A monoclonal antibody against the Gn protein of new bunyavirus and its application

Abstract

The present invention discloses a monoclonal antibody against the Gn protein of the novel bunyavirus. The antibody has unique light and heavy chain CDR regions, shows good in vitro neutralizing activity, with an IC 50 value of 0.16 μg / mL, exhibits high affinity for SFTSV Gn with a KD value of 13.3 pM, and shows a 100% protection rate in the treatment of challenged mice. The present invention also provides the application of the monoclonal antibody in the preparation of a detection kit for severe fever with thrombocytopenia syndrome, and the application in the preparation of a drug for preventing and / or treating severe fever with thrombocytopenia syndrome.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody against new bunyavirus Gn protein and application thereof. Background Art

[0002] Severe Fever with Thrombocytopenia Syndrome (SFTS) is an infectious disease caused by infection with Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) (new Bunyavirus (SFTSV)), with fever and thrombocytopenia as the main clinical features. The high morbidity and mortality caused by SFTS has caused serious public health problems. The clinical manifestations of infectious diseases caused by new Bunyavirus (SFTSV) infection are mainly characterized by fever and thrombocytopenia. A small number of patients have severe and rapidly progressive conditions and may die from multiple organ failure.

[0003] SFTSV infection can lead to various clinical manifestations such as asymptomatic infection, mild fever, severe bleeding or multiple organ failure, and even death. The pathogenesis of SFTS is very complex and has not yet been fully elucidated. In recent years, the number of SFTS cases has increased year by year, and the case fatality rate has always remained at a high level. Studies have found that the new bunyavirus is transmitted from person to person and from animal to person, which seriously threatens human health. However, there are still no licensed vaccines and specific antiviral drugs for human use. To prevent SFTSV infection, on the one hand, we should stay away from the source of the epidemic, take good protection, and avoid tick bites; on the other hand, it is of great significance to accelerate the development of vaccines and antiviral drugs. In recent years, the number of SFTS cases has increased year by year, and the case fatality rate has always remained at a high level. The development of effective vaccines and specific antiviral drugs is one of the most effective ways to prevent and treat SFTS.

[0004] SFTSV contains three genomic segments: L, M, and S. The L segment encodes RNA-dependent RNA polymerase (RdRp), and the M segment encodes Gn and Gc envelope glycoproteins, which play a role in cell attachment and membrane fusion, respectively, and are the main components of specific neutralizing antibodies on the virus surface. The S gene encodes nucleoprotein (NP) and non-structural proteins (NSs).

[0005] Antibodies play a key role in the treatment of a variety of viral diseases, such as those caused by Hantavirus, cytomegalovirus, rabies virus, and respiratory syncytial virus infection. Neutralizing antibodies are expected to be developed as drugs for the prevention and treatment of viral infectious diseases, and can provide a reference for the design of vaccines. Previous studies have shown that the plasma of recovered patients has a potential therapeutic effect on cases of fever with thrombocytopenia syndrome, and the key role is played by the neutralizing antibodies in the blood of recovered patients. At present, no specific therapeutic drugs for the new bunyavirus have been approved. Therefore, it is very important to find monoclonal antibodies that can effectively neutralize the new bunyavirus, which is of great significance for the clinical treatment of fever with thrombocytopenia syndrome.

[0006] Based on the technical problems existing in the prior art, the purpose of the present invention is to provide a monoclonal antibody against the new bunyavirus Gn protein, so as to effectively neutralize the fever with thrombocytopenia syndrome virus, and then provide its use in the preparation of a fever with thrombocytopenia syndrome detection kit, and its use in the preparation of a drug for preventing and / or treating fever with thrombocytopenia syndrome. Summary of the invention

[0007] Based on the above purpose, the present invention first provides a monoclonal antibody against the new bunyavirus Gn protein, the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO: 1 at positions 26-33, 51-58 and 97-106, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 3 at positions 27-36, 54-56 and 93-101.

[0008] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3. In the present invention, the antibody having a specific technical solution of the heavy chain variable region and the light chain variable region is named "MM04 antibody".

[0009] In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is as shown in SEQ ID NO: 5, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 7, and the light chain is a Kappa chain.

[0010] Secondly, the present invention provides a polynucleotide encoding the heavy chain and light chain of the above-mentioned monoclonal antibody, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2, and the polynucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 4.

[0011] In a preferred embodiment, the sequence of the polynucleotide encoding the heavy chain constant region of the monoclonal antibody is shown as SEQ ID NO:6, and the sequence of the polynucleotide encoding the light chain constant region of the monoclonal antibody is shown as SEQ ID NO:8.

[0012] Thirdly, the present invention provides a vector containing the above-mentioned polynucleotide encoding the heavy chain and light chain of the monoclonal antibody.

[0013] In a preferred embodiment, the vector is a eukaryotic expression vector. In a specific embodiment of the present invention, the expression vector is pcDNA3.1.

[0014] Fourthly, the present invention provides a host cell containing the vector.

[0015] The host cells include prokaryotic cells, fungal cells, insect cells, including but not limited to COS cells, CHO cells, BHK cells, HEK293 cells; preferably, the host cells are mammalian cells; in a specific embodiment of the present invention, the host cells are HEK 293F cells.

[0016] Fifth, the present invention provides the use of the above-mentioned monoclonal antibody in the preparation of a fever with thrombocytopenia syndrome detection kit. The kit is based on the immunological characteristics of antigen-antibody specific binding, and can be used to detect specific antigens in the specimen, that is, fever with thrombocytopenia syndrome virus particles, or whether its Gn protein exists. The detection method includes but is not limited to: enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence immunoassay, etc., and the detection can be in vitro detection or in situ detection.

[0017] Sixth, the present invention provides the use of the above-mentioned monoclonal antibody in the preparation of a drug for preventing and / or treating fever with thrombocytopenia syndrome.

[0018] Finally, the present invention provides a pharmaceutical composition, which contains the above monoclonal antibody.

[0019] The monoclonal antibody MM04 against the new Bunyavirus Gn protein provided by the present invention showed good in vitro neutralization activity against the virus, and its IC 50The value is 0.16 µg / mL, showing a high affinity for SFTSV Gn, and its KD value is 13.3 pM. A 100% protection rate was shown in the treatment of challenged mice. Moreover, the antibody MM04 provided by the present invention does not compete at all with the anti-SFTSV Gn antibodies JKL2, JKL8, JKL77, and JKL79 (CN 118373906A) disclosed in the prior art. Recognize different epitopes. Therefore, the antibody provided by the present invention and other antibodies in the prior art can be used in immunological detection technologies such as double antibody sandwiches, showing the application prospects of the antibodies provided by the present invention in the preparation of a fever with thrombocytopenia syndrome detection kit. In addition, the present invention also shows the application prospects of the antibodies in the preparation of drugs for the prevention and / or treatment of fever with thrombocytopenia syndrome, especially the antibodies provided by the present invention and other prior art antibodies that recognize different epitopes of the same antigen as an antibody combination, which can be applied to cocktail combination therapy for fever with thrombocytopenia syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE test result of the MM04 antibody purified in Example 2;

[0021] Figure 2 The results of the neutralization activity test of the MM04 antibody in Example 3;

[0022] Figure 3 The MM04 antibody affinity test results of Example 4;

[0023] Figure 4 This is the survival curve of the challenged mice in Example 6;

[0024] Figure 5 The change of serum viral load of mice on the 3rd day after the virus attack in Example 6;

[0025] Figure 6 The figure shows the change in serum viral load in mice on the 5th day after infection in Example 6. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0027] Meaning of English abbreviations:

[0028] SFTSV stands for fever with thrombocytopenia syndrome virus;

[0029] OD stands for optical density;

[0030] HRP stands for horseradish peroxidase;

[0031] TMB stands for 3 ,3',5,5'-tetramethylbenzidine;

[0032] PBS stands for phosphate-buffered saline;

[0033] ELISA stands for enzyme-linked immunosorbent assay;

[0034] FBS stands for fetal bovine serum;

[0035] IC 50 represents the half inhibitory concentration;

[0036] DMEM stands for Dulbecco's modified eagle medium;

[0037] PBST stands for phosphate-buffered saline containing Tween 20;

[0038] BLI stands for biofilm interferometry.

[0039] Example 1. Hybridoma cell preparation and monoclonal antibody screening

[0040] 1. Animal immunization

[0041] After the prepared SFTSV Gn protein was mixed with adjuvants, 6-8 week old SPF grade female C57 mice were immunized three times by intramuscular injection, with an interval of two weeks between each immunization and an immunization volume of 100 μL per mouse.

[0042] The serum titer was tested by indirect ELISA method, and the binding of antiserum to immunogen was tested at the same time. 450 -Blank greater than 1.0 is a qualified titer standard. Mice with high titers are preferred for hybridoma fusion.

[0043] SFTSV Gn antigen (prepared and stored in the laboratory) was coated onto the ELISA plate at 5 μg / mL at 4°C overnight. The cells were washed three times with 0.05% PBST, blocked in a 37°C wet box for 2 h with PBS containing 5% goat serum (purchased from Solarbio, Catalog No. SL038-10mL), and mouse serum was diluted with antibody diluent (purchased from Kangwei Century, Catalog No. CW23405), added to the wells, and incubated in a 37°C wet box for 1 h, washed three times with 0.05% PBST, and 50 µL of 4000-fold diluted HRP-labeled Goat Anti Mouse IgG (purchased from Prile, Catalog No. C1308) secondary antibody was added, and incubated in a 37°C wet box for 30 min, washed three times with 0.05% PBST, and 100 µL of TMB colorimetric solution (purchased from Polymer, Catalog No. MF142-01) was added; color was developed for 10 min at room temperature in the dark, and 50 µL of ELISA stop solution (purchased from Solarbio, Catalog No. C1058) was added to stop color development, and OD 450 Read the value.

[0044] 2. Hybridoma cell preparation and screening

[0045] Take the spleen cells of immunized mice, mix them with mouse myeloma cells at a ratio of 1:1, and fuse them using the electrofusion method to obtain hybridoma cells. Use HAT selection medium to change the cell medium once on the 6th and 8th day after fusion; take the supernatant of the main clone stage cells for screening and detection on the 10th day after cell fusion.

[0046] The hybridoma cell culture supernatant was tested by indirect ELISA to retain the antigen-binding clones. The test process is as follows:

[0047] SFTSV Gn antigen was coated onto ELISA plates at a concentration of 1 μg / mL (100 μL / well) at 4°C overnight. The cells were washed three times with 0.05% PBST, blocked in a 37°C wet box for 2 h with PBS containing 5% goat serum (purchased from Solarbio, Catalog No. SL038-10 mL), and the cell supernatant was diluted with antibody diluent (purchased from Kangwei Century, Catalog No. CW23405), added to the wells, and incubated in a 37°C wet box for 1 h, washed three times with 0.05% PBST, and 50 µL of 4000-fold diluted HRP-labeled Goat Anti Mouse IgG (purchased from Prile, Catalog No. C1308) secondary antibody was added, and incubated in a 37°C wet box for 30 min, washed three times with 0.05% PBST, and 100 µL TMB color development solution (purchased from Polymer, Catalog No. MF142-01) was added; color was developed for 10 min at room temperature in the dark, and 50 µL ELISA stop solution (purchased from Solarbio, Catalog No. C1058) was added to stop color development, and OD450 A total of 5 monoclonal antibody strains were screened and the corresponding cell supernatant OD 450 The values ​​were 2.46, 2.24, 3.00, 2.78, and 2.74, respectively; MM04 antibody was screened out from 5 monoclonal antibodies, and its corresponding cell supernatant OD 450 The value is 3.00.

[0048] Through screening, the present invention obtained a monoclonal antibody MM04 against the new Bunyavirus Gn protein. The sequencing results showed that the coding sequence of the heavy chain variable region of the monoclonal antibody MM04 was shown in SEQ ID NO:2, the amino acid sequence was shown in SEQ ID NO:1, the coding sequence of the heavy chain constant region was shown in SEQ ID NO:6, and the amino acid sequence was shown in SEQ ID NO:5; the coding sequence of the light chain variable region was shown in SEQ ID NO:4, the amino acid sequence was shown in SEQ ID NO:3, the coding sequence of the heavy chain constant region was shown in SEQ ID NO:8, and the amino acid sequence was shown in SEQ ID NO:7.

[0049] Example 2. Preparation of Antibodies

[0050] 1. Construction and purification of recombinant plasmid

[0051] (1) The heavy chain coding sequence of the MM04 antibody was cloned into the HindIII and BamH I restriction sites of pcDNA3.1 (+) (Invitrogen, V79020) by molecular biology techniques using molecular cloning methods. The resulting recombinant plasmid was sequenced and verified to be correct, and was named pcDNA3.1-MM04H, which is the heavy chain expression plasmid of the antibody MM04; wherein the N-terminus of the heavy chain coding sequence was added with a nucleic acid sequence as shown in SEQ ID NO.10 and an amino acid sequence as shown in SEQ ID NO.9 as a signal peptide.

[0052] (2) The light chain coding sequence of the MM04 antibody was cloned into the Hind III and BamH I restriction sites of pcDNA3.1 (+) using molecular biology techniques of molecular cloning methods. The resulting recombinant plasmid was sequenced and verified to be correct and named pcDNA3.1-MM04L, which is the light chain expression plasmid of the antibody MM04. The N-terminal of the light chain coding sequence is added with a nucleic acid sequence as shown in SEQ ID NO.12, and the amino acid sequence is a signal peptide as shown in SEQ ID NO.11.

[0053] 2. Antibody Expression

[0054] The antibody light and heavy chain recombinant plasmids pcDNA3.1-MM04H and pcDNA3.1-MM04L constructed in step 1 were transfected with the transfection reagent ExpiFectamine TM 293 transfection kit (Thermo Fisher, A14524) was used to co-transfect Expi 293F TM Cells (Thermo Fisher, A14527) were selected one day before transfection to obtain cells in good growth state at a density of 3×10 6 / mL of Expi 293F TM Cells were centrifuged and the supernatant was removed. TM Resuspend in cell culture medium (Thermo Fisher Scientific, A1435101) and adjust the cell density to 1.0×10 6 / mL, and divided into 30mL cell suspension / bottle, and cultured in a cell shaker at 37℃, 5% CO2, 125rpm; and expanded to 50 times the volume of 1.5L. On the day of transfection, transfection complex preparation: ExpiFectamine TM 293 transfection reagent diluted in Expi 293F TM Cell culture medium, gently mix, add 120μg of light and heavy chain plasmid DNA, mix well and leave at room temperature for 10min; then add the mixed solution to the prepared Expi 293F TM The cells were gently mixed and returned to the cell shaker for continued culture; cell activity was monitored 48 hours after transfection, and when the cell activity dropped to 80-85%, the culture supernatant was collected by centrifugation at 8,000 rpm for 10 min for purification.

[0055] 3. Antibody Purification

[0056] The cell expression supernatant was collected and filtered with a 0.45 μm filter membrane to remove cell debris and other impurities. The supernatant was purified using HiTrapMabSelectXtra chromatography filler (Cytia, 17526903) and loaded into an XK50 / 60 chromatography column (Cytia, 28988964). The elution peak when UV280>50 mAU was collected. The buffer was then replaced with a Hitrap Dedalting chromatography column, and the final buffer was 0.01 M citrate buffer (pH 7.0). Samples were taken for SDS-PAGE detection and concentration determination (concentration was 1.92 mg / mL). The samples were aliquoted and frozen at -80°C for later use. The SDS-PAGE detection results of the purified MM04 antibody are shown in the figure. Figure 1 shown.

[0057] 4. Quantification of Antibodies

[0058] After replacing the buffer, the antibody was sterilized by filtration with a 0.45 μm filter membrane, and the protein concentration was determined by a NanoDrop UV spectrophotometer, which was 1.68 mg / mL.

[0059] Example 3. Neutralization activity detection of MM04 antibody

[0060] Vero cells (frozen in the laboratory) in a medium containing 10% FBS (purchased from Gibco, catalog number 10099-141C) were mixed and counted and then plated into a 24-well plate (purchased from NEST, catalog number 702001-NEST) with 1×10 cells per well. 5Add 500 μL of DMEM medium containing 10% FBS (purchased from Gibco, catalog number D95701) to each well of cells. After the cells adhered for 12 hours, virus inoculation was performed. MM04 antibody was diluted to 2 µg / mL using DMEM, and then diluted 2-fold in sequence, with a total of 9 concentration gradients, and 2-3 replicates were set for each concentration gradient; SFTSV (100 FFU) was mixed with the diluted antibody, and the blank control group was added with antibody solvent; incubated at 37°C for 1.5 hours. After discarding the original culture medium in the plate, wash it 3 times with PBS (purchased from Gibco, catalog number C10010500BT), add the diluted virus solution to the 24-well plate, shake it slowly, and put it into the cell incubator, shake it once every 15 minutes, and aspirate the virus solution after incubation for 1.5 hours. Wash 3 times with 1 mL of PBS, cover with culture medium containing 1.25% methylcellulose (purchased from Calblochem, Catalog No. Aquacide II, Lct2891301, CAS 9004-32-4), and culture at 37°C for 3.5 days. After 3.5 days, add 1 mL of 3.7% formaldehyde (purchased from Sinopharm Group, Catalog No. A83030) directly to each well in the biosafety cabinet and shake horizontally for 30 min. Discard the formaldehyde and culture medium mixture, add 1-1.5 mL of formaldehyde to each well, shake well and aspirate, repeat 2-3 times, wash the methylcellulose clean, add 1-1.5 mL of formaldehyde to each well, shake horizontally for 30 min to fix the cells; wash the formaldehyde 4-5 times with 0.05% PBST (purchased from Solarbio, Catalog No. P1033). Add 200 μL of PBS containing 0.3% Triton-X100 (purchased from Solarbio, catalog number T8200) and 5% skim milk (purchased from BD, catalog number 232100) to each well, and shake gently on a horizontal shaker for 1.5 h. Discard the blocking permeabilization solution, add 200 μL of 0.05% PBST to each well for washing, shake gently on a horizontal shaker for 5 min, and repeat 3 times. Add Anti-mouse SFTSV NP antibody (prepared and stored in the laboratory) and incubate overnight at 4°C, wash three times with 0.05% PBST, and shake gently on a horizontal shaker for 5 min each time. Add goat anti-mouse IgG-HRP antibody (purchased from Prile, catalog number C1308) and incubate at room temperature for 1 hour, wash three times with 0.05% PBST, and shake gently on a horizontal shaker for 5 min each time. DAB color development solution (purchased from Tiangen, catalog number PA110) was used for color development. 200 μL per well, color development for 3-10 min. Discard the colorimetric solution; wash three times with 0.05% PBST, air-dry the plate, and count the spots. Inhibition rate = [(average number of spots in the blank control group - average number of spots in the experimental group) / average number of spots in the blank control group] × 100. Analyze using GraphPad prism 8 to calculate IC 50 , the results are as follows Figure 2 As shown, MM04 exhibited good in vitro neutralization activity, with an IC 50 The value is 0.16 µg / mL.

[0061] Example 4. BLI detection of antigen-antibody affinity

[0062] The Octet platform was used for antigen-antibody affinity testing. The AMC2 biosensor was selected, and the tip was hydrated in a buffer (PBS containing 0.02% Tween20 and 0.1% BSA) for 15 minutes. The MM04 antibody was diluted to 10µg / mL using the above buffer and immobilized on the AMC2 biosensor for 120s. SFTSV Gn was diluted in a 1.5-fold gradient (ranging from 46.7nM to 4.07 nM), bound for 180s, dissociated in the buffer for 180s, and the sensor was regenerated using 500 mM phosphate buffer. After the program was run, the Octet analysis software Data Analysis was used for data analysis and affinity constants were calculated.

[0063] The results are as follows Figure 3 As shown, the MM04 antibody exhibited a high affinity for SFTSV Gn, with a KD value of 13.3 pM.

[0064] Example 5. Antigenic epitopes recognized by detection antibodies

[0065] Before the experiment, Q Buffer (a kinetic buffer prepared with 1×PBS and a final concentration of 0.025% Tween20 and 0.2% BSA (IgG-Free, Protease-Free)) was used to dilute the MM04 antibody to 100 nM, Q Buffer was used to dilute the antigen (SFTSVGn) to 100 nM, and the test antibody was diluted to 300 nM, and 100 uL of antigen and 100 uL of test antibody were taken and mixed evenly. Then, the baseline was balanced for 30 s. After the balance was completed, the MFC probe (purchased from Gator, catalog number 160004), i.e., MouseFc (MFC) Probes, was used. It is a biosensor tool for binding mouse IgG antibody (Fc region) and mouse Fc fusion protein, to capture 100 nM MM04 antibody for 40 s; after 30 s of baseline balance, all binding sites on MFC were blocked with irrelevant antibodies for 10 min; MM04 was bound to the test antibody and antigen mixture for 5 min; and finally, the probe was regenerated. After the program was completed, the experimental data were analyzed using Analysis HT 12.2.0.2 software.

[0066] The results are shown in Table 1. MM04 does not compete with the prior art anti-SFTSV Gn antibodies JKL2, JKL8, JKL77, and JKL79 (CN 118373906A) at all and recognizes different epitopes. The percentages in Table 1 are the inhibition rates obtained by dividing the signal values ​​after the antigen is premixed with other antibodies and the signal values ​​without premixing with other antibodies.

[0067] Table 1. Detection results of antigen epitopes recognized by antibodies

[0068]

[0069] Example 6. Preventive protection results of MM04 antibody in mice

[0070] Five 6-week-old type I interferon receptor-deficient mice raised in an SPF environment were intraperitoneally inoculated with SFTSV HBMC16 strain, 20 FFU / mouse, and the body weight was recorded; 1 hour later, MM04 antibody was intraperitoneally injected at 0.05 mg / 20 g mouse body weight; the drug was administered once a day for the following 4 days, and the control group was given an equal amount of mouse IgG1 isotype control antibody; and the daily body weight changes and survival status were recorded; 100 μL of tail vein blood was collected on the 3rd, 5th and 7th days of infection, serum was separated, and viral RNA was extracted using a viral RNA extraction kit (purchased from Tiangen, catalog number DP315-R), and the specific steps were carried out according to the instructions of the kit; serum viral load was detected by probe qPCR. The system used was a one-step qPCR detection probe kit (purchased from Norvezan, catalog number Q225-C13), and the reaction system, procedure and primers are shown in Tables 2, 3 and 4.

[0071] Table 2. RT-qPCR reaction system

[0072]

[0073] Table 3. RT-qPCR reaction procedure

[0074]

[0075] Table 4. Probe-based qPCR primers

[0076]

[0077] The results are as follows Figure 4-Figure 6 As shown, Figure 4 This is the survival curve of mice. The final survival rate of mice in the drug-treated group was 100%, and all mice in the control group died; Figure 5The results of serum viral load on the third day after mice were infected with SFTSV were as follows: the serum viral load of the control group was 2409.9 Copies / μL, and the serum viral load of the treatment group was 168.3 Copies / μL, which was only 6.98% of the serum viral load of the control group; Figure 6 The results of serum viral load on the 5th day after mice were infected with SFTSV. The serum viral load of the control group was 6456.5 Copies / μL, and the serum viral load of the treatment group decreased to 181.9 Copies / μL, which was only 2.81% of the serum viral load of the control group. It can be seen that the viral load in the serum of mice in the drug-treated group was significantly lower than that of the control group on the 3rd and 5th days.

[0078] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the Gn protein of the new Bunyavirus, characterized in that: The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 of the monoclonal antibody are shown in SEQ ID NO:1 at positions 26-33, 51-58 and 97-106, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO:3 at positions 27-36, 54-56 and 93-101.

2. The monoclonal antibody against the new bunyavirus Gn protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

3. The monoclonal antibody against the new Bunyavirus Gn protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

7.

4. A polynucleotide encoding the heavy chain and light chain of the monoclonal antibody against the new bunyavirus Gn protein according to any one of claims 1 to 3, characterized in that: The polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2, and the polynucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

4.

5. The polynucleotide according to claim 4, characterized in that The sequence of the polynucleotide encoding the heavy chain constant region of the monoclonal antibody against the new Bunyavirus Gn protein is shown in SEQ ID NO:6, and the sequence of the polynucleotide encoding the light chain constant region of the monoclonal antibody against the new Bunyavirus Gn protein is shown in SEQ ID NO:

8.

6. An expression vector comprising the polynucleotide encoding the heavy chain and light chain of the monoclonal antibody against the new bunyavirus Gn protein according to claim 5.

7. A host cell containing the expression vector according to claim 6.

8. Use of the monoclonal antibody against the Gn protein of the new bunyavirus according to any one of claims 1 to 3 in the preparation of a test kit for fever with thrombocytopenia syndrome.

9. Use of the monoclonal antibody against the Gn protein of the new bunyavirus according to any one of claims 1 to 3 in the preparation of a medicament for preventing and / or treating fever with thrombocytopenia syndrome.

10. A pharmaceutical composition, characterized in that The composition contains the monoclonal antibody against the new bunyavirus Gn protein according to any one of claims 1 to 3.

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