Polyantibody as well as preparation method and application thereof

Abstract

The invention discloses a fusion protein, nano-particles formed by self-assembly of the fusion protein, and a polyantibody taking the nano-particles as a carrier, and also provides a preparation method and application of the product. The nanoparticles provided by the invention can efficiently load one or more monoclonal antibodies so as to prepare a polyantibody; the Fab antigen binding domain of the polyantibody is spatially ordered and radially arranged on the surface of the nanoparticle, the structural characteristic greatly improves the sensitivity of antigen detection, and the polyantibody has important clinical application value for rapid detection and early warning of related antigens or viruses, and has good industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fusion protein, nanoparticles self-assembled from the fusion protein, polyantibodies using the nanoparticles as carriers, and their preparation methods and applications. Background Technology

[0002] By using genetic engineering to cascade protein or peptide domains with different functions to achieve multifunctional integration of a single molecule, it has become one of the core technologies in fields such as biomaterials and drug delivery. Among them, fusion proteins with self-assembly capabilities can spontaneously form structurally stable nanoparticles through non-covalent interactions under mild conditions. These nanoparticles combine the biocompatibility of proteins with the size advantages of nanomaterials and have been widely developed as functional devices such as drug carriers and biosensors.

[0003] Self-assembled nanoparticle-based carrier systems have demonstrated immense application potential in the biomedical field due to their unique advantages. On one hand, modular design allows for the flexible introduction of specific functional domains, enabling nanoparticle carriers to possess customized functions such as specific recognition. On the other hand, nanoparticles exhibit size and surface effects, with their high specific surface area facilitating the directional display of affinity sites, thus offering possibilities for improving detection sensitivity.

[0004] Early, rapid, and accurate detection of SARS-CoV-2 is crucial for reducing the risk of disease transmission. Compared to nucleic acid testing, the gold standard for SARS-CoV-2 detection, antigen testing still faces a series of challenges, especially in the early stages of infection, where low sensitivity and high false negative rates pose significant obstacles to the field. Therefore, there is an urgent need to develop methods that improve the sensitivity and specificity of SARS-CoV-2 antigen testing to reduce missed and misdiagnosed cases.

[0005] Therefore, developing a fusion protein with high self-assembly efficiency, stable structure and easy large-scale production, as well as the nanoparticles formed by its self-assembly and the carrier complex based on the particles, is of great significance for promoting the practical application of biocarrier technology. Summary of the Invention

[0006] Purpose of the invention In view of the needs of the prior art, the purpose of this invention is to provide a fusion protein that can efficiently self-assemble into nanoparticles, nanoparticles self-assembled from the fusion protein, polyantibodies using the nanoparticles as carriers, and their preparation methods and applications.

[0007] Solution To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a fusion protein comprising an antibody Fc binding element and a self-assembled nanoparticle protein element.

[0008] In a feasible implementation, the fusion protein has the structure shown in formula (I) from the N-terminus to the C-terminus: A-L1-B-L2-C (I) In formula (I), A is the antibody Fc binding element; B represents a self-assembled nanoparticle protein element; C is an optional label element; L1 and L2 are each independent peptide elements, either non-linked or linked.

[0009] Preferably, the antibody Fc binding element is the immunoglobulin binding domain of a streptococcal G protein, more preferably the C2 domain of a streptococcal G protein, and even more preferably the amino acid sequence shown in SEQ ID NO:1. More preferably, it is encoded by the nucleotide sequence shown in SEQ ID NO:4. Preferably, the self-assembled nanoparticle protein element is selected from the following proteins or their peptides: heat shock protein, ferritin, virus-like particle protein, encapsulation protein and Mi3 protein, preferably a Mi3 protein peptide, more preferably an amino acid sequence as shown in SEQ ID NO:2; more preferably, it is encoded by a nucleotide sequence as shown in SEQ ID NO:5; Preferably, the linker peptide element is a GS linker peptide, preferably (GGGS)n or (GGGGS)n, where n is an integer between 1 and 10, preferably an integer between 1 and 5; Preferably, the tag element is a histidine tag, preferably a tag composed of 6-10 histidines in series, more preferably 8 histidines in series.

[0010] More preferably, the fusion protein has an amino acid sequence selected from the following: (1) The amino acid sequence as shown in SEQ ID NO:3; (2) An amino acid sequence with the same or similar function obtained by substituting, deleting and / or adding one or more amino acids as shown in SEQ ID NO:3; or (3) An amino acid sequence that has at least 90% sequence identity with the amino acid sequence defined in (1) or (2) and has the same or similar function.

[0011] In a second aspect, the present invention provides a polynucleotide encoding the fusion protein as described in the first aspect above; Preferably, the polynucleotide comprises a nucleotide sequence selected from the following: 1) A nucleotide sequence as shown in SEQ ID NO:6; or 2) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in SEQ ID NO:6, but is different from the nucleotide sequence shown in SEQ ID NO:6 due to the degeneracy of the genetic code.

[0012] Thirdly, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above and one or more expression regulatory elements operatively linked thereto.

[0013] Fourthly, the present invention provides a recombinant vector comprising the polynucleotides as described in the second aspect above or the nucleic acid constructs as described in the third aspect above.

[0014] Fifthly, the present invention provides a host cell wherein the cell is transformed or transfected with the polynucleotides described in the second aspect above, the nucleic acid constructs described in the third aspect above, or the recombinant vectors described in the fourth aspect above.

[0015] Sixthly, the present invention provides a method for preparing the fusion protein as described in the first aspect above, characterized in that the method comprises the following steps: Under conditions suitable for the expression of the fusion protein, host cells as described in the fifth aspect above are cultured to express the fusion protein; Preferably, the method further includes the steps of separating and purifying the fusion protein.

[0016] In a seventh aspect, the present invention provides a nanoparticle, said nanoparticle being self-assembled from the fusion protein described in the first aspect above; Preferably, the nanoparticles are hexadecimals.

[0017] In an eighth aspect, the present invention provides the use of the fusion protein as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the recombinant vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, and / or the nanoparticle as described in the seventh aspect above in the preparation of polyantibodies.

[0018] Ninthly, the present invention provides a polyantibody comprising: (1) Nanoparticles as described in aspect 7 above, serving as a carrier; and, (2) One or more monoclonal antibodies loaded on the nanoparticles.

[0019] In this field, "polyantibody" refers to a polymer structure formed by multiple antibodies linked by covalent or non-covalent bonds.

[0020] In feasible implementations, the polyantibody of the present invention can be a homologous polyantibody (i.e., the nanoparticles are loaded with one monoclonal antibody) or a heterologous polyantibody (i.e., the nanoparticles are loaded with two or more monoclonal antibodies).

[0021] In a feasible specific implementation, the polyantibody is a polyantibody against SARS-CoV-2, preferably a homologous polyantibody against SARS-CoV-2, wherein the nanoparticles are loaded with a monoclonal antibody against the SARS-CoV-2 antigen, preferably loaded with a monoclonal antibody against the SARS-CoV-2 N protein (e.g., Novizan's SARS-CoV-2 monoclonal antibody RM3146 or RM3147).

[0022] In a tenth aspect, the present invention provides a composition for detecting SARS-CoV-2 infection, the composition comprising the polyantibody as described in the ninth aspect above, wherein the monoclonal antibody is a monoclonal antibody against the SARS-CoV-2 antigen, preferably a monoclonal antibody against the SARS-CoV-2 N protein.

[0023] In the eleventh aspect, the present invention provides a method for preparing the polyclonal antibody as described in the ninth aspect above, the method comprising: incubating the monoclonal antibody with the nanoparticles as described in the seventh aspect above, thereby obtaining the antibody; Preferably, the molar ratio of the monoclonal antibody to the nanoparticles as described in claim 10 is (60-80):1, more preferably 60:1; Preferably, the incubation conditions are 22-27℃ for 40 min-1.5 h, and more preferably 24-26℃ for 1 h; Preferably, the preparation method further includes a step of removing unreacted components by size exclusion chromatography.

[0024] In a twelfth aspect, the present invention provides a method for detecting SARS-CoV-2 infection, the method comprising: using a polyantibody as described in the ninth aspect above or a composition as described in the tenth aspect above for detection, wherein the polyantibody comprises a monoclonal antibody against the SARS-CoV-2 antigen.

[0025] In a thirteenth aspect, the present invention provides a method for detecting SARS-CoV-2, the method comprising: detecting a sample using a polyantibody as described in the ninth aspect above. The polyantibody comprises a monoclonal antibody against the SARS-CoV-2 antigen. Preferably, the sample is a pharyngeal swab sample.

[0026] Beneficial effects The nanoparticle carrier provided by this invention can load one or more monoclonal antibodies for the preparation of polyantibodies. The Fab antigen-binding domains of the prepared polyantibodies are arranged in a spatially ordered radial pattern on the surface of the nanoparticles, thereby significantly improving their sensitivity and / or broad-spectrum application for antigen detection. This has important clinical application value and industrialization prospects for the rapid detection and early warning of related antigens or viruses. Attached Figure Description

[0027] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. Here, the specific term "illustrative" means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0028] Figure 1 This describes the construction and identification of the polyantibody described in the embodiments of the present invention.

[0029] Figure 2 Transmission electron microscopy images of SARS-CoV-2 monoclonal antibody RM3146 (Figure a), the prepared nanoparticles (referred to as "MG", Figure b), and the constructed SARS-CoV-2 polyantibodies (referred to as "MG-3146" and "MG-3147", Figure c and Figure d, respectively).

[0030] Figure 3 The antigen-binding affinity curves of SARS-CoV-2 monoclonal antibodies RM3146 (Figure a) and RM3147 (Figure c) and SARS-CoV-2 polyantibodies MG-3146 (Figure b) and MG-3147 (Figure d) detected in Example 3 are shown.

[0031] Figure 4 The results are the test results of the ELISA method described in Example 4, which tests the detection sensitivity of SARS-CoV-2 monoclonal antibody RM3147 and polyantibody MG-3147 antigen.

[0032] Figure 5 This is a schematic diagram of the structure of the SARS-CoV-2 colloidal gold lateral immunochromatographic test strip based on SARS-CoV-2 polyantibody described in Example 5.

[0033] Figure 6 The results show the sensitivity detection of the SARS-CoV-2 colloidal gold lateral immunochromatographic test strip described in Example 6. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including," "having," "containing," or "comprises" will be understood to include the stated elements or components without excluding other elements or other components, unless otherwise specifically stated or understood from the context.

[0036] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0037] Furthermore, unless otherwise expressly stated, all ranges, quantities, values, and percentages used in this disclosure are modified with the word "about". Here, "about" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0038] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0039] The present invention will be further illustrated by the following examples; unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Example 1: Expression of the fusion protein and preparation of nanoparticles according to the present invention In this embodiment, the fusion protein of the present invention with the amino acid sequence shown in SEQ ID NO:3 was prepared, and its elemental structure is as follows: C2-(GGGS)3-Mi3-(GGGS)-8*His.

[0041] Specifically, firstly, the codon-optimized DNA coding sequence of the fusion protein shown in SEQ ID NO:3 was obtained, as shown in SEQ ID NO:6; this DNA coding sequence was cloned into the *E. coli* expression vector PET30a plasmid using NdeI and BamHI double restriction sites to construct a recombinant expression vector; the recombinant expression vector was used to transform competent *E. coli*, and positive single clones were selected for expansion culture; the expanded culture solution was collected, ultrasonically disrupted, and then affinity chromatography was performed using AKTAPure (GE HealthCare, His Excel 5 mL column) to enrich the target protein, followed by size exclusion chromatography (Superose)... TM 6. Increase the 10 / 300 GL chromatography column to further purify the protein.

[0042] The protein products obtained in the above steps were identified by SDS-PAGE gel electrophoresis. The SDS-PAGE gel electrophoresis image is shown below. Figure 1 As shown; Figure 1 The results show that the molecular weight of the protein expressed above is consistent with the theoretical molecular weight of the fusion protein of the present invention, which indicates that the fusion protein of the present invention was successfully obtained after the above steps.

[0043] Furthermore, the product obtained in the above steps was observed using transmission electron microscopy, and its transmission electron microscopy image is shown below. Figure 2 Figure b in the figure shows that the product obtained through the above steps is a uniform nanoparticle structure with a diameter of approximately 28 nm.

[0044] Example 2: Preparation of SARS-CoV-2 Polyantibody The nanoparticles prepared in Example 1 were mixed with monoclonal antibodies RM3146 and RM3147 (purchased from Novizan) against the SARS-CoV-2 N protein at a molar ratio of 1:60 and incubated at room temperature for 1 h. Then, unreacted components were removed by size exclusion chromatography to obtain SARS-CoV-2 polyantibodies MG-3146 and MG-3147.

[0045] The SARS-CoV-2 polyantibodies MG-3146 and MG-3147 obtained were observed using transmission electron microscopy, and their transmission electron micrographs are shown below. Figure 2 As shown in Figures c and d, the SARS-CoV-2 polyantibodies MG-3146 and MG-3147 are uniform nanoparticle structures with a diameter of approximately 30 nm.

[0046] In addition, for comparison, a transmission electron microscope image of the monoclonal antibody RM3146 was also obtained. Figure 2 ).

[0047] Example 3: Affinity Detection of SARS-CoV-2 Polymeric Antibodies In this embodiment, the affinity of the SARS-CoV-2 polyantibodies MG-3146 and MG-3147 and their corresponding monoclonal antibodies prepared in Example 2 above was tested.

[0048] Specifically, the affinity between the SARS-CoV-2 polyantibody and the SARS-CoV-2 N protein was analyzed using the Biacore 8K biomolecular interaction system, as follows: First, a 50 nM solution of the target antibody (SARS-CoV-2 polyantibody or monoclonal antibody) was used as the mobile phase and passed through the SensorChip Protein G chip (GE Healthcare) to complete binding and elution sequentially, eliminating direct sample interference. Then, a PBST-diluted 2 μg / mL solution of the target antibody was coupled to the Protein G chip. After stable coupling, a serially diluted SARS-CoV-2 N protein solution (purchased from Sinocare) was injected as the mobile phase. The detection program was set as follows: antigen-antibody binding for 60 s, dissociation for 120 s, followed by chip regeneration with ammonium ethanol solution for 120 s. After each detection, the chip baseline was ensured to return to its initial level. The above experiments were independently repeated three times. The affinity constant (KD) between the antibody and antigen was calculated using Biacore 8K dedicated evaluation software (GE Healthcare) and a 1:1 Langmuir binding model. The affinity data obtained from the three repeated experiments were statistically analyzed, and Graphpad Prism 8.0 software was used for data fitting and graphing analysis.

[0049] See results Figure 3 ad graph in the middle; Figure 3 This indicates that, compared to SARS-CoV-2 monoclonal antibodies, polyclonal antibodies with the same molar amount of subunits have 2-4 times higher affinity.

[0050] Example 4: Sensitivity test for antigen detection of SARS-CoV-2 polyantibody In this embodiment, the antigen detection sensitivity of the SARS-CoV-2 polyantibody MG-3147 and its corresponding monoclonal antibody prepared in Example 2 above was tested by ELISA. The test method is as follows: 1. Coating antibody: SARS-CoV2 antibody Mab15 (purchased from Phytobio Biotechnology Co., Ltd., a mouse-derived antibody) was diluted to 2 μg / mL with coating buffer and plated for ELISA. After mixing, 200 μL was added to each well of a 96-well plate, with three replicates per sample. The plate was incubated overnight at 4 ℃ in a humidified chamber. 2. Washing the plate: Wash the 96-well plate three times with 1×PBST; 3. Blocking: Add 5% skim milk (diluted with 1×PBS), 300 μL / well, incubate at 37 ℃ in a humidified chamber for 2 h, then remove the skim milk; 4. Incubation of antigen: Dilute the antigen (such as the SARS-CoV-2 N protein described in Example 3) with 1×PBS at certain ratios (10 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.01 ng / mL, 0 ng / mL), 100 μL / well, incubate at 37 ℃ in a humidified chamber for 2 h; 5. Wash the plate, same as step 2; 6. Antibody incubation: SARS-CoV2 antibody RM3147 (Novozymes, rabbit-derived antibody) and polyantibody MG-3147 were diluted with 1×PBS to the same subunit molar amount, i.e., 2 μg / mL and 2.4 μg / mL, respectively, and incubated at 37℃ in a humidified chamber for 2 h. 7. Wash the plate, same as step 2; 8. Incubation of HRP-labeled secondary antibody: Dilute HRP-labeled secondary antibody 5000 times with 5% skim milk (diluted with 1×PBS), 100 μL / well, incubate at 37℃ in a humidified chamber for 1 h; 9. Wash the plate: Wash the 96-well plate 5 times with 1×PBST and 3-4 times with 1×PBS; 10. Color development: Add 100 μL of TMB one-step color development solution to each well. After ten minutes, add 100 μL of 2 M H2SO4 to terminate the reaction. Immediately measure the OD 450 nm absorbance using a microplate reader.

[0051] The results are as follows Figure 4 As shown; Figure 4 This indicates that, compared to SARS-CoV-2 monoclonal antibodies, polyantibodies with the same molar amount of subunits have a 5-fold increased antigen detection sensitivity.

[0052] Example 5: Preparation of a colloidal gold lateral immunochromatographic test strip based on SARS-CoV-2 polymeric antibody In this embodiment, based on the SARS-CoV-2 polyantibody MG-3147 or its corresponding monoclonal antibody prepared in Example 2, SARS-CoV-2 colloidal gold immunochromatographic test strips and test cards were prepared. The specific operations are as follows: (1) Colloidal gold labeling of antibodies: Take 5 ml of 0.04% colloidal gold, add 60-75 μL of 0.2M K2CO3 (pH 8.6-9.3), and stir for 5 min; add SARS-CoV-2 polyantibody MG-3147 or monoclonal antibody RM3147 (antibody volume = 50 μg / antibody concentration), continue stirring for 5 min, and then add 50 μL of blocking agent (5% BSA) to stop the labeling; then, centrifuge at 10000 rpm for 7 min, remove the supernatant, reconstitute the precipitate with gold reconstitution solution (0.2 M PBS + 1% BSA + 2% sucrose + 0.05% Tween-20), and make up to 0.5 mL with the reconstitution solution to prepare the colloidal gold antibody working solution.

[0053] The method for labeling sheep anti-chicken IgY for quality control is the same as above.

[0054] (2) Preparation of colloidal gold lateral immunochromatographic test strips The colloidal gold-labeled SARS-CoV-2 detection antibody and quality control antibody were prepared into a lateral immunochromatographic test strip. A schematic diagram of the test strip structure is shown below. Figure 5 Furthermore, for ease of use, the test strip was made into a test card.

[0055] Specifically, the test card includes an outer shell, which is formed by the snap-fitting of an upper shell and a lower shell. The upper shell presses the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper firmly onto a PVC plate. The conjugate pad treatment solution is 10 mM Tris buffer (pH 8.0), containing 1% BSA, 0.9% NaCl, and 0.5% Triton-100. The conjugate pad is sprayed with 10% concentrated colloidal gold-labeled SARS-CoV2 antibody (including colloidal gold-labeled SARS-CoV-2 polyantibody or its monoclonal antibody and colloidal gold-labeled goat anti-chicken IgY), and placed in a forced-air drying oven at 37°C overnight. Parallel test lines (T lines) and control lines (C lines) are sequentially drawn on the nitrocellulose membrane. The test line (T line) is SARS-CoV-2 polyantibody MG-3147 or its monoclonal antibody RM3147, diluted to 0.5-2.0 mg / mL for streaking, with a streaking volume of 0.1... 5 μL / cm, streaking speed is 5 100 mm / s; Control line C is goat anti-chicken IgY, diluted to 0.5-2.0 mg / mL for streaking, with a streaking volume of 5 μL / cm and a streaking speed of 5. The speed is 100 mm / s. The distance between the detection line (T-line) and the control line (C-line) is 5 mm. The detection line (T-line) is closer to the sample pad, and the control line (C-line) is closer to the absorbent paper. A sample application port is located on the upper shell at the position corresponding to the sample pad, and an observation window is located on the upper shell at the position corresponding to the nitrocellulose membrane.

[0056] Example 6: Antigen detection sensitivity test of colloidal gold lateral immunochromatographic test strip based on SARS-CoV-2 polymeric antibody The SARS-CoV-2 N protein (as described in Example 3) was used as an antigen and serially diluted 2-fold to concentrations of 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.6 ng / mL, 0.3 ng / mL, 0.15 ng / mL, and 0 ng / mL. 100 μL of each of the above-mentioned diluted SARS-CoV-2 antigen solutions was added to the sample wells of the colloidal gold lateral immunochromatographic test strips prepared in Example 5, based on the SARS-CoV-2 polyantibody MG-3147 or its monoclonal antibody RM3147. The detection limits of the two immunochromatographic test strips were determined as indicators for evaluating the antigen detection sensitivity of the two test strips.

[0057] The results are as follows Figure 6 As shown; Figure 6 The results showed that the detection limit of the immunochromatographic test strip based on the SARS-CoV-2 polyantibody MG-3147 was 0.3 ng / mL, while the detection limit of the immunochromatographic test strip based on the monoclonal antibody RM3147 was 2.5 ng / mL. This means that the antigen detection sensitivity of the test strip based on the polyantibody MG-3147 was more than four times higher than that of the test strip based on its corresponding monoclonal antibody. This result further demonstrates that the polyantibody of the present invention has a significantly improved antigen detection sensitivity compared to its corresponding monoclonal antibody.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0059] The sequences involved in this application are: SEQ ID NO:1 (Chain C, STREPTOCOCCAL PROTEIN G C2 FRAGMENT amino acid sequence) TTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE SEQ ID NO:2 (Mi3 amino acid sequence) KMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE* SEQ ID NO:3 ( (Amino acid sequence of MG) TTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGSGGGSGGGSKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTEGGGSHHHHHHHH* SEQ ID NO:4 (Nucleotide sequence of Chain C, STREPTOCOCCAL PROTEIN G C2 FRAGMENT) ACTACATATAAACTAGTAATAAATGGAAAGACCCTGAAAGGTGAAACCACGACGGAGGCGGTTGACGCGGCTACCGCAGAGAAGGTGTTCAAGCAGTACGCAAACGATAACGGCGTGGATGGTGAATGGACCTACGATGACGCTACCAAAACGTTTACCGTTACTGAA SEQ ID NO:5 (Nucleotide sequence of Mi3) AAAATGGAAGAGTTGTTCAAGAAGCACAAAATCGTGGCGGTGCTGCGCGCGAACAGCGTTGAGGAGGCAAAGAAAAAAGCGCTGGCTGTGTTCCTGGGCGGCGTTCATCTGATTGAAATCACCTTTACCGTTCCGGACGCGGATACCGTGATTAAAGAATTGAGCTTTTTAAAAGAGATGGGTGCAATCATCGGCGCGGGTACGGTTACCAGCGTTGAGCAAGCACGTAAAGCGGTGGAGAGCGGTGCGGAATTCATTGTTAGCCCGCATCTCGACGAAGAGATCAGCCAGTTCGCAAAAGAGAAGGGTGTTTTTTATATGCCGGGCGTGATGACCCCGACCGAACTGGTGAAAGCTATGAAGCTGGGTCATACCATTCTGAAGCTGTTCCCGGGTGAAGTGGTGGGCCCGCAATTCGTGAAGGCCATGAAGGGTCCGTTTCCGAATGTCAAATTCGTGCCAACTGGTGGTGTAAATCTGGACAACGTCTGTGAATGGTTTAAGGCCGGCGTCTTGGCGGTTGGCGTCGGTTCCGCGTTGGTAAAGGGCACGCCGGTCGAGGTTGCCGAAAAAGCGAAGGCGTTTGTTGAGAAGATCCGTGGTTGCACCGAG SEQ ID NO:6 ( MG nucleotide sequence) ACTACATATAAACTAGTAATAAATGGAAAGACCCTGAAAGGTGAAACCACGACGGAGGCGGTTGACGCGGCTACCGCAGAGAAGGTGTTCAAGCAGTACGCAAACGATAACGGCGTGGATGGTGAATGGACCTACGATGACGCTACCAAAACGTTTACCGTTACTGAAGGTGGTGGCTCTGGCGGTGGCTCTGGCGGAGGTTCCAAAATGGAAGAGTTGTTCAAGAAGCACAAAATCGTGGCGGTGCTGCGCGCGAACAGCGTTGAGGAGGCAAAGAAAAAAGCGCTGGCTGTGTTCCTGGGCGGCGTTCATCTGATTGAAATCACCTTTACCGTTCCGGACGCGGATACCGTGATTAAAGAATTGAGCTTTTTAAAAGAGATGGGTGCAATCATCGGCGCGGGTACGGTTACCAGCGTTGAGCAAGCACGTAAAGCGGTGGAGAGCGGTGCGGAATTCATTGTTAGCCCGCATCTCGACGAAGAGATCAGCCAGTTCGCAAAAGAGAAGGGTGTTTTTTATATGCCGGGCGTGATGACCCCGACCGAACTGGTGAAAGCTATGAAGCTGGGTCATACCATTCTGAAGCTGTTCCCGGGTGAAGTGGTGGGCCCGCAATTCGTGAAGGCCATGAAGGGTCCGTTTCCGAATGTCAAATTCGTGCCAACTGGTGGTGTAAATCTGGACAACGTCTGTGAATGGTTTAAGGCCGGCGTCTTGGCGGTTGGCGTCGGTTCCGCGTTGGTAAAGGGCACGCCGGTCGAGGTTGCCGAAAAAGCGAAGGCGTTTGTTGAGAAGATCCGTGGTTGCACCGAGGGCGGTGGCTCGCATCACCACCACCACCACCATCAC。

Claims

1. A fusion protein, characterized in that, The fusion protein includes an antibody Fc binding element and a self-assembled nanoparticle protein element.

2. The fusion protein according to claim 1, characterized in that, The fusion protein has the structure shown in formula (I) from the N-terminus to the C-terminus: A-L1-B-L2-C (I) In formula (I), A is the antibody Fc binding element; B represents a self-assembled nanoparticle protein element; C is an optional label element; L1 and L2 are each independent peptide elements, either non-linked or linked.

3. The fusion protein according to claim 1 or 2, characterized in that, The antibody Fc binding element is the immunoglobulin binding domain of streptococcal G protein, preferably the C2 domain of streptococcal G protein, and more preferably the amino acid sequence shown in SEQ ID NO:1; And / or, the self-assembled nanoparticle protein element is selected from the following proteins or their peptides: heat shock proteins, ferritin, virus-like particle proteins, encapsulation proteins and Mi3 proteins, preferably Mi3 protein peptides, and more preferably the amino acid sequence shown in SEQ ID NO:2; And / or, the linker peptide element is a GS linker peptide, preferably (GGGS)n or (GGGGS)n, where n is an integer between 1 and 10, preferably an integer between 1 and 5; And / or, the tag element is a histidine tag, preferably a tag consisting of 6-10 histidines in series, more preferably 8 histidines in series.

4. The fusion protein according to claim 3, characterized in that, The fusion protein has an amino acid sequence selected from the following: (1) The amino acid sequence as shown in SEQ ID NO:3; (2) An amino acid sequence with the same or similar function obtained by substituting, deleting and / or adding one or more amino acids as shown in SEQ ID NO:3; or (3) An amino acid sequence that has at least 90% sequence identity with the amino acid sequence defined in (1) or (2) and has the same or similar function.

5. A polynucleotide encoding the fusion protein as described in any one of claims 1-4; Preferably, the polynucleotide comprises a nucleotide sequence selected from the following: 1) A nucleotide sequence as shown in SEQ ID NO:6; or 2) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in SEQ ID NO:6, but is different from the nucleotide sequence shown in SEQ ID NO:6 due to the degeneracy of the genetic code.

6. A nucleic acid construct comprising the polynucleotide as described in claim 5 and one or more expression regulatory elements operatively linked thereto.

7. A recombinant vector comprising the polynucleotide of claim 5 or the nucleic acid construct of claim 6.

8. A host cell wherein the cell is transformed or transfected with the polynucleotide of claim 5, the nucleic acid construct of claim 6, or the recombinant vector of claim 7.

9. A method for preparing the fusion protein according to any one of claims 1-4, characterized in that, The method includes the following steps: Under conditions suitable for the expression of the fusion protein, the host cells as described in claim 8 are cultured to express the fusion protein; Preferably, the method further includes the steps of separating and purifying the fusion protein.

10. A nanoparticle, characterized in that, The nanoparticles are self-assembled from the fusion protein according to any one of claims 1-4; Preferably, the nanoparticles are hexadecimals.

11. The use of the fusion protein of any one of claims 1-4, the polynucleotide of claim 5, the nucleic acid construct of claim 6, the recombinant vector of claim 7, the host cell of claim 8, and / or the nanoparticle of claim 10 in the preparation of polyantibodies.

12. A polymeric antibody comprising: (1) The nanoparticles as described in claim 10 as a carrier; and, (2) One or more monoclonal antibodies loaded on the nanoparticles.

13. The polyantibody according to claim 11, characterized in that, The monoclonal antibody is a monoclonal antibody against the SARS-CoV-2 antigen, preferably a monoclonal antibody against the SARS-CoV-2 N protein.

14. A composition for detecting, preventing, and / or treating SARS-CoV-2 infection, characterized in that, The composition comprises the polyantibody according to claim 13.

15. The method for preparing the polyantibody according to claim 12 or 13, characterized in that, The preparation method includes: incubating the monoclonal antibody with the nanoparticles as described in claim 10 to obtain the product; Preferably, the molar ratio of the monoclonal antibody to the nanoparticles as described in claim 10 is (60-80):1, more preferably 60:1; Preferably, the incubation conditions are 22-27℃ for 40 min-1.5 h, and more preferably 24-26℃ for 1 h; Preferably, the preparation method further includes a step of removing unreacted components by size exclusion chromatography.

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