Recombinant antigen, reagent containing same, and use

Abstract

Provided are a recombinant antigen, a kit containing same, and a use, belonging to the technical field of immunodiagnostics. Specifically, the present invention relates to a recombinant antigen containing rraB or MysB as a fusion partner, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a labeling conjugate, a kit, and a use. Using the recombinant antigen for immunodiagnosis can significantly improve the activity and specificity of immunodiagnosis.

Description

Recombinant antigens, reagents containing same and uses thereof

[0001] Priority information

[0002] This application claims priority to and the benefit of Chinese Patent Application No. 202410895391.1, filed on July 4, 2024, and is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure belongs to the field of immunodiagnostic technology, and relates to a recombinant antigen containing a specific fusion partner, a reagent containing same and uses thereof; in particular, the present disclosure relates to a recombinant antigen, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a label conjugate, a kit, a detection method and uses thereof. BACKGROUND

[0004] In many immunodiagnostic applications, recombinantly produced recombinant antigens containing fusion partners are used as binding partners, for example as antigens in immunoassays designed to detect specific immunoglobulin analytes. Part of the recombinant antigen structure is a polypeptide of interest or antigenic polypeptide as an antigenic part, which is intended to recognize and bind to a specific analyte present in the sample being tested; another part of the recombinant antigen structure is a fusion partner, which is fused to the antigenic part conferring specificity, to facilitate its cloning, expression, overproduction, folding / re-folding and purification, and to facilitate increasing its solubility, its stability or its folding reversibility.

[0005] Immunochromatographic technology is a detection technology based on antigen-antibody specific binding reaction, and its basic principle is to fix specific antigen or antibody on a nitrocellulose membrane, and through chromatography, the antibody or antigen in the sample to be detected is combined with the fixed antigen or antibody to form a visible specific band, thereby realizing rapid detection of the antigen or antibody. At present, many research institutions and enterprises have developed infectious disease detection kits based on immunochromatographic technology, such as hepatitis C or syphilis detection kits. However, there are still some deficiencies in the detection process of immunochromatographic technology, such as low sensitivity, weak specificity, and inability to be directly labeled. SUMMARY

[0006] The inventors have surprisingly found that using rraB or MysB as a fusion partner can greatly improve the detection performance of the recombinant antigen as a diagnostic reagent, which is superior to existing fusion partners.

[0007] The first aspect of the present application provides a recombinant antigen, wherein the recombinant antigen contains at least one polypeptide of interest and at least one rraB or MysB as a fusion partner.

[0008] The second aspect of the present application provides a labeled conjugate, wherein the labeled conjugate comprises the recombinant antigen as described above, and a tracer label coupled to the recombinant antigen.

[0009] The third aspect of the present application provides a kit, wherein the kit comprises the recombinant antigen as described above, or the labeled conjugate as described above.

[0010] The fourth aspect of the present application provides use of the aforementioned recombinant antigen, labeled conjugate, or kit in preparation of an immunoassay product.

[0011] The fifth aspect of the present application provides a recombinant nucleic acid molecule encoding the aforementioned recombinant antigen, an expression vector containing the recombinant nucleic acid molecule, and a host cell containing the expression vector.

[0012] The sixth aspect of the present application provides a method for detecting an analyte in a sample, comprising: contacting the aforementioned recombinant antigen, labeled conjugate, or kit with the sample to form an immunocomplex; and detecting the presence or amount of the immunocomplex.

[0013] The sixth aspect of the present application provides use of the aforementioned recombinant antigen, labeled conjugate, or kit in improving the activity and specificity of an immunoassay.

[0014] The seventh aspect of the present application provides a method for diagnosing a disease in vitro, comprising: contacting the aforementioned recombinant antigen, labeled conjugate, or kit with an ex vivo sample to form an immunocomplex; and detecting the presence or amount of the immunocomplex. BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 shows a PE carrier map used in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0016] The word "a" or "an" when used in the claims and / or specification means "one," but also "one or more," "at least one," or "one or more than one."

[0017] As used in the claims and specification, the word "comprising" or "including" means "including but not limited to."

[0018] Throughout the application, the term "about" means a value includes the standard deviation of the error of the apparatus or method used to determine the value.

[0019] Although the disclosed content supports a definition of the term "or" as being either an alternative or in the alternative, the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to an alternative exclusive sense.

[0020] The term "pathogen polypeptide" refers to a polypeptide of an exogenous pathogen, such as bacteria, viruses, and other microorganisms that directly or indirectly cause disease. Exemplary pathogens include, for example, Bacillus, Clostridium, Corynebacterium, Listeria, Erysipelothrix, Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium, Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia, Rickettsia, Micrococcus, Mycobacterium, Neisseria, Campylobacter, pathogenic viruses such as, for example, papillomavirus, parvovirus, adenovirus, herpesvirus, vaccinia virus, arenavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus, paramyxovirus, reovirus, retrovirus, rhabdovirus, human immunodeficiency virus (HIV), adenovirus, coronavirus, human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza B, Middle East respiratory syndrome coronavirus (MERS-CoV), parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, Bordetella parapertussis, Bordetella pertussis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and combinations thereof. Preferably SARS-CoV, HIV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rubella virus, human cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, rabies virus, human T-lymphotrophic leukaemia virus, dengue virus, human papillomavirus, West Nile virus, encephalitis virus, measles virus, influenza virus, parainfluenza virus. varicella virus, ecotropic virus type, coxsackie virus, Japanese encephalitis virus, Epstein-Barr virus, mumps virus, Treponema pallidum, Borrelia burgdorferi infection, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Ureaplasma urealyticum, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Helicobacter pylori, Neisseria gonorrhoeae, Plasmodium, Trypanosoma cruzi, and Toxoplasma gondii.

[0021] The term "autoimmune disease antigen polypeptide" refers to an endogenous polypeptide that causes an autoimmune disease. Exemplary autoimmune diseases include, for example: type I diabetes mellitus, rheumatoid arthritis, graft versus host disease (GVHD), nephritis, multiple sclerosis, mixed connective tissue disease, pemphigus vulgaris, bullous pemphigoid, membranous glomerulonephritis, neuromyelitis optica, autoimmune encephalomyelitis, autoimmune hepatitis, chronic inflammatory demyelinating polyradiculoneuropathy, dermatomyositis, giant cell arteritis, granulomatosis with polyangiitis, Kawasaki disease, lupus nephritis, Takayasu arteritis, and Weber-Christian arteritis.

[0022] The term "rraB" refers to ribonuclease activity modulator B, derived from Escherichia coli. The amino acid sequence of rraB is exemplified as SEQ ID NO: 1.

[0023] The term "MysB" refers to MysB family protein, derived from Escherichia coli. The amino acid sequence of MysB is exemplified as SEQ ID NO: 2.

[0024] The term "Folden" refers to Folden domain, which is a domain of T4 phage fibritin protein, located at the C-terminal end of the protein, often used as a fusion partner, and the amino acid sequence thereof is exemplified as SEQ ID NO: 3.

[0025] The term "Bff217" refers to CN105838694B, and the amino acid sequence thereof is exemplified as SEQ ID NO: 4.

[0026] The term "fusion partner" refers to a short peptide that is fused or linked to a polypeptide of interest. The fusion partner can be fused or linked to the N-terminus and / or C-terminus of the polypeptide of interest (optionally through a linker or a protease cleavage site).

[0027] The term "peptide linker" or "linking peptide" refers to a short peptide used to link two molecules (e.g., proteins). Typically, a fusion protein, e.g., polypeptide 1-linker-polypeptide 2, more specifically, polypeptide of interest-linker-fusion partner, is obtained by introducing a polynucleotide sequence encoding the short peptide (e.g., by PCR amplification or ligase) between two DNA fragments encoding two proteins of interest to be linked, respectively, and performing protein expression.

[0028] As used in the present disclosure, the term "recombinant antigen" is a polypeptide obtained by using recombinant DNA or recombinant RNA technology, which can be obtained in vivo or in vitro.

[0029] As used herein, the term "polypeptide" refers to any molecule comprising three or more amino acid residues linked by peptide bonds. Polypeptides according to the present application include peptides (e.g., tripeptides, oligopeptides, etc.), as well as peptides that can include chemical modifications (e.g., glycosylation (glycopeptides), phosphorylation, hydroxylation, sulfation, palmitoylation, and disulfide bond formation). Polypeptide can also refer to a protein.

[0030] As used herein, the term "signal peptide" refers to a polypeptide attached to a target protein, which can facilitate the expression or transfer of the target protein. Exemplarily, a signal peptide can be attached to the N-terminus of a target protein, and is usually cleaved to be removed, and thus is not present in the mature protein secreted from a cell.

[0031] As used herein, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0032] As used herein, a "conservative substitution" typically exchanges one amino acid at one or more positions of a protein. Such substitution can be conservative. As substitutions that are considered to be conservative substitutions, specifically, there can be mentioned substitution of Ala to Ser or Thr, substitution of Arg to Gin, His or Lys, substitution of Asn to Glu, Gin, Lys, His or Asp, substitution of Asp to Asn, Glu or Gin, substitution of Cys to Ser or Ala, substitution of Gin to Asn, Glu, Lys, His, Asp or Arg, substitution of Glu to Gly, Asn, Gin, Lys or Asp, substitution of Gly to Pro, substitution of His to Asn, Lys, Gin, Arg or Tyr, substitution of He to Leu, Met, Val or Phe, substitution of Leu to He, Met, Val or Phe, substitution of Lys to Asn, Glu, Gin, His or Arg, substitution of Met to He, Leu, Val or Phe, substitution of Phe to Trp, Tyr, Met, He or Leu, substitution of Ser to Thr or Ala, substitution of Thr to Ser or Ala, substitution of Trp to Phe or Tyr, substitution of Tyr to His, Phe or Trp, and substitution of Val to Met, He or Leu. In addition, a conservative mutation also includes naturally occurring mutations due to individual differences, strain, species differences, etc., of a gene from which the mutation is derived.

[0033] "Sequence identity" and "percentage of identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are the same (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and scoring the number of positions that contain the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides and comparing it to the number of positions that contain different nucleotide or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing a different nucleotide (i.e., substitution) or by lacking a nucleotide (i.e., one or two nucleotide insertions or nucleotide deletions in one of the two polynucleotides). A polypeptide can differ at one position, for example, by containing a different amino acid (i.e., substitution) or by lacking an amino acid (i.e., one or two amino acid insertions or amino acid deletions in one of the two polypeptides). Sequence identity can be calculated by dividing the number of positions that contain the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage of identity can be calculated by dividing the number of positions that contain the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0034] Illustratively, two or more sequences or subsequences are "sequentially identical" or have a "percentage of sequence identity" to each other when the two or more sequences or subsequences, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection, comprise at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity over a region of the two or more sequences or subsequences. Determination of "sequence identity" or "percentage of identity" can be based on any suitable region of the sequences. For example, a region of at least about 50 residues, at least about 100 residues, at least about 200 residues, at least about 400 residues, or at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biological polymers (i.e., nucleic acid or polypeptide) being compared.

[0035] As used herein, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of an individual fragment, or can be part of a larger nucleotide sequence structure, which is derived from a nucleotide sequence that is isolated at least once in quantity or concentration, and is capable of being identified, manipulated, and the sequence and its component nucleotides recovered by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" is substituted for "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (individual fragments or entire fragments), or can be part of or a component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "Recombinant polynucleotide", "recombinant nucleic acid molecule" are a subset of "polynucleotide".

[0036] As used herein, the term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, "recombinant antigen", "recombinant protein", "fusion protein", etc.

[0037] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences for expression of a gene of interest in a suitable host.

[0038] As used herein, the term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional unit comprising i) a collection of genetic elements having a regulatory effect on gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical, and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used.

[0039] The term "host cell" in the present disclosure means any cell type susceptible to transformation, transfection, transduction, etc. with a gene editing element, a nucleic acid construct or a recombinant expression vector of the present disclosure. The term "recombinant host cell" encompasses a host cell that is altered by the introduction of a gene editing element, a nucleic acid construct or a recombinant expression vector, and is specifically achieved by transformation. The host cell of the present disclosure can be a prokaryotic cell or a eukaryotic cell, as long as it is a cell that can introduce a recombinant nucleic acid molecule or a recombinant expression vector of the present disclosure.

[0040] The term "transformation, transfection, transduction" in the present disclosure has the meaning commonly understood by those skilled in the art, i.e. the process of introducing foreign DNA into a host. The methods of transformation, transfection, transduction include any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaP04) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0041] The term "amplification carrier" in the present disclosure is used to load a tracer marker selected from at least one of polysaccharide, polylysine, protein carrier, PEG (polyethylene glycol), polyethylene imine, and dendrimer. Specifically, the polysaccharide includes at least one of cross-linked polysucrose, dextran; the protein carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin; the dendrimer includes polyethylene glycol and / or polypropylene imine. In some embodiments, the amplification carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin.

[0042] The term "solid phase carrier" in the present disclosure refers to a solid phase material having at least one surface capable of immobilizing a binding partner for capturing an analyte in a sample. The solid phase carrier can be in the form of a particle, microparticle, nanoparticle, metal colloid, fiber, nylon, paper, bead, membrane, filter paper, and other supports such as test tubes, microtiter plates, chips, slides, capillaries, and microarrays.

[0043] As used in the present disclosure, the term "sample", "specimen", "sample to be tested" or "test sample" relates to any kind of sample for which it is desired to determine whether it contains an antibody. Illustratively, the sample to be tested can be any product produced by a subject, or any product derived from a product produced by a subject. The sample can be taken from any tissue or body fluid, for example a blood sample (including samples derived from blood), a serum sample, a lymph sample, a saliva sample, synovial fluid. The sample derived from blood can be a selected portion of a patient's blood or a vaccinee's blood, for example a selected cell-containing portion, or a plasma or serum portion. In some embodiments, the sample can be any sample containing an antibody product of a humoral immune response.

[0044] As used in the present disclosure, "diagnosing" includes detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or likely progression or regression) of a subject with a disease or condition, and determining the effect of a treatment on a subject with a disease or condition. For example, a diagnosis can be used to detect the presence or likelihood of a subject being infected with Treponema pallidum, hepatitis C, or the human immunodeficiency virus or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, e.g., a pharmaceutical) or other treatment.

[0045] The terms "individual," "patient," or "subject" as used throughout this disclosure include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0046] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the products can exist in vitro, ex vivo, or in vivo. The term "in vitro" refers to experiments in which materials, biological substances, cells, and / or tissues are used in laboratory conditions or culture; while the term "in vivo" refers to experiments and procedures in which whole multicellular organisms are used. In some embodiments, methods performed in vivo can be performed on a non-human animal. "Ex vivo" refers to events that occur outside or in an organism, e.g., outside a human or animal body, e.g., events that can occur or take place on tissues (e.g., whole organs) or cells taken from an organism.

[0047] Sequence

[0048] In the technical solutions of the present disclosure, the meanings represented by the numbering of the sequence table in the specification are as follows:

[0049] The sequence shown in SEQ ID NO: 1 is the polypeptide sequence of rraB in the examples;

[0050] The sequence shown in SEQ ID NO: 2 is the polypeptide sequence of MysB in the examples;

[0051] The sequence shown in SEQ ID NO: 3 is the sequence of the fusion partner Folden in the examples;

[0052] The sequence shown in SEQ ID NO: 4 is the sequence of the fusion partner Bff217 in the examples;

[0053] The sequence shown in SEQ ID NO: 5 is the sequence of the HCV-NS3 antigen used in the examples;

[0054] The sequence shown in SEQ ID NO: 6 is the sequence of the HCV-Core antigen used in the examples;

[0055] The sequence shown in SEQ ID NO: 7 is the sequence of the HCV-Core-NS3 fusion antigen used in the examples;

[0056] The sequence shown in SEQ ID NO: 8 is the sequence of the TP antigen used in the examples;

[0057] The sequence shown in SEQ ID NO: 9 is the sequence of the TP antigen used in the examples;

[0058] The sequence shown in SEQ ID NO: 10 is the sequence of the TP antigen used in the examples;

[0059] The sequence shown in SEQ ID NO: 11 is the sequence of the HIV-2 gp36 antigen used in the examples;

[0060] Recombinant antigen

[0061] In one aspect, the present disclosure provides a recombinant antigen, wherein the recombinant antigen contains at least one target polypeptide, and at least one rraB or MysB as a fusion partner.

[0062] In some embodiments, the amino acid sequence of rraB is shown in SEQ ID NO: 1.

[0063] In some embodiments, the amino acid sequence of MysB is shown in SEQ ID NO: 2.

[0064] It should be understood that rraB with one or more amino acids substituted, repeated, deleted or added on the basis of the amino acid sequence shown in SEQ ID NO: 1, and having or partially having the polypeptide activity of the sequence shown in SEQ ID NO: 1, also belongs to the protection scope of the present application. MysB with one or more amino acids substituted, repeated, deleted or added on the basis of the amino acid sequence shown in SEQ ID NO: 2, and having or partially having the polypeptide activity of the sequence shown in SEQ ID NO: 2, also belongs to the protection scope of the present application.

[0065] In some embodiments, the polypeptide sequence of the rraB is as set forth in a truncated SEQ ID NO: 1. For example, the truncated SEQ ID NO: 1 comprises amino acids from position X to position Y of SEQ ID NO: 1, wherein X is selected from any integer between 1 and 20, and Y is selected from any integer between 100 and 138. Exemplary, non-limiting examples include, for example, amino acids 2-138, 3-138, 4-138, 5-138, 6-138, 7-138, 8-138, 9-138, 10-138, 18-138, 19-138, 20-138, etc. of SEQ ID NO: 1 (N-terminal truncation only); for example, amino acids 1-137, 1-136, 1-135, 1-134, 1-133, 1-132, 1-131, 1-130, 1-129, 1-128, etc. of SEQ ID NO: 1 (C-terminal truncation only); for example, amino acids 2-137, 5-133, 10-128, 15-123, 20-100, etc. of SEQ ID NO: 1 (both N-terminal and C-terminal truncation).

[0066] In some embodiments, the polypeptide sequence of the MysB is as set forth in a truncated SEQ ID NO: 2. For example, the truncated SEQ ID NO: 2 comprises amino acids from position M to position N of SEQ ID NO: 2, wherein M is selected from any integer between 1 and 20, and N is selected from any integer between 100 and 123. Exemplary, non-limiting examples include, for example, amino acids 2-123, 3-123, 4-123, 5-123, 6-123, 7-123, 8-123, 9-123, 10-123, 18-123, 19-123, 20-123, etc. of SEQ ID NO: 2 (N-terminal truncation only); for example, amino acids 1-122, 1-121, 1-120, 1-119, 1-118, 1-117, 1-116, 1-115, 1-114, 1-113, etc. of SEQ ID NO: 2 (C-terminal truncation only); for example, amino acids 2-122, 5-118, 10-113, 15-108, 20-100, etc. of SEQ ID NO: 2 (both N-terminal and C-terminal truncation).

[0067] The polypeptide sequence of the above-mentioned rraB or MysB can have a variant, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2. For example, the variant is a variant produced by conservative substitution or a naturally-occurring variant due to individual differences, strain differences, species differences, etc. of the gene source. These variants also belong to the protection scope of the present application.

[0068] In some embodiments, the recombinant antigen provided by the present disclosure comprises a target polypeptide and a fusion partner connected by a cleavage site.

[0069] In some embodiments, the recombinant antigen provided by the present disclosure comprises a target polypeptide and a fusion partner connected by a linker peptide.

[0070] In some embodiments, the target polypeptide, the linker peptide, and the fusion partner are sequentially connected to the expression vector in order.

[0071] In some embodiments, the fusion partner, the linker peptide, and the target polypeptide are sequentially connected to the expression vector in order.

[0072] In some embodiments, the target polypeptide, the cleavage site, and the fusion partner are sequentially connected to the expression vector in order.

[0073] In some embodiments, the fusion partner, the cleavage site, and the target polypeptide are sequentially connected to the expression vector in order.

[0074] In some embodiments, the target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. The polypeptide of an exogenous pathogen induces an immune response to produce a corresponding autoantibody in the individual, and in autoimmune disease patients, the autoimmune tolerance state is broken, and the endogenous antigen polypeptide also induces the production of autoantibodies. Immunodiagnosis of these autoantibodies is an effective means to determine whether an individual is infected with a corresponding pathogen / patients with a corresponding autoimmune disease.

[0075] In some embodiments, the target polypeptide is selected from the group consisting of: hepatitis C virus antigen, Treponema pallidum antigen, SARS-CoV-2 antigen, HIV antigen, HBV antigen (e.g. HBe antigen, HBS antigen), hepatitis A (HAV) antigen, hepatitis E (HEV) antigen, Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus (e.g. HSV I, HSV II), human T-cell lymphotropic virus (HTLV) antigen, Mycoplasma pneumoniae antigen, influenza antigen (e.g. influenza A, influenza B), Epstein-Barr virus antigen, Helicobacter pylori antigen, glutamate decarboxylase, tyrosine phosphatase protein, Plasmodium antigen, dengue virus antigen, Salmonella typhi antigen, Mycobacterium tuberculosis antigen, streptolysin O, cyclic citrullinated peptide, monkeypox virus antigen.

[0076] In some embodiments, the length of the fragment of the target polypeptide is not particularly limited in principle, and can be a full-length antigen polypeptide or an antigen fragment having epitope activity that can be used to detect the corresponding autoantibody. For example, in some embodiments, the antigen can be a full-length antigen or a variant thereof, and in some specific embodiments, the antigen can be a truncated antigen fragment.

[0077] In some embodiments, the target polypeptide is an antigen that can be coupled with a tracer label.

[0078] In some embodiments, the target polypeptide is a hepatitis C virus antigen (HCV antigen).

[0079] In some embodiments, the hepatitis C virus antigen is optionally selected from the group consisting of HCV Core, HCV NS3, and HCV NS4. The specific sequence of the HCV antigen is not limited, and many HCV antigens are disclosed in Patent Publication Nos. CN101287989B, CN112341527B, or CN112225783B, any of which is within the scope of the present application.

[0080] In some embodiments, the HCV antigen comprises an amino acid sequence as set forth in SEQ ID NO: 5, 6, or 7. But a truncation or an addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the N-terminus or C-terminus of SEQ ID NO: 5, 6, or 7. Or a variant of the amino acid sequence as set forth in SEQ ID NO: 5, 6, or 7, for example, a variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5, 6, or 7. For example, the variant is a variant resulting from conservative substitution or a naturally occurring variant due to individual differences, strain differences, species differences, etc. Also within the scope of the present application.

[0081] In some embodiments, the HCV antigen is optionally selected from the amino acid sequence as set forth in SEQ ID NO: 5, 6, or 7.

[0082] In some embodiments, the target polypeptide is a Treponema pallidum antigen (TP antigen).

[0083] In some embodiments, the Treponema pallidum antigen is optionally selected from Tp15, Tp17, and Tp47.

[0084] The specific sequence of the TP antigen is not limited, and patent publication No. CN105542014B discloses many TP antigens, any of which belongs to the protection scope of the present application.

[0085] In some embodiments, the amino acid sequence of the TP antigen comprises that as set forth in SEQ ID NO: 8, 9, or 10. Or a truncation or an addition of N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids based on SEQ ID NO: 8, 9, or 10. Or a variant of the amino acid sequence as set forth in SEQ ID NO: 8, 9, or 10, for example, having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8, 9, or 10. For example, the variant is a variant produced by conservative substitution or a naturally occurring variant due to individual differences, strain differences, species differences, etc. Also belongs to the protection scope of the present application.

[0086] In some embodiments, the TP antigen is optionally selected from the amino acid sequence as set forth in SEQ ID NO: 8, 9, or 10.

[0087] In some embodiments, the target polypeptide is a human immunodeficiency virus antigen (HIV antigen).

[0088] In some embodiments, the HIV antigen is optionally selected from HIV-1 gp41 and HIV-2 gp36.

[0089] The specific sequence of the HIV antigen is not limited, and any of the HIV antigens in patent publication Nos. CN107090019B, CN114605505B, CN118515733A, or CN118515734A belongs to the protection scope of the present application.

[0090] In some embodiments, the amino acid sequence of the HIV antigen comprises SEQ ID NO: 11. Or a truncation or addition of N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids based on SEQ ID NO: 11. Or a variant of the amino acid sequence of SEQ ID NO: 11, for example, having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 11. For example, the variant is a variant resulting from conservative substitution or a naturally occurring variant due to differences in the individual, strain, species, etc. from which the gene is derived. Also within the scope of the present disclosure.

[0091] In some embodiments, the amino acid sequence of the HIV antigen comprises SEQ ID NO: 11.

[0092] The addition of the above-mentioned addition refers to the addition of N-terminal or C-terminal to the corresponding amino acid sequence position of the HCV, TP or HIV full gene encoded protein.

[0093] In the present disclosure, there is no particular restriction in principle for the selection of the linker peptide. In some preferred embodiments, the linker peptide can be a flexible linker peptide. The linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n or (GGGGS)n; wherein n is selected from an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the linker peptide is a short peptide containing 1-20 amino acids, for example, a short peptide of 3-13 amino acids.

[0094] In some embodiments, the recombinant antigen further comprises a tag at its N-terminus and / or C-terminus. For example, the tag is a His tag, preferably a 6xHis tag, which is connected to the N-terminus of the recombinant antigen, and can facilitate the purification of the recombinant antigen protein. In some alternative embodiments, the recombinant antigen and the tag can be connected by a linker peptide.

[0095] In the present disclosure, exemplary recombinant antigens can have any one of the following structures:

[0096] (1) Tag - optional linker peptide - target polypeptide - optional linker peptide - fusion partner;

[0097] (2) target polypeptide - optional linker peptide - fusion partner - optional linker peptide - tag.

[0098] In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleotide sequence encoding any one of the recombinant receptor binding proteins described above, which can be DNA, RNA or a combination thereof.

[0099] In one aspect, the present disclosure provides a recombinant expression vector comprising the recombinant nucleic acid molecule described above, which can be used for the transfer of the recombinant nucleic acid molecule into a cell. In some embodiments, the recombinant expression vector comprises one or more regulatory elements, which can be a promoter, an enhancer, a silencer, an insulator, and the like elements commonly used in the art. The regulatory element is operably linked to the recombinant nucleic acid molecule to mediate the transcription and translation of the recombinant nucleic acid molecule.

[0100] The term "operably linked" can include the covalent linkage of a selected nucleic acid molecule sequence to a regulatory element sequence (e.g., a promoter and / or an enhancer) to place the expression of the nucleic acid sequence under the influence or control of the regulatory element (thereby forming an expression cassette). Thus, if the regulatory element is capable of affecting the transcription of a nucleic acid molecule, the regulatory element is operably linked to the nucleic acid molecule. The resulting transcript can be translated into the desired polypeptide, protein.

[0101] In the present disclosure, the vectors suitable for constructing the recombinant expression vector include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviruses (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, adenoviral vectors, adenovirus-associated viral vectors, vaccinia virus vectors, and herpes virus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), and the like.

[0102] In some embodiments, the vector is a prokaryotic vector comprising various elements for expression in prokaryotic cells. For example, the vector includes, but is not limited to, pET series vectors, Duet series vectors, pGEX series vectors, pHY300 vectors, pHY300PLK vectors, PE vectors. As a preferred, the vector is a PE vector.

[0103] In one aspect, the present disclosure provides a recombinant host cell comprising the recombinant nucleic acid molecule or the recombinant expression vector described above. The recombinant host cell is obtained by transforming, transfecting, or transducing the recombinant nucleic acid molecule or the recombinant expression vector into a host cell. The host cell in the present disclosure can be a eukaryotic cell or a prokaryotic cell, as long as it is capable of introducing the recombinant nucleic acid molecule or the recombinant expression vector of the present disclosure to achieve the expression of the recombinant receptor binding protein.

[0104] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The bacteria can be cocci (e.g., Micrococcus, Thermococcus, Streptococcus), bacilli (e.g., Enterobacter, Bacillus, Actinobacillus, Acidaminococcus, Lactobacillus), or spirochetes (e.g., Brachyspira). In some embodiments, the host cell is E. coli ER2529, E. coli ER2566; preferably, E. coli ER2566.

[0105] In some embodiments, the recombinant antigen can be prepared by expression in a recombinant host cell, which can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the recombinant host cell is a prokaryotic cell, such as a recombinant host cell introduced into E. coli by a recombinant expression vector.

[0106] The recombinant host cell is cultured under conditions suitable for expression of the protein, and after the end of the culture, the recombinant antigen protein is collected from the cell culture or fermentation broth, and then the recombinant antigen protein is subjected to protein purification treatment.

[0107] In some embodiments, the step of subjecting the recombinant antigen protein to protein purification treatment includes Ni-NTA affinity chromatography and protein dialysis.

[0108] Labeling conjugate or kit

[0109] In one aspect, the present disclosure provides a labeling conjugate comprising the recombinant antigen of the first aspect, and a tracer label coupled to the recombinant antigen. The present disclosure finds that, by using rraB or MysB and a target polypeptide to express in a fusion manner in an E. coli expression system, the recombinant antigen after fusion can maintain antigenicity during the labeling process, and the labeling conjugate prepared using the recombinant antigen of the present disclosure can significantly improve the detection activity and specificity, as compared to the recombinant antigen expressed in a fusion manner with other fusion partners.

[0110] In some embodiments, the recombinant antigen is coupled to a tracer label capable of generating a detection signal. In some embodiments, the tracer label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label. In some embodiments, the tracer label is selected from at least one of acridinium ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, colloidal gold, fluorescent microspheres, trispyridine ruthenium, quantum dot luminescent material, and upconversion luminescent material.

[0111] In some embodiments, the tracer label is colloidal gold.

[0112] The inventors find that the recombinant antigen expressed using rraB or MysB as a fusion partner has a significantly better effect than other tracer labels when used to label colloidal gold.

[0113] In some embodiments, the tracer label is directly conjugated to the recombinant antigen, or indirectly conjugated to the recombinant antigen through an intermediate agent. Direct conjugation of the tracer label to the recombinant antigen is preferred over indirect conjugation of the tracer label to the recombinant antigen through an intermediate agent.

[0114] In some embodiments, the tracer label is indirectly conjugated to the recombinant antigen based on an anti-fusion partner (or tag) antibody, i.e., the tracer label is directly conjugated to the anti-fusion partner (or tag) antibody, and the anti-fusion partner (or tag) antibody specifically binds to the fusion partner (or tag) portion in the conjugated recombinant antigen. In some embodiments, the tracer label is indirectly conjugated to the recombinant antigen based on a magnifying carrier, i.e., the tracer label is directly conjugated to the magnifying carrier, and the recombinant antigen is also directly conjugated to the same magnifying carrier.

[0115] In one aspect, the present disclosure provides a kit comprising the recombinant antigen or the label conjugate of the present disclosure. The term "kit" is not intended to limit the form of the product, e.g., the product does not require to be in a box shape, but can be in a plate shape, a strip shape, or other forms, as long as it is in a separate form. For example, the kit can be a lateral flow test strip. In some embodiments, the test strip comprises the recombinant antigen coated thereon, at least one detection antibody or at least one detection antigen with a tracer label (e.g., colloidal gold). In some embodiments, the test strip comprises the recombinant antigen conjugated with a tracer label, at least one detection antibody or at least one detection antigen coated thereon. In some embodiments, the kit can be prepared based on a double antigen sandwich principle.

[0116] In alternative embodiments, the kit further comprises at least one of a sample pre-treatment reagent (e.g., a sample purification and enrichment reagent, a lysis solution, etc.), a washing solution (e.g., water, etc.), a buffer solution (e.g., PBS or Tris, etc.), a stabilizing solution, and a color developing reagent for a signal substance.

[0117] Uses / Methods

[0118] In one aspect, the present disclosure provides uses of the aforementioned recombinant antigen, label conjugate, or kit in the preparation of an immunoassay product. In some embodiments, the immunoassay product is a detection product for detecting an antibody against hepatitis C virus, an antibody against Treponema pallidum, or an antibody against human immunodeficiency virus.

[0119] In one aspect, the present disclosure provides a method for detecting an analyte in a sample, comprising: contacting the aforementioned recombinant antigen, label conjugate, or kit with the sample to form an immunocomplex; and detecting the presence or amount of the immunocomplex. In some embodiments, the analyte is an antibody against hepatitis C virus, an antibody against Treponema pallidum, or an antibody against human immunodeficiency virus.

[0120] In one aspect, the present application provides use of the aforementioned recombinant antigen, label conjugate, or kit in improving the activity and specificity of an immunoassay product. In some embodiments, the immunoassay product is a detection product for detecting hepatitis C virus antibody, Treponema pallidum antibody, or human immunodeficiency virus antibody.

[0121] In one aspect, the present application provides a method for diagnosing a disease in vitro, comprising: contacting the aforementioned recombinant antigen, label conjugate, or kit with an ex vivo sample to form an immunocomplex; and detecting the presence or amount of the immunocomplex. In some embodiments, the disease is hepatitis C, syphilis infection, or AIDS.

[0122] Examples

[0123] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0124] The experimental techniques and experimental methods used in the present examples are all conventional techniques and methods, and for example, the experimental methods not specifically described in the following examples are generally performed according to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0125] The PE vector used in the following examples has a vector map as shown in Figure 1: the PE vector itself contains a 6xHis tag at the N-terminus, so that the recombinant antigen obtained by translation contains a 6-histidine tag, which can facilitate the NI-NTA assisted affinity purification.

[0126] The sequence of the HCV antigen is not limited in source, and many HCV antigen sequences are disclosed in the prior art, for example, see any HCV antigen in CN101287989B, CN112341527B or CN112225783B. The HCV antigen sequence selected in this embodiment is shown in SEQ ID NO: 5. The sequence of the TP antigen is not limited in source, for example, see any TP antigen in CN105542014B. The TP antigen sequence selected in this embodiment is shown in SEQ ID NO: 8. The sequence of the HIV antigen is not limited in source, for example, see any HIV antigen in CN107090019B, CN114605505B, CN118515733A or CN118515734A. The HIV antigen sequence selected in this embodiment is shown in SEQ ID NO: 11.

[0127] Preparation Example 1: Preparation of recombinant antigen

[0128] 1.1, Design of recombinant antigen

[0129] According to the design table of the recombinant antigen as above, the corresponding fusion partner is introduced at the N-terminus of the target polypeptide, and the expression plasmid is constructed according to the coding nucleic acid of the protein sequence. The gene sequence contains restriction enzyme cutting sites BamHI and EcoRI at both ends, the mutant gene is treated with restriction enzymes BamHI and EcoRI respectively, and the treated gene fragment is connected to the vector PE (as shown in Figure 1), to obtain the corresponding expression plasmid. The linker GS is provided between the target polypeptide and the fusion partner.

[0130] 1.2 Induction expression of recombinant antigen

[0131] The expression plasmid constructed in 1.1 is transformed into E. coli BL21 competent cells (NEB, item number: C2530H) by heat shock method, and is coated on LB plate containing 100 ug / ml antibiotic and cultured at 37°C for 16h. The colonies are picked, and the positive strains identified by bacterial liquid PCR and double enzyme digestion are selected for preservation and inoculation into LB medium containing 50 ug / ml Kan and cultured at 37°C with shaking. When the OD600 reaches 0.6-0.8, 1.0 mM IPTG is added, and the culture is induced at 37°C for 2-4h. The total protein is extracted, and the expression of the recombinant protein is identified by SDS-PAGE. The recombinant antigen is purified by affinity column and ion exchange chromatography.

[0132] Preparation Example 2: Preparation of labeled conjugate

[0133] 2.1, Direct labeling of antigen

[0134] 1) Labeling: Take 10 ml of 4 / 10,000 colloidal gold, add 100 ul of 0.2M K2CO3, stir for 5 min, add 100 ug of recombinant antigen prepared in Preparation Example 1, stir for 5 min, then add 100 ul of 10% BSA + 10% sodium caseinate blocking termination labeling; centrifuge at 10,000 rpm for 7 min, remove the supernatant, and resuspend the precipitate with gold resuspension solution to 1 ml (i.e. 1 / 10 volume of colloidal gold solution); 2) Preparation of gold working solution: dilute the labeled concentrated gold to OD = 6 with gold resuspension solution to prepare the gold working solution; spread on glass fiber;

[0135] 2) Preparation of gold working solution: dilute the labeled concentrated gold to OD = 6 with gold resuspension solution to prepare the gold working solution; spread on glass fiber;

[0136] 3) Preparation of dried gold: place the spread gold in a freeze dryer (1-2 hr) or in a 37°C drying room overnight;

[0137] 4) Preparation of gold label strip: cut the gold label strip to the desired width with a strip cutter.

[0138] 2.2, Indirect labeling of antigen

[0139] The method of indirect labeling of antigen is known, and can be specifically referred to CN101363848B.

[0140] 1) Labeling: Take 10 ml of 4 / 10,000 colloidal gold, add 100 ul of 0.2M K2CO3, stir for 5 min, add 100 ug of recombinant antigen prepared in Preparation Example 1, stir for 5 min, then add 100 ul of 10% BSA + 10% sodium caseinate blocking termination labeling; centrifuge at 10,000 rpm for 7 min, remove the supernatant, and resuspend the precipitate with gold resuspension solution to 1 ml (i.e. 1 / 10 volume of colloidal gold solution); 2) Preparation of gold working solution: dilute the labeled concentrated gold to OD = 6 with gold resuspension solution to prepare the gold working solution; spread on glass fiber;

[0141] 2) Preparation of gold working solution: dilute the labeled concentrated gold to OD = 6 with gold resuspension solution to prepare the gold working solution; spread on glass fiber;

[0142] 3) Preparation of dried gold: place the spread gold in a freeze dryer (1-2 hr) or in a 37°C drying room overnight;

[0143] 4) Preparation of gold label strip: cut the gold label strip to the desired width with a strip cutter.

[0144] Example 1: Detection of antibody by double antigen sandwich method

[0145] 1, Preparation of colloidal gold detection test paper

[0146] 1.1 Preparation of nitrocellulose membrane

[0147] Preparation of nitrocellulose membrane: dilute the coating antigen (purchased from Phoenix Biotech) to 1-5 mg / ml with coating buffer, draw a T line as the detection line, and the T line is close to the colloidal gold end; dilute the secondary antibody to 1-5 mg / ml with coating buffer, draw a C line as the control line, and the C line is close to the absorbent pad. Dry at 37°C, and package for use.

[0148] 1.2 Preparation of sample pad of test strip

[0149] After immersing the sample pad in blocking solution (containing BSA) for 30 min, dry at 37°C, package, and store at 4°C for standby.

[0150] 1.3 Assembly of test strip

[0151] Place the absorbent pad (purchased from Millipore), nitrocellulose membrane, gold-labeled strip prepared in Preparation Example 2, and sample pad on a non-water-absorbing support sheet, cut into small strips of 3 mm wide. Package ten small strips together, add a desiccant, vacuum seal, and obtain the test strip.

[0152] 2. Use the double-antigen sandwich method to detect whether the sample contains the antibody to be tested. The darkness of the red precipitate line indicates the strength of the reaction, and the darker the red color, the stronger the reactivity, and vice versa. The strength of the reaction is represented by a combination of letters C and numbers, and the smaller the number after C, the stronger the reactivity, and vice versa. No red precipitate line is represented by B.

[0153] Detection results: The results in Tables 1 and 2 show that the rraB fusion partner or MysB fusion partner can effectively improve the activity and specificity of detection compared to the antigen without the fusion partner. The performance of the rraB fusion partner is improved most significantly, and even better than the mainstream indirect labeling on the market.

[0154] Table 1. Test results of HCV recombinant antigen labeling

[0155] Table 3. Test results of HIV recombinant antigen labeling

[0156] Detection results: The results in Table 3 show that the rraB fusion partner or MysB fusion partner can effectively improve the activity of detection compared to the antigen without the fusion partner. The performance of the rraB fusion partner is improved most significantly. Industrial applicability

[0157] The present disclosure provides a recombinant antigen containing rraB or MysB as a fusion partner, and the recombinant antigen is used for immunodiagnosis, which can significantly improve the activity and specificity of immunodiagnosis. Therefore, the recombinant antigen provided by the present disclosure has excellent practical performance and broad market application prospects.

[0158] Although the embodiments of the present disclosure have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present disclosure, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present disclosure.

Claims

1. A recombinant antigen, wherein, The recombinant antigen contains at least one target polypeptide, and at least one rraB or MysB as a fusion partner.

2. The recombinant antigen of claim 1, wherein, The amino acid sequence of the rraB is shown in SEQ ID NO: 1 or has at least 90% identity thereto, and / or the amino acid sequence of the MysB is shown in SEQ ID NO: 2 or has at least 90% identity thereto.

3. The recombinant antigen of any one of claims 1-2, wherein, The target polypeptide is selected from a hepatitis C virus antigen, a Treponema pallidum antigen, or an HIV antigen; Optionally, the hepatitis C virus antigen is selected from HCV Core, HCV NS3, and HCV NS4; optionally, the hepatitis C virus antigen is selected from SEQ ID NO: 5-7, or has at least 90% identity thereto; Optionally, the Treponema pallidum antigen is selected from Tp15, Tp17, and Tp47; optionally, the Treponema pallidum antigen is selected from SEQ ID NO: 8-10, or has at least 90% identity thereto; Optionally, the HIV antigen is selected from HIV-1 gp41 and HIV-2 gp36; optionally, the HIV antigen is selected from SEQ ID NO: 11, or has at least 90% identity thereto.

4. A labeling conjugate, wherein, The labeled conjugate comprises the recombinant antigen of any one of claims 1-3, and a tracer label coupled to the recombinant antigen; Optionally, the tracer label is colloidal gold.

5. A kit, wherein, The kit comprises the recombinant antigen of any one of claims 1-3, or the labeled conjugate of claim 4.

6. Use of the recombinant antigen of any one of claims 1-3, the labeled conjugate of claim 4, or the kit of claim 5 in the preparation of an immunoassay product; Optionally, the immunoassay product is a detection product for detecting hepatitis C virus antibodies, Treponema pallidum antibodies, or HIV antibodies.

7. A recombinant nucleic acid molecule encoding the recombinant antigen of any one of claims 1-3, an expression vector comprising the recombinant nucleic acid molecule, and a host cell comprising the expression vector.

8. A method of detecting an analyte in a sample, comprising: contacting the recombinant antigen of any one of claims 1-3, the labeled conjugate of claim 4, or the kit of claim 5 with the sample to form an immunocomplex; detecting the presence or amount of the immunocomplex; Optionally, the analyte is hepatitis C virus antibodies, Treponema pallidum antibodies, or HIV antibodies.

9. Use of the recombinant antigen of any one of claims 1-3, the labeled conjugate of claim 4, or the kit of claim 5 in improving the activity and specificity of an immunoassay product; Optionally, the immunoassay product is a detection product for detecting hepatitis C virus antibodies, Treponema pallidum antibodies, or HIV antibodies.

10. A method of diagnosing a disease in vitro comprising: contacting the recombinant antigen of any one of claims 1-3, the labeled conjugate of claim 4, or the kit of claim 5 with an ex vivo sample to form an immunocomplex; and detecting the presence or amount of the immunocomplex. Optionally, the disease is hepatitis C, syphilis infection, or AIDS.

Images