CN117736316A

CN117736316A - Monoclonal antibody against new bunyavirus NP antigen, application and product thereof

Info

Publication number: CN117736316A
Application number: CN202311750858.5A
Authority: CN
Inventors: 张严峻; 陈科达; 魏文涛; 李建华; 颜浩; 黄志伟
Original assignee: Sure Biotech (hangzhou) Ltd
Current assignee: Sure Biotech (hangzhou) Ltd
Priority date: 2023-12-19
Filing date: 2023-12-19
Publication date: 2024-03-22

Abstract

The invention provides a monoclonal antibody for resisting a novel bunyavirus NP antigen, and application and a product thereof, and relates to the technical field of biology. According to the invention, a novel bunyavirus NP antigen is used for immunizing a Balb/c mouse, spleen cells of the mouse are fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large number of mouse ascites are obtained through culture and re-immunization, and then a monoclonal antibody of the novel bunyavirus NP antigen with high purity, high sensitivity and high specificity is obtained through multi-step separation and purification, so that a required raw material is provided for developing an immune test strip for detecting the novel bunyavirus NP antigen. The monoclonal antibody of the anti-new bunyavirus NP antigen can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has good specific binding capacity through verification.

Description

Monoclonal antibody against new bunyavirus NP antigen, application and product thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody for resisting a novel bunyavirus NP antigen, and application and a product thereof.

Background

New bunyavirus, known as severe fever with thrombocytopenia syndrome virus (Severe Fever with Thrombocytopenia Syndrome Bunyavirus, SFTSV), was first reported and isolated in Henan province of China. SFTSV is an enveloped negative-sense virus with one RNA strand. Three single-stranded fragments constitute the SFTSV genome: large fragment (L), medium fragment (M) and small fragment (S). The L fragment contains 6368 nucleotides, encodes an RNA-dependent RNA polymerase (RdRp) for viral replication, mediating viral RNA replication and mRNA synthesis; the M fragment contains 3378 nucleotides and the viral envelope glycoproteins (glycoproteins N Gn and C Gc) play a key role in viral assembly and viral particle formation; the S fragment consists of 1744 nucleotides and encodes nucleocapsid protein (N) and nonstructural proteins (NSs) using two reverse reading frames.

The patients infected by the new bunyavirus mainly have systemic poisoning symptoms such as fever, aversion to cold, hypodynamia, systemic ache and the like; digestive tract symptoms such as emesis, hematemesis, diarrhea, etc. Patients with rapid deterioration of the disease state quickly suffer from disturbance of consciousness, and bleeding of the respiratory tract and digestive tract, and more serious patients die due to multiple organ failure such as respiratory failure, disseminated intravascular coagulation and the like. While the pathogenesis of SFTSV is still further explored, preliminary researches show that infection of SFTSV may activate partial receptor tyrosine kinase, such as nerve growth factor receptor, vascular endothelial growth factor receptor and the like, and complete replication and release of virus through RTKs-PI3K/Akt-mTOR pathway.

Monoclonal antibodies, by virtue of their specificity and flexibility, are important tools for the diagnosis of infectious diseases. Based on the high affinity and high specificity of the binding of the antibody to the antigen, the monoclonal antibody of the novel bunyavirus NP antigen can specifically bind to NP protein, and can be used as an SFTSV-NP monoclonal antibody for prevention and treatment. However, up to now, no monoclonal antibodies against the NP protein of the new bunyavirus have been marketed, and therefore, development of an effective monoclonal antibody against the NP antigen of the new bunyavirus has great significance for the prevention and treatment of diseases.

In view of this, the present invention has been made.

Disclosure of Invention

It is a first object of the present invention to provide a monoclonal antibody against the NP antigen of the new bunyavirus to solve at least one of the above problems.

A second object of the present invention is to provide a biomaterial.

The third object of the present invention is to provide the application of the monoclonal antibody against the NP antigen of the novel bunyavirus in preparing a novel bunyavirus detection product.

A fourth object of the present invention is to provide a novel bunyavirus marker.

A fifth object of the present invention is to provide a kit for detection of a new bunyavirus.

In order to achieve the above object, the following solutions are proposed:

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

As a further embodiment, the variable region comprises a heavy chain variable region VH having an amino acid sequence as set forth in SEQ ID No. 6.

As a further embodiment, the variable region comprises a light chain variable region VL having an amino acid sequence as shown in SEQ ID No. 7.

As a further technical scheme, the monoclonal antibody against the new bunyavirus NP antigen is an IgG antibody.

In a second aspect, the present invention provides a biomaterial selected from any one of a-c:

a. a nucleotide comprising a nucleotide sequence encoding the monoclonal antibody against the new bunyavirus NP antigen;

b. a vector carrying the nucleotide in a;

c. a cell carrying the nucleotide of a, or containing the vector of b, or expressing the monoclonal antibody against the novel bunyavirus NP antigen.

In a third aspect, the invention provides the use of the monoclonal antibody against the novel bunyavirus NP antigen in the preparation of a novel bunyavirus detection product.

In a fourth aspect, the present invention provides a novel bunyavirus marker comprising said monoclonal antibody against a novel bunyavirus NP antigen and a marker;

the monoclonal antibody against the novel bunyavirus NP antigen is conjugated to a label.

As a further technical scheme, the marker comprises enzyme, fluorescent molecular marker, fluorescent microsphere, color microsphere, colloidal gold, biotin or streptavidin.

In a fifth aspect, the invention provides a kit for detection of a new bunyavirus, said kit comprising said monoclonal antibody against a NP antigen of a new bunyavirus or said marker of a new bunyavirus.

As a further technical scheme, the kit comprises an immunochromatography detection kit, an ELISA detection kit, an immunomagnetic particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, a novel bunyavirus NP antigen is used for immunizing a Balb/c mouse, spleen cells of the mouse are fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large number of mouse ascites are obtained through culture and re-immunization, and then a monoclonal antibody anti-SFTSV-NP-mab1 of the novel bunyavirus NP antigen with high purity, high sensitivity and high specificity is obtained through multi-step separation and purification, so that a required raw material is provided for developing an immune test strip for detecting the novel bunyavirus NP antigen. The monoclonal antibody anti-SFTSV-NP-mab1 of the anti-novel bunyavirus NP antigen can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has good specific binding capacity through verification.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of indirect ELISA detection of antibody titers;

FIG. 2 shows the Western Blot detection results;

FIG. 3 shows the results of indirect immunofluorescence assay.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

By "variable region" or "variable domain" of an antibody is meant a domain of an antibody that recognizes and binds an antigen at the amino terminus of the heavy or light chain of the antibody, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) connected by 4 Frameworks (FR). The CDRs in each chain are held closely together by the FR to form the variable region, and typically the variable regions VL/VH for the heavy and light chains are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The term "vector" refers to a nucleic acid vehicle into which nucleotides can be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise 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.).

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

TABLE 1

Hypervariable region	Sequence(s)	Numbering device
			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

In some alternative embodiments, the variable region comprises a heavy chain variable region VH having the amino acid sequence shown in SEQ ID No. 6.

EVQLEESGGGLVQPGGSRKLSCAVSGFTFSNFGIHWVRQAPEKGLEWVAYISSGGTTIYYADTVKGRFTISRDNPMNTLFLQMTSLRSEDTAMYHCARSDYFGMMDYWGQGTSVTVSS(SEQ ID NO.6)。

In some alternative embodiments, the variable region comprises a light chain variable region VL having the amino acid sequence shown in SEQ ID No. 7.

DIVITQTPSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLVYYTSRLHSGVPSRFSGSGSGTDFSLTISNLEQEDIATYFCQQAHTLLTFGAGTKLEIK(SEQ ID NO.7)。

In some alternative embodiments, the monoclonal antibody against the neobunyavirus NP antigen is an IgG antibody.

b. a vector carrying the nucleotide in a;

The monoclonal antibody of the anti-new bunyavirus NP antigen provided by the invention can specifically identify the new bunyavirus, so that the monoclonal antibody can be used for detecting the new bunyavirus.

The marker can be used for specific labeling of the novel bunyavirus.

In some alternative embodiments, the label includes, but is not limited to, an enzyme, a fluorescent molecular label, a fluorescent microsphere, a color microsphere, colloidal gold, biotin, or streptavidin.

In some alternative embodiments, the kit comprises an immunochromatographic assay kit, an ELISA assay kit, an immunomagnetic microparticle assay kit, an immunofluorescent assay kit, or an immunoblotting assay kit.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

EXAMPLE 1 preparation of monoclonal antibody anti-SFTSV-NP-mab1

1. Preparation of novel bunyavirus NP antigen

The NP protein is capable of packaging viral nucleic acids into ribonucleoprotein complexes (RNPs) that prevent degradation of the viral nucleic acids by exogenous nucleases or the immune system of the host. Thus, in the present invention, the inventors selected NP protein and the new bunyavirus in nature to screen for specific antibodies.

Wherein, the original sequence gene number of the selected new bunyavirus NP antigen is: nc_043452.1, sequence synthesis work was delegated to Shanghai Bioengineering Co., ltd.

1. Acquisition of plasmids

The DNA sequence of SFTSV-NP is constructed into an expression vector pET-30a through codon optimization, the SFTSV-NP/pET-30a plasmid is transferred into a cloning vector DH5 alpha competent cell, and the transformation is carried out by adopting a thermal shock method and PCR to identify whether the transformation is successful. And (3) picking up successfully transformed single colony for amplification, extracting ug-level recombinant plasmid by a plasmid small extraction kit, and confirming the correctness of the final recombinant plasmid by NdeI and XhoI double-enzyme digestion and sequencing.

2. Expression and acquisition of proteins

The amplified SFTSV-NP/pET30a plasmid was transferred into the expression strain BL21 (DE 3), plated on LB plates, inverted at 37℃and cultured overnight. The next day, single colonies are picked, shaken, and the colonies with high expression are preserved at-80 ℃.

Taking out SFTSV-NP/pET30a/BL21 (DE 3) strain seed liquid from a refrigerator at the temperature of-80 ℃, taking 45ul after thawing at room temperature, adding the seed liquid into a 500ml conical flask filled with 180ml LB culture medium, adding 180ul of 50mg/ml Kanamycin, and culturing at the temperature of 37 ℃ by a constant temperature shaking table at 200rpm for overnight;

induction of expression: the culture activated strain is inoculated to the strain according to the volume ratio of 1:40 were inoculated into 1000ml Erlenmeyer flasks containing 400ml LB medium with a simultaneous addition of 410ul of 50mg/ml Kanamycin; adding IPTG with the final concentration of 200ug/ml into each bottle, and continuously culturing at 25 ℃ and 200rpm for 5 hours; and (3) centrifuging at 6000rpm and 10 ℃ for 15min, and collecting the thalli. And after the thalli are crushed by ultrasonic, discarding cell sediment, and obtaining the supernatant containing the target protein.

3. Purification of target proteins

Since the recombinant protein obtained carries 6×His tag, it was affinity purified using a His-tagged nickel column. Finally, the target protein (HIS-SFTSV-NP antigen) is obtained and used for immunizing mice.

2. Preparation of monoclonal antibody anti-SFTSV-NP-mab 1.

Generally, balb/c healthy female mice of 6-8 weeks of age are selected for immunization according to a pre-specified immunization schedule. As immunogen, BALB/c mice are immunized, spleen lymphocytes of the mice which are successfully immunized are extracted, the lymphocytes are fused with myeloma cells SP2/0 of the mice by a cell fusion technology, and hybridoma cell strains which stably secrete monoclonal antibodies against the NP antigen of the new bunyavirus are obtained after two rounds of subcloning and screening, so that the monoclonal antibodies against the NP antigen of the new bunyavirus are obtained.

The HIS-SFTSV-NP antigen after prokaryotic expression and purification is subjected to staged immunization on experimental mice.

The animal immunity experiment comprises the following specific steps:

1. balb/c mice with consistent weight and age average were randomly divided into 2 groups, with aluminum adjuvant (aluminum hydroxide adjuvant) and without aluminum adjuvant.

2. Before the start of the experiment, mice were each collected preimmune serum (preimmune serum was collected on the fifth day, blood was taken through eyeballs, and a proper amount of blood was taken to ensure the normal state of the mice), and the collected serum was stored at 80 ℃.

3. The preparation mode of the aluminum adjuvant (aluminum hydroxide adjuvant) group comprises the following steps: prior to immunization, each antigen was diluted to the corresponding dose (75 μg/mouse) in 75 μl PBS, respectively, and mixed with alum adjuvant (1 mg/mouse) at a volume antigen: adjuvant=3:1 (i.e., 25 μl adjuvant was added to 75 μl immunogen dilution); shaking the adjuvant before use, and slowly dripping the injected adjuvant (25 μl) into the immunogen solution; after the adjuvant and the immunogen dilution were thoroughly mixed, the two were thoroughly mixed for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent experiments were performed according to the experimental procedure of immunized animals.

4. Group without aluminum adjuvant: the antigens were diluted in 100 μl PBS at the corresponding doses (75 μg/mouse) in the above table, 100 μl of immunogen, followed by the following immunization animal experimental procedure.

5. Subcutaneous injections at 2 week intervals: the experiment is designed into a 3-time immunization mode, but blood is taken from eyeballs after 7 days of each immunization injection, partial mouse supernatant is obtained by a centrifugation method, serum titer is detected firstly, the heart takes blood to obtain the maximum blood volume after 7 days of the last immunization, the supernatant is obtained by centrifugation, and the supernatant is stored at 80 ℃;

6. serum titers were measured.

The serum titers of immunized mice were detected by indirect ELISA using the novel bunyavirus NP antigen as coating antigen. Indirect ELISA method: after 1. Mu.g/ml of coating antigen diluted with coating liquid was added to each well of the ELISA plate, and after coating was performed overnight at 4 ℃, the plate was washed 3 times with a washing liquid (PBST) (the same applies below), 200. Mu.l of blocking liquid (5% nonfat milk powder) was added to each well, and the plate was left in a 37℃incubator for 2 hours, after the washing was taken out, 50. Mu.l of diluted serum was added to each well, and reacted in a 37℃incubator for 30 minutes, after the washing, 50. Mu.l of goat anti-mouse IgG-HRP solution was added, and reacted in a 37℃incubator for 30 minutes. Washing, adding 100 μl of substrate solution, developing in a 37 ℃ incubator for 10min in dark, and finally adding 2mol/L H ₂ SO ₄ The reaction was stopped at 50. Mu.L and the A450 values were read on a microplate reader. The three immunization mode of mice has the three post-immune orbital blood titers>62500.3 mice can reach more than 50% at 1:12500, and fusion can be arranged.

The immune spleen cells are fused with myeloma cell line SP2/0 cells, the fused cells are screened by a HAT selection medium (the HAT selection medium contains hypoxanthine, aminopterin and thymine), and ELISA positive screening and subcloning are carried out on the fused cells; the screened positive monoclonal antibody is used for taking ascites, and the Protein A/G antibody purification column is used for purifying the antibody to obtain a plurality of monoclonal antibodies, wherein the ELISA titer of the purified antibodies is more than 1:128,000, and the purity is more than 90%.

3. The binding activity of the monoclonal antibody to recognize the new bunyavirus NP antigen was examined.

The results of indirect ELISA test on the obtained monoclonal antibodies show that the SV03-6 has excellent titer, the working concentration can reach 15.625ng/ml, the detection limit on the NP protein of the novel bunyavirus can reach 1.95ng/ml, and the results refer to figure 1.

The monoclonal antibody-based high affinity can be used for related detection of the NP protein of the new bunyavirus, such as Western Blot, indirect immunofluorescence and other experimental detection.

In this example, the inventors used the new bunyas natural virus to infect Vero cells, after plaques had occurred, the infected cells were collected, and the cells were lysed for Western Blot detection.

As shown in FIG. 2, western Blot detection was performed using monoclonal antibody SV03-6 as the primary antibody, and the naturally occurring viral NP protein exhibited a clearly visible black band at 20-25 kDa, with no other non-specific bands.

In this example, vero cells in six well plates were infected with the new bunyas natural virus and fixed after virus-like lesions had occurred. Indirect immunofluorescence assay with monoclonal antibody SV03-6 as primary antibody:

1) Resuscitating Vero cells, and plating the cells into a 24-well plate after the cells are fully paved with T25 culture flasks;

2) Inoculating the strain of SFTSV after the cells are fully paved on a 24-hole plate, adding the virus, translating and shaking uniformly, and infecting for 48-72 hours;

3) When obvious lesions appear on the cells, the culture medium is sucked off, the cells are washed three times by 2% BSA, and 80% precooled acetone is added for fixation for 15min;

4) Absorbing the fixing solution, washing with 2% BSA for three times, adding triton-100, and incubating for 10min;

5) Absorbing triton-100, washing with 2% BSA for three times, adding monoclonal antibody SV03-6 prepared in advance as primary antibody, and incubating at 37 ℃ for 1 hour or overnight at 4 ℃;

6) The primary antibody was blotted and washed with 2% BSATraversing, adding Alexa configured in advance488 fluorescence labeled anti-mouse antibody is used as secondary antibody, and incubated for 1 hour at 37 ℃;

7) Absorbing the secondary antibody, washing with 2% BSA for three times, and adding a DAPI solution prepared in advance for color development for 10min;

8) The DAPI solution was blotted off, washed three times with 2% bsa, and the fluorescence observed under a fluorescence microscope. And a photographing record is made.

The results are shown in FIG. 3, and indirect immunofluorescence detection was performed using monoclonal antibody SV03-6 as the primary antibody. The results show that: DAPI staining appeared dark blue, showing the position of the Vero cell nucleus, alexa488 fluorescence shows that the antibody is tightly combined with SFTSV natural virus, and the DAPI staining result chart and the fluorescent secondary antibody staining result chart are combined to obtain a MERGE result chart, so that the distribution of the binding of the monoclonal antibody SV03-6 and SFTSV infected Vero cells can be seen through the MERGE. In summary, the tight binding of the monoclonal antibody to the native viral NP protein can be clearly seen under green fluorescence.

The SV03-6 antibody is named as anti-SFTSV-NP-mab1 according to the evaluation result of the product, and can be used for detecting a new bunyavirus antigen detection kit.

4. Monoclonal antibody anti-SFTSV-NP-mab1 heavy chain V region (VH) and light chain V region (VL) sequence analysis.

Primers were designed to amplify the heavy chain V region (VH) and light chain V region (VL) genes.

The primers were as follows:

heavy chain variable region forward primer (VH-FOR):

GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG(SEQ ID NO.10)；

heavy chain variable region reverse primer (VH-BACK):

GGAAGGTGTGCACACCGCTGGAC(SEQ ID NO.11)；

light chain variable region forward primer (VL-FOR):

CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC(SEQ ID NO.12)；

light chain variable region reverse primer (VL-BACK):

GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG(SEQ ID NO.13)。

taking hybridoma cell lines (about 107 cells) in the logarithmic growth phase of anti-SFTSV-NP-mab1, extracting total RNA of the cells according to the instruction of a Trizol RNA extraction kit, performing reverse transcription by taking the total RNA as a template to synthesize a cDNA first strand, and performing PCR (polymerase chain reaction) amplification on the VH/VL genes of the antibody by taking the amplified products as templates.

The heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-SFTSV-NP-mab1 were recovered and sequenced by the company.

The VH/VL gene sequences were then analyzed:

the sequence obtained is as follows:

variable region sequence of heavy chain: the blue tag region is the CDR region.

anti-SFTSV-NP-mab1 VH:354bp。

GAGGTTCAGCTGGAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCGGTCTCTGGATTCACTTTCAGTAACTTTGGAATCCACTGGGTTCGTCAGGCTCCAGAGAAGGGACTGGAGTGGGTCGCATACATTAGTAGTGGCGGCACTACCATCTACTATGCAGACACTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATCCCATGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAAGACACGGCCATGTATCACTGTGCAAGATCAGATTACTTCGGTATGATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.8)。

anti-SFTSV-NP-mab1 protein：118aa。

Variable region sequence of light chain: the blue tag region is the CDR region.

anti-SFTSV-NP-mab1 LVκ：318bp VK3。

GACATTGTGATCACCCAGACTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGGAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGGTCTATTACACATCAAGATTACATTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTTTTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGCTCATACGCTGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATAAAA(SEQ ID NO.9)。

anti-SFTSV-NP-mab1 LVκprotein:106aa。

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A monoclonal antibody against a neobunyavirus NP antigen, wherein the variable region of the monoclonal antibody against a neobunyavirus NP antigen comprises: complementarity determining regions CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, complementarity determining regions CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, complementarity determining regions CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, complementarity determining regions CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, complementarity determining regions CDR2-VL having the amino acid sequence shown in SEQ ID NO.14 and complementarity determining regions CDR3-VL having the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody against the NP antigen of the new bunyavirus according to claim 1, wherein the variable region comprises a heavy chain variable region VH having the amino acid sequence shown in SEQ ID No. 6.

3. The monoclonal antibody against the NP antigen of the new bunyavirus according to claim 1, wherein the variable region comprises a light chain variable region VL having the amino acid sequence shown in SEQ ID No. 7.

4. The monoclonal antibody against the neobunyavirus NP antigen of claim 1, wherein the monoclonal antibody against the neobunyavirus NP antigen is an IgG antibody.

5. A biomaterial, characterized in that the biomaterial is selected from any one of a-c:

a. a nucleotide comprising a nucleotide sequence encoding a monoclonal antibody to the new bunyavirus NP antigen of any one of claims 1-4;

b. a vector carrying the nucleotide in a;

c. a cell carrying the nucleotide of a or containing the vector of b or expressing a monoclonal antibody against the new bunyavirus NP antigen of any one of claims 1-4.

6. Use of a monoclonal antibody against a new bunyavirus NP antigen as claimed in any one of claims 1-4 for the preparation of a new bunyavirus detection product.

7. A novel bunyavirus marker comprising the monoclonal antibody of any one of claims 1-4 against a novel bunyavirus NP antigen and a marker;

8. The novel bunyavirus marker of claim 7, wherein said marker comprises an enzyme, a fluorescent molecular marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

9. A kit for detection of a new bunyavirus, characterized in that the kit comprises a monoclonal antibody against a new bunyavirus NP antigen according to any one of claims 1-4 or a new bunyavirus marker according to claim 7 or 8.

10. The kit of claim 9, wherein the kit comprises an immunochromatographic assay kit, an ELISA assay kit, an immunomagnetic microparticle assay kit, an immunofluorescent assay kit, or an immunoblotting assay kit.