Polyantigen and preparation method thereof

Abstract

The invention discloses a polyantigen and a preparation method thereof. Wherein the polyantigen is formed by self-assembly of fusion protein, and the fusion protein comprises capsid protein and C-reactive protein. The multi-antigen is of a virus-like particle structure, wherein the C-reactive protein is located on the outer side of the virus-like particle structure. The polymer antigen is an icosahedron polymer particle, partially simulates spatial sites of a natural antigen pentamer, and improves the recognition efficiency of the generated antibody on the natural antigen.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a multimeric antigen and a method for preparing the same. More specifically, the present invention relates to a fusion protein, a nucleic acid, a vector, a cell, and a method for preparing an anti-C-reactive protein antibody. Background Art

[0002] C-reactive protein (CRP) is a recognized marker of inflammation, and its normal level in the body is low. When the body is infected or tissues are damaged, its level in plasma rises sharply, and when the inflammatory response is severe, the high value can reach hundreds of milligrams per liter. It is usually used to distinguish between bacterial and viral infections. For cardiovascular patients, it is necessary to pay attention to the increase in C-reactive protein levels in the low value range of >3mg / L, which indicates the risk of cardiovascular abnormalities. This performance requirement, which requires taking into account both low-value sensitivity and high-value linearity, places very high potency requirements on the antibodies used for detection.

[0003] The recombinant CRP antigen monomer is 25 kd in size, a relatively small molecular weight. The antibodies generated after immunization do not recognize native CRP or have extremely low recognition efficiency. This is because the spatial conformation of the recombinant CRP monomer antigen differs significantly from that of the native pentameric protein, or the monomer antigen has low immunogenicity. Consequently, the antibodies generated are unable to form a normal antibody-antigen complex with native CRP, and thus 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 to at least a certain extent.

[0006] The present invention is completed based on the following findings of the inventors The inventors discovered that recombinant CRP has a molecular weight of 25 kDa. Compared to the native pentameric protein, recombinant CRP molecules are unable to spontaneously form pentamers. Their small molecular weight makes them small antigens, inherently low in immunogenicity and structurally distinct from the native protein. Consequently, CRP antibodies produced through conventional immunization have low titers and are unable to recognize native CRP molecules, making them unsuitable for detection. To overcome this problem, the inventors chose to link the recombinant CRP protein to the capsid protein to form a fusion protein. This fusion protein utilizes its in vitro self-assembly principle to form a multimeric antigen, increasing the antigen's own immunogenicity while partially mimicking the aggregated form of the native antigen. Furthermore, the VLP's ability to significantly stimulate an immune response further enhances antibody production efficiency. Furthermore, the individually expressed VLPs were chemically cross-linked to a gel matrix, and the resulting antibodies were affinity purified to remove the VLP antibodies. The results of ELISA experiments showed that the titer of antibodies against natural CRP antigen after immunization with multimeric antigens was about 30 times higher than that after immunization with monomeric antigens; after affinity purification, it was about 1 times higher than that before affinity purification, and about 60 times higher than that after immunization with monomeric antigens.

[0007] Based on this, the first aspect of the present invention provides 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 producing high-titer antibodies.

[0008] In a second aspect, the present invention provides a multimeric antigen. According to an embodiment of the present invention, the multimeric antigen is self-assembled from the fusion protein described in the first aspect of the present invention. The multimeric antigen according to an embodiment of the present invention can efficiently activate a humoral immune response and produce high-titer antibodies.

[0009] In a third aspect, the present invention provides a nucleic acid, which, according to an embodiment of the present invention, encodes the fusion protein described in the first aspect of the present invention and the multimeric antigen described in the second aspect of the present invention.

[0010] 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.

[0011] In a fifth aspect, the present invention provides a cell. According to an embodiment of the present 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 multimeric antigen described in the second aspect.

[0012] In a sixth aspect, the present invention provides a method for preparing the multimeric antigen described in the second aspect of the present invention. According to an embodiment of the present invention, the method comprises: subjecting the fusion protein described in the first aspect of the present invention to self-assembly to form the multimeric antigen. The method according to an embodiment of the present invention can prepare the multimeric antigen described in the second aspect of the present invention, thereby efficiently activating a humoral immune response and producing high-titer antibodies.

[0013] In its seventh aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the method comprises: immunizing an animal with the multimeric antigen described in the second aspect of the present invention, obtaining antiserum from the immunized animal; and purifying the antiserum to prepare an anti-C-reactive protein antibody. The method according to this embodiment of the present invention can produce high-titer antibodies.

[0014] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which: Figure 1 This is a flowchart of the technical implementation in Example 1 of the present invention.

[0016] Figure 2 This is a diagram of the molecular structure of CP-CRP in Example 1 of the present invention.

[0017] Figure 3 Schematic diagram of CRP-VLP self-assembly to form multimeric antigens in Example 1 of the present invention.

[0018] Figure 4 Schematic diagram of affinity purification of CRP antibody in Example 1 of the present invention.

[0019] Figure 5 This is a comparison chart of the antibody titers generated by different types of CRP antigens in Example 1 of the present invention.

[0020] Figure 6 This is a comparison chart of the titers of antibodies after VLP affinity purification after immunization with the multi-antigen in Example 1 of the present invention.

[0021] Figure 7 This is a comparison chart of the titers of antibodies obtained by conventional purification after immunization with the two multimeric antigens CRP-CP and CP-CRP in Example 1 of the present invention.

[0022] Figure 8This is a comparison chart of the titers of antibodies obtained by conventional purification after immunization with three multimeric antigens: CP-CRP-VLP, PCV2-CRP-VLP, and HPV58L1-CRP-VLP in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] 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 understood as limiting the present invention.

[0024] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0025] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0026] In the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0027] In the present invention, the term "antibody" is used in the broadest sense and includes full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies. The specific structure is not limited, as long as they exhibit the desired biological activity. Antibodies typically consist of a light chain (lighter in molecular weight) and a heavy chain (heavy in molecular weight), with the heavy chain (H chain) and light chain (L chain) linked by disulfide bonds to form an antibody molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region). The carboxyl terminus (C-terminus) is relatively stable and exhibits minimal variation, and is called the constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively.

[0028] In the present invention, the term "antigen" refers to a substance that can stimulate the immune system to produce antibodies or induce immune cell responses. In some optional embodiments of the present invention, "antigen" refers to natural antigens, monomeric antigens, and polymeric antigens. According to some optional embodiments of the present invention, for example, "antigen" refers to CP-CRP or CRP.

[0029] In the present invention, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between cells or hosts. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes vectors with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by culturing a suitable cell or host containing the vector.

[0030] In the present invention, the term "cell" generally refers to cells that have been modified, transformed, or recombined using genetic engineering or cell fusion techniques to modify or reorganize the genetic material of host cells to obtain cells with unique traits that are stably inherited. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. As used herein, the terms "transformed" or "transfected" 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 sequences of the present application and can be used for the expression and / or secretion of the target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0031] For the purposes of this invention, the term "VLP" refers to a synthetic nanoparticle whose structure and morphology mimic those of a natural virus, but which lacks viral genetic material (DNA / RNA) and, therefore, lacks infectivity or replication. Its core component is formed by self-assembly of one or more viral structural proteins (such as capsid proteins).

[0032] In the present invention, the term "polymeric antigen" refers to a high-molecular-weight complex formed by the assembly of multiple identical or different antigenic units in a specific manner. Specifically, it refers to a virus-like particle (VLP) structure formed by the self-assembly of a fusion protein of C-reactive protein (CRP) and hepatitis B virus core capsid protein (CP) (i.e., a CRP-VLP complex).

[0033] In the present invention, the term "capsid protein" refers to the primary structural protein that constitutes the viral capsid. Capsid proteins are a common structure shared by most viruses and are generally capable of self-assembly. However, the structures of capsid proteins vary significantly between different viral species. In specific embodiments of the present invention, capsid proteins specifically refer to the hepatitis B virus (HBV) core capsid protein, porcine circovirus type 2 (PCV2) capsid protein, and the human papillomavirus (HPV) type 58 L1 protein. The present invention provides a fusion protein, a multimeric antigen, a nucleic acid, a vector, a cell or a host, a method for preparing the multimeric antigen described in the second aspect of the present invention, and a method for preparing an antibody or an antigen-binding fragment thereof.

[0034] Fusion protein In its first aspect, the present invention provides 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 producing high-titer antibodies.

[0035] According to an embodiment of the present invention, the capsid protein is selected from at least one of hepatitis B virus core capsid protein, porcine circovirus type 2 capsid protein and human papillomavirus type 58 L1 protein.

[0036] 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.

[0037] 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.

[0038] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is connected to the C-terminus of the hepatitis B virus core capsid protein.

[0039] According to an embodiment of the present invention, the N-terminus of the C-reactive protein is connected to the C-terminus of the porcine circovirus type 2 capsid 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 human papillomavirus type 58 L1 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 hepatitis B virus core capsid protein.

[0042] According to an embodiment of the present invention, the C-reactive protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0043] QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASG IVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP (SEQ ID NO: 1) According to an embodiment of the present invention, the hepatitis B virus core capsid protein has an amino acid sequence as shown in SEQ ID NO: 2.

[0044] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 2) According to an embodiment of the present invention, the porcine circovirus type 2 capsid protein has an amino acid sequence as shown in SEQ ID NO: 3.

[0045] MTYPRRRRFRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPPLTVPFEYYRIRKVKVEFWPCSPITQGDRGV GSTAVILDDFVTKANALTYDPYVNYYRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK (SEQ ID NO: 3) According to an embodiment of the present invention, the human papillomavirus type 58 L1 protein has an amino acid sequence as shown in SEQ ID NO: 4.

[0046] (SEQ ID NO: 4) It should be noted that, based on the amino acid sequence of the fusion protein disclosed herein, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the fusion protein, for example, isolating and purifying the fusion protein from the culture product of recombinant cells. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the fusion protein disclosed herein, it falls within the scope of protection of this application.

[0047] Polymeric antigens In the second aspect of the present invention, a multimeric antigen is proposed. According to an embodiment of the present invention, the multimeric antigen is self-assembled by the fusion protein described in the first aspect of the present invention. The multimeric antigen according to the embodiment of the present invention can efficiently activate the humoral immune response and produce high-titer antibodies. According to an embodiment of the present invention, the multimeric antigen is self-assembled after C-reactive protein is fused with viral particles VLP and expressed. Compared with C-reactive protein expressed alone, the multimeric antigen is an icosahedral multimeric particle, which partially simulates the spatial site of the natural antigen pentamer and improves the efficiency of antibody recognition of natural antigens. The present invention proposes the use of VLPs that can participate in immune presentation and enhance humoral immunity as carriers of CRP. CRP molecules are located on the surface of the VLP icosahedron, and with the help of the VLP's activation effect on the immune system, the immune efficiency is improved and the antibody titer is increased. For the anti-VLP antibodies produced in the above process, the VLP-coupled affinity medium is used to remove them. This method further purifies the CRP antibodies, and in this design, the target antibody is not bound to the medium and flows through directly. This avoids the damage to the antibody functional region caused by elution under acidic conditions after the antigen and antibody functional region bind in the traditional antigen affinity process, and preserves the activity of the antibody to the greatest extent.

[0048] According to an embodiment of the present invention, the multimeric antigen is a virus-like particle structure, wherein the C-reactive protein is located on the outside of the virus-like particle structure.

[0049] According to a specific embodiment of the present invention, the present invention proposes a CRP protein multimer capable of enhancing the immunogenicity of recombinant CRP. Compared with recombinant CRP, the CRP multimer expresses the core protein of hepatitis B virus - capsid protein fused to the N-terminus of CRP.

[0050] According to an embodiment of the present invention, compared with 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 by the capsid protein, and the CRP is located on the outside of the VLP structure and is in a polymeric state.

[0051] According to an embodiment of the present invention, the titer of the antibodies obtained after immunizing animals with poly-CRP as an antigen is about 30 times higher than that of the antibodies obtained after immunizing animals with a recombinant monomeric CRP antigen against natural CRP protein; and after the anti-VLP antibodies are removed by a VLP-coupled affinity medium, the titer of the poly-CRP immune antibody is about 60 times higher than that of the monomeric CRP immune antibody against natural CRP antigen.

[0052] According to the embodiments of the present invention, a C-reactive protein multimeric antigen with a VLP structure was obtained by self-assembly, which improved the antigen stability and aggregation. At the same time, the presence of VLP also enhanced the effect of antigen presentation, stimulated the immune system, and increased the efficiency of antibody production. After the non-specific VLP antibodies were removed by affinity chromatography, the titer of the CRP antibody obtained was significantly improved compared with the control group immunized with the monomeric antigen.

[0053] It should be noted that, based on the amino acid sequence of the multimeric antigen disclosed herein, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the multimeric antigen, for example, isolating and purifying the multimeric antigen from the culture product of recombinant cells. This is easy to achieve for those skilled in the art. Based on this, regardless of the technology used to prepare the multimeric antigen disclosed herein, it falls within the scope of protection of this application.

[0054] Nucleic Acids In a third aspect, the present invention provides a nucleic acid, which, according to an embodiment of the present invention, encodes the fusion protein described in the first aspect of the present invention and the multimeric antigen described in the second aspect of the present invention.

[0055] According to an embodiment of the present invention, the nucleic acid includes DNA or RNA.

[0056] It should be noted that, for the nucleic acids mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in this application include DNA forms or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0057] Those skilled in the art will appreciate that the features and advantages described above for fusion proteins and multimeric antigens are also applicable to the nucleic acid and will not be described in detail here.

[0058] carrier 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. When the nucleic acid described in the third aspect is linked to the vector, the nucleic acid can be directly or indirectly linked to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid. Of course, these control elements can be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. Of course, it is sufficient that the nucleic acid and the control elements are operably linked.

[0059] As used herein, "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include plasmids, bacteriophages, and the like. After the vectors according to some specific embodiments of the present application are introduced into appropriate recipient cells, they can be mediated by a regulatory system to effectively express the aforementioned fusion protein, thereby enabling the in vitro production of large quantities of the fusion protein.

[0060] According to an embodiment of the present invention, the vector may refer to a cloning vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this application is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0061] In some optional embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0062] In some optional embodiments of the present invention, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0063] Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, multimeric antigens, and nucleic acids are also applicable to this vector and will not be elaborated here.

[0064] cell In a fifth aspect, the present invention provides a cell. According to an embodiment of the present 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 multimeric antigen described in the second aspect.

[0065] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fourth aspect into the cell.

[0066] It should be noted that the cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0067] In an optional embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0068] It should be noted that the "suitable conditions" described in the present invention refer to conditions suitable for the expression of the fusion protein and multimeric antigen of the present invention. It will be readily understood by those skilled in the art that conditions suitable for the expression of the fusion protein and multimeric antigen include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell status, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the fusion protein and multimeric antigen based on the specific environment of the laboratory.

[0069] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, multimeric antigen, nucleic acid, and vector are also applicable to the cell and will not be elaborated here.

[0070] Method for preparing multimeric antigens In a sixth aspect, the present invention provides a method for preparing a multimeric antigen. According to an embodiment of the present invention, the method comprises: subjecting the fusion protein described in the first aspect of the present invention to self-assembly to form a multimeric antigen. The method according to an embodiment of the present invention can prepare the multimeric antigen described in the second aspect of the present invention, thereby efficiently activating a humoral immune response and producing high-titer antibodies.

[0071] According to an embodiment of the present invention, the self-assembly process is performed in a buffer solution.

[0072] According to an embodiment of the present invention, the buffer solution is a phosphate buffer solution with a pH of 7.4.

[0073] According to an embodiment of the present invention, the phosphate buffer contains 20 mM phosphate and 150 mM NaCl.

[0074] According to an embodiment of the present invention, the self-assembly is performed under the condition of stirring at 4° C. for 48 hours.

[0075] Method for preparing antibodies or antigen-binding fragments thereof In its seventh aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the method comprises: immunizing an animal with the multimeric antigen described in the second aspect of the present invention, obtaining antiserum from the immunized animal; and purifying the antiserum to prepare an anti-C-reactive protein antibody. The method according to an embodiment of the present invention can produce high-titer antibodies or antigen-binding fragments thereof.

[0076] According to an embodiment of the present invention, the purification process is performed by an affinity medium coupled with the capsid protein.

[0077] According to an embodiment of the present invention, the affinity medium coupled with the capsid protein is prepared by the following method: subjecting the self-assembly to a solid-phase matrix coupling treatment.

[0078] According to an embodiment of the present invention, the solid phase matrix is ​​an NHS-activated gel medium.

[0079] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0080] Example Example 1: The technical implementation process of this embodiment is as follows: Figure 1 The specific process is as follows.

[0081] 1. Preparation of CP-CRP fusion protein, CRP-CP fusion protein, PCV2-CRP fusion protein, HPV58L1-CRP fusion protein, CP protein, CRP protein Gene synthesis was used to construct Figure 2 Shown are CP-CRP fusion protein, CRP-CP fusion protein (not shown in the picture), PCV2-CRP fusion protein (not shown in the picture), HPV58L1-CRP fusion protein (not shown in the picture) and CP and CRP expression vectors.

[0082] According to the conventional induction expression method, the expression vector was transformed into the host cell BL21 (DE3), and the clone was selected and amplified to OD 0.6; IPTG was added to a final concentration of 0.2 mM and induced at 16°C overnight.

[0083] The cells were collected, suspended in purification buffer, and then ultrasonically lysed. The target protein was extracted by combined chromatography.

[0084] 2. Preparation of CP-CRP-VLP complex, CRP-CP-VLP complex, PCV2-CRP-VLP complex, HPV58L1-CRP-VLP complex, CP-VLP 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 into 20 mM PB, 150 mM NaCl pH 7.4 buffer, and the protein concentration was adjusted to between 0.5 and 1 mg / mL by 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 until further use. 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 self-assembly of the CP protein were obtained, respectively.

[0085] 3. Preparation of VLP-coupled Affinity Media The CP-VLP solution formed by CP protein self-assembly in step 2 was exchanged into coupling buffer (0.2 M NaHCO3, 0.5 M NaCl pH 8.3), added to NHS-activated gel at a ratio of 10 mg / mL, and shaken at 160 rpm and 16°C overnight for reaction.

[0086] The reaction medium was blocked in blocking buffer (0.1 M Tris-HCl pH 8.3) with shaking at 16°C overnight.

[0087] After blocking, the activated gel was washed sequentially with 5 volumes of deionized water, acetic acid-sodium acetate buffer (0.1M acetic acid-sodium acetate, 0.5M NaCl, pH 4.0), boric acid-sodium borate buffer (0.1M boric acid-sodium borate, 0.5M NaCl, pH 8.0), and deionized water to fully remove unreacted ligands. The activated medium was stored in 20% ethanol at 4°C.

[0088] 4. Animal immunization New Zealand white rabbits were immunized with the CP-CRP-VLP complex, PCV2-CRP-VLP complex, HPV58L1-CRP-VLP complex, and CRP-CP-VLP complex obtained in step 2, along with the CRP monomer antigen obtained in step 1, to produce polyclonal antibodies according to the standard immunization protocol described by Wang Chuanwu et al. (2002). Five immunizations were performed, and after each immunization, 5 mL of ear blood was collected per tube, purified, and assayed for immune efficacy using ELISA.

[0089] The specific experimental procedures are as follows: 1) Preparation of immune antigen: adjust the concentration to 1 mg / mL using normal saline; For the first immunization, take 1.2 mL of the immune antigen from step 1, add 1.2 mL of Freund's complete adjuvant, and emulsify evenly; For the second to fifth immunizations, 1.2 mL of the immunizing antigen in 1) was taken, and 1.2 mL of Freund's incomplete adjuvant was added and emulsified evenly.

[0090] 2) Animal Immunization: Select healthy adult New Zealand white rabbits. For the first immunization, administer 0.8 mL / vial subcutaneously at multiple sites. The second to fifth immunizations were performed on the 15th, 30th, 45th, and 60th days after the first immunization, respectively, using 0.8 mL / vial for subcutaneous multi-point immunization. 3) Serological testing: Collect 10 mL of blood from rabbit ears on day 7 after immunization. After purification, titer was determined by ELISA.

[0091] 5. Antibody Purification Conventional purification method: The collected antiserum was added to saturated ammonium sulfate at a volume ratio of 1:1 and stirred overnight.

[0092] The precipitate was collected by centrifugation at 4000 rpm.

[0093] Add phosphate buffer (10 mM PB 150 mM NaCl pH 7.4) 10 times the volume of serum to fully dissolve the precipitate.

[0094] The excess ammonium sulfate was removed by dialysis at 4°C overnight.

[0095] Adjust the protein concentration to 1 mg / mL and store at 4°C.

[0096] VLP media affinity purification: The activated medium prepared in step 3 was filled into the purification column. Equilibrate with equilibration buffer (10 mM PB, 150 mM NaCl, pH 7.4) at 4°C for 10 column volumes.

[0097] Antibodies immunopurified from CP-CRP-VLP, PCV2-CRP-VLP, HPV58L1-CRP-VLP, and CRP-CP-VLP multimeric antigens were passed through the 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 bound to the purification column and removed.

[0098] The concentration of CRP antibody after affinity adjustment was 1 mg / mL for subsequent detection. The schematic diagram of CP-CRP-VLP self-assembly to form multimeric antigen in this embodiment is shown in FIG. Figure 3 , CRP antibody affinity purification diagram, please refer to Figure 4 .

[0099] 6.ELISA test The antibodies obtained by conventional purification after immunization with recombinant monomeric CRP antigen, the antibodies obtained by conventional purification after immunization with CP-CRP-VLP multimeric antigen, and the antibodies obtained by affinity purification after immunization with CP-CRP-VLP multimeric antigen were tested for titer by ELISA test in parallel using natural antigen and recombinant antigen. The results are shown in the table below. Figure 5 and Figure 6 The results showed that the titer of antibodies against natural antigens after immunization with multimeric antigens was approximately 30 times higher than that after immunization with monomeric antigens; the titer of antibodies against natural antigens after affinity purification was approximately 1 times higher than that before affinity purification, and approximately 60 times higher than that after immunization with monomeric antigens. This indicates that antibodies prepared after immunization with CP-CRP-VLP multimeric antigens can effectively recognize natural antigens. At the same time, after VLP affinity purification to remove impurities, the antibody titer was further improved.

[0100] The antibodies obtained by conventional purification after immunization with CP-CRP-VLP multi-antigen and the antibodies obtained by conventional purification after immunization with CRP-CP-VLP multi-antigen were tested for titer by ELISA test using natural antigen and recombinant antigen in parallel. Figure 7 .

[0101] The results showed that antibodies obtained after immunization with a recombinant antigen (CP-CRP) linking the N-terminus of C-reactive protein to the C-terminus of the hepatitis B virus core capsid protein showed a strong reaction to native CRP. Antibodies obtained after immunization with another recombinant antigen (CRP-CP) linking the C-terminus of C-reactive protein to the N-terminus of the capsid protein showed no significant reaction to native CRP. This suggests that the multimeric structure formed by the CP-CRP fusion protein is more similar to that of the native protein, making it more suitable for the preparation of antibodies that react with native CRP.

[0102] The antibodies obtained by conventional purification after immunization with CP-CRP-VLP multi-antigen, the antibodies obtained by conventional purification after immunization with PCV2-CRP-VLP multi-antigen, and the antibodies obtained by conventional purification after immunization with HPV58L1-CRP-VLP multi-antigen were tested for titer by ELISA test using natural antigen and recombinant antigen in parallel. Figure 8 .

[0103] The results showed that antibodies obtained by immunization with recombinant antigens (CP-CRP, PCV2-CRP, and HPV58L1-CRP) that linked the N-terminus of C-reactive protein to the C-terminus of capsid protein all showed strong reactivity against native CRP protein. This suggests that the multimeric structure of the fusion protein formed by fusion with capsid protein is closer to that of the native protein, making it more suitable for the preparation of antibodies that can react with native C-reactive protein.

[0104] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0105] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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 C-reactive protein is linked to the capsid protein; Optionally, the capsid protein is selected from at least one of hepatitis B virus core capsid protein, porcine circovirus type 2 capsid protein and human papillomavirus type 58 L1 protein.

2. The fusion protein according to claim 1, characterized in that The N-terminus of the C-reactive protein is connected to the C-terminus of the capsid protein; And / or, the C-terminus of the C-reactive protein is connected to the N-terminus of the capsid protein.

3. The fusion protein according to claim 1, characterized in that The N-terminus of the C-reactive protein is connected to the C-terminus of the hepatitis B virus core capsid protein; and / or, the N-terminus of the C-reactive protein is connected to the C-terminus of the porcine circovirus type 2 capsid protein; and / or, the N-terminus of the C-reactive protein is linked to the C-terminus of the human papillomavirus type 58 L1 protein; and / or, the C-terminus of the C-reactive protein is connected to the N-terminus of the hepatitis B virus core capsid protein; and / or, the C-reactive protein has the amino acid sequence shown in SEQ ID NO: 1; And / or, the hepatitis B virus core capsid protein has the amino acid sequence shown in SEQ ID NO: 2; And / or, the porcine circovirus type 2 capsid protein has the amino acid sequence shown in SEQ ID NO: 3; And / or, the human papillomavirus type 58 L1 protein has the amino acid sequence shown in SEQ ID NO:

4.

4. A multimeric antigen, characterized in that The multimeric antigen is formed by self-assembly of the fusion protein according to any one of claims 1 to 3.

5. The multimeric antigen according to claim 4, characterized in that The multimeric antigen is a virus-like particle structure, wherein the C-reactive protein is located on the outside of the virus-like particle structure.

6. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to any one of claims 1 to 3 or the multimeric antigen according to claim 4 or 5.

7. A carrier, characterized in that The vector comprises the nucleic acid of claim 6.

8. A cell, characterized in that The cell carries the nucleic acid of claim 6 or the vector of claim 7; or The cell expresses the fusion protein according to any one of claims 1 to 3 or the multimeric antigen according to claim 4 or 5.

9. A method for preparing the multimeric antigen according to claim 4 or 5, characterized in that: include: The fusion protein according to any one of claims 1 to 3 is subjected to self-assembly treatment to form a multimeric antigen.

10. The method according to claim 9, characterized in that The self-assembly treatment is carried out in a buffer solution; Optionally, the buffer solution is a phosphate buffer solution with a pH of 7.4; Optionally, the phosphate buffer comprises 20 mM phosphate and 150 mM NaCl; Optionally, the self-assembly is performed under stirring at 4° C. for 48 hours.

11. A method for preparing an antibody or an antigen-binding fragment thereof, characterized in that: include: Immunizing an animal with the multimeric antigen according to claim 4 or 5, Obtain antiserum from immunized animals; The antiserum is purified to prepare anti-C-reactive protein antibodies.

12. The method according to claim 11, characterized in that The purification process is carried out by using an affinity medium coupled with the capsid protein; Optionally, the affinity medium coupled with the capsid protein is prepared by the following method: The self-assembled capsid protein is coupled to a solid-phase matrix; And / or, the solid matrix is ​​an NHS-activated gel medium.

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