Antibody specifically targeting dengue virus NS1 protein and application thereof

Abstract

The invention discloses an antibody specifically targeting dengue virus NS1 protein and application of the antibody, amino acid sequences of heavy chain variable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody are respectively shown as SEQ ID NO: 4-6, amino acid sequences of light chain variable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody are respectively shown as SEQ ID NO: 7-9, and experimental verification shows that the antibody has the advantages that the antibody can be specifically targeted to the dengue virus NS1 protein; the antibody has high affinity and specificity to the dengue virus NS1 protein, can be used for effective detection of the dengue virus NS1 protein, lays a foundation for research and development of dengue virus related detection products, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to an antibody specifically targeting dengue virus NS1 protein and application thereof. BACKGROUND

[0002] Dengue virus (DENV) is a mosquito-borne virus of the Flaviviridae family, which is mainly transmitted through mosquito bites and can cause human dengue fever after infection. The virus also includes related viruses such as Zika virus (ZIKV) and West Nile virus (WNV), and there is a risk of cross-immune reaction between DENV and these viruses. There are currently five serotypes of DENV, of which DENV-1, 2, 3 and 4 are the main global epidemic strains at present; infection can progress to severe and fatal diseases such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), with a high mortality rate of 12%-44%, which is a major threat to human health.

[0003] Early diagnosis and timely intervention are the key to reducing the incidence and mortality of severe dengue fever. Current diagnostic methods for dengue fever infection mainly include virus isolation and culture, viral nucleic acid detection (RT-PCR), antigen and antibody detection, and combined application of multiple methods. Non-structural protein 1 (NS1 protein) is the only glycoprotein secreted into the host's blood during DENV infection, and is an early-established core diagnostic biomarker. The protein sequence is highly conserved, with a molecular weight of about 45 kDa, and can exist in monomer, dimer and hexamer forms in vivo; high concentrations of soluble NS1 protein (peak value up to 600 ng / mL) can appear in the blood of patients within 72 hours before the onset of the disease, and are earlier than IgM / IgG antibody production, and persist in the blood for 9-18 days, which makes it an ideal target for early diagnosis of dengue fever.

[0004] However, there are currently only a limited number of NS1 detection reagent products approved in China, and the market is mainly occupied by imported brands. Imported products not only have high prices, increasing the cost of clinical application and public health prevention and control, but also their supply stability is easily affected by international environment, logistics transportation and other factors, making it difficult to fully meet the domestic normal prevention and control needs. Therefore, the development of dengue virus NS1 protein-specific antibody raw materials with independent intellectual property rights and stable performance is of great significance for breaking the dependence on imports and improving the early diagnosis technology system for dengue fever, and the related needs are urgent and necessary.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The application aims to provide an antibody specifically targeting dengue virus NS1 protein and its application, and discloses a novel antibody 7D11 against dengue virus NS1 protein for the first time.

[0007] The application achieves the above-mentioned application purposes by adopting the following technical solutions. In one aspect, the application provides an antibody specifically targeting dengue virus NS1 protein.

[0008] Further, the antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the corresponding amino acid sequences are shown as SEQ ID NO: 4-6. The light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3, and the corresponding amino acid sequences are shown as SEQ ID NO: 7-9.

[0009] Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2.

[0010] Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 3.

[0011] In some embodiments, the amino acid sequences corresponding to CDR-H1, CDR-H2, CDR-H3 of the heavy chain variable region or CDR-L1, CDR-L2, CDR-L3 of the light chain variable region are not limited to the amino acid sequences shown above, and the CDR1, CDR2 and CDR3 in the heavy chain variable region or the light chain variable region of the antibody shown above are defined by using any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be generated in the future) to obtain the amino acid sequences or nucleotide sequences corresponding to the heavy chain variable region CDR1-3 or the light chain variable region CDR1-3, which are within the protection scope of the application.

[0012] In some embodiments, the CDR numbering scheme is any one or more combinations of IMGT numbering scheme, Chothia numbering scheme, AbM numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, Aho numbering scheme, the sequences of CDR1-3 of the heavy chain variable region or CDR1-3 of the light chain variable region defined by any one of the above CDR numbering schemes or any combination of two or more CDR numbering schemes are all within the scope of the present application.

[0013] In the present application, the Dengue fever is a viral infectious disease caused by Dengue virus and mainly transmitted by mosquito bites, is the most widely spread arboviral infectious disease in the world, and is prevalent in more than 100 countries in the tropics and subtropics. It is not directly transmitted from person to person. After infection with Dengue virus, the disease usually occurs 5-9 days later, the symptoms are mostly mild, and some infected persons can be asymptomatic. Typical clinical manifestations include sudden fever (up to more than 39℃), which can be accompanied by obvious fatigue, anorexia, nausea, severe headache, retro-orbital pain, muscle and joint pain (commonly known as "three pains"), and facial, neck, and chest flushing (i.e. "three reds"), sometimes accompanied by rash, bleeding manifestations (nosebleeds, gum bleeding, heavy menstrual bleeding, subcutaneous bleeding, etc.). Most patients can recover within 1-2 weeks, but a small number of patients develop rapidly after the onset of the disease, and can develop hemorrhage, shock, and even death. In addition, individual cases can develop into Dengue hemorrhagic fever and Dengue shock syndrome.

[0014] In another aspect, the present application provides a nucleic acid molecule.

[0015] Further, the nucleic acid molecule encodes the antibody as described above.

[0016] Further, the core function of the nucleic acid molecule is to specifically encode the antibody that specifically targets the Dengue virus NS1 protein as described above, which is the core genetic material basis for realizing the recombinant expression, industrial production and subsequent technology transformation of the antibody.

[0017] In some embodiments, the coding sequence of the nucleic acid molecule has a clear targeting relationship, specifically, the antibody encoded by the nucleic acid molecule comprises CDR-H1, CDR-H2, CDR-H3 (the amino acid sequences are shown in SEQ ID NO: 4-6, respectively), CDR-L1, CDR-L2, CDR-L3 (the amino acid sequences are shown in SEQ ID NO: 7-9, respectively), and preferably encodes the complete heavy chain variable region (the amino acid sequence is shown in SEQ ID NO: 2) and the light chain variable region (the amino acid sequence is shown in SEQ ID NO: 3) as described above.

[0018] In some embodiments, based on the degeneracy of codons, the nucleic acid molecules not only encompass nucleotide sequences that correspond exactly to the above-mentioned amino acid sequences, but also include all degenerate nucleotide sequences that can encode the same antibody amino acid sequences, i.e. by replacing synonymous bases in codons (such as codons UUA, UUG, CUU, CUC, CUA, CUG for leucine), without changing the amino acid composition and spatial conformation of the antibody, and can adapt to the codon bias of different host cells (such as mammalian cells, yeast cells, plant cells), to improve the expression efficiency of the antibody in a heterologous expression system.

[0019] In some embodiments, the nucleic acid molecules also include variant sequences that are highly homologous to the core coding sequences as described above, including nucleotide sequences with a homology of ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%, 98% or 99%) to the above-mentioned nucleotide sequences, and the encoded antibodies can still specifically bind to dengue virus type 1-4 NS1 protein, maintain high affinity (such as EC 50 ≤0.5 μg / mL) and high specificity (no cross-reaction). At the same time, in view of the diversity defined by the CDR regions, the nucleic acid molecules also encompass nucleotide sequences that correspond to the CDR sequences or complete variable region sequences after defining the heavy chain variable region or light chain variable region CDR1-3 of the antibodies described in the present application using any one or more of the CDR numbering schemes of IMGT, Chothia, AbM, Kabat, Martin (enhanced Chothia), Aho, etc.

[0020] In some embodiments, the nucleic acid molecules can be obtained by artificial synthesis, PCR amplification and other conventional molecular biology methods, and have clear sequences and strong operability, and can be stably inserted into suitable expression vectors (such as pcDNA3.4, pCDNA3.1, etc.), and after transformation into recombinant host cells, high-efficiency expression of the antibodies can be achieved. The nucleic acid molecules can provide a replicable and optimized genetic template for the large-scale production of antibodies, solve the problems of limited amount of natural antibodies and insufficient stability, and at the same time provide a flexible operation basis for subsequent molecular modification (such as humanization, affinity maturation, Fc segment modification) of the antibodies.

[0021] In another aspect, the present application provides an expression vector.

[0022] Further, the expression vector comprises the nucleic acid molecules as described above in the present application; Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Optionally, the expression vector is a viral vector, a plasmid vector or a bacteriophage vector; Optionally, the viral vector is a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a herpes virus vector.

[0023] In some embodiments, the expression vector is obtained by inserting the nucleic acid molecule (encoding the heavy chain variable region, the light chain variable region, or the complete antibody sequence) of the present application as described above into a multiple cloning site of a vector backbone by molecular cloning technology, forming a recombinant expression vector that can be stably replicated and expressed, which not only contains the core elements of the antibody coding sequence, but also integrates key regulatory sequences such as promoters (eukaryotic promoters such as CMV promoter, EF-1a promoter, or prokaryotic promoters such as T7 promoter, lac promoter) that ensure the expression efficiency, terminators, ribosome binding sites, polyA tail sequences, etc., and can also carry a screening marker gene (such as an antibiotic resistance gene, a fluorescent protein gene), which facilitates the rapid screening of successfully transformed host cells and ensures the positivity and stability of the recombinant vector.

[0024] In some embodiments, the corresponding vector type can be selected according to the actual application scenario (such as the demand for antibody expression amount, the type of host cell, and the subsequent application direction). Specifically, a eukaryotic expression vector or a prokaryotic expression vector can be selected, wherein the eukaryotic expression vector is suitable for mammalian cells (such as HEK293, CHO cells), yeast cells, etc. as hosts, and can realize the correct folding, glycosylation modification, and secretion expression of antibodies, thereby ensuring the natural biological activity of the antibodies; the prokaryotic expression vector is suitable for prokaryotic cells such as Escherichia coli, and can realize the efficient expression of antibody fragments (such as scFv), thereby meeting the requirements of rapid preparation and preliminary functional verification. In addition, a viral vector (lentivirus vector, adenovirus vector, adeno-associated virus vector, etc.), a plasmid vector, or a bacteriophage vector can also be selected, wherein the viral vector can be used for in vivo expression of antibodies or gene therapy related research, the plasmid vector is preferred for large-scale expression due to its simple construction and high stability, and the bacteriophage vector is suitable for antibody library construction and affinity maturation applications.

[0025] In some embodiments, the specific expression vector can be routinely selected by those skilled in the art according to the actual requirements (such as the selection of host cells, the purpose of protein function research, and the requirement for expression amount).

[0026] On the other hand, the present application provides a genetically modified host cell.

[0027] Further, the genetically modified host cell comprises the expression vector of the present application as described above; Optionally, the host cell is a eukaryotic cell; Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell; Optionally, the host cell is a CHO cell, a 293 cell, an NS / 0 myeloma cell, a HeLa cell, a Cap cell, or a COS cell.

[0028] In some embodiments, the genetically engineered host cell is obtained by introducing an expression vector comprising a nucleic acid encoding an antibody against the dengue virus NS1 protein into a primary host cell, and then screening and identifying.

[0029] Further, in the process of genetic engineering, the expression vector enters the host cell through transfection (such as liposome transfection, electroporation transfection), transformation or infection, and the antibody coding sequence carried by the expression vector is driven by the regulatory element in the host cell to start transcription and translation, and finally realizes the synthesis, modification and secretion (or intracellular expression) of the antibody. The genetically engineered host cell not only stably integrates the target expression vector, but also realizes the purification and maintenance of positive cells in long-term subculture through screening marker genes (such as antibiotic resistance, fluorescence screening), ensuring the persistence and stability of antibody expression.

[0030] In some embodiments, the genetically engineered host cell preferably uses eukaryotic cells as the original host cell, because it has a perfect protein processing and modification system (such as glycosylation, disulfide bond formation, folding assembly), which can ensure the correct spatial conformation and complete biological activity of the recombinant antibody, and avoid the problems of antibody folding error and activity loss caused by prokaryotic cell expression.

[0031] Further, the optional eukaryotic cells include mammalian cells, plant cells or yeast cells, wherein the mammalian cells are the preferred type, and further CHO cells (Chinese hamster ovary cells, which have the advantages of high expression, good product stability, and glycosylation pattern close to human, and are the classic host for antibody drug industrialization), 293 cells (human embryonic kidney cells, which have high transfection efficiency and strong transient expression ability, and are suitable for rapid preparation and preliminary function verification of antibodies), NS / 0 myeloma cells (which have strong antibody secretion ability and can realize high-purity antibody expression), and HeLa cells, Cap cells, COS cells and other mature engineering cell strains.

[0032] It should be noted that the present application does not have a particular limitation on the specific type of host cell, and those skilled in the art can make a routine selection according to the actual needs.

[0033] On the other hand, the present application provides a population of genetically engineered host cells.

[0034] Further, the population of genetically engineered host cells comprises the genetically engineered host cell as described above.

[0035] In another aspect, the present application provides any one of the following methods: (1) A method for preparing the antibody as defined above, comprising the steps of culturing the genetically modified host cell as defined above, and isolating the antibody from the culture product; (2) A method for detecting dengue virus NS1 protein for non-diagnostic purposes, comprising the steps of contacting a sample to be tested with the antibody as defined above, detecting the immunoreaction between the sample and the antibody, and determining the expression level of the dengue virus NS1 protein in the sample.

[0036] In another aspect, the present application provides any one of the following products: (1) A dengue virus NS1 protein detection reagent, comprising the antibody as defined above; (2) A dengue virus NS1 protein detection kit, comprising the antibody as defined above, or the detection reagent; (3) A dengue virus NS1 protein detection chip, comprising the antibody as defined above, or the detection reagent; (4) A dengue virus NS1 protein detection test strip, comprising the antibody as defined above, or the detection reagent; (5) A dengue virus infection disease diagnostic product, comprising the antibody as defined above, or the detection reagent; (6) A pharmaceutical composition, comprising the antibody as defined above; (7) A pharmaceutical preparation, comprising the antibody as defined above, or the pharmaceutical composition.

[0037] In some embodiments, the detection reagent takes the antibody specific to the dengue virus NS1 protein as the core functional component, which is a special reagent for early and accurate detection of dengue virus infection. The detection reagent takes the antibody (containing specific heavy chain variable region, light chain variable region and CDR sequences, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 3, and the sequences of CDR-H1 to CDR-H3 and CDR-L1 to CDR-L3 are shown as SEQ ID NO: 4-9, respectively) as the capture antibody or detection antibody, combines with the principle of immunodetection technology (such as ELISA, colloidal gold immunochromatography, chemiluminescence immunoassay, etc.), constructs a detection system, and uses the high specific binding ability (no cross reaction with Zika virus, influenza virus and other related or common viruses) and high affinity of the antibody to the dengue virus 1-4 type NS1 protein to efficiently recognize the soluble NS1 protein in the sample to be tested (such as blood, serum, etc.) and form a stable antigen-antibody immune complex. The detection reagent can convert the antigen-antibody binding event into a quantifiable detection signal through signal amplification, color development or fluorescence detection, realize the qualitative or quantitative analysis of the dengue virus NS1 protein, and has the advantages of high detection sensitivity, strong specificity, convenient operation, etc., which can meet the needs of clinical diagnosis, rapid screening on the spot of epidemic situation, public health monitoring and other scenes.

[0038] In addition, the present application also provides a method for detecting dengue virus NS1 protein in a sample to be tested or a method for diagnosing whether a subject has a dengue virus infection disease or has a risk of suffering from a dengue virus infection disease, which comprises the following steps: contacting the sample to be tested from the subject with the antibody, detection reagent, detection kit, detection chip, detection test strip or diagnostic product as described above, detecting the immune reaction of the sample and the antibody, determining the expression level of the dengue virus NS1 protein in the sample, and judging whether the subject has a dengue virus infection disease or has a risk of suffering from a dengue virus infection disease based on the result.

[0039] In some embodiments, the subject includes human and non-human animals. Among them, the non-human animals include all vertebrates (such as mammals and non-mammals), for example, non-human primates (such as cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians and reptiles. In some embodiments, the subject is preferably a human.

[0040] In some embodiments, the sample to be tested refers to a collection (e.g., fluid, cell or tissue) isolated from a subject, as well as fluids, cells or tissues present in the body of a subject. Exemplary samples to be tested are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, fecal matter, sputum, mucosal secretions of secretory tissues or secretory organs, ascites, pleural, pericardial, peritoneal, peritoneal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, e.g., culture media (including conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures or cell cultures.

[0041] In another aspect, the present application provides the use of any of the following: (1) the antibody as described above, the nucleic acid molecule as described above, the expression vector as described above or the genetically engineered host cell as described above in the preparation of a detection reagent for detecting dengue virus NS1 protein; (2) the antibody as described above, the nucleic acid molecule as described above, the expression vector as described above or the genetically engineered host cell as described above in the preparation of a detection kit, detection chip or detection test strip for detecting dengue virus NS1 protein; (3) the antibody as described above, the nucleic acid molecule as described above, the expression vector as described above or the genetically engineered host cell as described above in the preparation of a diagnostic product for diagnosing dengue virus infection disease; (4) the antibody as described above, the nucleic acid molecule as described above, the expression vector as described above or the genetically engineered host cell as described above in the preparation of a pharmaceutical composition for preventing and / or treating dengue virus infection disease; (5) the antibody as described above, the nucleic acid molecule as described above, the expression vector as described above or the genetically engineered host cell as described above in the preparation of a pharmaceutical preparation for preventing and / or treating dengue virus infection disease.

[0042] In the present application, the dengue virus infection disease refers to a series of diseases caused by infection of human body by each serotype of dengue virus (DENV), including but not limited to the currently globally prevalent DENV-1, DENV-2, DENV-3, DENV-4, and other discovered or potentially prevalent dengue virus serotypes, including the complete disease spectrum from asymptomatic infection, common dengue fever to severe dengue fever related conditions. Specifically, it includes common dengue fever with typical symptoms of fever, headache, muscle and joint pain, rash, etc., and severe types with rapid disease progression and high mortality risk, such as dengue hemorrhagic fever (DHF) with vascular leakage and bleeding tendency, and dengue shock syndrome (DSS) with shock and multiple organ dysfunction, etc. The core pathogenic mechanism of which is related to the replication of dengue virus in vivo and the pathological damage mediated by NS1 protein. The antibody and related products of the present application can achieve early diagnosis and targeted intervention of such diseases by targeting NS1 protein. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 : Electrophoresis result diagram corresponding to anti-dengue virus NS1 protein antibody 7D11; Figure 2 : HPLC result diagram corresponding to anti-dengue virus NS1 protein antibody 7D11; Figure 3 : ELISA detection of the binding activity of anti-dengue virus NS1 protein antibody 7D11. DETAILED DESCRIPTION

[0044] The present application will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present application and cannot be understood as a limitation of the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present application are easily obtained by those skilled in the art, and if not specifically stated, they can be obtained from commercial channels. The experimental methods of the present application not specified in the present application are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present application and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application and should not and will not limit the present application described in detail in the claims.

[0045] Example 1: Screening of antibodies targeting dengue virus NS1 protein 1. Immunogen recombinant expression Synthetic dengue virus NS1 protein sequence, construct to pCDNA3.1 vector; extract plasmid for transfection; transfection to HEK293 cells, culture cells for 7 days; harvest supernatant, Ni column purification, through concentration replacement buffer, obtain recombinant dengue virus NS1 protein.

[0046] Among them, the sequence of the recombinant dengue virus NS1 protein is shown as SEQ ID NO: 1, and the sequence information is shown in Table 1 below.

[0047] Table 1 Sequence of recombinant dengue virus NS1 protein

[0048] 2, immunize mice, SP2 / 0 fusion, screening, subclone Firstly, use Freund's complete adjuvant, 100 μg per mouse, intraperitoneal injection, total dose 0.5 mL per mouse, second immunization interval 3 weeks; the second time use Freund's incomplete adjuvant, the dose is 50 μg / 0.5 mL per mouse, the third immunization interval is 2 weeks; 10 days after the third injection, prepare cell fusion.

[0049] Take the feeder cells, which can be used at 10 5 cells per well, plate 10 5 cells per 100 μL per well one day before fusion; take the mouse immune spleen cells and the prepared myeloma cells to be fused with the fusion agent PEG, and plate into the 96 cell culture plate which has been added with feeder cells, 100 μL per well.

[0050] Screen positive wells by ELISA detection method, plate recombinant dengue virus NS1 protein overnight; wash the plate, block with skimmed milk powder, 37℃, 1 h; wash the plate, add 100 μL 96 well culture supernatant, 37℃, incubate for 1 h; wash the plate, add HRP labeled goat anti-mouse secondary antibody, 37℃, incubate for 30 min; wash the plate, add color developing solution, color develop for 10 min, add stop solution, read OD 450 value; screen high expression cell strains for subculture.

[0051] 3, sequence fishing Collect cells, extract total RNA with Trizol, use oligo (dT) 20 as primer, reverse transcription to generate cDNA. Then use specific primers to amplify the heavy and light chain variable region genes respectively. The PCR products are purified by electrophoresis, then inserted into the vector by TA cloning, transformed, and the positive clones are selected for sequencing.

[0052] 4, experimental results Screen out the antibody 7D11 targeting dengue virus NS1 protein, and the corresponding sequence information is shown in Tables 2 and 3.

[0053] Table 2 Heavy chain variable region and light chain variable region sequences of antibody 7D11 targeting dengue virus NS1 protein

[0054] Table 3 CDR sequences of antibody 7D11 targeting dengue virus NS1 protein

[0055] Example 2 Expression and verification of antibodies targeting dengue virus NS1 protein 1. Antibody expression The amino acid sequence of antibody 7D11 targeting dengue virus NS1 protein screened in Example 1 was optimized according to human codons, synthesized by Kingsway, and constructed into pcDNA3.4 vector. The plasmid was prepared for use.

[0056] One day before transfection, good 293 cells (adherent cell confluence reached 70-80%, and the density of suspension cells was 3x10 5 cells / mL) were inoculated in culture vessels. On the day of transfection, endotoxin-free plasmids were prepared in a ratio of HC:LC = 1:1-1.2, and the amount of PEI was calculated according to N / P = 3:1. DNA and PEI were diluted with serum-free medium and then mixed, and the complex was incubated at room temperature for 15-20 min. The complex was added to the cells, which were cultured at 37°C, 5% CO2. The medium was changed after 6-12 h, and the supernatant was collected after 48-96 h of continuous culture. The concentration and activity of the antibody were detected by ultraviolet UV 280 nm, ELISA, and SDS-PAGE methods.

[0057] The results showed that the concentration of the 7D11 clone was 403 mg / L.

[0058] 2. Physicochemical properties (1) Detection of antibody purity by SDS-PAGE method Main instruments: chemiluminescence imager, purchased from Tanon, model Tanon-5200; electrophoresis instrument, purchased from BIO-RAD, model powerpac basic; electrophoresis tank, purchased from BIO-RAD, model DYC-Mini4.

[0059] Main reagents: 1 M Tris-HCl buffer, purchased from Beijing Solyle Biotech Co., Ltd.; 1.5 M Tris-HCl buffer, purchased from Beijing Solyle Biotech Co., Ltd.; 10% SDS, purchased from Beijing Solyle Biotech Co., Ltd.; FastStain, purchased from Gene Universal; 30% gel preparation solution (29:1), purchased from Beijing Solyle Biotech Co., Ltd.; Rainbow 180 broad-spectrum protein marker, purchased from Beijing Solyle Biotech Co., Ltd.

[0060] Sample preparation: mix 20 μL of sample with 5 μL of 5×reducing buffer, heat at 95℃ for 5 min, and cool; mix 20 μL of sample with 5 μL of 5×non-reducing buffer.

[0061] Electrophoresis: configure the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0062] Staining and destaining: after electrophoresis, take the gel and put it into an appropriate amount of Coomassie brilliant blue staining solution, and stain at room temperature for 1 h or more; pour out the staining solution, add an appropriate amount of Coomassie brilliant blue staining and destaining solution, and destain at room temperature for 4-24 h. After destaining is completed, soak with ddH2O, refer to the marker protein, compare with the unstained gel, cut the gel of the desired protein component, collect, and separate the protein to be purified from the gel.

[0063] The electrophoresis result diagram of the antibody 7D11 targeting the dengue virus NS1 protein is shown in FIG. 1, and the bands from left to right are marker, reduced band, respectively. The results show that the detection purity of the 7D11 is greater than 95%. Figure 1

[0064] (2) HPLC is used to detect the purity of the antibody Main instruments: liquid chromatograph, purchased from Agilent Technologies, model Agilent 1100 / 1200 / 1260; liquid chromatography column, purchased from Tosoh Corporation, model WieTSKgel G3000SWxl, 7.8 mmI.D.×30 cm; pH meter, purchased from Sartorius Scientific Instruments Co., Ltd.; electronic balance, purchased from Sartorius Scientific Instruments Co., Ltd.

[0065] Main reagents: dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride, all purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0066] ​Mobile phase preparation: Add dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride into about 900 mL purified water, stir to dissolve, dilute to 1 L, measure the pH with a pH meter to ensure that the pH is between 6.2±0.1. Filter with a 0.22 μm filter membrane and store at room temperature.

[0067] Sample preparation: System suitability sample: dilute the standard to 2 mg / mL with the mobile phase; test sample: dilute the sample to be tested to 2 mg / mL with the mobile phase.

[0068] Chromatographic conditions: injection volume is 25 μL, injection temperature is 6℃, running time is 30 min, flow rate is 0.5 mL / min, column temperature is 30℃, detection wavelength is 280 nm (VWD) or detection wavelength is 280 nm, bandwidth is 4 nm, reference wavelength is 360 nm, bandwidth is 80 nm (DAD), peak width is 0.025 min (0.5 s).

[0069] The HPLC result chart of the antibody 7D11 targeting the dengue virus NS1 protein is shown in Figure 2 The results show that the detection purity of the 7D11 is greater than 95%.

[0070] 3. ELISA detection of the binding activity of the antibody Coating: dilute the antigen (dengue virus NS1 protein) to 2 μg / mL with the coating solution, mix well, add to a 96-well coating plate, 100 μL / well, seal and block at 4℃ overnight. Wash 3 times with a plate washer, and the last time should not have liquid remaining on the plate. Dry the liquid on the surface of the plate with a water-absorbing paper.

[0071] Blocking: add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37℃ for 1 h, and wash the plate 3 times according to the above procedure. Dilute the antibody in gradient, 100 μL / well, react at 37℃ for 1 h, and wash the plate 3 times according to the above procedure.

[0072] Add secondary antibody: dilute with DPBS at 1:2000, add to a 96-well plate, 100 μL / well, react at 37℃ for 1 h, and wash the plate 3 times according to the above procedure.

[0073] Color development: add TMB, 100 μL / well, develop at room temperature in the dark for 10 min.

[0074] Termination: add 2N H2SO4, 100 μL / well. Measure OD 450 within 10 min.

[0075] The result chart of the ELISA detection of the binding activity of the antibody 7D11 targeting the dengue virus NS1 protein is shown inFigure 3 As shown in the results, the antibody 7D11 targeting dengue virus NS1 protein can specifically bind to dengue virus NS1 protein, and shows a concentration-dependent manner, and the corresponding EC50 of 7D11 is 0.1151 μg / mL, which has a high affinity. 50 As shown in the results, the antibody 7D11 targeting dengue virus NS1 protein can specifically bind to dengue virus NS1 protein, and shows a concentration-dependent manner, and the corresponding EC50 of 7D11 is 0.1151 μg / mL, which has a high affinity.

[0076] 4. Specific detection of the antibody Coat dengue virus NS1 protein, Zika virus culture, SARS-CoV-2 virus culture, influenza A virus culture, influenza B virus culture, parainfluenza virus culture, adenovirus culture, syncytial virus culture at the same concentration, add antibody, add secondary antibody, develop, read, data analysis.

[0077] The results of the specific detection of the antibody 7D11 targeting dengue virus NS1 protein are shown in Table 4 below, and the results show that only dengue virus NS1 protein (dengue virus 1-4 type NS1 protein) develops normally, and the rest of the antigens have no positive reaction, i.e. the antibody 7D11 targeting dengue virus NS1 protein can specifically bind to dengue virus NS1 protein, which has high specificity.

[0078] Table 4. Results of specific detection of the antibody 7D11 targeting dengue virus NS1 protein

Claims

1. An antibody that specifically targets the dengue virus NS1 protein, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3, whose corresponding amino acid sequences are shown in SEQ ID NO:4-6, respectively. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose corresponding amino acid sequences are shown in SEQ ID NO:7-9, respectively.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4; Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Optionally, the expression vector is a viral vector, a plasmid vector, or a bacteriophage vector; Optionally, the viral vector is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, or herpesvirus vector.

6. A genetically modified host cell, characterized in that, The genetically modified host cell comprises the expression vector of claim 5; Optionally, the host cell is a eukaryotic cell; Optionally, the eukaryotic cell is a mammalian cell, plant cell, or yeast cell; Optionally, the host cell is a CHO cell, a 293 cell, an NS / 0 myeloma cell, a HeLa cell, a Cap cell, or a COS cell.

7. A genetically modified host cell population, characterized in that, The genetically modified host cell population comprises the genetically modified host cells of claim 6.

8. Any of the following methods, characterized in that, The method includes: (1) A method for preparing the antibody according to any one of claims 1-3, the method comprising the following steps: culturing the genetically modified host cells according to claim 6, and isolating the antibody from the culture product; (2) A method for detecting dengue virus NS1 protein for non-diagnostic purposes, the method comprising the following steps: contacting the sample to be tested with the antibody according to any one of claims 1-3, detecting the immune reaction between the sample and the antibody, and determining the expression level of dengue virus NS1 protein in the sample.

9. Any of the following products, characterized in that: The products include: (1) A dengue virus NS1 protein detection reagent, wherein the detection reagent comprises the antibody according to any one of claims 1-3; (2) A dengue virus NS1 protein detection kit, wherein the detection kit comprises the antibody of any one of claims 1-3, or the detection reagent; (3) A dengue virus NS1 protein detection chip, wherein the detection chip comprises the antibody or the detection reagent according to any one of claims 1-3; (4) A dengue virus NS1 protein detection strip, wherein the detection strip comprises the antibody of any one of claims 1-3, or the detection reagent; (5) A diagnostic product for dengue virus infection, wherein the diagnostic product comprises the antibody of any one of claims 1-3, or the detection reagent; (6) A pharmaceutical composition comprising the antibody according to any one of claims 1-3; (7) A pharmaceutical preparation comprising the antibody of any one of claims 1-3, or the pharmaceutical composition thereof.

10. The following application, characterized in that, The applications include: (1) The use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the genetically modified host cell of claim 6 in the preparation of a detection reagent for detecting dengue virus NS1 protein; (2) The use of any one of the antibodies of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the genetically modified host cell of claim 6 in the preparation of a detection kit, detection chip, or test strip for detecting dengue virus NS1 protein; (3) The use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the genetically modified host cell of claim 6 in the preparation of diagnostic products for diagnosing dengue virus infection; (4) The use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the genetically modified host cell of claim 6 in the preparation of a pharmaceutical composition for the prevention and / or treatment of dengue virus infection; (5) The use of any one of the antibodies of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the genetically modified host cell of claim 6 in the preparation of pharmaceutical formulations for the prevention and / or treatment of dengue virus infection.

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