Recombinant antigen and use thereof

Abstract

Provided are a recombinant antigen and a use thereof, relating to the technical field of immunodiagnosis. In particular, the present invention relates to a recombinant antigen containing a chlorella antifreeze protein or a reverse protein thereof as a fusion partner, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a reagent, a kit, and a use. The use of the recombinant antigen for immunodiagnosis can remarkably improve the sensitivity and signal-to-noise ratio of immunodiagnosis.

Description

Recombinant antigens and their uses

[0001] Priority information

[0002] This application claims priority to Chinese patent application number 202310848455.8, entitled “Recombinant Antigens and Their Uses,” filed with the Chinese Patent Office on July 11, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of immunodiagnosis technology, and relates to a recombinant antigen containing a specific fusion partner and its use; specifically, the present 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 Art

[0004] In many immunodiagnostic applications, recombinant antigens produced recombinantly and containing a fusion partner are used as binding partners, for example, in immunoassays designed to detect specific immunoglobulin analytes. The recombinant antigen structure contains as one part a target polypeptide or antigenic polypeptide as the antigenic portion, which is designed to recognize and bind to the specific analyte present in the sample being tested; the recombinant antigen structure also contains as another part a fusion partner, which is fused to the antigenic portion that confers specificity, in order to facilitate its cloning, expression, overproduction, folding / refolding and purification, and to increase its solubility, its stability or its reversibility of folding.

[0005] However, in immunodiagnostic applications, recombinant antigens need to be coupled to tracer markers or solid-phase carriers to form usable diagnostic reagents. During the coupling process, activators are introduced, which can easily cause conformational changes and epitope shielding in the antigen. In addition, tracer markers or solid-phase carriers may also shield and destroy the antigen's epitopes. Therefore, it is necessary to discover fusion partners suitable for immunodiagnosis to reduce antigenicity loss of recombinant antigens during the coupling process in reagent preparation.

[0006] Summary of the Invention

[0007] The inventors unexpectedly discovered that using Chlorella antifreeze protein or its reverse protein as a fusion partner can greatly improve the detection performance of recombinant antigens as diagnostic reagents, which is better than existing fusion partners.

[0008] The first aspect of the present 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 a Chlorella antifreeze protein or its reverse protein as a fusion partner.

[0009] The target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. Foreign pathogen polypeptides trigger an immune response in an individual, producing corresponding autoantibodies. In patients with autoimmune diseases, the state of autoimmune tolerance is broken, and endogenous antigen polypeptides can 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.

[0010] The second aspect of the present disclosure provides a detection reagent / detection kit or test strip, comprising the aforementioned recombinant antigen, and a tracing marker or a solid phase carrier.

[0011] The tracer marker or solid phase carrier is directly coupled to the recombinant antigen, or is indirectly coupled to the recombinant antigen through an intermediate medium.

[0012] The solid phase carrier serves as a separation medium to capture / separate the analyte present in the sample during the detection process, and the tracer marker serves as a signal molecule to characterize the presence and content of the analyte during the detection process.

[0013] The third aspect of the present disclosure provides the use of the aforementioned recombinant antigen in immunodiagnosis or as an immunogen to prepare antibodies against a target polypeptide.

[0014] The fourth aspect of the present disclosure provides use of the aforementioned recombinant antigen in producing a vaccine.

[0015] The fifth aspect of the present disclosure 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.

[0016] A sixth aspect of the present 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 partner sequence, with the sample to form a first recombinant antigen-analyte-second recombinant antigen immune binding complex; and detecting the presence or amount of the immune binding complex; wherein the fusion partner of one of the first recombinant antigen and the second recombinant antigen corresponds to a polypeptide sequence of a Chlorella antifreeze protein or a polypeptide sequence of its inverse protein, and the fusion partner of the other of the first recombinant antigen and the second recombinant antigen does not correspond to a polypeptide sequence of a Chlorella antifreeze protein or a polypeptide sequence of its inverse protein.

[0017] In a seventh aspect of the disclosure, a method for diagnosing whether an individual suffers from a pathogen infection and / or an autoimmune disease is provided, comprising: mixing a first recombinant antigen and a second recombinant antigen, each comprising a target polypeptide sequence and a fusion partner sequence, with a sample from a test individual to form a first recombinant antigen-disease analyte-second recombinant antigen immune binding complex; and judging whether the individual suffers from a pathogen infection and / or an autoimmune disease based on the presence or amount of the immune binding complex; wherein the fusion partner of one of the first recombinant antigen and the second recombinant antigen corresponds to a polypeptide sequence of Chlorella antifreeze protein or a polypeptide sequence of an inverted protein of Chlorella antifreeze protein, and the fusion partner of the other of the first recombinant antigen and the second recombinant antigen does not correspond to a polypeptide sequence of Chlorella antifreeze protein or a polypeptide sequence of an inverted protein of Chlorella antifreeze protein.

[0018] In an eighth aspect, the present disclosure provides use of the aforementioned recombinant antigen, detection reagent, kit or test strip in diagnosing whether an individual has a pathogen infection and / or an autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 shows a map of the PE carrier used in the examples of the present disclosure. DETAILED DESCRIPTION

[0020] The following embodiments of the present disclosure are described in detail. The embodiments described below are exemplary and are intended only to explain the present disclosure and are not to be construed as limiting the present disclosure. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or in the product instructions were used. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products.

[0021] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0022] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0024] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0025] The term "pathogen polypeptide" refers to polypeptides of exogenous pathogens such as bacteria, viruses, and other microorganisms that directly or indirectly cause disease. Exemplary pathogens include, for example, Yersinia, Klebsiella, Providencia, Erwinia, Enterobacter, Salmonella, Serratia, Aerobacter, Escherichia, Pseudomonas, Shigella, Vibrio, Aeromonas, , Streptococcus, Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium, Corynebacterium, Eberthella, Francisella, Haemophilus, Bacteroides, Listeria, Erysipelothrix sipelothrix), Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium, Legionella, Treponema, Borrelia, Leptospira, Actinomyces ctinomyces), Nocardia, Rickettsia, Micrococcus, Mycobacterium, Neisseria, Campylobacter, pathogenic viruses (such as, for example, papillomaviruses, parvoviruses, adenoviruses, herpesviruses, vacciniaviruses, arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial virus, influenza virus, picornaviruses, paramyxoviruses, reoviruses, retroviruses, rhabdoviruses, human immunodeficiency virus (HIV), tapeworms,Hymenolepsis, Diphyllobothrium, Echinococcus, Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fasciola, Paragonimus, Schistosoma, Enterobius, Trichuris, Ascaris, Ancylos toma), Necator, Wuchereria, Brugi, Loa, Onchocerca, Dracunculus, Naegleria, Acanthamoeba, Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora ra), Cryptosporidium, Enterocytozoa, Strongyloides, Trichinella), fungal causes (e.g., Ringworm, Histoplasmosis, Blastomycosis, Aspergillosis, Cryptococcosis, Sporotrichosis, Coccidiodomycosis, Paracoccidioidomycosis, idioidomycosis), Mucomycosis, Candidiasis, Dermatophytosis, Protothecosis, Pityriasis, Mycetoma, Paracoccidiodomycosis, Phaeohphomycosis, Pseudallescheriasis, Trichosporosis, Pneumocystis,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, Chlamydia pneumoniae, Mycoplasma pneumoniae, and combinations thereof. Preferred are novel coronavirus, 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-lymphotropic virus, dengue virus, human papillomavirus, West Nile virus, encephalitis virus, measles virus, influenza virus, 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.

[0026] 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, rheumatoid arthritis, graft-versus-host disease (GVHD), nephritis, multiple sclerosis, mixed connective tissue disorder, pemphigus vulgaris, bullous pemphigoid, membranous glomerulonephritis, neuromyelitis optica, autoimmune encephalomyelitis, autoimmune hepatitis, chronic inflammatory demyelinating polyneuropathy, dermatomyositis, giant cell arteritis, granulomatosis with polyangiitis, Kawasaki disease, lupus nephritis, polyarteritis nodosa, pyoderma gangrenosum, spondyloarthritis, systemic lupus erythematosus, and Takayasu's arteritis.

[0027] The term "Chlorella antifreeze protein" refers to an antifreeze protein from Chlorella vulgaris, as exemplified by SEQ NO. 1 (NCBI Accession No. PRW45461). The term "reverse version of Chlorella antifreeze protein" refers to the reverse coding sequence of the Chlorella antifreeze protein, i.e., the N-terminal amino acid is positioned at the C-terminus, and the C-terminal amino acid is positioned at the N-terminus, resulting in a polypeptide product encoded in reverse order, as exemplified by SEQ NO. 2. The phrase "polypeptide sequence corresponding to the Chlorella antifreeze protein" and "polypeptide sequence of the Chlorella antifreeze protein" are interchangeable. The polypeptide sequence of the Chlorella antifreeze protein can be the full-length 192 amino acid sequence as shown in SEQ NO. 1, or a truncated version of the antifreeze protein sequence, including amino acids X to Y of SEQ NO. 1, where X is selected from any integer between 1 and 20, and Y is selected from any integer between 173 and 192. Exemplary, non-limiting, 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, 20-141, etc. (only in the case of N-terminal truncation); for example, amino acids 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, 1-173, etc. (only in the case of C-terminal truncation) of SEQ NO. 1; for example, amino acids 1-173, 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, 1-173, etc. (only in the case of C-terminal truncation) of SEQ NO. 1; 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, 20-173 of SEQ ID NO: 1 (in the case of truncations at both the N-terminus and the C-terminus). 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 to SEQ ID NO: 1.

[0028] The “polypeptide sequence corresponding to the reverse protein of the Chlorella antifreeze protein” and the “polypeptide sequence of the reverse protein of the Chlorella antifreeze protein” can be interchangeable. The polypeptide sequence of the reverse protein of the Chlorella antifreeze protein can be the full-length reverse protein sequence of 192 aa as shown in SEQ NO. 2, or it can be a truncated reverse protein sequence including amino acids X to Y of SEQ NO. 2, wherein X is selected from any integer from 1 to 20, and Y is selected from any integer from 173 to 192. Exemplary, non-limiting, 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, 20-141, etc. (only in the case of N-terminal truncation); for example, amino acids 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, 1-173, etc. (only in the case of C-terminal truncation) of SEQ NO. 1; for example, amino acids 1-173, 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, 1-173, etc. (only in the case of C-terminal truncation) of SEQ NO. 1; 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, 20-173 of SEQ ID NO: 1 (in the case of truncations at both the N-terminus and the C-terminus). The polypeptide sequence corresponding to the reverse protein of Chlorella antifreeze protein may have variants, for example, a sequence 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: 2.

[0029] The terms "polypeptide sequence corresponding to a target polypeptide" and "polypeptide sequence of a target polypeptide" are interchangeable.

[0030] The term "fusion partner" can be fused or linked to the N-terminus and / or C-terminus of the polypeptide of interest (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, initiation factors (TFs), 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.

[0031] The terms "peptide linker" and "connecting peptide" refer to short peptides used to connect two molecules (e.g., proteins). Typically, a fusion protein is obtained by introducing (e.g., by PCR amplification or ligase) a polynucleotide sequence encoding the short peptide between two DNA fragments encoding the two target proteins to be connected, and expressing the proteins, such as polypeptide 1-connecting peptide-polypeptide 2, or more specifically, target polypeptide-connecting peptide-fusion partner.

[0032] As used in the present 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.

[0033] 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 the present application include peptides (e.g., tripeptides, oligopeptides, etc.), as well as peptides that may include chemical modifications such as saccharification (glycopolypeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation. Polypeptides may also refer to proteins.

[0034] As used herein, 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, the signal peptide can be attached to the N-terminus of the target protein and can usually be cleaved and removed, so that it is not present in the mature protein secreted by the cell.

[0035] As used in the present disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In the present disclosure, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In some embodiments, the "mutation" of the present disclosure can be selected from "conservative mutation", "semi-conservative mutation" and "non-conservative mutation". In the present disclosure, the term "non-conservative mutation" or "semi-conservative mutation" can be a mutation that causes loss of function or partial loss of protein function. The term "conservative mutation" refers to a mutation that can maintain the function of a protein normally. A representative example of a conservative mutation is a conservative substitution.

[0036] As used in the present disclosure, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions can be conservative. Specifically, substitutions considered to be conservative substitutions include the substitution of Ala to Ser or Thr, the substitution of Arg to Gln, His or Lys, the substitution of Asn to Glu, Gln, Lys, His or Asp, the substitution of Asp to Asn, Glu or Gln, the substitution of Cys to Ser or Ala, the substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, the substitution of Glu to Gly, Asn, Gln, Lys or Asp, the substitution of Gly to Pro, the substitution of His to Asn, Lys, Gln, Arg or Tyr, the substitution of Ile to Leu, Met, Val or Phe, the substitution of Leu to Ile, Substitution of Met, Val or Phe, substitution of Lys for Asn, Glu, Gln, His or Arg, substitution of Met for Ile, Leu, Val or Phe, substitution of Phe for Trp, Tyr, Met, Ile or Leu, substitution of Ser for Thr or Ala, substitution of Thr for Ser or Ala, substitution of Trp for Phe or Tyr, substitution of Tyr for His, Phe or Trp, and substitution of Val for Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations due to individual differences, strain differences, species differences, etc., from which the gene is derived.

[0037] "Sequence identity" and "percentage identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). A polypeptide can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing 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.

[0038] Illustratively, in the present disclosure, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residues when compared and aligned using a sequence comparison algorithm or as measured by visual inspection for maximum correspondence. The determination / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues in length, a region of at least about 200 residues in length, a region of at least about 400 residues in length, or a region of at least about 500 residues in length. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (i.e., nucleic acids or polypeptides).

[0039] As used in the present disclosure, the term "polynucleotide" refers to a polymer consisting of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, derived from a nucleotide sequence separated at least once in quantity or concentration, capable of identifying, manipulating, and recovering sequences and their component nucleotide sequences by standard molecular biology methods (e.g., using a cloning vector). 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), wherein "U" replaces "T." In other words, a "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragment or entire fragment), or can be a component or ingredient 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" belong to a type of "polynucleotide."

[0040] 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, a "recombinant antigen," "recombinant protein," "fusion protein," and the like.

[0041] As used in this disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences to express a gene of interest in a suitable host.

[0042] As used herein, the term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding 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 that is transcribed into mRNA and translated into protein; and iii) appropriate transcriptional and translational initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, bacteriophages, and transposons.

[0043] The term "host cell" in this disclosure refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a gene editing element, nucleic acid construct, or recombinant expression vector of the present disclosure. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the introduction of a gene editing element, nucleic acid construct, or recombinant expression vector, and recombinant host cells are 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 be introduced with a recombinant nucleic acid molecule or recombinant expression vector of the present disclosure.

[0044] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0045] The term "amplification carrier" as used herein is used to carry a tracer marker and is selected from at least one of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethyleneimine, and dendrimers. Specifically, polysaccharides include cross-linked polysucrose and dextran; protein carriers include at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole limpet hemocyanin, and thyroglobulin; and dendrimers 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 limpet hemocyanin, and thyroglobulin.

[0046] The term "solid support" in this disclosure refers to a solid phase material having at least one surface to which a binding partner can be immobilized, and is used to capture an analyte in a sample. Solid supports can be in the form of particles, microparticles, nanoparticles, metal colloids, fibers, nylon, paper, beads, membranes, filter paper, and other supports such as test tubes, microplates, chips, slides, capillaries, and microarrays.

[0047] As used in this disclosure, the terms "sample", "specimen", "test sample" or "test sample" refer to any type of sample for which it is desired to determine whether it contains antibodies to Treponema pallidum. Exemplary, the test sample 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, such as a blood sample (including a sample obtained from blood), a serum sample, a lymph sample, a saliva sample, or a synovial fluid. The sample obtained from blood can be a selected portion of the patient's blood or the vaccine recipient's blood, such as a selected cell-containing portion, or a plasma or serum portion. In some embodiments, the sample can be any sample containing antibody products of a humoral immune response.

[0048] As used herein, "diagnosis" includes the 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 of a subject with a disease or condition (or its likely progression or regression), and determining the effect of treatment on a subject with a disease or condition. For example, diagnosis can be used to detect the presence or likelihood of syphilis / infection with Treponema pallidum in a subject or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, e.g., pharmaceutical) or other treatment.

[0049] The terms "individual," "patient," or "subject" as used herein include mammals. Mammals include, but are not limited to, domestic 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).

[0050] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the products can exist in an in vitro, ex vivo, or in vivo form. The term "in vitro" refers to experiments using materials, biological substances, cells and / or tissues under laboratory conditions or in culture medium; while the term "in vivo" refers to experiments and procedures using intact multicellular organisms. In some embodiments, the methods performed in vivo can be performed on non-human animals. "Ex vivo" refers to an event that exists or occurs outside an organism, such as outside a human or animal, such as an event that can exist or occur on a tissue (such as a whole organ) or cell taken from an organism.

[0051] sequence

[0052] In the technical solution disclosed herein, the meanings represented by the numbers in the amino acid sequence listing in the specification are as follows:

[0053] The sequence shown in SEQ ID NO: 1 is the polypeptide sequence of the Chlorella antifreeze protein in the Examples;

[0054] The sequence shown in SEQ ID NO: 2 is the polypeptide sequence of the reverse protein of the Chlorella antifreeze protein in the embodiment;

[0055] The sequence shown in SEQ ID NO: 3 is the sequence of the fusion partner GST in the Examples;

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

[0057] The sequence shown in SEQ ID NO: 5 is the sequence of the fusion partner ubiquitin in the Examples;

[0058] The sequence shown in SEQ ID NO: 6 is the sequence of the fusion partner SlyD in the Examples;

[0059] The sequence shown in SEQ ID NO: 7 is the sequence of the fusion partner SKP in the Examples;

[0060] The sequence shown in SEQ ID NO: 8 is the HIV gp41 polypeptide sequence in the Examples;

[0061] The sequence shown in SEQ ID NO:9 is the TP17 polypeptide sequence in the Examples;

[0062] Recombinant antigens

[0063] In one aspect, the present 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 a Chlorella antifreeze protein or its reverse protein as a fusion partner.

[0064] In some embodiments, the Chlorella antifreeze protein is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide in which the amino acid sequence shown in SEQ ID NO: 1 is substituted, repeated, deleted, or added with one or more amino acids, and has or partially has the activity of the polypeptide shown in SEQ ID NO: 1.

[0065] In some embodiments, the reverse protein of the Chlorella antifreeze protein is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide in which the amino acid sequence shown in SEQ ID NO: 2 is substituted, repeated, deleted, or added with one or more amino acids, and has or partially has the activity of the polypeptide shown in SEQ ID NO: 2.

[0066] In some embodiments, the recombinant antigen provided by the present disclosure comprises a target polypeptide and a fusion partner connected by an enzyme cleavage site;

[0067] In some embodiments, the recombinant antigen provided by the present disclosure comprises a target polypeptide and a fusion partner connected by a linker peptide;

[0068] In some embodiments, the target polypeptide, linker peptide, and fusion partner are linked to the expression vector in this order;

[0069] In some embodiments, the fusion partner, the connecting peptide, and the target polypeptide are linked to the expression vector in this order;

[0070] In some embodiments, the target polypeptide, restriction site, and fusion partner are sequentially linked to the expression vector;

[0071] In some embodiments, the fusion partner, the restriction site, and the target polypeptide are sequentially linked to the expression vector;

[0072] In some embodiments, the target polypeptide is selected from: new coronavirus antigen, HIV antigen (such as HIV-1 antigen, HIV-2 antigen, P24 antigen, O type antigen), HCV antigen, HBV antigen (such as HBe antigen, HBS antigen), hepatitis A (HAV) antigen, hepatitis E (HEV) antigen, syphilis antigen, Toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus (such as HSV I, HSV II), human lymphoblastic T-cell leukemia (HTLV) antigen, Mycoplasma pneumonia antigen, influenza antigen (such as 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.

[0073] In some embodiments, there is no particular limitation 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 corresponding autoantibodies. 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.

[0074] In some embodiments, the target polypeptide is the gp41 antigen of HIV-1.

[0075] In some embodiments, the HIV-1 gp41 antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:8.

[0076] In some embodiments, the HIV-1 gp41 antigen comprises a portion of a polypeptide having an amino acid sequence as shown in SEQ ID NO:8.

[0077] In some embodiments, the HIV-1 gp41 antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:8, or a polypeptide having an amino acid sequence as shown in SEQ ID NO:8 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO:8.

[0078] In some embodiments, the target polypeptide is the TP17 antigen of Treponema pallidum.

[0079] In some embodiments, the TP17 antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:9.

[0080] In some embodiments, the TP17 antigen comprises a portion of a polypeptide having an amino acid sequence as shown in SEQ ID NO:9.

[0081] In some embodiments, the TP17 antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:9, or a polypeptide having an amino acid sequence as shown in SEQ ID NO:9 that is substituted, repeated, deleted, or added with one or more amino acids, and has or partially has the activity of the polypeptide having the sequence as shown in SEQ ID NO:9.

[0082] In the present disclosure, there is no particular limitation in principle on the selection of connecting peptides. In some preferred embodiments, the connecting peptide can be a flexible connecting peptide. The connecting peptide includes but is not limited to GS, GGSGG, KESGSVSSEQLAQFRSLD, GSAGSAAGSGEF, GGSGGEFGGSGG, etc. In some preferred embodiments, the connecting peptide is GGSGG. In some preferred embodiments, the connecting peptide is GS.

[0083] In some embodiments, the recombinant antigen further comprises a tag polypeptide at its N-terminus and / or C-terminus. Exemplarily, the tag polypeptide is a His tag, preferably a 6×His tag, which is attached to the N-terminus of the recombinant antigen to facilitate purification of the recombinant antigen protein by NI-NTA affinity chromatography. In some alternative embodiments, the recombinant antigen and the tag polypeptide can be linked by a linker peptide. In some embodiments, the recombinant antigen is also conjugated to a tracer marker.

[0084] In some embodiments, the tracking label is indirectly coupled to the recombinant antigen based on the anti-fusion partner antibody, that is, the tracking label is directly coupled to the anti-fusion partner antibody, and the anti-fusion partner antibody specifically binds to the fusion partner part of the coupled recombinant antigen.

[0085] In some embodiments, the tracking marker is indirectly coupled to the recombinant antigen based on the amplification vector, that is, the tracking marker is directly coupled to the amplification vector, and the recombinant antigen is also directly coupled to the same amplification vector.

[0086] In some embodiments, the recombinant antigen is further coupled to a solid phase carrier.

[0087] In some embodiments, the recombinant antigen is coupled to the solid support during the detection process based on a "transfer tag" and a "transfer tag receptor." For example, the recombinant antigen is labeled with biotin (transfer tag), and the solid support is coated with avidin (transfer tag receptor); the recombinant antigen carries a His tag (transfer tag), and the solid support has nickel on its surface (transfer tag receptor); the recombinant antigen is labeled with dinitrobenzene (transfer tag), and the solid support is coated with an anti-dinitrobenzene antibody (transfer tag receptor), etc.

[0088] In the present disclosure, exemplary recombinant antigens may have any of the following structures:

[0089] (1) Tag-optional linker peptide-target polypeptide-optional linker peptide-fusion partner;

[0090] (2) Target polypeptide-optional linker peptide-fusion partner-optional linker peptide-tag.

[0091] In one aspect, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned recombinant receptor binding proteins. The recombinant nucleic acid molecule can be DNA, RNA, or a combination thereof.

[0092] In one aspect, the present disclosure provides a recombinant expression vector comprising the aforementioned recombinant nucleic acid molecule, which can be used to transfer the recombinant nucleic acid molecule into a cell. In some embodiments, the recombinant expression vector comprises one or more regulatory elements, which can be promoters, enhancers, silencers, insulators, and other elements commonly used in the art. The regulatory elements are operably linked to the recombinant nucleic acid molecule to mediate transcription and translation of the recombinant nucleic acid molecule.

[0093] The term "operably linked" can include situations where a selected nucleic acid 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 (thereby forming an expression cassette). Thus, a regulatory element is operably linked to a nucleic acid molecule if the regulatory element is capable of effecting transcription of the nucleic acid molecule. The resulting transcript can be translated into a desired polypeptide or protein.

[0094] In the present disclosure, vectors suitable for constructing recombinant expression vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), etc.

[0095] In some embodiments, the vector is a prokaryotic vector comprising various elements for expression in prokaryotic cells. For example, vectors include, but are 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.

[0096] In one aspect, the present 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 the present disclosure may be a eukaryotic cell or a prokaryotic cell, as long as the recombinant nucleic acid molecule or recombinant expression vector of the present disclosure can be introduced into the host cell to achieve expression of the recombinant receptor binding protein.

[0097] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The bacterium can be a coccus (e.g., Micrococcus, Thermococcus, Streptococcus), a bacillus (e.g., Enterobacter, Bacillus, Actinomyces, Acetobacter, Lactobacillus), or a spirochete (e.g., Brachyspira). In some embodiments, the host cell is Escherichia coli ER2529 or Escherichia coli ER2566; preferably, Escherichia coli ER2566.

[0098] In some embodiments, the recombinant antigen can be prepared by expressing 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, a recombinant host cell is obtained by introducing a recombinant expression vector into Escherichia coli.

[0099] The 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 fluid or fermentation fluid, and then the recombinant antigen protein is purified.

[0100] In some embodiments, the step of subjecting the recombinant antigen protein to protein purification comprises Ni-NTA affinity chromatography and protein dialysis.

[0101] Reagents / Kits

[0102] In one aspect, the present disclosure provides a detection reagent comprising a recombinant antigen. The present disclosure discovered that by fusion-expressing a Chlorella vulgaris antibody protein or its reverse 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 fused with other fusion partners, the detection reagent prepared using the recombinant antigen of the present disclosure can significantly improve detection sensitivity and signal-to-noise ratio.

[0103] In some embodiments, the recombinant antigen is conjugated to a tracer marker capable of generating a detection signal. The tracer marker is selected from at least one of acridinium ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, colloidal gold, fluorescent microspheres, ruthenium terpyridine, quantum dot luminescent materials, and upconversion luminescent materials. Exemplarily, the signal molecule is acridinium ester (AE), which exhibits high luminescence efficiency when oxidized by H2O2 under alkaline conditions.

[0104] On the one hand, the present disclosure provides a kit comprising a recombinant antigen of the present disclosure, such as a kit for detecting the presence of corresponding autoantibodies from a subject's sample. In some embodiments, the kit includes reagents suitable for immunoassays. In some embodiments, the kit may include instructions for using an immunodiagnostic reagent of the present disclosure (e.g., a detection reagent comprising the corresponding recombinant antigen) in an immunoassay for detecting autoantibodies. In some embodiments, in some embodiments, the kit may include a calibrator or a control, such as a standard or control HIV antibody. In some embodiments, the HIV protein or conjugate of the present disclosure is included in a container such as a test tube, a microplate, or a test strip in the kit. In some embodiments, the kit may also include solid supports such as magnetic beads, test tubes, microplates, cuvettes, films, filter paper, syringes, pipettes, buffers (e.g., assay buffer, wash buffer), pretreatment reagents, tracer markers such as enzyme-labeled substrate solutions, etc.

[0105] On the one hand, the present disclosure includes test strips containing the recombinant antigens of the present disclosure, such as lateral flow test strips. In some embodiments, the test strips contain HIV proteins coated thereon, at least one detection antibody or at least one detection antigen with a tracer marker (such as colloidal gold). In some embodiments, the test strips contain recombinant antigens coupled to a tracer marker, at least one detection antibody or at least one detection antigen coated thereon. In some embodiments, the present disclosure can quickly and accurately detect autoantibodies in a sample by naked eye observation or a chemiluminescence fully automatic instrument. In some embodiments, a test kit can be prepared using the double antigen sandwich principle. For example, in some embodiments, autoantibodies in a sample are captured by HIV proteins coated on a solid phase carrier, or autoantibodies in a sample are detected by recombinant antigens labeled with a tracer marker. In some embodiments, a test kit can be prepared using the indirect method principle. For example, in some embodiments, autoantibodies in a sample are captured by recombinant antigens coated on a solid phase carrier. In some embodiments, an excitation solution is added and the luminescence value is measured using a fully automatic chemiluminescence instrument. The luminescence value is positively correlated with the total concentration of autologous antibodies in the sample and is compared with the critical value to determine whether it is positive or negative.

[0106] method

[0107] In one aspect, the present 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 partner sequence, with the sample to form a first recombinant antigen-analyte-second recombinant antigen immunological binding complex; detecting the presence or amount of the immunological binding complex;

[0108] 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 reversed protein of Chlorella antifreeze protein, and the fusion partner of the other of the first and second recombinant antigens does not correspond to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reversed protein of Chlorella.

[0109] In some embodiments, the first recombinant antigen is the recombinant antigen described in the first aspect above, which will not be repeated here.

[0110] In some embodiments, the target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. The types of the pathogen polypeptide and autoimmune disease antigen polypeptide are as described above and will not be repeated here.

[0111] In one aspect, the present disclosure provides a method for diagnosing whether an individual has a pathogen infection and / or an autoimmune disease, comprising: mixing a first recombinant antigen and a second recombinant antigen, each comprising a target polypeptide sequence and a fusion partner sequence, with a sample from a test subject to form a first recombinant antigen-disease analyte-second recombinant antigen immunological binding complex; and determining whether the individual has a pathogen infection and / or an autoimmune disease based on the presence or amount of the immunological binding complex; wherein the fusion partner of one of the first and second recombinant antigens corresponds to a polypeptide sequence of a Chlorella antifreeze protein or a polypeptide sequence of an inverted version of the Chlorella antifreeze protein, and the fusion partner of the other of the first and second recombinant antigens does not correspond to a polypeptide sequence of a Chlorella antifreeze protein or a polypeptide sequence of an inverted version of the Chlorella antifreeze protein. The present disclosure has discovered that by fusion-expressing a Chlorella antibody protein or a inverted version of the Chlorella antibody 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 expressed using other fusion partners, the recombinant antigens of the present disclosure are more effective for detecting samples from a test subject, thereby diagnosing whether an individual has a pathogen infection and / or an autoimmune disease.

[0112] In some embodiments, the first recombinant antigen is the recombinant antigen described in the first aspect above, which will not be repeated here.

[0113] In some embodiments, the target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. The types of the pathogen polypeptide and autoimmune disease antigen polypeptide are as described above and will not be repeated here.

[0114] Diagnostic uses

[0115] In one aspect, the present disclosure provides the use of the aforementioned recombinant antigens, detection reagents, or kits for diagnosing whether an individual has a pathogen infection and / or an autoimmune disease. The present disclosure has discovered that by fusion-expressing a Chlorella antibody protein or its reverse protein with a target polypeptide in an E. coli expression system, the fused recombinant antigen can maintain its antigenicity during the coupling process. Compared to recombinant antigens expressed by fusion with other fusion partners, the recombinant antigens disclosed herein, or detection reagents or kits containing the recombinant antigens, can effectively detect samples from a test individual and diagnose whether the individual has a pathogen infection and / or an autoimmune disease. In some embodiments, the diagnosis comprises: mixing a first recombinant antigen and a second recombinant antigen, each comprising a target polypeptide sequence and a fusion partner sequence, with a sample from a test individual to form a first recombinant antigen-disease analyte-second recombinant antigen immune binding complex; and judging whether the individual suffers from a pathogen infection and / or an autoimmune disease based on the presence or amount of the immune binding complex; wherein the fusion partner of one of the first recombinant antigen and the second recombinant antigen corresponds to a polypeptide sequence of Chlorella antifreeze protein or a polypeptide sequence of an inverted version of Chlorella antifreeze protein, and the fusion partner of the other of the first recombinant antigen and the second recombinant antigen does not correspond to a polypeptide sequence of Chlorella antifreeze protein or a polypeptide sequence of an inverted version of Chlorella antifreeze 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 the pathogen polypeptide and autoimmune disease antigen polypeptide are as described above and will not be repeated here.

[0118] Example

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

[0120] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples were obtained through commercial channels unless otherwise specified.

[0121] The PE vector used in the following examples has a vector map shown in FIG1 : The PE vector itself contains a 6×His tag at the N-terminus, so the recombinant antigens obtained by translation all contain a 6-histidine tag, which can promote NI-NTA-assisted affinity purification.

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

[0123] 1.1 Design of recombinant gp41 antigen

[0124] Table 1

[0125] According to the design table of the recombinant antigen as shown in Table 1, the corresponding fusion partner was introduced at the N-terminus of the target polypeptide gp41, and an expression plasmid was constructed based on 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 Feipeng's own vector PE (as shown in Figure 1) to obtain the corresponding expression plasmid. A linker GGSGG is provided between the target polypeptide and the fusion partner.

[0126] 1.2 Recombinant antigen induced expression

[0127] Transform the expression plasmids constructed in 1.1 into competent E. coli BL21 cells (NEB, Cat. No. C2530H) using the heat shock method. Plate the plasmids onto LB plates containing 100 μg / ml Amp and incubate at 37°C for 16 hours. Select colonies and select positive strains identified by PCR and double enzyme digestion. Inoculate the strains into LB medium containing 50 μg / ml Kan and incubate at 37°C with shaking. When the OD600 reaches 0.6-0.8, add 1.0 mM IPTG and induce the culture at 37°C for 2-4 hours. Extract the total protein and analyze the expression of the recombinant protein by SDS-PAGE.

[0128] (1) Expression of recombinant antigen in supernatant: The experiment found that gp41-1 and gp41-2 were basically expressed in the supernatant, and gp41-4 was partially expressed in the supernatant and partially expressed in the precipitate. The recombinant antigen was purified using affinity columns and ion exchange chromatography.

[0129] (2) Inclusion body expression of recombinant antigens: Experiments have shown that gp41-3, gp41-5, gp41-6, and gp41-7 are basically expressed in inclusion bodies. After washing with a buffer containing tween 20, the inclusion bodies are dissolved using 2-8 M urea. After renaturation by dialysis, the recombinant antigens are purified by affinity column and ion exchange chromatography.

[0130] Example 2: Preparation of chemiluminescent reagent

[0131] 2.1. Coating of gp41 antigen

[0132] 1) Activation of magnetic microspheres (using carboxyl magnetic beads as an example): Carboxyl magnetic microspheres with a diameter of approximately 2 μm were washed three times with 15 mM MES pH 5.5 buffer. MES buffer was then added to bring the magnetic bead concentration to 10 mg / mL. 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg of N-hydroxysuccinimide were added per 10 mg of magnetic microspheres. The mixture was then shaken or stirred on a shaker or mechanical stirrer at room temperature for 1 hour.

[0133] 2) Coating: Remove the supernatant from the magnetic microspheres activated in step 1), wash once with 15 mM MES pH 5.5 buffer, then add 15 mM MES pH 5.5 to a concentration of 10 mg / mL, then add 0.1 mg of HIV gp41 coating antigen (available from Feipeng Bio) per 10 mg of magnetic microspheres, and then shake or stir on a shaker or mechanical stirrer at room temperature for about 2.5 hours to allow the coating antigen to couple to the magnetic bead surface.

[0134] 3) Blocking: After the coating reaction is complete, perform magnetic separation, remove the supernatant, and wash three times with TBS (25mM Tris + 150mM NaCl, pH 7.5). Add blocking buffer containing 0.5% BSA in TBS (pH 7.5) to a concentration of 10 mg / mL of magnetic microspheres. Oscillate or stir for 3 hours at room temperature on a shaker or mechanical stirrer. After the reaction is complete, wash with PBST (25mM PB + 150mM NaCl + 0.1% Tween-20, pH 7.5) and store in 50mM HEPES buffer, pH 7.4. The resulting magnetic bead working solution should be stored at 2-8°C.

[0135] 2.2 Direct labeling of gp41 antigen

[0136] The six recombinant antigens prepared in Example 1 were diluted to a concentration of 1 mg / ml, and 1 ml of the sample was placed in a dialysis bag in 1×PBS (pH 7.2) and dialyzed overnight. The next day, the dialyzed antigen was removed and placed in a centrifuge tube, 16.7 μL of dissolved acridinium ester was added, mixed and rotated at room temperature for 15 minutes, and then 3.34 μL of glycine was added to terminate the reaction. After the reaction was terminated, the mixture was placed back in a dialysis bag in 1×PBS (pH 7.2) and dialyzed at 4°C overnight. After the dialysis was completed, an equal volume of glycerol was added to the mixture, mixed and stored at -20°C to obtain acridinium ester-labeled gp41 recombinant antigen (AE-Ag).

[0137] 2.3 Indirect labeling of gp41 antigen

[0138] Take 100 μL of 10 mg / mL BSA solution, add pre-dissolved acridinium ester solution, and react for 15 minutes. Then desalt with a desalting column, add Traut's reagent to the solution, and react for 15 minutes. After desalting, obtain the BSA intermediate for use. Take 100 μL of 2 mg / mL recombinant antigen, add pre-dissolved SMCC crosslinker solution, and react for 15 minutes. Mix the resulting solution with the BSA intermediate in a certain proportion and react for 24 hours. After the reaction is completed, the solution is dialyzed and purified to obtain the gp41 recombinant antigen (AE-BSA-Ag) indirectly labeled with acridinium ester.

[0139] Example 3: Chemiluminescence test

[0140] The reagent prepared in Example 2 was used to detect HIV clinical samples (from Feipeng Bio) using the double antigen sandwich method.

[0141] 3.1. Testing Process

[0142] 1) Place 100 μl of sample and 50 μl of magnetic bead working solution coated with gp41 recombinant antigen into the wells of the luminescence plate and mix and react at 37°C for 15 minutes;

[0143] 2) Then adsorb the beads using a magnetic plate and wash them with 1×PBST solution;

[0144] 3) Add 100 μl HIV AE labeling working solution into the wells of the luminescence plate and mix and react at 37°C for 10 minutes.

[0145] 4) Then adsorb the beads to the magnetic plate again and wash them with 1×PBST solution;

[0146] 5) Then, AE pre-excitation solution and AE excitation solution were added, and the luminescence value was read using a ThermoFisher chemiluminescence analyzer.

[0147] Test results:

[0148] Table 2. Test results of direct labeling of gp41 recombinant antigen

[0149] The results in Table 2 indicate that the Chlorella antifreeze protein or its reverse sequence as a fusion partner of the recombinant gp41 antigen has excellent detection activity or signal-to-noise ratio (P / N) for the sample compared with other fusion partners or no fusion partner.

[0150] Recombinant antigens gp41-1, gp41-2, gp41-6, and gp41-3 were indirectly labeled with acridinium ester according to Example 2.3 and then tested.

[0151] Table 3. Test results of indirect labeling of gp41 recombinant antigen

[0152] The results in Table 3 show that the detection performance advantage of gp41-1 and gp41-2 over gp41-6 and gp41-3 is further improved when indirect labeling is performed using an amplification vector (BSA).

[0153] The fusion partner effect was verified using the methods of Examples 1 to 3 for the target polypeptide, Treponema pallidum antigen TP17 (SEQ ID NO: 9). The results showed that the 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:

[0154] Table 4. Test results of indirect labeling of TP17 recombinant antigen

[0155] This demonstrates that the Chlorella antifreeze protein and its reverse protein have the advantage of improving detection performance as fusion partners in recombinant antigens. This is hypothesized to be because the Chlorella antifreeze protein and its reverse protein contain a large number of hydrophilic amino acids, which are distributed on the protein surface, resulting in a high hydrophilicity. Furthermore, the Chlorella antifreeze protein surface is rich in amino acids with similar charges, which can reduce the formation of protein aggregates and indirectly prevent the recombinant antigen from forming disordered multimers.

[0156] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. 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 a polypeptide sequence of a reverse protein thereof as a fusion partner.

2. The recombinant antigen according to claim 1, wherein The Chlorella antifreeze protein is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide in which the amino acid sequence shown in SEQ ID NO: 1 is substituted, repeated, deleted or added with one or more amino acids, and has or partially has the activity of the polypeptide of the sequence shown in SEQ ID NO: 1; The reverse protein of the Chlorella antifreeze protein is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide in which the amino acid sequence shown in SEQ ID NO: 2 is substituted, repeated, deleted or added with one or more amino acids, and has or partially has the activity of the polypeptide of the sequence shown in SEQ ID NO: 2; Optionally, the recombinant antigen comprises a target polypeptide and a fusion partner connected by a connecting peptide; Optionally, the target polypeptide is a pathogen polypeptide, or an autoimmune disease antigen polypeptide.

3. A detection reagent, wherein: The reagent comprises the recombinant antigen according to any one of claims 1 to 2, and a tracer marker or a solid phase carrier coupled to the recombinant antigen.

4. The detection reagent according to claim 3, wherein The recombinant antigen is indirectly coupled with a tracer marker.

5. A detection kit or test strip, wherein: The kit or test strip comprises the recombinant antigen according to any one of claims 1 to 2, or the reagent according to any one of claims 3 to 4.

6. Use of the recombinant antigen according to any one of claims 1 to 2 in immunodiagnosis or as an immunogen for preparing antibodies against a target polypeptide.

7. Use of the recombinant antigen according to any one of claims 1 to 2 in producing a vaccine.

8. A recombinant nucleic acid molecule encoding the recombinant antigen according to claim 1 or 2, an expression vector containing the recombinant nucleic acid molecule, and a host cell containing the expression vector.

9. A method for detecting an analyte in a sample, comprising: Mixing a first recombinant antigen and a second recombinant antigen, each of which comprises a target polypeptide sequence and a fusion partner sequence, with a sample to form a first recombinant antigen-analyte-second recombinant antigen immune binding complex; detecting the presence or amount of the immune binding complex; The fusion partner of one of the first recombinant antigen and the second recombinant antigen corresponds to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reverse protein of Chlorella antifreeze protein, and the fusion partner of the other of the first recombinant antigen and the second recombinant antigen does not correspond to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reverse protein of Chlorella antifreeze protein; Optionally, the first recombinant antigen is the recombinant antigen according to any one of claims 1 to 2.

10. The method according to claim 9, wherein the target polypeptide is a pathogen polypeptide, or an autoimmune disease antigen polypeptide.

11. A method for diagnosing whether an individual suffers from pathogen infection and / or autoimmune disease, wherein: include: Mixing a first recombinant antigen and a second recombinant antigen, each of which comprises a target polypeptide sequence and a fusion partner sequence, with a sample from a subject to be tested to form a first recombinant antigen-disease analyte-second recombinant antigen immune binding complex; Based on the presence or amount of the immune binding complex, determining whether the individual suffers from pathogen infection and / or autoimmune disease; The fusion partner of one of the first recombinant antigen and the second recombinant antigen corresponds to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reverse protein of Chlorella antifreeze protein, and the fusion partner of the other of the first recombinant antigen and the second recombinant antigen does not correspond to the polypeptide sequence of Chlorella antifreeze protein or the polypeptide sequence of the reverse protein of Chlorella antifreeze protein; Optionally, the first recombinant antigen is the recombinant antigen according to any one of claims 1 to 2.

12. The method according to claim 11, wherein: The target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide.

13. Use of the recombinant antigen according to claim 1 or 2, the detection reagent according to claim 3 or 4, or the kit or test strip according to claim 5 in diagnosing whether an individual suffers from pathogen infection and / or autoimmune disease.

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