An antibody against parainfluenza virus type 3 NP protein and its application

Abstract

The present invention discloses an antibody against parainfluenza virus type 3 NP protein and its application. The present invention belongs to the field of biotechnology, and the technical problem to be solved is to provide an antibody or an antigen-binding fragment thereof that binds to parainfluenza virus type 3 NP protein. The antibody or antigen-binding fragment thereof provided by the present invention binds to parainfluenza virus type 3 NP protein and exhibits more excellent properties.

Description

Technical Field

[0001] The invention belongs to the field of cell biotechnology and immunology, and relates to an antibody against parainfluenza virus type 3 NP protein and application thereof. Background Art

[0002] Human Parainfluenza Virus Type 3 (HPIV-3) is an RNA virus that infects the respiratory tract and belongs to the genus Paramyxoviridae of the family Paramyxoviridae. It is one of the common pathogens that cause upper respiratory tract infections and is highly contagious. After infection with parainfluenza virus type 3, symptoms may include cough, runny nose, sore throat, fever, sneezing, headache, muscle pain, etc. Some people may develop pneumonia or laryngitis, especially infants under 6 months old are at high risk. If the infection is not treated in time, it may induce severe respiratory diseases.

[0003] The detection methods of parainfluenza virus type 3 mainly include virus isolation and culture, serological examination, virus antigen detection, virus nucleic acid detection, etc. Among them, serological examination helps the early diagnosis of parainfluenza virus by detecting specific IgM antibodies in acute serum, and viral nucleic acid detection (such as PCR-fluorescent probe method) is often used in clinical testing due to its rapid and sensitive characteristics. Although nucleic acid testing is widely used in clinical practice, for hospitalized patients or patients at risk of serious illness, if the nucleic acid test is negative but the patient is suspected of parainfluenza virus infection, a more sensitive detection method is still needed.

[0004] Since nucleic acid testing has false negatives and takes a long time, and has high requirements for site equipment and technicians, immunological testing can meet on-site testing and diagnosis because of its rapidity, which is more accurate and can assist clinical diagnosis. Based on this, the present invention has discovered an antibody against parainfluenza virus type 3 NP protein, and provides a method for detecting parainfluenza virus or parainfluenza virus NP protein. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide an antibody against parainfluenza virus type 3 NP protein, wherein the antibody has a high activity of binding to parainfluenza virus type 3 NP protein.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that has binding specificity to the parainfluenza type 3 NP protein.

[0008] Further, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:2; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:3.

[0009] In the present invention, the term "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signal transduction). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, and the like.

[0010] In the present invention, the term "antigen binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen binding fragment" includes aptamers, spiegelmers, and bivalent antibodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0011] In the present invention, a "light chain variable region (VL)" or "heavy chain variable region (VH)" consists of a "framework" region interspersed with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0012] In the present invention, "CDR" refers to the "complementarity determining region" within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy chain and the light chain, which are specifically designated as "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" for each variable region. "Framework" (FR) or "framework sequence" in the present invention refers to the sequence of the variable region minus the CDR. Because the precise definition of CDR sequences can be determined by different systems, the meaning of framework sequences is subject to correspondingly different descriptions. The three complementarity determining regions HCDR1, HCDR2, HCDR3 of the heavy chain variable region, the six CDRs of the complementarity determining regions LCDR1, LCDR2, LCDR3 of the light chain variable region (also divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the absence of designation of specific subregions as FR1, FR2, FR3 and FR4, the framework regions as indicated by others represent the FRs combined within the variable region of a single, naturally occurring immunoglobulin chain. As used in the present invention, FR represents one of the four subregions, and FR represents two or more of the four subregions that make up the framework region.

[0013] A variety of methods are known in the art for determining the CDR sequences in a given VH or VL amino acid sequence: Kabat complementarity determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the position of structural loops (Chothia et al., (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883), AbM CDRs are a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software, and "Contact" CDRs are based on analysis of available complex crystal structures. Depending on different CDR determination methods, the sequences in these CDRs may be different.

[0014] The CDR of the antibody of the present invention can be manually assessed to determine the boundaries according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0015] Those skilled in the art will appreciate that the classes of antibody heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), among which there are also some subclasses (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody, IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been fully characterized and the functional specificity conferred is also known. All immunoglobulin classes are within the scope of protection disclosed in the present invention.

[0016] Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions or insertions or any combination thereof compared to the amino acid sequence shown in SEQ ID NO:2.

[0017] Further, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or insertions or any combination thereof compared to the amino acid sequence shown in SEQ ID NO:3.

[0018] In the present invention, the term "identity" refers to the sequence similarity with the amino acid sequence used in the present invention. In order to determine the sequence identity, a sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine the appropriate parameters for comparison, including any algorithm required for achieving optimal comparison in the full-length sequence compared. Therefore, amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequence of the present invention are within the scope of protection of the present invention.

[0019] In the present invention, the antibody sequence obtained by modification also belongs to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to the change in the amino acid sequence resulting in the increase of one or more amino acids compared to the naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0020] Furthermore, the antibody includes a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody.

[0021] In the present invention, the term "monoclonal antibody", sometimes also referred to as "monoantibody" or Ab, refers to an immunoglobulin obtained from a pure line of cells, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. This characteristic contrasts with polyclonal antibody preparations, which generally include antibodies against different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0022] In the present invention, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light chain and the heavy chain are both from the variable regions of antibodies of one animal species (such as mice, rats, etc.), while the constant portion is homologous to the antibody sequence from another species (such as humans). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, while the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response during injection is lower than that of using antibodies with non-human constant regions.

[0023] In the present invention, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of heterologous antibodies when introduced into humans, while maintaining the complete antigen binding affinity and specificity of the antibody. Antibody humanization is the process of increasing the proportion of human sequences in heterologous antibodies. Preferably, more than 85%, preferably 90%, further preferably 95%, such as 95%, 96%, 97%, 98%, or even more than 99% of human genes are humanized antibodies. It is understood that the framework region or constant region in a humanized antibody may be partially or entirely encoded by human antibody genes. When less than 85% of human genes are present in the variable region of a humanized antibody, it is considered to be a chimeric antibody.

[0024] In the present invention, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, the murine antibody may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo.

[0025] Furthermore, the antigen-binding fragment includes Fab, Fab′, F(ab′)2, Fv or single-chain Fv antibody.

[0026] In the present invention, "Fab fragment" is composed of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0027] In the present invention, the "Fc region" contains two heavy chain fragments including the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domain.

[0028] In the present invention, the "Fv region" comprises the variable regions from both the heavy chain and the light chain, but lacks the constant region.

[0029] In the present invention, "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally speaking, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0030] A second aspect of the present invention provides an antibody derivative.

[0031] Furthermore, the antibody derivative comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention and an antibody marker; the antibody marker comprises an enzyme, fluorescein, isotope, biotin, colloidal gold, chemiluminescent agent and the like.

[0032] In the present invention, the term "antibody marker" mainly refers to combining a specific antibody with some chemical substances or biological molecules, so that the optical, radioactive or chemical properties of the antibody are changed, thereby facilitating the detection, location or purification of the target molecule.

[0033] In the present invention, the enzyme labeling in the antibody labeling refers to combining an enzyme with an antibody and utilizing the catalytic action of the enzyme to detect the target molecule. Commonly used enzymes include horseradish peroxidase (HRP), alkaline phosphatase (AP), etc. HRP-labeled antibodies are commonly used in experiments such as ELISA (enzyme-linked immunosorbent assay) to perform quantitative detection by reacting with a substrate to produce a color change.

[0034] In the present invention, the fluorescent label in the antibody marker refers to combining a fluorescent dye with an antibody and using a fluorescent signal to detect the target molecule. Commonly used fluorescent dyes include fluorescein, rhodamine, autofluorescent protein, etc. Among them, fluorescein isothiocyanate (FITC) is one of the most commonly used fluoresceins and is used in experiments such as immunohistochemistry.

[0035] In the present invention, isotope labeling refers to combining a radioactive isotope with an antibody and using radioactive decay to detect the target molecule. Commonly used radioactive isotopes include iodine-125, iodine-131, deuterium-3, etc.

[0036] In the present invention, biotin labeling refers to combining biotin with an antibody, and using the combination of biotin and avidin or streptavidin to detect the target molecule. Biotin can also be used to purify the target molecule. The biotin-avidin system has been widely used in antigen analysis due to its high affinity and cascade amplification effect.

[0037] In the present invention, colloidal gold labeling refers to combining colloidal gold particles with antibodies and using the surface plasmon resonance of colloidal gold to detect target molecules. Colloidal gold can also be used for electron microscopy and optical microscopy detection. Due to its high electron density characteristics, colloidal gold labeling can be seen as dark brown particles under a microscope, and the detection results are intuitive and easy to interpret.

[0038] In the present invention, chemiluminescent labeling refers to directly labeling antibodies with chemiluminescent agents and detecting them through chemiluminescent reactions. Commonly used labeling luminescent agents are luminols.

[0039] In the present invention, the antibody marker also includes a magnetic marker, which combines magnetic particles with antibodies and uses magnetic signals to detect, separate or enrich target molecules. Commonly used magnetic particles include superparamagnetic ferrites and magnetic ions.

[0040] A third aspect of the present invention provides a biomaterial.

[0041] Furthermore, the biological material includes nucleic acid molecules, vectors, and host cells.

[0042] Furthermore, the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0043] In the present invention, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form, linear or circular, or mixtures thereof (including hybrid molecules). Therefore, the nucleic acid according to the present invention includes DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA. The term "isolated" generally refers to being substantially free of components (such as viruses, nucleic acids or proteins) that are normally associated with or interact with the environment in which it naturally occurs. The term "isolated nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides or their analogs in an isolated form of any length that are separated from their natural environment or artificially synthesized, which have been separated from at least about 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides or other materials that are naturally found together with the nucleic acid molecule when the total nucleic acid is isolated from the source cell. In some embodiments, an isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules found in the natural environment of the nucleic acid that could interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic, or research uses.

[0044] Furthermore, the vector comprises the nucleic acid molecule described in the third aspect of the present invention.

[0045] In the present invention, a vector refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention or a precursor thereof, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0046] Furthermore, the host cell comprises the nucleic acid molecule or vector described in the third aspect of the present invention.

[0047] In the present invention, "recombinant host cell", "host cell", "cell", "cell line", "cell culture" and other higher-level eukaryotic cell lines representing microorganisms or cultured as single-cell entities can be used interchangeably, which can be used as, or have been used as, recipients of recombinant vectors or other transfected DNA, and include the original progeny of the transfected original cells. The vector is used to express the antibody against parainfluenza type 3 Np protein described in the present invention or the antibody or its functional fragment described in the present invention. Host cells useful in the present invention include, but are not limited to, eukaryotic host cells. Preferred eukaryotic host cells include, but are not limited to, mammalian host cells, insect host cells, fungal host cells, eukaryotic algae host cells, nematode host cells, protozoan host cells and fish host cells. Preferably, mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells or MDCK cells. Preferred fungal host cells include Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida. In a specific embodiment of the present invention, the host cell is a HEK293 cell.

[0048] The fourth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0049] Furthermore, the method comprises culturing the host cell according to the third aspect of the present invention under conditions suitable for expressing the antibody, and optionally the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell or culture medium.

[0050] The fifth aspect of the present invention provides a method for detecting parainfluenza virus or detecting parainfluenza virus NP protein.

[0051] Furthermore, the method comprises contacting the sample to be tested with the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, and observing the immune reaction between the sample to be tested and the antibody or antigen-binding fragment thereof to determine the expression level of the parainfluenza virus NP protein or the parainfluenza virus infection status in the sample.

[0052] In the present invention, the term "sample" is used in its broadest sense. In certain cases, samples include cells (such as bacteria, yeast and fungi), organisms, specimens or cultures obtained from any source, and biological and environmental samples. Among them, biological samples can be obtained from animals (including humans) and refer to biological materials or compositions found therein, including but not limited to bone marrow, interstitial fluid, urine, cerebrospinal fluid, nucleic acids, DNA, blood, serum, platelets, plasma, tissues, and purified or filtered forms thereof; environmental samples include but are not limited to water samples, food samples, air samples, microbial samples, animal cell samples, surface materials, soil samples, crystal samples and industrial samples.

[0053] A sixth aspect of the present invention provides an application.

[0054] Further, the application includes any of the following:

[0055] 1) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody derivative according to the second aspect of the present invention, or the biological material according to the third aspect of the present invention in the preparation of a product for detecting parainfluenza virus or parainfluenza virus NP protein;

[0056] 2) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody derivative according to the second aspect of the present invention, or the biomaterial according to the third aspect of the present invention in the preparation of a product for diagnosing parainfluenza virus infection;

[0057] 3) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the antibody derivative according to the second aspect of the present invention, or the biomaterial according to the third aspect of the present invention in the preparation of products for diseases related to parainfluenza virus infection.

[0058] Furthermore, the parainfluenza virus includes parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3 and parainfluenza virus type 4.

[0059] Furthermore, the parainfluenza virus is selected from parainfluenza virus type 3.

[0060] Furthermore, the diseases associated with parainfluenza virus infection include fever, runny nose, pharyngitis, laryngitis, cough, dyspnea, respiratory failure, and bronchial asthma.

[0061] When the sample is processed in a laboratory environment, the most reliable results may be obtained. For example, the sample can be obtained from the subject in a doctor's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide instant results in the clinician's office or allow the subject to be tested at home. In some cases, the demand for portable, prepackaged, disposable, and testable by the subject without assistance or guidance, etc., is more important than high accuracy. In many cases, especially with a physician follow-up, it may be sufficient to perform preliminary tests, even tests with reduced sensitivity and / or specificity. Therefore, the assay provided in the form of a product may involve detecting and measuring a relatively small amount of parainfluenza virus or parainfluenza virus Np protein in a subject sample to reduce the complexity and cost of the assay. Any form of sample assay capable of detecting sample parainfluenza virus Np protein as described in the present invention can be used. Typically, the assay will quantify antibodies in the sample to a certain extent, such as whether their concentration or amount is above or below a predetermined threshold. Such kits include colloidal gold immunoassay kits, chemiluminescent kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits (ELISA), fluorescent immunoassay kits or microfluidic chips. It should be noted that the detection method and kit described in the present invention are not only used to detect samples from patients (humans, animals), but also include non-diagnostic detection of various samples in the environment, including but not limited to water samples, food samples, air samples, microbial samples, animal cell samples, surface materials, soil samples, crystal samples, industrial samples, etc.

[0062] Beneficial effects of the present invention:

[0063] The present invention provides an antibody against parainfluenza virus type 3 NP protein, which has the characteristics of high purity and high expression level, has good binding activity with parainfluenza virus type 3 NP protein, can be used as an effective detection method for screening parainfluenza viruses, and has a high detection accuracy rate for parainfluenza virus NP protein infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the electrophoresis diagram for detecting the parainfluenza type 3 NP protein antibody 5B9;

[0065] Figure 2 This is the HPLC chart for detecting the parainfluenza type 3 NP protein antibody 5B9;

[0066] Figure 3 This is a graph showing the binding activity of the parainfluenza type 3 NP protein antibody 5B9 detected by ELISA. DETAILED DESCRIPTION

[0067] Before describing the inventive method, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments, and are not intended to be restrictive, because the scope of the present invention is limited only by the appended claims.

[0068] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0069] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar to or equivalent to the methods and materials described in the present invention can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned in the present invention are incorporated into the present invention by reference in their entirety.

[0070] In the present invention, the term "EC50", also known as "half effective concentration", refers to the concentration of a drug, antibody or toxic agent that induces a response of 50% between baseline and maximum after a specific exposure time.

[0071] In the present invention, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0072] In the present invention, the term "affinity" or "binding affinity" refers to the intrinsic binding ability that reflects the interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Binding affinity can be measured by common methods known in the art.

[0073] In the present invention, the term "comprising" or "including" means including the described elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, the situation consisting of the described elements, integers or steps is also covered.

[0074] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0075] Example 1 Screening of 5B9 Antibody

[0076] 1. Synthesis of recombinant parainfluenza type 3 NP protein

[0077] Synthesize the parainfluenza type 3 NP protein sequence and construct it into the pet28a vector; extract the transformation plasmid; transform Escherichia coli BL21 (DE3) and ferment it; harvest the cells by centrifugation, break and centrifuge, resuspend the protein precipitate in buffer, purify it with Ni column, and obtain the parainfluenza type 3 NP protein after concentration and replacement of buffer. The recombinant parainfluenza type 3 NP protein sequence is from NCBI, and the sequence is shown in Table 1.

[0078] Table 1 Sequence of recombinant parainfluenza type 3 NP protein

[0079]

[0080]

[0081] 2. Immunity

[0082] Immunize mice with Freund's complete adjuvant, 100ug per mouse, intraperitoneal injection, total dose 0.5ml / mouse, and perform the second immunization 3 weeks later; Freund's incomplete adjuvant was used from the second time onwards, dose 50ug / 0.5ml / mouse, and perform the third immunization 2 weeks later; prepare for cell fusion 10 days after the third injection;

[0083] SP2 / 0 fusion, take feeder cells, use at 105 / well, and plate 10 on the day before fusion. 5 / 100μl / well; Mouse immune spleen cells were taken and fused with the prepared myeloma cells using the fusion agent PEG, and then plated into a 96-cell culture plate that had been added with feeder cells, 100μl / well.

[0084] 3. Screening and subcloning

[0085] Positive wells were screened by ELISA detection method, and recombinant parainfluenza type 3 NP protein was plated overnight; the plate was washed, and skimmed milk powder was added for blocking at 37°C for 1 hour; the plate was washed, and 100ul of 96-well culture supernatant was added, and the plate was incubated at 37°C for 1 hour; the plate was washed, and HRP-labeled goat anti-mouse secondary antibody was added, and the plate was incubated at 37°C for 30 minutes; the plate was washed, and the color developing solution was added, and the color was developed for 10 minutes, and the stop solution was added to read the OD450 value; the high-expression cell lines were screened for subcloning.

[0086] 4. Sequence Retrieval

[0087] Collect cells, extract RNA, reverse transcribe, design primers, PCR, transform, pick clones, and send for sequencing.

[0088] 5. Results

[0089] The anti-parainfluenza type 3 NP protein antibody 5B9 was screened out, and its sequence is shown below:

[0090] VH (heavy chain variable region), sequence number SEQ ID NO: 2

[0091] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISSGGGTYYPDSVRGRFTISRDSVRNILYLQMSSLRSEDTAMYYCAREGEIYYDYDVKDYYAMDYWGQGTSVTVSS

[0092] Among them, the sequences of HCDR1, HCDR2 and HCDR3 are GFTFSSYA, ISSGGGT and AREGEIYYDYDVKDYYAMDY, and the sequence numbers are SEQ ID NO: 4, 5, and 6, respectively.

[0093] VL (light chain variable region), sequence number SEQ ID NO: 3

[0094] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQLSRELPYTFGGGTKLEIK

[0095] Among them, the sequences of LCDR1, LCDR2 and LCDR3 are KSVSTSGYSY, LAS, QLSRELPYT, and the sequence numbers are SEQ ID NO: 7, 8, 9, respectively.

[0096] Example 25 Functional study of B9 antibody

[0097] 1. Antibody Expression

[0098] After HEK293 transient transfection and PEI transfection, the expression level of 5B9 antibody obtained was 225 mg / L.

[0099] 2. Physical and chemical properties of antibodies

[0100] 2.1 Gel electrophoresis to detect the purity of 5B9 antibody

[0101] The results are as follows Figure 1 As shown, the bands from left to right are marker and reduced bands respectively; the detection purity of 5B9 antibody is greater than 95%.

[0102] 2.2 HPLC detection of 5B9 antibody purity

[0103] The results are as follows Figure 2 As shown, the detected purity of 5B9 antibody was greater than 95%.

[0104] 3. Antibody binding activity detection

[0105] Coat NP protein, add antibody, add secondary antibody, develop color, read, and analyze data.

[0106] Binding activity results such as Figure 3 As shown, the 5B9 antibody can specifically bind to the parainfluenza virus type 3 NP protein with an EC50 of 5.65 ng / ml.

[0107] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity to parainfluenza type 3 NP protein, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:2; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:3; and the CDR sequence is determined by any one of the IMGT, Chothia, Kabat and AbM schemes.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

3.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2; The light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

3.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region comprises a sequence having one or more amino acid substitutions, deletions or insertions or any combination thereof compared to the amino acid sequence shown in SEQ ID NO: 2; The light chain variable region comprises a sequence having one or more amino acid substitutions, deletions or insertions or any combination thereof compared to the amino acid sequence shown in SEQ ID NO:

3.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies or mouse antibodies; The antigen-binding fragment includes Fab, Fab', F(ab')2, Fv or single-chain Fv antibody.

6. An antibody derivative, characterized in that: The antibody derivative comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and an antibody marker; the antibody marker comprises an enzyme, fluorescein, isotope, biotin, colloidal gold, or a chemiluminescent agent.

7. A biomaterial, characterized in that: It includes: 1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; 2) A vector comprising the nucleic acid molecule described in 1); 3) A host cell comprising the nucleic acid molecule described in 1) or the vector described in 2).

8. A method for preparing the antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The method comprises culturing the host cell of claim 7 under conditions suitable for expressing the antibody.

9. The method according to claim 8, characterized in that The method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell or culture medium.

10. A method for detecting parainfluenza virus type 3 NP protein in a sample for non-diagnostic purposes in vitro, characterized in that: The method comprises contacting the sample to be tested with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, observing the immune reaction between the sample to be tested and the antibody or antigen-binding fragment thereof to determine the expression level of the parainfluenza virus type 3 NP protein in the sample.

11. An application, characterized in that: The application includes any of the following: 1) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody derivative according to claim 6, or the biological material according to any one of claims 7 to 8 in the preparation of a product for detecting parainfluenza virus type 3 and / or parainfluenza virus type 3 NP protein; 2) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody derivative according to claim 6, or the biological material according to any one of claims 7 to 8 in the preparation of a product for diagnosing infection with parainfluenza virus type 3 and / or parainfluenza virus type 3 NP protein.

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