Antibody aiming at severe fever with thrombocytopenia syndrome virus glycoprotein and application thereof

Abstract

The monoclonal antibody of the glycoprotein Gn of the severe fever with thrombocytopenia syndrome virus is screened on the basis of immune repertoire sequencing, and the biological characteristics of the monoclonal antibody are identified. The method comprises the following steps: taking purified SFTSV-Gn as bait, taking virus specific B cells from an immunized female BALB / c mouse B cell bank, selecting an antibody sequence with potential binding activity by utilizing a 10 * immune repertoire sequencing method in combination with bioinformatics analysis, constructing the antibody sequence to an eukaryotic expression vector pcDNA3.4, expressing and purifying a monoclonal antibody, and identifying the biological activity of the monoclonal antibody. 14 antibodies are selected from 3544 captured B cells, functional verification shows that 12 antibodies can be combined with SFTSV-Gn glycoprotein, and the Gn-5 antibody and the Gn-7 antibody have high combining capacity; western blot, indirect immunofluorescence test and flow cytometry prove that the antibody can specifically recognize the SFTSV, the effective concentration is as low as 2 mu g / mL, and a foundation is laid for development of a rapid diagnosis reagent for the SFTSV.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antibody against a fever with thrombocytopenia syndrome virus and an application thereof, and in particular to an antibody against a fever with thrombocytopenia syndrome virus glycoprotein and an application thereof. Background Art

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

[0003] The SFTSV genome consists of three gene fragments: large fragment (L), medium fragment (M) and small fragment (S). The M gene encodes the viral glycoprotein precursor, which is cleaved into N-terminal glycoprotein (Gn) and C-terminal glycoprotein (Gc), which can induce the production of neutralizing antibodies and play an important role in the anti-SFTS infection process. In this study, purified SFTSV-Gn glycoprotein was used as bait to prepare spleen lymphocytes of SFTSV-immunized mice, sort virus-specific B cells, perform single-cell high-throughput sequencing, obtain transcriptome and immune repertoire, select antibodies with potential activity for cloning, expression and purification, verify biological activity, and obtain antibodies with binding activity, laying the foundation for the development of clinical SFTS rapid diagnosis technology. Summary of the invention

[0004] On the 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, and 7; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID No.2, 4, 6, and 8.

[0005] In one embodiment, 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.

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

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

[0008] In one embodiment, 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.

[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 murine antibody, or a chimeric antibody.

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

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

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

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

[0015] On the other hand, the present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody; optionally, further comprises a pharmaceutically acceptable carrier.

[0016] On the other hand, the present invention also provides the use of the above-mentioned antibody in preparing a reagent or a medicine for treating or preventing 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 a reagent or a drug for neutralizing the fever with thrombocytopenia syndrome virus.

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

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

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

[0021] The present invention screens the severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn monoclonal antibody based on immune repertoire sequencing and identifies its biological characteristics. Purified SFTSV-Gn is used as bait to fish virus-specific B cells from the B cell library of immune female BALB / c mice. The antibody sequence with potential binding activity is selected by 10× immune repertoire sequencing method combined with bioinformatics analysis, and constructed into the eukaryotic expression vector pcDNA3.4 to express and purify the monoclonal antibody, and identify its biological activity. 14 strains of antibodies are selected from the captured 3544 B cells, and functional verification shows that 12 strains of antibodies can bind to SFTSV-Gn glycoprotein, among which Gn-5 and Gn-7 antibodies have higher binding ability; Western blot, indirect immunofluorescence test and flow cytometry prove that the antibody can specifically recognize SFTSV, and the effective concentration is as low as 2μg / mL, which lays a foundation for the development of SFTSV rapid diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 . Specific B cell flow cytometry gating strategy. A: FSC and SSC are used to circle lymphocyte subsets; B: FSC-H is used to circle single lymphocyte subsets; C: AF700 is used to circle live lymphocyte subsets; D: CD45 is used to circle lymphocyte subsets; E: CD3 / 14 / 16 is removed from lymphocyte subsets; F: SFTSV-Gn positive lymphocyte subsets are circled.

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

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

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

[0027] Figure 6 .Identification of the active site of SFTSV recognized by 2 antibodies. 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 protein with His tag (M: protein marker; 1: SFTSV; 2: Gn protein with 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 with His tag). Note: The red box in the figure represents the position of the agarose gel mass spectrometry identification.

[0028] Figure 7 .IFA (×200) of Gn-5 and Gn-7 antibody activities; the upper picture is Gn-7 and the lower picture is Gn-5.

[0029] Figure 8 .Flow cytometry detection of THP1 cells infected with SFTSV. A: Lymphocyte subsets are circled by FSC and SSC; B: Single lymphocyte subsets are circled by FSC-H; C-E: Flow cytometry gate images of THP1 cells that were not infected and infected with 0.1 and 1 MOI SFTSV.

[0030] Fig. 9 .The proportion of positive THP1 cells at different infection doses. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention is 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 and human monocytic leukemia (THP-1) cells, Vero cells and SFTSV strain (SDTA-1, GenBank: KX641909) were deposited by the Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Colleges and Universities of Shandong Province; the expression vector pcDNA3.4 was purchased from Nanjing GenScript Biotechnology Co., Ltd.; and Escherichia coli DH5α was purchased from TaKaRa, Japan.

[0035] 1.2 Experimental animals

[0036] SPF female BALB / c mice, 12, 6-8 weeks old, weighing 16-24 g, were purchased from Jinan Pengyue Experimental Animal Co., Ltd., animal license number: SCXK (Lu) 20220006. The experiments were all carried out for the purpose of scientific research and were carried out in accordance with the relevant provisions of experimental animal ethics (W2023083000294).

[0037] 1.3 Main reagents and instruments

[0038] DMEM medium (Cat. No. C11995500BT) and fetal bovine serum (FBS, Cat. No. 10437-028) were purchased from Gibco, USA; penicillin-streptomycin dual antibody (Cat. No. P1400), DMSO (Cat. No. D8371), red blood cell lysis buffer (Cat. No. R1010) and 10×PBS (Cat. No. G4207), FITC-labeled goat anti-mouse IgG (Cat. No. SF131) were purchased from Beijing Solebao Technology Co., Ltd.; polyethyleneimine (PEI, Cat. No. 043896) was purchased from Polysciences, USA; HRP-labeled goat anti-mouse IgG (Cat. No. DP110087) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Alexa Fluor@700 (Cat. No. 564997) was purchased from BD Biotechnology, USA; anti-mouse CD3-FITC, CD16-FITC, CD45-PE, IgG-PecpCY5.5 and anti-His Monoclonal antibody labeled with tag protein-APC was purchased from Biolegend (Beijing) Biotechnology Co., Ltd.; QuickAntibody (Cat. No.: KX0210042) immune adjuvant was purchased from Beijing Biolong Immunotechnology Co., Ltd.; reverse transcription reagent (Cat. No.: FSQ101) was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; endotoxin-free plasmid extraction kit (Cat. No.: DP118-02) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; His affinity chromatography column (Cat. No.: 29048586) was purchased from Cytiva, USA; Protein A affinity chromatography column (Cat. No.: L00680) was purchased from Nanjing GenScript Biotechnology Co., Ltd.; FACS AriaⅢ flow cytometer was purchased from BD, USA; ELISA reader was purchased from BioTek; Agilent 2100 Bioanalyzer was purchased from Agilent; sequencing, probes and primers were synthesized by Qingke Biotechnology Co., Ltd.

[0039] 1.4 Mouse immunization and spleen single cell preparation

[0040] SFTSV (about 10 5 TCID 50 ) was mixed with adjuvant in a volume ratio of 1:1, and BALB / c mice were immunized with 100 μL / mouse for hind calf muscle, and secondary immunization was performed at an interval of 14 days; 3 days later, blood was collected from the tail of the mice to separate serum and detect neutralization titer. Qualified mice were killed by cervical dislocation and soaked in alcohol for 5 minutes; spleens were taken and soaked in serum-free RPMI1640 medium, ground and filtered through a sieve, centrifuged at 400g for 5 minutes to collect cells, and lysed on ice for 15 minutes by adding red blood cell lysis solution; cells were washed twice with PBS and RPMI1640 medium at 400g for 5 minutes, respectively, and cryopreservation solution (FBS+10% DMSO) was added to freeze the cells for later use.

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

[0042] 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), SFTSV-Gn was expressed. The plasmid pcDNA3.4-SFTSV-Gn was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and transformed into competent Escherichia coli DH5α. The target plasmid was extracted according to the instructions of the plasmid extraction kit and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmid was transfected into human embryonic kidney HEK293T cells with PEI and cultured in a cell culture incubator at 37 ° C and 5% CO2 for 72 hours; the supernatant was collected and the recombinant SFTSV-Gn protein was purified by His affinity chromatography column, and after 10% SDS-PAGE identification, it was stored at -80 ° C for future use.

[0043] Take frozen mouse spleen cells, wash them twice with 2% FBS, add SFTSV-Gn protein and incubate on ice for 30 minutes; according to the reagent instructions, mix mouse anti-CD3-FITC, CD16-FITC, CD45-PE, IgG-PecpCY5.5 and anti-His tag protein-APC in a volume ratio of 1:1, add them to the cell suspension, and stain for 30 minutes in the dark; wash twice, add 100 μL PBS containing Alexa Fluor@700 (working concentration: 66.7 ng / mL) cell viability dye to resuspend the cells, and incubate on ice in the dark for 10-15 minutes; after resuspension, sort out the live SFTSV-Gn + CD45 + CD3 - CD16 - B lymphocytes.

[0044] 1.6 Single-cell sequencing

[0045] The above-mentioned flow sorted cell fluid was concentrated to the loading volume, and Beijing Guokr Biotechnology Co., Ltd. completed the library construction and data analysis. Specifically, the 10×Single Cell 5′Reagent Kit v3.1 was used to complete the construction of the transcriptome and B lymphocyte antigen receptor repertoire (BCR). After passing the Agilent 2100Bioanalyzer test, the sequencing was completed using the IlluminaNovaseq 6000 platform, and each sample obtained 80Gb of sequencing transcriptome and 20Gb of BCR immune repertoire data. The generated data was quality checked using the software FastQC and analyzed using the software Cell Ranger to obtain the paired heavy and light chain sequences of the antibody.

[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' B Cells [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 rich clonal types or high numbers of unique molecular markers (UMI) sequences were selected for gene synthesis. The selected antibody variable region genes were synthesized by Nanjing GenScript Biotechnology Co., Ltd. and connected to the pcDNA3.4 vector containing the heavy and light chain constant region genes of mouse monoclonal antibodies. 14 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 mass ratio of heavy chain: light chain = 1:2. After 72 hours of transfection, the cell supernatant was collected, centrifuged, concentrated 10 times, and then analyzed by 10% SDS-PAGE. At the same time, the separated protein was transferred to PVDF and blocked with 5% skim milk at 4°C overnight; HRP-labeled goat anti-mouse IgG was used as the detection antibody (1:2000 dilution) and incubated at 37°C for 1 hour, then washed 5 times with PBST; ECL exposure method was used for development. The correct target antibody was purified and identified using a Protein A affinity chromatography column for subsequent functional tests.

[0050] 1.9 Antibody Function Verification

[0051] 1.9.1 Antibody binding test - ELISA method

[0052] Gn protein (200 ng / well) and SFTSV (about 10 4 TCID 50 ) was coated on the ELISA plate, and the sample was added and incubated at 4°C overnight; it was blocked with 5% skim milk at 37°C for 1h; it was washed with PBS, and the purified antibody was added to the plate at 50μg / mL, and incubated at 37°C for 30min; it was washed with PBST, and HRP-labeled goat anti-mouse IgG (1:2000 dilution) was added, and incubated at 37°C for 30min; it was washed with PBST, and 60μL TMB colorimetric solution was added, and it was incubated in a constant temperature incubator at 37°C for 10min; 60μL stop solution was added, and the A was detected by BioTek microplate reader 450 The experiment was repeated twice, with duplicate wells each time, and the final value was expressed as the mean.

[0053] 1.9.2 Antigenicity

[0054] Vero cells inoculated with SFTSV were transferred to PVDF membranes, and blocking solution (5% skim milk) was added, and the membranes were incubated at 4°C overnight. Purified antibodies were used as primary antibodies (50 μg / mL) and HRP-labeled goat anti-mouse IgG was used as secondary antibodies (1:2 000 dilution). The specific steps were the same as 1.8, and the target bands were identified by mass spectrometry.

[0055] 1.9.3 Specificity - using indirect immunofluorescence assay (IFA)

[0056] Vero cells were cultured at 1.5 × 10 4The cells were plated in 96-well plates at a MOI of 0.1 the next day. SFTSV was inoculated at a MOI of 0.1 and replaced with 1% maintenance solution in a cell culture incubator at 37°C and 5% CO2 for 96 h. Pre-cooled anhydrous ethanol was added and fixed at 4°C for 30 min. The cells were air-dried and the antibodies to be tested were diluted with 3% BSA to 10, 2, and 0.4 μg / mL, added to the fixed wells, and allowed to stand at 37°C for 30 min. The cells were washed with PBST, FITC-labeled goat anti-mouse IgG (1:150 dilution) was added, and the cells were incubated in the dark for 30 min. The cells were washed 3 times with PBST, 60 μL DAPI was added for staining, and the cells were incubated in the dark for 5 min. The cells were washed 3 times with PBST, 50 μL 20% glycerol was added, and the cells were observed under a fluorescence microscope in the dark.

[0057] 1.10 Detection of SFTSV-infected cells

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

[0059] 1.11 Statistical analysis

[0060] The two-group comparison of normally distributed quantitative data was performed using the independent sample t test or the one-way ANOVA test for multiple group comparisons. The inter-group comparison of skewed distributed quantitative data was performed using the Mann-Whitney U rank sum test, with P < 0.05 (two-sided test) as the test level. The false discovery rate (FDR) method was used for multiple comparisons. Graphpad Prism 8.0 software was used for statistical analysis and drawing.

[0061] Example 2. Antibody screening and functional verification results

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

[0063] After the secondary immunization of BALB / c mice, the neutralization titer of IFA test reached 1:640, and the spleen lymphocytes of mice were aseptically separated and frozen for future use. The purified SFTSV-Gn protein was analyzed by 10% SDS-PAGE, and the target protein band of about 55,000 was visible. Figure 1 Specific B cells were sorted from spleen lymphocytes of resuscitated mice. The sorting strategy is shown in Figure 2That is, after gating out CD45-positive single lymphocytes, T cells and monocytes represented by CD3, CD14 and CD16 were excluded, and SFTSV Gn + The mononuclear lymphocytes were sequenced by Beijing Guokr 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 had complete antibody light and heavy chains. Based on the abundance of clonal types or the number of UMI sequences, the top 14 antibodies were selected for gene synthesis and plasmid construction. The antibody plasmid was analyzed by 1% agarose gel electrophoresis, and a specific band of about 7,000 bp was observed, which was mainly in supercoil conformation and the size was consistent with expectations, as shown in Figure 2. Figure 3 ; Sequencing results were compared and it was shown that the target gene fragment was consistent with the selected antibody gene sequence, indicating that the plasmid was successfully constructed.

[0066] 2.3 Identification of expressed antibodies

[0067] After the correct antibody gene was transfected into HEK293T cells for 72 hours, the cell supernatant was identified by 10% SDS-PAGE and Western blot. The target protein band was visible at a relative molecular mass of about 55,000, indicating that the target antibody was successfully expressed. Figure 4 After Protein A affinity chromatography, a single symmetrical peak was observed, indicating that the purity of the antibody could meet the requirements of subsequent experiments. Figure 5 .

[0068] 2.4 Antibody activity identification

[0069] 2.4.1 Binding activity

[0070] The 14 selected SFTSV-Gn specific antibodies were tested by ELISA, and 3 were positive (>0.2). 12 strains had binding ability to Gn, among which Gn-5, Gn-7, Gn-12 and Gn-2 had higher binding ability to Gn. 450 They are 3.65, 2.3, 2.4 and 2.3 respectively.

[0071] Table 1. Binding ability 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 were selected to react specifically with the purified recombinant Gn protein and SFTSV. Western blot analysis showed that the former had a specific band at a relative molecular mass of about 55,000, and the size was consistent with expectations, see Figure 6 A and Figure 6 B, while SFTSV has three main bands at relative molecular masses of about 60,000, 50,000 and 35,000. Figure 6 C and Figure 6 D.

[0078] 2.4.3 Specificity

[0079] IFA results showed that the selected Gn-5 and Gn-7 antibodies could specifically bind to Vero cells inoculated with SFTSV and produce fluorescence reactions, while the Vero cells not infected with SFTSV showed no fluorescence, indicating that the prepared monoclonal antibodies could specifically recognize SFTSV-Gn protein; at the same time, Gn-5 and Gn-7 antibodies could still produce fluorescence reactions 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 was about 2 μg / mL. Figure 7 .

[0080] Virus infection rate of 2.5 cells

[0081] The Gn-5 antibody was used to detect THP-1 cells infected with different SFTSV doses. It was found that about 0.02% of positive cells could be detected in uninfected THP-1 cells, while typical strong positive cell colonies appeared in THP-1 cells infected with SFTSV. Figure 8 The infection rate of MOI=0.1 was 0.7%, and that of MOI=1 was 4.1%. There were significant differences among the groups (F=12.4, P<0.05). The infection rate of MOI=1 was significantly higher than that of uninfected cells (t=31.3, P=0.003) and also significantly higher than that of MOI=0.1 (t=22.9, P=0.01). However, the infection rate of MOI=0.1 was not significantly higher than that of uninfected cells (t=32.8, P=0.002). Fig. 9 , indicating that the SFTSV infection rate was positively correlated with the infection dose.

[0082] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. 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, and 7; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID No.2, 4, 6, and 8.

2. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody, a recombinant antibody or a polyclonal antibody.

3. The antibody according to claim 1, characterized in that The antibody is a human antibody, a murine antibody or a chimeric antibody.

4. The antibody according to claim 1, characterized in that The antibody also includes an IgG heavy chain constant region.

5. A polynucleotide encoding the antibody according to any one of claims 1 to 4. 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 to 4; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

9. Use of the antibody according to any one of claims 1 to 4 in the preparation of an agent targeting the Gn protein of HFTS virus.

10. Use of the antibody according to any one of claims 1 to 4 in the preparation of a reagent for detecting fever with thrombocytopenia syndrome virus.

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