Recombinant antigens and uses thereof

Abstract

The invention provides a recombinant antigen and application thereof, and belongs to the technical field of immunodiagnosis. The invention particularly relates to a recombinant antigen containing chlorella antifreeze protein or 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 application. When the recombinant antigen is used for immunodiagnosis, the sensitivity and the signal-to-noise ratio of the immunodiagnosis can be remarkably improved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310848455.8, filed with the Chinese Patent Office on July 11, 2023, and entitled “Recombinant Antigens and Uses Thereof,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure belongs to the field of immunodiagnosis technology and relates to a recombinant antigen containing a specific fusion partner and uses thereof; 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 uses thereof. Background Art

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

[0005] However, in immunodiagnosis applications, recombinant antigens need to be coupled with 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 of the antigen. In addition, tracer markers or solid phase carriers may also shield and destroy the epitopes of the antigen. Therefore, it is necessary to explore fusion partners suitable for immunodiagnosis so that the recombinant antigen can reduce the antigenicity loss during the coupling 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 greatly improve the detection performance of recombinant antigens as diagnostic reagents, which is better than existing fusion partners.

[0007] The 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 reverse protein as a fusion partner.

[0008] The target polypeptide is a pathogen polypeptide or an autoimmune disease antigen polypeptide. The polypeptide of the foreign pathogen triggers an immune response in the individual to produce corresponding autoantibodies. In the body of an autoimmune disease patient, the state of autoimmune tolerance is broken, and the endogenous antigen polypeptide also induces the production of autoantibodies. Immunodiagnosis of these autoantibodies is an effective means to determine whether an individual is infected with the corresponding pathogen / suffering from the corresponding autoimmune disease.

[0009] The second aspect of the present invention provides a detection reagent / detection kit or test strip, comprising the aforementioned recombinant antigen, and a tracing marker or a solid phase carrier.

[0010] 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.

[0011] 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.

[0012] The third aspect of the present invention provides the use of the aforementioned recombinant antigen in immunodiagnosis or as an immunogen to prepare antibodies against a target polypeptide.

[0013] The fourth aspect of the present invention provides the use of the aforementioned recombinant antigen in producing a vaccine.

[0014] The 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] The 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 partner 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 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 its inverse 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 its inverse protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A map of the PE carrier used in the embodiments of the present disclosure is shown. DETAILED DESCRIPTION

[0017] 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".

[0018] 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.

[0019] 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.

[0020] 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 the alternatives are mutually exclusive.

[0021] The term "pathogen polypeptide" refers to polypeptides of exogenous pathogens of 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, and the like. Streptococcus, Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium, Corynebacterium, Eberthella, Francisella, Haemophilus, Bacteroides, Listeria, Erysipelothria x), Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium, Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia Nocardia, Rickettsia, Micrococcus, Mycobacterium, Neisseria, Campylobacter, pathogenic viruses (such as, for example, papillomavirus, parvovirus, adenovirus, herpesvirus, vacciniavirus, arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus, paramyxovirus, reovirus, retrovirus, rhabdovirus, human immunodeficiency virus (HIV), tapeworm, Hymenolepsis,Diphyllobothrium, Echinococcus, Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fasciola, Paragonimus, Schistosoma, Enterobius, Trichuris, Ascaris, Ancylostoma, Necator , Wuchereria, Brugi, Loa, Onchocerca, Dracunculus, Naegleria, Acanthamoeba, Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora, Cryptosporidium idium, Enterocytozoa, Strongyloides, Trichinella), fungal causes (e.g., Ringworm, Histoplasmosis, Blastomycosis, Aspergillosis, Cryptococcosis, Sporotrichosis, Coccidiodomycosis, Paracoccidioidomycosis, Mucormycosis, 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-lymphocytic leukemia 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.

[0022] 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 polyradiculoneuropathy, dermatomyositis, giant cell arteritis, granulomatosis with polyangiitis, Kawasaki disease, lupus nephritis, polyarteritis nodosa, pyoderma gangrenosum, spondyloarthritis, systemic lupus erythematosus, and Takayasu's arteritis.

[0023] The term "Chlorella antifreeze protein" refers to the antifreeze protein of Chlorella, as shown in SEQ NO.1 (NCBI accession number: PRW45461). The term "reverse protein of Chlorella antifreeze protein" refers to the reverse coding sequence of the antifreeze protein of Chlorella, that is, the amino acid at the N-terminus is placed at the C-terminus, and the amino acid at the C-terminus is placed at the N-terminus, and the polypeptide product obtained by the overall reverse coding is shown in SEQ NO.2. The "polypeptide sequence corresponding to the Chlorella antifreeze protein" and the "polypeptide sequence of the Chlorella antifreeze protein" can be interchangeable, and the polypeptide sequence of the Chlorella antifreeze protein can be the full-length antifreeze protein sequence of 192aa as shown in SEQ NO.1, or it can be a truncated antifreeze protein sequence, including the amino acids from position X to position Y of SEQ NO.1, 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) of SEQ NO.1; 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 1-191, 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; 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, etc. (in the case of truncations at both the N-terminus and the C-terminus). The polypeptide sequence corresponding to the 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 with SEQ ID NO: 1.

[0024] “A polypeptide sequence corresponding to the reverse protein of Chlorella antifreeze protein” and “a polypeptide sequence of the reverse protein of Chlorella antifreeze protein” can be interchangeable. The polypeptide sequence of the reverse protein of Chlorella antifreeze protein can be a full-length reverse protein sequence of 192aa as shown in SEQ NO.2, or 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) of SEQ NO.1; 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 1-191, 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; 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, etc. (in the case of truncations at both the N-terminus and the C-terminus). The polypeptide sequence corresponding to the inverted 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 with SEQ ID NO: 2.

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

[0026] 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.

[0027] The terms "peptide linker" and "connecting peptide" refer to a short peptide 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 two target proteins to be connected, and performing protein expression, such as polypeptide 1-connecting peptide-polypeptide 2, more specifically, target polypeptide-connecting peptide-fusion partner.

[0028] 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.

[0029] As used in the present 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.), and peptides that may include chemical modifications (e.g., glycosylation (glycopolypeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation). Polypeptide may also refer to protein.

[0030] As used in the present disclosure, the term "signal peptide" refers to a polypeptide that is connected to a target protein and can promote the expression or transfer of the target protein. Exemplarily, the signal peptide can be connected to the N-terminus of the target protein and can usually be cut and removed, so it is not present in the mature protein secreted by the cell.

[0031] 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 a nucleotide sequence or an amino acid sequence. In some embodiments, the "mutation" of the present disclosure may 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" may 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.

[0032] As used in the present disclosure, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions may be conservative. Specifically, substitutions considered to be conservative substitutions include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, substitutions of Asp to Asn, Glu or Gln, substitutions of Cys to Ser or Ala, substitutions of Gln to Asn, Glu, Lys, His, Asp or Arg, substitutions of Glu to Gly, Asn, Gln, Lys or Asp, substitutions of Gly to Pro, substitutions of His to Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0033] "Sequence identity" and "percentage of 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. The polynucleotides may 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). The polypeptides may 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, the percentage of identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0034] Exemplary, in the present disclosure, two or more sequences or subsequences have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% "sequence identity" or "percent identity" of nucleotides or amino acid residues when compared and aligned using a sequence comparison algorithm or measured by visual inspection with 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, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).

[0035] As used in the present disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biological methods (e.g., using a cloning vector) and sequences and their component nucleotide sequences. 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 fragments or entire fragments), 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 one of "polynucleotides".

[0036] As used in the present disclosure, 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", etc.

[0037] As used in this disclosure, the term "vector" refers to a DNA construct that contains a DNA sequence operably linked to appropriate control sequences so as to express a gene of interest in a suitable host.

[0038] As used in this disclosure, the term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. 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 transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, bacteriophages, and transposons.

[0039] The term "host cell" in the present disclosure means any cell type that is easily transformed, transfected, transduced, etc. with a gene editing element, nucleic acid construct or recombinant expression vector comprising the present disclosure. The term "recombinant host cell" covers 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 the recombinant host cell is specifically achieved by transformation. The host cell of the present disclosure can be a prokaryotic cell or a eukaryotic cell, as long as it is a cell that can introduce a recombinant nucleic acid molecule or a recombinant expression vector of the present disclosure.

[0040] The terms "transformation, transfection, and transduction" in this disclosure 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 of introducing nucleic acid into a cell, 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 the present disclosure is used to load the tracer marker, and is selected from at least one of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethylene imine, 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; dendrimers include polyethylene glycol and / or polypropylene imine. In some embodiments, the amplification carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole limpet hemocyanin, and thyroglobulin.

[0042] The term "solid phase carrier" in the present disclosure refers to a solid phase material having at least one surface to which a binding partner can be fixed, which is used to capture the analyte in the sample. The solid phase carrier can be in the form of particles, microparticles, nanoparticles, metal colloids, fibers, nylon, paper, beads, membranes, filter papers and other supports such as test tubes, microplates, chips, slides, capillaries and microarrays.

[0043] As used in the present disclosure, the terms "sample", "specimen", "sample to be tested" or "test sample" refer to any type of sample in which it is desired to determine whether it contains Treponema pallidum antibodies. Exemplary, the sample to be tested can be any product produced by a subject, or any product derived from a product produced by a subject. The sample can be taken from any tissue or body fluid, 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.

[0044] As used in this disclosure, "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 a subject suffering from syphilis / infection with Treponema pallidum or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, e.g., pharmaceutical) or other treatment.

[0045] The terms "individual", "patient" or "subject" used in the context of this disclosure 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).

[0046] 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 events that exist or occur outside an organism, such as outside a human or animal, 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 scheme of the present disclosure, the meanings represented by the numbers in the amino acid sequence table of the specification are as follows:

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

[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 reverse protein of the Chlorella antifreeze protein in the embodiment; PKLRDAADQAAKSADGALDEGKAQARGLGEKADGKLESYKEKATDAVDEAHRRAEDTAGAGEAGKLGERAAGQADRGAGEAGGAADRVAREASGGLESATSKAGEAAESARQKASEYAEQAAAKVDGLTDAAKQEAYGLNQRQDQTVDRATEQVDAAAGTVTEKVKQAADSVAHKATEIASKAKDALSEDQM (SEQ ID NO: 2)

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

[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 Examples;

[0057] KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDN AGAKAGLTFLVDLIKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT(SEQ IDNO:4)

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

[0059] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG(SEQ ID NO:5)

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

[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 Examples;

[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] 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 a reverse protein thereof as a fusion partner.

[0070] 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 with the sequence shown in SEQ ID NO: 1.

[0071] 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 with the sequence shown in SEQ ID NO: 2.

[0072] 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.

[0073] In some embodiments, the recombinant antigen provided by the present disclosure comprises a target polypeptide and a fusion partner connected by a connecting peptide.

[0074] In some embodiments, the target polypeptide, the connecting peptide, and the fusion partner are linked to the expression vector in this order.

[0075] In some embodiments, the fusion partner, the connecting peptide, and the target polypeptide are connected to the expression vector in this order.

[0076] In some embodiments, the target polypeptide, the restriction site, and the fusion partner are linked to the expression vector in this order.

[0077] In some embodiments, the fusion partner, the restriction site, and the target polypeptide are linked to the expression vector in this order.

[0078] In some embodiments, the target polypeptide is selected from: new coronavirus antigen, HIV antigen (e.g., HIV-1 antigen, HIV-2 antigen, P24 antigen, O type antigen), HCV antigen, HBV antigen (e.g., HBe antigen, HBS antigen), hepatitis A (HAV) antigen, hepatitis E (HEV) antigen, syphilis antigen, Toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus (e.g., HSV I, HSV II), human lymphoblastic 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, Salmonella typhi antigen, Mycobacterium tuberculosis antigen, streptolysin O, cyclic citrullinated peptide, and monkeypox virus antigen.

[0079] In some embodiments, there is no particular limitation on the length of the target polypeptide fragment in principle, and 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, and in some specific embodiments, the antigen can be a truncated antigen fragment.

[0080] In some embodiments, the target polypeptide is the gp41 antigen of HIV-1.

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

[0083] 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 through 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.

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

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

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

[0087] 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 through 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:9.

[0088] In the present disclosure, there is no particular limitation in principle for the selection of connecting peptides. In some preferred embodiments, the connecting peptide can select 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.

[0089] 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 connected to the N-terminus of the recombinant antigen to facilitate NI-NTA affinity chromatography purification of the recombinant antigen protein. In some optional embodiments, the recombinant antigen and the tag polypeptide may be connected via a connecting peptide.

[0090] In some embodiments, the recombinant antigen is further coupled to a tracer marker.

[0091] In some embodiments, the tracking marker is indirectly coupled to the recombinant antigen based on the anti-fusion partner antibody, that is, the tracking marker 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.

[0092] In some embodiments, the tracing marker is indirectly coupled to the recombinant antigen based on the amplification vector, that is, the tracing marker is directly coupled to the amplification vector, and the recombinant antigen is also directly coupled to the same amplification vector.

[0093] In some embodiments, the recombinant antigen is further coupled to a solid phase carrier.

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

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

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

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

[0098] 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, and the recombinant nucleic acid molecule can be DNA, RNA or a combination thereof.

[0099] On the one hand, the present disclosure provides a recombinant expression vector, which comprises the above-mentioned 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 element is operably connected 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 a situation 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 (thereby forming an expression cassette). Thus, if the regulatory element is capable of acting on the transcription of the nucleic acid molecule, the regulatory element is operably linked to the nucleic acid molecule. The resulting transcript can be translated into a desired polypeptide or protein.

[0101] 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, adenovirus-associated viral vectors, vaccinia virus vectors and herpes virus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), etc.

[0102] In some embodiments, the vector is a prokaryotic vector, which contains 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, the present disclosure provides a recombinant host cell, which comprises the above-mentioned recombinant nucleic acid molecule or recombinant expression vector. The recombinant host cell is obtained by transforming, transfecting, or transducing a recombinant nucleic acid molecule or a recombinant expression vector into a host cell. The host cell in the present disclosure can be a eukaryotic cell or a prokaryotic cell, as long as the recombinant nucleic acid molecule or recombinant expression vector of the present disclosure can be introduced to achieve the expression of the recombinant receptor binding protein.

[0104] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The bacterium can be a coccus (e.g., Micrococcus, Thermococcus, Streptococcus), a bacillus (e.g., Enterobacter, Bacillus, Actinobacillus, Acetobacter, Lactobacillus) or a spirochete (e.g., Brachyspira). In some embodiments, the host cell is Escherichia coli ER2529, Escherichia coli ER2566; preferably, Escherichia coli ER2566.

[0105] 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, the recombinant host cell is obtained by introducing a recombinant expression vector into Escherichia coli.

[0106] 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.

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

[0108] Reagents / Kits

[0109] In one aspect, the present disclosure provides a detection reagent, which includes a recombinant antigen. The present disclosure found that by fusion expression of a Chlorella antibody protein or its reverse protein and a target polypeptide in an Escherichia coli expression system, the fused recombinant antigen can maintain antigenicity during the coupling process, and compared with recombinant antigens obtained by fusion expression with other fusion partners, the detection reagent prepared with the recombinant antigen of the present disclosure can significantly improve the detection sensitivity and signal-to-noise ratio.

[0110] In some embodiments, the recombinant antigen is coupled with a tracer marker that can generate 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, terpyridine ruthenium, quantum dot luminescent materials, and upconversion luminescent materials. Exemplarily, the signal molecule is acridinium ester (AE), which has a high luminescence efficiency when oxidized by H2O2 under alkaline conditions.

[0111] On the one hand, the present invention provides a kit comprising a recombinant antigen of the present invention, such as a kit for detecting the presence of corresponding autoantibodies from a subject 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 invention (e.g., a detection reagent comprising a 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 invention 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 a solid support such as magnetic beads, a test tube, a microplate, a cuvette, a film, a filter paper, a syringe, a pipette, a buffer such as an assay buffer, a wash buffer, a pretreatment reagent, a tracer marker such as an enzyme-labeled substrate solution, etc.

[0112] On the one hand, the present invention includes a test strip containing the recombinant antigen of the present invention, such as a lateral flow test strip. In some embodiments, the test strip comprises HIV protein 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 strip comprises a recombinant antigen coupled to a tracer marker, at least one detection antibody or at least one detection antigen coated thereon. In some embodiments, the present invention can quickly and accurately detect autoantibodies in a sample by naked eye observation or a chemiluminescent fully automatic instrument. In some embodiments, a 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 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 chemiluminescence automatic 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.

[0113] method

[0114] 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 immune binding complex; detecting the presence or amount of the immune binding complex;

[0115] 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 recombinant antigen 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 thereof.

[0116] In some embodiments, the first recombinant antigen is the recombinant antigen described in the first aspect above, which will not be described in detail 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 the autoimmune disease antigen polypeptide are as described above and will not be described in detail 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 representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

[0120] The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0121] The PE carrier used in the following examples has a carrier map as shown in Figure 1 As shown: The N-terminus of the PE vector itself contains a 6×His tag, so the recombinant antigens translated all contain a 6-histidine tag, which can promote NI-NTA-assisted affinity purification.

[0122] Example 1: Preparation of HIVgp41 recombinant antigen

[0123] 1.1. Design of gp41 recombinant antigen

[0124] Table 1

[0125]

[0126] According to the design table of the recombinant antigen as shown in Table 1, the corresponding fusion partner was introduced into the N-terminus of the target polypeptide gp41, and the expression plasmid was constructed according to the coding nucleic acid of the protein sequence. The gene sequence contained restriction enzyme sites BamHI and EcoRI at both ends. The mutant gene was treated with restriction enzymes BamHI and EcoRI respectively, and the treated gene fragment was connected to the PE vector of Feipeng (such as Figure 1 As shown), the corresponding expression plasmid is obtained, wherein a linker GGSGG is provided between the target polypeptide and the fusion partner.

[0127] 1.2 Recombinant antigen induced expression

[0128] The expression plasmids constructed in 1.1 were transformed into E. coli BL21 competent cells (NEB, catalog number: C2530H) by heat shock method, and spread on LB plates containing 100ug / ml Amp, and cultured at 37℃ for 16h. Pick the colonies, select the positive strains identified by bacterial liquid PCR and double enzyme digestion for seed preservation and inoculate them into LB medium containing 50ug / ml Kan, and culture at 37 degrees with shaking. When OD600 reaches 0.6-0.8, add 1.0mM IPTG, induce culture at 37℃ for 2-4h, extract total protein, and identify the expression of recombinant protein by SDS-PAGE.

[0129] (1) Expression of recombinant antigen in supernatant: The experiment found that gp41-1 and gp41-2 were basically expressed in supernatant, and gp41-4 was partially expressed in supernatant and partially expressed in precipitate. The recombinant antigen was purified using affinity column and ion exchange chromatography.

[0130] (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 dialysis and renaturation, the recombinant antigens are purified using affinity columns and ion exchange chromatography.

[0131] Example 2: Preparation of chemiluminescent reagent

[0132] 2.1. Coating of gp41 antigen

[0133] 1) Activation of magnetic microspheres (taking carboxyl magnetic beads as an example), carboxyl magnetic microspheres with a diameter of about 2 μm were washed three times with 15 mM MES pH 5.5 buffer, and then MES buffer was added to make the magnetic bead concentration reach 10 mg / mL, and then 2 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide was added per 10 mg of magnetic microspheres, and 2 mg of N-hydroxysuccinimide was added per 10 mg of magnetic microspheres, and then placed in a room temperature shaker or mechanical stirrer for shaking or stirring for 1 hour.

[0134] 2) Coating: remove the supernatant from the activated magnetic microspheres in step 1), wash once with 15 mM MES pH 5.5 buffer, then add 15 mM MES pH 5.5 to make its concentration reach 10 mg / mL, then add 0.1 mg HIV gp41 coating antigen (available from FiPen Biotech) per 10 mg of magnetic microspheres, and then place in a room temperature shaker or mechanical stirrer for shaking or stirring for about 2.5 hours to allow the coating antigen to couple to the surface of the magnetic beads.

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

[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, and 1 ml of the sample was placed in a dialysis bag in 1×PBS (pH7.2) and dialyzed overnight. The next day, the dialyzed antigen was taken out 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 mixed solution was placed back in a dialysis bag in 1×PBS (pH7.2) and dialyzed overnight at 4°C. After the dialysis was completed, an equal volume of glycerol was added to the mixed solution, mixed and stored at -20°C, and the acridinium ester-labeled gp41 recombinant antigen (AE-Ag) was obtained.

[0138] 2.3 Indirect labeling of gp41 antigen

[0139] Take 100uL of 10mg / mL BSA solution, add pre-dissolved acridinium ester solution, and react for 15 minutes. Then use a desalting column for desalting, add Traut reagent to the solution, and react for 15min. After desalting, obtain the BSA intermediate for use. Take 100uL of 2mg / mL recombinant antigen, add pre-dissolved SMCC crosslinker solution, and react for 15min. Mix the resulting solution and BSA intermediate in a certain proportion and react for 24h. 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.

[0140] Example 3: Chemiluminescence test

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

[0142] 3.1、Testing process

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

[0144] 2) Then adsorb with magnetic plate and wash the magnetic beads with 1×PBST solution;

[0145] 3) Add 100ul HIV AE labeling working solution into the well of the luminescent plate and mix and react at 37℃ for 10min.

[0146] 4) Then adsorb the beads to the magnetic plate again and wash them with 1×PBST solution;

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

[0148] Test results:

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

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

[0151] The results showed that the Chlorella antifreeze protein or its reverse sequence as a fusion partner of the recombinant gp41 antigen had excellent detection activity or signal-to-noise ratio (P / N) of the sample compared with other fusion partners or no fusion partner.

[0152] 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.

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

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

[0155] The results showed that the detection performance advantage of gp41-1 and gp41-2 over gp41-6 and gp41-3 was further improved when indirect labeling was performed using an amplification vector (BSA).

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

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

[0158]

[0159]

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

[0161] Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of 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 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 claim 1, 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 1-2, or the reagent according to any one of claims 3-4.

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

7. Use of the recombinant antigen according to claim 1 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 recombinant antigen 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.

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

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