Protein covalent coupling system Snoop8Catcher Snoop8Tag and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a protein covalent coupling system Snoop8Catcher Snoop8Tag and application of the protein covalent coupling system Snoop8Catcher Snoop8Tag. The system consists of Snoop8Catcher (the amino acid sequence is as shown in SEQ ID NO: 1) and Snoop8Tags (the amino acid sequence is as shown in SEQ ID NO: 2), and the Snoop8Catcher and the Snoop8Tags can be efficiently and specifically covalently linked through isopeptide bonds under mild conditions. Compared with an existing Snoop system, the coupling efficiency of the system is higher, the sequence identity is lower than 40%, and the limitation of existing patents can be effectively avoided. The system can be widely applied to the fields of vaccine construction (such as porcine deltacoronavirus multivalent nanoparticle vaccines), antibody-drug conjugate preparation, protein nanomaterial assembly, diagnostic reagent development and the like, and has extremely high industrialization value and application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biopharmaceuticals, and more specifically, to a novel protein covalent conjugation system Snoop8Catcher / Snoop8Tag obtained by screening metagenomic structure libraries, and its applications in vaccine construction (especially porcine deltacoronavirus multivalent nanoparticle vaccines), antibody-drug conjugate preparation, protein nanomaterial assembly, and diagnostic reagent development. Background Technology

[0002] Protein conjugation technology is one of the core technologies in the field of bioengineering. It enables the specific and stable connection of different functional protein molecules, thereby exerting a synergistic enhancement effect. It has been widely used in many fields such as vaccine design, targeted therapy, and protein chips. Currently, commonly used covalent conjugation systems mainly include the SpyCatcher / SpyTag system and the SnoopCatcher / SnoopTag system (hereinafter referred to as the "SnoopPC system").

[0003] The SnoopPC system is derived from the D4 domain of Streptococcus pneumoniae adhesin RrgA and was obtained through protein engineering. In this system, SnoopTag is a short peptide of 12-13 amino acids, and SnoopCatcher is a protein module of approximately 113 amino acids. The two can be spontaneously covalently linked under mild conditions through isopeptide bonds formed between lysine and aspartic acid residues, without the need for external enzymes or energy. Furthermore, it is orthogonal to the SpyCatcher / SpyTag system and exhibits no cross-reactivity.

[0004] In the vaccine field, the SnoopPC system has been used for antigen presentation and vaccine construction. For example, SnoopCatcher can be displayed on the surface of nanoparticles or virus-like particles and covalently linked to antigen proteins carrying SnoopTags, thereby achieving multivalent display of antigens and enhancing the immune response. Furthermore, this system can be used in conjunction with the SpyCatcher / SpyTag system to achieve simultaneous loading of two antigens.

[0005] However, the SnoopPC system has significant drawbacks: on the one hand, its covalent linking efficiency is generally lower than that of the Spy system, limiting its potential for independent applications; on the other hand, the protein sequence of this system is already covered by numerous foreign patents, severely restricting its further development and industrial application in China. Therefore, developing a novel protein conjugation system with independent intellectual property rights and higher linking efficiency has become an urgent technical problem to be solved in this field.

[0006] Porcine deltacoronavirus (PDCoV) is a novel porcine enteric coronavirus, and the disease it causes is classified as a Class II animal disease in my country. This virus invades cells by binding its spike protein to the aminopeptidase N receptor on the surface of host cells. The receptor-binding domain (RBD) of the spike protein is a key antigenic region for eliciting neutralizing antibodies and is an ideal target for subunit vaccine design. However, similar to most coronavirus RBDs, PDCoVRBD alone has weak immunogenicity and often requires adjuvant assistance or multiple immunizations. Multivalent presentation strategies (such as protein self-assembly or nanoparticle display) can significantly enhance its immunogenicity. Ferritin, a natural 24-meric nanocage-like protein, possesses excellent thermal stability, chemical stability, and pH tolerance. It can display antigenic epitopes on each subunit through gene fusion, achieving multi-site antigen presentation on a single particle surface, making it an ideal antigen carrier. Therefore, developing novel and efficient protein conjugation systems to achieve stable conjugation of PDCoVRBD and ferritin is of great significance for constructing highly effective PDCoV vaccines. Summary of the Invention

[0007] To address the technical problems of low coupling efficiency, patent restrictions, and weak immunogenicity of PDCoVRBD alone in existing SnoopPC systems, this invention provides a novel protein covalent conjugation system, Snoop8Catcher / Snoop8Tag. This system has higher coupling efficiency, can circumvent existing patent restrictions, and can efficiently achieve covalent conjugation of target proteins (such as PDCoVRBD and ferritin), providing a new technical solution for the development of vaccines, antibody-drug conjugates, and other products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a novel protein covalent coupling system Snoop8Catcher / Snoop8Tag, which includes two components, Snoop8Catcher and Snoop8Tag, wherein the amino acid sequence of Snoop8Catcher is shown in SEQ ID NO:1 and the amino acid sequence of Snoop8Tag is shown in SEQ ID NO:2.

[0009] SEQIDNO:1 (Snoop8Catcher): MEELEGAELTVKKADGTLIESWTSGTAQKVIGLGPGTYTMVETSAPQGYAVAPSITFRVEADGTVQIKEDGNWVDAANATIQMVDELTPY SEQIDNO:2(Snoop8Tag): TPSYGVTFSKVAA Furthermore, the Snoop8Catcher and Snoop8Tag can form heteropeptide bonds through the aspartic acid in Snoop8Catcher and the lysine in Snoop8Tag, achieving spontaneous and specific covalent linkage. This process does not require external enzymes or energy donors and the reaction conditions are mild.

[0010] In this invention, Snoop8Catcher and / or Snoop8Tag can fuse with target proteins to form fusion proteins, thereby achieving covalent linkage between target proteins through isopeptide bonds. The target proteins include, but are not limited to, antigen proteins (such as PDCoVRBD), enzymes, antibodies, receptors, structural proteins (such as ferritin), and reporter proteins.

[0011] In a preferred embodiment, the target protein is PDCoVRBD and ferritin, and the corresponding fusion protein is: (a) a fusion protein of RBD and Snoop8Tag (RBD-Snoop8Tag); (b) a fusion protein of ferritin and Snoop8Catcher (Ferritin-Snoop8Catcher). The two fusion proteins can self-assemble into an RBD-Ferritin nanoparticle complex through the interaction between Snoop8Tag and Snoop8Catcher. This complex can serve as the core active ingredient of the PDCoV vaccine.

[0012] The present invention also provides a nucleic acid molecule encoding the Snoop8Catcher or Snoop8Tag described above, an expression vector containing the nucleic acid molecule, and a host cell transformed or transfected with the expression vector. The host cell may be a prokaryotic cell (such as Escherichia coli BL21(DE3)) or a eukaryotic cell (such as 293F cells), and a suitable expression system may be selected according to the expression requirements of the target protein.

[0013] Furthermore, this invention provides a method for preparing covalently linked protein complexes, comprising the following steps: (a) constructing and expressing a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a fusion of a first target protein and Snoop8Catcher or Snoop8Tag, and the second fusion protein comprises a fusion of a second target protein and Snoop8Tag or Snoop8Catcher; (b) mixing the purified first fusion protein and the second fusion protein in a suitable buffer (e.g., 20 mM Tris-HCl, 150 mM NaCl, pH 8.0); and (c) incubating at 4°C to 37°C for 0.5 to 24 hours to allow the formation of heteropeptide bonds between Snoop8Catcher and Snoop8Tag, ultimately obtaining a covalently linked protein complex.

[0014] This invention also provides a vaccine composition comprising the above-described RBD-Ferritin nanoparticle complex and a pharmaceutically acceptable adjuvant or carrier. The adjuvant may be a commonly used vaccine adjuvant such as aluminum adjuvant or oil emulsion adjuvant, and the carrier may be physiological saline, phosphate buffer, etc.

[0015] The present invention also provides a method for detecting protein interactions. The method utilizes the specific covalent linkage characteristics of Snoop8Catcher and Snoop8Tag, fuses a first test protein with Snoop8Catcher, fuses a second test protein with Snoop8Tag, and detects whether a covalent complex is formed after co-incubation. If a covalent complex is formed, it indicates that there is an interaction between the two test proteins.

[0016] Meanwhile, the present invention provides a kit containing Snoop8Catcher and Snoop8Tag proteins (or nucleic acid molecules encoding both), and may also contain buffer, instructions for use and / or control proteins, which are convenient for use in protein coupling experiments or protein interaction detection experiments.

[0017] The beneficial effects of this invention are: 1. This patent has been verified through in vitro experiments: within the commonly used pH range in biological experiments (pH 6.4-pH 7.4, such as phosphate buffer and PBS buffer) and within the standard incubation time (3-24 hours), the covalent binding efficiency of the Snoop8 system is significantly higher than that of the SnoopPC system; even in temperature gradient experiments (4℃, 25℃, 37℃), the Snoop8 system can maintain an efficiency comparable to the SnoopPC system without significant activity decay. This advantage solves the problem of "incomplete conjugation and low product purity" caused by the low efficiency of the traditional Snoop system, and is particularly suitable for scenarios with high efficiency requirements, such as vaccine construction (e.g., the conjugation of PDCoVRBD and ferritin requires efficient connection to ensure nanoparticle uniformity) and antibody-drug conjugate (ADC) preparation (requiring precise control of the drug-antibody conjugation ratio).

[0018] 2. The Snoop8 system continues the characteristic of "no need for exogenous catalytic enzymes or additional energy donors": covalent linkage can be completed simply by spontaneously forming isopeptide bonds (amide exchange reaction) between aspartic acid in Snoop8Catcher and lysine in Snoop8Tag in a conventional buffer solution (such as 20mM Tris-HCl, 150mM NaCl, pH 8.0). This "enzyme-free and energy-free" reaction mode avoids the risk of degradation of the target protein by exogenous enzymes (such as ligases) and does not require harsh conditions (such as high temperature and high salt), thus maximizing the protection of the natural activity of biomolecules such as antigens, antibodies, and enzymes. After PDCoVRBD (antigen) and ferritin (carrier) are coupled through the Snoop8 system, the immunogenicity of RBD and the nanocage structure of ferritin are still maintained, and the resulting RBD-Ferritin nanoparticles can effectively induce specific antibodies. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0020] Figure 1 : A schematic diagram of the Snoop8 complex formed by the covalent connection of Snoop8Catcher and Snoop8Tag; Figure 2 Gel filtration chromatography patterns and SDS-PAGE electrophoresis verification results of Snoop8Catcher (a), Snoop8Tag (b) and their reaction complex (c); Figure 3 Comparison of binding efficiency between SnoopPC and Snoop8 systems under different pH (a), temperature (b), and reaction time (c) conditions; Figure 4 Gel filtration chromatography and SDS-PAGE analysis results of Snoop8Catcher-Ferritin (a), PDCoVRBD-Snoop8Tag (b) and their assembled complex (c), and negative stained electron micrograph of the Ferritin-PDCoVRBD complex (d). Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available products, and all experimental methods are conventional methods. Unless otherwise specified, all experiments were conducted in accordance with the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 4th Edition), or as recommended by the manufacturer.

[0023] Example 1: In vitro assembly and validation of Snoop8Catcher and Snoop8Tag The Snoop8Catcher / Snoop8Tag system was screened by obtaining the protein structure of the SnoopPC system (PDB:2WW8) from the PDB database (rcsb.org). Structural similarity searches were performed using the ESM metagenomic protein structure database to screen candidate proteins with an RMSD (root mean square deviation) less than 2 Å and sequence identity less than 40% with the SnoopPC system. The top 100 candidate proteins were manually reviewed, preserving the complete structures of key catalytic residues (lysine, aspartic acid, and glutamic acid), and then splitting them into Cater and Tag portions. Complex structure prediction was performed using AlphaFold software to screen for candidate proteins that could form stable complexes with an RMSD less than 2 Å. After preliminary in vitro screening, candidate protein number 8 (named Snoop8) efficiently formed isopeptide bonds, confirming its Cater portion as Snoop8Catcher (SEQ ID NO:1) and its Tag portion as Snoop8Tag (SEQ ID NO:2).

[0024] Purification of Snoop8Catcher / Snoop8Tag protein: Due to the short Snoop8Tag sequence, to facilitate purification and detection, it was linked to the C-terminus of maltose-binding protein (MBP) using the (Gly-Ser)2 linker to construct the Snoop8Tag-MBP fusion expression plasmid; at the same time, a Snoop8Catcher standalone expression plasmid was constructed.

[0025] The two expression plasmids were transformed into E. coli BL21(DE3) competent cells, and single colonies were picked and inoculated into LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C with shaking until OD500.600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 1 mM and induce expression at 16℃ for 18 hours.

[0026] After the induction of expression was completed, the bacterial cells were collected by centrifugation at 4℃ and 8000rpm for 10 minutes. The bacterial cells were resuspended in buffer A (20mM Tris-HCl, 150mM NaCl, pH 8.0), and sonicated (300W power, 3 seconds working, 5 seconds interval, total time 30 minutes). The supernatant was collected by centrifugation at 4℃ and 12000rpm for 20 minutes.

[0027] After filtering the supernatant through a 0.22 μm filter membrane, it was loaded onto a Ni column (pre-equilibrated with buffer A) for affinity chromatography. Impurities were washed away with buffer A containing 20 mM imidazole, followed by gradient elution with buffer A containing 1 M imidazole. The protein fractions corresponding to the elution peaks were collected.

[0028] The collected protein fractions were concentrated using ultrafiltration tubes with a molecular weight cutoff of 30 kDa, and then loaded onto Superdex 20010 / 300GL (Snoop8Catcher) and Hiload 16 / 60 Superdex 200PG (Snoop8Tag-MBP) gel filtration chromatography columns, respectively. Buffer A was used as the elution buffer, and the flow rate was 0.5 mL / min. The target protein peaks were collected. Protein purity was identified by 12% SDS-PAGE electrophoresis. The results showed that both Snoop8Catcher and Snoop8Tag-MBP columns yielded high-purity expression (…). Figure 2 a, 2b).

[0029] To verify the covalent binding of Snoop8Catcher / Snoop8Tag, purified Snoop8Catcher and Snoop8Tag-MBP were mixed at a molar ratio of 1:2 and incubated overnight at 4°C. The mixture was then loaded onto a Hiload 16 / 60 Superdex 200PG gel filtration chromatography column using buffer A as the elution buffer at a flow rate of 0.5 mL / min. Each chromatographic peak was collected and verified by 12% SDS-PAGE electrophoresis.

[0030] The results showed that, compared with Snoop8Catcher and Snoop8Tag-MBP alone, a new elution peak appeared after mixed incubation, and the corresponding molecular weight was significantly larger than that of the two proteins individually. SDS-PAGE electrophoresis showed new protein bands, indicating that Snoop8Catcher and Snoop8Tag successfully formed a covalent complex. Figure 2 c).

[0031] The reaction efficiency was compared under different conditions. Different pH, temperature and reaction time conditions were set to compare the reaction efficiency of the Snoop8 system and the traditional SnoopPC system. The specific conditions are as follows: (1) pH conditions: three gradients were set: pH 6.4 (phosphate buffer: 1.74g NaH2PO4, 2.7g Na2HPO4, 3.5g NaCl, and water was added to make up to 400mL), pH 7.4 (PBS buffer), and pH 8.0 (20mM Tris-HCl, 150mM NaCl); (2) Temperature conditions: three gradients were set: 4℃, 25℃ and 37℃; (3) Time conditions: seven gradients were set: 0.5h, 1h, 3h, 6h, 12h, 18h and 24h.

[0032] Dilute Snoop8Catcher and Snoop8Tag-MBP (or the Catcher and Tag of the SnoopPC system) to 10 nM and incubate under the conditions described above (sample at the set temperature for temperature gradient experiments, sample at the set time for time gradient experiments, and incubate at 4℃ for 3 hours for pH gradient experiments). After incubation, add 5×SDS loading buffer, boil at 100℃ for 8 minutes to terminate the reaction, and perform 12% SDS-PAGE electrophoresis followed by Coomassie Brilliant Blue staining.

[0033] ImageJ software was used to measure the gray values ​​of the complex band and the unreacted tag protein band in each lane, and the binding efficiency was calculated (binding efficiency = gray value of complex band / (gray value of complex band + gray value of unreacted tag protein band) × 100%). Statistical difference analysis was performed using GraphPadPrism software, and the experiment was repeated three times.

[0034] The results show that ( Figure 3 Within the pH range of 6.4 and 7.4, the binding efficiency of the Snoop8 system was significantly higher than that of the SnoopPC system (P<0.01). Under different temperature conditions, there was no significant difference in binding efficiency between the two systems (P>0.05). Within the reaction time range of 3–24 hours, the binding efficiency of the Snoop8 system was consistently higher than that of the SnoopPC system (P<0.01 or P<0.001), and the binding efficiency gradually increased and tended to stabilize with prolonged reaction time. This indicates that the Snoop8 system has superior reaction efficiency under mild conditions.

[0035] Example 2: In vitro assembly of Snoop8Catcher-Ferritin and PDCoVRBD-Snoop8Tag Purification of the Snoop8Catcher-Ferritin fusion protein: The Snoop8Catcher sequence was linked to the N-terminus of ferritin via a (GGGS)3 linker to construct the pET-28a-Snoop8Catcher-Ferritin recombinant expression plasmid. This plasmid was transformed into E. coli BL21(DE3) competent cells, and single colonies were picked and inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking until OD. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.3 mM and induce expression at 37°C for 6 hours.

[0036] Bacterial cells were collected, resuspended in buffer B (20 mM Tris-HCl, 50 mM NaCl, pH 8.0), sonicated, and centrifuged to collect the supernatant. The supernatant was heated in a 70°C metal bath for 15 minutes, centrifuged at 4°C and 12000 rpm for 20 minutes to remove denatured proteins. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a Superose 6 Increase 10 / 300 GL gel chromatography column. Buffer B was used as the elution buffer, and the flow rate was 0.5 mL / min. The target protein peak was collected. High-purity Snoop8 Catcher-Ferritin fusion protein was obtained by 12% SDS-PAGE electrophoresis. Figure 4 a).

[0037] Purification of the PDCoVRBD-Snoop8Tag protein: The Snoop8Tag sequence was linked to the C-terminus of PDCoVRBD via a (GGGS)3 linker to construct the pCDNA3.1-PDCoVRBD-Snoop8Tag eukaryotic expression plasmid. This plasmid was transfected into 293F cells (cultured in FreeStyle™ 293 Expression Medium) using the PEI transfection method. After transfection, the cells were cultured at 37°C, 5% CO2, and 120 rpm for 5 days with shaking. The cell culture supernatant was then collected.

[0038] The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a Ni column (pre-equilibrated with buffer A). Impurities were washed away with buffer A containing 20 mM imidazole, and the target protein was eluted with buffer A containing 500 mM imidazole. The eluted protein fraction was concentrated using an ultrafiltration tube with a molecular weight cutoff of 50 kDa and loaded onto a Superose 6 Increase 10 / 300 GL gel filtration chromatography column. Buffer A was used as the elution buffer, and the flow rate was 0.5 mL / min. The target protein peak was collected. High-purity PDCoVRBD-Snoop8Tag protein was obtained by 12% SDS-PAGE electrophoresis. Figure 4 b).

[0039] Complex assembly and validation: 0.6 mg of purified Snoop8Catcher-Ferritin was mixed with 2.4 mg PDCoVRBD-Snoop8Tag and incubated overnight on ice. The mixture was loaded onto a Superose 6 Increase 10 / 300 GL gel filtration chromatography column, using buffer A as the elution buffer at a flow rate of 0.5 mL / min. The elution peak was collected and validated by 12% SDS-PAGE electrophoresis.

[0040] The results showed that a new elution peak appeared after mixed incubation, with a molecular weight significantly larger than that of the two fusion proteins individually. SDS-PAGE electrophoresis showed new protein bands, indicating that Snoop8Catcher-Ferritin and PDCoVRBD-Snoop8Tag were successfully assembled to form an RBD-Ferritin nanoparticle complex. Figure 4 c).

[0041] For negative staining electron microscopy analysis, the assembled RBD-Ferritin nanoparticle complex was diluted to 0.02 mg / mL, dropped onto a copper grid, allowed to stand for 1 minute, stained three times with 2% uranium acetate solution (1 minute each time), and allowed to air dry. It was then observed using a Talos L120C transmission electron microscope (accelerating voltage 120 kV, magnification 57000×).

[0042] Electron micrographs show ( Figure 4 d) The complex consists of uniform spherical particles with a diameter of approximately 20-25 nm and visible RBD domain protrusions on the surface, indicating that RBD-Ferritin multivalent nanoparticles with good morphology have been successfully constructed and can be used for the preparation of PDCoV vaccines.

[0043] Example 3: Immunogenicity Detection of PDCoVRBD-Ferritin Nanoparticle Vaccine The RBD-Ferritin nanoparticle complex prepared in Example 2 was mixed with aluminum adjuvant (final concentration 0.5 mg / mL) to prepare a PDCoV vaccine composition. Six- to eight-week-old female BALB / c mice were randomly divided into three groups (n=10 per group): Experimental group: Immunize each mouse with the above vaccine composition by intraperitoneal injection of 100 μL (containing 10 μg RBD). Control group 1: A mixture of immunized PDCoVRBD protein (10 μg) and aluminum adjuvant; Control group 2: a mixture of immunophysiological saline and aluminum adjuvant.

[0044] Mice serum was collected on days 0, 14, and 28 post-immunization. The titer of PDCoVRBD-specific antibodies in the serum was detected using an indirect ELISA method: ELISA plates were coated with PDCoVRBD protein (2 μg / mL) and incubated overnight at 4°C; after blocking, serially diluted mouse serum was added and incubated at 37°C for 1 hour; HRP-labeled goat anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 hour; TMB colorimetry was performed, and the OD value was measured at 450 nm. 450 The highest dilution, greater than 0.2 and more than twice that of the negative control, is the antibody titer.

[0045] The results showed that on day 14 post-immunization, the serum RBD-specific antibody titer in the experimental group (1:12800-1:25600) was significantly higher than that in control group 1 (1:1600-1:3200). On day 28 post-immunization, the antibody titer in the experimental group further increased to 1:51200-1:102400, while the antibody titer in control group 1 was 1:6400-1:12800, and no specific antibodies were detected in control group 2. This indicates that the RBD-Ferritin nanoparticle vaccine constructed in this invention can significantly enhance the immunogenicity of PDCoVRBD and induce higher levels of specific antibodies.

[0046] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A protein covalent coupling system, characterized in that, The protein covalent coupling system includes Snoop8Catcher and Snoop8Tag, the amino acid sequence of Snoop8Catcher is shown in SEQ ID NO:1, and the amino acid sequence of Snoop8Tag is shown in SEQ ID NO:

2.

2. The protein covalent coupling system according to claim 1, characterized in that, The Snoop8Catcher and Snoop8Tag can be covalently linked by forming an isopeptide bond between the aspartic acid in the Snoop8Catcher and the lysine in the Snoop8Tag.

3. The protein covalent coupling system according to claim 1 or 2, characterized in that, The Snoop8Catcher and / or Snoop8Tag can fuse with the target protein to form a fusion protein, and achieve covalent linkage between the target proteins through the isopeptide bond; the target protein includes the porcine deltacoronavirus spike protein receptor-binding domain and ferritin.

4. The protein covalent coupling system according to claim 3, characterized in that, The fusion protein is: (a) A fusion protein of RBD and Snoop8Tag; as well as (b) A fusion protein of ferritin and Snoop8Catcher; The fusion proteins (a) and (b) are capable of self-assembling into an RBD-Ferritin nanoparticle complex through the interaction between Snoop8Tag and Snoop8Catcher.

5. A nucleic acid molecule, characterized in that, Its encoding is Snoop8Catcher or Snoop8Tag as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, It is transformed or transfected with the expression vector described in claim 6.

8. Use of the protein covalent coupling system according to any one of claims 1-4 in the preparation of pharmaceuticals, vaccines, diagnostic reagents or protein nanomaterials.

9. A vaccine composition, characterized in that, It comprises the RBD-ferritin nanoparticle complex of claim 4 and a pharmaceutically acceptable adjuvant or carrier.

10. A reagent kit, characterized in that, It includes the Snoop8Catcher and Snoop8Tag proteins as described in any one of claims 1-4, or the nucleic acid molecules encoding them.

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