Recombinant antigens and their uses

Abstract

This disclosure provides a recombinant antigen and its uses, belonging to the field of immunodiagnostic technology. Specifically, this disclosure relates to a recombinant antigen containing Chlorella antifreeze protein or its inverse protein as a fusion chaperone, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, reagents, a kit, and its uses. Using this recombinant antigen in immunodiagnostics can significantly improve the sensitivity and signal-to-noise ratio of immunodiagnostics.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202310848455.8, filed on July 11, 2023, entitled "Recombinant Antigen and Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of immunodiagnostic technology and relates to a recombinant antigen containing a specific fusion partner and its use; specifically, this disclosure relates to a recombinant antigen, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a reagent composition, a kit, a detection method, and its use. Background Technology

[0004] In many immunodiagnostic applications, recombinant antigens containing fusion partners are used as binding partners, for example, in immunoassays designed to detect specific immunoglobulin analytes. A portion of the recombinant antigen structure is a target polypeptide or antigenic polypeptide, which serves as the antigenic moiety and is designed to recognize and bind to a specific analyte present in the sample being tested. The other portion of the recombinant antigen structure is a fusion partner, which fuses to the specific antigenic moiety to facilitate its cloning, expression, overproduction, folding / refolding, and purification, and to increase its solubility, stability, or reversible folding.

[0005] However, in immunodiagnostic applications, recombinant antigens need to be conjugated with tracer labels or solid-phase carriers to form usable diagnostic reagents. The conjugation process introduces activators, which can easily cause conformational changes and epitope masking of the antigen. Furthermore, tracer labels or solid-phase carriers may also mask or destroy antigenic epitopes. Therefore, it is necessary to discover fusion partners suitable for immunodiagnostics to minimize antigenicity loss during the conjugation process of reagent preparation. Summary of the Invention

[0006] The inventors unexpectedly discovered that using Chlorella antifreeze protein or its reverse protein as a fusion partner can significantly improve the detection performance of recombinant antigens as diagnostic reagents, outperforming existing fusion partners.

[0007] A first aspect of the present invention provides a recombinant antigen, wherein the recombinant antigen contains at least one polypeptide sequence corresponding to a target polypeptide and at least one polypeptide sequence corresponding to a Chlorella antifreeze protein or its inverse protein as a fusion partner.

[0008] The target polypeptide is either a pathogen polypeptide or an autoimmune disease antigen polypeptide. In an individual, the polypeptide of an exogenous pathogen triggers an immune response, producing corresponding autoantibodies. In patients with autoimmune diseases, the state of autoimmune tolerance is broken, and endogenous antigen polypeptides also induce the production of autoantibodies. Immunodiagnosis of these autoantibodies is an effective means of determining whether an individual is infected with the corresponding pathogen or suffers from the corresponding autoimmune disease.

[0009] A second aspect of the invention provides a detection reagent / test kit or test strip, comprising the aforementioned recombinant antigen and a tracer label or solid-phase carrier.

[0010] The tracer or solid-phase carrier is directly coupled to the recombinant antigen, or indirectly coupled to the recombinant antigen through an intermediate medium.

[0011] In this process, the solid support acts as a separation medium to capture / separate analytes from the sample, while the tracer label acts as a signal molecule to characterize the presence and content of the analyte.

[0012] A third aspect of the invention provides the use of the aforementioned recombinant antigen in immunodiagnostics or as an immunogen in the preparation of antibodies against the target polypeptide.

[0013] A fourth aspect of the invention provides the use of the aforementioned recombinant antigen in the production of vaccines.

[0014] A fifth aspect of the present invention 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.

[0015] A sixth aspect of the present invention provides a method for detecting an analyte in a sample, comprising: mixing a first recombinant antigen and a second recombinant antigen, each comprising a target polypeptide sequence and a fusion chaperone sequence, with the sample to form a first recombinant antigen-analyte-second recombinant antigen immune-binding complex; detecting the presence or content of the immune-binding complex; wherein the fusion chaperone of the first recombinant antigen and the second recombinant antigen corresponds to a polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of its inverse protein, and the fusion chaperone of the other recombinant antigen does not correspond to a polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of its inverse protein. Attached Figure Description

[0016] Figure 1 The PE carrier spectrum used in the embodiments of this disclosure is shown. Detailed Implementation

[0017] When used in conjunction with the term “comprising” in the claims and / or specification, the words “a” or “an” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0018] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0019] Throughout the application, the term “about” means: a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0020] While the disclosure supports the definition of the term "or" as merely a substitute and "and / or", the term "or" in the claims means "and / or" unless expressly stated as merely a substitute or as mutually exclusive among substitutes.

[0021] The term "pathogen polypeptide" refers to polypeptides of exogenous pathogens, such as bacteria, viruses, and other microorganisms, that cause disease directly or indirectly. Exemplary pathogens include, for example, *Yersinia*, *Klebsiella*, *Providencia*, *Erwinia*, *Enterobacter*, *Salmonella*, *Serratia*, *Aerobacter*, *Escherichia*, *Pseudomonas*, *Shigella*, *Vibrio*, *Aeromonas*, and *Streptococcus*. (Streptococcus), Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium, Corynebacterium, Eberthella, Francisella, Haemophilus, Bacteroides, Listeria, Erysipelothrix x), Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium, Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia Genus *Nocardia*, Genus *Rickettsia*, Genus *Micrococcus*, Genus *Mycobacterium*, Genus *Neisseria*, Genus *Campylobacter*, pathogenic viruses (such as papillomavirus, parvovirus, adenovirus, herpesvirus, vaccine virus, arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, piconemavirus, paramyxovirus, reovirus, retrovirus, rhabdovirus, human immunodeficiency virus (HIV), genus *Taenia*, genus *Hymenolepsis*.Genus *Diphyllobothrium*, *Echinococcus*, *Fasciolopsis*, *Heterophyes*, *Metagonimus*, *Clonorchis*, *Fasciola*, *Paragonimus*, *Schistosoma*, *Enterobius*, *Trichuris*, *Ascaris*, *Ancylostoma*, *Necator* The genera *Wuchereria*, *Brugi*, *Loa*, *Onchocerca*, *Dracunculus*, *Naegleria*, *Acanthamoeba*, *Plasmodium*, *Trypanosoma*, *Leishmania*, *Toxoplasma*, *Entamoeba*, *Giardia*, *Isospora*, and *Cryptospora* are mentioned. *Idium*, *Enterocytozoa*, *Strongyloides*, *Trichinella*, fungal causes (e.g., ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidiodomycosis, paracoccidioidomycosis, mucormycosis) Mucomycosis, Candidiasis, Dermatophytosis, Protothecosis, Pityriasis, Mycetoma, Paracoccidiodomycosis, Phaeohhomycosis, Pseudallescheriasis, Trichosporosis, Pneumocystis, Adenovirus, Coronavirus, Human Metapneumovirus, Human RhinovirusEnteroviruses, 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 pertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and combinations thereof. Preferred viruses include SARS-CoV-2, 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-lymphoblastic leukemia virus, dengue virus, human papillomavirus, West Nile virus, encephalitis virus, measles virus, influenza virus, and parainfluenza virus. Varicella virus, ecovirus, Coxsackie virus, Japanese encephalitis virus, Epstein-Barr virus, mumps virus, Treponema pallidum, Borrelia burgdorferi infection, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Ureaplasma urealyticum, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Helicobacter pylori, Neisseria gonorrhoeae, Plasmodium, Trypanosoma cruzi, and Toxoplasma gondii.

[0022] The term "autoimmune disease antigen polypeptide" refers to endogenous polypeptides that cause autoimmune diseases. Exemplary autoimmune diseases include, for example: type 1 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, granulomatous disease with polyangiitis, Kawasaki disease, lupus nephritis, polyarteritis nodosa, pyoderma gangrenosa, vertebral arthritis, systemic lupus erythematosus, and taranoarteritis.

[0023] The term "Chlorella antifreeze protein" refers to the antifreeze protein of Chlorella, as exemplified by SEQ NO.1 (NCBI accession number: PRW45461). The term "reverse protein of Chlorella antifreeze protein" refers to the reverse coding sequence of the Chlorella antifreeze protein, i.e., placing the N-terminal amino acid at the C-terminus and the C-terminal amino acid at the N-terminus, thus reverse coding the entire polypeptide product, as exemplified by SEQ NO.2. "Polypeptide sequence corresponding to Chlorella antifreeze protein" and "polypeptide sequence of Chlorella antifreeze protein" can be interchanged. The polypeptide sequence of Chlorella antifreeze protein can be the full-length 192aa antifreeze protein sequence shown in SEQ NO.1, or it can be a truncated antifreeze protein sequence, including amino acids X to Y of SEQ NO.1, where X is selected from any integer from 1 to 20, and Y is selected from any integer from 173 to 192. Exemplary and non-limiting examples include, for example, amino acids 2-141, 3-141, 4-141, 5-141, 6-141, 7-141, 8-141, 9-141, 10-141, 18-141, 19-141, and 20-141 of SEQ NO.1 (only the N-terminus is truncated); for example, amino acids 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, and 1-173 of SEQ NO.1 (only the C-terminus is truncated); for example, SEQ NO.1 The amino acid sequences corresponding to the Chlorella antifreeze protein in SEQ ID NO:1 are amino acids 2-191, 15-191, 16-191, 17-191, 18-191, 19-191, 3-190, 4-189, 5-188, 6-187, 7-186, 8-185, 9-184, 10-183, 20-174, and 20-173 (in cases where both the N-terminus and C-terminus are truncated). Variants of the polypeptide sequence corresponding to the Chlorella antifreeze protein may exist, for example, sequences 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 with SEQ ID NO:1.

[0024] "Polypeptide sequence corresponding to the reverse protein of Chlorella antifreeze protein" and "Polypeptide sequence of the reverse protein of Chlorella antifreeze protein" can be interchanged. The polypeptide sequence of the reverse protein of Chlorella antifreeze protein can be the full-length reverse protein sequence of 192aa as shown in SEQ NO.2, or it can be a truncated reverse protein sequence, including amino acids from position X to position Y of SEQ NO.2, where X is selected from any integer from 1 to 20, and Y is selected from any integer from 173 to 192. Exemplary and non-limiting examples include, for example, amino acids 2-141, 3-141, 4-141, 5-141, 6-141, 7-141, 8-141, 9-141, 10-141, 18-141, 19-141, and 20-141 of SEQ NO.1 (only the N-terminus is truncated); for example, amino acids 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, and 1-173 of SEQ NO.1 (only the C-terminus is truncated); for example, SEQ NO.1 The amino acid sequences of NO.1, specifically amino acids 2-191, 15-191, 16-191, 17-191, 18-191, 19-191, 3-190, 4-189, 5-188, 6-187, 7-186, 8-185, 9-184, 10-183, 20-174, and 20-173 (with both the N-terminus and C-terminus truncated), can exist as variants of the inverse protein corresponding to the Chlorella antifreeze protein, for example, sequences 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 with SEQ ID NO:2.

[0025] The terms "the polypeptide sequence corresponding to the target polypeptide" and "the polypeptide sequence of the target polypeptide" are interchangeable.

[0026] The term "fusion partner" refers to a fusion or linkage to the N-terminus and / or C-terminus of a target polypeptide (optionally via a linker or protease cleavage site). Examples of fusion partners include, but are not limited to, histidine (His) tags, glutathione transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), NusA, disulfide isomerases (DsbA, DsbB, DsbC), SUMO tags, msyB tags, priming factors (TF), FkBP family factors (FkpA, SlyD, SlpA, Skp), hSOD factors, ubiquitin tags, Myc tags, Flag tags, fluorescent protein (e.g., GFP) tags, biotin tags, and avidin tags.

[0027] The terms "peptide linker" or "linker peptide" refer to short peptides used to link two molecules (e.g., proteins). Typically, fusion proteins are obtained by introducing (e.g., by PCR amplification or ligase) the polynucleotide sequence encoding the short peptide between two DNA fragments encoding the two target proteins to be linked, and then expressing the proteins, such as peptide 1-linker peptide-peptide 2, or more specifically, target peptide-linker peptide-fusion chaperone.

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

[0029] As used in this disclosure, the term "polypeptide" refers to any molecule comprising three or more amino acid residues linked by peptide bonds. Polypeptides according to this application include peptides (e.g., tripeptides, oligopeptides, etc.), and peptides that may contain chemical modifications (e.g., glycosylation (glycopeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation). Polypeptides may also refer to proteins.

[0030] As used in this disclosure, the term "signal peptide" refers to a polypeptide that is attached to a target protein and can promote the expression or transfer of the target protein. For example, a signal peptide may be attached to the N-terminus of a target protein and is typically cleaved away, thus not present in mature proteins secreted by the cell.

[0031] As used in this disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this disclosure, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In some embodiments, the "mutation" of this disclosure may be selected from "conserved mutation," "semi-conserved mutation," and "non-conserved mutation." In this disclosure, the term "non-conserved mutation" or "semi-conserved mutation" can be a mutation that causes loss or partial loss of protein function. The term "conserved mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conserved mutation is a conserved substitution.

[0032] As used in this disclosure, a “conservative substitution” generally refers to the exchange of one amino acid at one or more sites in a protein. This substitution can be conserved. Examples of substitutions considered conserved include, specifically, substitutions of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0033] In this disclosure, "sequence identity" and "identity percentage" refer to the percentage of identical (i.e., same) nucleotides or amino acids 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 polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitution or mutation) or deleted nucleotides (i.e., nucleotide insertion or deletion in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitution or mutation) or deleted amino acids (i.e., amino acid insertion or deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0034] Exemplary, in this disclosure, when comparing and aligning two or more sequences or subsequences with maximum correspondence using sequence comparison algorithms or by visual inspection, the sequences possess at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of nucleotide or amino acid residues of “sequence identity” or “identity percentage.” The determination / calculation of “sequence identity” or “identity percentage” can be based on any suitable region of the sequence. 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 some embodiments, the sequences are substantially identical along the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).

[0035] As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or as a component of a larger nucleotide sequence structure derived from a nucleotide sequence isolated at least once in number or concentration, capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, U, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), where "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (single fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "Recombinant polynucleotides" and "recombinant nucleic acid molecules" are types of "polynucleotides".

[0036] As used in this disclosure, the term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides may be contained in a suitable vector, and the vector may be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant antigen," a "recombinant protein," a "fusion protein," etc.

[0037] As used in this disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence for expressing a target gene in a suitable host.

[0038] As used in this disclosure, the term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used.

[0039] In this disclosure, the term "host cell" refers to any cell type that is easily transformed, transfected, or transduced using gene-editing elements, nucleic acid constructs, or recombinant expression vectors containing the gene-editing elements, nucleic acid constructs, or recombinant expression vectors of this disclosure. The term "recombinant host cell" encompasses a host cell that differs from the parent cell after the introduction of gene-editing elements, nucleic acid constructs, or recombinant expression vectors; recombinant host cells are specifically achieved through transformation. The host cells of this disclosure can be prokaryotic or eukaryotic cells, as long as they are cells capable of receiving the recombinant nucleic acid molecules or recombinant expression vectors of this disclosure.

[0040] The terms “transformation,” “transfection,” and “transduction” in this disclosure have the meanings commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) 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 this disclosure is used to load tracer markers and is selected from at least one of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethyleneimine, and dendritic polymers. Specifically, polysaccharides include cross-linked sucrose and dextran; protein carriers include at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin; and dendritic polymers include polyethylene glycol and / or polypropyleneimine. In some embodiments, the amplification carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin.

[0042] The term "solid support" as used in this disclosure refers to a solid material having at least one surface capable of immobilizing and binding a pair of analytes for capturing analytes in a sample. Solid supports can take the form of particles, microparticles, nanoparticles, metal colloids, fibers, nylon, paper, beads, membranes, filter paper, and other supports such as test tubes, microplates, chips, glass slides, capillaries, and microarrays.

[0043] As used in this disclosure, the terms "sample," "sample," "sample to be tested," or "test sample" refer to any type of sample for which it is necessary to determine whether it contains Treponema pallidum antibodies. Exemplarily, a sample to be tested can be any product generated by a subject or any product derived from a product generated by a subject. The sample can be taken from any tissue or body fluid, such as blood samples (including samples derived from blood), serum samples, lymph samples, saliva samples, or synovial fluid. Samples derived from blood can be selected portions of a patient's blood or a vaccinated recipient's blood, such as selected cellular portions, or plasma or serum portions. In some embodiments, the sample can be any sample containing antibody products of a humoral immune response.

[0044] As used in this disclosure, "diagnosis" includes the detection or identification of a subject's disease state or condition, determining the likelihood that a subject will have a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or possible progression or resolution) of a subject with a disease or condition, and determining the effect of 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 having syphilis / being infected with Treponema pallidum or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, such as a pharmaceutical product) or other treatment.

[0045] As used in the context of this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human 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 in vitro, ex vivo, or in vivo forms. The term "in vitro" refers to experiments using materials, biological substances, cells, and / or tissues under laboratory conditions or in a culture medium; while the term "in vivo" refers to experiments and procedures using an intact multicellular organism. In some embodiments, in vivo methods can be performed on non-human animals. "Ex vivo" refers to events that exist outside or occur outside an organism, such as events outside a human or animal body, such as events that can exist or occur on tissues (e.g., whole organs) or cells taken from an organism.

[0047] sequence

[0048] In the technical solution disclosed herein, the meanings of the amino acid sequence listing numbers are as follows:

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

[0050] MQDESLADKAKSAIETAKHAVSDAAQKVKETVTGAAADVQETARDVTQDQRQNLG

[0051] YAEQKAADTLGDVKAAAQEAYESAKQRASEAAEGAKSTASELGGSAERAVRDAAGG

[0052] AEGAGRDAQGAAREGLKGAEGAGATDEARRHAEDVADTAKEKYSELKGDAKEGLGRAQAKGEDLAGDASKAAQDAADRLKP(SEQ ID NO:1)

[0053] The sequence shown in SEQ ID NO:2 is the polypeptide sequence of the inverse protein of the Chlorella antifreeze protein in the examples; PKLRDAADQAAKSADGALDEGKAQARGLGEKADGKLESYKEKATDAVDEAHRRAEDTAGAGEAGKLGERAAGQADRGAGEAGGAADRVAREASGGLESATSKAGEAAESARQKASEYAEQAAAKVDGLTDAAKQEAYGLNQRQDQTVDRATEQVDAAAGTVTEKVKQAADSVAHKATEIASKAKDALSEDQM (SEQ ID NO:2)

[0054] The sequence shown in SEQ ID NO:3 is the sequence of the fusion partner GST in the embodiment;

[0055] SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYS KDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD(SEQ ID NO:3)

[0056] The sequence shown in SEQ ID NO:4 is the sequence of the fusion partner MBP in the embodiment;

[0057] KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDN AGAKAGLTFLVDLIKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQ IDNO:4)

[0058] The sequence shown in SEQ ID NO:5 is the sequence of the fusion chaperone ubiquitin in the embodiment;

[0059] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRRTLSDYNIQKESTLHLVLRLRGG(SEQ ID NO:5)

[0060] The sequence shown in SEQ ID NO:6 is the sequence of the fusion partner SlyD in the embodiment;

[0061] MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGCCGGHGHDHGHEHGGEGCCGGKGNGGCGCH(SEQ ID NO:6)

[0062] The sequence shown in SEQ ID NO:7 is the sequence of the fusion partner SKP in the embodiment;

[0063] ADKIAIVNMGSLFQQVAQKTGVSNTLENEFKGRASELQRMETDLQAKMKKLQS MKAGSDRTKLEKDVMAQRQTFAQKAQAFEQDRARRSNEERGKLVTRIQTAVKSVAN SQDIDLVVDANAVAYNSSDVKDITADVLKQVK(SEQ ID NO:7)

[0064] The sequence shown in SEQ ID NO:8 is the HIV gp41 polypeptide sequence in the examples;

[0065] TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQ QLLGIWGCSGKLICTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIE ESQNQQEKNEKDLLALDSWKNLWNWFDIT(SEQ ID NO:8)

[0066] The sequence shown in SEQ ID NO:9 is the TP17 polypeptide sequence in the examples;

[0067] CVSCTTVCPHAGKAEKVECALKGGIFRGTLPAADCPGIDTTVTFNADGTAQK VELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK(SEQ IDNO:9)

[0068] Recombinant antigen

[0069] On one hand, this disclosure provides a recombinant antigen, wherein the recombinant antigen contains at least one polypeptide sequence corresponding to a target polypeptide, and at least one polypeptide sequence corresponding to Chlorella antifreeze protein or its inverse protein as a fusion partner.

[0070] In some embodiments, the Chlorella antifreeze protein is a polypeptide containing the amino acid sequence shown in SEQ ID NO:1, or a polypeptide containing one or more amino acids substituted, repeated, deleted, or added to 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.

[0071] In some embodiments, the reverse protein of Chlorella antifreeze protein is a polypeptide containing the amino acid sequence shown in SEQ ID NO:2, or a polypeptide having one or more amino acids substituted, repeated, deleted, or added to 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.

[0072] In some embodiments, the recombinant antigen provided in this disclosure comprises a target polypeptide and a fusion chaperone linked via an enzyme cleavage site.

[0073] In some embodiments, the recombinant antigen provided in this disclosure comprises a target polypeptide and a fusion chaperone linked by a linker peptide.

[0074] In some implementations, the target peptide, linker peptide, and fusion chaperone are sequentially linked to the expression vector in the order of target peptide, linker peptide, and fusion chaperone.

[0075] In some implementations, the fusion chaperone, linker peptide, and target polypeptide are sequentially linked to the expression vector in the order of fusion chaperone, linker peptide, and target polypeptide.

[0076] In some implementations, the target peptide, restriction enzyme site, and fusion chaperone are sequentially ligated onto the expression vector in the order of target peptide, restriction enzyme site, and fusion chaperone.

[0077] In some implementations, the fusion chaperone, restriction enzyme site, and target peptide are sequentially ligated onto the expression vector.

[0078] In some embodiments, the target polypeptide is selected from: COVID-19 antigen, HIV antigen (e.g., HIV-1 antigen, HIV-2 antigen, P24 antigen, O antigen), HCV antigen, HBV antigen (e.g., HBe antigen, HBS antigen), hepatitis A (HAV) antigen, hepatitis E (HEV) antigen, syphilis antigen, toxoplasmosis antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus (e.g., HSV I, HSV II), human lymphocytic T-cell leukemia (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, tyrosine bacillus antigen, tuberculosis bacillus antigen, streptococcal hemolysin O, cyclic citrulline peptide, and monkeypox virus antigen.

[0079] In some embodiments, there are no particular restrictions on the length of the target polypeptide fragment; it can be a full-length antigen polypeptide or an antigen fragment with 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, while in some specific embodiments, the antigen can be a truncated antigen fragment.

[0080] In some implementations, the target peptide is the HIV-1 gp41 antigen.

[0081] In some embodiments, the HIV-1 gp41 antigen comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:8.

[0082] In some embodiments, the HIV-1 gp41 antigen comprises a portion of a polypeptide with the amino acid sequence shown in SEQ ID NO:8.

[0083] In some embodiments, the HIV-1 gp41 antigen comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:8, or a polypeptide with one or more amino acids substituted, repeated, deleted, or added to the amino acid sequence as shown in SEQ ID NO:8, and having or partially having the polypeptide activity of the sequence shown in SEQ ID NO:8.

[0084] In some implementations, the target polypeptide is the TP17 antigen of Treponema pallidum.

[0085] In some embodiments, the TP17 antigen comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:9.

[0086] In some embodiments, the TP17 antigen comprises a portion of a polypeptide with the amino acid sequence shown in SEQ ID NO:9.

[0087] In some embodiments, the TP17 antigen comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:9, or a polypeptide with one or more amino acids substituted, repeated, deleted, or added to the amino acid sequence as shown in SEQ ID NO:9, and having or partially having the polypeptide activity of the sequence shown in SEQ ID NO:9.

[0088] In this disclosure, there are no particular restrictions on the selection of the linker peptide. In some preferred embodiments, the linker peptide can be a flexible linker peptide. Linker peptides include, but are not limited to, GS, GGSGG, KESGSVSSEQLAQFRSLD, GSAGSAAGSGEF, GGSGGEFGGSGG, etc. In some preferred embodiments, the linker peptide is GGSGG. In some preferred embodiments, the linker peptide is GS.

[0089] In some embodiments, the recombinant antigen further comprises a tag peptide at its N-terminus and / or C-terminus. Exemplarily, the tag peptide is a His tag, preferably a 6×His tag, which is linked to the N-terminus of the recombinant antigen to facilitate NI-NTA affinity chromatography purification of the recombinant antigen protein. In some alternative embodiments, the recombinant antigen and the tag peptide can be linked by a linker peptide.

[0090] In some implementations, the recombinant antigen is also conjugated with a tracer.

[0091] In some implementations, the tracer is based on indirect coupling between the anti-fusion chaperone antibody and the recombinant antigen, i.e., the tracer is directly coupled to the anti-fusion chaperone antibody, which specifically binds to the fusion chaperone portion of the coupled recombinant antigen.

[0092] In some implementations, the tracer is indirectly coupled to the recombinant antigen via an amplification carrier, meaning the tracer is directly coupled to the amplification carrier, and the recombinant antigen is also directly coupled to the same amplification carrier.

[0093] In some implementations, the recombinant antigen is also coupled with a solid-phase carrier.

[0094] In some implementations, the recombinant antigen is coupled to a solid-phase carrier during detection based on a "transfer tag" and a "transfer tag receptor." For example, the recombinant antigen is labeled with biotin (transfer tag), while the solid-phase carrier is coated with avidin (transfer tag receptor); the recombinant antigen carries a His tag (transfer tag), while the surface of the solid-phase carrier has nickel (transfer tag receptor); the recombinant antigen is labeled with dinitrobenzene (transfer tag), while the solid-phase carrier is coated with anti-dinitrophenyl antibody (transfer tag receptor), etc.

[0095] In this disclosure, exemplary recombinant antigens may have any of the following structures:

[0096] (1) Tag - Optional linker peptide - Target peptide - Optional linker peptide - Fusion partner;

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

[0098] On the one hand, this disclosure provides a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a recombinant receptor-binding protein of any of the above, wherein the recombinant nucleic acid molecule may be DNA, RNA or a combination thereof.

[0099] On one hand, this disclosure provides a recombinant expression vector containing the aforementioned recombinant nucleic acid molecule, which can be used to transfer the recombinant nucleic acid molecule into cells. In some embodiments, the recombinant expression vector contains one or more regulatory elements, which can be elements commonly used in the art such as promoters, enhancers, silencers, and insulators. The regulatory elements are operatively 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 situations where a selected nucleic acid molecule sequence is covalently linked to a regulatory element sequence (e.g., a promoter and / or enhancer) to place the expression of the nucleic acid sequence under the influence or control of the regulatory element (thus forming an expression cassette). Therefore, if a regulatory element can act on 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 or protein.

[0101] In this disclosure, vectors suitable for constructing recombinant expression vectors 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, adenovirus vectors, adenovirus-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0102] In some embodiments, the vector is a prokaryotic vector containing various elements expressed 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, and PE vectors. Preferably, the vector is a PE vector.

[0103] On the one hand, this disclosure provides a recombinant host cell comprising the aforementioned recombinant nucleic acid molecule or recombinant expression vector. The recombinant host cell is obtained by transforming, transfecting, or transducing the recombinant nucleic acid molecule or recombinant expression vector into a host cell. The host cell in this disclosure can be a eukaryotic cell or a prokaryotic cell, as long as it can accommodate the introduction of the recombinant nucleic acid molecule or recombinant expression vector of this disclosure to achieve recombinant receptor-binding protein expression.

[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, Actinobacter, Acetobacter, Lactobacillus), or spirilla (e.g., Spirochetes breve). In some embodiments, the host cell is Escherichia coli ER2529 or Escherichia coli ER2566; preferably, it is Escherichia 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, for example, obtained by introducing a recombinant expression vector into *E. coli*.

[0106] Recombinant host cells are cultured under conditions suitable for protein expression. After the culture is completed, the recombinant antigen protein is collected from the cell culture medium or fermentation broth and then purified.

[0107] In some embodiments, the steps of purifying the recombinant antigen protein include Ni-NTA affinity chromatography and protein dialysis.

[0108] Reagents / Reagent Kits

[0109] On the one hand, this disclosure provides a detection reagent comprising a recombinant antigen. This disclosure discovers that by fusing Chlorella antibody protein or its inverse protein with a target polypeptide in an Escherichia coli expression system, the fused recombinant antigen can maintain its antigenicity during the coupling process. Compared to recombinant antigens obtained by fusion expression using other fusion partners, the detection reagent prepared using the recombinant antigen of this disclosure can significantly improve detection sensitivity and signal-to-noise ratio.

[0110] In some embodiments, the recombinant antigen is conjugated with a tracer label capable of generating a detection signal. The tracer label is selected from at least one of acridine ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, colloidal gold, fluorescent microspheres, ruthenium terpyridine, quantum dot luminescent materials, and upconversion luminescent materials. For example, the signal molecule is acridine ester (AE), which exhibits high luminescence efficiency when oxidized by H₂O₂ under alkaline conditions.

[0111] On one hand, the present invention provides kits containing recombinant antigens of the present invention, such as kits for detecting the presence of corresponding autoantibodies from subject samples. In some embodiments, the kit includes reagents suitable for immunoassays. In some embodiments, the kit may include instructions for using immunodiagnostic reagents of the present invention (e.g., detection reagents containing the corresponding recombinant antigens) in immunoassays for detecting autoantibodies. In some embodiments, the kit may contain calibrators or controls, such as standard or control HIV antibodies. In some embodiments, the HIV protein or conjugate of the present invention is contained in a container such as a test tube, microplate, or test strip in the kit. In some embodiments, the kit may also contain solid-phase supports such as magnetic beads, test tubes, microplates, cuvettes, membranes, filter paper, syringes, pipettes, buffers such as assay buffers, wash buffers, pretreatment reagents, tracer labels such as enzyme-labeled substrate solutions, etc.

[0112] On one hand, the present invention includes test strips containing the recombinant antigen of the present invention, such as lateral chromatography test strips. In some embodiments, the test strip contains HIV protein coated thereon, at least one detection antibody or at least one detection antigen with a tracer label (such as colloidal gold). In some embodiments, the test strip contains a recombinant antigen conjugated with a tracer label, at least one detection antibody or at least one detection antigen coated thereon. In some embodiments, the present invention can rapidly and accurately detect autoantibodies in a sample by visual inspection or by a fully automated chemiluminescence instrument. In some embodiments, the kit can be prepared using a double-antigen sandwich principle. For example, in some embodiments, autoantibodies in the sample are captured by HIV protein coated on a solid-phase support, or autoantibodies in the sample are detected by recombinant antigen labeled with a tracer label. In some embodiments, the kit can be prepared using an indirect method principle. For example, in some embodiments, autoantibodies in the sample are captured by recombinant antigen coated on a solid-phase support. In some implementations, an excitation solution is added, and the luminescence value is measured using a fully automated chemiluminescence instrument. The luminescence value is positively correlated with the total concentration of autoantibodies in the sample, and compared with a critical value to determine whether the result is positive or negative.

[0113] method

[0114] On one hand, this disclosure provides a method for detecting an analyte in a sample, comprising: mixing a first recombinant antigen and a second recombinant antigen, each comprising a target polypeptide sequence and a fusion chaperone sequence, with the sample to form a first recombinant antigen-analyte-second recombinant antigen immune binding complex; and detecting the presence or content of the immune binding complex.

[0115] The fusion partner of one of the first and second recombinant antigens corresponds to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of its inverse protein, while the fusion partner of the other recombinant antigen does not correspond to the polypeptide sequence of Chlorella antifreeze protein or its inverse protein.

[0116] In some embodiments, the first recombinant antigen is the recombinant antigen described in the first aspect above, which will not be repeated here.

[0117] In some embodiments, the target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. The types of pathogen polypeptides and autoimmune disease antigen polypeptides are as described above and will not be repeated here.

[0118] Example

[0119] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0120] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those 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. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0121] The PE carrier used in the following embodiments has the following carrier spectrum: Figure 1 As shown: The PE vector itself contains a 6×His tag at its N-terminus, so the recombinant antigens obtained by translation all contain 6 histidine tags, which can promote NI-NTA-assisted affinity purification.

[0122] Example 1: Preparation of HIV gp41 recombinant antigen

[0123] 1.1 Design of gp41 recombinant antigen

[0124] Table 1

[0125]

[0126] Based on the design table in Table 1 above for the recombinant antigen, a corresponding fusion chaperone was introduced at the N-terminus of the target polypeptide gp41, and an expression plasmid was constructed according to the coding nucleic acid of the protein sequence. The gene sequence contains restriction enzyme sites BamHI and EcoRI at both ends. The mutant gene was treated with restriction endonucleases BamHI and EcoRI, respectively, and the treated gene fragment was ligated into Phypon's proprietary vector PE (e.g., ...). Figure 1 As shown in the figure, the corresponding expression plasmid was obtained. A linker GGSGG was set between the target peptide and the fusion chaperone.

[0127] 1.2 Recombinant antigen-induced expression

[0128] The constructed expression plasmids were transformed into *E. coli* BL21 competent cells (NEB, catalog number: C2530H) using the heat shock method and plated on LB agar plates containing 100 μg / ml Amp, and incubated at 37°C for 16 h. Colonies were picked, and positive strains identified by PCR and double enzyme digestion were selected for preservation and inoculated into LB medium containing 50 μg / ml Kan, and cultured with shaking at 37°C. Once the OD600 reached 0.6-0.8, 1.0 mM IPTG was added, and the cells were induced at 37°C for 2-4 h. Total protein was extracted, and SDS-PAGE was used to identify the expression of recombinant proteins.

[0129] (1) Expression of recombinant antigen in supernatant: Experiments showed that gp41-1 and gp41-2 were mainly expressed in supernatant, while gp41-4 was partially expressed in supernatant and partially expressed in precipitate. Protein purification of recombinant antigen was carried out using affinity column and ion exchange chromatography.

[0130] (2) Expression of recombinant antigen inclusion bodies: Experiments showed that gp41-3, gp41-5, gp41-6, and gp41-7 were basically inclusion bodies. After washing with a buffer containing Tween 20, the inclusion bodies were dissolved with 2-8M urea, and after dialysis and refolding, the recombinant antigens were purified by affinity column and ion exchange chromatography.

[0131] Example 2: Preparation of chemiluminescent reagents

[0132] 2.1 Coating of gp41 antigen

[0133] 1) Activation of magnetic microspheres (taking carboxyl magnetic beads as an example): Take carboxyl magnetic microspheres with a diameter of about 2 μm and wash them three times with 15 mL MES pH 5.5 buffer. Then add MES buffer to make the magnetic bead concentration reach 10 mg / mL. Then add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide for every 10 mg of magnetic microspheres and 2 mg of N-hydroxysuccinimide for every 10 mg of magnetic microspheres. Then place them in a shaker or mechanical stirrer at room temperature for 1 hour.

[0134] 2) Coating: Remove the supernatant from the activated magnetic microspheres in step 1), wash once with 15mM MES pH5.5 buffer, then add 15mM MES pH5.5 to achieve a concentration of 10mg / mL, and add 0.1mg of HIV gp41 coating antigen (available from Feipeng Biotechnology) per 10mg of magnetic microspheres. Then place the mixture in a shaker or mechanical stirrer at room temperature for about 2.5 hours to allow the coating antigen to be coupled to the surface of the magnetic beads.

[0135] 3) Blocking: After the coating reaction is complete, magnetic separation is performed. The supernatant is removed, and the mixture is washed three times with TBS (25 mM Tris + 150 mM NaCl) at pH 7.5. Blocking buffer containing 0.5% BSA (TBS, pH 7.5) is added to bring the magnetic microsphere concentration to 10 mg / mL. The mixture is then placed in a shaker or mechanical stirrer at room temperature for 3 hours. After the reaction is complete, the mixture is washed with PBST (25 mM PB + 150 mM NaCl + 0.1% Tween-20, pH 7.5) and finally stored in 50 mM HEPES pH 7.4 buffer to obtain the magnetic bead working solution, which is then stored at 2–8 degrees Celsius.

[0136] 2.2 Direct labeling of gp41 antigen

[0137] The six recombinant antigens prepared in Example 1 were diluted to a concentration of 1 mg / ml. 1 ml of each sample was placed in a dialysis bag and dialyzed overnight in 1×PBS (pH 7.2). The next day, the dialyzed antigen was removed and placed in a centrifuge tube. 16.7 μL of dissolved acridine ester was added, and the mixture was vortexed at room temperature for 15 min. Then, 3.34 μL of glycine was added to terminate the reaction. After terminating the reaction, the mixture was placed back in a dialysis bag and dialyzed overnight in 1×PBS (pH 7.2) at 4°C. After dialysis, an equal volume of glycerol was added, mixed well, and stored at -20°C to obtain the acridine ester-labeled gp41 recombinant antigen (AE-Ag).

[0138] 2.3 Indirect labeling of gp41 antigen

[0139] Take 100 μL of 10 mg / mL BSA solution and add pre-dissolved acrid ester solution, reacting for 15 minutes. Then, desalt the solution using a desalting column, add Traut reagent, and react for 15 minutes. The desalted BSA intermediate is obtained for later use. Take 100 μL of 2 mg / mL recombinant antigen and add pre-dissolved SMCC crosslinker solution, reacting for 15 minutes. Mix the resulting solution with the BSA intermediate in a specific ratio and react for 24 hours. After the reaction, dialysis is performed to purify the solution, yielding the acrid ester-labeled gp41 recombinant antigen (AE-BSA-Ag).

[0140] Example 3: Chemiluminescence test

[0141] The reagents prepared in Example 2 were used to detect HIV clinical samples (Feipeng Biotechnology) using the double antigen sandwich method.

[0142] 3.1 Testing Process

[0143] 1) Place 100 μL of sample and 50 μL of magnetic bead working solution coated with gp41 recombinant antigen into the well of the luminescent plate and mix and react at 37°C for 15 min;

[0144] 2) Then, the magnetic beads are adsorbed by a magnetic plate and washed with 1×PBST solution;

[0145] 3) Add 100 μL of HIV AE-labeled working solution to the wells of the luminescent plate and mix and react at 37°C for 10 min.

[0146] 4) Then, the magnetic beads are adsorbed again using a magnetic plate and washed with 1×PBST solution;

[0147] 5) Then add AE pre-excitation solution and AE excitation solution, and read the luminescence value using a ThermoFisher chemiluminescence analyzer.

[0148] Test results:

[0149] Table 2. Test results for direct labeling of gp41 recombinant antigen

[0150] Gp41-1 Gp41-2 Gp41-6 Gp41-3 Gp41-4 Gp41-5 Gp41-7 Negative control 923 899 817 822 835 1021 907 Low-value positive quality control 245132 274733 200433 203621 193011 190133 163384 High-value positive quality control 517801 523233 468900 451834 430883 434800 317664 Low P / N value 265.5 305.5 245.3 247.7 231.1 186.2 180.1 High P / N value 560.9 582 573.9 549.6 516 425.8 350.2

[0151] The results showed that Chlorella antifreeze protein or its reverse sequence, as a fusion partner for recombinant gp41 antigen, exhibited superior detection activity or signal-to-noise ratio (P / N) compared to other fusion partners or no fusion partner.

[0152] Recombinant antigens gp41-1, gp41-2, gp41-6, and gp41-3 were used for indirect labeling with acridine esters according to Example 2.3, and then tested.

[0153] Table 3. Test results of indirect labeling with gp41 recombinant antigen

[0154] gp41-1 gp41-2 gp41-6 gp41-3 Negative control 605 594 567 622.5 Low-value positive quality control 250972 275794 197372 143563 Median positive quality control 338879 356192 256882 211634 High-value positive quality control 915484 984367 701409 551328 Low P / N value 414.8 464.2 348 230.6 Median P / N 560.1 599.6 453 339.9 High P / N value 1513.1 1657.1 1237 885.6

[0155] The results show that, when using amplifying carrier (BSA) for indirect labeling, gp41-1 and gp41-2 further enhance the detection performance advantage over gp41-6 and gp41-3.

[0156] Following the methods described in Examples 1 to 3, the fusion partner effect was verified on the target polypeptide, Treponema pallidum antigen TP17 (SEQ ID NO: 9). The results showed that Chlorella antifreeze protein or its reverse sequence, as a fusion partner for the recombinant TP17 antigen, exhibited superior detection activity and signal-to-noise ratio (P / N) compared to other fusion partners or no fusion partner. Exemplary data are shown in Table 4.

[0157] Table 4. Test results of indirect labeling with TP17 recombinant antigen

[0158]

[0159]

[0160] This demonstrates that Chlorella antifreeze protein and its reverse protein, as fusion partners in recombinant antigens, have the advantage of improving detection performance. It is speculated that this is because Chlorella antifreeze protein and its reverse protein contain a large number of hydrophilic amino acids, which are distributed on the protein surface, resulting in good hydrophilicity. Furthermore, the surface of Chlorella antifreeze protein is rich in amino acids with the same charge, which can weaken the formation of protein aggregates and indirectly prevent the formation of disordered multimers of recombinant antigens.

[0161] Although embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present disclosure. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present disclosure.

Claims

1. A recombinant antigen, wherein the structure of the recombinant antigen is: N-fusion chaperone-linking peptide-target polypeptide-C; wherein, The fusion partner is Chlorella antifreeze protein or its inverse protein, the target polypeptide is HIV-1 gp41 antigen or Treponema pallidum TP17 antigen, the polypeptide sequence of HIV-1 gp41 antigen is shown in SEQ ID NO:8, the polypeptide sequence of Treponema pallidum TP17 antigen is shown in SEQ ID NO:9, the polypeptide sequences of Chlorella antifreeze protein and its inverse protein are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, and the linker peptide is a flexible linker peptide.

2. A detection reagent, wherein, The reagent includes the recombinant antigen according to claim 1, and a tracer label or solid carrier conjugated to the recombinant antigen.

3. The detection reagent according to claim 2, wherein, The recombinant antigen is indirectly conjugated with a tracer.

4. A test kit or test strip, wherein, The kit or test strip contains the recombinant antigen of claim 1, or the reagent of any one of claims 2-3.

5. The use of the recombinant antigen of claim 1 in the preparation of immunodiagnostic agents for HIV infection, wherein the target polypeptide in the recombinant antigen of claim 1 is the HIV-1 gp41 antigen; or The use of the recombinant antigen of claim 1 in the preparation of an immunodiagnostic agent for Treponema pallidum infection, wherein the target polypeptide in the recombinant antigen of claim 1 is the TP17 antigen of Treponema pallidum.

6. A recombinant nucleic acid molecule encoding the recombinant antigen of claim 1.

7. An expression vector containing the recombinant nucleic acid molecule of claim 6.

8. A host cell containing the expression vector of claim 7.

9. A method for detecting an analyte in a sample for non-diagnostic purposes, comprising: The first and second recombinant antigens, each containing the target polypeptide sequence and the fusion chaperone sequence, are mixed with the sample to form a first recombinant antigen-analyte-second recombinant antigen immune binding complex. Detect the presence or content of the immune-binding complex; The fusion partner of one of the first and second recombinant antigens corresponds to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reverse protein of Chlorella antifreeze protein, while the fusion partner of the other recombinant antigen in the first and second recombinant antigens does not correspond to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of its reverse protein. The target polypeptide is either the HIV-1 gp41 antigen or the Treponema pallidum TP17 antigen. The polypeptide sequence of the HIV-1 gp41 antigen is shown in SEQ ID NO:8, the polypeptide sequence of the Treponema pallidum TP17 antigen is shown in SEQ ID NO:9, and the polypeptide sequences of the Chlorella antifreeze protein and its inverse protein are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The structure of the recombinant antigen is: N-fusion chaperone-linker peptide-target polypeptide-C, and the linker peptide is a flexible linker peptide.

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