Metal ion induced protein nanoparticles and application thereof in vaccine preparation

Abstract

The invention relates to metal ion induced protein nanoparticles and application thereof in vaccine preparation. The protein nanoparticles are formed by self-assembly of protein monomers through induction of the protein monomers by metal ions, and combination of the protein monomers and the metal ions is achieved through amino acid on the surface of protein. According to the application, the capability of the Cage-i53-Zn1-HEHE-5 protein nanoparticles for simultaneously displaying a plurality of protein antigens is proved by taking the Cage-i53-Zn1-HEHE-5 protein nanoparticles as an example. On the basis, more types of protein monomers reversibly combined with metal ions are further designed, and nanoparticles formed by self-assembly of the protein monomers under induction of the metal ions have wide application prospects in vaccine development.

Description

Technical Field

[0001] The present application belongs to the field of protein engineering, and specifically relates to metal ion-induced protein nanoparticles and their application in vaccine preparation. Background Art

[0002] Protein design combines knowledge from computational technology, biology, physics, and chemistry to create proteins with specific three-dimensional structures and special functions by manipulating amino acid sequences. The design of proteins with specific structures, especially the design of protein nanoparticles, has important applications in protein vaccine design, drug delivery, and other fields. Compared with natural protein nanoparticles, such as ferritin, de novo designed protein nanoparticles do not have specific structures and biological functions, have a larger design space, and also have the advantages of small protein molecular weight and high stability. The design of protein nanoparticles achieves powerful combination capabilities and precise designability, provides a new solution for in vivo delivery, and has important application prospects and value.

[0003] The new protein nanoparticle design method has greatly expanded the combination mode of protein nanoparticles. Compared with the spontaneous and two-component de novo designed nanoparticles of natural protein nanoparticles, the new single-component protein nanoparticles that achieve self-aggregation by ion induction can achieve more precise protein aggregation and dissociation control, as well as simpler protein composition. At the same time, the single-component characteristics of the single-component inducible aggregation protein nanoparticles make it easier to modify protein subunits and more feasible for commercial production. The design and development of single-component, inducible protein nanoparticles is of great value.

[0004] Vaccines have saved billions of lives since their birth. Today, vaccines can be divided into four major types: attenuated vaccines, inactivated vaccines, nucleic acid vaccines, and recombinant protein vaccines. Attenuated vaccines and inactivated vaccines are produced by screening mutant strains with lower toxicity to humans and by physical or chemical treatment of existing strains, respectively. Although attenuated vaccines can induce strong immune responses, pathogens can still reproduce and mutate in the host, which is unsafe. Although inactivated vaccines are safe, they often only induce moderate immune responses and have limitations. Existing nucleic acid vaccines, such as mRNA vaccines, have been widely used in the prevention of the new crown epidemic, but such vaccines usually require a cold environment for long-term storage and are not convenient for long-distance transportation. In contrast, vaccines based on recombinant proteins can induce strong immune responses and have the characteristics of high stability, showing great application value. Protein nanoparticles have become excellent carriers for delivering vaccine antigens due to their ideal shape and size. At present, the development of vaccine delivery systems based on protein nanoparticles has become a new direction for vaccine development, which will accelerate the development of more important pathogen vaccines.

[0005] In protein vaccine development, single-molecule soluble antigens have poor immunogenicity. This is because low-molecular-weight antigens are easily eliminated from the organism and produce almost no immune response. When antigens are coupled to particles, their immunogenicity and physical and metabolic stability are enhanced. Compared with soluble antigens, nanoparticle-associated antigens diffuse more effectively from the injection site to the lymph nodes and remain longer. Multivalent antigens on nanoparticles also enhance the internalization of antigen-presenting cells through multiple mechanisms and can effectively induce B cell receptor (BCR) cross-linking, thereby greatly enhancing B cell uptake and presentation. Antigens coupled to nanoparticles are also more stable and easier to maintain antigen conformation.

[0006] Some self-assembling proteins such as ferritin and two-component de novo designed nanoparticles such as I53-50 have been successfully used as scaffolds to display complex glycoprotein antigens of influenza hemagglutinin, HIV envelope, and Epstein-Barr virus. [1][2][3] . However, due to the limitations of the few types of natural protein particles available and the fixed basic structure, all widely used self-assembling particle proteins will spontaneously assemble after being expressed in recombinant cells. Two-component de novo designed nanoparticles require two components to achieve assembly, which increases the complexity of assembly. These factors hinder the development of nanoparticles in vaccine design. Therefore, it is necessary to develop a new protein nanoparticle composed of a single component to reduce the complexity of assembly while improving the safety of assembled nanoparticles, thereby improving the applicability of nanoparticles as carriers for vaccine delivery.

[0007] References

[0008] [1] Kanekiyo M, Wei CJ, Yassine H et al. Self-assembling influenzananoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499,102–106 (2013).

[0010] [2]Abbott RK,Lee JH,Menis S et al.Precursor Frequency and AffinityDetermine B Cell Competitive Fitness in Germinal Centers,Tested withGermline-Targeting HIV Vaccine Immunogens.Immunity.2018Jan

[0011] 16;48(1):133-146.e6.

[0012] [3]Kanekiyo M,Bu W,Joyce MG et al.Rational Design of an Epstein-BarrVirus Vaccine Targeting the Receptor-Binding Site.Cell.2015Aug

[0013] 27;162(5):1090-100. Summary of the invention

[0014] The technical purpose of the present invention is to provide a self-assembled protein nanoparticle for vaccine carrier and its use as a vaccine carrier.

[0015] The present invention is dedicated to providing a self-assembled protein nanoparticle, which is formed by self-assembly of protein monomers through metal ion induction, wherein the metal ions are selected from zinc ions, nickel ions and calcium ions. For the protein nanoparticles formed by self-assembly of protein monomers through zinc ion induction, zinc ions interact with histidine and aspartic acid on the surface of protein monomers to form tetrahedral coordination, thereby forming the protein nanoparticles; for the protein nanoparticles formed by self-assembly of protein monomers through nickel ion induction, nickel ions interact with histidine on the surface of protein monomers to form octahedral coordination, thereby forming the protein nanoparticles; for the protein nanoparticles formed by self-assembly of protein monomers through calcium ion induction, calcium ions interact with aspartic acid and asparagine on the surface of protein monomers to form octahedral coordination, thereby forming the protein nanoparticles.

[0016] In a specific embodiment, the protein monomer has the following amino acid sequence:

[0017] SEQ ID No.: 1, any one of 4-23, 28-30.

[0018] In a specific embodiment, the self-assembled protein nanoparticles are polymers or protein microfilaments formed by the protein monomers, and the polymers can be 2 to 60 polymers of protein monomers.

[0019] In a specific embodiment, the self-assembled protein nanoparticles are polymers formed by the protein monomers, for example, dimers, trimers, tetramers, heterodimers, dodecamers, hexamers, or protein microfilaments.

[0020] In a specific embodiment, the multimers include homomultimers and heteromultimers.

[0021] In a specific embodiment, the zinc ion-induced self-assembled protein nanoparticles are selected from the following:

[0022] A homogenous hexameric protein monomer having the sequence SEQ ID No.: 1 (named Cage-i53-Zn1-HEHE-5);

[0023] A homodimer of a protein monomer having a sequence of SEQ ID No.: 4 (named as C2-Zn1-HEHE-2);

[0024] A homodimer of a protein monomer having a sequence of SEQ ID No.: 5 (named as C2-Zn2-HEHE-2);

[0025] A homotrimer of a protein monomer having a sequence of SEQ ID No.: 6 (named C3-Zn2-HEHE-19);

[0026] A homotetramer of a protein monomer having a sequence of SEQ ID No.: 7 (named as C4-Zn1-HEHE-1);

[0027] A homopentamer of a protein monomer having a sequence of SEQ ID No.: 8 (named C5-Zn1-HEHE-1);

[0028] A heterodimer of a protein monomer having a sequence of SEQ ID No.: 9 (named Heter-Zn2-HEHE-22a) and a protein monomer having a sequence of SEQ ID No.: 10 (named Heter-Zn2-HEHE-22b);

[0029] A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 11 (named Cage-t32-Zn1-HEHE-34);

[0030] A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 12 (named Cage-t32-Zn1-HEHE-35);

[0031] A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 13 (named Cage-t32-Zn1-HEHE-36);

[0032] A homologous hexameric protein monomer having a sequence of SEQ ID No.: 14 (named Cage-i53-Zn1-HEHE-14);

[0033] A homogenous hexameric protein monomer having a sequence of SEQ ID No.: 15 (named Cage-i53-Zn1-HEHE-16);

[0034] A homogenous hexameric protein monomer having a sequence of SEQ ID No.: 16 (named Cage-i53-Zn1-HEHE-6);

[0035] A homologous protein microfilament of a protein monomer having the sequence SEQ ID No.: 17 (named Fiber-n6-Zn1-HEHE-5);

[0036] A homologous protein microfilament of a protein monomer having the sequence SEQ ID No.: 18 (named Fiber-n6-Zn1-HEHE-74);

[0037] A homologous protein microfilament of a protein monomer having the sequence SEQ ID No.: 19 (named Fiber-n5-Zn1-HEHE-917);

[0038] A homologous protein microfilament of a protein monomer having the sequence SEQ ID No.: 20 (named Fiber-n6-Zn1-HEHE-43);

[0039] A homologous protein microfilament of a protein monomer having the sequence SEQ ID No.: 21 (named Fiber-n6-Zn1-HEHE-44);

[0040] A homologous protein microfilament of a protein monomer (named Fiber-n6-Zn1-HEHE-46) with the sequence SEQ ID No.: 22;

[0041] The homologous protein microfilament of the protein monomer with the sequence SEQ ID No.: 23 (named as Fiber-n6-Zn1-HEHE-410).

[0042] In a specific embodiment, the nickel ion-induced self-assembled protein nanoparticles are homotrimers of a protein monomer with a sequence of SEQ ID No.: 28 (named C3-Ni1-HH*3-18).

[0043] In a specific embodiment, the calcium ion-induced self-assembled protein nanoparticles are selected from the following:

[0044] A homotrimer of a protein monomer having a sequence of SEQ ID No.: 29 (named C3-Cal-DN*3-20), and

[0045] A homotrimer of a protein monomer (named C3-Cal-DDDD-48) with a sequence of SEQ ID No.: 30. In another aspect, the present invention provides a nucleic acid molecule encoding the above protein monomer.

[0046] In another aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0047] In yet another aspect, the present invention provides a host cell comprising the above-mentioned recombinant vector.

[0048] In another aspect, the present invention provides a method for preparing the self-assembled protein nanoparticles, comprising:

[0049] S1: providing a solution containing the above protein monomer;

[0050] S2: adding zinc salt, nickel salt or calcium salt solution to the solution of step S1 and letting it stand;

[0051] S3: Purification to obtain protein nanoparticles.

[0052] In a specific implementation, the zinc salt solution is a zinc sulfate solution, the nickel salt solution is a nickel sulfate solution, and the calcium salt solution is a calcium chloride solution.

[0053] In a specific embodiment, for the preparation of Cage-i53-Zn1-HEHE-5, Cage-t32-Zn1-HEHE-35, Cage-i53-Zn1-HEHE-14, Cage-i53-Zn1-HEHE-16, Cage-i53-Zn1-HEHE-6, Fiber-n6-Zn1-HEHE-5, Fiber-n7-Zn1-HEHE-74, Fiber-n5-Zn1-HEHE-917, Fiber-n6-Zn1-HEHE-43, Fiber-n6-Zn1-HEHE-44, Fiber-n6-Zn1-HEHE-46, and Fiber-n6-Zn1-HEHE-410 protein nanoparticles, The molar ratio of zinc salt to protein monomer is 2:1; for the preparation of C2-Zn1-HEHE-2, C2-Zn2-HEHE-2, C3-Zn2-HEHE-19, C4-Zn1-HEHE-1, C5-Zn1-HEHE-1, Heter-Zn2-HEHE-22, Cage-t32-Zn1-HEHE-34, and Cage-t32-Zn1-HEHE-36 protein nanoparticles, the protein solution contains 1 mM zinc salt; for the preparation of C3-Ni1-HH*3-18 protein nanoparticles, the protein solution contains 1 mM nickel salt; for the preparation of C3-Ca1-DN*3-20 and C3-Ca1-DDDD-48 protein nanoparticles, the protein solution contains 10 mM calcium salt.

[0054] In a specific embodiment, after adding the metal salt solution in step S2, the mixture is allowed to stand for 2-15 minutes.

[0055] In a specific embodiment, in step S3, protein nanoparticles are purified by molecular sieve chromatography.

[0056] In another aspect, the present invention provides use of the self-assembled protein nanoparticles in preparing a vaccine scaffold.

[0057] In another aspect, the present invention provides a vaccine scaffold comprising the self-assembled protein nanoparticles.

[0058] In yet another aspect, the present invention provides a conjugate vaccine, which comprises at least the self-assembled protein nanoparticles as described above as a vaccine scaffold to display one or more antigens.

[0059] Beneficial Effects

[0060] The protein monomer Cage-i53-Zn1-HEHE-5 designed in this application can be induced by zinc ions to self-assemble to form protein nanoparticles. In the absence of zinc ions in the solution, Cage-i53-Zn1-HEHE-5 exists in the form of protein monomers. When zinc ions are present in the solution and the amount of zinc ions is twice that of Cage-i53-Zn1-HEHE-5, Cage-i53-Zn1-HEHE-5 will bind to the zinc ions to form an aggregate of 60 protein monomers (60-mers), also referred to herein as protein nanoparticles.

[0061] The binding of Cage-i53-Zn1-HEHE-5 with zinc ions is achieved through amino acids on the surface of Cage-i53-Zn1-HEHE-5 protein. In nature, zinc ions can interact with histidine and aspartic acid on proteins and form tetrahedral coordination. With the help of this principle, the present invention designs histidine and aspartic acid that can interact with zinc ions on the surface of proteins, while simulating the assembly form of target proteins with zinc ions, and further designs amino acids in other regions, and finally obtains protein monomer Cage-i53-Zn1-HEHE-5. Cage-i53-Zn1-HEHE-5 can present protein monomer form in the absence of zinc ions, and assemble into protein nanoparticles in the presence of zinc ions.

[0062] At the same time, the present application also designs more types of proteins with reversible zinc ion binding, including dimers C2-Zn1-HEHE-2, C2-Zn2-HEHE-2, trimer C3-Zn2-HEHE-19, tetramer C4-Zn1-HEHE-1, pentamer C5-Zn1-HEHE-1, heterodimer Heter-Zn2-HEHE-22, dodecamer Cage-t32-Zn1-HEHE-34, Cage-t32-Zn1-HEHE-35, Cage-t32-Zn1-HEHE-36, and hexadecamer Cage e-i53-Zn1-HEHE-14, Cage-i53-Zn1-HEHE-16, Cage-i53-Zn1-HEHE-6, protein microfilaments Fiber-n6-Zn1-HEHE-5, Fiber-n7-Zn1-HEHE-74, Fiber-n5-Zn1-HEHE-917, Fiber-n6-Zn1-HEHE-43, Fiber-n6-Zn1-HEHE-44, Fiber-n6-Zn1-HEHE-46, Fiber-n6-Zn1-HEHE-410. In addition, the present application also designs more types of proteins with reversible nickel ion binding, C3-Ni1-HH*3-18 trimer and proteins with reversible calcium ion binding, C3-Ca1-DN*3-20 trimer, C3-Ca1-DDDD-48 trimer. These proteins can exist as monomers in the absence of metal ions and assemble in the presence of metal ions.

[0063] The metal ion-induced protein nanoparticles constructed by protein design in this application perfectly solve the problem of uncontrollable aggregation and dissociation of conventional protein nanoparticles. At the same time, as single-component protein nanoparticles, protein monomers are easier to assemble, and there is no need to add other protein components that may cause immunity, which greatly improves safety. Therefore, these protein nanoparticles can be developed as vaccine scaffolds for displaying one or more protein antigens, which has broad prospects in vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 : SDS-PAGE gel image of Cage-i53-Zn1-HEHE-5 protein.

[0065] Figure 2: Molecular sieve chromatography spectrum of Cage-i53-Zn1-HEHE-5. The blue solid line in the figure is the molecular sieve chromatography spectrum of Cage-i53-Zn1-HEHE-5 mixed with EDTA, the red solid line is the molecular sieve chromatography spectrum of protein nanoparticles formed by Cage-i53-Zn1-HEHE-5 and zinc ions, and the gray dotted line is the molecular sieve chromatography spectrum of protein nanoparticles formed by Cage-i53-Zn1-HEHE-5 and zinc ions and then dissociated into monomers by adding EDTA.

[0066] Figure 3 : Analytical ultracentrifugation (AUC) analysis of nanoparticles and monomers of Cage-i53-Zn1-HEHE-5.

[0067] Figure 4 : Cryo-electron microscopy image of Cage-i53-Zn1-HEHE-5 nanoparticles.

[0068] Figure 5 : Density map of reconstructed electron microscopy results of Cage-i53-Zn1-HEHE-5 protein nanoparticles.

[0069] Figure 6 : Resolution image of reconstructed electron microscopy results of Cage-i53-Zn1-HEHE-5 protein nanoparticles.

[0070] Figure 7 : Negative staining photos of CFP-Cage-i53-Zn1-HEHE-5 protein nanoparticles.

[0071] Figure 8 : Emission spectra of Cage-i53-Zn1-HEHE-5 protein nanoparticles linked with different fluorescent proteins under excitation light of 433 nm. Red is the emission spectrum of protein nanoparticles composed of CFP-Cage-i53-Zn1-HEHE-5 and YFP-Cage-i53-Zn1-HEHE-5 (FRET); black is the emission spectrum of protein nanoparticles composed of CFP-Cage-i53-Zn1-HEHE-5 and YFP-Cage-i53-Zn1-HEHE-5 mixed with EDTA (FRET+EDTA); purple is the emission spectrum of protein nanoparticles composed of CFP-Cage-i53-Zn1-HEHE-5 alone and protein nanoparticles composed of YFP-Cage-i53-Zn1-HEHE-5 alone (control); orange is the emission spectrum of protein nanoparticles composed of CFP-Cage-i53-Zn1-HEHE-5 alone and protein nanoparticles composed of YFP-Cage-i53-Zn1-HEHE-5 alone and EDTA (control+EDTA).

[0072] Fig. 9 : Crystal structures of dimers, trimers, tetramers, pentamers, and heterodimers constructed in Example 6. A is the crystal structure of a C2-Zn1-HEHE-2 dimer, B is the crystal structure of a C2-Zn2-HEHE-2 dimer, C is the crystal structure of a C3-Zn2-HEHE-19 trimer, D is the crystal structure of a C4-Zn1-HEHE-1 tetramer, E is the crystal structure of a C5-Zn1-HEHE-1 pentamer, F is the crystal structure of a Heter-Zn2-HEHE-22 heterodimer, G is the crystal structure of a C3-Ni1-HH*3-18 trimer, and H is the crystal structure of a C3-Ca1-DN*3-20 trimer. In the figure, the colored spiral structures are protein molecules, purple is zinc ions, blue is nickel ions, and green is calcium ions.

[0073] Fig.10 : Density maps of cryo-EM reconstruction of dodecamers, hexamers, and protein microfilaments constructed in Example 6. A is the electron microscopy density map of Cage-t32-Zn1-HEHE-34 dodecamers, B is the electron microscopy density map of Cage-t32-Zn1-HEHE-35 dodecamers, C is the electron microscopy density map of Cage-t32-Zn1-HEHE-36 dodecamers, D is the electron microscopy density map of Cage-i53-Zn1-HEHE-14 hexamers, E is the electron microscopy density map of Cage-i53-Zn1-HEHE-16 hexamers, F is the electron microscopy density map of Cage-i53-Zn1-HEHE-6 hexamers, G is the electron microscopy density map of Fiber-n6-Zn1-HEHE The electron microscope density map of HE-5 protein filaments, H is the electron microscope density map of Fiber-n6-Zn1-HEHE-74 protein filaments, I is the electron microscope density map of Fiber-n6-Zn1-HEHE-917 protein filaments, J is the electron microscope density map of Fiber-n6-Zn1-HEHE-43 protein filaments, K is the electron microscope density map of Fiber-n6-Zn1-HEHE-44 protein filaments, L is the electron microscope density map of Fiber-n6-Zn1-HEHE-46 protein filaments, and M is the electron microscope density map of Fiber-n6-Zn1-HEHE-410 protein filaments.

[0074] Fig.11 : Designed structure of C3-Ca1-DDDD-48 trimer protein.

[0075] Fig.12: AUC data graphs of dimers, trimers, tetramers, pentamers, heterodimers, dodecamers, and hexamers constructed in Example 6. A is the AUC data graph of C2-Zn1-HEHE-2 and its dimer, B is the AUC data graph of C2-Zn2-HEHE-2 and its dimer, C is the AUC data graph of C3-Zn2-HEHE-19 and its trimer, D is the AUC data graph of C4-Zn1-HEHE-1 and its tetramer, E is the AUC data graph of C5-Zn1-HEHE-1 and its pentamer, F is the AUC data graph of Heter-Zn2-HEHE-22a, Heter-Zn2-HEHE-22b and their heterodimer Heter-Zn2-HEHE-22, G is the AUC data graph of Cage-t32-Zn1-HEHE-34 and its dodecamer AUC data graph, H is the AUC data graph of Cage-t32-Zn1-HEHE-35 and its dodecamer, I is the AUC data graph of Cage-t32-Zn1-HEHE-36 and its dodecamer, J is the AUC data graph of Cage-i53-Zn1-HEHE-14 and its sixty-mer, K is the AUC data graph of Cage-i53-Zn1-HEHE-16 and its sixty-mer, L is the AUC data graph of Cage-i53-Zn1-HEHE-6 and its sixty-mer, M is the AUC data graph of C3-Ni1-HH*3-18 and its trimer, and N is the AUC data graph of C3-Ca1-DN*3-20 and its trimer.

[0076] Fig.13: SEC data graphs of dimers, trimers, tetramers, pentamers, heterodimers, dodecamers, and hexamers constructed in Example 6. A is the SEC data graph of C2-Zn1-HEHE-2 and its dimer, B is the SEC data graph of C2-Zn2-HEHE-2 and its dimer, C is the SEC data graph of C3-Zn2-HEHE-19 and its trimer, D is the SEC data graph of C4-Zn1-HEHE-1 and its tetramer, E is the SEC data graph of C5-Zn1-HEHE-1 and its pentamer, F is the SEC data graph of Heter-Zn2-HEHE-22a, Heter-Zn2-HEHE-22b and their heterodimer Heter-Zn2-HEHE-22, G is the SEC data graph of Cage-t32-Zn1-HEHE-34 and its dodecamer, H is the SEC data graph of Cage-t SEC data chart of Cage-t32-Zn1-HEHE-35 and its dodecamer, I is the SEC data chart of Cage-t32-Zn1-HEHE-36 and its dodecamer, J is the SEC data chart of Cage-i53-Zn1-HEHE-14 and its sixty-mer, K is the SEC data chart of Cage-i53-Zn1-HEHE-16 and its sixty-mer, L is the SEC data chart of Cage-i53-Zn1-HEHE-6 and its sixty-mer, M is the SEC data chart of C3-Ni1-HH*3-18 and its trimer, N is the SEC data chart of C3-Ca1-DN*3-20 and its trimer, and O is the SEC data chart of C3-Ca1-DDDD-48 and its trimer.

[0077] Fig.14 : Schematic diagram of the protein nanoparticles of Cage-i53-Zn1-HEHE-5 displaying CFP in Example 4. DETAILED DESCRIPTION

[0078] Hereinafter, the technical solution of the present application is described in detail through specific embodiments so that those skilled in the art can better understand the present invention. However, these embodiments are not intended to limit the scope of the present application.

[0079] the term

[0080] In this article, protein monomer refers to a single protein that is not assembled.

[0081] In this article, protein nanoparticles refer to protein aggregates formed by self-assembly of protein monomers under the induction of metal ions.

[0082] Example 1: Expression and purification of Cage-i53-Zn1-HEHE-5 protein and its self-assembly to form nanoparticles

[0083] The gene encoding Cage-i53-Zn1-HEHE-5 (SEQ ID No.: 1) was cloned into the pET-28a (+) E. coli expression vector with an 8×His tag and an HRV3C protease cleavage site (MGHHHHHHHHSSGLEVLFQGPGGT, SEQ ID No.: 2) at the N-terminus. The cloned plasmid was transformed into E. coli BL21 (DE3) (EC1002, WEIDIBio), cultured in 1L LB broth at 37°C, 220RPM until OD600 reached 0.6-0.8, IPTG (206-703-0, MACKLIN) was added at a final concentration of 1mM, and cultured overnight at 37°C 220RPM. The cells were collected by centrifugation at 5000g for 5 minutes, resuspended in 40ml protein buffer (25mM Tris-HCL (pH 7.0) + 150mM NaCl), and lysed by ultrasonication for 10 minutes. The whole cell lysate was separated by centrifugation at 14000RPM for 30 minutes, and the target protein was purified from the supernatant using Ni-NTA resin. The target protein was sampled and run on 15% SurePAGE (M00719, GenScript). See the protein gel for the protein gel image. Figure 1 .

[0084] The target protein solution was mixed with HRV 3C protease, dialyzed and digested overnight in 1L protein buffer to cut the His tag at the N-terminus of the protein. The dialyzed protein solution was centrifuged at 4000g for 10 minutes to obtain the supernatant, and the protein not cleaved by the protease was removed from the supernatant using Ni-NTA resin. EDTA with a final concentration of 1mM was added to the treated protein solution, and after concentration, the target protein was further purified in the protein buffer using a molecular sieve chromatography Superdex 200Increase 10 / 300GL column (28990944, Cytiva). The protein solution obtained by the molecular sieve was measured for protein concentration using NanoPhotometer N50 (implen), and the protein molar concentration was calculated by the protein molecular weight. Zinc sulfate solution was added to the protein solution so that the final concentration of zinc sulfate in the solution was equal to twice the protein molar concentration. After waiting for several minutes, the protein nanoparticles formed by Cage-i53-Zn1-HEHE-5 and zinc ions (hereinafter referred to as Cage-i53-Zn1-HEHE-5 protein nanoparticles) were collected in protein buffer using molecular sieve chromatography Superose 6 Increase 10 / 300GL column (29091596, Cytiva). The chromatogram is shown in Figure 2 .

[0085] Cage-i53-Zn1-HEHE-5 was mixed with EDTA (which can react with Mg 2+ , Ca2+ , Mn 2+ 、Zn 2+ The binding of divalent metal ions such as Cage-i53-Zn1-HEHE-5 to divalent ions can be prevented) and the peak position of size exclusion chromatography (SEC) is the molecular weight of a single protein molecule Cage-i53-Zn1-HEHE-5. Figure 2 The results show that when Cage-i53-Zn1-HEHE-5 is mixed with zinc ions, the SEC peak position moves forward, proving that Cage-i53-Zn1-HEHE-5 forms nanoparticles. Cage-i53-Zn1-HEHE-5 aggregates after adding zinc ions, and then depolymerizes by adding EDTA, which shows that the protein has controllable aggregation and depolymerization capabilities.

[0086] Example 2: Determination of protein molecular weight and sedimentation coefficient using analytical ultracentrifuge (AUC).

[0087] The Cage-i53-Zn1-HEHE-5 protein nanoparticles and Cage-i53-Zn1-HEHE-5 mixed with EDTA were used to measure the protein sedimentation coefficient by analytical ultracentrifuge (AUC). The AUC results are shown in Figure 3 shown.

[0088] from Figure 3 It can be seen that the Cage-i53-Zn1-HEHE-5 protein nanoparticles are uniform. According to AUC calculation, in the presence of EDTA, the molecular weight of Cage-i53-Zn1-HEHE-5 is 29.6kDa, which is close to the molecular weight of protein monomers. At the same time, the molecular weight of nanoparticles formed by the combination of Cage-i53-Zn1-HEHE-5 and zinc ions measured by AUC is 1.53MDa, which is close to the molecular weight of sixty-mer protein. This shows that under liquid conditions, Cage-i53-Zn1-HEHE-5 can be induced by zinc ions to form nanoparticles.

[0089] Example 3: Cryo-electron microscopy observation of Cage-i53-Zn1-HEHE-5 protein nanoparticles

[0090] Cryo-EM samples were prepared using the FEI Vitrobot Mark IV semi-automatic sample preparation device. The temperature of the FEI Vitrobot Mark IV was set to 8°C, the humidity was set to 100%, and the Blot Time was 3 seconds. In the Vitrobot, 3 μL of 10 mg / ml Cage-i53-Zn1-HEHE-5 protein nanoparticles were spotted onto the glow-discharged Quantifoil R1.2 / 1.3Cu 200 grid, quickly frozen and transferred, and stored in liquid nitrogen. The cryo-data were collected on a Glacios 2 cryo-EM with a Falcon 4i direct electron detector. The data collection conditions were: the electron microscope magnification was 120,000X; the Pixel Size was in super-resolution mode. Cryo-EM images are shown in Figure 4 .

[0091] Using cryoSPARC to process cryo-EM data, the resolution is The density map of the reconstructed electron microscopy results is shown in Figure 5 , resolution results are shown in Figure 6 .

[0092] Example 4: Determination of the ability of Cage-i53-Zn1-HEHE-5 protein nanoparticles to display proteins

[0093] To confirm that Cage-i53-Zn1-HEHE-5 has the potential to display other proteins, the gene encoding CFP (amino acid sequence SEQ ID No.: 25) (nucleotide sequence: SEQ ID No.: 24) and the gene of Cage-i53-Zn1-HEHE-5 were connected through 3*GGGS (SEQ ID No.: 3) sequences and cloned into the pET-28a (+) E. coli expression vector with an 8×His tag and HRV3C protease cleavage site at the N-terminus. The cloned plasmid was transformed into E. coli BL21 (DE3) (EC1002, WEIDIBio), cultured in 1L LB broth at 37°C, 220RPM until OD600 reached 0.6-0.8, the culture was cooled to 18°C, IPTG (206-703-0, MACKLIN) was added at a final concentration of 0.3mM, and cultured overnight at 18°C ​​220RPM. The CFP-Cage-i53-Zn1-HEHE-5 protein was obtained by the protein purification method mentioned in Example 1, and CFP-Cage-i53-Zn1-HEHE-5 protein nanoparticles were obtained by adding zinc sulfate solution.

[0094] Negatively stained samples were observed by Talos L120C electron microscope. The negatively stained photos are shown in Figure 7, confirming the ability of Cage-i53-Zn1-HEHE-5 to display other proteins. The schematic diagram of protein nanoparticles of Cage-i53-Zn1-HEHE-5 displaying CFP can be found in Fig.14 .

[0095] Example 5: Determination of the ability of Cage-i53-Zn1-HEHE-5 protein nanoparticles to display multiple proteins

[0096] This example uses fluorescence resonance energy transfer experiments to verify whether a single protein nanoparticle can display multiple proteins simultaneously.

[0097] Fluorescence resonance energy transfer (FRET) is when the fluorescence spectrum of a fluorescent molecule (also called a donor molecule) overlaps with the excitation spectrum of another fluorescent molecule (also called an acceptor molecule), the excitation energy of the donor fluorescent molecule induces the acceptor molecule to emit fluorescence, and at the same time the fluorescence intensity of the donor fluorescent molecule itself decays. Taking CFP and YFP in this experiment as an example, the fluorescence emitted by the donor CFP can be absorbed by YFP and excite YFP to emit yellow fluorescence. If FRET does not occur, only CFP emits light, and the emission spectrum will produce a fluorescence emission peak near 458nm; if FRET occurs, not only CFP will produce fluorescence, but YFP will also emit yellow fluorescence, that is, a fluorescence emission peak will be produced near 527nm.

[0098] The gene encoding CFP (amino acid sequence SEQ ID No.: 25, nucleotide sequence: SEQ ID No.: 24) or YFP (amino acid sequence SEQ ID No.: 27, nucleotide sequence: SEQ ID No.: 26) was connected to the gene of Cage-i53-Zn1-HEHE-5 through a 3*GGGS sequence and cloned into the pET-28a (+) E. coli expression vector with an 8×His tag and an HRV3C protease cleavage site at the N-terminus. The cloned plasmid was transformed into E. coli BL21 (DE3) (EC1002, WEIDIBio), cultured in 1L LB broth at 37°C, 220RPM until OD600 reached 0.6-0.8, the culture was cooled to 18°C, IPTG (206-703-0, MACKLIN) was added at a final concentration of 0.3mM, and cultured overnight at 18°C ​​220RPM. CFP-Cage-i53-Zn1-HEHE-5 protein and YFP-Cage-i53-Zn1-HEHE-5 protein were obtained by the protein purification method mentioned in Example 1.

[0099] The proteins of Cage-i53-Zn1-HEHE-5 linked to CFP and Cage-i53-Zn1-HEHE-5 linked to YFP were mixed at equimolar concentrations, and zinc ions in an amount twice that of the protein substance were added. After waiting for a few minutes, the protein nanoparticles composed of CFP-Cage-i53-Zn1-HEHE-5 and YFP-Cage-i53-Zn1-HEHE-5 were collected in protein buffer using a molecular sieve chromatography Superose 6Increase 10 / 300GL column (29091596, Cytiva), which were used as the FRET group.

[0100] Add zinc ions twice the amount of protein substance to the protein of Cage-i53-Zn1-HEHE-5 connected to CFP, wait for a few minutes, and collect protein nanoparticles connected only to CFP in protein buffer using molecular sieve chromatography Superose 6Increase 10 / 300GL column (29091596, Cytiva). Add zinc ions twice the amount of protein substance to the protein of Cage-i53-Zn1-HEHE-5 connected to YFP, wait for a few minutes, and collect protein nanoparticles connected only to YFP in protein buffer using molecular sieve chromatography Superose6Increase 10 / 300GL column (29091596, Cytiva). Afterwards, the above protein nanoparticles connected only to CFP and the protein nanoparticles connected only to YFP were mixed and used as the control group.

[0101] The emission spectrum of the protein nanoparticles was measured using an ELISA reader under an excitation light of 433 nm.

[0102] Add EDTA to the protein solution of the FRET group and the Control group at a final concentration of 1 mM, mix well, and let stand for 20 minutes (FRET+DETA and Control+EDTA groups, respectively), and then measure the emission spectrum. Figure 8 .

[0103] The emission spectrum showed that the protein nanoparticles composed of a mixture of two proteins (FRET group) produced a fluorescence emission peak near 527 nm compared with the other experimental groups (including FRET+DETA, Control, and Control+EDTA groups), indicating that fluorescence resonance energy transfer can occur, indicating that a single protein nanoparticle contains Cage-i53-Zn1-HEHE-5 connected to two fluorescent proteins at the same time.

[0104] Example 6. Construction and characterization of other protein nanoparticles

[0105] By means of protein design, the following protein aggregates in various aggregation forms were further obtained in a similar manner to that in Example 1 (wherein the nickel salt was nickel sulfate and the calcium salt was calcium chloride):

[0106] Dimer of C2-Zn1-HEHE-2, dimer of C2-Zn2-HEHE-2, trimer of C3-Zn2-HEHE-19, tetramer of C4-Zn1-HEHE-1, pentamer of C5-Zn1-HEHE-1, heterodimer of Heter-Zn2-HEHE-22, dodecamer of Cage-t32-Zn1-HEHE-34, dodecamer of Cage-t32-Zn1-HEHE-35, dodecamer of Cage-t32-Zn1-HEHE-36, hexamer of Cage-i53-Zn1-HEHE-14, hexamer of Cage-i53-Zn1-HEHE-16, hexamer of Cage-i5 3-Zn1-HEHE-6 sixtymer, Fiber-n6-Zn1-HEHE-5 protein microfilament, Fiber-n7-Zn1-HEHE-74 protein microfilament, Fiber-n5-Zn1-HEHE-917 protein microfilament, Fiber-n6-Zn1-HEHE-43 protein microfilament, Fiber-n6-Zn1-HEHE-44 protein microfilament, Fiber-n6-Zn1-HEHE-46 protein microfilament, Fiber-n6-Zn1-HEHE-410 protein microfilament, C3-Ni1-HH*3-18 trimer, C3-Ca1-DN*3-20 trimer, C3-Ca1-DDDD-48 trimer.

[0107] The amino acid sequences of the above protein monomers are shown in SEQ ID No.: 1, 5-23, 28-30 respectively:

[0108] Cage-i53-Zn1-HEHE-5:

[0109]

[0110] C2-Zn1-HEHE-2:

[0111]

[0112] C2-Zn2-HEHE-2:

[0113]

[0114] C3-Zn2-HEHE-19:

[0115]

[0116] C4-Zn1-HEHE-1:

[0117]

[0118] C5-Zn1-HEHE-1:

[0119]

[0120] Heter-Zn2-HEHE-22a:

[0121]

[0122] Heter-Zn2-HEHE-22b:

[0123]

[0124] Cage-t32-Zn1-HEHE-34:

[0125]

[0126] Cage-t32-Zn1-HEHE-35:

[0127]

[0128] Cage-t32-Zn1-HEHE-36:

[0129]

[0130]

[0131] Cage-i53-Zn1-HEHE-14:

[0132]

[0133] Cage-i53-Zn1-HEHE-16:

[0134]

[0135] Cage-i53-Zn1-HEHE-6:

[0136]

[0137] Fiber-n6-Zn1-HEHE-5:

[0138]

[0139] Fiber-n6-Zn1-HEHE-74:

[0140]

[0141] Fiber-n5-Zn1-HEHE-917:

[0142]

[0143] Fiber-n6-Zn1-HEHE-43:

[0144]

[0145] Fiber-n6-Zn1-HEHE-44:

[0146]

[0147] Fiber-n6-Zn1-HEHE-46:

[0148]

[0149] Fiber-n6-Zn1-HEHE-410:

[0150]

[0151] C3-Ni1-HH*3-18:

[0152]

[0153] C3-Ca1-DN*3-20:

[0154]

[0155] C3-Ca1-DDDD-48:

[0156]

[0157] CFP:

[0158]

[0159] CFP:

[0160]

[0161] YFP:

[0162]

[0163]

[0164] YFP:

[0165]

[0166] Among them, 50-60 mg / ml of protein containing equimolar concentration of zinc ions was added to the crystallization buffer to obtain crystals. The crystal diffraction data was obtained by diffraction using an ultra-high brightness X-ray single crystal diffractometer. The crystal structures of C2-Zn1-HEHE-2 dimer, C2-Zn2-HEHE-2 dimer, C3-Zn2-HEHE-19 trimer, C4-Zn1-HEHE-1 tetramer, C5-Zn1-HEHE-1 pentamer, Heter-Zn2-HEHE-22 heterodimer, C3-Ni1-HH*3-18 trimer, and C3-Ca1-DN*3-20 trimer were calculated. Their crystal structures are shown in Fig. 9 The figure shows that protein monomers can aggregate into polymeric forms in the presence of metal ions.

[0167] In the same manner as in Example 3, the cryo-EM reconstruction results of Cage-t32-Zn1-HEHE-34, Cage-t32-Zn1-HEHE-35, and Cage-t32-Zn1-HEHE-36 dodecamers, Cage-i53-Zn1-HEHE-14, Cage-i53-Zn1-HEHE-16, and Cage-i53-Zn1-HEHE-6 hexamers, and Fiber-n6-Zn1-HEHE-5, Fiber-n7-Zn1-HEHE-74, Fiber-n5-Zn1-HEHE-917, Fiber-n6-Zn1-HEHE-43, Fiber-n6-Zn1-HEHE-44, Fiber-n6-Zn1-HEHE-46, and Fiber-n6-Zn1-HEHE-410 protein microfilaments were obtained. The density map is shown in the figure. Fig.10 , which clearly shows that protein monomers aggregate into dodecamers, hexamers, and protein filaments in the presence of metal ions.

[0168] Fig.11 The structure of the designed C3-Ca1-DDDD-48 trimer protein is shown, and the figure shows that in the presence of calcium ions, the protein monomers aggregate into trimers.

[0169] In the same manner as in Examples 1 and 2, the AUC and SEC data of the above different polymers were obtained as shown in Fig.12 and 13 . Fig.12 The AUC data plot shows the change in protein molecular weight in the presence or absence of metal ions. Fig.13The SEC data of the graph shows that in the presence of metal ions, the protein peak shifts forward significantly, and when EDTA is added to the protein aggregate, the protein peak returns to the control group level (blue line). This indicates that protein monomers can achieve reversible binding of metal ions. Therefore, Fig.12 and 13 It was confirmed that the present application successfully obtained protein nanoparticles in various aggregate forms.

Claims

1. A self-assembled protein nanoparticle, which is formed by self-assembly of protein monomers by metal ion-induced protein monomers, The metal ions are selected from zinc ions, nickel ions and calcium ions, For protein nanoparticles formed by self-assembly of protein monomers induced by zinc ions, The zinc ions interact with histidine and aspartic acid on the surface of the protein monomer and form tetrahedral coordination, thereby forming the protein nanoparticles; Protein nanoparticles formed by self-assembly of protein monomers induced by nickel ions, wherein the nickel ions interact with histidine on the surface of the protein monomers to form octahedral coordination, thereby forming the protein nanoparticles; and Regarding the protein nanoparticles formed by self-assembly of protein monomers induced by calcium ions, calcium ions interact with aspartic acid and asparagine on the surface of the protein monomers and form octahedral coordination, thereby forming the protein nanoparticles.

2. The protein nanoparticle according to claim 1, wherein The protein monomer has the following amino acid sequence: SEQ ID No.: any one of 1, 4-23, 28-30.

3. The protein nanoparticle according to claim 1, wherein The self-assembled protein nanoparticles are polymers or protein microfilaments formed by the protein monomers, and the polymers are 2 to 60 polymers of protein monomers.

4. The protein nanoparticle according to claim 1, wherein (i) Zinc ion induced self-assembled protein nanoparticles are selected from the following: A homogenous hexameric protein monomer having the sequence SEQ ID No.: 1; A homodimer of a protein monomer having a sequence of SEQ ID No.: 4; A homodimer of a protein monomer having a sequence of SEQ ID No.: 5; A homotrimer of a protein monomer having a sequence of SEQ ID No.: 6; A homotetramer of a protein monomer having a sequence of SEQ ID No.: 7; A homopentamer of a protein monomer having a sequence of SEQ ID No.: 8; A heterodimer of protein monomers having sequences of SEQ ID No.: 9 and SEQ ID No.: 10; A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 11; A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 12; A homologous dodecamer of a protein monomer having a sequence of SEQ ID No.: 13; A homogenous hexameric protein monomer having the sequence SEQ ID No.: 14; A homogenous hexameric protein monomer having the sequence SEQ ID No.: 15; A homogenous hexameric protein monomer having the sequence SEQ ID No.: 16; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 17; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 18; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 19; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 20; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 21; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 22; A homologous protein microfilament of a protein monomer having a sequence of SEQ ID No.: 23, (ii) the protein nanoparticles self-assembled by nickel ion are homotrimers of the protein monomers with the sequence of SEQ ID No.: 28, (iii) The calcium ion-induced self-assembled protein nanoparticles are homotrimers of protein monomers whose sequences are selected from SEQ ID No.: 29 and SEQ ID No.:

30.

5. A nucleic acid molecule encoding the protein monomer according to claim 4. A recombinant vector comprising the nucleic acid molecule according to claim 5 . A host cell comprising the recombinant vector according to claim 6 .

8. A method for preparing self-assembled protein nanoparticles according to any one of claims 1 to 4, comprising: S1: providing a solution containing the protein monomer; S2: adding zinc salt, nickel salt or calcium salt solution to the solution of step S1 and letting it stand; S3: Purification to obtain protein nanoparticles.

9. Use of the self-assembled protein nanoparticles according to any one of claims 1 to 4 in the preparation of vaccine scaffolds.

10. A vaccine scaffold comprising the self-assembled protein nanoparticles according to any one of claims 1 to 4.

11. A conjugate vaccine, comprising at least the self-assembled protein nanoparticles according to any one of claims 1 to 4 as a vaccine scaffold to display one or more antigens.

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