Parainfluenza type 3 NP protein antibody 6D3

Abstract

The present invention discloses an antibody and an antigen-binding fragment thereof to the NP protein of human parainfluenza virus type 3, comprising HCDR1‑3 of the heavy chain variable region and LCDR1‑3 of the light chain variable region; discloses an antibody derivative, a nucleic acid molecule encoding the antibody and the antigen-binding fragment thereof, discloses a method for detecting a virus or protein with the antibody, a method for preparing the antibody, and the application of the antibody and the antigen-binding fragment thereof in preparing a product. The present invention provides an antibody with high specificity and high sensitivity against the NP protein of HPIV‑3, which has broad application prospects and practical value.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to an antibody against human parainfluenza virus type 3 NP protein. Background Art

[0002] Human parainfluenza virus (HPIV) is a type of virus that can cause respiratory tract infections. It can cause severe recurrent infections of the lower respiratory tract, especially in children, the elderly and immunocompromised people. Its pathogenicity is second only to respiratory syncytial virus (RSV). More than half of the cases are caused by human parainfluenza virus type 3, which poses a great threat to the life and health of newborns. From a serological point of view, human parainfluenza virus can be divided into 4 types (types 1-4), of which type 4 is divided into two subtypes, a and b. In 2018, the International Committee on Classification of Virology classified HPIV-1 and HPIV-3 as the genus Respirovirus of the family Paramyxoviridae, and HPIV-2 and HPIV-4 as the genus Mumps virus of the family Paramyxoviridae. Among them, HPIV-3 has the highest infection rate and causes the heaviest disease burden. There is currently no effective vaccine to prevent infection with human parainfluenza virus.

[0003] Human parainfluenza virus type 3 mainly infects the distal airways, usually causing pneumonia and bronchiolitis. Serological monitoring shows that 90% to 100% of children aged 5 years and above have antibodies against human parainfluenza virus type 3. There are no vaccines and drugs against HPIV-3 on the market at home and abroad. Given that this virus is more likely to cause serious infections in children under 5 years old, it is particularly necessary to develop targeted vaccines and drugs.

[0004] The nucleoprotein (NP) of influenza virus and parainfluenza virus is the main component of the viral nucleocapsid and is involved in the assembly of viral ribonucleoprotein vRNP, RNA replication and viral particle assembly. NP protein is the main antigenic protein of parainfluenza virus, has multiple functions, is conservative in structure, and has certain homology between different types of parainfluenza viruses. This makes it one of the hot targets for anti-influenza virus and parainfluenza virus drug research. The current viral antigen detection lacks highly sensitive and highly specific antibodies. The development of antibodies against human parainfluenza virus type 3 NP protein plays an important role in the preparation of products for detecting viruses or proteins. It can achieve early detection, diagnosis and timely treatment, and can effectively prevent and reduce diseases and even deaths caused by severe infection of human parainfluenza virus type 3 in infected people (especially children under 5 years old). 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 human parainfluenza virus type 3 NP protein and an antigenic fragment thereof.

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

[0007] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof to human parainfluenza virus type 3 NP protein, wherein the antibody comprises HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 1; and the antibody comprises LCDR1-LCDR3 of the light chain variable region as shown in SEQ ID NO: 2.

[0008] Furthermore, the antibody includes a monoclonal antibody, a polyclonal antibody, a humanized antibody, a mouse antibody, a chimeric antibody or a bivalent antibody.

[0009] The HCDR of the heavy chain variable region and the LCDR of the light chain variable region may be numbered in the order of IMGT, Chothia, and Kabat.

[0010] Furthermore, the amino acid sequences of HCDR1-HCDR3 of the heavy chain variable region are shown in SEQ ID NOs:3-5; the amino acid sequences of LCDR1-LCDR3 of the light chain variable region are shown in SEQ ID NOs:6-8, wherein the CDR numbering is according to the IMGT numbering scheme.

[0011] The second aspect of the present invention provides an antibody derivative, comprising the antibody and antigen-binding fragment thereof according to the first aspect of the present invention and a detectable marker bound to the antibody.

[0012] The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0013] Furthermore, the detectable marker is an enzyme.

[0014] Furthermore, the detectable marker is horseradish peroxidase.

[0015] The method of binding to the antibody is at least one of radioisotope labeling, colloidal gold labeling, fluorescein labeling, biotinylation labeling, chemiluminescence labeling, electron-dense labeling, and enzyme labeling.

[0016] Furthermore, the method of binding to the antibody is through enzyme labeling.

[0017] The third aspect of the present invention provides a nucleic acid molecule, a vector comprising the nucleic acid molecule, or a recombinant host cell comprising the nucleic acid molecule or the vector, wherein the nucleic acid molecule comprises a protein capable of encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0018] The fourth aspect of the present invention provides a method for detecting parainfluenza virus type 3 NP protein or detecting parainfluenza virus, using the antibody or antigen-binding fragment thereof described in the first aspect of the present invention and / or the antibody derivative described in the second aspect of the present invention to detect human parainfluenza virus type 3 NP protein or parainfluenza virus.

[0019] The fifth 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, wherein the method comprises artificial synthesis or preparation by genetic engineering technology.

[0020] Furthermore, the preparation by genetic engineering technology refers to culturing a recombinant host cell containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect of the present invention under conditions suitable for expressing the antibody.

[0021] The sixth aspect of the present invention provides a product for detecting human parainfluenza virus type 3 NP protein, the product comprising a first antibody, the first antibody comprising: the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the antibody derivative according to the second aspect of the present invention.

[0022] Furthermore, the product also includes a second antibody, a solid phase carrier loaded with the first antibody and / or the second antibody, an eluent, and a color developing solution.

[0023] Furthermore, the second antibody is a labeled antibody to which the detectable marker described in the second aspect of the present invention is linked.

[0024] Furthermore, the second antibody is a goat anti-mouse IgG antibody labeled with horseradish peroxidase.

[0025] Furthermore, the solid phase carrier includes but is not limited to a reagent strip, a detection pad, and a test paper strip.

[0026] The seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention and / or the antibody derivative according to the second aspect of the present invention in the preparation of a product, wherein the use includes any one of the following:

[0027] 1) Application in the preparation of products for detecting parainfluenza virus or parainfluenza virus NP protein.

[0028] Furthermore, the product is used to detect parainfluenza virus type 3.

[0029] Furthermore, the product is used to detect parainfluenza virus type 3 NP protein.

[0030] 2) Application in the preparation of products for diagnosing parainfluenza virus infection.

[0031] Furthermore, the product is used to diagnose parainfluenza virus type 3 infection.

[0032] 3) Application in the preparation of products for diagnosing diseases caused by parainfluenza virus infection.

[0033] Furthermore, the diseases include pneumonia, tracheobronchitis, pharyngitis, laryngitis, rhinitis, sinusitis, otitis media, myocarditis, meningitis, Guillain-Barré syndrome, and acute disseminated encephalomyelitis.

[0034] Advantages and beneficial effects of the present invention:

[0035] The present invention provides an antibody or an antigen-binding fragment thereof with high specificity and high sensitivity against the NP protein of HPIV-3. The antibody obtained after purification is used for preparing products for detecting parainfluenza virus or its protein, diagnosing parainfluenza virus infection and diagnosing diseases caused by infection, which greatly improves the sensitivity and specificity of parainfluenza virus detection and disease diagnosis, and has broad application prospects and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the antibody electrophoresis detection diagram;

[0037] Figure 2 This is a graph of antibody high performance liquid chromatography detection;

[0038] Figure 3 The graph is a graph of antibody binding activity. DETAILED DESCRIPTION

[0039] The present invention provides an antibody and an antigen-binding fragment thereof with high specificity and high sensitivity against the NP protein of HPIV-3, which can be used to prepare products for detecting parainfluenza virus or its protein, products for diagnosing parainfluenza virus infection and products for diagnosing diseases caused by infection.

[0040] In the present invention, "parainfluenza virus" refers to human parainfluenza virus (HPIV), which is a single-stranded, enveloped RNA virus. Human parainfluenza virus RNA encodes six structural proteins in a conservative order: nucleoprotein or nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase glycoprotein (HN) and RNA polymerase (L). HPIV is divided into types 1-4 according to serological classification, of which type 4 has two subtypes, a and b. Type 3 of parainfluenza virus belongs to the genus Respiratory virus of the family Paramyxoviridae and is associated with a series of diseases, including common otitis media, pharyngitis, conjunctivitis, pharyngitis, tracheobronchitis and pneumonia. HPIV not only causes a variety of serious respiratory diseases in children, but also causes serious diseases and fatal pneumonia in the elderly and immunocompromised people. In the present invention, "NP protein" refers to nucleocapsid protein, which is the main antigenic protein in parainfluenza virus, has certain homology in different serotypes of parainfluenza virus, and participates in viral ribonucleoprotein vRNP assembly, RNA replication and viral particle assembly.

[0041] In the context of the present invention, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0042] 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 form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to the change in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence. In the present invention, the modification preferably occurs in an area outside the variable region, such as the constant region or framework region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of the present invention, or has improved antigen binding properties.

[0043] In the present invention, the term "antigen binding fragment" includes a part of a complete antibody, generally referring to one or more fragments of an antibody that specifically binds to an antigen. The antigen binding function of an antibody can be achieved by a full-length fragment of an antibody. In the present invention, the antigen binding fragment preferably includes its heavy chain variable region VH and light chain variable region VL, more preferably includes its heavy chain variable region HCDR1-3 and light chain variable region LCDR1-3, more preferably includes the sequences shown in SEQ ID NO:3-5 and SEQ ID NO:6-8. The binding function of an antibody can also be achieved by: a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2. The term "binding" refers to a binding effect that is selective for an antigen and can be distinguished from an undesirable or non-specific interaction. 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.

[0044] In the present invention, the terms "heavy chain variable region" and "light chain variable region" refer to the structure of an antibody from N-terminus to C-terminus, where each antibody heavy chain has a heavy chain variable region VH, also known as a heavy chain variable domain, followed by three heavy chain constant domains CH1, CH2 and CH3; similarly, from N-terminus to C-terminus, each antibody light chain has a light chain variable region VL, also known as a light chain variable domain, followed by a light chain constant domain CL. The heavy chain variable region and the light chain variable region can be further divided into hypervariable regions (i.e., CDRs) and relatively conservative regions (i.e., framework regions FRs) interposed between the hypervariable regions.

[0045] In the present invention, the term "CDR" refers to the "hypervariable region" or "complementarity determining region" of an antibody. Both the heavy chain variable region and the light chain variable region have three CDRs, which together constitute the antigen binding site of the antibody and can form precise complementarity with the antigenic determinant in terms of spatial structure. Examples of other antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The position of CDR in the variable region is marked differently according to different numbering systems, and the numbering schemes include IMGT, Chothia, Kabat, etc. The IMGT numbering scheme is based on the amino acid sequence alignment of germline V genes; the Chothia numbering scheme is based on crystal structure; and the Kabat numbering scheme defines the variability parameter as the number of different amino acids at a given position divided by the frequency of the most amino acids at that position. According to different CDR determination schemes, the residues in the CDR are as described in Table 1. In the present invention, the sequences of HCDR1-3 and LCDR1-3 are shown in SEQ ID NO:3-5, 6-8, and the numbering scheme is IMGT.

[0046] Table 1. CDR residues with different numbering schemes

[0047] CDR IMGT Program Chothia Solution Kabat regimen HCDR1 <![CDATA[H26-H33 / H35B 1 ]]> <![CDATA[H26-H32..34 1 ]]> <![CDATA[H31-H35 / H35B 1 ]]> HCDR2 H51-H56 H52-H56 H50-H65 HCDR3 H93-H102 H95-H102 H95-H102 LCDR1 L27-L32 L24-L34 L24-L34 LCDR2 L50-L51 L50-L56 L50-L56 LCDR3 L89-L97 L89-L97 L89-L97

[0048] Note 1: If both H35A and H35B do not exist, HCDR1 ends at position 32; if only H35A exists, HCDR1 ends at position 33; if both H35A and H35B exist, HCDR1 ends at position 34.

[0049] In the present invention, the term "antibody derivative" refers to a derivative comprising the antibody of the present invention and a detectable marker bound to the antibody, which can exhibit the desired biological activity and can be detected by the detectable marker attached thereto. The detectable marker includes at least one of a radioactive isotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0050] In the present invention, the combination of the antibody and the detectable marker includes at least one of radioisotope labeling, colloidal gold labeling, fluorescein labeling, biotinylation labeling, chemiluminescence labeling, electron-dense labeling, and enzyme labeling.

[0051] The term "radioactive isotope labeling" refers to a labeled immunoassay method that combines the high sensitivity of isotope analysis with the specificity of antigen-antibody reaction, using radioactive isotopes as tracers. This technology has many advantages, such as high sensitivity (can detect ultra-trace amounts of substances from nanograms (ng) to picograms (pg), or even femtograms (fg)), strong specificity (can distinguish antigens with similar structures), strong repeatability, small amounts of samples and reagents, and easy standardization and automation of the assay method.

[0052] The term "colloidal gold labeling method" refers to the technology of combining antigen-antibody immune reaction with colloidal gold labeling for antigen and antibody detection. Colloidal gold technology uses colloidal gold particles as a marker, which binds to antigens or antibodies and displays red strips on the test strip. It has three methods: double antibody sandwich method, competitive method and indirect method.

[0053] The term "fluorescein labeling method" refers to the technology of using fluorescent substances to label antibodies to detect corresponding antigens or antibodies in cells or tissues. The types of fluorescent substances generally include fluorescein isothiocyanate, rhodamine fluorescein, dichlorotriazine aminofluorescein, etc. Generally, the specimen to be tested is fixed on the surface of the slide, and a known fluorescent antibody is added and then rinsed with a buffer solution. After drying, it is observed under a fluorescence microscope. If it is positive, the fluorescent antigen-antibody complex can be seen; if it is negative, there is no fluorescence (because the fluorescent antibody cannot bind to the antigen and is washed away).

[0054] The term "biotinylation" refers to the detection of antibodies that have their epsilon-amino groups covalently attached to acylated biotin via an arm. Biotinylation is the labeling of a target molecule such as a protein, antibody, peptide, or other biomolecule with biotin. Biotinylated proteins generally retain biological activity because the biotin group is relatively small. Biotin is a natural vitamin that binds with high affinity to avidin and streptavidin proteins. The binding between biotin and avidin is very rapid and once formed is not affected by extreme pH, temperature, organic solvents, and other denaturing agents. Biotin labeling reagents can be targeted to specific functional groups or residues on biomolecules, such as primary amines, sulfhydryls, carboxyls, or carbohydrates. Biotinylated proteins can be captured or detected by enzyme-linked immunosorbent assays (ELISA), Western blotting, immunohistochemistry (IHC), immunoprecipitation (IP), or flow cytometry applications.

[0055] The term "chemiluminescent labeling method" refers to the method of directly labeling antigens or antibodies (chemiluminescent markers) with chemiluminescent agents, reacting with corresponding antibodies or antigens in the sample to be tested, and magnetic particle antigens or antibodies, separating the bound state (precipitated part) and free state chemiluminescent markers through a magnetic field, and then adding a luminescent promoter to carry out a luminescent reaction, and performing quantitative or qualitative detection by detecting the luminescent intensity. The term "chromophore", also known as "chromophore", refers to the unsaturated group and its related chemical bonds contained in the molecule that can absorb light radiation and have transitions. Some organic compound molecules contain groups containing unsaturated bonds, which can absorb in the ultraviolet and visible light regions (200-800nm) and have a large absorption coefficient. This absorption has wavelength selectivity, absorbing light of a certain wavelength (color) but not absorbing light of another wavelength (color), thereby making the substance appear colored.

[0056] The term "electron-dense labeling method" refers to a method in which antibodies are labeled with electron-dense substances such as ferritin, and then reacted with biological specimens containing corresponding antigens. The location of the electron-dense substances can be observed under an electron microscope to identify the sites of antigen-antibody reaction.

[0057] The basic principle of the term "enzyme labeling method" is to bind the antigen or antibody to the surface of a solid phase carrier and maintain its immune activity, while the corresponding antibody or antigen is connected to an enzyme to form an enzyme-labeled antibody or antigen. The enzyme-labeled antibody or antigen retains both the immune activity and the ability to bind to the antigen or antibody on the surface of the solid phase carrier, and the enzyme activity and the ability to use the enzyme as a detection signal. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. The amount of the product is proportional to the amount of the antibody or antigen being tested, so it can be qualitatively or quantitatively analyzed based on the color depth. Depending on the purpose of detection, the enzyme-labeled antibody method includes the indirect method, the double antibody sandwich method, the competitive method, and the antibody capture method. Commonly used enzymes in the enzyme labeling method are alkaline phosphatase and horseradish peroxidase.

[0058] In the present invention, "nucleic acid molecule" refers to any polymer form having any length and consisting of ribonucleotides or deoxyribonucleotides. Generally, a nucleic acid molecule is a coding sequence, as used herein, which refers to a DNA sequence that is transcribed and translated into a polypeptide in a host cell when placed under the control of a suitable regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription termination sequence will usually be located at the 3' of the coding sequence.

[0059] In a specific embodiment of the present invention, the nucleic acid molecule refers to a nucleic acid sequence capable of encoding the antibody and antigen-binding fragment thereof described in the first aspect of the present invention. Once the coding sequence of the antibody and antigen-binding fragment thereof described in the present invention is isolated, recombinant technology can be used to obtain the antibody or recombinant antibody containing antigen-binding activity in large quantities.

[0060] In the present invention, a vector comprising nucleic acid molecules of the present invention is provided. The term "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 can be a plasmid vector, a viral vector, etc. In some embodiments, a vector refers to a straight or circular nucleic acid molecule that comprises a nucleic acid of the present invention that is operably connected to other segments of autonomous replication provided in a recombinant host cell, or an expression cassette according to a nucleic acid molecule. "Operably connected" means that the nucleic acid sequence of interest is connected to a regulatory sequence (e.g., in an in vitro transcription / translation system or when a vector is introduced into a host cell) in a manner that allows the expression of the nucleotide sequence.

[0061] In the present invention, the term "recombinant host cell" refers to the introduction of a vector into a host cell, so that the recombinant host cell can transcribe the target nucleic acid sequence and / or translate the target protein. In the present invention, the recombinant host cell can be made to express the antibody or antigen-binding fragment thereof described in the first aspect of the present invention by such genetic engineering technology, thereby achieving the method described in the fifth aspect of the present invention.

[0062] The present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, wherein the method comprises preparation by artificial synthesis or genetic engineering technology.

[0063] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention or antigen-binding fragments thereof are obtained by direct amino acid synthesis.

[0064] In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0065] In the context of the present invention, the term "subclone" refers to the insertion of a target DNA fragment into a specific region of a cloning vector, which usually involves fine processing of the target DNA, such as cutting or modifying it with a special enzyme. The purpose of subclone is to locate the target DNA fragment to a specific position on the vector so that it can be analyzed, studied or recombinantly modified. Subclone is divided into cell clone and molecular clone. Cell clone refers to the selection of cells with certain characteristics from the original clones for culture; molecular cloning refers to the selection of specific small fragments from the clones of large fragments for cloning. In a specific embodiment of the present invention, subclone refers to cell clone.

[0066] In the present invention, the term "ELISA" or "enzyme-linked immunosorbent assay" refers to a method for quantitatively or semi-quantitatively determining the protein concentration of a sample (e.g., hybridoma cells) in a multi-well plate format (usually 96 wells per plate). In brief, proteins in solution are adsorbed onto ELISA plates. Antibodies specific for the target protein can be used to probe the plates. Background is minimized by optimizing blocking and washing methods, and specificity is ensured by the presence of positive and negative controls. Detection methods are typically based on colorimetry or chemiluminescence.

[0067] In the present invention, the term "transient transfection" is one of the ways to introduce DNA into eukaryotic cells. In transient transfection, recombinant DNA is introduced into a highly infectious cell line to obtain temporary but high-level expression of the target gene. The transfected DNA does not have to be integrated into the host chromosome, and the transfected cells can be harvested in a shorter time than stable transfection, and the expression of the target gene in the lysate can be detected.

[0068] In the present invention, the term "PEI" refers to polyethyleneimine, which is a cationic polymer that is usually used to bind negatively charged DNA molecules to form a complex and transfect into adherent or suspended cells. PEI is currently the most widely used gene carrier and transfection reagent in the field of industrialized, large-scale transient transfection to express recombinant proteins or viral vectors. Cationic polymers also include polyamino esters (PBAE), chitosan, polyacrylamine (PAH), diethylaminoethyl dextran (DEAE-dextran), polyamine dendrimers (PAMAM), etc. The common principle of cationic polymer transfection is: DNA is wrapped to form a complex under physiological pH conditions to prevent degradation by DNA enzymes, and then attached to the cell membrane to be endocytosed into the cell. After rupture and release, the DNA enters the cytoplasm, thereby exerting a certain function. The biggest difference between cationic polymers and cationic liposomes is that they do not contain hydrophobic parts and are completely soluble in water, and can be easily chemically modified.

[0069] In the present invention, the term "EC 50 "Also known as the "half effective concentration", in quantitative reactions it refers to the dose of antibodies, drugs or poisons that can cause 50% of the maximum reaction intensity; in qualitative reactions it refers to the dose of drugs that causes 50% of the experimental animals to have a positive reaction.

[0070] 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. Simple improvements to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0071] Example 1 Preparation and physicochemical property detection of parainfluenza virus type 3 NP protein antibody

[0072] 1. Antibody Preparation

[0073] 1. Recombinant Expression of Immunogens

[0074] 1) Synthesize the parainfluenza type 3 NP protein sequence and construct it into the pet28a vector;

[0075] 2) Extracting transformation plasmid;

[0076] 3) Transformation into Escherichia coli BL21 (DE3) and fermentation culture;

[0077] 4) The cells were harvested by centrifugation, crushed and centrifuged, the protein precipitate was resuspended in a buffer, purified by a Ni column, and the parainfluenza type 3 NP protein was obtained after concentration and replacement of the buffer. The protein sequence is shown in SEQ ID NO: 9 in Table 2.

[0078] 2. Animal Immunization

[0079] 1) Immunize mice with the recombinant parainfluenza type 3 NP protein obtained in step 1, using Freund's complete adjuvant for the first time, intraperitoneally injected at a dose of 100 μg / 0.5 ml / mouse;

[0080] 2) The second immunization was performed three weeks later. Freund's incomplete adjuvant was used for intraperitoneal injection at a dose of 50 μg / 0.5 ml / mouse. The third immunization was performed two weeks later.

[0081] 3) Prepare cell fusion 10 days after the third injection of immunization. Take feeder cells and press 10 5 / well, plate 10 one day before fusion 5 pcs / 100μl / well;

[0082] 4) Mouse immune spleen cells were taken and fused with the prepared myeloma cells using the fusion agent PEG, and plated into a 96-well cell culture plate to which feeder cells had been added, at 100 μl / well.

[0083] 3. Screening of high-expressing cell lines

[0084] 1) Screening positive wells by ELISA detection method, and plating the recombinant parainfluenza type 3 NP protein obtained in step 1 overnight;

[0085] 2) Wash the plate, add skim milk powder, and incubate at 37°C for 1 hour;

[0086] 3) Wash the plate, add 100 μl of 96-well culture supernatant, and incubate at 37°C for 1 h;

[0087] 4) Wash the plate, add HRP-labeled goat anti-mouse secondary antibody, and incubate at 37°C for 30 min;

[0088] 5) Wash the plate, add color developing solution, and develop color for 10 minutes;

[0089] 6) Add stop solution and read the OD450 value;

[0090] 7) Screen high-expressing cell lines for subcloning.

[0091] 4.Sequence Retrieval

[0092] 1) Collect the cells obtained by subcloning the high-expression cell line obtained in step 3, extract RNA, perform reverse transcription, design primers, and perform PCR amplification;

[0093] 2) The amplified product was transformed, and after culture, a single clone was picked and sent for sequencing to obtain the antibody sequence of clone number 6D3, whose amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 1-2 in Table 2.

[0094] Table 2. List of amino acid sequences corresponding to SEQ IDs

[0095]

[0096]

[0097] 5. Antibody Expression

[0098] 1) Transiently transfect the antibody in step 4 into HEK293 cells using PEI to construct cells expressing the antibody;

[0099] 2) After testing, the antibody expression level of clone 6D3 was 198 mg / L.

[0100] 2. Antibody physicochemical properties testing

[0101] 1) The purity of the prepared antibody is greater than 95% as determined by electrophoresis. Figure 1 As shown;

[0102] 2) The purity of the prepared antibody was greater than 95% as determined by HPLC. Figure 2 As shown;

[0103] 3) After coating the recombinant parainfluenza type 3 NP protein, the prepared antibody and horseradish peroxidase-labeled goat anti-mouse secondary antibody were added in sequence, the readings were read after color development, the data were analyzed and the curve was fitted. The EC of the antibody clone 6D3 was 50 is 0.395ng / ml, the results are as follows Figure 3 shown.

[0104] 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 against human parainfluenza virus type 3 NP protein, characterized in that: The antibody or antigen-binding fragment thereof comprises HCDR1-HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 1; and The antibody or antigen-binding fragment thereof comprises LCDR1-LCDR3 of the light chain variable region as shown in SEQ ID NO:2; The amino acid sequence of HCDR1 of the heavy chain variable region is GFSITSNYY; The amino acid sequence of HCDR2 of the heavy chain variable region is ISFDGSN; The amino acid sequence of HCDR3 of the heavy chain variable region is APYQLGTMDY; The amino acid sequence of LCDR1 of the light chain variable region is QDIKIY; The amino acid sequence of LCDR2 in the light chain variable region is RVN; The amino acid sequence of LCDR3 in the light chain variable region is LQYDEFPLT.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibodies include monoclonal antibodies, humanized antibodies, chimeric antibodies or bivalent antibodies.

3. An antibody derivative, characterized in that: The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and a detectable marker bound to the antibody.

4. The antibody derivative according to claim 3, characterized in that The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

5. The antibody derivative according to claim 3, characterized in that The detectable label is an enzyme.

6. The antibody derivative according to claim 3, characterized in that The detectable marker is horseradish peroxidase.

7. The antibody derivative according to claim 3, characterized in that: The detectable marker is combined with the antibody by at least one of radioisotope labeling, colloidal gold labeling, fluorescein labeling, biotinylation labeling, chemiluminescence labeling, electron-dense labeling, and enzyme labeling.

8. The antibody derivative according to claim 3, characterized in that: The detectable marker is bound to the antibody by enzyme labeling.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid that can encode the antibody or antigen-binding fragment thereof according to claim 1.

10. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 9.

11. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method for detecting parainfluenza virus type 3 NP protein or parainfluenza virus in vitro for non-therapeutic purposes, characterized in that: The antibody or antigen-binding fragment thereof according to claim 1 and / or the antibody derivative according to claim 3 are used to detect human parainfluenza virus type 3 NP protein or parainfluenza virus.

13. A method for preparing the antibody or antigen-binding fragment thereof according to claim 1, said method comprising preparation by artificial synthesis or genetic engineering technology.

14. The method according to claim 13, characterized in that The preparation by genetic engineering technology refers to culturing a recombinant host cell containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 under conditions suitable for expressing the antibody.

15. A product for detecting human parainfluenza virus type 3 NP protein, characterized in that: The product comprises a first antibody, the first antibody include: The antibody or antigen-binding fragment thereof according to claim 1; or The antibody derivative according to claim 3.

16. The product according to claim 15, characterized in that The product also includes a second antibody, a solid phase carrier loaded with the first antibody and / or the second antibody, an eluent, and a color developing solution.

17. The product according to claim 16, characterized in that The second antibody is a labeled antibody, to which the detectable label described in any one of claims 3-4 is linked.

18. The product according to claim 16, characterized in that The second antibody is a goat anti-mouse IgG antibody labeled with horseradish peroxidase.

19. The product according to claim 16, characterized in that The solid phase carrier includes a reagent strip, a detection pad, and a test paper strip.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and / or the antibody derivative according to any one of claims 3 to 8 in the preparation of a product for detecting parainfluenza virus or parainfluenza virus NP protein.

21. The use according to claim 20, characterized in that The product is used to detect parainfluenza virus type 3.

22. The use according to claim 20, characterized in that The product is used to detect parainfluenza virus type 3 NP protein.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and / or the antibody derivative according to any one of claims 3 to 8 in the preparation of a product for diagnosing parainfluenza virus infection.

24. The use according to claim 23, characterized in that The product is used to diagnose parainfluenza virus type 3 infection.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and / or the antibody derivative according to any one of claims 3 to 8 in the preparation of a product for diagnosing a disease caused by parainfluenza virus infection.

26. The use according to claim 25, characterized in that The diseases include pneumonia, tracheobronchitis, pharyngitis, laryngitis, rhinitis, sinusitis, otitis media, myocarditis, meningitis, Guillain-Barré syndrome, and acute disseminated encephalomyelitis.

Images