CN110437333B - SFTSV inhibitors and uses thereof - Google Patents

Abstract

The present invention discloses an inhibitor of SFTSV which is a monoclonal antibody against SFTSV wherein the variable domain of the heavy chain comprises the sequence of CDR-H1 given in SEQ ID NO. 1, the sequence of CDR-H2 given in SEQ ID NO. 2 and the sequence of CDR-H3 given in SEQ ID NO. 3 and the variable domain of the light chain comprises the sequence of CDR-L1 given in SEQ ID NO. 4, the sequence of CDR-L2 given in SEQ ID NO. 5 and the sequence of CDR-L3 given in SEQ ID NO. 6. The invention also extends to therapeutic uses of the inhibitors, compositions and methods for detecting SFTSV.

Description

SFTSV inhibitors and uses thereof

Technical Field

The invention belongs to the field of molecular immunology, and relates to an SFTSV inhibitor and an application thereof.

Background

Fever with thrombocytopenia syndrome (SFTS) belongs to a novel tick borne hemorrhagic fever, is a new and natural epidemic-derived and acute infectious disease caused by infection of newly discovered and named bunyavirus phlebovirus, phlebovirus of phlebovirus family, namely fever with thrombocytopenia syndrome virus (SFTSV), is known as tick disease, and since 2009 is discovered in the eastern area of China, with the enhancement of monitoring intensity, confirmed cases are successively reported in many places, and Japanese, Korea, United states and Arabic Union Ministry and the like also have cases reported, and SFTS has already made a serious threat to the global human health.

Members of the order bunyaviridae are mostly transmitted by vector organisms such as ticks, mites, mosquitoes, mice, etc., causing regional or global epidemics. In China, Hantaan viruses (Hantaviridae) and Xinjiang hemorrhagic fever viruses (Nairoviridae) mainly cause local epidemics. In 2011, the chinese disease prevention and control center isolated SFTSV from blood of SFTS acute stage patients, identified the virus as a phlebovirus through gene sequence alignment and homology analysis, and the viral genome has been analyzed: consists of three single-stranded negative-strand RNA fragments, small (S), medium (M) and large (L), and is similar to other viruses of the order bunyaviridae, with sequences complementary to the 3 'and 5' ends of the viral genome. The S segment belongs to a double-sense RNA and mainly encodes a nucleoprotein NP and a non-structural protein NSS, and the L segment encodes an RNA-dependent RNA polymerase consisting of 2084 amino acids; the M segment encodes a membrane protein precursor with 1073 amino acids, and Gn and Gc glycoproteins formed by protease modification in host cells after translation mediate the whole process of virus infection of the host, are key antigen molecules for stimulating the host to generate neutralizing antibodies, and Gn and Gc become important targets for the research of SFTS vaccines at present.

SFTS patients usually have a history of tick bites, the typical manifestations after infection are acute, high fever is accompanied with systemic hypodynamia, headache and muscle and joint ache, and the typical clinical characteristics are that leucocyte and platelet are obviously reduced, transaminase is increased, serum lactate dehydrogenase is obviously increased, prothrombin time is prolonged, and electrolytes such as sodium, potassium, chlorine and the like are low. Most cases live in hilly areas, the first case is the middle-aged and elderly people with field work experience, the average fatality rate is about 10%, and the death cause is mainly multi-organ functional failure. The case is sporadic and can also cause household aggregation outbreak, SFTS has serious human transmission phenomenon, and SFTSV can be infected by blood or secretion of a patient.

Because the disease is a new natural epidemic infectious disease, the epidemic situation cannot disappear in a short time; at present, no effective vaccine is used for prevention, people infect SFTSV, no specific medicine is used clinically, and symptomatic treatment is mainly used; the initial symptoms of the disease are the same as those of the common influenza, patients are mostly concentrated in rural areas, the traffic is inconvenient, the health and medical level is weak, the disease condition is mostly developed into viremia and multi-organ failure after definite diagnosis, and only symptomatic treatment means can be adopted clinically at the moment. As a supplement to chemotherapy, antibody-mediated measures for preventing and treating viral infection have shown good effects, and the application prospect thereof is accepted by experts. The antibody is one of the most important antiviral immune mediators in human body, and the antibody molecule can kill and eliminate virus particles and infected cells by blocking the combination of the virus particles and receptors thereof, activating killer cells such as macrophages and NK cells, activating complement and other various mechanisms. The antibody preparation can neutralize a large amount of viruses in a patient body, reduce load, transform the disease condition, carry out emergency passive immunity on patients in close contact, such as accompanying and nursing staff, and prevent the second generation and third generation of infected persons.

Research shows that clinical use of virus-specific recovered human plasma can effectively neutralize virus, prevent the virus from diffusing in various organs in vivo, avoid lethal multiple organ failure, and play an important role in the outcome of the disease course of patients. However, not only is the source of polyclonal plasma limited, but clinical use is also limited by conditions such as poor quality control, mismatch of blood types of donor and recipient, potential infectious agents, etc. The murine monoclonal antibody is simple to prepare, has a clear treatment mechanism, but the heterogeneity of the murine monoclonal antibody hinders the application of the murine monoclonal antibody in a human body. However, human monoclonal antibodies are effective in overcoming the above problems.

At present, no anti-SFTSV antibody is on the market at home and abroad, so that detection and diagnosis and treatment articles based on the antibody with independent intellectual property rights are established and developed, and the method has important practical significance for the intervention of various related diseases.

Disclosure of Invention

The method comprises the steps of constructing a high-capacity human immune phage antibody library, screening a human single-chain antibody fragment (scFv) by taking the purified SFTSV-Gn protein as a target, and obtaining three human scFv antibody molecules which are named as 4-6, 2F6 and 1B 2. The three single-chain antibody fragments are subjected to full molecular gene construction by a molecular biology method, and are eukaryon expressed 4-6IgG1, 2F6IgG1 and 1B2IgG 1. Further research shows that the three monoclonal antibodies have stronger binding activity and neutralization effect on SFTS viruses.

Based on the above research, the present invention has the following protection objects:

the invention provides an SFTSV inhibitor which is a specific antibody aiming at an SFTSV protein. In a specific embodiment of the invention, the SFTSV inhibitor is a monoclonal antibody specific for the SFTSV protein.

The SFTSV inhibitors of the invention include:

(1) heavy chain CDR1 shown in SEQ ID NO. 1, heavy chain CDR2 shown in SEQ ID NO. 2, and heavy chain CDR3 shown in SEQ ID NO. 3; and/or

(2) Light chain CDR1 shown in SEQ ID NO. 4, light chain CDR2 shown in SEQ ID NO. 5, and light chain CDR3 shown in SEQ ID NO. 6.

As one aspect of the invention, the SFTSV inhibitors of the invention comprise:

(1) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 7; and/or

(2) And a light chain variable region having an amino acid sequence set forth in SEQ ID NO 8.

For convenience in explaining the functional variants of the inhibitors below, the SFTSV inhibitors described above are referred to as parent inhibitors.

The SFTSV inhibitors of the invention may be whole immunoglobulin molecules, which may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass. In a specific embodiment of the invention, the intact immunoglobulin molecule of the SFTSV inhibitor is of the IgG1 type.

The SFTSV inhibitors of the invention may also be antigen binding fragments, including but not limited to Fab fragments, Fab '-SH fragments, F (ab')²A fragment, an Fd fragment, an Fv fragment, a diabody, a single chain Fv (scfv), a single chain polypeptide comprising only one light chain variable region, a single chain polypeptide comprising three CDRs of a light chain variable region, a single chain polypeptide comprising only one heavy chain variable region, a single chain polypeptide comprising three CDRs of a heavy chain variable region, and a VHH. In particular embodiments of the invention, the SFTSV inhibitor may be an scFv.

The SFTSV inhibitors of the invention also include functional variants of the SFTSV inhibitors described previously, including, but not limited to, derivatives that have substantially similar primary structural sequences, but contain chemical and/or biochemical modifications in vitro or in vivo, e.g., not found in the parent inhibitor. Such modifications include phthalylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation, and the like. In other words, modifications in the amino acid and/or nucleotide sequence of the parent inhibitor do not significantly affect or alter the binding properties of the inhibitor encoded by or containing the nucleotide sequence, i.e., the inhibitor is still able to recognize and bind its target.

The functional variants may have conservative sequence modifications, including amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural as well as unnatural amino acids.

Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having similar structural or chemical properties. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chain amino acids (e.g., aspartic acid, glutamic acid), uncharged polar side chain amino acids (e.g., aspartic acid, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chain amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan). Those skilled in the art will appreciate that other amino acid residue family classifications besides the above-described families may also be used. In addition, a variant may have a non-conservative amino acid substitution, e.g., an amino acid is replaced with another amino acid residue having a different structure or chemical property. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted or deleted without abolishing immunological activity can be found using computer programs well known in the art.

Furthermore, functional variants may comprise a truncation of the amino acid sequence at the amino terminus or the carboxy terminus or both. The functional variants of the invention may have the same or different, higher or lower binding affinity than the parent inhibitor, but still bind to the SFTSV protein. Hereinafter, when the term "inhibitor" is used, it also covers functional variants of the inhibitor.

The functional variants also comprise modifications to hypervariable regions comprising amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Functional variants within the scope of the present invention have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, particularly at least about 95% to about 99%, and particularly at least about 97% to about 99% amino acid sequence homology with the parent inhibitor described herein.

Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally align amino acid sequences for comparison and to identify similar or identical amino acid residues. Functional variants can be obtained by altering the parent inhibitor or a portion thereof using common molecular biology methods known in the art, including but not limited to error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and heavy and/or light chain shuffling methods.

The present invention provides nucleic acid molecules encoding the SFTSV inhibitors described above. The nucleic acid molecule sequence encoding the heavy chain CDR1 is shown in SEQ ID NO. 9, the nucleic acid molecule sequence encoding the heavy chain CDR2 is shown in SEQ ID NO. 10, the nucleic acid molecule sequence encoding the heavy chain CDR3 is shown in SEQ ID NO. 11, and the nucleic acid molecule sequence encoding the heavy chain variable region is shown in SEQ ID NO. 12; the nucleic acid molecule sequence encoding light chain CDR1 is set forth in SEQ ID NO. 13, the nucleic acid molecule sequence encoding light chain CDR2 is set forth in SEQ ID NO. 14, the nucleic acid molecule sequence encoding light chain CDR3 is set forth in SEQ ID NO. 15, and the nucleic acid molecule sequence encoding light chain variable region is set forth in SEQ ID NO. 16.

The nucleic acid molecule may also comprise a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid molecule encoding an antibody comprising a heavy or light chain sequence.

Those skilled in the art will appreciate that functional variants of these nucleic acid molecules are also part of the present invention. A functional variant is a nucleic acid sequence that can be directly translated using standard genetic code to provide the same amino acid sequence as the sequence translated from the parent nucleic acid molecule.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, nucleic acid sequences encoding the inhibitors of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The nucleic acid sequence can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the sequences of the inhibitors of the invention by chemical synthesis.

The present invention also provides a recombinant vector comprising a nucleic acid molecule as described above, in addition to the nucleic acid molecule as described above, a regulatory sequence operably linked to the sequence of said nucleic acid molecule.

These recombinant vectors may be used to transform an appropriate host cell so that it can express the nucleic acid molecule or protein.

The vector includes a cloning vector or an expression vector. Cloning vectors are used to amplify nucleic acid molecules and expression vectors are used to express proteins encoded by nucleic acid molecules.

The invention also provides a recombinant cell comprising a nucleic acid molecule as described above or a vector as described above.

The recombinant cell can be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are Escherichia coli, Streptomyces; bacterial cells of salmonella typhimurium: fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS,293 cells, or Bowes melanoma cells.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the SFTSV inhibitor described above.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the inhibitors of the present invention without substantially reducing the effectiveness of the composition as is often the case.

Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

The compositions of the present invention may be formulated into various dosage forms as desired, and may be administered by a physician in a dosage amount beneficial to the patient, depending on such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like. Administration may be by injection or other therapeutic means, for example.

Drugs that may be used in combination with the pharmaceutical compositions of the present invention include other anti-SFTSV drugs.

The invention also provides an immunoconjugate comprising at least one inhibitor as described herein and further comprising at least one label-detectable moiety/substance. Detectable moieties/substances include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.

The labeling used to label the inhibitor for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, e.g., immunohistochemical staining of (tissue) samples, flow cytometry, laser scanning cytometry detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), bioassays (e.g., phagocytosis assays), western blot applications, and the like. Suitable labels are well known to those skilled in the art for detection/analysis/diagnostic techniques and/or methods known in the art.

In addition to the chemical generation of the immunoconjugate by direct or indirect (e.g. via a linker), the immunoconjugate may be generated as a fusion protein comprising the inhibitor of the invention and a suitable label. Fusion proteins can be produced by methods known in the art, e.g., recombinantly by constructing a nucleic acid molecule comprising an in-frame nucleotide sequence encoding an inhibitor and a nucleotide sequence encoding a suitable marker, and then expressing the nucleic acid molecule.

The invention also provides a test product for determining SFTSV in a sample, the product comprising an SFTSV inhibitor as described above.

Further, the product includes, but is not limited to, a detection reagent, a kit, a chip or a strip. All assay products capable of detecting SFTSV which include the inhibitors described above are included within the scope of the present invention.

The SFTSV inhibitors of the invention, which may also be referred to as anti-SFTSV antibodies, may be prepared by any of a variety of techniques. Typically, the antibody may be produced by cell culture techniques, including the production of monoclonal antibodies by conventional techniques, or by transfection of antibody genes, heavy and/or light chains into a suitable bacterial or mammalian cell host to allow for the production of the antibody, which may be recombinant. The term "transfection" of various forms is intended to include usually used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in various techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although the antibodies of the invention may be expressed in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, and most preferably in mammalian host cells, since such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody.

Exemplary mammalian host cells for expression of recombinant antibodies, the invention includes chinese hamster ovary (CHO cells), NS0 myeloma cells, COS cells, HEK 293T cells and SP2 cells used with a DHFR selectable marker. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is secreted into the medium in which the host cell is cultured, by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, to secrete the antibody into the medium in which the host cell is cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedure are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light and/or heavy chain of an antibody of the invention. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains, which is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the invention. In addition, bifunctional antibodies can be produced in which one heavy and one light chain is an antibody of the invention (i.e., binds to the SFTSV protein), and the other heavy and light chains are specific for an antigen other than the SFTSV protein.

The method of making monoclonal antibodies involves the preparation of immortalized cells, a cell line capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. The inhibitors or fragments and/or variants thereof as described above may be produced in an immunized animal.

Monoclonal antibodies can be isolated from the supernatant of growing hybridomas. In addition, various techniques can be employed to increase yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (e.g., a mouse). The monoclonal antibodies can then be harvested from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

The present invention provides a method of modulating SFTSV activity or levels comprising administering an SFTSV inhibitor as described above, or a pharmaceutical composition comprising an SFTSV inhibitor as described above, or a nucleic acid molecule encoding said SFTSV inhibitor as described above and a vector comprising said nucleic acid molecule.

The invention provides a method for preventing or treating SFTSV infection. The methods comprise administering an SFTSV inhibitor of the invention, or administering a pharmaceutical composition comprising an SFTSV inhibitor, or administering a composition comprising a nucleic acid molecule encoding an SFTSV inhibitor and a vector or cell comprising the nucleic acid molecule.

The invention also provides a method for detecting or measuring SFTSV in a sample. The method comprises contacting a sample with an SFTSV inhibitor of the invention.

The invention also provides application of the SFTSV inhibitor in preparation of products for detecting or measuring SFTSV.

The product comprises the SFTSV inhibitor described above; such products include, but are not limited to, detection reagents, kits, chips, or test strips. All products that include the SFTSV inhibitors described above that are capable of detecting SFTSV are included within the scope of the present invention.

The invention also provides the use of an SFTSV inhibitor as hereinbefore described in the preparation of an immunoconjugate as hereinbefore described.

The invention also provides the use of an SFTSV inhibitor as hereinbefore described in the preparation of a pharmaceutical composition as hereinbefore described.

The invention also provides the application of the SFTSV inhibitor in the preparation of the following medicaments:

(1) agents that modulate SFTSV activity or level;

(2) the application in the preparation of the medicament for neutralizing SFTSV virulence;

(3) the application in preparing the medicament for resisting SFTSV infection;

(4) the application of the compound in preparing a medicament for preventing or treating diseases caused by SFTSV infection.

The invention also provides the application of the pharmaceutical composition in the preparation of the following drugs:

(1) agents that modulate SFTSV activity or level;

(2) the application in the preparation of the medicament for neutralizing SFTSV virulence;

(3) the application in preparing the medicament for resisting SFTSV infection;

(4) the application of the compound in preparing a medicament for preventing or treating diseases caused by SFTSV infection.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population in which the individual antibodies comprised are identical except for a few naturally occurring mutations that may be present. The modifier "monoclonal" indicates only the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The term "administering" as used herein refers to contacting and/or delivering an SFTSV inhibitor to obtain a desired effect. SFTSV inhibitors can be administered to a subject in a variety of ways, including but not limited to oral, ocular, nasal, intravenous, topical, aerosol, suppository, and the like, and can be used in combination.

The term "effective amount" as used herein refers to a dosage of a drug that is effective over a desired period of time to achieve a desired dosage therapeutic result. An effective dose may be determined by one skilled in the art and may vary depending on the disease state, age, sex and weight of the individual, etc., the ability of the drug to elicit a desired response in the individual. The term as used herein also refers to reducing and/or inhibiting the function of an estrogen receptor, for example, in an animal, mammal, or human. A therapeutically effective amount may be administered in one or more administrations (e.g., the agent may be administered as a prophylactic treatment or therapeutically at any stage of disease progression, before or after symptoms, etc.), applications, or dosages, and is not intended to be limited to a particular formulation, combination, or route of administration. The number of administrations and the dosage depend on several factors, such as the target of treatment, the subject, etc., and can be easily determined by one skilled in the art.

The "sample" of the present invention may be a sample of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes or monocytes. The sample may be obtained directly from the patient or may be pretreated, for example, by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the sample.

Any cell type, tissue or body fluid may be used to obtain the sample. Such cell types, tissues and fluids may include tissue sections, such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (e.g., whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, bronchoalveolar lavage BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebrospinal fluid, sweat, nasal fluid, synovial fluid, menses, amniotic fluid, semen, and the like. Cell types and tissues may also include lymphatic fluids, gastrointestinal fluids, gynecological fluids, urine, peritoneal fluids, cerebrospinal fluids, fluids collected by vaginal irrigation or fluids collected by vaginal irrigation. The tissue or cell type may be provided by removing a cell sample from the animal, but may also be achieved by using cells previously isolated (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those with a history of treatment or outcome, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.

As used herein, "patient" refers to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates, monkeys, e.g., cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans).

"treating" as used herein describes reversing, alleviating or inhibiting the progression of the disease to which the term applies or one or more symptoms of the disease. Treatment may be performed in an acute or chronic manner. The term also refers to reducing the severity of a disease or symptom associated with a disease prior to having the disease.

As used herein, "prevention" includes preventing the onset of a disease or preventing symptoms associated with a disease.

Drawings

FIG. 1 is a diagram showing the results of PCR identification of V.kappa.gene;

FIG. 2 is a diagram showing the results of PCR identification of the Vlambda gene;

FIG. 3 is a diagram showing the results of PCR identification of VH genes;

FIG. 4 is a diagram showing the results of PCR identification of scFv genes;

FIG. 5 is a graph showing the results of identifying the binding specificity of an anti-SFTSV-Gn protein single-chain antibody using Phage-ELISA;

FIG. 6 is a graph showing the results of detection of antibody expression using SDS-PAGE;

FIG. 7 shows a fluorescence plot of antibody binding to virus;

FIG. 8 shows a fluorescence plot for detection of antibody microneutralization using indirect immunofluorescence;

FIG. 9 shows a graph of optimal dilution factor determination of HRP-labeled 4-5IgG 1;

FIG. 10 shows a graph investigating antibody competitive inhibition using ELISA experiments.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 screening of anti-SFTSV-Gn protein Single chain antibodies

1. Purification of JS-2010-014 Virus particles

1.1 materials

JS-2010-014, isolated from peripheral blood of Jiangsu patient in acute phase in 2010 by patent applicant.

1.2 methods and results

The virus was inoculated into Vero cells and then incubated at 37 ℃ with 5% CO₂The virus suspension is inactivated by 1:4000 β -propiolactone at 4 ℃ for 24h, cell debris is removed by low-speed centrifugation, the virus suspension is suspended by PBS after being super-separated for 2h, and is further purified by a molecular sieve chromatography technology, JS-2010-014 virus particles with higher purity can be obtained by the steps, and all virus operations are carried out in a biosafety level 2 (BSL-2) laboratory.

2. scFv humanized antibody library construction and anti-SFTSV-Gn protein single-chain antibody screening

2.1 materials

Primer: family-specific light chain (V κ and V λ), IgG heavy chain (VH) and overlap-PCR primers were designed according to the book Phage Display, with V κ 12, V λ 24, VH 6 and overlap-PCR 1 pairs.

V κ forward primer:

5’-GGGCCCAGGCGGCCGAGCTCCAGATGACCCAGTCTCC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGATGACYCAGTCTCC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGWTGACRCAGCTCC-3’。

v κ reverse primer:

5’-GGAAGATCTAGAGGAACCACCTTTGATYTCCACCTTGGTCCC-3’；

5’-GGAAGATCTAGAGGAACCACCTTTGATCTCCAGCTTGGTCCC-3’；

5’-GGAAGATCTAGAGGAACCACCTTTAATCTCCAGTCGTGTCCC-3’；

5’-GGAAGATCTAGAGGAACCACCTTTGATATCCACTTTGGTCCC-3’。

v λ forward primer:

5’-GGGCCCAGGCGGCCGAGCTCGTGBTGACGCAGCCGCCCTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCCTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGCCCTGACTCAGCCTCCCTCCGT-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAATCGCCCTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCATGCTGACTCAGCCCCACTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGGTGACYCAGGAGCCMTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCTTC-3’；

5’-GGGCCCAGGCGGCCGAGCTCGGGCAGACTCAGCAGCTCTC-3’。

v λ reverse primer:

5’-GGAAGATCTAGAGGAACCACCGCCTAGGACGGTCASCTTGGTS-3’；

5’-GGAAGATCTAGAGGAACCACCGCCTAAAATGATCAGCTGGGTT-3’；

5’-GGAAGATCTAGAGGAACCACCGCCGAGGACGGTCAGCTSGGTS-3’。

VH forward primer:

5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGGGCGGTGGCTCGGGC-3’；

5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGG-3’；

5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGTGGCGGTGGCTCGGGC-3’；

5’-GGTGGTTCCTCTGATCTTCCTCCTCGGTGGCGGTGGCTCGGGCG-3’；

5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGC-3’；

5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGTCGGGC-3’。

VH reverse primer:

5’-CCTGGCCGGCCTGGCCACTAGTGACCGATGGGCCCTTGGTGGAR-3’。

overlap-PCR forward primer:

5’-GAGGAGGAGGAGGAGGAGGCGGGGCCCAGGCGGCCGAGCTC-3’。

overlap-PCR reverse primer:

5’-GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCCACTAGTG-3’。

2.2 methods

2.2.1 isolation of peripheral blood lymphocytes and Total RNA extraction

Peripheral blood of 8 SFTS patients in convalescent period (with written consent of the ethical committee of the unit of the patent applicant and the relevant blood donors) was mixed with an equal amount of physiological saline, mononuclear cells were aspirated according to the instruction of lymphocyte separation solution, and RNA was extracted after three-time washing with physiological saline with reference to the instruction of total RNA extraction kit.

2.2.2 PCR amplification of antibody variable region genes

Mixing the extracted 8 parts of total RNA, and performing reverse transcription to obtain a first cDNA chain, wherein the reverse transcription conditions are as follows: 30min at 55 ℃, 5min at 85 ℃ and 30min at 4 ℃; then using cDNA as template to make PCR amplification of humanized antibody V kappa, V lambda and VH gene, and the PCR reaction condition is as follows: pre-denaturation at 94 deg.C for 10min, then at 94 deg.C for 20s, at 57 deg.C for 45s, at 72 deg.C for 1min, for 25 cycles, finally at 72 deg.C for 20min extension, gel electrophoresis and gel cutting, purification and recovery.

2.2.3 splicing of scFv genes

Mixing the purified V kappa gene fragment and the V lambda gene fragment in an equimolar way, then mixing the mixture with the VH gene fragment in an equivalent way, splicing the scFv gene by utilizing overlap-PCR, wherein the reaction conditions of the overlap-PCR are as follows: pre-denaturation at 94 deg.C for 10min, then at 94 deg.C for 20s, at 57 deg.C for 45s, at 72 deg.C for 1min, for 25 cycles, finally at 72 deg.C for 20min extension, gel electrophoresis and gel cutting, purification and recovery.

2.2.4 construction and quality identification of phage Single chain antibody library

The purified scFv gene and pComb3XSS plasmid are respectively subjected to sfII enzyme digestion, the target fragment obtained after the purification and recovery of the connecting gel is transferred into competent escherichia coli XL1-Blue, the competent escherichia coli XL1-Blue is added into 20mL of 2YT culture solution to be cultured for 45min at 37 ℃, and then the mixture is centrifuged, and the precipitate is smeared on a 2YT plate to be cultured overnight at 30 ℃. The following day, the lawn grown on the plates was collected in 2YT medium and cultured at 37 ℃ to an OD600 of 0.7. Adding the mixture to a final concentration of 1X 10⁹PFU/mL of helper phage VCSM 1337 ℃ for 45 min. Adding kanamycin with final concentration of 50 μ g/mL, culturing at 37 deg.C for 7 hr, centrifuging at 900g for 15min, discarding precipitate, adding 5 × PEG/NaCl into supernatant, mixing, and placing on ice for 3%h, centrifuging at 900g for 45min, suspending the precipitate in 2mL of PBS, filtering with a 0.45-micron filter membrane to obtain a filtrate, namely the human phage single-chain antibody library, and calculating the library capacity and diversity.

2.2.5 screening of anti-SFTSV-Gn protein-specific Single-chain antibodies

Taking 100 mu L of amplified phage library and solid-phase coated SFTSV-Gn protein to incubate together, carrying out 4 rounds of 'adsorption-elution-amplification' affinity screening, taking the 4 th round of eluent to infect escherichia coli XL1-Blue in logarithmic growth phase, coating a2 XYT culture plate, culturing overnight at 37 ℃, randomly selecting 200 single colonies, respectively inoculating a 96-hole deep-well plate (containing 100 mu g/mL ampicillin, 12.5 mu g/mL tetracycline and 1g/mL glucose), carrying out overnight shaking culture at 37 ℃, and carrying out 1:10 were separately inoculated into new 96-well deep-well plates (containing 100. mu.g/mL ampicillin and 12.5. mu.g/mL tetracycline) and incubated at 37 ℃ with shaking for 5h, and helper phage VCSM13 (final concentration 1X 10)⁹PFU/mL), incubation for 1h at 37 ℃, adding kanamycin (with the final concentration of 50 mug/mL) and shaking for culture at 30 ℃ overnight to prepare Phage single-chain antibody, coating an ELISA plate with 0.1 mug/hole SFTSV-Gn protein, using PBS buffer (containing 5g/mL skimmed milk powder) to dilute HRP-labeled anti-M13 antibody at a ratio of 1:2000 for Phage-ELISA identification and determination of OD450 value, determining to be Positive when Positive/Negative is more than or equal to 2.1, and sending the Positive cloned bacterial liquid to a marine company for sequencing.

2.3 results

2.3.1 identification of the genes for the humanized antibodies V.kappa.Vlambda.and VH

The 12V kappa gene primers were used to amplify 12 target fragments of about 350bp in size, as shown in FIG. 1, where M: DNAmarker; 1-12: PCR products; 13: and (5) negative control. The 24V lambda gene primers were used to amplify 24 target fragments of about 350bp in size, as shown in FIG. 2, where M: DNA marker; 1: negative control; 2-25: and (3) PCR products. 6 target fragments with the size of about 400bp are amplified by using 6 pairs of VH gene primers, and the result is shown in FIG. 3, wherein M: DNA marker; 1-6: PCR products; 7: and (5) negative control. The results were in agreement with expectations.

2.3.2 splicing of scFv antibody genes

The objective fragment of about 750bp is obtained by random splicing by using overlap-PCR, and the result is shown in FIG. 4, wherein M: DNAmarker; 1-3: (iii) an overlap-PCR product; 4: and (5) negative control. The results were in agreement with expectations.

2.3.4 screening of anti-SFTSV-Gn protein Single chain antibodies

The humanized SFTSV virus single-chain antibody library is subjected to 4 rounds of affinity screening by taking the SFTSV-Gn protein as an antigen, the anti-SFTSV-Gn protein specific single-chain antibody is selectively enriched, and the output/input ratio is improved by 30 times (Table 1). 200 Phage single clones were randomly picked for Phage-ELISA assay and OD450 values were determined, showing that 19 single chain antibodies specifically bind to SFTSV-Gn protein (FIG. 5). Sequencing analysis is carried out on 19 positive clone bacterial liquids to obtain 3scFv antibodies with different amino acid sequences, which are respectively named as 4-6, 2F6 and 1B 2; 4-6 antibody heavy and light chain variable region nucleic acid and protein sequences are shown below:

the amino acid sequence of the 4-6 antibody heavy chain variable region is shown as SEQ ID NO. 7, and the nucleic acid sequence is shown as SEQ ID NO. 12; the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO. 1, and the nucleic acid sequence is shown in SEQ ID NO. 9; the amino acid sequence of CDR2 in the heavy chain variable region is shown in SEQ ID NO. 2, and the nucleic acid sequence is shown in SEQ ID NO. 10; the amino acid sequence of CDR3 in the heavy chain variable region is shown in SEQ ID NO. 3, and the nucleic acid sequence is shown in SEQ ID NO. 11.

The amino acid sequence of the variable region of the 4-6 antibody light chain is shown as SEQ ID NO. 8, and the nucleic acid sequence is shown as SEQ ID NO. 16; the amino acid sequence of CDR1 in the variable region of the light chain is shown in SEQ ID NO. 4, and the nucleic acid sequence is shown in SEQ ID NO. 13; the amino acid sequence of CDR2 in the variable region of the light chain is shown in SEQ ID NO. 5, and the nucleic acid sequence is shown in SEQ ID NO. 14; the amino acid sequence of CDR3 in the variable region of the light chain is shown in SEQ ID NO. 6, and the nucleic acid sequence is shown in SEQ ID NO. 15.

TABLE 1 enrichment Effect of affinity screening against scFv antibodies specific for the SFTSV-Gn protein

Number of screening rounds	Titer of scFv (PFU)	Titer of produced scFv (PFU)	Input/output
				First wheel	5×1012	6×105	1.20×10-7
Second wheel	3.6×1012	4×105	1.11×10-7
				Third wheel	2.4×1012	8×105	3.33×10-7
Fourth wheel	2.1×1012	7.5×106	3.57×10-6

Example 2scFv antibody Whole molecular construction and eukaryotic expression

1. Construction of baculovirus recombinant plasmid

Three single-chain antibody plasmids of 4-6, 2F6 and 1B2 are taken as templates to respectively carry out PCR amplification on VH and VL genes, and each amplification system comprises the following contents: 0.1. mu.g of scfv plasmid, 60pmol of forward primer, 60pmol of reverse primer, 10. mu.L of 10 XPCR buffer, dNTP8μL，MgCl ₂6 μ L of Ex Taq 0.5 μ L, water was added to 100 μ L, reaction conditions were 94 ℃ 5min, 94 ℃ 15sec, 56 ℃ 30sec, 72 ℃ 1min, 30 cycles, 72 ℃ 10min, and a band of interest was recovered by gel recovery kit, the PCR products were digested with XhoI/NheI (VH), SacI/NheI (Vk), respectively, while the eukaryotic baculovirus expression vector plasmid pAc-K-CH3 was first digested with XhoI/NheI, after insertion of the VH fragment, it was digested with SacI/HindIII, and a VL fragment was inserted, HindIII was 10 μ g of DNA, XhoI/NheI or SacI/HindIII, each 10u, 10 × digestion buffer, 10 μ L, water was added to 100 μ L, 37 ℃ to digest 20 h.1% agarose gel electrophoresis, and a band of interest was cut, and gel recovery was recovered, the ligation product was 16 ℃ C, the ligation product was transformed into E.coli, and the plasmid was purified by PCR, cloning with the above VL plasmid cloning, PCR, cloning was performed overnight, and the plasmid was purified by the above plasmid was approximately 5 α.

2. Expression of whole antibody molecules in insect cells

The recombinant plasmid was transfected into 293T cells using Baculogold co-transfection kit from pharmingen. After culturing for 4-5 days at 27 ℃, observing infection conditions; the infection supernatant was collected after 5 days to obtain recombinant virus. Plaque purification and recombinant virus amplification 293T cells were infected with recombinant virus after transfer to 24-well plates. The supernatant was harvested after culturing at 27 ℃ for 4-5 days. Centrifugation was carried out at 2000rpm for 10min to remove cell debris. Loading the harvested protein expression supernatant to a proteinA affinity chromatography column of GEHealthcare after 0.45 mu L of microporous filter membrane; PBS was washed to baseline. Eluting with eluent (0.1mol/L Gly-HCl, pH2.7), and neutralizing with 1mol/L Tris to pH7.0; the purified samples were checked for purity by SDS-PAGE. The results are shown in FIG. 6, where 1: 4-6 monoclonal antibody non-reduction, 2: 4-6 monoclonal antibody reduction, 3: 1B2 monoclonal antibody non-reducing, 4: 1B2 monoclonal antibody reduction, 5: 2F6 mab non-reducing, 6: 2F6 mab reduction, 7: and (3) protein Marker, wherein the purified whole molecular antibodies are named 4-6IgG1, 2F6IgG1 and 1B2IgG 1.

Example 3 binding of Whole molecule antibodies to SFTSV

1. Method of producing a composite material

(1) Vero cells were inoculated in 24-well plates, cultured at 37 ℃ and inoculated with virus solutions per well when the fusion rate reached 90%.

(2) After two days, the virus solution was removed and 400. mu.l of 4% paraformaldehyde was added to each well and fixed at room temperature for 30 min.

(3) Paraformaldehyde is discarded, washed 3 times with PBS, and 400. mu.l of 0.2% Triton X-100 is added to permeate the cell membrane for 15min at room temperature.

(4) Triton X-100 was discarded, washed 3 times with PBS, blocked by addition of 5mg/ml BSA at room temperature for 30 min.

(5) The blocking solution was discarded, 300. mu.l of purified whole-molecule monoclonal antibody was added to each well, multiple wells were prepared, a blank control group in which no antibody was added and a positive control group of 4-5IgG1 monoclonal antibody (see patent document: humanized antibody against SFTSV, Notification No. CN102942629B) were prepared, and incubation was carried out at 37 ℃ for 1 hour.

(6) Remove primary antibody, add 500. mu.l PBST, wash 3 times at 500 rpm, shake 5 min.

(7) FITC-labeled anti-human IgG was added and incubated at 37 ℃ for 30min in the absence of light.

(8) The secondary antibody was removed, washed 3 times with 500. mu.l PBST, 500 rpm, and shaken for 5 min.

(9) And (4) observing under a fluorescence microscope.

2. Results

4-6IgG1, 2F6IgG1, and 1B2IgG1 have higher binding activity to SFTSV (FIG. 7).

Example 4 Whole molecule monoclonal antibody micro neutralization assay

1. Method of producing a composite material

(1) The Vero cells are inoculated on a 96-well plate, and the virus antibody compound is inoculated when the cell fusion rate reaches 90 percent.

(2) Equal amounts of 100TCID50 virus and antibody (100. mu.g/ml) were mixed well and incubated for 1h at 37 ℃.

(3) Discarding the culture solution, adding 100 μ l of the complex into each well, incubating at 37 deg.C for 2h, and setting multiple wells, and setting antibody-free group, virus-free group and 4-5IgG1 positive control group.

(4) The complex was discarded, 100. mu.l of the maintenance medium was added to each well, and the cells were incubated at 37 ℃ for 48 hours.

(5) The maintenance solution was removed and 200. mu.l of 4% paraformaldehyde was added to each well and fixed at room temperature for 30 min.

(6) Paraformaldehyde is discarded, washed 3 times with PBS, and 200. mu.l of 0.2% Triton X-100 is added to permeate the cell membrane for 15min at room temperature.

(7) Triton X-100 was discarded, washed 3 times with PBS, blocked by addition of 5mg/ml BSA at room temperature for 30 min.

(8) The blocking solution was discarded and primary anti-NP antibody was added at 100. mu.l per well and incubated at 37 ℃ for 1 h.

(9) Remove primary antibody, add 200. mu.l PBST to wash 3 times at 500 rpm, shake for 5 min.

(10) Mu.l of FITC-labeled anti-human secondary antibody was added and incubated at 37 ℃ for 30min in the absence of light.

(11) The secondary antibody was removed, washed 3 times with 500. mu.l PBST, 500 rpm, and shaken for 5 min.

(12) And (4) observing under a fluorescence microscope.

2. Results

The results of the micro-neutralization experiments show that 4-6IgG1, 2F6IgG1 and 1B2IgG1 all have a neutralizing effect on SFTSV (figure 8).

EXAMPLE 5 preliminary analysis of monoclonal antibody epitopes

1. Optimal dilution factor determination of HRP-labeled 4-5IgG1

Coating an enzyme label plate (100 ng/hole) with purified SFTSV-Gn protein, diluting HRP-labeled 4-5IgG1 from 1: 100-1: 600, incubating at the temperature of 100 mu L/hole for 1h, washing the plate, developing TMB, measuring an OD450 value, and selecting the dilution of the HRP-labeled 4-5IgG1 with the OD450 value of 1.0-1.5 as an optimal dilution multiple. As a result, it was found that according to 1: at 300 dilution, the OD450 (1.363) is between 1.0 and 1.5 (FIG. 9), so 1:300 is the optimal dilution factor of HRP-labeled 4-5IgG 1.

2. Preliminary analysis of 3 monoclonal antibody epitopes in competitive ELISA experiment

Working solutions prepared by using HRP labeled 4-5IgG1 according to a ratio of 1:300 are respectively used for carrying out competitive ELISA experiments on 4-5IgG1, 4-6IgG1, 2F6IgG1, 1B2IgG1 and anti-SFTSV-NP monoclonal antibodies (irrelevant antibodies are used as controls) by carrying out dilution in multiple ratios from 100ng, and reading OD450 to draw a competitive inhibition curve, wherein the experimental results show (figure 10) that the OD450 values are not obviously changed along with the dilution in multiple ratios of 4-6IgG1, 2F6IgG1 and 1B2IgG1, which indicates that 4-6IgG1, 2F6IgG1 and 1B2IgG1 are not mutually exclusive with 4-5IgG1 in combination with SFTSV-Gn protein and have no competitive relationship, and indicate that the epitope of 4-6IgG1, 2F6IgG1 and 1B2IgG1 is not identical with that 4-5IgG 1.

Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.

Sequence listing

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Claims (12)

1. An isolated SFTSV inhibitor, wherein the SFTSV inhibitor comprises:

(1) heavy chain CDR1 shown in SEQ ID NO. 1, heavy chain CDR2 shown in SEQ ID NO. 2, and heavy chain CDR3 shown in SEQ ID NO. 3; and

(2) light chain CDR1 shown in SEQ ID NO. 4, light chain CDR2 shown in SEQ ID NO. 5, and light chain CDR3 shown in SEQ ID NO. 6; the inhibitor is an antibody.

2. The SFTSV inhibitor of claim 1, wherein the SFTSV inhibitor comprises:

(1) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7; and

(2) and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

3. A nucleic acid molecule encoding the SFTSV inhibitor of claim 1.

4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule encoding the heavy chain CDR1 has the sequence shown in SEQ ID NO. 9, the nucleic acid molecule encoding the heavy chain CDR2 has the sequence shown in SEQ ID NO. 10, the nucleic acid molecule encoding the heavy chain CDR3 has the sequence shown in SEQ ID NO. 11, and the nucleic acid molecule encoding the heavy chain variable region has the sequence shown in SEQ ID NO. 12; the nucleic acid molecule sequence encoding light chain CDR1 is set forth in SEQ ID NO. 13, the nucleic acid molecule sequence encoding light chain CDR2 is set forth in SEQ ID NO. 14, the nucleic acid molecule sequence encoding light chain CDR3 is set forth in SEQ ID NO. 15, and the nucleic acid molecule sequence encoding light chain variable region is set forth in SEQ ID NO. 16.

5. A recombinant vector comprising the nucleic acid molecule of claim 3 or 4.

6. A recombinant cell comprising the nucleic acid molecule of claim 3 or 4 or the recombinant vector of claim 5.

7. A pharmaceutical composition comprising a therapeutically effective amount of the SFTSV inhibitor of claim 1 or 2.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

9. A detection product or immunoconjugate comprising the SFTSV inhibitor of claim 1 or 2.

10. A method of detecting the level of SFTSV for non-diagnostic purposes comprising contacting a sample containing SFTSV with an SFTSV inhibitor of claim 1 or 2.

11. The use of the SFTSV inhibitor of claim 1 or 2, comprising any one of:

(1) use in the preparation of a test product or immunoconjugate according to claim 9;

(2) use in the preparation of a pharmaceutical composition according to claim 7 or 8;

(3) use in the manufacture of a medicament for modulating the activity or level of SFTSV;

(4) the application in the preparation of the medicament for neutralizing SFTSV virulence;

(5) the application in preparing the medicament for resisting SFTSV infection;

(6) the application of the compound in preparing a medicament for preventing or treating diseases caused by SFTSV infection.

12. The pharmaceutical composition for use of claim 7 or 8, comprising any one of:

(1) use in the manufacture of a medicament for modulating the activity or level of SFTSV;

(2) the application in the preparation of the medicament for neutralizing SFTSV virulence;

(3) the application in preparing the medicament for resisting SFTSV infection;

(4) the application of the compound in preparing a medicament for preventing or treating diseases caused by SFTSV infection.

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