Monoclonal antibodies against novel bunyavirus np antigens, uses and products thereof

Abstract

This invention provides a monoclonal antibody against the novel Bunyavirus NP antigen, its applications, and related products, belonging to the field of biotechnology. This invention utilizes the novel Bunyavirus NP antigen to immunize Balb / c mice. Mouse spleen cells are fused with myeloma cells, and highly specific hybridoma cells are obtained through specific high-throughput screening. Large amounts of mouse ascites are obtained through culture and re-immunization. High-purity, high-sensitivity, and high-specificity monoclonal antibodies against the novel Bunyavirus NP antigen are then obtained through multi-step separation and purification, providing the necessary raw materials for developing immunoassay strips for detecting the novel Bunyavirus NP antigen. The monoclonal antibody against the novel Bunyavirus NP antigen of this invention can be used for immunological detection such as immunoblotting and immunofluorescence. The obtained antibody has been verified to have good specific binding ability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody against the novel Bunyavirus NP antigen and its applications and products. Background Technology

[0002] Severe Fever with Thrombocytopenia Syndrome Bunyavirus (SFTSV) is a virus first reported and isolated in Henan Province, China. SFTSV is an enveloped negative-sense virus with a single RNA strand. The SFTSV genome consists of three single-stranded segments: a large segment (L), a medium segment (M), and a small segment (S). The L segment, containing 6368 nucleotides, encodes RNA-dependent RNA polymerase (RdRp) for viral replication, mediating viral RNA replication and mRNA synthesis. The M segment, containing 3378 nucleotides, contains viral envelope glycoproteins (glycoproteins NGn and CGc), which play a crucial role in viral assembly and viral particle formation. The S segment, consisting of 1744 nucleotides, uses two reverse reading frames to encode nucleocapsid proteins (N) and non-structural proteins (NSs).

[0003] Patients infected with the novel Bunyavirus (SFTSV) mainly experience systemic poisoning symptoms such as fever, chills, fatigue, and body aches; gastrointestinal symptoms such as vomiting, hematemesis, and diarrhea. Patients with rapidly deteriorating conditions quickly develop altered consciousness, respiratory and gastrointestinal bleeding, and in more severe cases, death due to respiratory failure, disseminated intravascular coagulation, and multiple organ failure. The pathogenic mechanism of SFTSV is still under investigation. Preliminary studies suggest that SFTSV infection may activate certain receptor tyrosine kinases, such as nerve growth factor receptor and vascular endothelial growth factor receptor, and complete viral replication and release through the RTKs-PI3K / Akt-mTOR pathway.

[0004] Monoclonal antibodies have become an important tool for infectious disease diagnosis due to their specificity and flexibility. Based on the high affinity and specificity of antibody-antigen binding, monoclonal antibodies against the novel Bunyavirus NP antigen can specifically bind to the NP protein and could be used as SFTSV-NP monoclonal antibodies for both prevention and treatment. However, to date, no monoclonal antibodies targeting the novel Bunyavirus NP protein are commercially available. Therefore, developing effective monoclonal antibodies against the novel Bunyavirus NP antigen is of great significance for disease prevention and treatment.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a monoclonal antibody against the novel Bunyavirus NP antigen to address at least one of the aforementioned problems.

[0007] A second objective of this invention is to provide biological materials.

[0008] A third objective of this invention is to provide the application of the above-mentioned monoclonal antibody against the novel Bunyavirus NP antigen in the preparation of novel Bunyavirus detection products.

[0009] A fourth objective of this invention is to provide a novel Bunyavirus marker.

[0010] The fifth objective of this invention is to provide a kit for detecting the new Bunyavirus.

[0011] To achieve the above objectives, the following solutions are proposed:

[0012] In a first aspect, the present invention provides a monoclonal antibody against the novel Bunyavirus NP antigen, wherein the variable region of the monoclonal antibody against the novel Bunyavirus NP antigen comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.14, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.5.

[0013] As a further technical solution, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.6.

[0014] As a further technical solution, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.7.

[0015] As a further technical solution, the monoclonal antibody against the novel Bunyavirus NP antigen is an IgG antibody.

[0016] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0017] a. Nucleotides, said nucleotides comprising nucleotide sequences encoding the monoclonal antibody against the novel Bunyavirus NP antigen;

[0018] b. A carrier carrying the nucleotides in a;

[0019] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing a monoclonal antibody against the novel Bunyavirus NP antigen.

[0020] Thirdly, the present invention provides the application of the above-mentioned monoclonal antibody against the novel Bunyavirus NP antigen in the preparation of novel Bunyavirus detection products.

[0021] Fourthly, the present invention provides a novel Bunyavirus marker, comprising the aforementioned monoclonal antibody against the novel Bunyavirus NP antigen and the marker;

[0022] The monoclonal antibody against the novel Bunyavirus NP antigen was conjugated with a marker.

[0023] As a further technical solution, the markers include enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0024] Fifthly, the present invention provides a kit for detecting the novel Bunyavirus, the kit comprising the monoclonal antibody against the novel Bunyavirus NP antigen or the novel Bunyavirus marker.

[0025] As a further technical solution, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit, or an immunoblotting detection kit.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention utilizes the novel Bunyavirus NP antigen to immunize Balb / c mice. Mouse spleen cells are then fused with myeloma cells. Highly specific hybridoma cells are obtained through specific high-throughput screening. Large quantities of mouse ascites are obtained through culture and re-immunization. Subsequent multi-step separation and purification yields a high-purity, high-sensitivity, and high-specificity monoclonal antibody against the novel Bunyavirus NP antigen, anti-SFTSV-NP-mab1. This provides the necessary raw material for developing immunoassay strips for detecting the novel Bunyavirus NP antigen. The monoclonal antibody anti-SFTSV-NP-mab1 of this invention can be used for immunological detection such as immunoblotting and immunofluorescence. The obtained antibody has been verified to have excellent specific binding ability. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 For indirect ELISA detection of antibody titer results;

[0030] Figure 2 This refers to the results of Western Blot analysis.

[0031] Figure 3 This is the result of indirect immunofluorescence detection. Detailed Implementation

[0032] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0034] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." These domains are usually the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held tightly together by the FRs to form the variable region. Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0035] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0036] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0037] In a first aspect, the present invention provides a monoclonal antibody against the novel Bunyavirus NP antigen, wherein the variable region of the monoclonal antibody against the novel Bunyavirus NP antigen comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.14, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.5. The amino acid sequences of SEQ ID NO.1-SEQ ID NO.5 and SEQ ID NO.14 are shown in Table 1.

[0038] Table 1

[0039] High-variable area sequence serial number CDR1-VH GFTFSNFG SEQ ID NO.1 CDR2-VH ISSGGTTI SEQ ID NO.2 CDR3-VH ARSDYFGMMDY SEQ ID NO.3 CDR1-VL QDIRNY SEQ ID NO.4 CDR2-VL YTS SEQ ID NO.14 CDR3-VL QQAHTLLT SEQ ID NO.5

[0040] In some alternative embodiments, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 6.

[0041] EVQLEESGGGLVQPGGSRKLSCAVSGFTFSNFGIHWVRQAPEKGLEWVAYISSGGTTIYYADTVKGRFTISRDNPMNTLFLQMTSLRSEDTAMYHCARSDYFGMMDYWGQGTSVTVSS (SEQ ID NO. 6).

[0042] In some alternative embodiments, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.7.

[0043] DIVITQTPSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLVYYTSRLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQAHTLLTFGAGTKLEIK (SEQ ID NO. 7).

[0044] In some alternative embodiments, the monoclonal antibody against the novel Bunyavirus NP antigen is an IgG antibody.

[0045] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0046] a. Nucleotides, said nucleotides comprising nucleotide sequences encoding the monoclonal antibody against the novel Bunyavirus NP antigen;

[0047] b. A carrier carrying the nucleotides in a;

[0048] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing a monoclonal antibody against the novel Bunyavirus NP antigen.

[0049] Thirdly, the present invention provides the application of the above-mentioned monoclonal antibody against the novel Bunyavirus NP antigen in the preparation of novel Bunyavirus detection products.

[0050] The monoclonal antibody against the new Bunyavirus NP antigen provided by this invention can specifically recognize the new Bunyavirus and therefore can be used for the detection of the new Bunyavirus.

[0051] Fourthly, the present invention provides a novel Bunyavirus marker, comprising the aforementioned monoclonal antibody against the novel Bunyavirus NP antigen and the marker;

[0052] The monoclonal antibody against the novel Bunyavirus NP antigen was conjugated with a marker.

[0053] This marker can be used for specific labeling of the new Bunyavirus.

[0054] In some alternative embodiments, the markers include, but are not limited to, enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0055] Fifthly, the present invention provides a kit for detecting the novel Bunyavirus, the kit comprising the monoclonal antibody against the novel Bunyavirus NP antigen or the novel Bunyavirus marker.

[0056] In some alternative implementations, the kit includes an immunochromatographic assay kit, an ELISA kit, an immunomagnetic microparticle assay kit, an immunofluorescence assay kit, or an immunoblotting assay kit.

[0057] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0058] Example 1: Preparation of monoclonal antibody anti-SFTSV-NP-mab1

[0059] I. Preparation of Novel Bunyavirus NP Antigen

[0060] The NP protein can package viral nucleic acid into ribonucleoprotein complexes (RNPs), preventing the viral nucleic acid from being degraded by exogenous nucleases or the host's immune system. Therefore, in this invention, the inventors chose the NP protein and the novel Bunyavirus to screen for specific antibodies.

[0061] The original sequence number of the selected new Bunyavirus NP antigen is: >NC_043452.1. After the sequence was optimized, the sequence synthesis was entrusted to Shanghai Sangon Biotech Co., Ltd.

[0062] 1. Plasmid Acquisition

[0063] The SFTSV-NP DNA sequence was codon-optimized and constructed into the expression vector pET-30a. The SFTSV-NP / pET-30a plasmid was transformed into DH5α competent cells using the cloning vector. Transformation was performed using the heat shock method, and PCR was used to confirm successful transformation. Successfully transformed single colonies were picked and amplified. μg-level recombinant plasmids were extracted using a plasmid miniprep kit, and the correctness of the final recombinant plasmid was confirmed by NdeI and XhoI double digestion and sequencing.

[0064] 2. Protein Expression and Acquisition

[0065] The propagated SFTSV-NP / pET30a plasmid was transformed into the expression strain BL21(DE3), plated on LB agar plates, and incubated overnight at 37°C with kanamycin at 50.0 ug / ml, inverted. The next day, single colonies were picked, shaken, and high-expression colonies were preserved at -80°C.

[0066] Take the SFTSV-NP / pET30a / BL21(DE3) strain seed culture from the -80℃ freezer, thaw it at room temperature, take 45ul and add it to a 500ml Erlenmeyer flask containing 180ml LB medium, then add 180ul of 50mg / ml Kanamycin, and incubate overnight at 37℃ and 200rpm on a constant temperature shaker.

[0067] Induction of expression: The activated bacterial strain was inoculated into 1000ml Erlenmeyer flasks containing 400ml LB medium at a volume ratio of 1:40, with 410ul of 50mg / ml Kanamycin added simultaneously. IPTG was added to each flask to a final concentration of 200ug / ml, and the flasks were incubated at 25℃ and 200rpm for 5 hours. The cells were then collected by centrifugation at 6000rpm and 10℃ for 15 minutes. After sonication to disrupt the cells, the cell pellet was discarded, yielding the supernatant containing the target protein.

[0068] 3. Purification of the target protein

[0069] Since the obtained recombinant protein carried a 6×His tag, it was purified by affinity using a nickel column with a His tag. The final target protein (HIS-SFTSV-NP antigen) was obtained and used to immunize mice.

[0070] II. Preparation of monoclonal antibody anti-SFTSV-NP-mab1.

[0071] Generally, healthy female Balb / c mice aged 6-8 weeks are selected and immunized according to a pre-specified immunization protocol. Using these mice as immunogens, BALB / c mice are immunized, and lymphocytes from the spleen of successfully immunized mice are extracted. These lymphocytes are then fused with mouse myeloma cells SP2 / 0 using cell fusion technology. After two rounds of subclonal selection, a hybridoma cell line stably secreting monoclonal antibodies against the novel Bunyavirus NP antigen is obtained, thus yielding the monoclonal antibody against the novel Bunyavirus NP antigen.

[0072] The purified HIS-SFTSV-NP antigen was expressed in prokaryotes and used to immunize experimental mice in stages.

[0073] The specific steps of animal immunization experiments include:

[0074] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0075] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at 80℃.

[0076] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (75 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl of immunogen dilution). The adjuvant was shaken well before use, and the 25 μl adjuvant was slowly added dropwise to the immunogen solution. After thorough mixing of the adjuvant and immunogen dilution, the two solutions were allowed to mix thoroughly for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0077] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in the table above (75 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the animal immunization experiment procedures.

[0078] 5. Subcutaneous injection at 2-week intervals: The experiment was designed as a 3-immunization method, but blood was collected from the eyeballs 7 days after each immunization injection. Part of the mouse supernatant was obtained by centrifugation and the serum titer was tested first. 7 days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at 80℃.

[0079] 6. Detect serum titer.

[0080] Indirect ELISA was performed to detect the serum titer of immunized mice using the novel Bunyavirus NP antigen as the coating antigen. Indirect ELISA method: 50 μl of coating antigen diluted with coating buffer (1 μg / ml) was added to each well of the ELISA plate. After coating overnight at 4°C, the plate was washed three times with washing buffer (PBST). 200 μl of blocking buffer (5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 hours. After washing, 50 μl of diluted serum was added to each well, and the plate was incubated at 37°C for 30 minutes. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated at 37°C for 30 minutes. After washing, 100 μl of substrate solution was added, and the plate was incubated at 37°C in the dark for 10 minutes. Finally, 50 μL of 2 mol / L H₂SO₄ was added to stop the reaction, and the A450 value was read using an ELISA reader. After three immunizations, the orbital blood titer of mice was >62500. Only 3 mice achieved a titer of over 50% at a ratio of 1:12500, indicating that fusion could be planned.

[0081] Immunosplenic cells were fused with myeloma cell line SP2 / 0. Fusion cells were screened using HAT selective medium (containing hypoxanthine, aminopterin, and thymine), and ELISA was performed to screen for positive results and subcloning. Ascites fluid was collected from the selected positive monoclonal cells, and antibodies were purified using a Protein A / G antibody purification column to obtain multiple monoclonal antibodies. The purified antibodies had an ELISA titer >1:128,000 and a purity >90%.

[0082] III. Detection of the binding activity of monoclonal antibodies to the new Bunyavirus NP antigen.

[0083] Indirect ELISA was used to test the titers of several obtained monoclonal antibodies. The results showed that SV03-6 exhibited excellent titers, achieving a working concentration of 15.625 ng / ml, and a detection limit of 1.95 ng / ml for the novel Bunyavirus NP protein. (See attached table for details.) Figure 1 .

[0084] Based on the high affinity of monoclonal antibodies, they can be used for the detection of the new Bunyavirus NP protein, such as in Western blotting and indirect immunofluorescence assays.

[0085] In this embodiment, the inventors infected Vero cells with a novel Bunyavirus. After plaques appeared, the infected cells were collected, lysed, and then used for Western blotting.

[0086] The results are attached. Figure 2 As shown, when Western blotting was performed using the monoclonal antibody SV03-6 as the primary antibody, the natural viral NP protein showed a clearly visible black band at 20–25 kDa, with no other non-specific bands.

[0087] In this embodiment, Vero cells in six-well plates were infected with neo Bunyavirus and fixed after viral-like lesions appeared. Indirect immunofluorescence detection was performed using monoclonal antibody SV03-6 as the primary antibody.

[0088] 1) Resuscitate Vero cells and, once the cells have filled the T25 culture flask, plate them into 24-well plates;

[0089] 2) After the cells have filled the 24-well plate, inoculate with the SFTSV strain. After adding the virus, transfer and shake well. Infect for 48-72 hours.

[0090] 3) Once obvious lesions appear in the cells, remove the culture medium, wash three times with 2% BSA, and fix with 80% pre-cooled acetone for 15 min;

[0091] 4) Remove the fixative, wash three times with 2% BSA, and incubate with Triton-100 for 10 min;

[0092] 5) Remove Triton-100, wash three times with 2% BSA, add pre-prepared monoclonal antibody SV03-6 as primary antibody, and incubate at 37°C for 1 hour or at 4°C overnight;

[0093] 6) Remove the primary antibody, wash three times with 2% BSA, and add the pre-prepared Alexa. 488 fluorescently labeled anti-mouse antibody was used as a secondary antibody and incubated at 37°C for 1 hour;

[0094] 7) Remove the secondary antibody, wash three times with 2% BSA, and add the pre-prepared DAPI solution for color development for 10 min;

[0095] 8) Remove the DAPI solution, wash three times with 2% BSA, and observe the fluorescence results under a fluorescence microscope. Record the observations by taking photos.

[0096] The results are attached. Figure 3 As shown, indirect immunofluorescence detection was performed using the monoclonal antibody SV03-6 as the primary antibody. Results showed that DAPI staining appeared deep blue, indicating the location of the Vero cell nucleus. Alexa... 488 fluorescence showed that the antibody tightly bound to the natural SFTSV virus. Combining the DAPI staining results with the fluorescent secondary antibody staining results yielded a MERGE image, which revealed the distribution of the monoclonal antibody SV03-6 binding to SFTSV-infected Vero cells. In summary, under green fluorescence, the tight binding of the monoclonal antibody to the natural viral NP protein was clearly visible.

[0097] Based on the product evaluation results, antibody SV03-6 was named anti-SFTSV-NP-mab1, and this antibody can be used for detection in the new Bunyavirus antigen detection kit.

[0098] IV. Sequence analysis of the V region (VH) of the heavy chain and the V region (VL) of the monoclonal antibody anti-SFTSV-NP-mab1.

[0099] Design primers to amplify the heavy chain V region (VH) and light chain V region (VL) genes.

[0100] The primers are as follows:

[0101] Heavy chain variable region forward primer (VH-FOR):

[0102] GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO. 10);

[0103] Heavy chain variable region reverse primer (VH-BACK):

[0104] GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO. 11);

[0105] Light chain variable region forward primer (VL-FOR):

[0106] CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC (SEQ ID NO. 12);

[0107] Light chain variable region reverse primer (VL-BACK):

[0108] GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG (SEQ ID NO. 13).

[0109] Take a hybridoma cell line of anti-SFTSV-NP-mab1 in the logarithmic growth phase (about 107 cells), extract total RNA from the cells according to the instructions of the Trizol RNA extraction kit, use the total RNA as a template to reverse transcribe and synthesize the first strand of cDNA, and use the above amplification product as a template to PCR amplify the VH / VL gene of the antibody.

[0110] The heavy chain VH (approximately 360 bp) and light chain VL (approximately 300 bp) fragments of anti-SFTSV-NP-mab1 were recovered and sent to the company for sequencing.

[0111] Then, the VH / VL gene sequences were analyzed:

[0112] The resulting sequence is as follows:

[0113] Variable region sequence of heavy chain: the blue marked region is the CDR region.

[0114] anti-SFTSV-NP-mab1 VH:354bp.

[0115] GAGGTTCAGCTGGAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCGGTCTCTGGATTCACTTTCAGTAACTTTGGAATCCACTGGGTTCGTCAGGCTCCAGAGAAGGGACTGGAGTGGGTCGCATACATTAGTAGTGGCGGCACTACCATCTACTA TGCAGACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATCCCATGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAAGACACGGCCATGTATCACTGTGCAAGATCAGATTACTTCGGTATGATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.8).

[0116] anti-SFTSV-NP-mab1 protein: 118aa.

[0117] EVQLEESGGGLVQPGGSRKLSCAVSGFTFSNFGIHWVRQAPEKGLEWVAYISSGGTTIYYADTVKGRFTISRDNPMNTLFLQMTSLRSEDTAMYHCARSDYFGMMDYWGQGTSVTVSS (SEQ ID NO. 6).

[0118] Variable region sequence of light chain: the blue marked area is the CDR region.

[0119] anti-SFTSV-NP-mab1 LVκ: 318bp VK3.

[0120] GACATTGTGATCACCCAGACTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGGAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGGTCTATTACACATCAAGATT ACATTCAGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGCTCATACGCTGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATAAAA(SEQ ID NO.9).

[0121] anti-SFTSV-NP-mab1 LVκprotein:106aa.

[0122] DIVITQTPSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLVYYTSRLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQAHTLLTFGAGTKLEIK (SEQ ID NO. 7).

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against a novel Bunyavirus NP antigen, characterized in that, The variable region of the monoclonal antibody against the novel Bunyavirus NP antigen includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the complementarity-determining region CDR2-VL of the amino acid sequence YTS, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody against the new Bunyavirus NP antigen according to claim 1, characterized in that, The variable region includes the heavy chain variable region VH of the amino acid sequence shown in SEQ ID NO.

6.

3. The monoclonal antibody against the novel Bunyavirus NP antigen according to claim 1, characterized in that, The variable region includes the light chain variable region VL of the amino acid sequence shown in SEQ ID NO.

7.

4. The monoclonal antibody against the novel Bunyavirus NP antigen according to claim 1, characterized in that, The monoclonal antibody against the novel Bunyavirus NP antigen is an IgG antibody.

5. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, wherein the nucleic acid is a nucleic acid sequence encoding a monoclonal antibody against the novel Bunyavirus NP antigen as described in any one of claims 1-4; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a monoclonal antibody against the novel Bunyavirus NP antigen as described in any one of claims 1 to 4.

6. The use of the monoclonal antibody against the novel Bunyavirus NP antigen as described in any one of claims 1-4 in the preparation of novel Bunyavirus detection products.

7. A novel Bunyavirus marker, characterized in that, Includes the monoclonal antibody and marker against the novel Bunyavirus NP antigen as described in any one of claims 1-4; The monoclonal antibody against the novel Bunyavirus NP antigen was conjugated with a marker.

8. The novel Bunyavirus marker according to claim 7, characterized in that, The label is selected from enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

9. A kit for detecting novel Bunyavirus, characterized in that, The kit comprises a monoclonal antibody against the novel Bunyavirus NP antigen as described in any one of claims 1-4 or a novel Bunyavirus marker as described in claim 7 or 8.

10. The reagent kit according to claim 9, characterized in that, The kit is selected from immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

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