Fusion antigen and application thereof

Abstract

The invention provides a fusion antigen and application thereof, and belongs to the technical field of immunodiagnosis. The invention specifically discloses a fusion antigen, a nucleic acid molecule, a vector, a recombinant cell, a test strip or a kit and application thereof. The fusion antigen provided by the invention can realize accurate detection of the antibody corresponding to the antigen, and has improved detection activity or sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a fusion antigen, nucleic acid molecule, vector, recombinant cell, labeled conjugate, test strip or reagent kit, and their uses and methods for detecting antibodies. Background Technology

[0002] Hepatitis C is a serious liver disease caused by the hepatitis C virus (HCV). There are currently 170 million people infected with HCV worldwide, with over 40 million in my country alone. Approximately 50-85% of HCV-infected individuals will develop chronic hepatitis, and about 10-15% will develop cirrhosis. Hepatitis C virus poses a significant threat to human health and imposes a substantial burden on society and the economy.

[0003] Since Choo et al. successfully cloned the HCV gene in 1989, diagnostic and detection technologies for HCV have made great strides. Currently, there are three main methods for detecting hepatitis C: PCR detection of HCV RNA; detection of HCV antigen using monoclonal antibodies; and detection of HCV antibodies using enzyme-linked immunosorbent assay (ELISA), protein staining, and other methods.

[0004] HCV ELISA testing has undergone several generations of evolution and improvement. The first-generation ELISA-1 kit was developed in 1990 by Ortho Diagnostics in the UK, and was subsequently replaced by the second-generation ELISA-2. ELISA-2 kits were developed separately by Ortho Diagnostics in the UK and Abbott Diagnostics in the US. The third-generation ELISA-3 kit was first developed and manufactured by Ortho Diagnostics in the UK, Abbott Diagnostics in the US, and Sanofi Diagnostics in France. All three generations of ELISA tests utilize indirect methods, but indirect methods for detecting HCV antibodies have significant risks of false negatives, false positives, or uncertainty.

[0005] To overcome the shortcomings of the aforementioned indirect methods for HCV detection, a new double-antigen sandwich assay has been developed. This assay uses a coated antigen and a labeled antigen, which bind immunoassay to form an antigen-antibody-labeled antigen complex, thus detecting the antibody being tested. However, due to the unique characteristics of HCV antigens, kits using the double-antigen sandwich assay for HCV antibody detection still have limitations. Furthermore, the major epitope region of HCV, NS3 antigen, is conformationally dependent. When labeled with substances such as horseradish peroxidase or alkaline phosphatase, its conformation changes significantly, and the active epitope is not easily exposed, leading to a substantial decrease in the activity of the labeled NS3 antigen, or even inactivation. Early mainstream methods involved conjugating HCV antigen with activated long-arm biotin to improve conjugation efficiency, achieving market-standard analytical sensitivity and accuracy. Subsequently, Abbott invented an indirect labeling method using BSA to conjugate AE to the HCV antigen, forming an HCVAg-BSA-AE complex for detection. However, these indirect antigen labeling methods are cumbersome and costly.

[0006] CN112707968A discloses a recombinant receptor-binding protein comprising the SARS-CoV-2 receptor-binding domain RBD and a Foldon domain fused to the RBD. The purpose of this patent is to maintain the natural trimeric state of the RBD by fusing the Foldon, thereby mimicking the natural binding process of the trimer RBD to ACE2. This allows for the evaluation of neutralizing antibody titers by utilizing the competitive binding principle between ACE2 and SARS-CoV-2 neutralizing antibodies to the RBD protein. However, no reports have been found of fusing Foldon with antigens that naturally exist stably in a monomeric structure. Summary of the Invention

[0007] As disclosed in reference CN112707968A, ACE2 on the surface of host cells mediates SARS-CoV-2 infection of host cells by recognizing the trimer RBD. In the cited reference, Foldon's fusion with RBD aims to obtain the RBD trimer, thereby mimicking its binding to ACE2. However, the fusion effect of Foldon against antigens that are naturally stable in monomeric structures is unknown, and the labeling activity of such a fusion protein on antigens and its impact on diagnostic efficacy are also unknown and unpredictable.

[0008] This invention aims to provide a fusion antigen and its applications. The fusion antigen of this invention enables accurate detection of antibodies, exhibiting high detection activity, sensitivity, or positive / negative discrimination.

[0009] In a first aspect, the present invention provides a fusion antigen. According to embodiments of the invention, the fusion antigen comprises an antigen and a Foldon domain, wherein the antigen is selected from naturally occurring antigens that are stable in a monomeric structure.

[0010] In a second aspect, the present invention provides an HCV fusion antigen. According to an embodiment of the invention, the fusion antigen comprises an HCV antigen and a Foldon domain.

[0011] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the fusion antigen described in the first or second aspect.

[0012] In a fourth aspect, the present invention provides a carrier. According to embodiments of the invention, the carrier carries the nucleic acid molecule described in the third aspect.

[0013] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell comprises the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect; or expresses the fusion antigen described in the first or second aspect.

[0014] In a sixth aspect, the present invention provides a labeled conjugate. According to embodiments of the invention, the labeled conjugate comprises the fusion antigen described in the first or second aspect.

[0015] In a seventh aspect, the present invention provides a test strip or reagent kit. According to embodiments of the present invention, the test strip or reagent kit comprises: the fusion antigen described in the first or second aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the labeled conjugate described in the sixth aspect.

[0016] In an eighth aspect of the invention, the invention proposes the use of the fusion antigen described in the first or second aspect, the labeled conjugate described in the sixth aspect, or the test strip or kit described in the seventh aspect in the preparation of products for detecting the presence of HCV antibodies, measuring the amount or concentration of HCV antibodies, monitoring the progression of hepatitis C, monitoring the effectiveness of hepatitis C treatment, and / or determining the incidence or risk of hepatitis C in a subject.

[0017] In a ninth aspect of the invention, a method for detecting anti-HCV antibodies in a sample is provided. According to an embodiment of the invention, the method includes: contacting a sample to be tested with a fusion antigen as described in the first or second aspect, a labeled conjugate as described in the sixth aspect, or a test strip or kit as described in the seventh aspect to form an immune complex and detecting the presence of the complex, wherein the presence of the complex indicates the presence of HCV antibodies in the sample.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0020] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0023] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0024] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0025] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0026] In this document, the term "having at least 90% sequence similarity" can mean having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence similarity. The sequence similarity described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are all shown in N-terminus to C-terminus arrangement.

[0027] Without substantially affecting the activity of the HCV antigen or Foldon domain (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of the present invention to obtain variants of the above-mentioned HCV antigen or Foldon domain. These variants are all considered to be included within the scope of protection of the present invention.

[0028] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid differences (mutations) that differ from a reference sequence. This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0029] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and / or between cells or hosts. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0030] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0031] This invention proposes a method for detecting antibodies by fusing antigens, nucleic acid molecules, vectors, recombinant cells, labeled conjugates, test strips or kits, their uses, and other methods, which will be described in detail below.

[0032] Fusion antigen

[0033] In a first aspect, the present invention provides a fusion antigen. According to embodiments of the present invention, the fusion antigen comprises an antigen and a Foldon domain, wherein the antigen is selected from naturally occurring antigens that are stable in a monomeric structure. The fusion antigen of the present invention enables accurate detection of antigen-corresponding antibodies, exhibiting high detection activity, sensitivity, or positive / negative discrimination.

[0034] In this paper, the term "Foldon domain" refers to the domain of the T4 phage fibritin protein, located at the C-terminus of the protein.

[0035] In one alternative embodiment, the Foldon domain may be the 27 residues at the C-terminus of phage T4 fibrin or a mutant thereof, with the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0036] In an alternative embodiment, the Foldon domain can be a truncated or extended form of the 27 residues at the C-terminus of phage T4 fibrin, either by shortening or adding 10 or more amino acids from the N-terminus or C-terminus. Therefore, the truncated or extended form of GYIPEAPRDGQAYVRKDGEWVLLSTFL is also considered an equivalent substitution falling within the scope of this invention.

[0037] In an alternative implementation, the Foldon domain has at least 90% sequence similarity to SEQ ID NO:1.

[0038] According to an embodiment of the present invention, the Foldon domain has an amino acid sequence as shown in SEQ ID NO:1.

[0039] In this paper, the term "monomer structure" refers to the protein molecule structure formed by a single polypeptide chain. Its quaternary and tertiary structures are at the same level. Complexes formed by two or more monomers are collectively called multimers, and if there are two subunits, they are called dimers or dimers.

[0040] In this article, the term "natural" refers to proteins that are naturally formed through evolution in nature. Natural structures are the basis for the stability of the composition and the performance of functions in biological systems. For example, proteins on natural cell membranes, proteins secreted by natural cells, or structural proteins of natural viruses are natural proteins.

[0041] In this article, the term "antigen that exists naturally in a monomeric structure" refers to an antigen that exists stably in its monomeric structure in its natural state, such as being stably present on the cell membrane in monomeric form. It effectively performs its function in its monomeric structure, for example, by binding to receptors and initiating cascade amplification or immune responses.

[0042] In this document, the term "antigen selected from antigens that exist stably in a monomeric structure naturally" does not imply that the antigen is necessarily a naturally obtained antigen. The antigen may also be a non-naturally obtained antigen, such as an antigen obtained through recombinant expression. The key requirement is that the antigen exists stably in a monomeric structure in its natural state.

[0043] For example, the receptor-binding domain (RBD) of SARS-CoV-2 naturally exists as a trimer on the viral membrane. ACE2 on the surface of host cells mediates the infection of host cells by recognizing the trimer RBD. Therefore, RBD is a typical protein that naturally exists stably in a trimer structure (Liu Zezhong, Zhou Jie, Zhu Yun, et al. Application of the trimer antigen vaccine strategy based on recombinant human type III collagen in COVID-19 and influenza vaccines [J]. Synthetic Biology, 2024, 5(2):385.).

[0044] For example, HCV’s non-structural proteins, structural proteins, or core proteins all exist stably in their monomeric form and trigger related immune responses in their monomeric form (Yang Wenxiang, Xiong Wei, Yang Jie, Fan Bolin. Research progress on the infection mechanism of hepatitis C virus. World Chinese Journal of Gastroenterology 2011; 19(20):2133-2140 [DOI:10.11569 / wcjd.v19.i20.2133]).

[0045] According to an embodiment of the present invention, the Foldon domain is connected to the N-terminus or C-terminus of the antigen.

[0046] According to embodiments of the present invention, the antigen and the Foldon domain are directly linked or connected via a linker peptide.

[0047] According to embodiments of the present invention, the linker peptide comprises cleavage amino acids at enzyme cleavage sites and / or a linker.

[0048] According to an embodiment of the present invention, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n or (GGGGS)n; wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a larger integer.

[0049] According to an embodiment of the present invention, the linker peptide is a short peptide containing 1 to 20 amino acids, for example, a short peptide containing 3 to 13 amino acids.

[0050] According to an embodiment of the present invention, the fusion antigen comprises N-terminal and / or C-terminal modifications.

[0051] According to embodiments of the present invention, the modification is selected from protein tags and / or detectable label modifications.

[0052] According to embodiments of the present invention, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0053] According to an embodiment of the present invention, the antigen is selected from infectious disease-related antigens.

[0054] According to an embodiment of the present invention, the antigen is selected from hepatitis C virus antigen (HCV antigen).

[0055] The specific sequence of HCV antigen is not restricted. HCV includes HCV genotypes 1a, 1b, 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 5a, or 6a.

[0056] Patent publication numbers CN101287989B, CN 112341527B, or CN 112225783B disclose a number of HCV antigens, and any HCV antigen falls within the protection scope of this invention.

[0057] According to an embodiment of the present invention, the HCV antigen includes the NS3 protein, which is widely used in HCV diagnosis.

[0058] According to an embodiment of the present invention, the NS3 protein comprises an NS3 polypeptide selected from the amino acid sequence of the HCV whole-genome encoded protein at positions 1075-1657, and the polypeptide has a length of 41-583 amino acids.

[0059] According to an embodiment of the present invention, the NS3 protein comprises a polypeptide at positions 1202-1465 of the amino acid sequence of the HCV whole-genome encoded protein.

[0060] According to an embodiment of the present invention, the HCV antigen further includes the Core protein.

[0061] According to an embodiment of the present invention, the Core protein comprises an NS3 polypeptide selected from the amino acid sequence of the HCV whole-genome encoded protein from positions 1 to 59, wherein the polypeptide is 28 to 59 amino acids in length.

[0062] According to an embodiment of the present invention, the Core protein comprises a polypeptide at positions 1-34 of the amino acid sequence of the HCV whole-genome encoded protein.

[0063] According to an embodiment of the present invention, the HCV antigen further includes the NS4 protein.

[0064] According to an embodiment of the present invention, the NS4 protein comprises a polypeptide at positions 1698-1931 of the amino acid sequence of the HCV whole-genome encoded protein.

[0065] According to embodiments of the present invention, the amino acid sequence of the NS3 protein is shown in SEQ ID NO:4. The amino acid sequence of the Core protein is shown in SEQ ID NO:5.

[0066] In an alternative embodiment, the NS3 protein can be truncated or augmented by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-terminus or C-terminus of SEQ ID NO:4, as long as the binding function of the NS3 protein to the antibody is maintained.

[0067] In an alternative embodiment, the Core protein can be truncated or augmented by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-terminus or C-terminus of SEQ ID NO:5, as long as the binding function of the Core protein to the antibody is maintained.

[0068] In one alternative implementation, the truncation or extension refers to truncating or extending the corresponding amino acid sequence position at the N-terminus or C-terminus of the HCV whole-genome encoded protein.

[0069] According to embodiments of the present invention, the proteins (such as NS3 protein, Core protein, and NS4 protein) are directly connected or linked through linker peptides.

[0070] According to embodiments of the present invention, the linker peptide comprises cleavage amino acids at enzyme cleavage sites and / or a linker.

[0071] According to an embodiment of the present invention, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n or (GGGGS)n; wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a larger integer.

[0072] According to embodiments of the present invention, the amino acid sequence of the HCV antigen is shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0073] According to embodiments of the present invention, the amino acid sequence of the HCV antigen has at least 90% sequence similarity to SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0074] According to an embodiment of the present invention, the amino acid sequence of the HCV fusion antigen is shown in SEQ ID NO:3.

[0075] In a second aspect, the present invention provides an HCV fusion antigen. According to embodiments of the present invention, the fusion antigen comprises an HCV antigen and a Foldon domain. The HCV fusion antigen of the present invention enables accurate detection of anti-HCV antibodies, exhibiting high detection activity, sensitivity, or positive / negative discrimination.

[0076] In one alternative embodiment, the Foldon domain may be the 27 residues at the C-terminus of phage T4 fibrin or a mutant thereof, with the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0077] In an alternative embodiment, the Foldon domain can be a truncated or extended form of the 27 residues at the C-terminus of phage T4 fibrin, either by shortening or adding 10 or more amino acids from the N-terminus or C-terminus. Therefore, the truncated or extended form of GYIPEAPRDGQAYVRKDGEWVLLSTFL is also considered an equivalent substitution falling within the scope of this invention.

[0078] In an alternative implementation, the Foldon domain has at least 90% sequence similarity to SEQ ID NO:1.

[0079] According to an embodiment of the present invention, the Foldon domain has an amino acid sequence as shown in SEQ ID NO:1.

[0080] According to an embodiment of the present invention, the Foldon domain is connected to the N-terminus or C-terminus of the antigen.

[0081] According to embodiments of the present invention, the antigen and the Foldon domain are directly linked or connected via a linker peptide.

[0082] According to embodiments of the present invention, the linker peptide comprises cleavage amino acids at enzyme cleavage sites and / or a linker.

[0083] According to an embodiment of the present invention, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n or (GGGGS)n; wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a larger integer.

[0084] According to an embodiment of the present invention, the linker peptide is a short peptide containing 1 to 20 amino acids, for example, a short peptide containing 3 to 13 amino acids.

[0085] According to an embodiment of the present invention, the fusion antigen comprises N-terminal and / or C-terminal modifications.

[0086] According to embodiments of the present invention, the modification is selected from protein tags and / or detectable label modifications.

[0087] According to embodiments of the present invention, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0088] The specific sequence of HCV antigen is not restricted. HCV includes HCV genotypes 1a, 1b, 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 5a, or 6a.

[0089] Patent publication numbers CN101287989B, CN 112341527B, or CN 112225783B disclose a number of HCV antigens, and any HCV antigen falls within the protection scope of this invention.

[0090] According to an embodiment of the present invention, the HCV antigen includes the NS3 protein, which is widely used in HCV diagnosis.

[0091] According to an embodiment of the present invention, the NS3 protein comprises an NS3 polypeptide selected from the amino acid sequence of the HCV whole-genome encoded protein at positions 1075-1657, and the polypeptide has a length of 41-583 amino acids.

[0092] According to an embodiment of the present invention, the NS3 protein comprises a polypeptide at positions 1202-1465 of the amino acid sequence of the HCV whole-genome encoded protein.

[0093] According to an embodiment of the present invention, the HCV antigen further includes the Core protein.

[0094] According to an embodiment of the present invention, the Core protein comprises an NS3 polypeptide selected from the amino acid sequence of the HCV whole-genome encoded protein from positions 1 to 59, wherein the polypeptide is 28 to 59 amino acids in length.

[0095] According to an embodiment of the present invention, the Core protein comprises a polypeptide at positions 1-34 of the amino acid sequence of the HCV whole-genome encoded protein.

[0096] According to an embodiment of the present invention, the HCV antigen further includes the NS4 protein.

[0097] According to an embodiment of the present invention, the NS4 protein comprises a polypeptide at positions 1698-1931 of the amino acid sequence of the HCV whole-genome encoded protein.

[0098] According to embodiments of the present invention, the amino acid sequence of the NS3 protein is shown in SEQ ID NO:4. The amino acid sequence of the Core protein is shown in SEQ ID NO:5.

[0099] In an alternative embodiment, the NS3 protein can be truncated or augmented by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-terminus or C-terminus of SEQ ID NO:4, as long as the binding function of the NS3 protein to the antibody is maintained.

[0100] In an alternative embodiment, the Core protein can be truncated or augmented by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-terminus or C-terminus of SEQ ID NO:5, as long as the binding function of the Core protein to the antibody is maintained.

[0101] In one alternative implementation, the truncation or extension refers to truncating or extending the corresponding amino acid sequence position at the N-terminus or C-terminus of the HCV whole-genome encoded protein.

[0102] According to embodiments of the present invention, the proteins (such as NS3 protein, Core protein, and NS4 protein) are directly connected or linked through linker peptides.

[0103] According to embodiments of the present invention, the linker peptide comprises cleavage amino acids at enzyme cleavage sites and / or a linker.

[0104] According to an embodiment of the present invention, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n or (GGGGS)n; wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a larger integer.

[0105] According to embodiments of the present invention, the amino acid sequence of the HCV antigen is shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0106] According to embodiments of the present invention, the amino acid sequence of the HCV antigen has at least 90% sequence similarity to SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0107] According to an embodiment of the present invention, the amino acid sequence of the HCV fusion antigen is shown in SEQ ID NO:3.

[0108] Nucleic acid molecules, vectors, and cells or hosts

[0109] In the process of preparing or obtaining the fusion antigens described in the first or second aspect, nucleic acid molecules expressing these fusion antigens can be linked to different vectors and then expressed in different cells to obtain the corresponding fusion antigens.

[0110] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the fusion antigen described in the first or second aspect. The nucleic acid molecule according to embodiments of the invention can encode the aforementioned fusion antigen.

[0111] According to an embodiment of the present invention, the above-mentioned nucleic acid molecule is DNA.

[0112] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0113] In a fourth aspect, the present invention provides a carrier. According to an embodiment of the present invention, the carrier carries the nucleic acid molecule described in the third aspect. When the nucleic acid molecule is attached to the carrier, the nucleic acid molecule can be directly or indirectly connected to control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the carrier itself, or they can be exogenous, i.e., not derived from the carrier itself. Naturally, the nucleic acid molecule and the control elements can be operably connected.

[0114] In this article, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages. After the vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned fusion antigen can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the fusion antigen.

[0115] In some specific embodiments of the present invention, the above-mentioned vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0116] In some specific embodiments of the present invention, the above-mentioned vector is a lentivirus vector.

[0117] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector.

[0118] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell comprises the nucleic acid molecule described in the third aspect or the vector described in the fourth aspect; or expresses the fusion antigen described in the first or second aspect. Using this cell, under suitable conditions, the aforementioned fusion antigen can be efficiently expressed intracellularly.

[0119] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the vector described in the fourth aspect into the recombinant cells.

[0120] It should be noted that the recombinant cells of this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The aforementioned eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0121] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0122] According to an embodiment of the present invention, the recombinant cells are mammalian cells, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0123] It should be noted that the "suitable conditions" mentioned in this invention refer to conditions suitable for the expression of the fusion antigen described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the fusion antigen include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion antigen according to the specific environment of their laboratory.

[0124] Labeled conjugates, test strips or kits

[0125] In a sixth aspect, the present invention provides a labeled conjugate. According to embodiments of the present invention, the labeled conjugate comprises the fusion antigen described in the first or second aspect. The labeled conjugate of the present invention can specifically bind to antibodies corresponding to the antigen and can be used for qualitative or quantitative detection of antigen-corresponding antibodies.

[0126] According to embodiments of the present invention, the above-mentioned marked conjugate may further include at least one of the following additional technical features:

[0127] According to an embodiment of the present invention, the labeled conjugate further comprises a marker conjugated to the fusion antigen.

[0128] According to embodiments of the present invention, the fusion antigen in the conjugate can be directly or indirectly conjugated with the marker.

[0129] In this paper, the term "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target.

[0130] According to embodiments of the present invention, the markers include at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0131] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0132] According to embodiments of the present invention, the fluorescent dyes mentioned above include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0133] According to embodiments of the present invention, the enzymes mentioned above include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0134] According to embodiments of the present invention, the aforementioned radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0135] According to embodiments of the present invention, the chemiluminescent reagents mentioned above include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0136] According to embodiments of the present invention, the above-mentioned nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0137] According to embodiments of the present invention, the above-mentioned colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0138] According to embodiments of the present invention, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0139] In an optional embodiment of the present invention, the above-mentioned markers can directly label fusion antigens or indirectly label fusion antigens. The specific labeling method is not limited and is within the protection scope of the present invention.

[0140] In an optional embodiment of the present invention, the labeled conjugate can directly label the fusion antigen. Compared with indirect labeling, the preparation method of the labeled conjugate obtained by directly labeling the fusion antigen is simpler and more convenient, and the resulting labeled conjugate has better clinical specificity, especially when detecting HCV antibodies, its positive and negative differentiation is more obvious.

[0141] In an optional embodiment of the present invention, the indirect connection in the labeled conjugate may be that the label is directly coupled to a hydrophilic carrier (exemplary bovine serum albumin BSA, KLH, OVA, etc.) and indirectly linked to the fusion antigen through a heterobifunctional linker.

[0142] In a seventh aspect, the present invention provides a test strip or kit. According to embodiments of the present invention, the test strip or kit comprises: the fusion antigen described in the first or second aspect, the nucleic acid molecule described in the third aspect, the carrier described in the fourth aspect, the recombinant cell described in the fifth aspect, or the labeled conjugate described in the sixth aspect. As previously mentioned, the fusion antigen in some specific embodiments or examples of the present invention can bind to the corresponding antibody; therefore, the test strip or kit containing the above-mentioned fusion antigen can effectively perform qualitative or quantitative detection of the corresponding antibody. The test strip or kit provided by the present invention can be used, for example, for detections involving the specific binding properties of fusion antigens and antibodies, such as immunoblotting and immunoprecipitation. As previously mentioned, the test strip or kit containing the above-mentioned fusion antigen has higher detection accuracy and a clear positive / negative differentiation.

[0143] According to embodiments of the present invention, the test strip comprises a fusion antigen with a detectable label (such as colloidal gold), at least one detection antibody or at least one detection antigen coated on a solid phase. In some embodiments, the present invention can rapidly and accurately detect antibodies in a subject by visual observation or by a fully automated chemiluminescence instrument. In some embodiments, the kit can be prepared using the double-antigen sandwich principle. For example, in some embodiments, antibodies in the sample are captured by an antigen coated on a solid phase, or antibodies in the sample are detected by a fusion antigen labeled with a detectable label. In some embodiments, an excitation solution is added, and the luminescence value is measured using a fully automated chemiluminescence instrument. The luminescence value is positively correlated with the total antibody concentration in the sample, and compared with a threshold value to determine whether the result is positive or negative.

[0144] According to embodiments of the present invention, the kit includes reagents suitable for performing immunoassays. In some embodiments, the kit may include instructions for use of the immunodiagnostic reagents of the present invention (e.g., labeled conjugates containing fusion antigens) in immunoassays for detecting corresponding antibodies. In some embodiments, the kit may include calibrators or controls, such as standards or control antibodies. In some embodiments, the fusion antigens or labeled conjugates of the present invention are contained in containers such as test tubes, microplates, or test strips within the kit. In some embodiments, the kit may also include solid-phase supports such as magnetic beads, test tubes, microplates, cuvettes, membranes, filter paper, syringes, pipettes, buffers such as assay buffers, wash buffers, pretreatment reagents, detectable labeling such as enzyme-labeled substrate solutions, etc.

[0145] use

[0146] In an eighth aspect of the invention, the invention proposes the use of the fusion antigen described in the first or second aspect, the labeled conjugate described in the sixth aspect, or the test strip or kit described in the seventh aspect in the preparation of products for detecting the presence of HCV antibodies, measuring the amount or concentration of HCV antibodies, monitoring the progression of hepatitis C, monitoring the effectiveness of hepatitis C treatment, and / or determining the incidence or risk of hepatitis C in a subject.

[0147] method

[0148] In a ninth aspect, the present invention provides a method for detecting anti-HCV antibodies. According to an embodiment of the present invention, the method includes: contacting a sample to be tested with a fusion antigen as described in the first or second aspect, a labeled conjugate as described in the sixth aspect, or a test strip or kit as described in the seventh aspect to form an immune complex; and detecting the presence of the complex, wherein the presence of the complex indicates the presence of HCV antibodies in the sample. The detection method of the present invention can detect anti-HCV antibodies and has advantages such as high detection accuracy and clear differentiation between positive and negative results.

[0149] According to an embodiment of the present invention, the method is a double-antigen sandwich method.

[0150] In a tenth aspect of the invention, a method is provided for monitoring the progression of hepatitis C, monitoring the efficacy of hepatitis C treatment, and / or determining the incidence or risk of hepatitis C in a subject. According to an embodiment of the invention, the method includes: contacting a sample to be tested with the fusion antigen described in the first or second aspect, the labeled conjugate described in the sixth aspect, or the test strip or kit described in the seventh aspect to form an immune complex; and detecting the presence of the complex, wherein the presence of the complex indicates the presence of HCV antibodies in the sample. The detection method of the present invention has advantages such as high detection accuracy and significant positive / negative differentiation.

[0151] In an eleventh aspect of the invention, the invention provides a method for preparing the fusion antigen described in the first or second aspect, comprising expressing recombinant cells of the fifth aspect.

[0152] The amino acid sequence listing of the present invention is shown below:

[0153]

[0154]

[0155]

[0156] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0157] Preparation Example 1: Plasmid construction, protein expression and purification of HCV fusion antigen

[0158] 1. Construction of a fusion tag carrier

[0159] The fusion tag shown in SEQ ID NO:1 was selected as the specific protein for cloning and construction. Its DNA sequence can be obtained using E. coli, PET32A, gene synthesis, etc., and codon optimization can be performed to achieve the optimal expression state. An expression vector containing the HIS tag was constructed, and a GGSGG adapter was introduced at the 5' end of the fusion tag sequence, with reserved restriction enzyme sites.

[0160] 2. Construction of HCV gene-engineered fusion antigen expression plasmid

[0161] The source of HCV antigen sequences is not limited. Many HCV antigen sequences have been disclosed in the prior art, such as any HCV antigen described in any of the embodiments in CN101287989B, CN 112341527B or CN 112225783B.

[0162] The HCV antigens selected in this embodiment include the NS3 protein shown in SEQ ID NO:4 and the Core protein shown in SEQ ID NO:5. The ligation of the NS3 and Core protein gene fragments can be performed by restriction enzyme digestion and T4 DNA ligase, or by bridge PCR using primer ligation. The segments can be ligated with or without linkers. All the gene fragments were constructed into the prepared expression vector using restriction enzyme digestion and T4 DNA ligase.

[0163] 3. HCV fusion antigen induction and purification fusion antigen induction expression

[0164] The constructed expression plasmid was transformed into *E. coli* BL21 competent cells (NEB (New England Biolabs), catalog number: C2530H) using a heat shock method and plated on LB agar plates containing antibiotics, incubated at 37°C for 16 h. Single colonies were picked, and positive strains identified by bacterial PCR and double enzyme digestion were preserved and inoculated into LB medium containing 50 μg / ml Kans, and cultured with shaking at 37°C. After the OD600 reached 0.6–0.8, 1.0 mM IPTG was added, and the culture was induced at 37°C for 2–4 h. Total protein was extracted, and SDS-PAGE was used to identify the expression of recombinant protein. The protein was purified to 96% purity by nickel ion chelation and SP column purification using the 6*HIS tag at the N-terminus of the recombinant protein. The sequence of the HCV fusion antigen is shown in SEQ ID NO:3.

[0165] Preparation Example 2: Preparation of Fusion Antigen-Labeled Conjugates by Direct Labeling Method

[0166] The specific steps are as follows:

[0167] 1.1 Dialysis antigen: The antigen (HCV fusion antigen in Preparation Example 1 or unfused Folden HCV antigen) was diluted to 1 mg / mL in advance with dialysis buffer, and then dialyzed overnight at 4°C with 20 mM PB + 150 mM NaCl; after about 14 to 16 hours, the buffer was changed.

[0168] 1.2. Labeling: Take out the activated commercially available NHS-labeled substance (e.g., NHS-AE), add the activated NHS-labeled substance to the antigen according to the labeling ratio in the instructions, and react with shaking in the dark for 0.5 h.

[0169] 1.3 Free label treatment: The labeled antigen-label was dialyzed with 20mM PB + 150mM NaCl at 4℃. The process should be kept away from light. The solution was changed every hour for a total of 5 times. The antigen-label was then recovered.

[0170] 1.4 Recover the antigen-labeled material, add an equal volume of glycerol, and store it in a light-proof tube or brown bottle at -20 degrees Celsius for later use.

[0171] Preparation Example 3: Preparation of Fusion Antigen-Labeled Conjugates by Indirect Labeling Method

[0172] The specific steps are as follows: Weigh common conjugation carrier proteins (e.g., BSA, KLH, OVA, etc.) in solid form, add buffer solution, add activated label (e.g., AE-NHS) to the carrier solution, react at room temperature, block with glycine, dialyze the sample overnight using a dialysis bag, and recover the label from the dialysis bag to obtain the carrier-labeled conjugate (e.g., BSA-AE). The carrier-labeled conjugate is activated with a heterobifunctional linker (SMCC, LC-SMCC, or Sulfo-SMCC, etc.), while the HCV antigen is activated with 2-IT. The activated HCV antigen is mixed with the SMCC-BSA-AE sample, reacted at room temperature using a rotary mixer, and then reacted overnight at low temperature to obtain the indirectly labeled antigen-labeled conjugate.

[0173] Example 1: Diagnostic activity and sensitivity of HCV fusion antigen on the AE labeling platform

[0174] The detection was performed using a double-antigen sandwich method. The reaction process involved mixing the test sample and HCV antigen (purchased from Feipeng Biotechnology) coated magnetic beads in the wells of a luminescent plate at 37°C. After adsorption by the magnetic plate, the magnetic beads were washed with 1×PBST solution. Then, the antigen-labeled conjugate working solution prepared in Example 2 was added and mixed in the wells of the luminescent plate at 37°C. At this point, a double-antigen sandwich system was formed in the wells. After adsorption by the magnetic plate, the magnetic beads were washed with 1×PBST solution. Then, AE excitation solution was added, and the luminescence value was read using a Thermo Fisher chemiluminescence analyzer. The results showed that the detection activity and sensitivity of the HCV antigen fused with the Folden domain tag were higher than those of the HCV antigen without the Folden domain tag.

[0175] Table 1: Detection data of HCV fusion antigen on the AE labeling platform

[0176]

[0177] Example 2: Diagnostic activity and sensitivity of HCV fusion antigens with different labeling methods on the AE labeling platform

[0178] The detection was performed using a double-antigen sandwich method. The reaction process involved mixing the sample and HCV antigen (purchased from Feipeng Biotechnology) coated magnetic beads in the wells of a luminescent plate at 37°C. The mixture was then adsorbed onto the magnetic plate, washed with 1×PBST solution, and the antigen-labeled conjugate prepared in Preparation Example 2 or Preparation Example 3 was added to the wells and mixed again at 37°C. This formed a double-antigen sandwich system within the wells. The mixture was then adsorbed onto the magnetic plate, washed with 1×PBST solution, and finally, AE excitation solution was added. The luminescence values ​​were read using a ThermoFisher chemiluminescence analyzer. The results are as follows:

[0179] Table 2: Detection data of HCV fusion antigens with different labeling methods on the AE labeling platform

[0180]

[0181] It is evident that HCV antigens fused with the Folden domain, whether directly or indirectly labeled, can be distinguished between positive and negative results. Among them, the experimental group with HCV antigens directly labeled with the Folden domain exhibited higher activity and better background.

[0182] Example 3: Diagnostic activity and sensitivity of recombinant HCV protein on HRP or AP labeling platforms

[0183] 1. HRP tagging and HRP tagging platform detection steps

[0184] Dissolved HRP and freshly prepared NaIO4 solution were stirred at room temperature in the dark for 20 minutes, and ethylene glycol was added and reacted for 30 minutes. Activated HRP was added to antigen that had been dialyzed with 0.1 mM pH 9.51 carbonate buffer and dialyzed overnight at 4°C. Freshly prepared NaBH4 solution was added, mixed, and incubated at 4°C for 2 hours. Then an equal volume of saturated ammonium sulfate was added, and the mixture was incubated at 4°C for 1 hour. After centrifugation for half an hour, the supernatant was discarded. The precipitate was dissolved in 0.2 M pH 7.4 PBS, and then 50% glycerol was added to obtain the HRP-labeled antigen, which was stored at -20°C.

[0185] The double-antigen sandwich method was used for detection. The reaction process involved mixing the sample and HCV antigen (purchased from Phytobio) coated magnetic beads in the wells of an ELISA plate at 37°C for a period of time, during which a double-antigen sandwich system would form. The plate was washed with 1×PBST solution, and then HRP chromogenic solution was added. The results showed that the detection activity and sensitivity of the HCV antigen fused with the Folden domain tag were higher than those of the HCV antigen without the Folden domain tag.

[0186] Table 3: Detection data of HCV fusion antigen on the HRP labeling platform

[0187] Fusion antigen: HCV antigen fused with Folden domain HCV antigen without fused Folden domain Positive quality control #1 2.012 1.683 Positive quality control #2 1.014 0.606 Positive quality control #3 0.459 0.333 Positive quality control #4 0.180 0.152 Positive quality control #5 0.658 0.436 Negative serum 1 0.013 0.032 Negative serum 2 0.019 0.055 Negative serum 3 0.021 0.046 P-mean: 0.865 0.642 N-mean: 0.02 0.04 P / N value: 48.39 14.50

[0188] 2. AP Labeling and Detection Procedure of the AP Labeling Platform: Antigen and AP Enzyme Processing: Take two equilibrated desalted columns and perform buffer replacement on HCV antigen and AP enzyme separately; Antigen Activation: Add 2-IT (2-iminothionane hydrochloride) solution to the antigen and react at room temperature for 15 minutes; add glycine to stop the reaction, then desalt again; AP Enzyme Activation: Add SMCC solution to the AP enzyme and react at room temperature for 15 minutes; add glycine to stop the reaction, then desalt again; Coupling: Mix the activated AP enzyme with the antigen, incubate overnight at 4°C, add ethanolamine solution, mix thoroughly, and incubate at room temperature for 10 minutes. Pass through a Superdex-200 column and collect the target protein peak. Add 50% glycerol and store at -20°C for later use.

[0189] Table 4: Detection data of HCV fusion antigen on the AP labeling platform

[0190]

[0191]

[0192] Following the sequence and antigen preparation method disclosed in CN101287989B, HCV antigens with the amino acid sequences shown in SEQ ID NO:6 or SEQ ID NO:7 were prepared. Following the sequence and antigen preparation method disclosed in CN112225783B, HCV antigens with the amino acid sequences shown in SEQ ID NO:8 or SEQ ID NO:9 were prepared. HCV fusion antigens were prepared according to Preparation Example 1 above, and antigen-labeled conjugates were prepared according to Preparation Examples 2 and 3. The diagnostic activity and sensitivity of the above-mentioned recombinant HCV proteins on different labeling platforms were tested according to Examples 1 to 3. The results showed that the detection activity and sensitivity of the HCV antigen fused with the Folden domain tag were higher than those of the HCV antigen without the Folden domain tag, and the activity of direct labeling of the HCV antigen fused with the Folden domain was better than that of indirect labeling.

[0193] Comparative Example 1: Following the preparation method in Preparation Example 1, the Folden domain was fused with an HIV antigen to obtain an HIV fusion antigen, and the performance of HIV antibodies was detected using the HIV fusion antigen. However, after the HIV antigen was fused with the Folden domain to obtain the HIV fusion antigen, it was prone to precipitation, and the positive detection signal was actually lower than that obtained by using the HIV antigen alone as the labeling antigen reagent.

[0194] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples, without contradiction. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion antigen, characterized in that, It includes an antigen and a Foldon domain, wherein the antigen is selected from naturally occurring antigens that exist stably in a monomeric structure.

2. The fusion antigen according to claim 1, characterized in that, The Foldon domain has the amino acid sequence shown in SEQ ID NO:1; Optionally, the antigen and the Foldon domain are directly linked or linked via a linker peptide; Optionally, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n, or (GGGGS)n; wherein n is selected from an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or larger.

3. The fusion antigen according to claim 1, characterized in that, The Foldon domain is attached to the N-terminus or C-terminus of the antigen; Optionally, the fusion antigen includes N-terminal and / or C-terminal modifications; Optionally, the modification is selected from protein tags and / or detectable label modifications; Optionally, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag; Optionally, the antigen is selected from infectious disease-related antigens; Optionally, the antigen is selected from hepatitis C virus antigens.

4. An HCV fusion antigen, characterized in that, Including HCV antigen and Foldon domain; Optionally, the Foldon domain has an amino acid sequence as shown in SEQ ID NO:1; Optionally, the antigen and the Foldon domain are directly linked or linked via a linker peptide; Optionally, the linker peptide comprises a sequence module selected from (G)n, (GS)n, (SG)n, (GGGS)n, (GGSGG)n, or (GGGGS)n; wherein n is selected from an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or larger. Optionally, the Foldon domain is connected to the N-terminus or C-terminus of the antigen; Optionally, the fusion antigen includes N-terminal and / or C-terminal modifications; Optionally, the modification is selected from protein tags and / or detectable label modifications; Optionally, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

5. A nucleic acid molecule, vector, or recombinant cell, characterized in that, The nucleic acid molecule encodes the fusion antigen according to any one of claims 1 to 4; the vector carries the above-mentioned nucleic acid molecule; the recombinant cell includes the above-mentioned nucleic acid molecule, vector, or expresses the fusion antigen according to any one of claims 1 to 4.

6. A labeled conjugate, characterized in that, Includes the fusion antigen as described in any one of claims 1 to 4; Optionally, the conjugate further comprises a marker conjugated to the fusion antigen; Optionally, the fusion antigen in the conjugate can be directly or indirectly conjugated to the marker; Optionally, the marker includes at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

7. A test strip or reagent kit, characterized in that, include: The fusion antigen according to any one of claims 1 to 4, the nucleic acid molecule, vector or recombinant cell according to claim 5, or the label conjugate according to claim 6.

8. Use of the fusion antigen of any one of claims 1 to 4, the labeled conjugate of claim 6, or the test strip or kit of claim 7 in the preparation of products for detecting the presence of HCV antibodies, measuring the amount or concentration of HCV antibodies, monitoring the progression of hepatitis C, monitoring the efficacy of hepatitis C treatment, and / or determining the incidence or risk of hepatitis C in a subject.

9. A method for detecting anti-HCV antibodies in a sample, characterized in that, include: The fusion antigen of any one of claims 1 to 4, the labeled conjugate of claim 6, or the test strip or kit of claim 7 are brought into contact with the sample to be tested to form an immune complex; and the presence of the complex is detected, wherein the presence of the complex indicates the presence of HCV antibodies in the sample.

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