U1-70k binding proteins, methods of making and uses

Abstract

The application provides a U1-70K binding protein, a preparation method and application, and relates to the technical field of biotechnology.The U1-70K 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 includes 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 includes 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 U1-70K, and can be used for identifying anti-U1-70K antibodies or U1-70K antigens, and assisting in diagnosing and detecting anti-U1-70K antibody positive diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a U1-70K 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] U1-70K belongs to the small ribonucleoprotein family and is composed of 437 amino acid residues. It is a key protein in the U1 small ribonucleoprotein (U1snRNP). The actual molecular weight of U1-70K is approximately 52 kDa, but due to the presence of a highly charged repetitive sequence of arginine-(glutamate / aspartic acid) and arginine-serine residues at its C-terminus, its migration rate in SDS-PAGE is similar to that of 70 kDa, hence the name U1-70K. Between amino acid residues 103 and 180 of the U1-70K protein is a single-copy RNP recognition motif, also known as the RNA binding domain (RBD). This is a common domain in eukaryotes and is closely related to post-transcriptional gene expression of mRNA and rRNA, RNA export, and RNA stability. Meanwhile, U1-70K can regulate the splicing of mRNA precursors by binding to stem-loop I of U1snRNA (U1 small nuclear RNA) through its RNA domain. This may be because U1-70K is involved in the formation and alternative splicing of mRNA precursors. This protein can directly interact with splicing factors (ASF / SF2 and SC-35) of the SR family that have alternative splicing sites.

[0004] Abnormal expression or dysfunction of the U1-70K protein may be associated with the development of certain diseases, such as autoimmune diseases. In some autoimmune diseases, such as mixed connective tissue disease (MCTD), autoantibodies against U1-snRNP can be detected at high titers. Therefore, high titers of anti-U1-snRNP antibodies are an important serological basis for diagnosing MCTD. U1-70K is the primary target antigen recognized by anti-U1-snRNP antibodies, and studies have shown that almost 100% of MCTD patients develop anti-U1-70K antibodies in the early stages.

[0005] Therefore, the development of high-affinity anti-U1-70K monoclonal antibodies is of great significance and value for the development of clinical diagnostic 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 U1-70K binding protein that exhibits good specific binding ability to U1-70K. Based on the U1-70K binding protein provided by this invention, another objective is to provide its applications. A further objective of this invention is to develop a U1-70K binding protein with good binding activity using a simpler method, which can be used for the identification of U1-70K antigens and to assist in the diagnosis or auxiliary diagnosis of diseases positive for anti-U1-snRNP antibodies.

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

[0009] Definition of noun:

[0010] In this paper, U1-70K (U1 small nuclear ribonucleoprotein-70K) is a component of the U1 small ribonucleoprotein (U1-snRNP) complex, consisting of 437 amino acid residues. Its migration rate in SDS-PAGE is about 70 KD, hence the name U1-70K.

[0011] The U1-70K binding protein provided by this invention can specifically bind to U1-70K. The U1-70K bound by the U1-70K binding protein includes U1-70K expressed naturally in cells or expressed in cells transfected with the U1-70K gene, as well as U1 small ribonucleoprotein (U1-snRNP) containing U1-70K. The U1-70K bound by the U1-70K binding protein also includes artificially modified U1-70K, 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 article, "anti-U1-70K antibody" refers to an antibody that can specifically bind to U1-70K.

[0013] 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."

[0014] 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.

[0015] 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.

[0016] 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.

[0017] This invention does not limit the method of obtaining the U1-70K binding protein. In some optional embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the U1-70K 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 U1-70K binding protein. In other optional 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.

[0018] 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 concentration 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.

[0019] 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 U1-70K 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.

[0020] 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.

[0021] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, episome plasmids, microcircular DNA, phage particles, and Cosmids; 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.).

[0022] 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.

[0023] 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).

[0024] In this article, "anti-U1-70K antibody positive disease" refers to a disease that can cause patients to have higher levels of anti-U1-70K antibodies and / or anti-U1-snRNP antibodies than before they have the disease. Exemplary anti-U1-70K antibody positive diseases include, but are not limited to, one or more of mixed connective tissue disease, systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, Sjögren's syndrome, and dermatomyositis.

[0025] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbers (Ⅰ), (Ⅱ)...(Ⅶ).

[0026] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0027] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0028] Firstly, a U1-70K binding protein is provided, which includes a heavy chain variable region and a light chain variable region.

[0029] The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3.

[0030] The VH-CDR1, VH-CDR2, and VH-CDR3 comprise 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 comprise 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.

[0031] 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 U1-70K binding protein provided by this invention. For example, if the U1-70K 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 in the heavy chain variable region of SEQ ID NO.1 and the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region of SEQ ID NO.2 are shown in Table 1:

[0032] Table 1

[0033]

[0034] In an optional embodiment, the U1-70K 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 STS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.25.

[0035] 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.

[0036] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the U1-70K 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.

[0037] In an optional implementation, the U1-70K binding protein is an antibody or antigen-binding fragment including a constant region.

[0038] In an optional implementation, at least a portion of the constant region sequence of the U1-70K binding protein is a human constant region sequence.

[0039] In an optional embodiment, the constant region sequence of the U1-70K binding protein is selected from the sequence of part or all of the constant regions 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.

[0040] In an optional embodiment, the U1-70K binding protein contains a heavy chain constant region.

[0041] In an optional embodiment, the heavy chain constant region sequence of the U1-70K 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.

[0042] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3.

[0043] In an optional embodiment, the U1-70K binding protein contains a light chain constant region.

[0044] In an optional implementation, the light chain constant region sequence is selected from the light chain constant region of a mouse.

[0045] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4.

[0046] In an optional embodiment, the U1-70K binding protein is a human-mouse chimeric antibody, and the heavy chain amino acid sequence of the U1-70K binding protein is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0047] In a second aspect, a biological material is also provided, comprising a polynucleotide, a carrier, or a cell; wherein the polynucleotide encodes the aforementioned U1-70K binding protein; the carrier carries the polynucleotide; and the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the U1-70K binding protein.

[0048] By linking the vector with a polynucleotide encoding the U1-70K binding protein, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line that can express the U1-70K binding protein, and the corresponding protein can be obtained through cell expression.

[0049] In an optional embodiment, the cells used to express the U1-70K binding protein are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0050] In an optional implementation, the cells used to express the U1-70K binding protein are CHO cells (Chinese hamster ovary cells).

[0051] Thirdly, a method for preparing the U1-70K binding protein of the first aspect is also provided, including culturing cells capable of expressing the U1-70K binding protein.

[0052] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the U1-70K binding protein into cells, and obtaining the U1-70K binding protein through purification.

[0053] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the U1-70K 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 U1-70K binding protein through purification.

[0054] In an optional embodiment, the cell is prepared by converting a polynucleotide encoding a U1-70K 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.

[0055] 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.

[0056] In an optional embodiment, the constant region segment includes one or more of CH1, CH2 and CH3, preferably including CH1, CH2 and CH3.

[0057] In an optional embodiment, the cell is a eukaryotic cell, preferably a mammalian cell.

[0058] In an optional embodiment, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

[0059] Fourthly, the application of the U1-70K binding protein of the first aspect or the biomaterial of the second aspect in any of the following (I) to (VII) is also provided:

[0060] (I) Testing for anti-U1-70K antibodies or anti-U1-snRNP antibodies for non-diagnostic and non-therapeutic purposes;

[0061] (II) Prepare products for detecting anti-U1-70K antibody or anti-U1-snRNP antibody;

[0062] (III) Prepare products for the diagnosis and / or auxiliary diagnosis of diseases positive for anti-U1-70K antibodies or anti-U1-snRNP antibodies;

[0063] (IV) Non-diagnostic and treatment-oriented testing of U1-70K;

[0064] (V) Prepare products for the detection of U1-70K;

[0065] (VI) is used to purify U1-70K;

[0066] (VII) Prepare products for purifying U1-70K.

[0067] In the applications of (I) to (III) above, U1-70K binding protein can be used as a standard and / or quality control material for detection to provide a reliable reference for the detection results, and can also be used to construct a standard curve.

[0068] In an optional embodiment, the anti-U1-70K antibody positive disease or the anti-U1-snRNP antibody positive disease independently includes one or more of mixed connective tissue disease and systemic lupus erythematosus.

[0069] The application described in aspect (IV) above can utilize the ability of the U1-70K binding protein to specifically target and bind to U1-70K, enabling the detection of U1-70K, U1-70K-expressing cells, or U1-snRNPs based on immunoassay techniques. For example, when the U1-70K binding protein is an immunoconjugate, such as one linked to a fluorescent group, a fluorescence detection device can be used to locate or detect U1-70K in real time. It can be used in techniques such as immunoblotting, immunoprecipitation, or flow cytometry that involve the specific binding properties of U1-70K antigens and antibodies to detect U1-70K. Based on the specific targeting and binding ability of the U1-70K binding protein to U1-70K, it can also be used for the purification of U1-70K as described in aspect (VI).

[0070] 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.

[0071] Fifthly, a reagent or kit is also provided, the reagent or kit comprising the U1-70K binding protein of the first aspect, or the biological material of the second aspect.

[0072] In an optional embodiment, the kit is used to detect anti-U1-70K antibody, anti-U1-snRNP antibody, anti-U1-70K antibody-positive disease, or anti-U1-snRNP antibody-positive disease; the kit includes standards and / or quality control products containing the U1-70K binding protein.

[0073] In an optional implementation, the kit is used to detect one or more of mixed connective tissue disease and systemic lupus erythematosus.

[0074] In an optional embodiment, the kit further includes detection reagents, which include detection reagents for anti-U1-70K antibody or anti-U1-snRNP antibody.

[0075] In an optional embodiment, the kit further includes at least one of the following components for detecting autoantibodies: dsDNA, nucleosomes, histones, Sm, ribosomal P protein, Scl-70, SSA / Ro52, SSA / Ro60, CENP-B, AMA M2, ssb, Jo-1, PM-Scl, Mi-2, and PCNA.

[0076] In an optional embodiment, the kit further includes a solid support.

[0077] In optional embodiments, the U1-70K binding protein in the reagent or kit is coupled to a solid-phase support; or the U1-70K binding protein in the reagent or kit and the solid-phase support are packaged separately. By coupling the U1-70K binding protein to the solid-phase support, it can be used to capture the U1-70K protein in the sample to be tested. Alternatively, by coupling the U1-70K binding protein to the solid-phase support, it can be used to purify the U1-70K protein.

[0078] In an optional embodiment, the anti-U1-70K antibody detection reagent includes a solid-phase carrier coupled with the U1-70K antigen.

[0079] In an optional implementation, the kit may further include a tracer marker.

[0080] In optional embodiments, the U1-70K binding protein in the reagent or kit is coupled to a tracer label; or the U1-70K binding protein and the tracer label in the reagent or kit are packaged separately. By coupling the U1-70K binding protein to the tracer label, it can be used for localized detection of U1-70K protein or for detecting U1-70K protein in a sample via Western blotting.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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).

[0085] 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.

[0086] This invention discloses a U1-70K binding protein that specifically recognizes and binds to the U1-70K antigen, and further prepares a human-mouse chimeric recombinant monoclonal antibody. This invention has the following beneficial effects:

[0087] (1) This method prepares U1-70K binding proteins with known sequences, which can be recombinantly expressed in vitro. It has the advantages of simple operation, short time consumption, controllable production process, small batch-to-batch variation of products, and good stability, and has good application prospects.

[0088] (2) The U1-70K binding protein can be coupled to a chromatographic matrix for immunoaffinity chromatography purification of the U1-70K antigen, thereby improving protein purity. The U1-70K binding protein can be coupled to a label for the detection of the U1-70K antigen, including but not limited to the use of liquid chromatography-mass spectrometry, immunofluorescence staining, flow cytometry, fluorescent microspheres, and other techniques to identify the U1-70K antigen.

[0089] (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

[0090] 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.

[0091] Figure 1 This is an SDS-PAGE protein electrophoresis image of the anti-U1-70K recombinant monoclonal antibody in Example 1. M represents the marker. "Reduced" indicates the reduced SDS-PAGE electrophoresis result of the U1-70K recombinant antibody, with heavy and light chains of 50 kDa and 25 kDa, respectively. "Non-reduced" indicates the non-reduced SDS-PAGE electrophoresis result of the U1-70K recombinant antibody, with a size of approximately 150 kDa.

[0092] Figure 2 The results of the ELISA assay for the binding of recombinant monoclonal antibody to U1-70K protein in Example 2 are shown. Ab-U1-70K represents the binding activity of the recombinant monoclonal antibody to the U1-70K antigen, and Ab-BSA represents the non-specific binding activity of the recombinant monoclonal antibody to the control protein BSA. Detailed Implementation

[0093] 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.

[0094] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0095] Example 1: Preparation of U1-70K chimeric antibody

[0096] A U1-70K-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.

[0097] Heavy chain variable region (SEQ ID NO.1)

[0098] QMQLQESGPAVIKPSQSLSLTCIVSGFSITSSSYCWHWIRQPPGKGLEWMGRICYEGSIYYSPSI KRSTISRDTSLNKFFIQLNSVTDEDTAMYYCSREDQGWYFDVWGAGTPVTVSS

[0099] Light chain variable region (SEQ ID NO.2)

[0100] DNVLTQSPAIMSASPGEKVTITCSASSSVSYIHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSG SGTSYSLTISRMEAEDAATYYCQQRTSYPFTFGSGTKLEIK

[0101] Table 1

[0102]

[0103] Nucleic acid fragments containing complete light chain and heavy chain genes were synthesized and inserted into the PTT5 vector, respectively. These fragments were then transformed into competent cells, and bacterial cultures were conducted 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.

[0104] 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 0.1 M Glycine (pH 3.0) and neutralized with 1 M 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.

[0105] Example 2: ELISA determination of the binding activity of recombinant monoclonal antibody to U1-70K antigen.

[0106] (1) Coat the U1-70K 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.

[0107] (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.

[0108] (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.

[0109] (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.

[0110] (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.

[0111] (6) Plot a nonlinear fit graph with OD value as the vertical axis and the logarithm of antibody molar concentration as the horizontal axis.

[0112] The results are as follows Figure 2 As shown, the EC50 value of the antibody is approximately 0.47 nM.

[0113] Example 3: Specificity of the recombinant monoclonal antibody against U1-70K

[0114] The humanized U1-70K antibody prepared in Example 1 was reacted with magnetic barcodes conjugated with U1-A antigen, U1-C antigen, and U1-70K antigen, respectively, and detected by a fully automated multiplex immunoassay analyzer from Zhuhai Lizhu Reagent Co., Ltd.

[0115] The testing process is as follows:

[0116] Step 1: The fully automated multiplex immunoassay analyzer aspirates 20 μL of sample and dilutes it to 2 μg / mL with sample diluent.

[0117] Step 2: Take 15 μL of the diluted sample, add 100 μL of antigen-conjugated magnetic barcode, incubate at 37°C for 15 min, then perform magnetic separation and wash 3 times.

[0118] 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.

[0119] 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.

[0120] Table 2 Antibody Specificity Experiment

[0121]

[0122] As can be seen from the data in Table 2, the humanized U1-70K antibody specifically binds to U1-70K and has no cross-reaction with U1-C and U1-A, indicating that the antibody has excellent specificity.

[0123] Example 4: Stability of the recombinant monoclonal antibody against U1-70K

[0124] The humanized U1-70K antibody prepared in Example 1 was diluted to 2 mg / mL, and then diluted 2000 times using this concentration as the initial concentration. After being placed at -80℃, 4℃, and 37℃ for 7 days, the antibody was reacted with magnetic barcodes conjugated with the U1-70K antigen. The results were then detected using a fully automated multiplex immunoassay analyzer from Zhuhai Lizhu Reagent Co., Ltd. (the procedure is the same as in Example 3). The test results are shown in Table 3.

[0125] Table 3. Signal retention rate after antibody thermal acceleration

[0126]

[0127] As shown in Table 3, after dilution 2000 times and treatment at 4°C for 7 days, the signal retention rate of the antibody of the present invention was 100%. After treatment at 37°C for 7 days, the signal retention rate of the antibody of the present invention was 89.7%. Overall, the antibody of the present invention exhibits good stability.

[0128] 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. A U1-70K binding protein, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, and VH-CDR3 comprise 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 comprise 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.

2. The U1-70K binding protein 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, 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 STS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

25.

3. The U1-70K binding protein according to claim 1, characterized in that, The heavy chain variable region further includes a backbone region, and / or the light chain variable region further includes a backbone region.

4. The U1-70K binding protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the U1-70K 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.

5. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, The U1-70K binding protein is an intact antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, or a bispecific antibody.

6. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, The antibody sequence, excluding the CDR region, is derived from one or more species including mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys, and humans.

7. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, It also includes constant regions.

8. The U1-70K binding protein according to claim 7, characterized in that, At least a portion of the constant region sequence is a human constant region sequence.

9. The U1-70K binding protein according to claim 7, characterized in that, The constant region sequence is selected from a portion or all of the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

10. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, The U1-70K binding protein contains a heavy chain constant region, and the sequence of the heavy chain constant region is selected from the human heavy chain constant region. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

3.

11. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, The U1-70K binding protein contains a light chain constant region, the sequence of which is selected from the light chain constant region of mice; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.

4.

12. The U1-70K binding protein according to any one of claims 1 to 4, characterized in that, The U1-70K binding protein is an intact antibody, with the heavy chain amino acid sequence shown in SEQ ID NO.5 and the light chain amino acid sequence shown in SEQ ID NO.

6.

13. A biomaterial, characterized in that, Including polynucleotides, carriers, or cells; The polynucleotide encodes the U1-70K binding protein according to 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 U1-70K binding protein as described in any one of claims 1 to 12.

14. The method for preparing the U1-70K binding protein 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 cell is prepared by converting a polynucleotide encoding a U1-70K binding protein as described in any one of claims 1 to 12 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.

16. The preparation method according to claim 14, characterized in that, The cells in question are eukaryotic cells.

17. The preparation method according to claim 14, characterized in that, The cells in question 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 17, characterized in that, The mammalian cells in question are 293F cells.

20. The use of the U1-70K binding protein according to any one of claims 1 to 12, or the biomaterial according to claim 13, in any one of the following (I) to (VII): (I) Testing for anti-U1-70K antibodies or anti-U1-snRNP antibodies for non-diagnostic and non-therapeutic purposes; (II) Prepare products for detecting anti-U1-70K antibody or anti-U1-snRNP antibody; (III) To prepare products for the diagnosis and / or auxiliary diagnosis of diseases positive for anti-U1-70K antibodies or anti-U1-snRNP antibodies; (IV) Non-diagnostic and treatment-oriented testing of U1-70K; (V) Prepare products for the detection of U1-70K; (VI) Used for purifying U1-70K; (VII) Prepare products for purifying U1-70K.

21. The application according to claim 20, characterized in that, The anti-U1-70K antibody positive disease or the anti-U1-snRNP antibody positive disease independently includes one or more of mixed connective tissue disease and systemic lupus erythematosus.

22. The application according to claim 20, characterized in that, In any of (I) to (III), the U1-70K binding protein is used as a standard or quality control product.

23. A reagent or kit, characterized in that, The reagent or kit comprises the U1-70K binding protein as described in any one of claims 1 to 12, or the biological material as described in claim 13.

24. The reagent or kit according to claim 23, characterized in that, The kit is used to detect anti-U1-70K antibody, anti-U1-snRNP antibody, anti-U1-70K antibody-positive disease, or anti-U1-snRNP antibody-positive disease; the kit includes standards and / or quality control products containing the U1-70K binding protein.

25. The reagent or kit according to claim 24, characterized in that, The kit also includes a detection reagent for anti-U1-70K antibody or anti-U1-snRNP antibody.

26. The reagent or kit according to claim 24, characterized in that, The kit also includes a detection reagent for detecting autoantibodies against at least one of the following components: dsDNA, nucleosomes, histones, Sm, ribosomal P protein, Scl-70, SSA / Ro52, SSA / Ro60, CENP-B, AMA M2, ssb, Jo-1, PM-Scl, Mi-2, and PCNA.

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