U1-70K binding protein, preparation method and application

Abstract

The invention provides a U1-70K binding protein as well as a preparation method and application thereof, and relates to the technical field of biology. 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, wherein the complementarity determining region of the heavy chain variable region comprises amino acid sequences consistent with VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region as shown in SEQ ID NO.1; the complementarity determining region of the light chain variable region comprises amino acid sequences which are consistent with the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region as shown in SEQ ID NO.2. The binding protein has good specific binding capacity with U1-70K, and can be used for identification of an anti-U1-70K antibody or a U1-70K antigen and auxiliary diagnosis and detection of positive diseases of the anti-U1-70K antibody.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a U1-70K binding protein, a preparation method and an application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] U1-70K belongs to the small nuclear ribonucleoprotein family and is composed of 437 amino acid residues. It is a key protein in U1 small nuclear ribonucleoprotein (U1snRNP). The actual molecular weight of U1-70K is about 52KD, but because its C-terminal tail contains a highly charged repeating sequence structure composed of arginine-(glutamic acid / aspartic acid) and arginine-serine, its migration speed in SDS-PAGE is similar to that of 70KD, so it is called U1-70K. There is a single copy of the RNP recognition motif between the 103rd and 180th amino acid residues of the U1-70K protein, also known as the RNA Binding Domain (RBD), which is a domain that often exists in eukaryotes and is closely related to the post-transcriptional gene expression of mRNA and rRNA, RNA output, and RNA stability. At the same time, U1-70K can bind to the stem-loop I of U1snRNA (U1 small nuclear RNA) through the RNA domain to regulate the splicing of mRNA precursors. This may be because U1-70K is related to the composition and selective splicing of mRNA precursors, and the protein can directly interact with splicing factors (ASF / SF2 and SC-35) of the SR family with selective splicing sites.

[0004] Abnormal expression or dysfunction of U1-70K protein may be related to the occurrence 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, and their titers are very high. Therefore, high titer anti-U1-snRNP antibodies are an important serological basis for the diagnosis of MCTD. U1-70K is the most important target antigen recognized by anti-U1-snRNP antibodies. Studies have shown that almost 100% of MCTD patients will 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 detection kits.

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

[0007] The object of the present invention is to provide a U1-70K binding protein, which has good specific binding ability with U1-70K. Based on the U1-70K binding protein provided by the present invention, the object of the present invention is also to provide its application. Another object of the present invention is to develop a U1-70K binding protein with good binding activity by a simpler method, which can be used for the identification of U1-70K antigens and assist in the diagnosis or auxiliary diagnosis of anti-U1-snRNP antibody positive diseases.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] Definition of terms:

[0010] In this article, U1-70K (U1 small nuclear ribonucleoprotein-70K) is a component of the U1 small nuclear ribonucleoprotein (U1-snRNP) complex, composed of 437 amino acid residues. Its migration speed in SDS-PAGE is about 70KD, so it is called U1-70K.

[0011] The U1-70K binding protein provided by the present invention can specifically bind to U1-70K, and the U1-70K bound by the U1-70K binding protein includes U1-70K naturally expressed by cells or expressed by cells transfected with the U1-70K gene, and U1 small nuclear ribonucleoprotein (U1-snRNP) containing U1-70K. The U1-70K bound by the U1-70K binding protein also includes artificially modified U1-70K, and the artificial modification includes but is not limited to a polypeptide or protein after mutation, truncation or fusion with other domains, and retains the necessary antigenic epitope for binding to the antibody.

[0012] The "anti-U1-70K antibody" herein refers to an antibody that can specifically bind to U1-70K.

[0013] The technical term "binding protein" herein refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments that contain a complementary determining region (CDR region). The proteins and protein fragments may be antibodies, and the terms "antibody" and "full-length antibody" include polyclonal antibodies and monoclonal antibodies. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the term "antibody" can be used interchangeably with "immunoglobulin."

[0014] Proteins and protein fragments can also be antigen-binding fragments that contain part or all of the antibody CDRs, which lack at least some of the amino acids present in the full-length chain but are still able to specifically bind to the antigen. 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. Such fragments are selected from, but not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody, VH and VL are connected by a connecting peptide), dsFv (disulfide stabilized Fv fragments, dsFv), bispecific antibodies, nanobodies, and any one of the minimum recognition units of antibodies. In addition to the above functional fragments, any fragments with increased half-life are also included.

[0015] The "variable region" or "variable domain" of a binding protein refers to the domain of the amino terminus of the heavy chain or light chain of an antibody that recognizes and binds to an antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable domain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDR) (also known as hypervariable regions) connected by four framework regions (FR). The extent of the framework region and CDR 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 a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. Generally, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0016] The term "constant region" or "constant domain" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination. The heavy chain of an antibody has one variable domain (VH), followed by a number of constant domains or regions, such as one or more of a hinge, CH1, CH2, CH3, and CH4, the CH1 domain being adjacent to the VH domain and at the amino terminus of the hinge region of the antibody heavy chain, and not forming part of the Fc region of the antibody; the hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is usually the CH3 domain, which usually forms the C-terminal portion of the antibody, and in some antibody types, such as IgM and IgE, the constant region also includes a CH4 domain. The constant region of an antibody can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as subclasses and mutant forms of the constant regions thereof.

[0017] The present invention does not limit the method of obtaining the U1-70K binding protein. In some optional embodiments, the corresponding antibody can be obtained by connecting the polynucleotide encoding the U1-70K binding protein to a vector and expressing it in a cell. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a protein 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 an automatic peptide synthesizer (for example, an automatic peptide synthesizer sold by Applied BioSystems, etc.); the antigen-binding fragment can also be optionally produced by enzymatic cleavage of the antigen-binding molecule (including a complete antibody), such as pepsin or papain cleavage; or produced by chemical cleavage, for example, the above-mentioned antigen-binding fragment is obtained by a method of chemical reduction splitting disulfide bonds.

[0018] The terms "specific recognition", "selective binding", "selectively binds" and "specifically binds" or similar expressions refer to the binding of a binding protein to a predetermined epitope on an antigen. Typically, the binding protein is present at a rate of about less than 10 -5 M, for example, less than about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K d The K value of an antibody can be determined using methods well established in the art. d Other standard assays for evaluating the binding ability of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis.

[0019] The term "polynucleotide" herein refers to a polymer 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 natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the above-mentioned U1-70K binding protein, and the encoding is optionally for encoding the sense strand or the antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0020] The term "vector" herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the cell.

[0021] The vector is well known to those skilled in the art, including but not limited to: plasmid, episomal plasmid, minicircle DNA, phagemid, cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0022] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Progeny may not necessarily be identical to the original cell due to natural, accidental or deliberate mutation, and may differ from the original cell in morphology and / or in genomic DNA. "Transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom.

[0023] As used herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, such as if it is naturally present, from its natural environment. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally associated with it or are normally mixed with it or in solution. Isolated polypeptides include naturally produced polypeptides contained in cell lysates, polypeptides in purified or partially purified form, recombinant polypeptides, polypeptides expressed or secreted by cells, and polypeptides in heterologous cells or cultures. In connection with nucleic acids, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, connected to a promoter, or artificially introduced into a heterologous cell).

[0024] The "anti-U1-70K antibody-positive disease" herein refers to a disease that can cause the patient's anti-U1-70K antibodies and / or anti-U1-snRNP antibodies to be higher than the level when the patient is not suffering from 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, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbers (I), (II)...(VII).

[0026] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0027] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0028] In a first aspect, a U1-70K binding protein is provided, which comprises a heavy chain variable region and a light chain variable region.

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

[0030] The VH-CDR1, VH-CDR2 and VH-CDR3 include amino acid sequences consistent with 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 include amino acid sequences consistent with 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 other regions except the CDR region in the variable region shown in SEQ ID NO.1 or 2 are not used to limit the U1-70K binding protein provided by the present invention. For example, if the U1-70K binding protein provided by the present application contains a framework region, it may be different from the framework 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 can be divided according to an optional method known in the art. Optionally, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the variable region are defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems. Taking the Kabat, Chothia, IMGT, ABM or Contact definitions 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:

[0032] Table 1

[0033]

[0034] In an optional embodiment, the U1-70K binding protein has VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region defined in Table 1, and VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region defined in Table 1. Taking the IMGT definition as an example: the amino acid sequence of the VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of the VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of the VH-CDR3 is shown in SEQ ID NO.19; the amino acid sequence of the VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of the VL-CDR2 is STS, and the amino acid sequence of the VL-CDR3 is shown in SEQ ID NO.25.

[0035] In an optional embodiment, the heavy chain variable region further comprises a framework region, and / or the light chain variable region further comprises a framework region. The species origin of the framework region includes, but is not limited to, one or more of rabbit, cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human and their mutants.

[0036] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the U1-70K binding protein is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2.

[0037] In an alternative embodiment, the U1-70K binding protein is an antibody or antigen binding fragment comprising a constant region.

[0038] In an alternative embodiment, 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 a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, wherein the IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD include their subclasses and mutant forms.

[0040] In an alternative 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 a portion 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 embodiment, 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, 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 the second aspect, a biological material is also provided, including a polynucleotide, a vector or a cell; wherein the polynucleotide encodes the above-mentioned U1-70K binding protein; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector or is capable of expressing the U1-70K binding protein.

[0048] After the polynucleotide encoding the U1-70K binding protein is connected to the vector, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line capable of expressing the U1-70K binding protein, and the corresponding protein can be obtained by 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 alternative embodiment, the cells used to express the U1-70K binding protein are CHO cells (Chinese Hamster Ovary cells).

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

[0052] In an optional embodiment, the preparation method further comprises transforming the polynucleotide encoding the U1-70K binding protein into cells and expressing the polynucleotide, and obtaining the U1-70K binding protein by purification.

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

[0054] In an optional embodiment, the cell is obtained by transforming a polynucleotide encoding the U1-70K binding protein as described in the first aspect into the cell, the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0055] In an optional embodiment, the constant region fragment is fused to the C-terminus of the heavy chain variable region to construct a complete heavy chain expression plasmid.

[0056] In an optional embodiment, the constant region fragment includes one or more of CH1, CH2 and CH3, preferably includes 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] In a fourth aspect, there is also provided a use of the U1-70K binding protein of the first aspect or the biomaterial of the second aspect in any one of the following (I) to (VII):

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

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

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

[0063] (IV) Non-diagnostic and therapeutic destination testing U1-70K;

[0064] (V) preparing products for detecting U1-70K;

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

[0066] (VII) Preparation of products for purification of U1-70K.

[0067] In the applications of aspects (I) to (III) above, the U1-70K binding protein can be used as a standard and / or quality control product during 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 of aspect (IV) above can utilize the ability of U1-70K binding protein to specifically target and bind to U1-70K, and realize the detection of U1-70K, cells expressing U1-70K or U1-snRNP based on immunodetection technology. For example, when U1-70K binding protein is an immunoconjugate, for example, it is connected with a fluorescent group, and a fluorescent detection device can be used to realize the positioning or real-time detection of U1-70K. For example, it can be used for immunoblotting, immunoprecipitation or flow cytometry technology, etc., which involve the use of the specific binding properties of U1-70K antigen and antibody to detect U1-70K. Based on the ability of U1-70K binding protein to specifically target and bind to U1-70K, it can also be used for purifying U1-70K in aspect (VI).

[0070] In an optional embodiment, in the above aspects (II), (III), (V) or (VII), those skilled in the art can prepare corresponding products (such as the above-mentioned immunoconjugates) according to actual uses, and select other reagents in the product, including but not limited to one or more of tracer markers, solid phase carriers, buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality control products and markers.

[0071] In a fifth aspect, a reagent or a kit is also provided, wherein the reagent or the kit comprises 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 antibodies, anti-U1-snRNP antibodies, anti-U1-70K antibody-positive diseases or anti-U1-snRNP antibody-positive diseases; the kit includes standards and / or quality control substances, and the standards and / or the quality control substances contain the U1-70K binding protein.

[0073] In an optional embodiment, 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 comprises a detection reagent, wherein the detection reagent comprises an anti-U1-70K antibody or an anti-U1-snRNP antibody detection reagent.

[0075] In an optional embodiment, the kit further comprises a detection reagent for autoantibodies of at least one of the following components: at least one of 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 comprises a solid phase carrier.

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

[0079] In an optional embodiment, the kit further comprises a tracing marker.

[0080] In an optional embodiment, the U1-70K binding protein in the reagent or kit is coupled with a tracer marker; or the U1-70K binding protein and the tracer marker in the reagent or kit are packaged separately. By coupling the U1-70K binding protein with the tracer marker, it can be used to locate and detect the U1-70K protein or detect the U1-70K protein in a sample by Western blotting.

[0081] The above reagents or kits may also optionally include reagents and / or consumables used in the art for detection reactions or protein purification, which are well known to those skilled in the art, including but not limited to one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality control products and markers.

[0082] The above reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow sorting, immunoblotting, immunohistochemistry, ELISA, immunochromatography or immunomagnetic beads. Those skilled in the art can prepare reagents or other reagents in the kit according to the corresponding detection means, and the present invention is not limited to this.

[0083] The tracer markers in any of the above embodiments include, but are not limited to, one or more of enzymes, luminescent markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals, and photosensitizers.

[0084] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Examples of luminescent markers include, but are not limited to, fluorescent proteins, synthetic small molecules or polymer dyes, and specific examples include, but are not limited to, Alexa350, Alexa 405, Alexa430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY630 / 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, 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 Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyl diamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin One or more of 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. Fluorescent microspheres, colored microspheres and latex microspheres are independently selected from products acceptable in the art, such as commercially available products. Radionuclides include but are not limited to 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 Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), 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 carrier in any of the above embodiments includes but is not limited to microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices or lateral flow devices; more specifically, for example, but 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 an optional embodiment, the chromatography matrix includes gel particles.

[0086] The present 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. The present invention has the following beneficial effects:

[0087] (1) This method prepares U1-70K binding proteins with known sequences, can be recombinantly expressed in vitro, is simple to operate, takes a short time, has controllable production processes, small product batch differences, and good stability, and has good application prospects.

[0088] (2) The U1-70K binding protein can be coupled to a chromatography matrix for immunoaffinity chromatography purification of the U1-70K antigen to improve protein purity. The U1-70K binding protein can be coupled to a marker for detection of the U1-70K antigen, including but not limited to the use of liquid phase chips, immunofluorescence staining, flow sorting, fluorescent microspheres and other technologies to identify the U1-70K antigen.

[0089] (3) As a quality control product in the detection kit, it can alleviate the problems of complicated operation of immune polyclonal antibodies and low subsequent coupling efficiency, reduce production costs, stabilize product quality, and significantly improve the reaction value; on the other hand, compared with the direct use of human serum, it can also avoid the problem of difficult sample sources and high costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0091] Figure 1 The SDS-PAGE protein electrophoresis diagram of the anti-U1-70K recombinant monoclonal antibody in Example 1, M represents Marker, reduced represents the reduced SDS-PAGE electrophoresis result of the U1-70K recombinant antibody, and the heavy chain and light chain are 50 kDa and 25 kDa, respectively, and non-reduced represents the non-reduced SDS-PAGE electrophoresis result of the U1-70K recombinant antibody, and the size is about 150 kDa;

[0092] Figure 2 The ELISA analysis results of the binding between the recombinant monoclonal antibody and the U1-70K protein in Example 2, Ab-U1-70K is the binding activity of the recombinant monoclonal antibody to the U1-70K antigen, and Ab-BSA is the non-specific binding activity of the recombinant monoclonal antibody to the control protein BSA. DETAILED DESCRIPTION

[0093] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0094] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

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

[0096] A U1-70K-specific high-affinity monoclonal strain was screened using phage display technology, and the Fab region sequence was obtained after sequencing. The heavy chain variable region is shown in SEQ ID NO.1, the light chain variable region is shown in SEQ ID NO.2, and the CDR region is shown in Table 1.

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

[0098] QMQLQESGPAVIKPSQSLSLTCIVSGFSITSSSYCWHWIRQPPGKGLEWMGRICYEGSIYYSPSI KSRTISRDTSLNKFFIQLNSVTDEDTAMYYCSREDQGWYFDVWGAGTPVTVSS

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

[0100] DNVLTQSPAIMSASPGEKVTITCSASSSVSYIHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSG SGTSYSLTISRMEAEDAATYYCQQRTSYPFTFGSGTKLEIK

[0101] Table 1

[0102]

[0103] The nucleic acid fragments containing the complete light chain gene and heavy chain gene were synthesized and inserted into the vector PTT5 respectively, and the competent cells were transformed and cultured to obtain the corresponding expression plasmids. Among them, the light chain constant region sequence is shown in SEQ ID NO.4, which is a mouse sequence; the light chain complete 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 sequence, and the heavy chain complete sequence is shown in SEQ ID NO.5.

[0104] The heavy chain expression plasmid and the light chain expression plasmid were mixed with PEI at a molar ratio of 1:2 and transfected into 293F suspension cells in the logarithmic growth phase. The cells were cultured in a shaking incubator at 37°C and 120 rpm. After 5 days, the cell supernatant was collected, the antibody was purified with Protein A, eluted with 0.1M Glycine (pH3.0), and neutralized with 1M Tris (pH8.0). After the elution was completed, the ultrafiltration centrifuge tube was replaced with PBS buffer and concentrated, and the protein concentration was determined. The obtained product was verified by SDS-PAGE, and the results are as follows: Figure 1 shown.

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

[0106] (1) U1-70K protein (2 μg / mL) was coated on an ELISA plate and BSA was coated as a nonspecific binding control and incubated at 4°C overnight.

[0107] (2) Discard the coating solution, wash the plate three times with PBST, pat dry, add 3% milk, and block at 37°C for 2 h.

[0108] (3) The blocking solution was discarded, the plate was washed 3 times with PBST, patted dry, and the antibody prepared in Example 1 was added (multiple concentration gradient experimental groups were set up, starting at 1 μM, and 3-fold gradient dilution), and incubated 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 h.

[0110] (5) Discard the secondary antibody, wash the plate 5 times with PBST, pat dry, add the colorimetric substrate for color development, add the stop solution after 15 min to stop the reaction, and measure the OD value with an enzyme reader.

[0111] (6) A nonlinear fitting plot was performed with the OD value as the ordinate and the logarithm of the antibody molar concentration as the abscissa.

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

[0113] Example 3 Specificity of anti-U1-70K recombinant monoclonal antibody

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

[0115] The detection process is as follows:

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

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

[0119] Step 4: The instrument automatically identifies each magnetic barcode and detects the fluorescence intensity of the corresponding complex, and then automatically converts it into an antibody index. The test results are shown in Table 2.

[0120] Table 2 Antibody specificity experiments

[0121]

[0122] It can be seen from the data in Table 2 that the U1-70K humanized 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 anti-U1-70K recombinant monoclonal antibody

[0124] The U1-70K humanized antibody prepared in Example 1 was diluted to 2 mg / mL, and then diluted 2000 times with this concentration as the initial concentration, and placed at -80°C, 4°C, and 37°C for 7 days, respectively, and then reacted with the magnetic barcode coupled with the U1-70K antigen, and detected by the fully automatic multiple immunoassay analyzer of Zhuhai Lizhu Diagnostics Co., Ltd. (the process refers to Example 3). The test results are shown in Table 3.

[0125] Table 3 Signal retention rate after thermal acceleration of antibodies

[0126]

[0127] It can be seen from Table 3 that after the antibody of the present invention was diluted 2000 times and treated at 4°C for 7 days, the signal retention rate was 100%. After being treated at 37°C for 7 days, the signal retention rate of the antibody of the present invention was 89.7%. In summary, the stability of the antibody of the present invention is good.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. U1-70K binding protein, characterized in that comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes complementary determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region includes complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3; The VH-CDR1, VH-CDR2 and VH-CDR3 include amino acid sequences consistent with 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 include amino acid sequences consistent with 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 The VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the U1-70K binding protein are defined by any one of the Kabat, Chothia, IMGT, ABM or Contact systems or a combination of multiple definition systems; Optionally, according to the IMGT definition: the amino acid sequence of the VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of the VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of the VH-CDR3 is shown in SEQ ID NO.19; The amino acid sequence of the VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of the VL-CDR2 is STS, and the amino acid sequence of the 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 comprises a framework region, and / or the light chain variable region further comprises a framework region; Optionally, the amino acid sequence of the heavy chain variable region of the U1-70K binding protein is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

2.

4. The U1-70K binding protein according to any one of claims 1 to 3, characterized in that The U1-70K binding protein is a complete antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody or antibody minimum recognition unit; Optionally, the species from which the remaining sequences of the antibody minus the CDR region originate include one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans.

5. The U1-70K binding protein according to any one of claims 1 to 3, characterized in that It also contains a constant region; Optionally, at least a portion of the constant region sequence is a human constant region sequence; Optionally, the constant region sequence is selected from a sequence of a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the U1-70K binding protein contains a heavy chain constant region, and the heavy chain constant region sequence is selected from a human heavy chain constant region; Optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3; Optionally, the U1-70K binding protein contains a light chain constant region, and the light chain constant region sequence is selected from a mouse light chain constant region; Optionally, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4; Optionally, the U1-70K binding protein is a complete antibody, the heavy chain amino acid sequence is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.

6.

6. Biomaterial, characterized in that including a polynucleotide, a vector or a cell; The polynucleotide encodes the U1-70K binding protein according to any one of claims 1 to 5; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the U1-70K binding protein according to any one of claims 1 to 5.

7. The method for preparing the U1-70K binding protein according to any one of claims 1 to 5, characterized in that: Comprising culturing the cell as claimed in claim 6; Optionally, the cell is obtained by transforming a polynucleotide encoding the U1-70K binding protein according to any one of claims 1 to 5 into a cell, wherein the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell; Optionally, the cell is a eukaryotic cell, preferably a mammalian cell; Optionally, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

8. Use of the U1-70K binding protein according to any one of claims 1 to 5, or the biomaterial according to claim 6 in any one of the following (I) to (VII): (I) Detection of anti-U1-70K antibodies or anti-U1-snRNP antibodies for non-diagnostic and therapeutic purposes; (II) preparing products for detecting anti-U1-70K antibodies or anti-U1-snRNP antibodies; (III) Preparation of products for diagnosis and / or auxiliary diagnosis of anti-U1-70K antibody positive diseases or anti-U1-snRNP antibody positive diseases; (IV) Non-diagnostic and therapeutic destination testing U1-70K; (V) preparing products for detecting U1-70K; (VI) used to purify U1-70K; (VII) preparing a product for purification of U1-70K; Optionally, 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; Optionally, in any one of (I) to (III), the U1-70K binding protein is used as a standard or quality control product.

9. A reagent or a kit, characterized in that The reagent or kit comprises the U1-70K binding protein according to any one of claims 1 to 5, or the biological material according to claim 6.

10. The reagent or kit according to claim 9, characterized in that The kit is used for detecting anti-U1-70K antibodies, anti-U1-snRNP antibodies, anti-U1-70K antibody-positive diseases or anti-U1-snRNP antibody-positive diseases; the kit comprises a standard substance and / or a quality control substance, and the standard substance and / or the quality control substance contain the U1-70K binding protein; Optionally, the kit further comprises an anti-U1-70K antibody or an anti-U1-snRNP antibody detection reagent; Optionally, the kit further comprises a detection reagent for detecting autoantibodies to at least one of the following components: at least one of 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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