Dengue virus NS1 antibody and application thereof
By developing a dengue virus NS1 antibody and its antigen-binding fragment, the problem of rapid, sensitive, and low-cost dengue fever detection in existing technologies has been solved, enabling early and accurate detection that is suitable for screening in primary healthcare institutions and at epidemic sites.
Patent Information
- Application Number
- CN202511940926.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-01-27
AI Technical Summary
Current diagnostic technologies cannot detect dengue fever quickly, sensitively, and at low cost, and their application is limited, especially in primary healthcare institutions and on-site screening during outbreaks, and they are prone to false negative results.
Antibodies targeting dengue virus NS1 and their antigen-binding fragments were developed, containing specific HCDR1-HCDR3 and LCDR1-LCDR3 sequences, for highly specific binding to dengue virus NS1 antigen, and detected by methods such as ELISA.
It enables early and accurate detection of dengue virus, reducing missed diagnoses and misdiagnoses. It is suitable for rapid screening in primary healthcare institutions and at epidemic sites, and has good binding activity and high specificity.
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Figure CN121405798A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to dengue virus NS1 antibodies and their applications. Background Technology
[0002] Dengue fever, the most widespread and morbid mosquito-borne infectious disease globally, has become a serious public health challenge in tropical and subtropical regions. Dengue virus (DENV) comprises four serotypes, DENV-1 to DENV-4, which can cause dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) after infection, with a mortality rate exceeding 20% in severe cases. Current diagnostic technologies cannot simultaneously meet the core requirements of being "rapid, sensitive, specific, and low-cost," particularly limiting their application in primary healthcare institutions and on-site screening during outbreaks, which significantly hinders dengue fever prevention and control efforts.
[0003] Viral isolation and culture was once considered the "gold standard" for dengue fever diagnosis, but the process is lengthy, requires specialized laboratory equipment and skilled technicians, limiting its application in primary healthcare institutions. Currently, the more commonly used methods for dengue fever diagnosis are serological testing or nucleic acid testing, but these methods are prone to false negatives leading to missed diagnoses and cannot achieve rapid on-site screening.
[0004] Dengue virus nonstructural protein 1 (NS1) is a highly conserved glycoprotein that is present in large quantities in the serum of patients in the early stages of viral infection. NS1 antigen detection has high sensitivity and specificity, enabling accurate detection of the virus in the early stages of infection and reducing missed diagnoses and misdiagnoses. Given the advantages of dengue virus NS1 antigen in diagnosis, the development of specific antibodies against NS1 antigen is of great significance. Summary of the Invention
[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide dengue virus NS1 antibodies and their applications.
[0006] To achieve the above objectives, the present invention provides the following technical solution: A first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting dengue virus NS1, said antibody or antigen-binding fragment comprising HCDR1-HCDR3 in the heavy chain variable region as shown in SEQ ID NO:2, and LCDR1-LCDR3 in the light chain variable region as shown in SEQ ID NO:3.
[0007] In the context of this invention, the term "antibody" is used in the broadest sense and explicitly covers monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two intact antibodies, provided they exhibit the desired biological activity. Modified antibody sequences are also within the scope of this invention. The term "modification" refers to any form of modification to an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with a different amino acid. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence. In this invention, modifications preferably occur in regions other than variable regions, such as the constant region or frame region of the antibody, and the modified antibody retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.
[0008] In this invention, the term "antigen-binding fragment" encompasses a portion of the complete antibody, typically referring to one or more fragments within the antibody that specifically bind to antigens. The antigen-binding function of an antibody can be achieved through the full-length fragment of the antibody. The term "binding" indicates that the binding is selective for the antigen and can be distinguished from unintended or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.
[0009] Furthermore, HCDR1-HCDR3 and LCDR1-LCDR3 can be numbered according to IMGT, Chothia, and Kabat.
[0010] In this invention, the term "CDR" refers to the "hypervariant region" or "complementarity-determining region" of an antibody. Both the heavy chain and light chain variable regions have three CDRs, which together constitute the antigen-binding site of the antibody and can form precise complementarity with the antigenic determinant in spatial structure. Other examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from different antibody fragments. The position of the CDR in the variable region is labeled differently according to different numbering systems, including IMGT, Chothia, and Kabat.
[0011] In some embodiments, the amino acid sequences of HCDR1-3 are shown in SEQ ID NO:4-6, and the amino acid sequences of LCDR1-LCDR3 are shown in SEQ ID NO:7-9, respectively.
[0012] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:3.
[0013] In some embodiments, the antibody includes a monoclonal antibody, a chimeric antibody, a bivalent or multivalent antibody.
[0014] A second aspect of the present invention provides a nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.
[0015] In this invention, the terms "nucleic acid" or "nucleic acid molecule" are used interchangeably and refer to any polymeric form of any length and composed of ribonucleotides or deoxyribonucleotides. Typically, a nucleic acid is a coding sequence, as used herein, referring to a DNA sequence that, when placed under appropriate regulatory sequence control, is transcribed and translated into a polypeptide in a host cell. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. The coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription termination sequence will typically be located at the 3' end of the coding sequence. Once the coding sequence of the antibody or its antigen-binding fragment described in this invention, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, can be obtained in large quantities using recombinant techniques.
[0016] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:10-11.
[0017] A third aspect of the present invention provides a recombinant vector comprising the nucleic acid described in the second aspect of the present invention.
[0018] In this invention, the term "recombinant vector" refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The recombinant vectors of this invention can be plasmid vectors, viral vectors, etc. In some embodiments, a vector refers to a linear or circular nucleic acid molecule containing the nucleic acid of this invention operably linked to other segments provided for autonomous replication in a recombinant host cell, or according to an expression cassette of the nucleic acid molecule. "Operably linked" means that the nucleic acid sequence of interest is linked to a regulatory sequence in a manner that allows for the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or when introducing the vector into a host cell). Vectors can be known vectors or self-constructed vectors. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors can include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include lactopolyvacuolar viral vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, and may include bovine papillomavirus vectors and EB virus vectors, retroviral vectors, etc.
[0019] A fourth aspect of the present invention provides a recombinant host cell comprising the nucleic acid described in the second aspect of the present invention and / or the recombinant vector described in the third aspect of the present invention.
[0020] In this invention, the terms "recombinant host cell" and "recombinant cell" are used interchangeably and refer to any cell type suitable for transformation, transfection, transduction, etc., using an expression vector containing nucleic acid molecules provided by this invention. Recombinant cells include any offspring of the parent cell that differ from the parent cell due to mutations occurring during replication. The recombinant cells include prokaryotic cells and eukaryotic cells; the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof.
[0021] The fifth aspect of the present invention provides an antibody derivative comprising the antibody or antigen-binding fragment described in the first aspect of the present invention, and a detectable marker and / or solid-phase carrier directly or indirectly coupled to the antibody or antigen-binding fragment.
[0022] In this invention, the term "antibody derivative" refers to a derivative comprising the antibody described in this invention and a detectable marker bound to the antibody, which can both exhibit the desired biological activity and be detected by the detectable marker attached thereto.
[0023] In some embodiments, the detectable marker includes at least one of a radioactive isotope, a metallic nanomaterial, fluorescein, biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.
[0024] In some embodiments, the detectable marker is an enzyme.
[0025] In some embodiments, the enzyme is horseradish peroxidase.
[0026] A sixth aspect of this invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect of this invention, the method comprising the steps of: artificially synthesizing the antibody or antigen-binding fragment thereof; or, culturing the recombinant host cells described in the fourth aspect of this invention, and isolating and purifying the antibody or antigen-binding fragment thereof described in the first aspect of this invention from the culture product.
[0027] In some embodiments, the antibodies or antigen-binding fragments of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, by direct amino acid synthesis. In some embodiments, the antibodies or antigen-binding fragments of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.
[0028] The seventh aspect of the present invention provides a method for preparing recombinant host cells as described in the fourth aspect of the present invention, the method comprising the steps of: introducing the nucleic acid molecule described in the second aspect of the present invention or the recombinant vector described in the third aspect of the present invention into the cells, wherein the introduction method includes calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.
[0029] The eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention in the preparation of a product for detecting dengue virus NS1 protein, said product comprising the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, and / or the antibody derivative described in the fifth aspect of the present invention.
[0030] In some embodiments, the product includes a reagent kit, test strip, immunomagnetic beads, microarray chip, drug, or fusion protein.
[0031] The ninth aspect of the present invention provides a method for in vitro detection of dengue virus NS1 protein, the method comprising the steps of co-incubating the dengue virus NS1 protein with the antibody or antigen fragment thereof described in the first aspect of the present invention.
[0032] Advantages and benefits of the present invention: The present invention provides a dengue virus NS1 antibody or its antigen-binding fragment, which has good binding activity and high specificity with dengue virus NS1 protein and has broad application prospects. Attached Figure Description
[0033] Figure 1 This is an image from antibody electrophoresis detection.
[0034] Figure 2 This is a chromatogram of antibody detection by high performance liquid chromatography.
[0035] Figure 3 This is a curve showing the antibody binding activity. Detailed Implementation
[0036] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.
[0037] Example 1. Immunogen Recombinant Expression The dengue virus NS1 protein sequence was synthesized and constructed into the pCDNA3.1 vector; plasmid for transfection was extracted; the cells were transfected into HEK293 cells and cultured for 7 days; the supernatant was harvested, purified by Ni column, and concentrated and replaced with buffer to obtain recombinant dengue virus NS1 protein (sequence shown in SEQ ID NO:1).
[0038] 2. Immunize mice, SP2 / 0 fusion, select, subclone. The first immunization was administered with Freund's complete adjuvant, 100 μg per animal, via intraperitoneal injection, with a total dose of 0.5 ml per animal. A second immunization was administered 3 weeks later. From the second immunization onwards, Freund's incomplete adjuvant was administered at a dose of 50 μg / 0.5 ml per animal, with a third immunization administered 2 weeks later. Cell fusion was prepared 10 days after the third injection.
[0039] To obtain feeder cells, use 10 5 / Hole application, lay 10 plates the day before fusion. 5 100 μl / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μl / well.
[0040] Positive wells were screened using ELISA. Recombinant dengue virus NS1 protein was plated overnight. After washing, the plates were blocked with skim milk powder and incubated at 37°C for 1 h. After washing, 100 μl of 96-well culture supernatant was added and incubated at 37°C for 1 h. After washing, HRP-labeled goat anti-mouse secondary antibody was added and incubated at 37°C for 30 min. After washing, chromogenic solution was added and chromogenic solution was developed for 10 min. Stop solution was added, and the OD450 value was read. High-expression cell lines were screened for subcloning.
[0041] 3. Sequence Fishing Cells were collected, RNA was extracted, reverse transcribed, primers were designed, PCR was performed, transformation was carried out, clones were selected, sequencing was performed, amplification culture was conducted, and recombinant dengue virus NS1 protein antibody with clone number 2E10 was obtained (the corresponding amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO:2-3, respectively).
[0042] 4. Antibody Expression and Optimization Using the amino acid sequence of the 2E10 protein as the core template, a systematic optimization was performed based on the codon usage preferences of human cells: First, the human genome codon frequency database was searched, and low-frequency codons in the sequence were replaced with high-frequency synonymous codons of the corresponding amino acids from human sources; at the same time, redundant elements that affect transcription, such as poly(A) signals and hidden splicing sites, were avoided, and the GC content was adjusted to the conventional 40%–60% range of human genes; codon combinations were optimized through RNA secondary structure prediction to avoid the formation of stem-loop structures in mRNA that hinder ribosome binding; key amino acid regions such as the active site and functional binding site of the 2E10 protein were strictly preserved throughout the process to ensure that the protein structure and function were not affected, and finally, an optimized coding sequence adapted to human expression systems was obtained.
[0043] The optimized heavy and light chain corresponding nucleic acid sequences are shown in SEQ ID NO:10-11, respectively.
[0044] The optimized nucleic acid sequence was sent to GenScript for synthesis and constructed into the pcDNA3.4 vector. Large-scale plasmid preparation was then completed. The antibody and optimized nucleic acid sequence are shown in Table 1.
[0045] Table 1. Sequence List
[0046] One day before transfection, healthy 293 cells (70-80% adherent cells and 3×10⁶ suspension cells) were transfected. 5DNA (cells / mL) was seeded into culture dishes. On the day of transfection, endotoxin-free plasmids were prepared at an HC:LC ratio of 1:1-1.2. The amount of PEI was calculated with N / P = 3:1. DNA and PEI were diluted separately with serum-free medium and mixed. The mixture was incubated at room temperature for 15-20 min to form a complex. The complex was added to the cells and cultured at 37°C and 5% CO2. The medium was changed after 6-12 h. The supernatant was collected after 48-96 h of culture. The antibody concentration and activity were detected by UV 280 nm, ELISA, and SDS-PAGE. The 2E10 clone was 428 mg / L.
[0047] 5. Antibody physicochemical property detection The purity of the prepared 2E10 antibody was greater than 95% as determined by electrophoresis and liquid chromatography. The electrophoresis results are as follows: Figure 1 As shown. The HPLC detection results are as follows. Figure 2 As shown.
[0048] 6. Binding activity After coating the recombinant dengue virus NS1 protein, the prepared antibody and horseradish peroxidase-labeled secondary antibody were added sequentially. After color development, the data were read, analyzed, and a curve was fitted. The antibody with clone number 2E10 was EC50. 50 The concentration was 0.1024 μg / ml, as shown in the results. Figure 3 As shown.
[0049] 7. Specificity Cultures of dengue virus NS1 protein, Zika virus, SARS-CoV-2, influenza A, influenza B, parainfluenza virus, adenovirus, and respiratory syncytial virus were coated with antibodies and horseradish peroxidase-labeled secondary antibodies, respectively. After color development, the data were read and analyzed.
[0050] Table 2. Specificity detection results of antibody 2E10 targeting dengue virus NS1 protein
[0051] The results are shown in Table 2. The test results show that only the dengue virus NS1 protein of dengue virus types 1-4 showed normal color development, while the other antigens did not show positive reactions. That is, the antibody 2E10, which targets the dengue virus NS1 protein, can specifically bind to the dengue virus NS1 protein of dengue virus types 1-4, and has high specificity against the dengue virus NS1 protein.
[0052] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.
Claims
1. An antibody or its antigen-binding fragment targeting dengue virus NS1, characterized in that, The antibody or its antigen-binding fragment comprises HCDR1-HCDR3 in the heavy chain variable region as shown in SEQ ID NO:2, and LCDR1-LCDR3 in the light chain variable region as shown in SEQ ID NO:3; Preferably, HCDR1-HCDR3 and LCDR1-LCDR3 can be numbered according to IMGT, Chothia, and Kabat. Preferably, the amino acid sequences of HCDR1-3 are as shown in SEQ ID NO:4-6, and the amino acid sequences of LCDR1-LCDR3 are as shown in SEQ ID NO:7-9, respectively.
2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:3; Preferably, the antibody includes monoclonal antibodies, chimeric antibodies, bivalent or multivalent antibodies.
3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-2; Preferably, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:10-11.
4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid described in claim 3.
5. Recombinant host cells, characterized in that, The recombinant host cell comprises the nucleic acid of claim 3 and / or the recombinant vector of claim 4.
6. An antibody derivative, characterized in that, The antibody derivative includes the antibody or antigen-binding fragment as described in any one of claims 1-2, and a detectable marker and / or solid-phase carrier directly or indirectly coupled to the antibody or antigen-binding fragment; Preferably, the detectable marker includes at least one of the following: radioactive isotopes, metal nanomaterials, fluorescein, biotin, avidin, biotin / avidin protein complex, biotin / avidin protein complex, chromophores, electron-dense substances, and enzymes. Preferably, the detectable marker is an enzyme; Preferably, the enzyme is horseradish peroxidase.
7. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The method includes the following steps: artificially synthesizing an antibody or its antigen-binding fragment; or culturing the recombinant host cell of claim 5 and separating and purifying the antibody or its antigen-binding fragment of any one of claims 1-2 from the culture product.
8. A method for preparing recombinant host cells according to claim 5, wherein the method comprises the following steps: The nucleic acid molecule described in claim 3 or the recombinant vector described in claim 4 is introduced into cells, wherein the introduction method includes calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.
9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2 in the preparation of a product for detecting dengue virus NS1 protein, characterized in that, The product comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-2, the nucleic acid molecule as described in claim 3, the recombinant vector as described in claim 4, the recombinant host cell as described in claim 5, and / or the antibody derivative as described in claim 6; Preferably, the product includes reagent kits, test strips, immunomagnetic beads, microarray chips, drugs, and fusion proteins.
10. A method for in vitro detection of dengue virus NS1 protein, characterized in that, The method includes the steps of co-incubating the dengue virus NS1 protein with the antibody or antigen fragment thereof as described in any one of claims 1-2.