Multimeric antigens and methods of making the same

Abstract

The application discloses a kind of multimeric antigens and preparation method thereof, wherein the multimeric antigen is formed by self-assembly of fusion protein, and the fusion protein includes capsid protein and C-reactive protein.The multimeric antigen in the application is virus-like particle structure, and the C-reactive protein is located on the outside of the virus-like particle structure.The multimeric antigen in the application is a icosahedral multimeric particle, which partially simulates the spatial site of natural antigen pentamer, and improves the recognition efficiency of the generated antibody to natural antigen.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a polymeric antigen and its preparation method. More specifically, this invention relates to a fusion protein, nucleic acid, vector, cell, and a method for preparing anti-C-reactive protein antibodies. Background Technology

[0002] C-reactive protein (CRP) is a well-known marker of inflammation, with normal levels in the body being low. When the body is infected or tissue is damaged, plasma levels rise sharply, reaching hundreds of milligrams per liter in severe inflammatory responses. It is commonly used to differentiate between bacterial and viral infections. For cardiovascular patients, elevated CRP levels (>3 mg / L) are of particular interest, as this indicates a risk of cardiovascular abnormalities. This balancing of low-value sensitivity and high-value linearity places very high demands on the titer of the antibodies used for detection.

[0003] The recombinant CRP antigen monomer has a small molecular weight of 25 kDa, and the antibodies obtained after immunization do not recognize natural C-reactive protein or have extremely low recognition efficiency. This is because the recombinant CRP monomer antigen has a large difference in spatial conformation from the natural pentamer protein, or the monomer antigen has low immunogenicity. As a result, the antibodies produced cannot form normal antibody-antigen complexes with natural C-reactive protein, and therefore cannot be recognized by the detection system.

[0004] Therefore, there is an urgent need to prepare new CRP antigens to generate antibodies that can form normal antibody-antigen complexes with natural C-reactive protein. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0006] This invention is based on the following discoveries of the inventors.

[0007] The inventors discovered that recombinant CRP has a molecular weight of 25 KD. Compared to the native pentamer protein, recombinant CRP molecules cannot spontaneously form a pentamer, and their small molecular weight classifies them as small-molecule antigens. This results in low immunogenicity and significant structural differences from the native protein, leading to low titers of CRP antibodies prepared via conventional immunization methods, which cannot recognize native CRP molecules and are therefore unsuitable for detection. To overcome this problem, the inventors chose to link recombinant CRP protein with a capsid protein to form a fusion protein. Utilizing its in vitro self-assembly principle, a polyantigen is formed, increasing the antigen's own immunogenicity while partially mimicking the aggregation form of the native antigen. Furthermore, the ability of VLP to significantly stimulate the immune response is utilized to further increase antibody production efficiency. On the other hand, the separately expressed VLP is chemically cross-linked onto a gel medium, and the resulting antibody is then subjected to affinity purification to remove the VLP. ELISA results showed that the titer of antibodies immunized with polyantigens against natural CRP antigens was about 30 times higher than that of antibodies immunized with monomeric antigens; the titer of antibodies after affinity purification was about 1 time higher than that before affinity purification, and about 60 times higher than that of antibodies immunized with monomeric antigens.

[0008] Based on this, in a first aspect, the present invention proposes a fusion protein. According to an embodiment of the present invention, the fusion protein comprises a capsid protein and a C-reactive protein; wherein the C-reactive protein is linked to the capsid protein. The fusion protein according to an embodiment of the present invention can spontaneously assemble into a virus-like particle structure under in vitro buffer conditions, thereby efficiently activating a humoral immune response and generating high-titer antibodies.

[0009] In a second aspect, the present invention provides a polyantigen. According to embodiments of the invention, the polyantigen is formed by the self-assembly of the fusion protein described in the first aspect of the invention. The polyantigen according to embodiments of the invention can efficiently activate humoral immune responses and generate high-titer antibodies.

[0010] In a third aspect, the present invention provides a nucleic acid. According to embodiments of the present invention, the nucleic acid encodes the fusion protein described in the first aspect of the present invention and the polyantigen described in the second aspect of the present invention.

[0011] In a fourth aspect, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the nucleic acid described in the third aspect of the present invention.

[0012] In a fifth aspect, the present invention provides a cell. According to embodiments of the invention, the cell carries the nucleic acid described in the third aspect or the vector described in the fourth aspect; or the cell expresses the fusion protein described in the first aspect or the polymeric antigen described in the second aspect.

[0013] In a sixth aspect, the present invention provides a method for preparing the polyantigen described in the second aspect of the invention. According to an embodiment of the invention, the method includes: performing a self-assembly treatment on the fusion protein described in the first aspect of the invention to form a polyantigen. The method according to the embodiment of the invention enables the preparation of the polyantigen described in the second aspect of the invention, thereby efficiently activating humoral immune responses and generating high-titer antibodies.

[0014] In a seventh aspect, the present invention provides a method for preparing antibodies or antigen-binding fragments thereof. According to an embodiment of the present invention, the method includes: immunizing an animal with the polyantigen described in the second aspect of the present invention; obtaining antiserum from the immunized animal; and purifying the antiserum to prepare anti-C-reactive protein antibodies. The method according to an embodiment of the present invention can produce high-titer antibodies.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0017] Figure 1 This is a flowchart illustrating the technical implementation of Embodiment 1 of the present invention.

[0018] Figure 2 This is a molecular structure diagram of CP-CRP in Example 1 of the present invention.

[0019] Figure 3 This is a schematic diagram of the CRP-VLP self-assembly to form a polyantigen in Example 1 of the present invention.

[0020] Figure 4 This is a schematic diagram of CRP antibody affinity purification in Example 1 of the present invention.

[0021] Figure 5 This is a comparison chart of antibody titers generated by different types of CRP antigens in Example 1 of the present invention.

[0022] Figure 6 This is a comparison chart of the titers of antibodies after VLP affinity purification following immunization with the polyantigen in Example 1 of the present invention.

[0023] Figure 7 This is a comparison chart of the titers of antibodies obtained by conventional purification after immunization with two polyantigens, CRP-CP and CP-CRP, in Example 1 of this invention.

[0024] Figure 8This is a comparison chart of the titers of antibodies obtained by conventional purification after immunization with three polyantigens, CP-CRP-VLP, PCV2-CRP-VLP, and HPV58L1-CRP-VLP, in Example 1 of this invention. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0027] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0028] In this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0029] In this invention, the term "antibody" is used in the broadest sense, encompassing full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain, linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and changes very little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively.

[0030] In this invention, the term "antigen" refers to a substance capable of stimulating the immune system to produce antibodies or inducing an immune cell response. In some alternative embodiments of the invention, "antigen" refers to natural antigens, monomeric antigens, and polymeric antigens. In some alternative embodiments of the invention, for example, "antigen" refers to CP-CRP or CRP.

[0031] In this invention, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and / or between cells or hosts. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce a desired expression product.

[0032] In this invention, the term "cell" generally refers to a cell whose genetic material has been modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0033] In this invention, the term "VLP" refers to a synthetically produced nanoparticle whose structure and morphology mimic natural viruses, but which does not contain viral genetic material (DNA / RNA) and therefore has no infectivity or replication ability. Its core components are formed by the self-assembly of one or more structural proteins of the virus (such as capsid proteins).

[0034] In this invention, the term "polyantigen" refers to a high molecular weight complex formed by the assembly of multiple identical or different antigen units in a specific manner. Specifically, it refers to the virus-like particle (VLP) structure (i.e., the CRP-VLP complex) formed by the self-assembly of a fusion protein of C-reactive protein (CRP) and the hepatitis B virus core capsid protein (CP).

[0035] In this invention, the term "capsid protein" refers to the main structural protein that constitutes the viral capsid. Capsid proteins are a common structure in most viruses and most possess self-assembly capabilities. However, the structure of capsid proteins varies considerably among different types of viruses. In specific embodiments of this invention, capsid proteins specifically refer to the core capsid protein of hepatitis B virus (HBV), the capsid protein of porcine circovirus type 2 (PCV2), and the L1 protein of human papillomavirus (HPV) type 58.

[0036] This invention provides a fusion protein, a polyantigen, a nucleic acid, a vector, a cell or host, a method for preparing the polyantigen described in the second aspect of this invention, and a method for preparing an antibody or an antigen-binding fragment thereof.

[0037] Fusion protein

[0038] In a first aspect, the present invention provides a fusion protein. According to embodiments of the invention, the fusion protein comprises a capsid protein and a C-reactive protein; wherein the C-reactive protein is linked to the capsid protein. The fusion protein according to embodiments of the invention can spontaneously assemble into a virus-like particle structure under in vitro buffer conditions, thereby efficiently activating a humoral immune response and generating high-titer antibodies.

[0039] According to an embodiment of the present invention, the capsid protein is selected from at least one of the hepatitis B virus core capsid protein, porcine circovirus type 2 capsid protein, and human papillomavirus type 58 L1 protein.

[0040] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is connected to the C-terminus of the capsid protein.

[0041] According to an embodiment of the present invention, the C-terminus of the C-reactive protein is connected to the N-terminus of the capsid protein.

[0042] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is linked to the C-terminus of the hepatitis B virus core capsid protein.

[0043] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is linked to the C-terminus of the porcine circovirus type 2 capsid protein.

[0044] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is linked to the C-terminus of the human papillomavirus type 58 L1 protein.

[0045] According to an embodiment of the present invention, the C-terminus of the C-reactive protein is linked to the N-terminus of the hepatitis B virus core capsid protein.

[0046] According to an embodiment of the present invention, the C-reactive protein has the amino acid sequence shown in SEQ ID NO: 1.

[0047] QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASG IVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP (SEQ ID NO: 1)

[0048] According to an embodiment of the present invention, the hepatitis B virus core capsid protein has the amino acid sequence shown in SEQ ID NO: 2.

[0049] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 2)

[0050] According to an embodiment of the present invention, the porcine circovirus type 2 capsid protein has the amino acid sequence shown in SEQ ID NO: 3.

[0051] MTYPRRRRFRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPPLTVPFEYYRIRKVKVEFWPCSPITQGDRGV GSTAVILDDFVTKANALTYDPYVNYYRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK (SEQ ID NO: 3)

[0052] According to an embodiment of the present invention, the human papillomavirus type 58 L1 protein has the amino acid sequence shown in SEQ ID NO: 4.

[0053] (SEQ ID NO: 4)

[0054] It should be noted that, based on the amino acid sequence of the fusion protein disclosed herein, those skilled in the art will readily conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the fusion protein, such as isolating and purifying the fusion protein from the culture product of recombinant cells. This is easily achievable for those skilled in the art. Therefore, regardless of the technique used to prepare the fusion protein disclosed herein, it falls within the protection scope of this application.

[0055] Polyantigen

[0056] In a second aspect, the present invention proposes a polyantigen. According to embodiments of the present invention, the polyantigen is formed by the self-assembly of the fusion protein described in the first aspect of the present invention. The polyantigen according to embodiments of the present invention can efficiently activate humoral immune responses and generate high-titer antibodies. According to embodiments of the present invention, the polyantigen is formed by the self-assembly of a C-reactive protein fused with a viral particle (VLP). Compared to a separately expressed C-reactive protein, the polyantigen is an icosahedral multimeric particle, partially mimicking the spatial sites of the natural antigen pentamer, thus improving the antibody's recognition efficiency of the natural antigen. The present invention proposes using a VLP that can participate in immune presentation and enhance humoral immunity as a carrier for CRP. The CRP molecule is located on the surface of the icosahedron of the VLP, and the activation effect of the VLP on the immune system improves immune efficiency and increases antibody titer. Anti-VLP antibodies generated in the above process are removed using an affinity medium conjugated with the VLP. This method further purifies the CRP antibody, and in this design, the target antibody does not bind to the medium and flows directly through it. This avoids the damage to the antibody functional regions caused by acidic elution after the antigen and antibody functional regions bind during the traditional antigen affinity process, thus preserving the antibody activity to the greatest extent.

[0057] According to an embodiment of the present invention, the polyantigen is a virus-like particle structure, wherein the C-reactive protein is located on the outer side of the virus-like particle structure.

[0058] According to a specific embodiment of the present invention, the present invention proposes a CRP protein multimer that can enhance the immunogenicity of recombinant CRP. Compared with recombinant CRP, the CRP multimer expresses the core protein of hepatitis B virus—capsid protein—at the N-terminus of CRP.

[0059] According to an embodiment of the present invention, relative to the recombinant CRP expressed alone, the fusion protein can self-assemble into a CRP-VLP complex in vitro after expression, wherein the VLP is an icosahedral granular structure spontaneously assembled from capsid proteins, and the CRP is located on the outside of the VLP structure, exhibiting a polymeric state.

[0060] According to an embodiment of the present invention, the antibody obtained by immunizing animals with polyCRP as an antigen has a titer approximately 30 times higher than that obtained by immunizing animals with recombinant monomeric CRP antigen relative to natural CRP protein; furthermore, after removing the anti-VLP antibody by a VLP-conjugated affinity medium, the titer of the polyCRP immune antibody is approximately 60 times higher than that of the monomeric CRP immune antibody against natural CRP antigen.

[0061] According to embodiments of the present invention, C-reactive protein polyantigens with VLP structures were obtained through self-assembly, which improved antigen stability and aggregation. At the same time, the presence of VLPs also enhanced the antigen presentation effect, stimulated the immune system, and increased the efficiency of antibody production. After removing non-specific VLP antibodies by affinity chromatography, the titer of the obtained CRP antibodies was significantly higher than that of the control group immunized with monomeric antigens.

[0062] It should be noted that, based on the amino acid sequence of the polyantigen disclosed herein, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, recombinant expression) to prepare the polyantigen, such as isolating and purifying the polyantigen from the culture product of recombinant cells. This is easily achievable for those skilled in the art. Therefore, regardless of the technique used to prepare the polyantigen disclosed herein, it falls within the protection scope of this application.

[0063] Nucleic acid

[0064] In a third aspect, the present invention provides a nucleic acid. According to embodiments of the present invention, the nucleic acid encodes the fusion protein described in the first aspect of the present invention and the polyantigen described in the second aspect of the present invention.

[0065] According to embodiments of the present invention, the nucleic acid includes DNA or RNA.

[0066] It should be noted that, for the nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0067] Those skilled in the art will understand that the features and advantages described above for fusion proteins and polyantigens also apply to this nucleic acid, and will not be repeated here.

[0068] carrier

[0069] In a fourth aspect, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the nucleic acid described in the third aspect. When the nucleic acid described in the third aspect is ligated to the vector, the nucleic acid can be directly or indirectly connected to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the nucleic acid and the control elements need to be operably connected.

[0070] In this article, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the expression of the aforementioned fusion protein can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the fusion protein.

[0071] According to embodiments of the present invention, the vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector used in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0072] In some optional embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0073] In some optional embodiments of the present invention, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0074] Those skilled in the art will understand that the features and advantages described above for fusion proteins, polyantigens, and nucleic acids also apply to this vector, and will not be repeated here.

[0075] cell

[0076] In a fifth aspect, the present invention provides a cell. According to embodiments of the invention, the cell or host carries the nucleic acid described in the third aspect or the vector described in the fourth aspect; or the cell expresses the fusion protein described in the first aspect or the polymeric antigen described in the second aspect.

[0077] According to an embodiment of the present invention, the cell is obtained by introducing the carrier described in the fourth aspect into the cell.

[0078] It should be noted that the cells used in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0079] In an optional embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, but do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0080] It should be noted that the "suitable conditions" mentioned in this invention refer to conditions suitable for the expression of the fusion protein and polyantigen described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the fusion protein and polyantigen include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion protein and polyantigen according to the specific environment of their laboratory.

[0081] Those skilled in the art will understand that the features and advantages described above for fusion proteins, polyantigens, nucleic acids, and vectors also apply to this cell, and will not be repeated here.

[0082] Methods for preparing polymeric antigens

[0083] In a sixth aspect, the present invention provides a method for preparing a polyantigen. According to an embodiment of the invention, the method includes: performing a self-assembly treatment on the fusion protein described in the first aspect of the invention to form a polyantigen. The method according to an embodiment of the invention enables the preparation of the polyantigen described in the second aspect of the invention, thereby efficiently activating humoral immune responses and generating high-titer antibodies.

[0084] According to an embodiment of the present invention, the self-assembly process is performed in a buffer solution.

[0085] According to an embodiment of the present invention, the buffer solution is a phosphate buffer solution with a pH of 7.4.

[0086] According to an embodiment of the present invention, the phosphate buffer contains 20 mM phosphate and 150 mM NaCl.

[0087] According to an embodiment of the present invention, the self-assembly is carried out under the condition of stirring at 4°C for 48 hours.

[0088] Methods for preparing antibodies or their antigen-binding fragments

[0089] In a seventh aspect, the present invention provides a method for preparing antibodies or antigen-binding fragments thereof. According to an embodiment of the present invention, the method includes: immunizing an animal with the polyantigen described in the second aspect of the present invention; obtaining antiserum from the immunized animal; and purifying the antiserum to prepare anti-C-reactive protein antibodies. The method according to an embodiment of the present invention is capable of producing high-titer antibodies or antigen-binding fragments thereof.

[0090] According to an embodiment of the present invention, the purification process is carried out by an affinity medium coupled with the capsid protein.

[0091] According to an embodiment of the present invention, the affinity medium coupled with the capsid protein is prepared by means of a solid-phase matrix coupling treatment of the self-assembly.

[0092] According to an embodiment of the present invention, the solid matrix is ​​an NHS-activated gel medium.

[0093] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0094] Example

[0095] Example 1:

[0096] The technical implementation process of this embodiment is as follows: Figure 1 As shown, the specific process is as follows.

[0097] 1. Preparation of CP-CRP fusion protein, CRP-CP fusion protein, PCV2-CRP fusion protein, HPV58L1-CRP fusion protein, CP protein, and CRP protein.

[0098] Gene synthesis methods were used to construct, such as Figure 2 The images show the CP-CRP fusion protein, CRP-CP fusion protein (not shown in the image), PCV2-CRP fusion protein (not shown in the image), HPV58L1-CRP fusion protein (not shown in the image), and CP and CRP expression vectors.

[0099] Following standard induction methods, the expression vector was transformed into host cells BL21(DE3), clones were selected and cultured to an OD of 0.6; then 0.2 mM IPTG was added and the cells were induced overnight at 16°C.

[0100] Bacterial cells were collected, purified, suspended in a buffer, and lysed by sonication. The target protein was then extracted by combinatorial chromatography.

[0101] 2. Preparation of CP-CRP-VLP complex, CRP-CP-VLP complex, PCV2-CRP-VLP complex, HPV58L1-CRP-VLP complex, and CP-VLP

[0102] The CP-CRP fusion protein, CRP-CP fusion protein, PCV2-CRP fusion protein, HPV58L1-CRP fusion protein, and CP protein purified in step 1 were dialyzed and replaced with buffer solutions of 20 mM PB, 150 mM NaCl, and pH 7.4. The protein concentration was adjusted to between 0.5 and 1 mg / mL using the Coomassie Brilliant Blue method. The mixture was stirred at 4°C for 48 hours, centrifuged at 12,000 rpm, concentrated, aliquoted, and stored at -80°C for later use. This yielded the CP-CRP-VLP complex prepared from the CP-CRP fusion protein, the CRP-CP-VLP complex prepared from the CRP-CP fusion protein, the PCV2-CRP-VLP complex prepared from the PCV2-CRP fusion protein, the HPV58L1-CRP-VLP complex prepared from the HPV58L1-CRP fusion protein, and the CP-VLP formed by the self-assembly of the CP protein.

[0103] 3. Preparation of VLP Coupling Affinity Media

[0104] The CP-VLP solution formed by the self-assembly of CP protein in step 2 was replaced with coupling buffer (0.2M NaHCO3, 0.5M NaCl pH 8.3), and added to the NHS activated gel at a ratio of 10 mg / mL. The reaction was carried out overnight at 160 rpm and 16°C with shaking.

[0105] The reaction medium was blocked overnight at 16°C with shaking in blocking buffer (0.1 M Tris-HCl pH 8.3).

[0106] The activated gel after blocking was washed sequentially with 5 volumes of deionized water, acetate-sodium acetate buffer (0.1M acetate-sodium acetate, 0.5M NaCl pH 4.0), borate-sodium borate buffer (0.1M borate-sodium borate, 0.5M NaCl pH 8.0), and deionized water to thoroughly remove unreacted ligands. The activated medium was stored in 20% ethanol at 4°C.

[0107] 4. Animal immunization

[0108] The CP-CRP-VLP complex, PCV2-CRP-VLP complex, HPV58L1-CRP-VLP complex, CRP-CP-VLP complex obtained in step 2, and the CRP monomer antigen obtained in step 1 were used to immunize New Zealand white rabbits according to the standard immunization procedure described by Wang Chuanwu et al. (2002) to prepare polyclonal antibodies. A total of five immunizations were performed. After each immunization, 5 mL of ear blood was collected, purified, and the immunization effect was detected by ELISA.

[0109] The specific experimental procedure is as follows: 1) Preparation of immunoantigen: Adjust the concentration to 1 mg / mL using physiological saline;

[0110] For the first immunization, take 1.2 mL of the immunogen from 1), add 1.2 mL of Freund's complete adjuvant, and emulsify evenly;

[0111] For the second to fifth immunizations, take 1.2 mL of the immunogen from 1) and add 1.2 mL of Freund's incomplete adjuvant, then emulsify until homogeneous.

[0112] 2) Animal immunization: Select healthy adult New Zealand white rabbits. For the first immunization, administer 0.8 mL / vial subcutaneously at multiple sites.

[0113] The second to fifth immunizations were administered on days 15, 30, 45, and 60 after the first immunization, respectively, using 0.8 mL / vial, administered subcutaneously at multiple sites.

[0114] 3) Serum detection: 10 mL of rabbit ear blood was collected on day 7 post-immunization. The titer was determined by ELISA after purification.

[0115] 5. Antibody purification

[0116] Purification using conventional methods:

[0117] The collected antiserum was added to saturated ammonium sulfate at a volume ratio of 1:1 and stirred overnight.

[0118] Collect the precipitate by centrifugation at 4000 rpm.

[0119] Add 10 times the serum volume of phosphate buffer (10mM PB + 150mM NaCl, pH 7.4) to fully dissolve the precipitate.

[0120] Excess ammonium sulfate was removed by overnight dialysis at 4°C.

[0121] Adjust the protein concentration to 1 mg / mL and store at 4°C.

[0122] VLP media affinity purification:

[0123] The activation medium prepared in step 3 was packed into the purification column. Ten column volumes were equilibrated at 4°C using equilibration buffer (10 mM PB, 150 mM NaCl, pH 7.4).

[0124] Antibodies obtained from immunopurification of CP-CRP-VLP, PCV2-CRP-VLP, HPV58L1-CRP-VLP, and CRP-CP-VLP polymeric antigens were slowly passed through a purification column at a flow rate of 1 mL / min. The flow-through was collected and concentrated to obtain high-titer CRP antibodies, while anti-VLP antibodies were removed from the purification column.

[0125] The concentration of the affinity-adjusted CRP antibody was set to 1 mg / mL for subsequent detection. For a detailed schematic diagram of the CP-CRP-VLP self-assembly to form a polyantigen in this embodiment, please refer to [link to schematic diagram]. Figure 3 For a detailed schematic diagram of CRP antibody affinity purification, please refer to [link / reference needed]. Figure 4 .

[0126] 6. ELISA testing

[0127] Antibodies obtained by routine purification after immunization with recombinant monomeric CRP antigen, antibodies obtained by routine purification after immunization with CP-CRP-VLP polymeric antigen, and antibodies obtained by affinity purification after immunization with CP-CRP-VLP polymeric antigen were simultaneously tested in parallel using natural and recombinant antigens via ELISA assays to detect titers. For detailed results, please refer to [link to ELISA test]. Figure 5 and Figure 6 The results showed that the antibody titer against the natural antigen increased approximately 30 times after immunization with the polyantigen compared to that after immunization with the monomeric antigen; the antibody titer against the natural antigen increased approximately 1 time after affinity purification of the polyantigen compared to before affinity purification, and approximately 60 times compared to that after immunization with the monomeric antigen. This indicates that the antibody prepared after CP-CRP-VLP polyantigen immunization can effectively recognize the natural antigen. Furthermore, the antibody titer was further improved after VLP affinity purification to remove impurities.

[0128] The antibodies obtained by routine purification after immunization with CP-CRP-VLP polymeric antigen and the antibodies obtained by routine purification after immunization with CRP-CP-VLP polymeric antigen were simultaneously tested for titer using parallel ELISA assays with both natural and recombinant antigens. For detailed results, please refer to [link to ELISA test]. Figure 7 .

[0129] The results showed that antibodies obtained from immunization with the recombinant antigen (CP-CRP) linking the N-terminus of C-reactive protein to the C-terminus of the hepatitis B virus core capsid protein (CP-CRP) exhibited a strong response against native CRP protein. Antibodies obtained from immunization with another group of recombinant antigens linking the C-terminus of C-reactive protein to the N-terminus of the capsid protein (CRP-CP) showed no significant response against native CRP protein. This indicates that the polymeric structure formed by the CP-CRP fusion protein is closer to the structure of the native protein, making it more suitable for preparing antibodies that can interact with native C-reactive protein.

[0130] Antibodies obtained by routine purification after immunization with CP-CRP-VLP polymeric antigen, antibodies obtained by routine purification after immunization with PCV2-CRP-VLP polymeric antigen, and antibodies obtained by routine purification after immunization with HPV58L1-CRP-VLP polymeric antigen were simultaneously subjected to parallel ELISA assays using both natural and recombinant antigens to detect titers. For detailed results, please refer to [link to ELISA test]. Figure 8 .

[0131] The results showed that antibodies obtained by immunization with recombinant antigens (CP-CRP, PCV2-CRP, HPV58L1-CRP) linking the N-terminus of C-reactive protein to the C-terminus of capsid protein all exhibited strong responses against natural CRP proteins. This indicates that the polymeric structure of the fusion protein formed by fusing the capsid protein is closer to the structure of the natural protein, which is more conducive to the preparation of antibodies that can react with natural C-reactive protein.

[0132] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," 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 present invention. In this specification, the 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. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0133] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that, include: Capsid protein and C-reactive protein; wherein the N-terminus of the C-reactive protein is connected to the C-terminus of the capsid protein; The capsid protein is selected from any one of the following: hepatitis B virus core capsid protein, porcine circovirus type 2 capsid protein, and human papillomavirus type 58 L1 protein. The amino acid sequence of the C-reactive protein is shown in SEQ ID NO: 1; The amino acid sequence of the hepatitis B virus core capsid protein is shown in SEQ ID NO: 2; The amino acid sequence of the porcine circovirus type 2 capsid protein is shown in SEQ ID NO: 3; The amino acid sequence of the human papillomavirus type 58 L1 protein is shown in SEQ ID NO:

4.

2. A polymeric antigen, characterized in that, The polyantigen is formed by the self-assembly of the fusion protein according to claim 1.

3. The polymeric antigen according to claim 2, characterized in that, The polyantigen is a virus-like particle structure, wherein the C-reactive protein is located on the outer side of the virus-like particle structure.

4. A nucleic acid, characterized in that, The nucleic acid encodes the fusion protein of claim 1 or the polyantigen of claim 2 or 3.

5. A carrier, characterized in that, The vector comprises the nucleic acid as described in claim 4.

6. A cell, characterized in that, The cell carries the nucleic acid of claim 4 or the vector of claim 5; or The cells express the fusion protein of claim 1 or the polyantigen of claim 2 or 3.

7. A method for preparing the polymeric antigen according to claim 2 or 3, characterized in that, include: The fusion protein of claim 1 is subjected to self-assembly to form a polyantigen.

8. The method according to claim 7, characterized in that, The self-assembly process is performed in a buffer solution.

9. The method according to claim 8, characterized in that, The buffer solution is a phosphate buffer solution with a pH of 7.

4.

10. The method according to claim 9, characterized in that, The phosphate buffer contains 20 mM phosphate and 150 mM NaCl.

11. The method according to claim 8, characterized in that, The self-assembly was carried out under conditions of stirring at 4°C for 48 hours.

12. A method for preparing an antibody or its antigen-binding fragment, characterized in that, include: Animals were immunized using the polymeric antigen described in claim 2 or 3. Obtain antiserum from immunized animals; The antiserum was purified to prepare anti-C-reactive protein antibody.

13. The method according to claim 12, characterized in that, The purification process was carried out using an affinity medium coupled with the capsid protein.

14. The method according to claim 13, characterized in that, The affinity medium coupled with the capsid protein is prepared by the following method: The self-assembled capsid protein was coupled to a solid-phase matrix. The solid matrix is ​​an NHS-activated gel medium.

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