Application of polyantibody in multiple detection of pathogens

Abstract

The invention provides a fusion protein, a nano-particle formed by self-assembly of the fusion protein, a polyantibody taking the nano-particle as a carrier, and preparation methods and applications of the fusion protein, the nano-particle and the polyantibody. The nanoparticles can load one or more monoclonal antibodies so as to be used for preparing a polyantibody; the Fab antigen binding domain of the prepared polyantibody is spatially ordered and radially arranged on the surface of the nanoparticle, so that the sensitivity and broad spectrum of the polyantibody for antigen detection are greatly improved, and the polyantibody has important clinical application value and industrialization prospect for rapid detection and early warning of related antigens or viruses.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanoparticle-based polymeric antibody, its preparation method, and its application. Background Technology

[0002] Over millions of years of evolution, nature has produced a variety of self-assembled protein nanostructures. Protein nanocages are a type of self-assembling biocage. Protein nanoparticles possess high symmetry, modifiability, and structural uniformity, giving them an advantage in constructing controllable self-assembled superstructures. They form highly ordered architectures with various sizes and shapes. The structure of a protein nanocage can be divided into three parts: the interior, the exterior, and the subunit interfaces. The interior can be loaded with therapeutic agents, while the exterior surface can be modified with functional ligands that have targeting effects or improve biodistribution. Finally, interactions at the subunit interfaces can induce cage disintegration / reassembly, enabling drug encapsulation. These characteristics make protein nanocages suitable carriers for the delivery of nanovaccines, nanoadjuvants, antigens, and immune stimuli.

[0003] Through genetic engineering, protein or peptide domains with different functions can be tandemly integrated to achieve multifunctionality within a single molecule. Among these, fusion proteins with self-assembly capabilities can spontaneously form structurally stable nanoparticles through non-covalent interactions under mild conditions. Their directional display and high affinity significantly improve the sensitivity of immunoassays.

[0004] Respiratory viruses are characterized by high morbidity and strong infectivity worldwide. With the spread of various respiratory pathogens, people of all ages face a high risk of infection. Specifically, common viruses causing acute respiratory infections include influenza viruses, coronaviruses, and respiratory syncytial viruses; in addition, rhinoviruses, adenoviruses, parainfluenza viruses, and human metapneumoviruses have also been documented to have spread. Respiratory pathogens often exhibit the characteristic of multiple pathogens spreading simultaneously, leading to overlapping infections of varying scales.

[0005] For most respiratory viruses, the early symptoms after infection are often similar, and frequent co-transmission further increases the difficulty of accurate diagnosis. Rapid antigen testing technology can diagnose infection by detecting viral proteins with paired antibodies. This method usually only takes 5-15 minutes and has the advantages of being simple to operate and time-saving. However, currently available rapid respiratory virus test kits only cover some viruses (such as SARS-CoV-2, influenza virus, and respiratory syncytial virus), and compared with nucleic acid testing, their sensitivity is lower and the false negative rate is higher, making it easy to miss diagnoses. Especially in the early stages of infection, due to the low viral load, antigen recognition becomes significantly more difficult, making it difficult to meet the minimum standards for antigen testing recommended by the World Health Organization (positive rate ≥80%, specificity ≥97%). During respiratory virus epidemics, primary healthcare institutions often experience strain on medical resources due to a lack of screening methods, which further exacerbates the risk of virus transmission and the difficulty of disease prevention and control. Therefore, there is an urgent need to improve the detection range and sensitivity of rapid antigen detection technology for respiratory viruses, so that it can accurately and efficiently detect and screen common epidemic pathogens, reduce the rate of missed diagnosis and misdiagnosis, and thus meet the needs of early diagnosis and treatment in grassroots settings such as homes and bedsides where professional technical support is lacking. Summary of the Invention

[0006] Purpose of the invention In view of the problems and needs of the existing technology, the purpose of this invention is to provide a fusion protein that can efficiently self-assemble into structurally stable 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 having 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 the A137R protein peptide of African swine fever virus; C is an optional label element; L1 and L2 are each independent peptide elements, either non-linked or linked.

[0008] In a feasible implementation, the antibody Fc binding element is the immunoglobulin binding domain of a streptococcal G protein, preferably the C2 domain of a streptococcal G protein, and more preferably the amino acid sequence shown in SEQ ID NO:1.

[0009] In a feasible implementation, the African swine fever virus A137R protein peptide has the amino acid sequence shown in SEQ ID NO:2.

[0010] In a feasible implementation, 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; In a feasible implementation, the tag element is a histidine tag, preferably a tag consisting of 6-10 histidines in series, more preferably 8 histidines in series.

[0011] In a preferred embodiment, 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] Sixthly, the present invention provides a method for preparing the fusion protein as described in the first aspect above, the method comprising 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In this field, "polyantibody" refers to a polymer structure formed by multiple monomeric antibodies linked by covalent or non-covalent bonds.

[0021] 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).

[0022] In a preferred embodiment, the polyantibody is a heterologous polyantibody targeting multiple respiratory pathogens.

[0023] In a preferred embodiment, the nanoparticles are loaded with monoclonal antibodies against SARS-CoV-2 (e.g., SARS-CoV-2 monoclonal antibodies RM3146 and RM3147 from Nanjing Novizan Biotechnology Co., Ltd.), monoclonal antibodies against influenza A virus (e.g., anti-influenza A virus monoclonal antibodies MI00905 and MI00906 from Hangzhou LONGi Biotechnology Co., Ltd.), and monoclonal antibodies against respiratory syncytial virus (e.g., anti-respiratory syncytial virus monoclonal antibodies A00401 and A00402 from Beijing Aigim Biotechnology Co., Ltd.).

[0024] In a tenth aspect, the present invention provides a detection composition comprising the polyantibody as described in the ninth aspect above.

[0025] In a feasible implementation, the detection composition is a detection reagent, such as a multiplex detection reagent for multiple respiratory pathogens.

[0026] 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 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.

[0027] In a twelfth aspect, the present invention provides a method for detecting respiratory pathogen infection, the method comprising: using a polyantibody as described in the ninth aspect above or a detection composition as described in the tenth aspect above for detection, wherein the polyantibody comprises a monoclonal antibody against one or more respiratory pathogens. Preferably, the polyantibody comprises a monoclonal antibody against SARS-CoV-2 (e.g., SARS-CoV-2 monoclonal antibodies RM3146 and RM3147 from Nanjing Novizan Biotechnology Co., Ltd.), a monoclonal antibody against influenza A virus (e.g., anti-influenza A virus monoclonal antibodies MI00905 and MI00906 from Hangzhou LONGi Biotechnology Co., Ltd.), and / or a monoclonal antibody against respiratory syncytial virus (e.g., anti-respiratory syncytial virus monoclonal antibodies A00401 and A00402 from Beijing Aigim Biotechnology Co., Ltd.).

[0028] Beneficial effects The fusion protein of this invention can efficiently self-assemble into structurally stable and uniformly sized nanoparticles. These nanoparticles can serve as carriers to load one or more monoclonal antibodies, thereby enabling the preparation of homologous or heterologous polyantibodies. The antigen-binding domains of the prepared polyantibodies can exhibit a spatially ordered radial arrangement on the surface of the nanoparticles, thus significantly improving their sensitivity and / or broad-spectrum application for antigen detection (in the case of heterologous polyantibodies). This has significant clinical application value and industrialization prospects for rapid, multiplex, and highly sensitive detection of relevant viruses or antigens. Attached Figure Description

[0029] 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.

[0030] Figure 1 The diagram shows the structure of the fusion proteins based on different streptococcal G protein C2 regions and A137R binding to antibody Fc (ac diagram) and their binding curves with antibodies F906 and RM3146 (df diagram), as described in Example 1.

[0031] Figure 2 This is a schematic diagram illustrating the expression, assembly, and conjugation with homologous and heterologous antibodies of the fusion protein of the present invention.

[0032] Figure 3 Transmission electron microscopy images of SARS-CoV-2 monoclonal antibody RM3146 (Figure a), nanoparticle carrier (hereinafter referred to as "GA", Figure b), and constructed polyantibodies (including multivalent polyantibody GA-SFR and monovalent polyantibodies GA-3147, GA-F906, and GA-R402, respectively, shown in Figure cf).

[0033] Figure 4 The binding affinity curves of the nanoparticle carrier GA to various viral antigens are shown.

[0034] Figure 5 Figure 1 shows the affinity curves for each antibody to the viral antigen. Figure a shows the affinity curve between the monoclonal antibody RM3146 and the SARS-CoV-2 recombinant N protein antigen; Figure b shows the affinity curve between the multivalent polymeric antibody GA-SFR and the SARS-CoV-2 recombinant N protein antigen; Figure c shows the affinity curve between the monoclonal antibody RM3147 and the SARS-CoV-2 recombinant N protein antigen; Figure d shows the affinity curve between the multivalent polymeric antibody GA-3147 and the SARS-CoV-2 recombinant N protein antigen; and Figure e shows the affinity curve between the monoclonal antibody F905 and the influenza A virus antigen. Figure f shows the affinity curves of the polyvalent antibody GA-SFR with the H1N1 influenza virus antigen; Figure g shows the affinity curves of the monoclonal antibody F906 with the H1N1 influenza virus antigen; Figure h shows the affinity curves of the polyvalent antibody GA-F906 with the H1N1 influenza virus antigen; Figure i shows the affinity curves of the monoclonal antibody R401 with the RSV antigen; Figure j shows the affinity curves of the polyvalent antibody GA-SFR with the RSV antigen; Figure k shows the affinity curves of the monoclonal antibody R402 with the RSV antigen; and Figure l shows the affinity curves of the polyvalent antibody GA-R402 with the RSV antigen.

[0035] Figure 6This is a schematic diagram of the POCT flow chromatography detection card based on polyantibody or monoclonal antibody described in Example 4.

[0036] Figure 7 The results are the sensitivity detection results of the POCT flow chromatography detection card based on polyantibodies or monoclonal antibodies described in Example 5. Detailed Implementation

[0037] To clearly illustrate the purpose, technical solutions, and advantages of this invention, the technical solutions in the embodiments of this invention will be described clearly and comprehensively below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. All other embodiments that can be obtained by those skilled in the art based on the disclosed embodiments of this invention without creative effort are within the protection scope of this invention.

[0038] Unless otherwise expressly stated in the specification and claims, the term "comprising" and its variations (such as "including," "having," "containing," "comprises," etc.) shall be understood as an open-ended limitation, that is, covering the stated elements or components but not excluding other unmentioned elements or components, unless otherwise specifically stated or the context clearly contradicts it.

[0039] All instances or exemplary expressions (such as "e.g.," "including") used herein are for the purpose of more fully illustrating the invention and do not constitute a limitation on the scope of the invention unless otherwise defined in the claims. Nothing in this specification should be construed as essential to the implementation of the invention for any element not listed in the claims.

[0040] Furthermore, unless otherwise expressly stated to the contrary, all ranges, quantities, values, and percentages mentioned in this disclosure should be understood as being modified by “about”. Here, “about” generally means that the actual value falls within ±10%, ±5%, ±1%, or ±0.5% of a specific value or range.

[0041] To illustrate the invention more thoroughly, numerous specific details are provided in the following detailed embodiments. Those skilled in the art will understand that the invention can still be practiced even without some of these details. In some embodiments, materials, components, methods, and means well-known to those skilled in the art are not described in detail, in order to highlight the core essence of the invention.

[0042] The present invention will be further described below with reference to the embodiments; unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0043] Example 1: Design and Screening of Antibody Fc Binding Elements In this embodiment, the antibody Fc binding elements in the fusion protein of the present invention were screened, and the selection criteria were that they could broadly bind to the Fc fragments of mouse and rabbit IgG antibodies.

[0044] It is known in the art that both Staphylococcus aureus protein A and Streptococcus sp. protein G can bind IgG Fc, with Streptococcus protein G exhibiting higher affinity and a wider binding range. The C2 region of the Streptococcus G protein is known to contain 65 amino acids, including four β chains and one α helix. Studies have shown that a polypeptide fragment at residues 34-44 of protein G prevents further binding of protein G to Fc. These 11 residues are located at the C-terminal portion of the α helix, the N-terminal portion of the β chain, and the extended loop region connecting these two structural elements. The amino acid residues in protein G involved in Fc interaction include: Glu27, Lys28, Lys31, Gln32, Asn35, Asp40, Glu42, and Trp43. Since the main functional regions of proteins G and Fc are the third α-helix and the fourth β-chain, the inventors designed three truncated variants: (1) the truncated variant Gtag, which involves amino acids 9-61 of the C2 region and contains 4 β chains and 1 α-helix (see the schematic diagram of its binding with antibody Fc for details). Figure 1 a); (2) The truncated G tag-2 involves amino acids 9-50 in the C2 region, contains 3 β chains and 1 α helix, and omits the last β chain (see the schematic diagram of its binding with antibody Fc). Figure 1 b); (3) The truncated G tag-3 involves amino acids 29-62 of the C2 region, containing two β chains and one α helix, with the first two β chains missing (see schematic diagram of its binding with antibody Fc for details). Figure 1 c).

[0045] These truncated variants were fused to the N-terminus of A137R for expression. The components of each fusion protein are as follows: C2 truncated variant-(GGGGS)3-A137R-8*His (wherein, the amino acid sequence of the A137R peptide is shown in SEQ ID NO:2). The expressed fusion proteins were named GA, GAS2, and GAS3, respectively. The fusion expression process is as follows: First, the codon-optimized DNA coding sequences of each fusion protein were obtained. These sequences were then 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 clones were selected for amplification culture. The culture medium was collected, sonicated, and then subjected to affinity chromatography using an AKTAPure (GE HealthCare, His Excel 5 mL column) to enrich the target protein. Finally, the protein was subjected to size exclusion chromatography (Superose)... TM 6. The protein was further purified using a 10 / 300 GL chromatography column (increased). The proteins obtained in the above steps were identified by SDS-PAGE gel electrophoresis. The consistency between the molecular weight of each protein shown in the SDS-PAGE gel electrophoresis pattern and its theoretical molecular weight confirmed that the expression of each fusion protein was successful. In addition, subsequent morphological examination showed that these fusion proteins spontaneously assembled to form hexammeric nanoparticles after expression.

[0046] To verify the broad-spectrum affinity of the polymeric vectors, rabbit antibodies (RM3146, Novizan) and mouse antibodies (MI00906, Hangzhou LONGi) were selected to detect the affinity of the fusion vectors GA, GAS2, and GAS3. Specifically, antibodies F906 and RM3146 were diluted to 200 nM. The fusion proteins GA, GAS2, and GAS3 were biotinylated and immobilized on an SA chip. Solutions of antibodies F906 and RM3146 were used as the mobile phase, respectively, to detect the antibody Fc binding ability of each fusion protein. A baseline of 60 s was set, followed by a 60 s capture time for the biotinylated polymeric vectors, and then a 60 s baseline leveling time. The antibody binding time and dissociation time were both 120 s.

[0047] The affinity test results for the three fusion proteins and the two antibodies are as follows: Figure 1 The results are shown in the df figure. The figure shows that the fusion protein GA has a strong affinity for both test antibodies, while GAS2 and GAS3 have no affinity for either test antibody. It is speculated that the reason may be that although the β chains of the 1st, 2nd and 5th chains do not directly contribute to the affinity of IgG Fc, they maintain a specific conformation.

[0048] In view of the above results, the truncated G tag (whose amino acid sequence is shown in SEQ ID NO:1 and its DNA coding sequence is shown in SEQ ID NO:4) was selected as the antibody Fc binding element for the preparation of the fusion protein and nanoparticle carrier of the present invention; and the fusion protein GA (whose amino acid sequence is shown in SEQ ID NO:3) prepared above was used as a representative example of the fusion protein of the present invention for subsequent experiments.

[0049] Example 2: Preparation and morphology detection of polyantibodies In this embodiment, capture-detection antibody pairs against SARS-CoV-2 (anti-SARS-CoV-2 monoclonal antibodies RM3146 and RM3147 from Nanjing Novizan Biotechnology Co., Ltd.), capture-detection antibody pairs against influenza A virus (anti-influenza A virus monoclonal antibodies MI00905 and MI00906 from Hangzhou LONGi Biotechnology Co., Ltd.), and capture-detection antibody pairs against respiratory syncytial virus (RSV) (anti-RSV monoclonal antibodies A00401 and A00402 from Beijing Aigim Biotechnology Co., Ltd.) were selected to construct monovalent polyantibodies carrying a single antibody for capture and polyvalent polyantibodies carrying three different detection antibodies for detection. A schematic diagram of their construction is shown below. Figure 2 .

[0050] Specifically, monoclonal antibodies RM3147, MI00906, and A00402 were conjugated with the nanoparticle carrier formed by the fusion protein GA prepared in Example 1 (hereinafter also referred to as "nanoparticle carrier GA") to prepare monovalent polyantibodies carrying a single antibody, which were named GA-3147, GA-906, and GA-402, respectively; monoclonal antibodies RM3146, MI00905, and A00401 were simultaneously conjugated with the nanoparticle carrier prepared in Example 1 to prepare a multivalent polyantibody carrying three antibodies, which was named GA-SFR.

[0051] The specific preparation method of the above-mentioned polymeric antibodies is as follows: The monoclonal antibody used for conjugation (for GA-SFR, a mixture of three antibodies in a 1:1:1 molar ratio) was mixed with the nanoparticle carrier GA in a 60:1 molar ratio and incubated at room temperature for 1 h; then, unreacted components were removed by size exclusion chromatography to obtain the polyantibody.

[0052] Next, the morphology of the monoclonal antibody RM3146, the nanoparticle carrier GA, and the prepared polyantibodies GA-SFR, GA-3147, GA-906, and GA-402 were analyzed. Specifically, a negative staining method was used. The target protein was diluted to 0.1 mg / mL with 20 mM Tris pH 8.0; a 230-mesh carbon support membrane (Zhongxiao Keyi) was evacuated for 3 min, and glow discharge was performed for 20 s to remove impurities from the surface of the carbon support membrane; then, 5 μL of the sample to be tested was dropped into the carbon support membrane and incubated for 1 min; then, it was rinsed twice with 2% uranyl acetate staining solution, and then incubated with 5 μL of staining solution for 1 min to negatively stain the protein. After removing excess staining solution, imaging observation was performed using a transmission electron microscope (Spirit 120 kV).

[0053] The results are as follows Figure 3 As shown in the af diagram. Figure 3 The data shows that the nanoparticle carrier GA (Figure b) and each polyantibody (Figure cf) both have nanoparticle structures with uniform and dispersed particle sizes. Furthermore, the particle size of each polyantibody is larger than that of the nanoparticle carrier, indicating that the nanoparticle carrier and each antibody have been successfully coupled.

[0054] Example 3: Affinity test of polyantibodies In this embodiment, the affinity test was performed on the polyantibody prepared in Example 2, as follows: The affinity of polyantibodies against SARS-CoV-2 antigens was analyzed using the Biacore 8K biomolecular interaction system.

[0055] First, the SARS-CoV-2 antigen S0, influenza A antigen F0, and respiratory syncytial virus antigen R0 were amino-conjugated onto a chip (SensorChip CM5, GE Health Care), respectively. Serially diluted nanoparticle carriers GA (400 nM, 200 nM, 100 nM, 50 nM, 25 nM) were used as the mobile phase for binding and elution to eliminate interference from the carrier on the specific binding of antigens and antibodies. The elution curves are shown below. Figure 4 As shown. Figure 4 The results show that the GA nanoparticle carrier does not bind nonspecifically to any of the viral antigens, thus eliminating its interference with subsequent antibody-antigen affinity tests.

[0056] Next, the stationary phase was coupled. Specifically, the polyantibodies and monoclonal antibodies prepared in Example 2 were coupled to the chip (SensorChip Protein G, GE Health Care) at 1-5 μg / mL. Then, the SARS-CoV-2 recombinant N protein antigen (40588-V08B, Sinocare), influenza A virus antigen (AI00901, Hangzhou LONGi), and respiratory syncytial virus antigen (A00411, Agilent) were serially diluted with PBST (to 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM, respectively), and then coupled to the stationary phase. The program was set as follows: binding for 60 s, dissociation for 120 s, and regeneration with ammonium ethanol for 120 s; the experiment was repeated 3 times. The obtained data were fitted to calculate affinity, and the exported data were plotted using GraphpadPrism 8.0. The results are shown below. Figure 5 As shown.

[0057] Figure 5 middle: (1) Figure a shows the affinity curve of monoclonal antibody RM3146 to its corresponding antigen SARS-CoV-2 recombinant N protein antigen, with an affinity KD = 6.30E-12; Figure b shows the affinity curve of polyvalent polymer antibody GA-SFR (which carries antibody RM3146) to SARS-CoV-2 recombinant N protein antigen, with an affinity KD = 3.06E-13; This result shows that under the same conditions, the affinity of polyvalent polymer antibody GA-SFR to the antigen is one order of magnitude higher than that of SARS-CoV-2 capture antibody RM3146. (2) Figure c shows the affinity curve of monoclonal antibody RM3147 to its corresponding antigen SARS-CoV-2 recombinant N protein antigen, with an affinity KD = 8.86E-12; Figure d shows the affinity curve of polyantibody GA-3147 to SARS-CoV-2 recombinant N protein antigen, with an affinity KD = 2.59E-12; This result shows that under the same conditions, the affinity of polyantibody GA-3147 is 3 times higher than that of SARS-CoV-2 detection antibody RM3147 to the antigen; (3) Figure e shows the affinity curve between monoclonal antibody F905 and its corresponding antigen, influenza A virus antigen, with an affinity KD = 1.35E-8; Figure f shows the affinity curve between polyvalent polymeric antibody GA-SFR (which carries antibody F905) and influenza A virus antigen, with an affinity KD = 1.32E-9; This result indicates that under the same conditions, the affinity of polyvalent polymeric antibody GA-SFR is one order of magnitude higher than that of influenza A capture antibody F905 to the antigen; (4) Figure g shows the affinity curve between monoclonal antibody F906 and its corresponding antigen, influenza A virus antigen, with an affinity KD = 3.52E-8; Figure h shows the affinity curve between polyantibody GA-F906 and influenza A virus antigen, with an affinity KD = 3.20E-10; This result indicates that under the same conditions, the affinity of polyantibody GA-F906 to the antigen is two orders of magnitude higher than that of influenza A detection antibody F906. (5) Figure i shows the affinity curve of monoclonal antibody R401 to its corresponding antigen RSV antigen, with an affinity KD = 2.43E-9; Figure j shows the affinity curve of polyvalent antibody GA-SFR (which carries antibody R401) to RSV antigen, with an affinity KD = 1.40E-10; This result shows that under the same conditions, the affinity of polyvalent antibody GA-SFR to antigen is one order of magnitude higher than that of RSV capture antibody R401. (6) Figure k shows the affinity curve between monoclonal antibody R402 and its corresponding antigen RSV antigen, with an affinity KD = 9.42E-9; Figure l shows the affinity curve between polyantibody GA-R402 and RSV antigen, with an affinity KD = 8.57E-9; This result indicates that under the same conditions, the affinity of polyantibody GA-R402 to the antigen is higher than that of RSV detection antibody R402.

[0058] Example 4: Preparation and sensitivity testing of POCT flow chromatography test strips based on polyantibodies In this embodiment, POCT flow chromatographic test strips based on polyantibodies or monoclonal antibodies were prepared, and the sensitivity of the two for viral antigen detection was compared.

[0059] The experimental group used GA-SFR antibody as the capture antibody and GA-3147, GA-F906, and GA-R402 as the detection antibodies, hereinafter referred to as the "polyantibody group" or "GA-SFR group"; the control group used a mixed antibody of RM3146, F905, and R401 as the capture antibody and RM3147, F906, and R402 as the detection antibodies, hereinafter referred to as the "monoclonal antibody group" or "SFR group".

[0060] The specific experimental method is as follows: 1. Label the capture antibody with latex microspheres; Take 0.0125 mL of microsphere suspension (DR0400CA, Suzhou, 4% solids content) into a 2 mL centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, sonicate to mix; add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution, sonicate to mix; place the centrifuge tube on a turntable, activate at 37℃ and 40 r / min for 15-30 min; centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, repeat the washing step once. Add 0.75 mL of conjugation buffer and sonicate to mix. Add 50 μg of the antibodies to be labeled (GA-SFR, RM3146, F905, R401, and chicken IgY for quality control) to 0.25 mL of conjugation buffer respectively to prepare the conjugation solution. Add the antibody-containing conjugation solution to the mixed microspheres and vortex to mix. Place the centrifuge tube on a turntable and conjugate at 37℃ and 40 r / min for 2 h. Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and block at 37℃ and 40 r / min for 1 h. Centrifuge at 15℃ and 20000 g for 10 min and remove the supernatant. Add 1.5 mL of microsphere washing solution, sonicate to mix, centrifuge at 15℃ and 20000 g for 10 min and remove the supernatant. Add 1.5 mL of microsphere washing solution and repeat the washing step once. Finally, add 0.5 mL of microsphere washing solution to the centrifuge tube. Microsphere preservation solution (final microsphere concentration 1 mg / mL) was sonicated to homogenize. The labeled antibodies were mixed in two ways: GA-SFR group at a ratio of GA-SFR:IgY = 3:1 (mixed solution 1); and SFR group at a ratio of RM3146:F905:R401:IgY = 1:1:1:1 (mixed solution 2). The prepared latex microsphere-labeled antibody solutions were sprayed onto glass fibers at a concentration of 1.5–2 μL / cm and dried overnight at 37°C.

[0061] Solution preparation: (1) Coupling buffer: 10 mM MES (pH 6.2±0.05), containing 0.05% ProClin 300; (2) EDC solution: 10 mg / mL, prepared with coupling buffer, and used immediately; (3) NHS solution: 10 mg / mL, prepared with coupling buffer, and used immediately; (4) Microsphere blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20 (pH 9.0±0.05), 1% BSA, 0.24% ethanolamine; (5) Microsphere washing solution: 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300; (6) Microsphere preservation solution: 25mM Tris (pH 7.2±0.05), 150mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0062] 2. Coating nitrocellulose membranes with detection antibodies: Dilute the detection antibodies from the experimental or control groups to 0.5-1.5 mg / ml using coating diluent, and then coat the membranes with 0.1 ml of the solution. 5 μL / cm, streaking speed is 5 100 mm / s; dry overnight in an oven at 37℃. Control line C is sheep anti-chicken IgY, diluted to 0.5-1.5 mg / mL for streaking, with a streaking volume of 0.1-5 μL / cm and a streaking speed of 5. 100 mm / s.

[0063] 3. Prepare and package goat anti-chicken IgY (A10003, Aigim) colloidal gold and chicken IgY (A10002, Aigim) for quality control, using the same method as above.

[0064] 4. Assembly of the test strips; For ease of use, this embodiment prepares the above-mentioned test strip into a test card for multiplex detection of respiratory viruses. The test card includes a 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 onto a PVC board; the test line T is close to the conjugate pad, and the control line C is close to the absorbent paper. A sample application hole is provided at the position of the upper shell corresponding to the sample pad, and an observation window is provided at the position of the upper shell corresponding to the nitrocellulose membrane. The structure of the entire test card is as follows. Figure 6 As shown.

[0065] The assembly plan is as follows: The experimental test card (GA-LFA) consists of glass fiber sprayed with mixed solution 1 and nitrocellulose membrane coated with polyantibodies (GA-3147, GA-F906, GA-R402).

[0066] The control group test card (SFR-LFA) consists of a glass fiber coated with mixed solution 2 and a nitrocellulose membrane coated with antibodies (RM3147, F906, R402).

[0067] 5. Perform Ct value detection and sensitivity testing on respiratory pathogen pharyngeal swabs to identify the detection limits of the two test cards.

[0068] The results are as follows Figure 7 As shown; Figure 7 The results show that the detection limit of the GA-LFA test card based on polyantibody is Ct = 28, and the detection limit of the SFR-LRA test card based on monoclonal antibody is Ct = 25; that is, the sensitivity of the test card based on polyantibody is more than 8 times higher than that of the test card based on monoclonal antibody.

[0069] Example 5: Detection case of POCT flow chromatography test strips based on polyantibodies or monoclonal antibodies In this embodiment, two respiratory virus multiplex detection cards prepared in Example 4 are used to detect clinical case samples.

[0070] Specifically, 40 pharyngeal swab samples of SARS-CoV-2 with different Ct values, 45 pharyngeal swab samples of influenza A with different Ct values, 47 pharyngeal syncytial virus with different Ct values, and 11 pharyngeal swab samples of healthy individuals were collected as test samples. The positive rate and specificity of the test cards GA-LFA and SFR-LFA prepared in Example 4 were tested respectively.

[0071] The results are shown in Table 1 below.

[0072] Table 1

[0073] The results in Table 1 show that: (1) Regarding SARS-CoV-2: When the viral load Ct value was in the range of 20-25, the positive rate of samples in both the GA-LFA and SFR-LFA groups was 100%. When the viral load Ct value was in the range of 25-30, the positive rate of the GA-LFA group was 90%, and the positive rate of the SFR-LFA group was 80%. When the viral load (Ct) value was in the range of 30-35, the positive rate of the GA-LFA group was 78.9%, and the positive rate of the SFR-LFA group was 63.2%. When the viral load Ct value was higher than 35, the positive rate of the GA-LFA group was 42.9%, and the positive rate of the SFR-LFA group was 28.6%.

[0074] Without considering viral load, the positivity rate and specificity of the GA-LFA group were 77.5% and 90.9%, respectively, while those of the SFR-LFA group were 65% and 90.9%, respectively.

[0075] (2) Regarding influenza A virus: When the viral load Ct value was less than 20, the positive rate of samples in both the GA-LFA and SFR-LFA groups was 100%. When the viral load Ct value was in the range of 20-25, the positivity rate of the GA-LFA group was 100%, and the positivity rate of the SFR-LFA group was 88.9%. When the viral load Ct value was in the range of 25-30, the positivity rate of the GA-LFA group was 85%, and the positivity rate of the SFR group was 75%. When the viral load Ct value was higher than 30, the positivity rate of the GA-LFA group was 45.5%, and the positivity rate of the SFR-LFA group was 27.3%.

[0076] Without considering viral load, the positivity rate and specificity of the GA-LFA group were 80% and 100%, respectively, while those of the SFR-LFA group were 68.9% and 100%, respectively.

[0077] (3) For respiratory syncytial virus: When the viral load Ct value was in the range of 20-25, the positive rate of samples in the GA-LFA group was 95.2%, and the positive rate of samples in the SFR-LFA group was 90.5%. When the viral load Ct value was in the range of 25-30, the positivity rate of the GA-SFR group was 29.4%, and the positivity rate of the SFR group was 17.6%. When the viral load Ct value was higher than 30, the positivity rate of the GA-SFR group was 22%, while the positivity rate of the SFR group was 0%.

[0078] Without considering viral load, the positivity rate and specificity of the GA-SFR group were 57.4% and 100%, respectively, while those of the SFR group were 46.8% and 100%, respectively.

[0079] The results above demonstrate that integrated nanoprobes can significantly improve the positive rate of pathogen detection without reducing specificity, especially for samples with low viral load.

[0080] 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.

[0081] The sequences involved in this application are: SEQ ID NO:1 ( (SPG C2 truncated amino acid sequence) YKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVT SEQ ID NO:2 ( (A137R amino acid sequence) MEAVLTKLDQEEKKALQNFHRCAWEETKNIINDFLEIPEERCTYKFNSYTKKMELLFTPEFHTAWHEVPECREFILNFLRLISGHRVVLKGPTFVFTKETKNLGIPSTINVDFQANIENMDDLQKGNLIGKMNIKEG* SEQ ID NO:3 (Amino acid sequence of the fusion protein GA) YKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTGGGSGGGSGGGSMEAVLTKLDQEEKKALQNFHRCAWEETKNIINDFLEIPEERCTYKFNSYTKKMELLFTPEFHTAWHEVPECREFILNFLRLISGHRVVLKGPTFVFTKETKNLGIPSTINVDFQANIENMDDLQKGNLIGKMNIKEGHHHHHHH* SEQ ID NO:4 ( (SPG C2 truncated nucleotide sequence) TATAAGCTAGTAATAAATGGAAAAACACTTAAGGGCGAGACTACAACCGAAGCGGTCGATGCGGCAACCGCCGAAAAGGTATTTAAACAGTATGCGAACGACAACGGTGTTGATGGTGAATGGACTTACGATGACGCTACGAAGACCTTCACCGTTACC SEQ ID NO:5(A137R nucleotide sequence) ATGGAGGCGGTGCTGACCAAGCTGGATCAAGAAGAGAAGAAAGCCTTGCAAAACTTCCATCGTTGCGCTTGGGAGGAAACGAAAAATATCATTAACGACTTCCTGGAGATCCCGGAAGAGCGCTGTACCTACAAATTCAACTCCTATACCAAGAAGATGGAACTGCTGTTTACCCCGGAGTTCCACACCGCGTGGCATGAAGTTCCAGAGTGCCGTGAATTTATCTTAAATTTTTTGCGTCTGATCAGCGGCCATCGCGTTGTGTTGAAAGGTCCGACGTTTGTGTTCACCAAGGAGACCAAGAACCTGGGCATCCCGAGCACCATTAACGTGGATTTCCAGGCAAATATTGAAAATATGGACGACCTGCAGAAAGGTAACCTCATTGGTAAAATGAATATCAAAGAGGGT SEQ ID NO:6 (GA nucleotide sequence) TATAAGCTAGTAATAAATGGAAAAACACTTAAGGGCGAGACTACAACCGAAGCGGTCGATGCGGCAACCGCCGAAAAGGTATTTAAACAGTATGCGAACGACAACGGTGTTGATGGTGAATGGACTTACGATGACGCTACGAAGACCTTCACCGTTACCGGCGGTGGCTCGGGCGGTGGTTCTGGCGGAGGCAGCATGGAGGCGGTGCTGACCAAGCTGGATCAAGAAGAGAAGAAAGCCTTGCAAAACTTCCATCGTTGCGCTTGGGAGGAAACGAAAAATATCATTAACGACTTCCTGGAGATCCCGGAAGAGCGCTGTACCTACAAATTCAACTCCTATACCAAGAAGATGGAACTGCTGTTTACCCCGGAGTTCCACACCGCGTGGCATGAAGTTCCAGAGTGCCGTGAATTTATCTTAAATTTTTTGCGTCTGATCAGCGGCCATCGCGTTGTGTTGAAAGGTCCGACGTTTGTGTTCACCAAGGAGACCAAGAACCTGGGCATCCCGAGCACCATTAACGTGGATTTCCAGGCAAATATTGAAAATATGGACGACCTGCAGAAAGGTAACCTCATTGGTAAAATGAATATCAAAGAGGGTCACCACCATCACCACCATCACCAC。

Claims

1. A fusion protein, 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 the A137R protein peptide of African swine fever virus; C is an optional label element; L1 and L2 are each independent peptide elements, either non-linked or linked.

2. The fusion protein according to claim 1, 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 African swine fever virus A137R protein peptide has 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 or more, more preferably 8 histidines connected in series.

3. The fusion protein according to claim 2, 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.

4. A polynucleotide encoding the fusion protein as described in any one of claims 1-3; 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.

5. A nucleic acid construct comprising the polynucleotide as described in claim 4 and one or more expression regulatory elements operatively linked thereto.

6. A recombinant vector comprising the polynucleotide of claim 4 or the nucleic acid construct of claim 5.

7. A host cell wherein the cell is transformed or transfected with the polynucleotide of claim 4, the nucleic acid construct of claim 5, or the recombinant vector of claim 6.

8. A method for preparing the fusion protein according to any one of claims 1-3, 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 7 are cultured to express the fusion protein; Preferably, the method further includes the steps of separating and purifying the fusion protein.

9. A nanoparticle, characterized in that, The nanoparticles are self-assembled from the fusion protein according to any one of claims 1-3; Preferably, the nanoparticles are hexadecimals.

10. Use of the fusion protein of any one of claims 1-3, the polynucleotide of claim 4, the nucleic acid construct of claim 5, the recombinant vector of claim 6, the host cell of claim 7, and / or the nanoparticle of claim 9 in the preparation of polyantibodies.

11. A polymeric antibody comprising: (1) The nanoparticles as described in claim 9 as a carrier; and, (2) One or more monoclonal antibodies loaded on the nanoparticles.

12. The polyantibody according to claim 11, characterized in that, The monoclonal antibodies include: monoclonal antibodies against SARS-CoV-2, monoclonal antibodies against influenza A virus, and / or monoclonal antibodies against respiratory syncytial virus.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polyantibody according to claim 11 or 12.

14. The method for preparing the polyantibody according to claim 11 or 12, characterized in that, The preparation method includes: incubating the monoclonal antibody with the nanoparticles as described in claim 9 to obtain the product; Preferably, the molar ratio of the monoclonal antibody to the nanoparticles as described in claim 9 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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