Chimeric antigen, kit comprising same, and use thereof

Abstract

Disclosed in the present application is a chimeric antigen, comprising a dengue virus NS5 antigen and an E antigen. The present application specifically relates to a chimeric antigen, a nucleic acid molecule, an expression vector, a host cell, a reagent, a kit, and use thereof. The chimeric antigen or a conjugate comprising the chimeric antigen of the present application has improved activity, and can be used for preparing a kit, such that accurate detection of anti-dengue virus antibodies can be achieved.

Description

Chimeric antigens, reagent kits containing them, and their uses

[0001] Priority information

[0002] This application claims priority to Chinese Patent Application No. 202411760476.5, filed on December 2, 2024, entitled "Chimeric Antigen, Reagent Kit Containing the Same and Use Therein", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of immunoassay technology, specifically relating to a chimeric antigen, nucleic acid molecule, vector, recombinant cell, conjugate, test strip or kit, and their uses and methods for detecting dengue virus antibodies in samples. Background Technology

[0004] Dengue fever is an acute vector-borne infectious disease caused by the dengue virus, a serotype subgroup belonging to the Flaviviridae family and Flavivirus genus. The virus particles are spherical, approximately 55 nanometers in diameter. The virus particles are enveloped by a lipoprotein membrane and possess envelope spikes. The outer layer of the viral envelope contains envelope protein E, and the inner layer contains membrane protein M. The viral core is an icosahedral nucleocapsid structure composed of a single-stranded, positive-sense RNA (+ssRNA) and a viral capsid protein C. The dengue virus genome RNA contains approximately 11,000 nucleotides, with a type I cap structure at its 5′ end and lacking a poly(A) tail at its 3′ end. Both the 5′ and 3′ ends of the genome contain a non-coding region, while the genome contains only one open reading frame (LOF). The 5′ quarter of this LEF encodes three structural proteins (C, PrM, and E), and the 3′ three-quarters encodes seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).

[0005] The primary mode of transmission for dengue fever is mosquito transmission. Aedes mosquitoes are the main vectors of the dengue virus. After biting a virus-carrying host (such as a human or monkey), the virus replicates and multiplies within the mosquito, then spreads to a new host by biting a healthy human. In addition, the dengue virus can also be transmitted through blood, direct contact (such as contact with the blood, urine, or saliva of an infected person), and indirect contact (such as contact with virus-contaminated objects). However, these modes of transmission are relatively less common and less widespread and effective than mosquito transmission. Dengue virus is prevalent in tropical and subtropical regions, particularly in the Far East, Southeast Asia, and the western Pacific Ocean.

[0006] Dengue fever is highly contagious and pathogenic, and there is currently no specific antiviral treatment. The main principles of treatment are early detection, early diagnosis, early mosquito control and isolation, and early treatment. Therefore, the development of high-performance dengue detection materials is urgently needed to effectively control its spread and epidemic. Summary of the Invention

[0007] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a chimeric antigen that enables accurate detection of antibodies with high sensitivity.

[0008] In a first aspect of this application, a chimeric antigen is proposed. According to an embodiment of this application, the chimeric antigen comprises dengue virus NS5 antigen and E antigen.

[0009] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the chimeric antigen described in the first aspect.

[0010] In a third aspect of this application, a vector or recombinant cell is provided. According to embodiments of this application, the vector carries the nucleic acid molecules described in the second aspect;

[0011] The recombinant cells include the nucleic acid molecules described in the second aspect, or the aforementioned vectors; or express the chimeric antigens described in the first aspect.

[0012] In a fourth aspect of this application, a conjugate is proposed. According to an embodiment of this application, the conjugate comprises the chimeric antigen and the conjugate portion described in the first aspect.

[0013] In a fifth aspect of this application, a test strip or reagent kit is provided. According to embodiments of this application, the test strip or reagent kit comprises: the chimeric antigen described in the first aspect, the nucleic acid molecule described in the second aspect, the vector or recombinant cell described in the third aspect, or the conjugate described in the fourth aspect.

[0014] In a sixth aspect of this application, the application discloses the use of the chimeric antigen described in the first aspect, the conjugate described in the fourth aspect, or the test strip or kit described in the fifth aspect in the preparation of a product for detecting dengue virus antibodies or diagnosing dengue virus infection-related diseases.

[0015] In a seventh aspect of this application, a method for detecting anti-dengue virus antibodies in a sample is provided. According to an embodiment of this application, the method includes: contacting the sample to be tested with the chimeric antigen described in the first aspect, the conjugate described in the fourth aspect, or the test strip or kit described in the fifth aspect to form an immune complex; and determining whether dengue virus antibodies are present in the sample based on the detection result of the complex.

[0016] Additional aspects and advantages of this application 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 this application. Detailed Implementation

[0017] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0018] 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 technical features indicated. 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 application, unless otherwise stated, "multiple" means two or more.

[0019] 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.

[0020] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application 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.

[0021] 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.

[0022] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0023] 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.

[0024] 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.

[0025] 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 application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. All amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0026] In this document, without substantially affecting the chimeric antigen (retaining at least 90% of its activity), those skilled in the art may 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 this application to obtain variants of the aforementioned antigen. These are all considered to be included within the scope of protection of this application.

[0027] As used in this disclosure, the term "polypeptide" refers to any molecule comprising three or more amino acid residues linked by peptide bonds. Polypeptides according to this application include peptides (e.g., tripeptides, oligopeptides, etc.), and peptides that may contain chemical modifications (e.g., glycosylation (glycopeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation). Polypeptides may also refer to proteins.

[0028] 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.

[0029] 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 sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0030] This application discloses a chimeric antigen, nucleic acid molecule, vector, recombinant cell, conjugate, test strip or kit, and their uses and methods for detecting anti-dengue virus antibodies in samples, which will be described in detail below.

[0031] chimeric antigen

[0032] In a first aspect, this application proposes a chimeric antigen. According to embodiments of this application, the chimeric antigen comprises dengue virus NS5 antigen and E antigen. The chimeric antigen of this application enables accurate detection of antibodies against the chimeric antigen and has the advantage of high sensitivity.

[0033] The term "chimeric antigen" refers to a recombinant antigen that combines different antigens into a single polypeptide through chemical synthesis or recombinant expression.

[0034] According to embodiments of this application, the chimeric antigen may further include at least one of the following technical features:

[0035] According to an embodiment of this application, the E antigen comprises the m-n amino acid region of the dengue virus E protein.

[0036] According to an embodiment of this application, m is taken from any integer from 1 to 307.

[0037] According to embodiments of this application, m is 1, 277, 296, 297, or 307.

[0038] According to an embodiment of this application, n is taken from any integer from 384 to 495.

[0039] According to embodiments of this application, n is 384, 394, 421, 434, or 495.

[0040] According to embodiments of this application, the E antigen includes the immunoglobulin-like domain of the dengue virus E protein. The immunoglobulin-like domain of the dengue virus E protein, as a primary target for antibody binding, contains antigenic epitopes capable of inducing the production of specific antibodies. The immunoglobulin-like domain segment of the dengue virus E protein is well known to those skilled in the art.

[0041] According to an embodiment of this application, m is taken from any integer from 277 to 307, and n is taken from any integer from 384 to 434.

[0042] According to embodiments of this application, the E antigen includes amino acid segments from position 297 to 394, position 277 to 434, position 307 to 384, or position 296 to 421 of the dengue virus E protein.

[0043] According to an embodiment of this application, the position of the E antigen is determined using the amino acid sequence shown in SEQ ID NO:1 as a reference sequence.

[0044] According to an embodiment of this application, the NS5 antigen comprises a full-length or truncated segment of the dengue virus NS5 protein; the number of amino acids in the truncated segment is any integer from 11 to 623.

[0045] According to an embodiment of this application, the number of amino acids in the truncated segment is any integer from 31 to 593.

[0046] According to embodiments of this application, the number of amino acids in the truncated segment is 11, 12, 13, 14, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 265, 300, 350, 400, 450, 500, 550, 593, or 623. According to embodiments of this application, the NS5 antigen comprises the x-th to y-th amino acid segment of the dengue virus NS5 protein, and the position of the NS5 antigen is determined with reference to the amino acid sequence shown in SEQ ID NO:3.

[0047] According to an embodiment of this application, x is taken from any integer from 1 to 869.

[0048] According to embodiments of this application, x is 1, 266, 277, 849, 869, or 878.

[0049] According to an embodiment of this application, y is taken from any integer from 2 to 899.

[0050] According to an embodiment of this application, y is taken from any integer from 265 to 899.

[0051] According to embodiments of this application, y is 265, 276, 869, 890, or 899.

[0052] According to an embodiment of this application, x is taken from any integer from 849 to 878, and y is taken from any integer from 890 to 899.

[0053] According to embodiments of this application, the NS5 antigen includes at least one of the following amino acid segments of the dengue virus NS5 protein: amino acid segments 869-899, 1-265, 266-276, 277-869, 849-890, and 878-899. The position of the NS5 antigen is determined with reference to the amino acid sequence shown in SEQ ID NO:3.

[0054] (SEQ ID NO:3). It should be noted that the positions of the dengue virus E protein (e.g., amino acids 297 to 394) and the positions of the dengue virus NS5 protein (e.g., amino acids 869 to 899) in this application were determined with reference to a reference sequence.The amino acid positions 297–394 and 869–899 are relative positions. For polypeptides related to the reference sequence (e.g., natural dengue virus strains from different species or different subtypes of natural dengue virus strains), the specific position is determined by the corresponding position of the relevant polypeptide. For example, publicly available computer software such as BLAST, BLAST2, ALIGN, or MEGALIGN™ (DNASTAR) can be used to align the relevant polypeptide with the reference sequence to determine its position. Therefore, antigens obtained from natural dengue virus strains from different species or different subtypes of natural dengue virus strains are all within the scope of protection of this application.

[0055] For example, the amino acid sequences corresponding to the "297th to 394th amino acid segment of the dengue virus E protein" described in this application are shown in SEQ ID NO:2 or SEQ ID NO:30 on type I dengue virus, respectively; and the amino acid sequences corresponding to type II, III, and IV dengue viruses are shown in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively.

[0056] For example, the amino acid sequences corresponding to the "869th to 899th amino acid segment of the dengue virus NS5 protein" described in this application on dengue virus types I, II, III, and IV are shown in SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

[0057] According to an embodiment of this application, the E antigen is composed of the m-n amino acid region of the dengue virus E protein.

[0058] According to an embodiment of this application, m is taken from any integer from 1 to 307.

[0059] According to embodiments of this application, m is 1, 277, 296, 297, or 307.

[0060] According to an embodiment of this application, n is taken from any integer from 384 to 495.

[0061] According to embodiments of this application, n is 384, 394, 421, 434, or 495.

[0062] According to an embodiment of this application, m is taken from any integer from 277 to 307, and n is taken from any integer from 384 to 434.

[0063] According to embodiments of this application, the E antigen is composed of the immunoglobulin-like domain of the dengue virus E protein. The immunoglobulin-like domain segment of the dengue virus E protein is well known to those skilled in the art.

[0064] According to an embodiment of this application, the E antigen is composed of amino acid segments from position 297 to 394, position 277 to 434, position 307 to 384, or position 296 to 421 of the dengue virus E protein.

[0065] According to an embodiment of this application, the NS5 antigen is composed of a full-length or truncated segment of the dengue virus NS5 protein; the number of amino acids in the truncated segment is any integer from 11 to 623.

[0066] According to an embodiment of this application, the number of amino acids in the truncated segment is any integer from 31 to 593.

[0067] According to embodiments of this application, the number of amino acids in the truncated segment is 11, 12, 13, 14, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 265, 300, 350, 400, 450, 500, 550, 593, or 623.

[0068] According to an embodiment of this application, the NS5 antigen is composed of amino acid segments from position x to position y of the dengue virus NS5 protein, and the position of the NS5 antigen is determined with reference to the amino acid sequence shown in SEQ ID NO:3.

[0069] According to an embodiment of this application, x is taken from any integer from 1 to 869.

[0070] According to embodiments of this application, x is 1, 266, 277, 849, 869, or 878.

[0071] According to an embodiment of this application, y is taken from any integer from 2 to 899.

[0072] According to an embodiment of this application, y is taken from any integer from 265 to 899.

[0073] According to embodiments of this application, y is 265, 276, 869, 890, or 899.

[0074] According to an embodiment of this application, x is taken from any integer from 849 to 878, and y is taken from any integer from 890 to 899.

[0075] According to an embodiment of this application, the NS5 antigen is composed of amino acid segments from position 869 to 899, position 1 to 265, position 266 to 276, position 277 to 869, position 849 to 890, or position 878 to 899 of the dengue virus NS5 protein.

[0076] The chimeric antigen derived from the fusion of NS5 antigen and E antigen exhibits superior activity compared to either a single E antigen or NS5 antigen.

[0077] The chimeric antigen of this application enables accurate detection of antibodies against chimeric antigens, and has the advantages of high sensitivity and high specificity.

[0078] According to embodiments of this application, the dengue virus NS5 antigen and E antigen are directly or indirectly fused; for example, through linker peptide fusion.

[0079] The term "linker peptide" refers to a short peptide used to link two molecules (e.g., proteins). The chimeric antigens of this invention may contain linker peptides. Typically, fusion proteins are obtained by introducing (e.g., by PCR amplification or ligase) a polynucleotide sequence encoding the short peptide between two DNA fragments encoding the two target proteins to be linked, and then expressing the proteins. The linker peptide may be a flexible linker, such as a G- or S-rich flexible linker.

[0080] According to embodiments of this application, the dengue virus NS5 antigen is fused to the N-terminus or C-terminus of the E antigen.

[0081] According to embodiments of this application, the dengue virus is selected from type I, II, III, or IV.

[0082] According to embodiments of this application, the amino acid sequence of the NS5 antigen has at least 90% sequence similarity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:14, or SEQ ID NO:15; the amino acid sequence of the E antigen has at least 90% sequence similarity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:25, or SEQ ID NO:30; and the sequence of the chimeric antigen has at least 90% sequence similarity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 to SEQ ID NO:11, or SEQ ID NO:26.

[0083] According to embodiments of this application, the amino acid sequence of the NS5 antigen has at least 90% sequence similarity to SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21; the amino acid sequence of the E antigen has at least 90% sequence similarity to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; and the sequence of the chimeric antigen has at least 90% sequence similarity to SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.

[0084] According to embodiments of this application, the amino acid sequence of the NS5 antigen is shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:14, or SEQ ID NO:15; the amino acid sequence of the E antigen is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:25, or SEQ ID NO:30; and the sequence of the chimeric antigen is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 to SEQ ID NO:11, or SEQ ID NO:26.

[0085] According to embodiments of this application, the amino acid sequence of the NS5 antigen is shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21; the amino acid sequence of the E antigen is shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; and the sequence of the chimeric antigen is shown in SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.

[0086] It should be noted that in this application, "the amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A or the amino acid sequence of SEQ ID NO:A with conservative modifications, all of which are within the scope of protection of this application. For example, "the amino acid sequence of the NS5 antigen as shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:7" means that the NS5 antigen is the amino acid sequence shown in SEQ ID NO:3, 4, or 7, or is the amino acid sequence of SEQ ID NO:3, 4, or 7 with conservative modifications, all of which are within the scope of protection of this application.

[0087] In this document, "conserved modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of the amino acid sequence to dengue virus antibodies. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antigens of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the antigen of this application can be substituted with other amino acid residues from the same side chain family, and the retention function of the modified antigen can be tested using the functional assay methods described herein. Exemplarily, conservative modifications are limited to no more than 80% of the total number, preferably no more than 90%. In this article, "conserved modified amino acid sequences" also include naturally occurring amino acid modifications. "Naturally occurring mutations" refer to mutations caused by changes in alleles due to individual differences or other reasons during the natural mutation process.

[0088] In some optional embodiments of this application, the chimeric antigen includes an antigen derived from dengue virus, which is composed of dengue virus NS5 antigen and E antigen.

[0089] It should be understood that the chimeric antigen of this application may contain one or more NS5 antigens, wherein the subtypes of the one or more NS5 antigens may be the same or different, and the sequences of the one or more NS5 antigens may be the same or different. The chimeric antigen of this application may contain one or more E antigens, wherein the subtypes of the one or more E antigens may be the same or different, and the sequences of the one or more E antigens may be the same or different. When multiple subtypes or multiple sequences of NS5 antigens or E antigens form a chimeric antigen, it has a further enhanced sensitivity compared to a single subtype or single sequence of NS5 antigen or E antigen.

[0090] It should be understood that the chimeric antigen of this application may also contain non-antigen functional segments, such as fusion partners.

[0091] The term "fusion partner" is sometimes also referred to as a tagged polypeptide, which is fused or linked to a target protein (e.g., a dengue virus-derived antigen of this disclosure) and thereby facilitates the soluble expression, stabilization, and / or purification of the recombinant protein. The tag may be fused to or linked to the N-terminus and / or C-terminus of the target protein (optionally via a linker or protease cleavage site). Such tags are well known to those skilled in the art and have been described in detail in the prior art literature. Examples of such tags include, but are not limited to, histidine (His) tags, glutathione transferase (GST) tags, maltose-binding protein (MBP) tags, thioredoxin (Trx) tags, NusA tags, disulfide isomerase DsbA tags, DsbC tags, SUMO tags, msyB tags, TF tags, priming factor tags, ubiquitin tags, Myc tags, Flag tags, fluorescent protein (e.g., GFP) tags, biotin tags, avidin tags, and Foldon domains.

[0092] Nucleic acid molecules, vectors, and cells or hosts

[0093] In the process of preparing or obtaining the chimeric antigens described in the first aspect, nucleic acid molecules expressing these chimeric antigens can be linked to different vectors and then expressed in different cells to obtain the corresponding chimeric antigens.

[0094] In a second aspect of this application, a nucleic acid molecule is provided. According to an embodiment of this application, the nucleic acid molecule encodes the chimeric antigen described in the first aspect. The nucleic acid molecule according to an embodiment of this application can encode the aforementioned chimeric antigen.

[0095] According to the embodiments of this application, the above-mentioned nucleic acid molecule is DNA.

[0096] 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 application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0097] In a third aspect, this application proposes a vector. According to an embodiment of this application, the vector carries the nucleic acid molecule described in the second aspect. When the nucleic acid molecule is attached to the vector, it can be directly or indirectly connected to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the nucleic acid molecule and the control elements only needs to be operably established.

[0098] 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. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the expression of the aforementioned dengue virus E antigen can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of dengue virus E antigen.

[0099] In some specific embodiments of this application, the above-mentioned vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0100] In some specific embodiments of this application, the above-mentioned vector is a lentiviral vector.

[0101] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0102] In a fourth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell comprises the nucleic acid molecule described in the second aspect, or the aforementioned vector; or expresses the chimeric antigen described in the first aspect. Using this cell, under suitable conditions, the aforementioned chimeric antigen can be effectively expressed intracellularly.

[0103] According to an embodiment of this application, the recombinant cells are obtained by introducing the vector described in the fourth aspect into the recombinant cells.

[0104] It should be noted that the recombinant cells in this application 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.

[0105] According to an embodiment of this application, the recombinant cells are eukaryotic cells.

[0106] According to embodiments of this application, 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.

[0107] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the chimeric antigen described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the chimeric 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 conditions for the expression of the chimeric antigen according to the specific environment of their laboratory.

[0108] Conjugates, test strips or kits

[0109] In a fifth aspect of this application, a conjugate is proposed. According to embodiments of this application, the conjugate comprises the chimeric antigen described in the first aspect and the conjugate moiety. The conjugate of this application can specifically bind to antibodies corresponding to the antigen and can be used for qualitative or quantitative detection of antigen-corresponding antibodies.

[0110] According to embodiments of this application, the above-mentioned conjugate may further include at least one of the following additional technical features:

[0111] According to embodiments of this application, the joining portion is selected from solid phases, markers, or other materials.

[0112] In this application, the term "solid phase" can refer to a substance that can be suspended or dispersed in a liquid phase (e.g., solid carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns).

[0113] According to embodiments of this application, the solid phase is selected from microspheres, plates, and membranes.

[0114] According to embodiments of this application, the solid phase is selected from magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes.

[0115] 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.

[0116] According to embodiments of this application, the label is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes. In some embodiments, the label is selected from fluorescent microspheres, colored latex microspheres, acridine esters, alkaline phosphatase, horseradish peroxidase, or colloidal gold.

[0117] 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 application.

[0118] According to embodiments of this application, the overseas Chinese federation material is selected from inert proteins, biotin, or avidin.

[0119] In a sixth aspect of this application, a test strip or kit is provided. According to embodiments of this application, the test strip or kit comprises: the chimeric antigen described in the first aspect, the nucleic acid molecule described in the second aspect, the carrier described in the third aspect, the recombinant cell described in the third aspect, or the conjugate described in the fifth aspect. As previously mentioned, the chimeric antigen in some specific embodiments or examples of this application can bind to corresponding antibodies; therefore, the test strip or kit containing the aforementioned chimeric antigen can effectively perform qualitative or quantitative detection of the corresponding antibody. The test strip or kit provided by this application can be used, for example, for detections involving the specific binding properties of chimeric antigens and antibodies. As previously mentioned, the test strip or kit containing the aforementioned chimeric antigen has the advantage of high sensitivity.

[0120] In this article, "kit" and "reagent" are used interchangeably. Kits or reagents do not need to have a box structure; they only require relative independence and suitable loading or containers, such as tubes, boxes, bottles, or cards. Some components may be placed in different containers, while others may be combined into one container if permissible.

[0121] According to embodiments of this application, the test strip comprises a chimeric 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, antibodies in a subject can be rapidly and accurately detected 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 antigens coated on a solid phase, or antibodies in the sample are detected by chimeric antigens 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.

[0122] According to embodiments of this application, the kit includes reagents suitable for performing immunoassays. In some embodiments, the kit may include instructions for use of the immunodiagnostic reagents of this application (e.g., labeled conjugates containing chimeric antigens) in an immunoassay for detecting the corresponding antibody. In some embodiments, the kit may include calibrators or controls, such as standards or control antibodies. In some embodiments, the chimeric antigens or labeled conjugates of this application 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 labels such as enzyme-labeled substrate solutions, etc.

[0123] use

[0124] In a sixth aspect of this application, the application discloses the use of the chimeric antigen described in the first aspect, the conjugate described in the fourth aspect, or the test strip or kit described in the fifth aspect in the preparation of a product; said product is used to detect dengue virus antibodies or diagnose dengue virus infection-related diseases.

[0125] According to embodiments of this application, the dengue virus infection-related diseases include dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.

[0126] method

[0127] In a seventh aspect of this application, a method for detecting dengue virus antibodies in a sample is provided. According to embodiments of this application, the method includes: contacting the sample to be tested with the chimeric antigen described in the first aspect, the conjugate described in the fourth aspect, or the test strip or kit described in the fifth aspect to form an immune complex; and determining the presence of dengue virus antibodies in the sample based on the detection result of the complex. The method of this application has the advantage of high sensitivity.

[0128] According to an embodiment of this application, the method is a capture method.

[0129] In an eighth aspect of this application, a method for diagnosing dengue virus infection-related diseases is proposed. According to embodiments of this application, the method includes: contacting a sample to be tested with the chimeric antigen described in the first aspect, the conjugate described in the fourth aspect, or the test strip or kit described in the fifth aspect to form an immune complex; and determining the presence of dengue virus in the sample based on the detection result of the complex. The method of this application has the advantage of high sensitivity.

[0130] According to an embodiment of this application, the method is a capture method.

[0131] According to embodiments of this application, the dengue virus infection-related diseases include dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.

[0132] The following will explain the solution of this application 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 this application. 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 art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0133] Example 1: Construction of chimeric antigen expression plasmid

[0134] The following clone containing dengue virus antigen (type I) was constructed:

[0135] Table 1 Design of chimeric antigens

[0136] Based on the chimeric antigens designed in Table 1, codon optimization and plasmid construction were performed on the gene fragments. The corresponding gene fragments could be obtained through template amplification or bridge PCR. The gene fragments and expression vectors were ligated by restriction endonucleases, T4 DNA ligase, and other enzyme tools. The E protein gene fragment was fused to the N-terminus of the NS5 protein gene fragment. The gene fragments were ligated with the adapter GGSGG. The expression plasmid was then constructed.

[0137] Example 2: Induction and purification of chimeric antigen

[0138] The expression plasmid constructed in Example 1 was transformed into the *E. coli* expression strain using a heat shock method and plated on LB agar plates containing 100 μg / ml kanamycin sulfate, and incubated at 37°C for 16 h. Single colonies were picked, and positive strains identified by bacterial culture PCR were sequenced. After successful sequencing, the strains were inoculated into LB agar plates containing 50 μg / ml kanamycin sulfate and incubated with shaking at 37°C. OD... 600After reaching 0.6-0.8, 1.0 mM IPTG was added, and the mixture was induced and cultured at 37°C for 2-4 h. The expression of the recombinant protein was identified by SDS-PAGE, and the purified proteins were named DN-1 to DN-8, respectively.

[0139] Example 3: Evaluation of the sensitivity and specificity of chimeric antigens using the capture method on a chromatography platform.

[0140] The purified chimeric antigen was evaluated using a colloidal gold chromatography platform, as detailed below:

[0141] 1. Chimeric antigen labeling

[0142] Take 5 ml of colloidal gold at 40,000 g / L, add an appropriate amount of 0.2 M K2CO3, stir for 5 min, add chimeric antigen, stir for 5 min, and then add an appropriate amount of 10% BSA blocking terminator; centrifuge at 10,000 rpm for 10 min, remove the supernatant, reconstitute the precipitate with gold reconstitution solution, and finally adjust the volume to 0.5 ml (i.e., 1 / 10 of the colloidal gold solution volume) with gold reconstitution solution; finally dilute the labeled antigen concentrated gold by a certain factor with gold reconstitution solution to prepare gold working solution, and spread the gold; freeze-dry the spread gold in a freeze dryer (1-2 h) or dry it overnight in a drying room at 37°C.

[0143] 2. Secondary antibody coating

[0144] Goat anti-human IgG secondary antibody and goat anti-human IgM secondary antibody were diluted to final concentrations of 1.0 mg / ml and 1.0 mg / ml respectively using coating diluent, coated, and then incubated overnight at 50°C.

[0145] 3. Preparation of gold bar

[0146] The gold standard strips are cut into strips of the required width using a strip cutter, assembled, and then sampled for testing.

[0147] 4. Application of colloid test strips

[0148] During detection, the antibody first binds to the colloidal gold-labeled antigen to form an antibody-colloidal gold-labeled antigen complex. Due to capillary action, the antibody-colloidal gold-labeled antigen complex migrates forward along the nitrocellulose membrane. Upon reaching the detection line, the antibody-colloidal gold-labeled antigen complex binds to the stripped secondary antibody, forming a secondary antibody-antibody-colloidal gold-labeled antigen complex, which accumulates on the detection line, forming a red precipitate line. The intensity of the red precipitate line indicates the strength of the reaction; the deeper the red, the stronger the reaction, and vice versa. The strength of the reaction is represented by the letter C and a number, with smaller numbers after C indicating stronger reactivity. No red precipitate line is indicated by B. (In Tables 2 and 3, the line to the left of the " / " indicates the IgG detection line, and the line to the right of the " / " indicates the IgM detection line.)

[0149] The test results showed that the detection sensitivity of chimeric antigens DN-2 to DN-8 was superior to that of existing antigen products on the market (DN-1). The detection sensitivity was DN-2, DN-4, DN-8 > DN-5, DN-6, DN-7 > DN-3.

[0150] Table 2 Detection Results

[0151] Example 4: Detection of chimeric antigens of different dengue virus subtypes

[0152] Referring to Example 1, chimeric antigens of different dengue virus subtypes were constructed, and exemplary clones are shown below:

[0153] Table 3 Design of chimeric antigens

[0154] Expression plasmids were constructed according to Example 1. Chimeric antigens were induced, expressed, and purified according to Example 2. The sensitivity and specificity of the chimeric antigens were evaluated using a capture method on a chromatography platform according to Example 3. The results showed that the chimeric antigens all exhibited high detection sensitivity and specificity, demonstrating improved detection accuracy compared to existing products on the market. Exemplary results are shown in the table below:

[0155] Table 4. Detection results of dengue virus type II chimeric antigen.

[0156] Table 5. Detection results of dengue virus type III chimeric antigen.

[0157] The amino acid sequence involved in this application is as follows:

[0158] 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 this application. 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, without contradiction, 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.

[0159] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application. Industrial applicability

[0160] The chimeric antigen disclosed herein enables accurate detection of dengue virus antibodies with high sensitivity. Therefore, the chimeric antigen disclosed herein possesses excellent practical performance and broad market application prospects.

Claims

1. A chimeric antigen, characterized in that, This includes dengue virus NS5 antigen and E antigen.

2. The chimeric antigen of claim 1, wherein, The E antigen includes the m-n amino acid region of the dengue virus E protein, where m is any integer from 277 to 307 and n is any integer from 384 to 434. Optionally, the E antigen includes the immunoglobulin-like domain of the dengue virus E protein; Optionally, the E antigen includes amino acid segments from position 297 to 394, position 277 to 434, position 307 to 384, or position 296 to 421 of the dengue virus E protein. Optionally, the position of the E antigen is determined using the amino acid sequence shown in SEQ ID NO:1 as a reference sequence; Optionally, the NS5 antigen comprises a full-length or truncated segment of the dengue virus NS5 protein; the number of amino acids in the truncated segment is any integer from 11 to 623. Optionally, the NS5 antigen includes the x-th to y-th amino acid segment of the dengue virus NS5 protein, where x is any integer from 849 to 878 and y is any integer from 890 to 899. Optionally, the NS5 antigen includes at least one of the following amino acid segments: amino acid segments from position 869 to 899, amino acid segments from position 1 to 265, amino acid segments from position 266 to 276, amino acid segments from position 277 to 869, amino acid segments from position 849 to 890, and amino acid segments from position 878 to 899 of the dengue virus NS5 protein. Optionally, the position of the NS5 antigen is determined with reference to the amino acid sequence shown in SEQ ID NO:

3.

3. The chimeric antigen of claim 1 or 2, wherein The E antigen is composed of the m-n amino acid region of the dengue virus E protein, where m is any integer from 277 to 307 and n is any integer from 384 to 434. Optionally, the E antigen is composed of the immunoglobulin-like domain of the dengue virus E protein; Optionally, the E antigen is composed of amino acid segments from position 297 to 394, position 277 to 434, position 307 to 384, or position 296 to 421 of the dengue virus E protein. Optionally, the NS5 antigen is composed of the full-length or truncated segment of the dengue virus NS5 protein; the number of amino acids in the truncated segment is any integer from 11 to 623. Optionally, the NS5 antigen is composed of the amino acid segments from position x to position y of the dengue virus NS5 protein, where x is any integer from 849 to 878 and y is any integer from 890 to 899. Optionally, the NS5 antigen is composed of amino acid segments from position 869 to 899, position 1 to 265, position 266 to 276, position 277 to 869, position 849 to 890, or position 878 to 899 of the dengue virus NS5 protein.

4. The chimeric antigen according to claim 1, characterized in that, The dengue virus is selected from type I, II, III or IV.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen as described in any one of claims 1 to 4.

6. A vector or recombinant cell, characterized in that, The carrier carries the nucleic acid molecule as described in claim 5; The recombinant cells comprise the nucleic acid molecules of claim 5 or the aforementioned vector; or express the chimeric antigens of any one of claims 1 to 4.

7. A conjugate, characterized in that, Includes the chimeric antigen and conjugated portion as described in any one of claims 1 to 4; Optionally, the joining portion is selected from solid phases, markers, or overseas Chinese association materials; Optionally, the solid phase is selected from microspheres, plates, and membranes; Optionally, the solid phase is selected from magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes; Optionally, the label is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes. Optionally, the marker is selected from fluorescent microspheres, colored latex microspheres, acridinium ester, alkaline phosphatase, horseradish peroxidase, or colloidal gold; Alternatively, the Overseas Chinese Federation may select inert proteins, biotin, or avidin.

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

9. Use of the chimeric antigen according to any one of claims 1 to 4, the conjugate according to claim 7, or the test strip or kit according to claim 8 in the preparation of the product; The product is used to detect dengue virus antibodies or diagnose dengue virus infection-related diseases.

10. A method of detecting dengue virus antibodies in a sample, comprising: include: The chimeric antigen as described in any one of claims 1 to 4, the conjugate as described in claim 7, or the test strip or kit as described in claim 8 are brought into contact with the sample to be tested to form an immune complex. Based on the detection results of the complex, it is determined whether dengue virus antibodies are present in the sample.

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