into nanoparticles; Preferably, the nanoparticles formed by the scaffold protein have a regular or irregular three-dimensional structure; Preferably, the nanoparticles formed by the scaffold protein have a cubic, polyhedral, granular, tubular, linear, dendritic and combinations thereof structure, more preferably a virus-like particle (VLP) structure; Preferably, the scaffold protein comprises one or more of i3-01 protein, ferritin, encapsulin protein, 03-33 protein, SL protein, IMX313 protein, DPSL protein, I53-50 protein, Dodecin protein, AP205 protein, PD protein or mi3 protein, or variants or fragments thereof, preferably including mi3 protein or variants or fragments thereof.

9. A method for preparing a protein labeled with a peptide tag according to claim 7 or 8, characterized in that: The method comprises the following steps: In the presence of transpeptidase, the carboxyl terminus carries LPXT(G) n The peptide tag is amino-terminal with (G) m Protein coupling connection, or carboxyl-terminal with LPXT (G) n Protein with amino terminal (G) m The peptide tag is coupled to obtain a peptide-tagged protein. wherein n and m are each independently selected from integers of 1 to 10.

10. The method according to claim 9, characterized in that: The transpeptidase includes sortase; Preferably, the transpeptidase comprises one or more of sortase A, sortase B, sortase C, sortase D, sortase E or sortase F; More preferably, the transpeptidase is sortaseA.

11. The method according to claim 9 or 10, characterized in that: The peptide tag is chemically synthesized, and / or the protein is encoded by a gene or chemically synthesized.

12. A pharmaceutical composition comprising: The conjugate according to any one of claims 1 to 5 or the nanoparticle according to claim 6; and pharmaceutically acceptable excipients.

13. Use of the conjugate according to any one of claims 1 to 5, the nanoparticle according to claim 6 or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing and / or treating a disease, preferably, the medicament comprises a vaccine; more preferably, the pharmaceutical composition comprises a nanoparticle vaccine.

14. Use of the nanoparticles according to claim 6 as a delivery carrier for delivering drugs, preferably, the drug is encapsulated in the nanoparticles.

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