PCNA-binding proteins, methods of making and uses

Abstract

The application provides a PCNA binding protein, a preparation method and application, and relates to the technical field of biology. The PCNA binding protein contains a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, the complementarity determining region of the heavy chain variable region comprises an amino acid sequence identical to VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO. 1, and the complementarity determining region of the light chain variable region comprises an amino acid sequence identical to VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO. 2. The binding protein has good specific binding capacity with PCNA, and can be used for identification of PCNA antibodies or PCNA antigens, and auxiliary diagnosis and detection of PCNA antibody positive diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a PCNA-binding protein, its preparation method, and its application. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Proliferating cell nuclear antigen (PCNA), also known as cyclin 2, was first discovered in 1978 by Miyachi et al. using double diffusion immunoprecipitation in the serum of SLE patients. It is named for its presence in actively proliferating cells. The traditional PCNA protein, with a molecular weight of 34 kDa, is a co-protein or processing factor of DNA polymerase delta. Highly conserved in eukaryotic cells, it consists of three interconnected monomers forming a homotrimeric circular structure, acting as a sliding clamp around double-stranded DNA. It plays a role in DNA replication, chromatin remodeling, DNA damage repair, and cell cycle progression. PCNA protein is one of the core molecules determining cell life and death. Its expression is regulated by p53, and PCNA protein interacts with p53 regulatory proteins Gadd45, MyD118, CR6, and most importantly, p21 in determining cell fate. On the one hand, if PCNA protein is abundant in cells when p53 is absent, DNA replication will occur. On the other hand, if p53 is present and PCNA protein levels are high in cells, DNA repair will occur. If PCNA loses function, is absent in cells, or is present in small amounts, apoptosis will occur.

[0004] Anti-PCNA antibodies are specific antibodies for systemic lupus erythematosus (SLE) and can serve as markers for the disease. A positive test may indicate SLE, although the positivity rate is low. Recent studies have also confirmed that anti-PCNA antibodies are not unique to SLE and can be found in many other autoimmune diseases. Anti-PCNA antibodies are also found in patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Furthermore, anti-PCNA antibodies have been detected in patients with malignant tumors (such as small cell lung cancer).

[0005] Currently, calibrators and quality control samples in anti-PCNA antibody detection kits mainly use patient serum. However, obtaining positive serum is difficult, the source is unstable, and the performance of each serum sample is inconsistent. Therefore, developing anti-PCNA recombinant monoclonal antibodies that can be mass-produced, have small batch-to-batch variability, and high affinity is of great significance and value for the development of clinical detection kits.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a PCNA-binding protein that exhibits good specific binding ability to PCNA. Based on the PCNA-binding protein provided by this invention, another objective is to provide its applications. A further objective of this invention is to develop a PCNA-binding protein with good binding activity using a simpler method, which can be used for the identification of PCNA antigens and to assist in the diagnosis or auxiliary diagnosis of PCNA antibody-positive diseases.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] Definition of noun:

[0010] In this article, PCNA refers to proliferating cell nuclear antigen (PCNA), also known as cyclin 2. It has a molecular weight of 34 kDa and is highly conserved in eukaryotic cells. It consists of three interconnected monomers forming a homotrimeric ring structure. PCNA is closely related to cellular DNA synthesis, plays a crucial role in the initiation of cell proliferation, and participates in many important cellular events, such as DNA damage repair, cell cycle regulation, chromosome recombination, DNA methylation, and apoptosis.

[0011] The PCNA-binding protein provided by this invention can specifically bind to PCNA. The PCNA bound by the PCNA-binding protein includes PCNA expressed naturally in cells or expressed in cells transfected with the PCNA gene. The PCNA bound by the PCNA-binding protein also includes artificially modified PCNA, wherein the artificial modification includes, but is not limited to, mutated, truncated, or fused peptides or proteins with other structural domains, and retains the necessary antigenic epitopes for antibody binding.

[0012] In this document, the technical term "binding protein" refers to a protein that binds to a specific antigen, broadly encompassing all proteins and protein fragments containing a complementarity-determining region (CDR). Proteins and protein fragments can be antibodies. The terms "antibody" and "full-length antibody" include both polyclonal and monoclonal antibodies. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. Non-naturally occurring antibodies are also referred to as "recombinant antibodies" in this document. The term "antibody" is used interchangeably with "immunoglobulin."

[0013] Proteins and protein fragments can also be antigen-binding fragments containing part or all of an antibody CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to antigens. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. These fragments are selected from, but are not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments, dsFv)), bispecific antibodies, nanobodies, and the smallest recognition unit of an antibody. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0014] The "variable region" or "variable domain" of a binding protein refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing the antigen. The heavy chain variable domain can be referred to as the "VH," and the light chain variable domain as the "VL." These domains are typically the most variable parts of the antibody and contain the antigen-binding site. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0015] The term "constant region" or "constant domain" of an antibody refers to the constant region of the antibody light chain, either alone or in combination, or the constant region of the antibody heavy chain. The antibody heavy chain has a variable domain (VH), followed by one or more constant domains or regions, such as hinges, CH1, CH2, CH3, and CH4. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the antibody heavy chain, and does not form a portion of the antibody's Fc region. The hinge region includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The N-terminus of CH2 is typically the CH3 domain, which usually forms the C-terminal portion of the antibody. In some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can originate from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms.

[0016] This invention does not limit the method of obtaining the PCNA-binding protein. In some alternative embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the PCNA-binding protein to a vector and expressing it in cells. The vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a structure capable of expressing the PCNA-binding protein. In other alternative embodiments, the binding protein can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as automated peptide synthesizers (e.g., automated peptide synthesizers sold by Applied BioSystems, etc.); the antigen-binding fragment can also optionally be generated by enzymatic cleavage of antigen-binding molecules (including intact antibodies), such as pepsin or papain cleavage; or by chemical cleavage, such as by chemical reduction of disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0017] The terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of a binding protein to an epitope on a pre-defined antigen. Typically, binding proteins bind at a rate of approximately less than 10... -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K d Value binding. The K value of the antibody can be determined using methods well-established in the art. d Values. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry.

[0018] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides encode the aforementioned PCNA-binding proteins, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0019] In this article, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the cells.

[0020] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0021] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Transformation” and “transformed cell” include primary test cells and cultures derived from them.

[0022] In this document, the terms “purified” or “isolated” associated with peptides or nucleic acids mean that the peptide or nucleic acid is not in its native medium or in its native form. Therefore, the term “isolated” includes peptides or nucleic acids removed from their original environment, such as if they are naturally occurring. For example, isolated peptides typically do not contain at least some proteins or other cellular components that are normally bound to or mixed with or in solution with them. Isolated peptides include naturally produced peptides contained in cell lysates, peptides in purified or partially purified forms, recombinant peptides, peptides expressed or secreted by cells, and peptides in heterologous cells or cultures. As associated with nucleic acids, the terms “isolated” or “purified” indicate, for example, that the nucleic acid is not in its native genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into heterologous cells).

[0023] In this article, "PCNA antibody positive disease" refers to a disease that causes a patient's PCNA antibody levels to be higher than those before the disease. Exemplary PCNA antibody positive diseases include, but are not limited to, autoimmune diseases (such as lupus), viral infections (such as chronic hepatitis B virus infection or hepatitis C virus infection), or cancers (such as small cell lung cancer).

[0024] In one aspect, a PCNA-binding protein is provided, comprising a heavy chain variable region and a light chain variable region.

[0025] The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the variable region of the light chain includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3.

[0026] The VH-CDR1, VH-CDR2, and VH-CDR3 comprise amino acid sequences identical to those of the heavy chain variable regions shown in SEQ ID NO.1; the VL-CDR1, VL-CDR2, and VL-CDR3 comprise amino acid sequences identical to those of the light chain variable regions shown in SEQ ID NO.2.

[0027] It is understood that the amino acid sequences of the variable regions shown in SEQ ID NO. 1 or 2, excluding the CDR region, are not intended to limit the PCNA-binding protein provided by this invention. For example, if the PCNA-binding protein provided by this application contains a backbone region, it may differ from the backbone region in the variable region shown in SEQ ID NO. 1 or 2. The CDR region in the variable region shown in SEQ ID NO. 1 or 2 may be divided according to any optional manner known in the art. Optionally, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the variable region may be defined by any one or a combination of multiple definition systems such as Kabat, Chothia, IMGT, ABM, or Contact. Taking Kabat, Chothia, IMGT, ABM, or Contact as examples, the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.1 and the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable region shown in SEQ ID NO.2 are shown in Table 1:

[0028] Table 1

[0029]

[0030]

[0031] In an optional embodiment, the PCNA-binding protein has VH-CDR1, VH-CDR2, and VH-CDR3, the heavy chain variable regions defined in Table 1, and VL-CDR1, VL-CDR2, and VL-CDR3, the light chain variable regions defined in Table 1. Taking the IMGT definition as an example: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19; the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is ATS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.25.

[0032] In an optional embodiment, the heavy chain variable region further comprises a backbone region, and / or the light chain variable region further comprises a backbone region. The species source of the backbone region includes, but is not limited to, one or more of the following: rabbit, cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human, and mutants thereof.

[0033] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the PCNA-binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0034] In an optional implementation, the PCNA-binding protein is an antibody or antigen-binding fragment including a constant region.

[0035] In an optional implementation, at least a portion of the constant region sequence of the PCNA-binding protein is a human constant region sequence.

[0036] In an optional embodiment, the constant region sequence of the PCNA-binding protein is selected from a portion or all of the constant region sequences of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD, wherein IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD include their subclasses and mutant forms.

[0037] In an optional embodiment, the PCNA-binding protein contains a heavy chain constant region.

[0038] In an optional embodiment, the heavy chain constant region sequence of the PCNA binding protein is selected from part or all of the constant region sequence of human IgG1, preferably including CH1, CH2 and CH3 of the constant region of human IgG1.

[0039] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3.

[0040] In an optional embodiment, the PCNA-binding protein contains a light chain constant region.

[0041] In an optional implementation, the light chain constant region sequence is selected from the light chain constant region of a mouse.

[0042] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4.

[0043] In an optional embodiment, the PCNA-binding protein is a human-mouse chimeric antibody, and the heavy chain amino acid sequence of the PCNA-binding protein is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0044] In a second aspect, a biological material is also provided, comprising a polynucleotide, a carrier, or a cell; wherein the polynucleotide encodes the aforementioned PCNA-binding protein; the carrier carries the polynucleotide; and the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the PCNA-binding protein.

[0045] By linking the vector with a polynucleotide encoding the PCNA-binding protein, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line that can express the PCNA-binding protein, and the corresponding protein can be obtained through cell expression.

[0046] In an optional embodiment, the cells used to express PCNA-binding protein are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0047] In an optional implementation, the cells used to express PCNA-binding protein are CHO cells (Chinese hamster ovary cells).

[0048] Thirdly, a method for preparing the PCNA-binding protein of the first aspect is also provided, including culturing cells capable of expressing the PCNA-binding protein.

[0049] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the PCNA-binding protein into cells, and obtaining the PCNA-binding protein through purification.

[0050] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the PCNA-binding protein as needed, and / or preparing a suitable expression vector as needed, transforming the expression vector into the desired cells and expressing it, and obtaining the PCNA-binding protein through purification.

[0051] In an optional embodiment, the cell is prepared by converting a polynucleotide encoding a PCNA-binding protein as described in the first aspect into the cell, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the conversion comprising co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0052] In an optional implementation, the C-terminus of the heavy chain variable region is fused with a constant region fragment to construct a complete heavy chain expression plasmid.

[0053] In an optional embodiment, the constant region segment includes one or more of CH1, CH2 and CH3, preferably including CH1, CH2 and CH3.

[0054] In an optional embodiment, the cell is a eukaryotic cell, preferably a mammalian cell.

[0055] In an optional embodiment, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

[0056] Fourthly, the application of the PCNA-binding protein of the first aspect or the biomaterial of the second aspect in any of the following (I) to (VII) is also provided:

[0057] (I) Detection of PCNA antibodies for non-diagnostic and treatment purposes;

[0058] (II) Preparation of products for detecting PCNA antibodies;

[0059] (III) Preparation of products for diagnosing PCNA antibody-positive diseases;

[0060] (IV) Non-diagnostic and treatment-oriented PCNA testing;

[0061] (VII) Prepare products for detecting PCNA;

[0062] (VI) Used for purifying PCNA;

[0063] (VII) Prepare products for purifying PCNA;

[0064] For detecting PCNA antibodies or diagnosing PCNA antibody-positive diseases, PCNA binding proteins can be used as standards and / or quality controls to provide a reliable reference for test results and can also be used to construct standard curves.

[0065] In an optional embodiment, the preparation of products for diagnosing PCNA antibody-positive diseases includes the preparation of products for diagnosing or assisting in the diagnosis of autoimmune diseases. The PCNA antibody-positive diseases include products for diagnosing or assisting in the diagnosis of autoimmune diseases, viral infections, or cancer. The autoimmune diseases may optionally include systemic lupus erythematosus; the viral infections may optionally include chronic hepatitis B virus infection or hepatitis C virus infection; and the cancers may optionally include small cell lung cancer.

[0066] The application described in aspect (IV) above can utilize the PCNA-binding protein's ability to specifically target and bind to PCNA, enabling the detection of PCNA or PCNA-expressing cells based on immunoassay techniques. For example, when the PCNA-binding protein is an immunoconjugate, such as one linked to a fluorescent group, a fluorescence detection device can be used to locate or detect PCNA in real time. It can be used in techniques such as immunoblotting, immunoprecipitation, or flow cytometry that involve the specific binding properties of PCNA antigens and antibodies to detect PCNA. Based on the PCNA-binding protein's ability to specifically target and bind to PCNA, it can also be used for the purification of PCNA as described in aspect (VI).

[0067] In optional embodiments, in aspects (II), (III), (V) or (VII) above, those skilled in the art can prepare corresponding products (such as the immunoconjugates mentioned above) according to actual uses, and select other reagent components in the product, including but not limited to one or more of the following: tracer markers, solid-phase carriers, buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, conjugates, negative controls, positive controls, standards, quality control products, and markers.

[0068] Fifthly, a reagent or kit is also provided, the reagent or kit comprising the PCNA-binding protein of the first aspect or the biological material of the second aspect.

[0069] In an optional implementation, the kit is used to detect PCNA antibodies or to detect PCNA antibody-positive diseases; the autoantibody-positive diseases include, but are not limited to, one or more of idiopathic inflammatory myopathy, antisynthetic enzyme syndrome, myositis, lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis, as well as other autoimmune diseases. The kit includes standards and / or quality controls containing the PCNA binding protein.

[0070] In an optional embodiment, the kit further includes detection reagents, including PCNA antibody detection reagents.

[0071] In an optional embodiment, the kit further includes a detection reagent containing antibodies against at least one of the following substances: dsDNA, nucleosomes, histones, Sm, ribosomal P protein, Scl-70, SSA / Ro52, SSA / Ro60, CENP-B, AMAM2, ssb, Jo-1, PM-Scl, Mi-2, Ku, and RNP / sm.

[0072] In an optional embodiment, the kit further includes a solid support.

[0073] In optional embodiments, the PCNA-binding protein in the reagent or kit is coupled to a solid-phase support; or the PCNA-binding protein in the reagent or kit and the solid-phase support are packaged separately. By coupling the PCNA-binding protein to the solid-phase support, it can be used to capture PCNA protein in a sample to be tested. Alternatively, by coupling the PCNA-binding protein to the solid-phase support, it can be used to purify PCNA protein.

[0074] In an optional embodiment, the PCNA antibody detection reagent includes a solid-phase carrier conjugated with PCNA antigen.

[0075] In an optional implementation, the kit may further include a tracer marker.

[0076] In optional embodiments, the PCNA-binding protein in the reagent or kit is coupled to a tracer label; or the PCNA-binding protein and the tracer label in the reagent or kit are packaged separately. By coupling the PCNA-binding protein to the tracer label, it can be used to locate and detect PCNA protein or to detect PCNA protein in a sample via Western blotting.

[0077] The reagents or kits described above may also optionally include reagents and / or consumables well known to those skilled in the art for use in detecting reactions or purifying proteins, including but not limited to one or more of buffers, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality controls, and markers.

[0078] The above-described reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, or immunomagnetic beads. Those skilled in the art can formulate other reagents in the reagents or kits according to the corresponding detection methods, and the present invention does not limit this.

[0079] The tracer markers in any of the above embodiments include, but are not limited to, one or more of the following: enzymes, luminescent markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radioactive contrast agents, paramagnetic ions, metals, and photosensitizers.

[0080] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Luminescent labels include, but are not limited to, fluorescent proteins, synthetic small molecules, or polymer dyes. Specific examples include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C, Phycocyanin R, Thiamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red, one or more of these. Fluorescent microspheres, colored microspheres, and latex microspheres are each independently selected from products acceptable in the art, such as those derived from commercially available products. Radionuclides include, but are not limited to, those derived from... 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0081] The solid-phase support in any of the above embodiments includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or side-flow devices; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic beads. The chromatography matrix can be any known chromatography matrix acceptable in the art, including but not limited to polystyrene, polysaccharide polymers, or silica gel. In optional embodiments, the chromatography matrix includes gel particles.

[0082] This invention discloses a PCNA-binding protein that specifically recognizes and binds to the PCNA antigen, and further prepares a human-mouse chimeric recombinant monoclonal antibody. This invention has the following beneficial effects:

[0083] (1) This method has the advantages of preparing PCNA-binding protein with known sequence, recombination expression in vitro, simple operation, short time consumption, controllable production process, small batch-to-batch variation of product, and good stability, and has good application prospects.

[0084] (2) This PCNA-binding protein can be coupled to a chromatography matrix for immunoaffinity chromatography purification of PCNA antigens, thereby improving protein purity. This PCNA-binding protein can also be coupled to a marker for the detection of PCNA antigens, including but not limited to the use of liquid chromatography chips, immunofluorescence staining, flow cytometry, fluorescent microspheres, and other techniques to identify PCNA antigens.

[0085] (3) As a quality control material in the test kit, it can alleviate the problems of complicated operation of polyclonal antibodies and low subsequent conjugation efficiency, reduce production costs, stabilize product quality, and significantly improve reaction values; on the other hand, compared with the direct use of human serum, it can also avoid the problems of difficult sample sources and high costs. Attached Figure Description

[0086] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0087] Figure 1 This is an SDS-PAGE protein electrophoresis image of the anti-PCNA recombinant monoclonal antibody in Example 1. M represents Maeker, "reduced" indicates the SDS-PAGE electrophoresis result of the PCNA recombinant antibody, with heavy and light chains of 50 kDa and 25 kDa, respectively, and "non-reduced" indicates the SDS-PAGE electrophoresis result of the PCNA recombinant antibody, with a size of approximately 150 kDa.

[0088] Figure 2 The results of the ELISA assay for the binding of recombinant monoclonal antibody to PCNA protein in Example 2 are shown. Ab-PCNA represents the binding activity of the recombinant monoclonal antibody to the PCNA antigen, and Ab-BSA represents the non-specific binding activity of the recombinant monoclonal antibody to the control protein BSA. Detailed Implementation

[0089] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0090] Example 1: Preparation of PCNA chimeric antibody

[0091] A PCNA-specific high-affinity monoclonal strain was obtained by screening using phage display technology. After sequencing, the Fab region sequence was obtained. Its heavy chain variable region is shown in SEQ ID NO.1, its light chain variable region is shown in SEQ ID NO.2, and its CDR region is shown in Table 1.

[0092] Heavy chain variable region (SEQ ID NO.1)

[0093] EVKIEESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIYPGSGGSYNPSL KSRISITRDTSKNQFFLQLNSVTAEDTATYYCTRANYGNYFDYWGQGTTLTVSS.

[0094] Light chain variable region (SEQ ID NO.2)

[0095] DVQMNQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGS RSGSDYSLTISSLESEDFVDYYCLQYASSPRTFGGGTKLEIK.

[0096] Table 1

[0097]

[0098] Complete light chain and heavy chain genes were synthesized and inserted into the PTT5 vector, respectively. The vectors were then transformed into competent cells, and bacterial cultures were selected to obtain the corresponding expression plasmids. The light chain constant region sequence is shown in SEQ ID NO.4, which is a mouse-derived sequence; the complete light chain sequence is shown in SEQ ID NO.6. The heavy chain constant region sequence is shown in SEQ ID NO.3, which is a human-derived sequence; the complete heavy chain sequence is shown in SEQ ID NO.5.

[0099] Heavy chain expression plasmid and light chain expression plasmid were mixed with PEI at a molar ratio of 1:2 and transfected into 293F suspension cells in logarithmic growth phase. The cells were cultured in a 37°C shaking incubator at 120 rpm. After 5 days, the cell supernatant was collected, and the antibody was purified with Protein A. The cells were eluted with 0.1M Glycine (pH 3.0) and neutralized with 1M Tris (pH 8.0). After elution, the ultrafiltration centrifuge tubes were replaced with PBS buffer and concentrated, and the protein concentration was determined. The obtained product was validated by SDS-PAGE, and the results are shown below. Figure 1 As shown.

[0100] Example 2: ELISA determination of the binding activity of recombinant monoclonal antibody to PCNA antigen.

[0101] (1) Coat PCNA protein (2 μg / mL) onto an ELISA plate, and simultaneously coat it with BSA as a non-specific binding control. Incubate overnight at 4°C.

[0102] (2) Discard the coating solution, wash the plate 3 times with PBST, pat dry, add 3% milk, and block at 37°C for 2 hours.

[0103] (3) Discard the blocking solution, wash the plate 3 times with PBST, pat dry, add the antibody prepared in Example 1 (set up multiple concentration gradient experimental groups, starting with 1 μM and serially diluted 3 times), and incubate at 37°C for 1.5 h.

[0104] (4) Discard the primary antibody, wash the plate 5 times with PBST, pat dry, add horseradish peroxidase (HRP) labeled mouse anti-human IgG secondary antibody, and incubate at 37°C for 1 hour.

[0105] (5) Discard the secondary antibody, wash the plate 5 times with PBST, pat dry, add the chromogenic substrate for color development, add the stop solution after 15 min to stop the reaction, and measure the OD value with an ELISA reader.

[0106] (6) Plot a nonlinear fit graph with OD value as the vertical axis and the logarithm of antibody molar concentration as the horizontal axis.

[0107] The results are as follows Figure 2 As shown, the EC50 value of the antibody is approximately 4.94 nM.

[0108] Example 3: Stability of the anti-PCNA recombinant monoclonal antibody

[0109] The control antibody was prepared according to the method of Example 1: VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region shown in SEQ ID NO.1 of Example 1 and VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region shown in SEQ ID NO.2 were replaced with the corresponding CDRs (HCDR1: VYAFSSSW, HCDR2: IYPADGDT, HCDR3: ARWLRAMDY; LCDR1: QNVGTN, LCDR2: SAS, LCDR3: QQYNSYPYT) in CN110300761B (application number CN201780086509.4, invention name: anti-PCNA monoclonal antibody and its use) to obtain the control antibody. The control antibody differs from the antibody prepared in Example 1 only in the CDR region.

[0110] The PCNA humanized antibody and control antibody from Example 1 were diluted to 2 mg / mL, and then diluted 2000 times with this concentration as the initial concentration. After being placed at -80℃, 4℃ and 37℃ for 7 days, they were detected by the fully automated multiplex immunoassay analyzer of Zhuhai Lizhu Reagent Co., Ltd.

[0111] The testing process is as follows:

[0112] Step 1: The fully automated multiplex immunoassay analyzer aspirates 20 μL of sample and automatically dilutes it 15 times.

[0113] Step 2: Take 15 μL of the diluted sample, add 100 μL of PCNA antigen-conjugated magnetic barcode, incubate at 37°C for 15 min, then perform magnetic separation and wash 3 times.

[0114] Step 3: Add 50 μL of phycoerythrin-labeled mouse anti-human IgG antibody, incubate at 37°C for 15 min, then perform magnetic separation and wash 3 times.

[0115] Step 4: The instrument automatically identifies each magnetic barcode and detects the fluorescence intensity of the corresponding complex, then automatically converts it into an antibody index. The test results are shown in Table 2.

[0116] Table 2

[0117]

[0118] In Table 2, PCNA-Ab01 is the antibody prepared in Example 1, and PCNA-Ab02 is the control antibody.

[0119] As shown in Table 2, the antibody prepared in Example 1, diluted 2000 times and treated at 4°C for 7 days, retained 100% of its signal. The control antibody, however, showed a 14% decrease. After treatment at 37°C for 7 days, the antibody prepared in Example 1 retained nearly 93% of its signal, while the control antibody showed a 31% decrease. Overall, the antibody prepared in this invention exhibits significantly better stability than the control antibody.

[0120] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-PCNA antibody or its antigen-binding fragment, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the variable region of the light chain includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, and VH-CDR3 are amino acid sequences identical to those of the VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.1; the VL-CDR1, VL-CDR2, and VL-CDR3 are amino acid sequences identical to those of the VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable region shown in SEQ ID NO.

2. The variable regions VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the anti-PCNA antibody or its antigen-binding fragment are defined by any one of the systems Kabat, Chothia, IMGT, ABM, or Contact.

2. The anti-PCNA antibody or its antigen-binding fragment according to claim 1, characterized in that, According to the IMGT definition: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19, the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is ATS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

25.

3. The anti-PCNA antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-PCNA antibody or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

4. The anti-PCNA antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The anti-PCNA antibody or its antigen-binding fragment is one of the following: intact antibody, F(ab')2, Fab', Fab, Fv, scFv, or dsFv.

5. The anti-PCNA antibody or its antigen-binding fragment according to claim 4, characterized in that, The species from which the backbone region of the anti-PCNA antibody or its antigen-binding fragment is derived include one or more of the following: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

6. The anti-PCNA antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, It also includes constant regions.

7. The anti-PCNA antibody or its antigen-binding fragment according to claim 6, characterized in that, At least a portion of the constant region sequence is a human constant region sequence.

8. The anti-PCNA antibody or its antigen-binding fragment according to claim 6, characterized in that, The constant region sequence is selected from the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

9. The anti-PCNA antibody or its antigen-binding fragment according to claim 8, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

3.

10. The anti-PCNA antibody or its antigen-binding fragment according to claim 6, characterized in that, The anti-PCNA antibody or its antigen-binding fragment contains a light chain constant region, the sequence of which is selected from the light chain constant region of a mouse.

11. The anti-PCNA antibody or its antigen-binding fragment according to claim 10, characterized in that, The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.

4.

12. The anti-PCNA antibody or its antigen-binding fragment according to claim 10, characterized in that, The heavy chain amino acid sequence of the anti-PCNA antibody or its antigen-binding fragment is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.

6.

13. A biomaterial relating to the anti-PCNA antibody or its antigen-binding fragment as described in any one of claims 1-12, characterized in that, Including polynucleotides, carriers, or cells; The polynucleotide encodes the anti-PCNA antibody or its antigen-binding fragment as described in any one of claims 1 to 12; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the anti-PCNA antibody or its antigen-binding fragment as described in any one of claims 1 to 12.

14. The method for preparing the anti-PCNA antibody or its antigen-binding fragment according to any one of claims 1 to 12, characterized in that, This includes culturing the cells as described in claim 13.

15. The preparation method according to claim 14, characterized in that, The cells are prepared by converting cells with a polynucleotide encoding an anti-PCNA antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 12, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

16. The preparation method according to claim 15, characterized in that, The cells in question are eukaryotic cells.

17. The preparation method according to claim 16, characterized in that, The cells are mammalian cells; 18. The preparation method according to claim 17, characterized in that, The mammalian cells include 293 cells or CHO cells.

19. The preparation method according to claim 18, characterized in that, The mammalian cells in question are 293F cells.

20. The use of the anti-PCNA antibody or its antigen-binding fragment according to any one of claims 1 to 12, or the biomaterial according to claim 13, in any one of the following (I) to (VI): (I) Detection of PCNA antibodies for non-diagnostic and non-therapeutic purposes; (II) Preparation of products for detecting PCNA antibodies; (III) To prepare products for the auxiliary diagnosis of PCNA antibody-positive diseases; (IV) PCNA testing for non-diagnostic and non-treatment purposes; (V) Prepare products for detecting PCNA; (VI) Used for purifying PCNA; (VII) Prepare products for purifying PCNA; The PCNA antibody-positive disease is systemic lupus erythematosus.

21. A reagent or kit, characterized in that, The reagent or kit contains the anti-PCNA antibody or its antigen-binding fragment as described in any one of claims 1 to 12, or the biological material as described in claim 13.

22. The reagent or kit according to claim 21, characterized in that, The kit is used to detect PCNA antibodies; the kit includes standards and / or quality control products, the standards and / or quality control products containing the anti-PCNA antibody or its antigen-binding fragment.

23. The kit or reagent kit according to claim 22, characterized in that, The kit also includes a PCNA antibody detection reagent.

24. The kit or reagent kit according to claim 23, characterized in that, The kit also includes detection reagents for antibodies against at least one of the following substances: dsDNA, nucleosomes, histones, Sm, ribosomal P protein, Scl-70, SSA / Ro52, SSA / Ro60, CENP-B, AMA M2, ssb, Jo-1, PM-Scl, Mi-2, Ku, and RNP / sm.

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