Binding peptide capable of enhancing binding rate and application thereof

Abstract

The present invention relates to a polypeptide forming one part of a two-part linker wherein the polypeptide (protein) spontaneously forms an isopeptide bond with a peptide tag, i.e. The other part of the two-part linker. The invention also relates to a method for producing a polypeptide of the invention and to the use of said polypeptide. According to the present invention, the reaction rate of the Sdy-Catcher variant obtained through mutation is significantly improved compared to the reaction rate of Sdy-Tag, such that the ideal direction is provided for the searching of polypeptides with characteristics of high binding rate and good binding effect in the field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a polypeptide forming one part of a two-part linker, wherein the polypeptide (protein) spontaneously forms an isopeptide bond with a peptide tag, i.e. the other part of the two-part linker. It also relates to a method for producing the polypeptide of the present application and the use of the polypeptide. BACKGROUND

[0002] Isopeptide bonds found in nature are usually catalyzed by glutamine transferase, ubiquitin ligase or transpeptidase. The covalent bond formed by the spontaneous combination of the lysine (Lys) side chain amino group with the asparagine (Asn) side chain amide group or the aspartic acid (Asp) side chain carboxyl group has high specificity and stability. In 2007, Kang et al. first discovered an intramolecular isopeptide bond that can spontaneously form in the Pilin protein (Pilin) Spy0128 of the gram-positive bacterium Streptococcus pyogenes. According to the differences in species origin and peptide segment properties, the commonly used isopeptide bond molecular adhesives are mainly divided into isopeptag-N / pilin-N, isopeptag / pilin-C, SnoopTag / SnoopCatcher, Spy-Tag / SpyCatcher and SdyTag / SdyCatcher. Among them, pilin-N, pilin-C, SnoopCatcher, SpyCatcher and SdyCatcher belong to peptide trapper, as one part of the two-part linker, isopepag-N, isopeptag, SnoopTag, Spy-Tag and SdyTag belong to peptide tag, as the other part of the two-part linker, the above-mentioned one part of the two-part linker as peptide trapper spontaneously forms an isopeptide bond with the other part of the two-part linker.

[0003] Polypeptides are connected by catalyzing covalent bonds, and this coupling method has been widely used in protein fusion, modification, etc. Isopeptide bonds are widely found in the pilin protein of gram-positive bacteria, among which Spy has been found in the fibronectin of the gram-positive bacterium Streptococcus pyogenes. This protein contains an N-terminal domain carrying an active lysine residue and a C-terminal domain carrying an asparagine, which are called Spy-Catcher and Spy-Tag, respectively.

[0004] Subsequently, the Wong team (Fong T. Wong et al., Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly, PLOS ONE, 2016 Oct. 26: 1-15) isolated SdyTag and SdyCatcher from Streptococcus dysgalactiae, which can also form isopeptide bonds through lysine residues and aspartic acid. Studies have shown that the selectivity of SpyCatcher for Spy-Tag is 320 times that of SdyTag, and the selectivity of SdyTag for SdyCatcher is 75 times that of SpyCatcher.

[0005] Currently, although the Spy-Tag / SpyCatcher technology has been used for various applications, its reaction speed and efficiency need to be improved, and the research and optimization of SdyTag and SdyCatcher discovered later are not deep enough.

[0006] Therefore, in order to solve the above-mentioned problems existing at present, it is urgent to find a more efficient isopeptide bond molecular adhesive in terms of binding rate and other aspects, such as a binding polypeptide formed by modifying and optimizing the existing SdyTag and SdyCatcher, so as to better meet the needs of establishing a more optimal connection system. SUMMARY

[0007] Based on the above-mentioned problems, the purpose of the present application is to provide a polypeptide which forms an isopeptide bond with a peptide tag, i.e. another part of the two-part linker, spontaneously as one part of the two-part linker. Wherein, the polypeptide is also called binding peptide 2, and the "peptide tag" or "another part of the two-part linker" is also called binding peptide 1.

[0008] In a first aspect, the present application provides a polypeptide, the polypeptide sequence as one part of a two-part linker, also known as "peptide catcher", the polypeptide comprising a reference sequence SdyCatcher or a variant (mutant molecule) of SdyCatcher.

[0009] As used herein, the term "variant (mutant molecule)" refers to a peptide or polypeptide whose amino acid sequence is changed by insertion, deletion or substitution of one or more amino acids compared with the reference sequence SdyCatcher, which improves at least one aspect of biological activity.

[0010] In some embodiments, the reference sequence is SdyCatcher.

[0011] In some embodiments, the variant (mutant molecule) of SdyCatcher has an amino acid sequence that differs from the reference sequence SdyCatcher by one or more insertions, deletions or substitutions of amino acids, and wherein the variant has improved at least one aspect of biological activity relative to the reference sequence SdyCatcher.

[0012] In some embodiments, the variant (mutant molecule) of SdyCatcher has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 92%; more preferably at least 94%; more preferably at least 95%, 96%, 98%, 99%, or 99.5% sequence identity with the reference sequence SdyCatcher.

[0013] In some embodiments, the variant (mutant molecule) of SdyCatcher has an amino acid sequence that differs from the reference sequence SdyCatcher by one or more mutations (insertions, deletions or substitutions) in the amino acid sequence of the reference sequence, while improving at least one aspect of biological activity of the reference sequence. The one or more typically means 1-10, preferably 9, more preferably 8, more preferably 7, more preferably 6, more preferably 5, more preferably 4, more preferably 3, more preferably 2, or 1. The substitutions can be non-conservative substitutions or conservative substitutions.

[0014] In some embodiments, the variant (mutant molecule) has an amino acid sequence that can contain conservative substitutions, while retaining the improved at least one aspect of biological activity of the variant relative to the reference sequence.

[0015] As used herein, the term "conservative substitutions" means that in the art, conservative substitutions are typically made with amino acids of similar or analogous properties, which do not substantially alter the function of the protein or polypeptide. "Amino acids of similar or analogous properties" include, for example, families of amino acid residues with similar side chains, including families with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more sites in a polypeptide of the application with another amino acid residue from the same side chain class will not substantially affect its activity.

[0016] Preferably, the amino acid structure of the SdyCatcher is as shown in SEQ ID NO: 1, which is named as "Sdy-Catcher-FL":

[0017] MIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDANGKELAGAMIELRNLSGQTIQSWISDGTVKVFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDS (SEQ ID NO: 1).

[0018] Further, for purification consideration, a 6xHis tag can be added to the N-terminus of the SdyCatcher molecule, including but not limited to adding a 6xHis tag to the N-terminus of the above "Sdy-Catcher-FL" molecule, the amino acid structure of which is adding a 6xHis tag to the N-terminus of the sequence of SEQ ID NO: 1, i.e. SEQ ID NO: 2, the sequence structure of which is as follows:

[0019] MHHHHHHIDTMSGLSGETGQSGNTTIEEDSTTHVKFSKRDANGKELAGAMIELRNLSGQTIQSWISDGTVKVFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDS (SEQ ID NO: 2).

[0020] In a second aspect, the present application provides a polypeptide, the sequence of which comprises SEQ ID NO: 1 or a variant sequence thereof (mutant molecule).

[0021] Preferably, the variant sequence of SEQ ID NO: 1 comprises 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, 30, 31, 32 amino acid deletions at the N-terminus or C-terminus of SEQ ID NO: 1, more preferably 10, 21, 22, 23, 24, 25, 26 amino acid deletions at the N-terminus of SEQ ID NO: 1.

[0022] Preferably, the variant sequence of SEQ ID NO: 1 comprises 10, 21, 22, 23, 24, 25, 26 amino acid deletions at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3, 4, or 5 amino acid mutations, or a combination of the above mutations. The mutations can be any mutation that improves or enhances at least one aspect of the biological activity of the polypeptide.

[0023] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 22 amino acids at the N-terminus of SEQ ID NO: 1.

[0024] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1.

[0025] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2 or 3 amino acid mutations, or a combination thereof. The mutations can be any mutation that results in an improvement or an amelioration of at least one biological activity of the polypeptide.

[0026] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3 amino acid mutations, or a combination thereof, at positions corresponding to positions 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 of the sequence of SEQ ID NO: 1.

[0027] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3 amino acid mutations, or a combination thereof, at positions corresponding to positions 29, 41, 45, 50, 66, 70, 87, 88, 89 or 98 of the sequence of SEQ ID NO: 1. The mutations can be any mutation that results in an improvement or an amelioration of at least one biological activity of the polypeptide.

[0028] Preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3 amino acid mutations, or a combination thereof. The mutations can be any mutation that results in an improvement or an amelioration of at least one biological activity of the polypeptide.

[0029] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains 1, 2, 3 amino acid mutations or combinations thereof, the mutations being at positions corresponding to 29, 41, 45, 50, 66, 70, 87, 88, 89 or 98 of the sequence of SEQ ID NO: 1, the mutations being selected from the group consisting of mutating isoleucine (I) at said position to threonine (T), mutating leucine (L) at said position to serine (S), mutating valine (V) at said position to aspartic acid (D), mutating lysine (K) at said position to arginine (R), mutating lysine (K) at said position to aspartic acid (D), mutating alanine (A) at said position to threonine (T), mutating methionine (M) at said position to serine (S), and mutating leucine (L) at said position to threonine (T).

[0030] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating I at position 45 of the sequence of SEQ ID NO: 1 to T.

[0031] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating L at position 50 of the sequence of SEQ ID NO: 1 to S.

[0032] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating V at position 66 of the sequence of SEQ ID NO: 1 to D.

[0033] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating K at position 29 and K at position 98 of the sequence of SEQ ID NO: 1 to R and D, respectively.

[0034] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating A at position 41 of the sequence of SEQ ID NO: 1 to T.

[0035] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 23 amino acids are deleted from the N-terminus of SEQ ID NO: 1, and further contains mutating M at position 70 of the sequence of SEQ ID NO: 1 to S.

[0036] Preferably, the variant sequence of SEQ ID NO: 1 is that 23 amino acids are deleted from N-terminus of SEQ ID NO: 1, while L at position 87 of the sequence of SEQ ID NO: 1 is mutated to T.

[0037] Preferably, the variant sequence of SEQ ID NO: 1 is that 23 amino acids are deleted from N-terminus of SEQ ID NO: 1, while A at position 88 of the sequence of SEQ ID NO: 1 is mutated to T.

[0038] Preferably, the variant sequence of SEQ ID NO: 1 is that 23 amino acids are deleted from N-terminus of SEQ ID NO: 1, while A at position 89 of the sequence of SEQ ID NO: 1 is mutated to T.

[0039] Preferably, the variant sequence of SEQ ID NO: 1 is that 10, 21, 22, 23, 24, 25, 26 amino acids are deleted from N-terminus of SEQ ID NO: 1, while another functional polypeptide sequence is fused to N-terminus or C-terminus (the "another functional polypeptide sequence fused to N-terminus or C-terminus" can also be referred to as "substitution").

[0040] More preferably, the another functional polypeptide sequence comprises a molecule capable of binding peptide function selected from SEQ GQSGDM, etc.

[0041] Further, the SdyCatcher and the polypeptide sequence optionally comprise a signal peptide at N-terminus, and a histidine tag at N-terminus or C-terminus.

[0042] Further, the variant sequence of SEQ ID NO: 1 comprises a sequence as set forth in SEQ ID NO: 3 (Sdy-Catcher-01), a sequence as set forth in SEQ ID NO: 4 (Sdy-Catcher-02), a sequence as set forth in SEQ ID NO: 5 (Sdy-Catcher-03), a sequence as set forth in SEQ ID NO: 6 (Sdy-Catcher-04), a sequence as set forth in SEQ ID NO: 7 (Sdy-Catcher-05), a sequence as set forth in SEQ ID NO: 8 (Sdy-Catcher-06), a sequence as set forth in SEQ ID NO: 9 (Sdy-Catcher-07), a sequence as set forth in SEQ ID NO: 10 (Sdy-Catcher-08), a sequence as set forth in SEQ ID NO: 11 (Sdy-Catcher-09), a sequence as set forth in SEQ ID NO: 12 (Sdy-Catcher-10), a sequence as set forth in SEQ ID NO: 13 (Sdy-Catcher-11), a sequence as set forth in SEQ ID NO: 14 (Sdy-Catcher-12), a sequence as set forth in SEQ ID NO: 15 (Sdy-Catcher-13), a sequence as set forth in SEQ ID NO: 16 (Sdy-Catcher-14), a sequence as set forth in SEQ ID NO: 17 (Sdy-Catcher-15), a sequence as set forth in SEQ ID NO: 18 (Sdy-Catcher-16), a sequence as set forth in SEQ ID NO: 19 (Sdy-Catcher-17), a sequence as set forth in SEQ ID NO: 20 (Sdy-Catcher-18), a sequence as set forth in SEQ ID NO: 21 (SdCdn-Catcher).

[0043] In some embodiments, the variant of SEQ ID NO: 1 comprises conservative substitutions based on a sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and retains improved biological activity of the reference sequence.

[0044] The Sdy-Catcher mutant molecule and Spy-Tag binding rate constant (Rate constant, (M -1 min -1 )10 2 ) is 2-400, preferably 50-350, more preferably 100-300.

[0045] Further, the variant according to any of the embodiments herein comprises a polypeptide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 92%; more preferably at least 94%; more preferably at least 95%, such as at least 96%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21) and retains the improved biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp of NCBI.

[0046] In a third aspect, the present application provides a polynucleotide encoding the polypeptide described above.

[0047] In a fourth aspect, the present application provides an expression vector comprising the polynucleotide described above.

[0048] In a fifth aspect, the present application provides a host cell comprising the expression vector described above, wherein the host cell is selected from a eukaryotic cell or a prokaryotic cell.

[0049] The eukaryotic cell is preferably a CHO cell, a yeast cell, a HEK293, a BHK-21, a C127, a MDCK, a NAMALWA, a VERO, a SP2 / 0, a COS, and the yeast cell is more preferably a Pichia pastoris or a Saccharomyces cerevisiae.

[0050] The prokaryotic cell is preferably an E. coli cell, and the E. coli cell is more preferably a BL21(DE3), a BL21(DE3)pLysS, a BL21 Star(DE3), a BL21-CodonPlus(DE3)-RIPL, a Rosetta 2(DE3) or a Rosetta 2(DE3)pLysS.

[0051] In a sixth aspect, the present application provides a method for preparing the polypeptide:

[0052] (1) linking the nucleotide sequence expressing the polypeptide into an expression vector respectively to construct an expression recombinant plasmid vector;

[0053] (2) transforming or transfecting a host cell;

[0054] (3) expressing the recombinant protein by using the host cell and purifying the recombinant protein;

[0055] The nucleotide can be codon optimized according to the codon bias of the expression system.

[0056] The host cell is selected from a eukaryotic cell or a prokaryotic cell, the eukaryotic cell is preferably a CHO cell, a yeast cell, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS, the yeast cell is more preferably Pichia pastoris or Saccharomyces cerevisiae, and the prokaryotic cell is preferably an Escherichia coli cell, and the Escherichia coli cell is more preferably BL21 (DE3), BL21 (DE3) pLysS, BL21 Star (DE3), BL21-CodonPlus (DE3)-RIPL, Rosetta 2 (DE3) or Rosetta 2 (DE3) pLysS.

[0057] In a seventh aspect, the present application provides a method for self-assembling two molecules or components by the polypeptide as the binding peptide 2 forming an isopeptide bond with the binding peptide 1 and through the isopeptide bond.

[0058] Preferably, the molecule or component can be: the molecule or component (i.e. entity) can be a nucleic acid molecule, a protein (e.g. an antibody or antigen-binding fragment thereof, an antigenic protein or immunogen thereof), a peptide, a small molecule organic compound, a fluorophore, a metal ligand complex, a polysaccharide, a nanoparticle, a 2D monolayer (e.g. graphene), a nanotube, a polymer, a cell, a virus, a virus-like particle or any combination of these.

[0059] Preferably, the nanoparticle comprises a nanoparticle protein, which can be a virus-like particle protein formed by a virus structural protein, preferably by a bacteriophage coat protein AP205.

[0060] Preferably, the nanoparticle comprises a nanoparticle protein, which can be further selected from: NPM particle, Ferritin, I53-50 particle, etc.

[0061] In some embodiments, the peptide tag (binding peptide 1) and / or the polypeptide (binding peptide 2) to which it is fused or conjugated is a component or entity of a solid support, i.e. a solid substrate or solid phase.

[0062] In a eighth aspect, the present application provides use of the polypeptide as a binding peptide 2 to form an isopeptide bond with the binding peptide 1 and self-assemble two molecules or components through the isopeptide bond.

[0063] In some embodiments, the binding peptide 1 is selected from Spy-Tag, Sdy-Tag.

[0064] In a ninth aspect, the present application provides an immunogenic complex comprising:

[0065] (1) an antigen component comprising an antigenic protein or an immunogenic fragment thereof, a binding peptide 1;

[0066] (2) a granulin component comprising a nanogranulin, a binding peptide 2;

[0067] The antigen component and the granulin component are covalently bound through the binding peptide 2 and the binding peptide 1.

[0068] Further, the present application provides an immunogenic complex comprising:

[0069] (1) an antigen component comprising an antigenic protein or an immunogenic fragment thereof, a linker peptide 1, a binding peptide 1;

[0070] (2) a granulin component comprising a nanogranulin, a linker peptide 2, a binding peptide 2;

[0071] The antigen component and the granulin component are covalently bound through the binding peptide 2 and the binding peptide 1.

[0072] In some embodiments, the antigenic protein or the immunogenic fragment thereof can correspond to a component or a portion thereof of a pathogen or a vector. For example, for ease of preparation, the antigen component can lack a transmembrane domain.

[0073] In some embodiments, the antigen protein or immunogenic fragment thereof can be a varicella-zoster virus (VZV) gE protein, which plays an important role in viral replication and cell-to-cell spread, which is the pathogenesis of viral transmission and skin infection. Most importantly, the VZV gE protein contains B cell and CD4+ T cell epitopes, which can induce neutralizing antibodies and CD4+ T cell immune responses, and is the most ideal potential vaccine antigen for preventing varicella-zoster virus. The recombinant varicella-zoster virus vaccine (CHO cell) belongs to the recombinant protein vaccine, which is expressed in Escherichia coli by gene recombination to express virus-like particle carrier Sdy-Catcher-NPM or Sdy-Catcher variant-NPM, and express varicella-zoster virus gE-Spy-Tag protein in CHO cells. The C-terminus of the VZV gE protein is modified by binding to the Spy-Tag end of the polypeptide, and the theoretical molecular weight of the integrated amino acid is about 60980.10 Dalton.

[0074] In some embodiments, the antigen protein or immunogenic fragment thereof can be a new crown RBD protein. A number of research data show that the most potent neutralizing antibodies against the new crown are mainly directed against the RBD-ACE2 binding epitope, and because the RBD has a small molecular weight and limited structural space, the binding antibodies of RBD basically have the ability to compete and block the combination of RBD and ACE2. Therefore, the new crown RBD protein as a vaccine antigen will induce more precise epitope and more potent neutralizing antibodies. The recombinant broad-spectrum new coronavirus vaccine (CHO cell) belongs to the recombinant protein vaccine, which is expressed in Escherichia coli by gene recombination to express virus-like particle carrier Sdy-Catcher-NPM or Sdy-Catcher variant-NPM, and express RBD-Sdy-Tag fusion protein in CHO cells. The C-terminus of the RBD protein is modified by binding to the Spy-Tag end of the polypeptide.

[0075] In some embodiments, an "immunogenic fragment" refers to a portion of an oligopeptide, polypeptide, or protein that is immunogenic and elicits a protective immune response when administered to a subject.

[0076] In some embodiments, the present application provides an immunogenic complex, which comprises:

[0077] (1) an antigen component comprising an antigen protein or an immunogenic fragment thereof, a linking peptide 1, and a binding peptide 1;

[0078] (2) a granule protein component comprising a nanoparticle protein, a linking peptide 2, and a binding peptide 2;

[0079] The antigen component and the granule protein component are covalently bound by the binding peptide 2 and the binding peptide 1.

[0080] All reagents used in the present application can be commercially purchased.

[0081] Compared with the prior art, the present application has the beneficial effect that the reaction rate of Sdy-Catcher with Spy-Tag is higher than that of Sdy-Tag. It is found that the reaction rate of the mutant Sdy-Catcher variant with Spy-Tag is further significantly improved compared with the reaction rate of Sdy-Catcher with Spy-Tag. The binding rate of the Sdy-Catcher variant (especially the N-terminal truncated Sdy-Catcher) with the particle protein component and the antigen component formed by the nanoparticle protein such as NPM particle is significantly improved compared with the control, i.e. the binding rate of Sdy-Catcher with the particle protein component and the antigen component formed by the nanoparticle protein such as NPM particle.

[0082] The above findings provide an ideal direction for finding polypeptides with higher binding rate and better binding effect in the art. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 The three-dimensional structure of Sdy-Catcher obtained by α-fold prediction is shown.

[0084] Figure 2 The expression and purification effect of Sdy-Catcher-FL is shown: a is the SDS-PAGE result of the sample collected during the purification process of Sdy-Catcher-FL; b is the result of SEC purification of Sdy-Catcher-FL.

[0085] Figure 3 The expression and purification effect of Sdy-Catcher-02 is shown: a is the SDS-PAGE result of the sample collected during the purification process of Sdy-Catcher-02; b is the result of Ni column purification of Sdy-Catcher-02.

[0086] Figure 4 The expression and purification effect of Sdy-Catcher-03 is shown: a is the SDS-PAGE result of the sample collected during the purification process of Sdy-Catcher-03; b is the result of SEC purification of Sdy-Catcher-03.

[0087] Figure 5 The expression and purification effect of Sdy-Catcher-04 is shown: a is the SDS-PAGE result of the sample collected during the purification process of Sdy-Catcher-04; b is the result of Ni column purification of Sdy-Catcher-04.

[0088] Figure 6 Expression and purification results of Sdy-Catcher-05 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-05; b is the result of Ni column purification of Sdy-Catcher-05.

[0089] Figure 7 Expression and purification results of Sdy-Catcher-06 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-06; b is the result of SEC purification of Sdy-Catcher-06.

[0090] Figure 8 Expression and purification results of Sdy-Catcher-07 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-07; b is the result of SEC purification of Sdy-Catcher-07.

[0091] Figure 9 Expression and purification results of Sdy-Catcher-08 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-08; b is the result of SEC purification of Sdy-Catcher-08.

[0092] Figure 10 Expression and purification results of Sdy-Catcher-09 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-09; b is the result of SEC purification of Sdy-Catcher-09.

[0093] Figure 11 Expression and purification results of Sdy-Catcher-10 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-10; b is the result of SEC purification of Sdy-Catcher-10.

[0094] Figure 12 Expression and purification results of Sdy-Catcher-11 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-11; b is the result of Ni column purification of Sdy-Catcher-11.

[0095] Figure 13Expression and purification results of Sdy-Catcher-12 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-12; b is the result of SEC purification of Sdy-Catcher-12.

[0096] Figure 14 Expression and purification results of Sdy-Catcher-13 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-13; b is the result of SEC purification of Sdy-Catcher-13.

[0097] Figure 15 Expression and purification results of Sdy-Catcher-14 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-14; b is the result of SEC purification of Sdy-Catcher-14.

[0098] Figure 16 Expression and purification results of Sdy-Catcher-15 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-15; b is the result of SEC purification of Sdy-Catcher-15.

[0099] Figure 17 Expression and purification results of Sdy-Catcher-16 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-16; b is the result of Ni column purification of Sdy-Catcher-16.

[0100] Figure 18 Expression and purification results of Sdy-Catcher-17 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-17; b is the result of SEC purification of Sdy-Catcher-17.

[0101] Figure 19 Expression and purification results of Sdy-Catcher-18 are shown: a is the result of SDS-PAGE of samples collected during the purification process of Sdy-Catcher-18; b is the result of SEC purification of Sdy-Catcher-18.

[0102] Figure 20 Expression and purification results of SdCdn-Catcher are shown: a is the result of SDS-PAGE of samples collected during the purification process of SdCdn-Catcher; b is the result of SEC purification of SdCdn-Catcher.

[0103] Figure 21 Figure 22 shows the results of the detection of the binding rate of Sdy-Catcher-03 and Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points in the binding reaction process; b is the gray value of Sdy-catcher-03 which does not bind tag in the a figure, as the ordinate, the standard curve is drawn with its mass as the abscissa; c is the binding rate curve of Sdy-Catcher-03 and Spy-Tag according to the residual molar quantity of Sdy-Catcher-03 1 / [Sdy-Catcher] (μΜ"1) as the ordinate, with time as the abscissa.

[0104] Figure 22 shows the results of the detection of the binding rate of Sdy-Catcher-03 and Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points in the binding reaction process; b is the gray value of Sdy-catcher-03 which does not bind tag in the a figure,

[0105] as the ordinate, the standard curve is drawn with its mass as the abscissa; c is the binding rate curve of Sdy-Catcher-03 and Spy-Tag according to the residual molar quantity of Sdy-Catcher-03 1 / [Sdy-Catcher] (μΜ"1) as the ordinate, with time as the abscissa.

[0106] Figure 23 Figure 22 shows the results of the detection of the binding rate of Sdy-Catcher-03 and Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points in the binding reaction process; b is the gray value of Sdy-catcher-03 which does not bind tag in the a figure,

[0107] as the ordinate, the standard curve is drawn with its mass as the abscissa; c is the binding rate curve of Sdy-Catcher-03 and Spy-Tag according to the residual molar quantity of Sdy-Catcher-03 1 / [Sdy-Catcher] (μΜ"1) as the ordinate, with time as the abscissa.

[0108] Figure 24 Figure 22 shows the results of the detection of the binding rate of Sdy-Catcher-03 and Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points in the binding reaction process; b is the gray value of Sdy-catcher-03 which does not bind tag in the a figure,

[0109] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-07 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-07 residues 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0110] Figure 25 Detection results of the binding rate of Sdy-Catcher-08 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-08 that does not bind tag in the a figure,

[0111] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-08 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-08 residues 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0112] Figure 26 Detection results of the binding rate of Sdy-Catcher-09 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-09 that does not bind tag in the a figure,

[0113] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-09 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-09 residues 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0114] Figure 27 Detection results of the binding rate of Sdy-Catcher-10 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-10 that does not bind tag in the a figure,

[0115] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-10 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-10 residues 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0116] Figure 28Results of detection of the binding rate of Sdy-Catcher-11 to Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-11 which does not bind tag in the a figure,

[0117] Standard curve taken as the ordinate and the mass as the abscissa; c is the binding rate curve of Sdy-Catcher-11 and Spy-Tag taken as the ordinate and the time as the abscissa according to the residual molar quantity of Sdy-Catcher-11 1 / [Sdy-Catcher] (μΜ"1).

[0118] Figure 29 Results of detection of the binding rate of Sdy-Catcher-12 to Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-12 which does not bind tag in the a figure,

[0119] Standard curve taken as the ordinate and the mass as the abscissa; c is the binding rate curve of Sdy-Catcher-12 and Spy-Tag taken as the ordinate and the time as the abscissa according to the residual molar quantity of Sdy-Catcher-12 1 / [Sdy-Catcher] (μΜ"1).

[0120] Figure 30 Results of detection of the binding rate of Sdy-Catcher-13 to Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-13 which does not bind tag in the a figure,

[0121] Standard curve taken as the ordinate and the mass as the abscissa; c is the binding rate curve of Sdy-Catcher-13 and Spy-Tag taken as the ordinate and the time as the abscissa according to the residual molar quantity of Sdy-Catcher-13 1 / [Sdy-Catcher] (μΜ"1).

[0122] Figure 31 Results of detection of the binding rate of Sdy-Catcher-14 to Spy-Tag: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-14 which does not bind tag in the a figure,

[0123] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-14 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-14 remaining 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0124] Figure 32 Detection results of the binding rate of Sdy-Catcher-15 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-15 not combined with tag in the a figure,

[0125] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-15 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-15 remaining 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0126] Figure 33 Detection results of the binding rate of Sdy-Catcher-17 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-17 not combined with tag in the a figure,

[0127] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-17 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-17 remaining 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0128] Figure 34 Detection results of the binding rate of Sdy-Catcher-18 and Spy-Tag are shown: a is the result of SDS-PAGE of the binding product samples selected at 9 time points during the binding reaction process; b is the gray value of Sdy-catcher-18 not combined with tag in the a figure,

[0129] Standard curve plotted as ordinate with its mass as abscissa; c is the binding rate curve of Sdy-Catcher-18 and Spy-Tag plotted as ordinate with the molar amount of Sdy-Catcher-18 remaining 1 / [Sdy-Catcher] (μΜ"1) as abscissa.

[0130] Figure 35Results of detection of the binding rate of SdCdn-catcher and Spy-Tag: a is the result of SDS-PAGE of the binding product sample selected at 9 time points in the binding reaction process; b is the gray value of SdCdn-catcher without binding tag in the a figure, as the ordinate, the standard curve is drawn with its mass as the abscissa; c is the binding rate curve of SdCdn-catcher and Spy-Tag drawn with time as the abscissa according to the residual molar mass 1 / [Sdy-Catcher] (μM-1) as the ordinate.

[0131] Figure 36 Results of binding effect of Sdy-Catcher-FL-NPM, SdCdN-Catcher-NPM and Spy-Tag-gE fusion peptide: a is the SDS-PAGE result of the binding product sample (Sdy-NPM+spy-tag-gE) based on the binding reaction of Sdy-Catcher-FL-NPM and Spy-Tag-gE fusion peptide; b is the standard curve drawn with the gray value of Sdy-Catcher-FL-NPM as the ordinate and its mass as the abscissa; c is the SDS-PAGE result of the binding product sample (SdCdn-NPM+spy-tag-gE) based on the binding reaction of SdCdN-Catcher-NPM and Spy-Tag-gE fusion peptide; d is the standard curve drawn with the gray value of SdCdN-Catcher-NPM as the ordinate and its mass as the abscissa.

[0132] Figure 37 Results of binding effect of Sdy-Catcher-FL-NPM, SdCdN-Catcher-NPM and Spy-Tag-RBD fusion peptide: a is the SDS-PAGE result of the binding product sample (Sdy-NPM+spy-tag-RBD) based on the binding reaction of Sdy-Catcher-FL-NPM and Spy-Tag-RBD fusion peptide; b is the standard curve drawn with the gray value of Sdy-Catcher-FL-NPM as the ordinate and its mass as the abscissa; c is the SDS-PAGE result of the binding product sample (SdCdn-NPM+spy-tag-RBD) based on the binding reaction of SdCdN-Catcher-NPM and Spy-Tag-RBD fusion peptide; d is the standard curve drawn with the gray value of SdCdN-Catcher-NPM as the ordinate and its mass as the abscissa. DETAILED DESCRIPTION

[0133] The principles and features of the present application are described below in connection with examples, which are only used to explain the present application and are not used to limit the scope of the present application. Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are used to describe the specific embodiments, but are not used to limit the scope of protection of the present application. The test methods in the following examples are not specified, and are generally carried out according to the conventional conditions or the conditions recommended by the manufacturers. When the examples give a numerical range, it should be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the examples, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application. The experimental materials used in the following examples, unless otherwise specified, are purchased from conventional reagent companies.

[0134] Example 1: Design of Sdy-Catcher mutant molecules

[0135] 1. Experimental methods:

[0136] According to the existing structure in the database and the amino acid sequence of Sdy-Catcher, the three-dimensional structure of Sdy-Catcher is predicted by α-fold, which is shown in Figure 1 .

[0137] According to the predicted three-dimensional structure, various variants of Sdy-Catcher are generated by sequence design. The amino acid sequence structures of these various variants of Sdy-Catcher are shown in Table 1, and the nucleotide sequences are shown in Table 2.

[0138] According to the predicted three-dimensional structure, the sequence design is carried out in the following specific ways:

[0139] Sdy-Catcher-02, Sdy-Catcher-03, Sdy-Catcher-04, Sdy-Catcher-05, Sdy-Catcher-11, Sdy-Catcher-12, Sdy-Catcher-13, SdCdn-Catcher are different truncations of the N-terminal of Sdy-Catcher, and the expression and purification results and the reaction rate with Spy-Tag are detected.

[0140] According to the expression and purification results and the comparison of the reaction rate with Spy-Tag, Sdy-Catcher-03 among the above mutant molecules was selected as the basis to further design the following sequences: Sdy-Catcher-06, Sdy-Catcher-07, Sdy-Catcher-08, Sdy-Catcher-14, Sdy-Catcher-15, Sdy-Catcher-16, Sdy-Catcher-17, Sdy-Catcher-18.

[0141] Sdy-Catcher-9 is based on Sdy-catcher-FL, which is point mutated and N-terminally replaced.

[0142] Table 1 Amino acid sequences of various Sdy-Catcher mutant molecules in Example 1 of the application

[0143]

[0144]

[0145] Table 2 Nucleotide sequences of various Sdy-Catcher mutant molecules in Example 1 of the application

[0146]

[0147]

[0148]

[0149] Example 2: Expression and purification of Sdy-Catcher mutant molecules in Example 1

[0150] 1. Experimental materials:

[0151] HisTrap Excel was purchased from Cytiva, and molecular sieve (Superdex 200 increase 10 / 300 GL) was purchased from Cytiva.

[0152] 2. Experimental method:

[0153] (1) Induction expression condition: inoculate Sdy-Catcher BL21(DE3) monoclonal bacteria in 300 mL TB(Kana+) medium, cultivate at 37°C, 220 rpm for 4-5 h, when the culture solution OD600 is about 0.6-0.8, transfer the bacterial solution to 18°C, add IPTG to a final concentration of 1 mM, and induce protein expression at 220 rpm for 16 h (Sdy-Catcher-(16-18) is induced to express for 24 h).

[0154] (2) Centrifugation: centrifuge the bacterial solution at 6000g at room temperature to collect the bacterial cells, discard the culture solution, and resuspend the bacterial cells in 60 mL of 150 mM NaCl, 20 mM Tris, pH 7.4 solution.

[0155] (3) Ultrasonic disruption: place the resuspended bacterial solution in an ice water bath and perform ultrasonic disruption. Use an amplitude bar No. 10, 50% power, ultrasonic opening for 3 s and stopping for 7 s, and the total ultrasonic time is 20 min.

[0156] (4) Centrifugation to collect the target protein: collect the cell disruption supernatant at 13000g, 4°C, for 30 min.

[0157] (5) Histrap purification of the target protein: the wash buffer is 20 mM Tris-HCl, 150 mM NaCl, pH 7.4; the elution buffer is 20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 7.4. Use Histrap Excel-5 mL Ni column for purification; equilibrate the Histrap Excel-5 mL with the wash buffer for 10 CV, then load the sample; after the sample loading is completed, rinse the column with the wash buffer for 10 CV; rinse the column with 4% elution buffer for 10 CV to wash away the impurities; linearly elute the target protein with 4%-100% elution buffer for 10 CV; elute the target protein with 100% elution buffer for 5 CV, and after the elution is completed, detect the protein purity by SDS-PAGE.

[0158] (6) SEC purification of target protein: After the target protein purified by Ni column was concentrated to 1 mL by a concentration tube, it was separated and purified by molecular sieve (Superdex 200 increase 10 / 300 GL), and the purification buffer was 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. After elution, the protein purity was detected by SDS-PAGE. The purified Sdy-Catcher mutant molecules Sdy-Catcher-FL, Sdy-Catcher-03, Sdy-Catcher-06, Sdy-Catcher-07, Sdy-Catcher-08, Sdy-Catcher-09, Sdy-Catcher-10, Sdy-Catcher-12, Sdy-Catcher-13, Sdy-Catcher-14, Sdy-Catcher-15, Sdy-Catcher-17, Sdy-Catcher-18 and SdCdn-Catcher molecules were identified by SDS-PAGE. The target protein was obtained by Ni column and molecular sieve purification. Sdy-Catcher-02, Sdy-Catcher-04, Sdy-Catcher-05, Sdy-Catcher-11 and Sdy-Catcher-16 were almost not obtained by Ni column purification, so further purification was not carried out. The expression and purification results of Sdy-Catcher mutant molecules are shown in FIG. 2. Figures 2 to 19

[0159] Example 3: Test of binding effect of Sdy-Catcher mutant molecules and Spy-Tag

[0160] 1. Experimental materials:

[0161] Main instruments used in the experiment: Thermo Mixer C constant temperature mixer, Amersham ImageQuant 800 molecular biology imager, dry and wet thermostat.

[0162] 2. Experimental method:

[0163] (1) Binding method:

[0164] All binding reactions were carried out in a TBS 7.4 buffer system at 25°C, and the final concentration of Sdy-Catcher mutant molecules and Spy-Tag was 10 μM. Samples were taken at 1 min, 2 min, 3 min, 4 min, 5 min, 10 min, 15 min, 30 min and 60 min, respectively, and terminated by adding LDS (with DTT).

[0165] (2) Calibration, SDS-PAGE, calculation​

[0166] ①Standard curve making:

[0167] From 1 μg Sdy-Catcher mutant molecules, 2-fold dilution was performed, with a total of 7 gradients.

[0168] ②SDS-PAGE operation:

[0169] SDS-PAGE was performed using PowerEase Touch 350W touch screen power supply and XCell4 SureLock medium gel electrophoresis tank at 180V, NuPAGE Bis-Tris precast gel (4-12%), and the gel was stained with staining solution for 2h, then decolorized overnight, and scanned and photographed by a molecular biology imager.

[0170] ③Calculation method:

[0171] Image QuantTL was used to analyze all bands in the SDS-PAGE gel of the previous step to obtain band gray values. The gray value of Sdy-Catcher was taken as the vertical coordinate, and its mass was taken as the horizontal coordinate to draw a standard curve. The mass of Sdy-Catcher remaining in the binding reaction was calculated by the equation formula of the standard curve, and the molar mass 1 / [Sdy-Catcher] (μM-1) was obtained according to its molecular weight, which was taken as the vertical coordinate, and the time was taken as the horizontal coordinate to draw a curve, which was the binding rate curve of Sdy-Catcher mutant molecules and Spy-Tag, and the slope was the binding rate ((M -1 min -1 )10 2 ).

[0172] (3) Binding rate results:

[0173] The binding rate data results are shown in Figures 20-33 and Table 3.

[0174] Table 3 Binding rate of Sdy-Catcher mutant molecules and Spy-Tag

[0175]

[0176] Example 4: Application effect test of Sdy-Catcher mutant molecules (SdCdn-Catcher) on nanoparticle proteins: binding effect test of the fusion peptide of Sdy-Catcher mutant molecules (SdCdn-Catcher) and NPM nanoparticle protein with the fusion peptide of Spy-Tag and antigen molecules

[0177] 1. Experimental materials:

[0178] Main instruments used in the experiment: Thermo Mixer C constant temperature mixer, Amersham ImageQuant 800 molecular biology imager, dry and wet thermostat.

[0179] Molecules involved in the experiment: SdCdn-Catcher, NPM nanoparticle protein, antigen molecule RBD, antigen molecule VZVgE protein, sequence structure as shown in Table 4. Among them, the Sdy-Catcher mutant SdCdn-Catcher is obtained by deleting 22 amino acids at the N-terminus of SEQ ID NO: 1 (Sdy-Catcher-FL). SdCdn-Catcher has one more amino acid D at the N-terminus than Sdy-Catcher03.

[0180] 2. Experimental method:

[0181] (1) Binding method:

[0182] The binding reaction was carried out in a buffer system of 20 mM Tris pH 7.4, 25% sucrose, and the final concentration of Sdy-Catcher-FL-NPM / SdCdN-Catcher-NPM and Spy-Tag-antigen molecule fusion peptide was 1:6 at 25°C. After 24 hours of reaction at 22°C, the reaction was terminated by adding LDS (with DTT).

[0183] (2) Standard curve preparation, SDS-PAGE operation, and binding effect calculation

[0184] ① Standard curve preparation:

[0185] Starting from 2 μg of Sdy-Catcher-FL-NPM / SdCdN-Catcher-NPM, 2-fold dilution was performed, with a total of 6 gradients.

[0186] ② SDS-PAGE operation:

[0187] SDS-PAGE was performed using PowerEase Touch 350W touch screen power supply and XCell4 SureLock medium gel electrophoresis tank at 180V on NuPAGE Bis-Tris precast gel (4-12%), and staining liquid was used for staining for 2h, followed by overnight decolorization. Scanning and photographing were performed by molecular biology imager.

[0188] ③ Calculation method:

[0189] Image QuantTL was used to analyze all bands in the SDS-PAGE gel image from the previous step to obtain their grayscale values. A standard curve was plotted with the grayscale values ​​of Sdy-Catcher-FL-NPM / SdCdN-Catcher-NPM on the ordinate and their mass on the x-axis. The residual mass of Sdy-Catcher-FL-NPM / SdCdN-Catcher-NPM in the binding reaction was calculated using the equation of the standard curve. The molar amount was determined based on its molecular weight, and the binding ratio was then calculated based on the residual molar amount.

[0190] (3) Based on the results of the proportional calculation:

[0191] See the data results separately. Figures 34-35 As shown in Table 5, the binding rate of SdCdN-Catcher-NPM to the Spy-Tag antigen fusion peptide is significantly higher than that of Sdy-Catcher-FL-NPM to the Spy-Tag antigen fusion peptide.

[0192] Table 4. Amino acid sequences of SdCdn-Catcher, NPM nanoparticle protein, Spy-Tag-RBD, and Spy-Tag-VZV gE protein in the embodiments of this application.

[0193]

[0194] Table 5. Binding rates of Sdy-Catcher-FL-NPM / SdCdn-Catcher-NPM and Spy-Tag-antigen fusion peptides

[0195]

[0196] In summary, the above embodiments and accompanying drawings are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide comprises a variant of SdyCatcher, which has one or more insertions, deletions or substitutions of amino acids relative to SdyCatcher, and which has improved at least one aspect of biological activity; preferably, the SdyCatcher comprises the sequence as set forth in SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein, (1) the variant of SdyCatcher has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 92%; more preferably at least 94%; more preferably at least 95%, 96%, 98%, 99%, or 99.5% sequence identity to SdyCatcher; (2) the variant of SdyCatcher has 1-10 mutations relative to SdyCatcher; (3) the substitutions can be non-conservative substitutions or conservative substitutions; or (4) the variant sequence of SEQ ID NO: 1 comprises deletion of 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, 30, 31, or 32 amino acids at the N-terminus or C-terminus of SEQ ID NO: 1, more preferably, deletion of 10, 21, 22, 23, 24, 25, or 26 amino acids at the N-terminus of SEQ ID NO: 1, and optionally, fusion of another functional polypeptide sequence at the N-terminus or C-terminus. ​ ​ 3. The polypeptide of claim 2, wherein the variant sequence of SEQ ID NO: 1 comprises a deletion of 10, 21, 22, 23, 24, 25 or 26 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3, 4 or 5 amino acid mutations, or a combination thereof; preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2 or 3 amino acid mutations, or a combination thereof; more preferably, the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3 amino acid mutations, or a combination thereof, at positions corresponding to positions 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 of the sequence of SEQ ID NO:

1.

4. The polypeptide of any one of claims 1-3, wherein the variant sequence of SEQ ID NO: 1 comprises a deletion of 23 amino acids at the N-terminus of SEQ ID NO: 1, and further comprises 1, 2, 3 amino acid mutations, or a combination thereof, at positions corresponding to positions 29, 41, 45, 50, 66, 70, 87, 88, 89 or 98 of the sequence of SEQ ID NO: 1, and the mutations are selected from the group consisting of mutating isoleucine (I) at said position to threonine (T), mutating leucine (L) at said position to serine (S), mutating valine (V) at said position to aspartic acid (D), mutating lysine (K) at said position to arginine (R), mutating lysine (K) at said position to aspartic acid (D), mutating alanine (A) at said position to threonine (T), mutating methionine (M) at said position to serine (S), and mutating leucine (L) at said position to threonine (T); preferably, the mutations or combination thereof are selected from any one of the following groups: (1) mutating I at position 45 of the sequence of SEQ ID NO: 1 to T; (2) mutating L at position 50 of the sequence of SEQ ID NO: 1 to S; (3) mutating V at position 66 of the sequence of SEQ ID NO: 1 to D; (4) mutating K at position 29 and K at position 98 of the sequence of SEQ ID NO: 1 to R and D, respectively; (5) mutating A at position 41 of the sequence of SEQ ID NO: 1 to T; (6) mutating M to S at position 70 corresponding to the sequence of SEQ ID NO: 1 ; (7) mutating L to T at position 87 corresponding to the sequence of SEQ ID NO: 1 ; (8) mutating A to T at position 88 corresponding to the sequence of SEQ ID NO: 1 ; (9) mutating A to T at position 89 corresponding to the sequence of SEQ ID NO:

1.

5. The polypeptide of any one of claims 1-4, wherein the variant sequence of SEQ ID NO: 1 comprises a polypeptide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% sequence identity to the sequence of SEQ ID NO: 3, the sequence of SEQ ID NO: 4, the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, the sequence of SEQ ID NO: 8, the sequence of SEQ ID NO: 9, the sequence of SEQ ID NO: 10, the sequence of SEQ ID NO: 11, the sequence of SEQ ID NO: 12, the sequence of SEQ ID NO: 13, the sequence of SEQ ID NO: 14, the sequence of SEQ ID NO: 15, the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 17, the sequence of SEQ ID NO: 18, the sequence of SEQ ID NO: 19, the sequence of SEQ ID NO: 20, or the sequence of SEQ ID NO: 21, and retains the improved biological activity of the reference sequence. Preferably, the variant sequence of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3, the sequence of SEQ ID NO: 4, the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, the sequence of SEQ ID NO: 8, the sequence of SEQ ID NO: 9, the sequence of SEQ ID NO: 10, the sequence of SEQ ID NO: 11, the sequence of SEQ ID NO: 12, the sequence of SEQ ID NO: 13, the sequence of SEQ ID NO: 14, the sequence of SEQ ID NO: 15, the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 17, the sequence of SEQ ID NO: 18, the sequence of SEQ ID NO: 19, the sequence of SEQ ID NO: 20, or the sequence of SEQ ID NO:

21.

6. An article of manufacture selected from the group consisting of: (1) a polynucleotide encoding the polypeptide of any one of claims 1-5; (2) an expression vector comprising (1); (3) a host cell comprising (2) or (3).

7. A method of making the polypeptide of any one of claims 1-5: (1) linking the nucleotide sequence expressing the polypeptide into an expression vector respectively to construct an expression recombinant plasmid vector; (2) transforming or transfecting a host cell; (3) applying the host cell to express the recombinant protein and purifying the recombinant protein.

8. A method characterized by, The polypeptide of any one of claims 1-5 as a binding peptide 2 forms an isopeptide bond with a binding peptide 1 containing Spy-Tag or Sdy-Tag and self-assembles two molecules or components through the isopeptide bond.

9. An application, characterized in that The polypeptide of any one of claims 1-5 as a binding peptide 2 forms an isopeptide bond with a binding peptide 1 containing Spy-Tag or Sdy-Tag and self-assembles two molecules or components through the isopeptide bond.

10. An immunogenic complex comprising: (1) an antigen component comprising an antigen protein or an immunogenic fragment thereof and a binding peptide 1; (2) a granule protein component comprising a nanoparticle protein and a binding peptide 2; The binding peptide 2 is selected from the polypeptide of any one of claims 1-5, and the binding peptide 1 contains Spy-Tag or Sdy-Tag; The antigen component and the granule protein component are covalently bound through the binding peptide 2 and the binding peptide 1; Optionally, the antigen component further comprises a linker peptide 1 connecting the antigen protein or the immunogenic fragment thereof and the binding peptide 1, and the granule protein component further comprises a linker peptide 2 connecting the nanoparticle protein and the binding peptide 2.

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