Antibodies against glycoproteins of the fever with thrombocytopenia syndrome virus and uses thereof

Abstract

The application is based on immune repertoire sequencing to screen fever with thrombocytopenia syndrome virus glycoprotein Gn monoclonal antibody and identify its biological characteristics. With purified SFTSV-Gn as a decoy, virus-specific B cells are fished from the B cell library of immunized female BALB / c mice, and 10x immune repertoire sequencing method is used in combination with bioinformatics analysis to select antibody sequences with potential binding activity, which are constructed into eukaryotic expression vector pcDNA3.4 for monoclonal antibody expression and purification, and biological activity is identified. From the 3544 captured B cells, 14 antibodies are selected, and functional verification shows that 12 antibodies can bind SFTSV-Gn glycoprotein, of which Gn-5 and Gn-7 antibodies have higher binding capacity; Western blot, indirect immunofluorescence test and flow cytometry prove that the antibody can specifically recognize SFTSV, and the effective concentration is as low as 2 μg / mL, which lays a foundation for the development of SFTSV rapid diagnostic reagent.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antibody against fever with thrombocytopenia syndrome virus and its application, and more particularly to an antibody against the glycoprotein of fever with thrombocytopenia syndrome virus and its application. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging tick-borne infectious disease, first discovered in Henan Province and other regions of China in 2009. Its pathogen is the severe fever with thrombocytopenia syndrome virus (SFTSV). SFTSV belongs to the order Bunyavirales, family Picornaviridae, genus Bandavirus, and subgenus Dabie bandavirus.

[0003] The SFTSV genome comprises three gene segments: a large segment (L), a medium segment (M), and a small segment (S). The M gene encodes a viral glycoprotein precursor, which is cleaved by enzymes into an N-terminal glycoprotein (Gn) and a C-terminal glycoprotein (Gc), inducing the production of neutralizing antibodies and playing a crucial role in combating SFTS infection. This study used purified SFTSV-Gn glycoprotein as bait to prepare SFTSV-immunized mouse spleen lymphocytes. Virus-specific B cells were sorted, and single-cell high-throughput sequencing was performed to obtain transcriptome and immunohistomic libraries. Antibodies with potential activity were selected, cloned, expressed, and purified, and their biological activity was verified to obtain antibodies with binding activity, laying the foundation for the development of rapid clinical diagnostic technologies for SFTS. Summary of the Invention

[0004] On one hand, the present invention provides an antibody that binds to SFTSV glycoprotein Gn, the antibody having a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No. 1, 3, 5, 7; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID No. 2, 4, 6, 8.

[0005] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1; and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0006] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 3; the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4.

[0007] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 5; the amino acid sequence of the light chain variable region is shown in SEQ ID No. 6.

[0008] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

[0009] In one embodiment, the antibody is a monoclonal antibody, a recombinant antibody, or a polyclonal antibody, preferably a monoclonal antibody.

[0010] In one embodiment, the antibody is a human antibody, a mouse antibody, or a chimeric antibody.

[0011] In one embodiment, the antibody further includes a heavy chain constant region comprising IgG, for example, a heavy chain constant region of IgG selected from IgG1, IgG2, IgG3 or IgG4.

[0012] On the other hand, the present invention also provides a polynucleotide encoding the above-mentioned antibody.

[0013] On the other hand, the present invention also provides a recombinant vector comprising the above-mentioned polynucleotides.

[0014] On the other hand, the present invention also provides a host cell comprising the above-described recombinant vector.

[0015] On the other hand, the present invention also provides a pharmaceutical composition comprising the antibody described above; optionally, it further comprises a pharmaceutical carrier.

[0016] On the other hand, the present invention also provides the use of the above-mentioned antibody in the preparation of reagents or drugs for the treatment or prevention of fever with thrombocytopenia syndrome.

[0017] On the other hand, the present invention also provides the use of the above-mentioned antibody in the preparation of reagents or drugs for fever with thrombocytopenia syndrome virus.

[0018] On the other hand, the present invention also provides the use of the above-mentioned antibody in the preparation of a reagent targeting the Gn protein of fever with thrombocytopenia syndrome virus.

[0019] On the other hand, the present invention also provides the application of the above-mentioned antibody in the detection of fever with thrombocytopenia syndrome virus.

[0020] On the other hand, the present invention also provides the application of the above-mentioned antibody in the preparation of reagents for detecting fever with thrombocytopenia syndrome virus.

[0021] This invention screens monoclonal antibodies against the SFTSV-Gn glycoprotein based on immune repertoire sequencing and identifies their biological characteristics. Using purified SFTSV-Gn as bait, virus-specific B cells were extracted from a library of immunized female BALB / c mice. Using 10× immune repertoire sequencing combined with bioinformatics analysis, antibody sequences with potential binding activity were selected and constructed into the eukaryotic expression vector pcDNA3.4 for monoclonal antibody expression, purification, and identification of their biological activity. Fourteen antibodies were selected from 3544 captured B cells. Functional validation showed that 12 antibodies could bind to the SFTSV-Gn glycoprotein, with Gn-5 and Gn-7 antibodies exhibiting high binding capacity. Western blot, indirect immunofluorescence assay, and flow cytometry demonstrated that these antibodies specifically recognize SFTSV at effective concentrations as low as 2 μg / mL, laying the foundation for the development of rapid diagnostic reagents for SFTSV. Attached Figure Description

[0022] Figure 1 SDS-PAGE analysis of purified SFTSV-Gn protein; where M: protein marker; 1-3: elution with 2%, 10%, and 30% imidazole, respectively.

[0023] Figure 2 Specific B-cell flow cytometry gating strategy. A: Gating lymphocyte subsets using FSC and SSC; B: Gating single lymphocyte subsets using FSC-H; C: Gating live lymphocyte subsets using AF700; D: Gating lymphocyte subsets using CD45; E: Gating lymphocyte subsets after removing CD3 / 14 / 16; F: Gating SFTSV-Gn positive lymphocyte subsets.

[0024] Figure 3 Electrophoresis diagram of partial antibody gene heavy chain identification. M: DNA marker DL15000; 1-6: Gn-1, Gn-2, Gn-3, Gn-4, Gn-5, and Gn-6 antibody heavy chain genes, respectively.

[0025] Figure 4 Identification of some antibody proteins by SDS-PAGE (A) and Western blot (B). M: protein marker; 1-6: antibodies against Gn-1, Gn-2, Gn-3, Gn-4, Gn-5, and Gn-6, respectively.

[0026] Figure 5 Protein A affinity chromatography pattern of antibody purification.

[0027] Figure 6 Identification of the active site for SFTSV recognition by two antibody strains. Figure 6 A: SDS-PAGE identification of SFTSV and Gn proteins (M: protein marker; 1: SFTSV; 2: Gn protein containing His tag); Figure 6 B: Western blot identification of Gn proteins with His tags (M: protein marker; 1: SFTSV; 2: Gn protein containing His tag); Figure 6 C: Western blot identification of Gn-7 antibody activity (M: protein marker; 1: SFTSV; 2: Gn protein containing His tag); Figure 6 D: Western blot identification of Gn-5 antibody activity (M: protein marker; 1: SFTSV, 2: Gn protein containing a His tag). Note: The red boxes in the figure represent the locations identified by gel mass spectrometry.

[0028] Figure 7 IFA (×200) of Gn-5 and Gn-7 antibody activities; the top figure is for Gn-7 and the bottom figure is for Gn-5.

[0029] Figure 8 Flow cytometry detection of THP1 cells infected with SFTSV. A: Lymphocyte subsets circled by FSC and SSC; B: Single lymphocyte subsets circled by FSC-H; C-E: Flow cytometry gating plots of uninfected and 0.1 and 1 MOI SFTSV-infected THP1 cells.

[0030] Figure 9 The proportion of positive THP1 cells at different infection doses. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0032] Example 1, Materials and Methods

[0033] 1.1 Cells, Viruses and Plasmids

[0034] Human embryonic kidney cells HEK293T, HEK293F, human monocytic leukemia (THP-1) cells, Vero cells and SFTSV strain (SDTA-1, GenBank: KX641909) were preserved by the Key Laboratory of Etiological Epidemiology of Emerging Infectious Diseases in Shandong Higher Education Institutions; the expression vector pcDNA3.4 was purchased from Nanjing Genscript Biotech Co., Ltd.; Escherichia coli DH5α was purchased from TaKaRa Company, Japan.

[0035] 1.2 Experimental animals

[0036] Twelve SPF-grade female BALB / c mice, 6 - 8 weeks old, weighing 16 - 24 g, were purchased from Jinan Pengyue Experimental Animal Co., Ltd., with the animal license number: SCXK (Lu) 20220006. All experiments in this study were conducted for scientific research purposes in the breeding and use of experimental animals, and were carried out in accordance with the relevant regulations of experimental animal ethics (W2023083000294).

[0037] 1.3 Main reagents and instruments

[0038] DMEM medium (Catalog No.: C11995500BT) and fetal bovine serum (FBS, Catalog No.: 10437-028) were purchased from Gibco, USA; penicillin-streptomycin antibiotics (Catalog No.: P1400), DMSO (Catalog No.: D8371), erythrocyte lysis buffer (Catalog No.: R1010), 10×PBS (Catalog No.: G4207), and FITC-labeled goat anti-mouse IgG (Catalog No.: SF131) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; polyethyleneimine (PEI, Catalog No.: 043896) was purchased from Polysciences, USA; HRP-labeled goat anti-mouse IgG (Catalog No.: DP110087) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Alexa Fluor@700 (Catalog No.: 564997) was purchased from BD Biosciences, USA; anti-mouse CD3-FITC, CD16-FITC, CD45-PE, IgG-PecpCY5.5, and anti-His... Tag-tagged protein-APC-labeled monoclonal antibody was purchased from Biolegend (Beijing) Biotechnology Co., Ltd.; QuickAntibody (Catalog No.: KX0210042) immunoadjuvant was purchased from Beijing Bio-Long Immunotherapy Co., Ltd.; reverse transcription reagent (Catalog No.: FSQ101) was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; endotoxin-free plasmid extraction kit (Catalog No.: DP118-02) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; His affinity chromatography column (Catalog No.: 29048586) was purchased from Cytiva, USA; Protein A affinity chromatography column (Catalog No.: L00680) was purchased from Nanjing Genscript Biotech Co., Ltd.; FACS AriaⅢ flow cytometer was purchased from BD Biosciences, USA; microplate reader was purchased from BioTek; Agilent 2100 Bioanalyzer was purchased from Agilent Technologies; sequencing, probes and primers were synthesized by Qingke Biotechnology Co., Ltd.

[0039] 1.4 Mouse immunization and preparation of single-cell spleen cells

[0040] SFTSV (approximately 10) 5 TCID 50 The mixture of 100 μL of the agent and adjuvant was administered to the hind leg muscles of BALB / c mice at a volume ratio of 1:1. A second immunization was performed 14 days later. Three days later, blood was collected from the tail of the mice to separate serum and determine the neutralizing titer. Immunized mice were euthanized by cervical dislocation and soaked in alcohol for 5 min. The spleen was collected and soaked in serum-free RPMI 1640 medium, ground, rinsed and filtered through a sieve, centrifuged at 400g for 5 min to collect cells, and lysed on ice for 15 min with erythrocyte lysis buffer. The cells were washed twice with PBS and RPMI 1640 medium, centrifuged at 400g for 5 min respectively, and then cryopreserved in freezing buffer (FBS + 10% DMSO) for later use.

[0041] 1.5 Flow cytometry sorting of SFTSV-specific B cells

[0042] SFTSV-Gn expression was performed according to the literature (WU Y, ZHU YH, GAO F, et al. Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope[J]. PNATL Acad Sci USA, 2017, 114(36): E7564-E7573. DOI: 10.1073 / pnas.1705176114). The plasmid pcDNA3.4-SFTSV-Gn was synthesized by Nanjing Genscript Biotech Co., Ltd., and transformed into competent Escherichia coli DH5α. The target plasmid was extracted according to the plasmid extraction kit instructions and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmid was transfected into human embryonic kidney cells HEK293T with PEI and cultured at 37℃ in a 5% CO2 cell culture incubator for 72 h. The supernatant was collected, and the recombinant SFTSV-Gn protein was purified by His affinity chromatography. After identification by 10% SDS-PAGE, it was stored at -80℃ for later use.

[0043] Frozen mouse spleen cells were washed twice with 2% FBS and incubated on ice for 30 min with SFTSV-Gn protein. Following the reagent instructions, mouse anti-CD3-FITC, CD16-FITC, CD45-PE, IgG-PecpCY5.5, and anti-His tag protein-APC were mixed at a 1:1 volume ratio and added to the cell suspension. The cells were stained in the dark for 30 min. After washing twice, the cells were resuspended in 100 μL of PBS containing Alexa Fluor@700 (working concentration: 66.7 ng / mL) cell viability dye and incubated on ice for 10–15 min in the dark. Viable SFTSV-Gn cells were then sorted after resuspending. + CD45 + CD3 - CD16 - B lymphocytes.

[0044] 1.6 Single-cell sequencing

[0045] The flow cytometry-sorted cell slurry was concentrated to the loading volume, and complete library construction and data analysis were performed by Beijing Guoke Biotechnology Co., Ltd. Specifically, the transcriptome and B lymphocyte antigen receptor (BCR) library were constructed using the 10×Single Cell 5′Reagent Kit v3.1. After passing the Agilent 2100 Bioanalyzer test, sequencing was performed using the Illumina Novaseq 6000 platform, yielding 80Gb of sequenced transcriptome and 20Gb of BCR immune library data for each sample. The generated data underwent quality control using FastQC software and analysis using Cell Ranger software to obtain the paired heavy and light chain sequences of the antibodies.

[0046] 1.7 Selection and Construction of Anti-SFTSV-Gn Monoclonal Antibody Plasmid

[0047] According to the antibody selection principles reported in the literature (CAO Y, SU B, GUO X, et al. Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' BCells[J]. Cell, 2020, 182(1):73-84e16. DOI::10.1073 / pnas.1705176114. SCHEID JF, BARNES CO, ERASLAN B, et al. B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV[J]. Cell, 2021, 184(12):3205-3221.), 14 antibody sequences with abundant clonal types or high number of unique molecular marker (UMI) sequences were selected for gene synthesis. The selected antibody variable region genes were synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated into the pcDNA3.4 vector containing the heavy chain and light chain constant region genes of mouse monoclonal antibodies. Fourteen antibodies were screened and named Gn-1, Gn-2, Gn-3, Gn-4, Gn-5, Gn-6, Gn-7, Gn-8, Gn-9, Gn-10, Gn-11, Gn-12, Gn-13, and Gn-14.

[0048] 1.8 Antibody purification and identification

[0049] The synthesized antibody sequence plasmid was transfected into HEK293T or HEK293F cells at a heavy chain:light chain ratio of 1:2. After 72 hours of transfection, the cell supernatant was collected, centrifuged, concentrated 10-fold, and analyzed by 10% SDS-PAGE. Simultaneously, the isolated protein was transferred to PVDF and blocked overnight at 4°C with 5% skim milk. HRP-labeled goat anti-mouse IgG (1:2000 dilution) was used as the detection antibody and incubated at 37°C for 1 hour, followed by washing five times with PBST and development using ECL. The correctly identified target antibody was purified and identified using a Protein A affinity chromatography column for subsequent functional assays.

[0050] 1.9 Antibody Function Validation

[0051] 1.9.1 Antibody Binding Ability Assay - ELISA Method

[0052] Using Gn protein (200 ng / well) and SFTSV (approximately 10 ng / well) respectively 4 TCID 50 The ELISA plate was coated with the sample and incubated overnight at 4°C. It was then blocked with 5% skim milk at 37°C for 1 hour. After washing with PBS, purified antibody was added at 50 μg / mL and incubated at 37°C for 30 minutes. After washing with PBST, HRP-labeled goat anti-mouse IgG (1:2000 dilution) was added and incubated at 37°C for 30 minutes. After washing with PBST, 60 μL of TMB chromogenic solution was added and the plate was incubated at 37°C for 10 minutes. Finally, 60 μL of stop solution was added, and the A titer was detected using a BioTek microplate reader. 450 The experiment was repeated twice, with duplicate wells each time, and the final results are expressed as the mean.

[0053] 1.9.2 Antigenicity

[0054] Vero cells inoculated with SFTSV were transferred onto a PVDF membrane, and blocking buffer (5% skim milk) was added and incubated overnight at 4°C. The purified antibody (50 μg / mL) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (1:2000 dilution) was used as the secondary antibody. The specific steps were the same as in 1.8, and the target band was identified by mass spectrometry.

[0055] 1.9.3 Specificity - Indirect immunofluorescence assay (IFA) was used.

[0056] Vero cells were loaded at 1.5 × 10⁻⁶. 4Seeds were seeded per well in a 96-well plate. The next day, SFTSV was seeded at an MOI of 0.1, replaced with 1% maintenance medium, and cultured at 37°C in a 5% CO2 incubator for 96 h. Pre-cooled anhydrous ethanol was added, and the cells were fixed at 4°C for 30 min. The cells were then air-dried, and the antibody was diluted with 3% BSA to 10, 2, and 0.4 μg / mL, added to the fixation wells, and incubated at 37°C for 30 min. The cells were washed with PBST, and FITC-labeled goat anti-mouse IgG (1:150 dilution) was added and incubated in the dark for 30 min. The cells were washed three times with PBST, stained with 60 μL of DAPI, and incubated in the dark for 5 min. The cells were washed three times with PBST, and 50 μL of 20% glycerol was added. The cells were observed under a fluorescence microscope in the dark.

[0057] 1.10 Detection of SFTSV-infected cells

[0058] The prepared antibody was used to analyze SFTSV-infected THP-1 cells: THP-1 cells were infected with 0.1 and 1 MOI and cultured at 37°C for 96 h in a 5% CO2 cell culture incubator; cells were fixed with pre-cooled anhydrous ethanol, and a working concentration of 50 μg / mL monoclonal antibody was added and incubated at 4°C for 30 min; FITC-labeled goat anti-mouse IgG diluted 1:100 was added and incubated at 4°C in the dark for 30 min; cells were detected by BD FACS AriaⅢ flow cytometry and the results were analyzed using FlowJo-V10 software.

[0059] 1.11 Statistical Analysis

[0060] For comparisons of normally distributed quantitative data between two groups, the independent samples t-test or the one-way ANOVA test was used for multiple group comparisons. For comparisons of skewed quantitative data between groups, the Mann-Whitney U rank-sum test was used, with a significance level of P < 0.05 (two-tailed test). False discovery rate (FDR) corrections were applied to multiple comparisons. Statistical analysis and graphing were performed using Graphpad Prism 8.0 software.

[0061] Example 2: Antibody Screening and Functional Validation Results

[0062] 2.1 SFTSV-specific B cell sorting and sequencing

[0063] After secondary immunization, BALB / c mice showed a neutralizing titer of 1:640 according to IFA. Splenic lymphocytes from these mice were aseptically isolated and cryopreserved for later use. Purified SFTSV-Gn protein was analyzed by 10% SDS-PAGE, revealing a target protein band of approximately 55,000. Figure 1 Specific B cells were sorted from the spleen lymphocytes of resuscitated mice. The sorting strategy is described in [link to strategy]. Figure 2After identifying CD45-positive single lymphocytes, T cells and monocytes represented by CD3, CD14, and CD16 are excluded, and SFTSV Gn is sorted. + The mononuclear lymphocytes were sequenced by Beijing Guoke Biotechnology Co., Ltd.

[0064] 2.2 Single-cell data and identification of anti-SFTSV-Gn monoclonal antibody plasmid

[0065] After alignment, splicing, and assembly of single-cell transcriptome and BCR sequencing data, a total of 3544 B cells were obtained, with an average of 10882 reads per cell, of which 3289 cells possessed complete antibody light and heavy chains. Based on clonogenic abundance or UMI sequence number, the top 14 antibodies were selected for gene synthesis and plasmid construction. 1% agarose gel electrophoresis analysis of the antibody plasmids revealed a specific band of approximately 7000 bp, predominantly in a supercoiled conformation, and its size was consistent with expectations. Figure 3 Sequence alignment results showed that the target gene fragment was consistent with the selected antibody gene sequence, indicating successful plasmid construction.

[0066] 2.3 Identification of expressed antibodies

[0067] After 72 hours of transfection of the correctly identified antibody gene into HEK293T cells, the cell supernatant was analyzed by 10% SDS-PAGE and Western blot. The target protein band was visible at approximately 55,000 relative molecular masses, indicating successful expression of the target antibody. Figure 4 After Protein A affinity chromatography, a single symmetrical peak was observed, indicating that the antibody purity met the requirements for subsequent experiments. Figure 5 .

[0068] 2.4 Antibody activity identification

[0069] 2.4.1 Binding Activity

[0070] Fourteen selected SFTSV-Gn specific antibodies were tested by ELISA. Three were positive (>0.2), and 12 showed binding affinity to Gn. Among them, Gn-5, Gn-7, Gn-12, and Gn-2 showed higher binding affinity to Gn. 450 The values ​​are 3.65, 2.3, 2.4 and 2.3 respectively.

[0071] Table 1. Binding affinity of selected antibodies to SFTSV and Gn proteins (A) 450 )

[0072] Antibody SFTSV Gn Gn-1 0.53 0.48 Gn-2 0.23 2.3 Gn-3 0.17 0.65 Gn-4 0.18 0.85 Gn-5 0.23 2.3 Gn-6 0.14 1.8 Gn-7 0.3 3.65 Gn-8 0.15 0.85 Gn-9 0.09 0.12 Gn-10 0.08 0.13 Gn-11 0.13 0.46 Gn-12 0.15 2.4 Gn-13 0.11 0.35 Gn-14 0.15 0.32

[0073] The heavy chain variable region and light chain variable region sequences of the above-mentioned Gn-5, Gn-7, Gn-12 and Gn-2 antibodies are shown below:

[0074]

[0075]

[0076] 2.4.2 Antigenicity

[0077] The anti-SFTSV-Gn monoclonal antibodies Gn-5 and Gn-7 specifically reacted with purified recombinant Gn protein and SFTSV. Western blot analysis showed that the former exhibited a specific band at a relative molecular mass of approximately 55,000, consistent with the expected size. Figure 6 A and Figure 6 B, while SFTSV shows three main bands at relative molecular masses of approximately 60,000, 50,000, and 35,000, see [reference needed]. Figure 6 C and Figure 6 D.

[0078] 2.4.3 Specificity

[0079] IFA results showed that both the selected Gn-5 and Gn-7 antibodies specifically bound to SFTSV-inoculated Vero cells, producing a fluorescent reaction, while no fluorescence was observed in uninfected SFTSV Vero cells (control). This indicates that the prepared monoclonal antibodies can specifically recognize the SFTSV-Gn protein. Furthermore, both Gn-5 and Gn-7 antibodies still produced a fluorescent reaction at a concentration of 2 μg / mL, but no fluorescence was observed at a concentration of 0.4 μg / mL, indicating that the minimum effective concentration of the two antibodies is approximately 2 μg / mL. (See...) Figure 7 .

[0080] 2.5% Virus infection rate in cells

[0081] When THP-1 cells were infected with different doses of SFTSV, detection with Gn-5 antibody revealed that approximately 0.02% of uninfected THP-1 cells were positive, while SFTSV-infected THP-1 cells showed typical strong positive cell colonies. Figure 8 The infection rate was 0.7% for MOI=0.1 and 4.1% for MOI=1. The differences among multiple groups were statistically significant (F=12.4, P<0.05). The infection rate for MOI=1 was significantly higher than that for uninfected cells (t=31.3, P=0.003) and also significantly higher than that for MOI=0.1 (t=22.9, P=0.01). However, the infection rate for MOI=0.1 was not significantly different from that for uninfected cells (t=32.8, P=0.002). (See [link to relevant documentation]). Figure 9 This indicates that the SFTSV infection rate is positively correlated with the infection dose.

[0082] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody binding to a glycoprotein Gn of SFTSV, the antibody having a heavy chain variable region and a light chain variable region; characterized in that, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2; or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 4; or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 6; or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.

8.

2. The antibody of claim 1, wherein The antibody is a monoclonal antibody or a recombinant antibody.

3. The antibody of claim 1, wherein The antibody is a human antibody, a murine antibody or a chimeric antibody.

4. The antibody of claim 1, wherein The antibody further comprises a heavy chain constant region of IgG. 5.A polynucleotide encoding the antibody according to any one of claims 1-4. 6.A recombinant vector comprising the polynucleotide according to claim 5. 7.A host cell comprising the recombinant vector according to claim 6. 8.A pharmaceutical composition comprising the antibody according to any one of claims 1-4.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. 10.Use of the antibody according to any one of claims 1-4 in the preparation of a reagent for detecting fever with thrombocytopenia syndrome virus.

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