Metapneumovirus M protein antibody and application thereof

Abstract

The invention discloses a metapneumovirus M protein antibody and application thereof. The amino acid sequences of the complementary determining regions of the heavy chain variable region and the light chain variable region of the metapneumovirus M protein antibody provided by the invention are clear and are respectively shown as SEQ ID NO: 1-8, and the metapneumovirus M protein antibody has relatively high affinity and specificity to the metapneumovirus M protein. The metapneumovirus M protein antibody disclosed by the invention can effectively avoid cross reaction and remarkably improve the accuracy and sensitivity of detection, is suitable for early antigen detection of metapneumovirus infection, provides a reliable technical means for screening of metapneumovirus and prevention and control of diseases, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a metapneumovirus M protein antibody and application thereof. BACKGROUND

[0002] Metapneumovirus is an enveloped negative-sense single-stranded RNA virus and is one of the main pathogens of acute respiratory infection. The virus has strong infectivity and is mainly transmitted through respiratory droplets. Mild patients mainly involve the upper respiratory tract and generally last for about 1 week, with clinical manifestations of nasal congestion, cough and the like. Severe patients can involve the lower respiratory tract and develop into bronchitis, pneumonia and other diseases, with clinical manifestations of fever, shortness of breath, wheezing, dyspnea and the like. Metapneumovirus is a respiratory virus that can cause infection in people of all ages, and is mostly manifested as mild self-limiting disease, but in people with low immune function, infection can progress to severe pneumonia, multiple organ failure and even death.

[0003] At present, there are few specific antiviral drugs for metapneumovirus infection, and treatment mainly relies on symptomatic support such as fever reduction, cough relief and oxygen inhalation. In recent years, breakthrough progress has been made in targeted antibody technology, and various new neutralizing antibodies not only exhibit long-term protection in passive immunization prevention, but also show precise targeting advantages in the treatment of severe infection, providing an innovative solution for the whole process of viral infection prevention and control. Metapneumovirus (MPV) M protein (matrix protein) is part of the virus structure, responsible for virus assembly and maturation, and it plays an important role in the process of viral infection. Therefore, developing specific detection antibodies or antibody therapeutic drugs that can target metapneumovirus M protein is an important direction for future metapneumovirus detection and treatment. SUMMARY

[0004] In order to make up for the deficiencies of the prior art, the application provides a metapneumovirus M protein antibody and application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: The application provides a metapneumovirus M protein antibody, a heavy chain variable region CDR-H1 of the antibody is shown as SEQ ID NO: 1; a CDR-H2 is shown as SEQ ID NO: 2; a CDR-H3 is shown as SEQ ID NO: 3; a light chain variable region CDR-L1 of the antibody is shown as SEQ ID NO: 5; a CDR-L2 is shown as SEQ ID NO: 6; and a CDR-L3 is shown as SEQ ID NO: 7.

[0006] Further, the heavy chain variable region of the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 75% homology with SEQ ID NO: 4; and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% homology with SEQ ID NO: 8.

[0007] In some embodiments, the antibody is an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be assigned to a kind of class, kappa and lambda. Heavy chains can be assigned to a kind of class, mu, delta, gamma, alpha, or epsilon, and define a kind of antibody through its distinctive sequence. In a light chain and a heavy chain, a variable region and a constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair form the antibody binding site.

[0008] In some embodiments, the amino acid sequences corresponding to the CDR-H1-3 of the heavy chain variable region of the present application are not limited to the amino acid sequences as set forth in SEQ ID NO: 1-3, and the amino acid sequences corresponding to the CDR-L1-3 of the light chain variable region of the present application are not limited to the amino acid sequences as set forth in SEQ ID NO: 5-7. The CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region as set forth in SEQ ID NO: 4 and the CDR-L1, CDR-L2, CDR-L3 in the light chain variable region as set forth in SEQ ID NO: 8 are defined by using any CDR numbering scheme (existing CDR numbering scheme or new CDR numbering scheme to be generated in the future), respectively, and the amino acid sequences or nucleotide sequences corresponding to the CDRs obtained by the definition are within the scope of the present application.

[0009] In some embodiments, the CDR numbering scheme is any one or more combinations of IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, Aho numbering scheme, the sequences of CDR-H1-3 of the heavy chain variable region or CDR-L1-3 of the light chain variable region defined by any one of the above CDR numbering schemes or any combination of two or more CDR numbering schemes are encompassed within the scope of the present application.

[0010] In some embodiments, "homologous" can be determined using methods known in the art, such as sequence comparison algorithms, two or more sequences have a specified percentage of nucleotides identical on a specified region when compared and aligned for maximum correspondence. In some embodiments, a polymeric molecule is considered "homologous" to another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the monomers in the molecule are identical (exactly the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0011] In some embodiments, functional variants of the metapneumovirus M protein antibodies described herein are also encompassed within the scope of the present application, the "functional variant" refers to a protein having substantial or significant sequence identity or similarity to a parent antibody, the functional variant retains the biological activity of the parent antibody. A functional variant encompasses, for example, a variant of a metapneumovirus M protein antibody (parent antibody) described herein that retains the ability to recognize a target cell to a similar, the same or a higher extent as compared to the parent antibody. With reference to the parent antibody, a functional variant may, for example, have at least about 75%, 80%, 85%, 90%, 95% or more identity in the amino acid sequence to the parent antibody.

[0012] In some embodiments, a functional variant may, for example, comprise the amino acid sequence of a parent antibody with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may, for example, comprise the amino acid sequence of a parent antibody with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may, for example, enhance the biological activity of the functional variant such that the biological activity of the functional variant is improved as compared to the parent antibody.

[0013] In some embodiments, conservative amino acid substitutions are known in the art and include substitutions in which one amino acid having a particular physical and / or chemical property is exchanged for another amino acid having the same or similar chemical or physical property. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituted for another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0014] The second aspect of the application provides a bispecific antibody comprising an antibody according to the first aspect of the application.

[0015] Further, the bispecific antibody comprises a second antibody which specifically binds to a further antigen.

[0016] In some embodiments, the bispecific antibody comprises binding specificities for two different antigens (one of which is a metapneumovirus M protein according to the application and the other of which is an antigen other than a metapneumovirus M protein). In other embodiments, the bispecific antibody comprises two different binding specificities for the same antigen (e.g., different binding affinities and / or specific epitopes for the same antigen).

[0017] The third aspect of the application provides a nucleic acid molecule encoding an antibody according to the first aspect of the application or a bispecific antibody according to the second aspect of the application.

[0018] In the present application, the term "nucleic acid" or "nucleic acid molecule" is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and hybrids between DNA and RNA that include DNA or RNA analogs such as those containing modified backbones (e.g., peptide nucleic acids (PNAs)) or modified bases. Thus, nucleic acids of the present application include DNA, cDNA, mRNA, recombinant nucleic acids, and the like. Once the coding sequence for the antibody of the present application has been isolated, recombinant techniques can be employed to obtain the antibody in large quantities. An exemplary method is to clone its encoding gene into a vector, which is then introduced into cells, and the antibody is then isolated from the propagated host cells by conventional methods.

[0019] The term "encoding" as used herein in the context of a nucleic acid, such as a gene, cDNA, or mRNA, refers to the inherent property of specific sequences of nucleotides in a nucleic acid to serve as templates for synthesis of other polymers and macromolecules having a specified sequence of nucleotides (e.g., a rRNA, tRNA, and mRNA) or a specific sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose sequence is identical to the mRNA and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0020] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of a nucleic acid molecule can be obtained using conventional techniques. Once the relevant sequence has been obtained, it can be obtained in large quantities using recombinant methods. This is typically done by cloning the sequence into a vector, which is then introduced into cells, and the sequence is then isolated from the propagated host cells by conventional methods. In addition, the relevant sequence can be synthesized using artificial synthesis methods, particularly for short fragments. Typically, longer fragments are obtained by first synthesizing a number of small fragments, which are then ligated.

[0021] In a particular embodiment of the present application, the nucleic acid molecule is codon-optimized from the amino acid sequence of the antibody of the first aspect of the present application, wherein the heavy chain nucleotide sequence is set forth in SEQ ID NO: 10 and the light chain nucleotide sequence is set forth in SEQ ID NO: 11.

[0022] In the present application, the codon optimization method is known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to match codon frequencies in the target and host organism to ensure proper folding; to bias the GC content to increase mRNA stability or reduce secondary structure; to minimize tandem repeat codons or base runs that can impair gene architecture or expression; to tailor transcription and translation control regions; to insert or remove protein trafficking sequences; to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; to add, remove, or substitute protein domains; to insert or delete restriction sites; to modify ribosome binding sites and mRNA degradation sites; to modulate translation rates so that various domains of the protein can fold properly; or to reduce or eliminate problematic secondary structures in the nucleic acid.

[0023] The fourth aspect of the present application provides an expression vector comprising the nucleic acid molecule of the third aspect of the present application.

[0024] The term "expression" used in the present application refers to the process by which a nucleic acid is transcribed into mRNA and translated into a peptide, polypeptide, or protein.

[0025] In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and expressed. The vector can be transformed, transduced, or transfected into a host cell, so that the genetic material elements carried by the vector can be expressed in the host cell.

[0026] In some embodiments, various vectors known in the art can be selected, for example, commercially available vectors, and then the nucleic acid encoding the antibody of the first aspect of the present application can be operably linked to the expression regulatory sequence to form an expression vector. In some embodiments, the vector includes, but is not limited to, plasmid, phagemid, cosmid, artificial chromosome, virus-derived vector.

[0027] In some embodiments, the vector can be constructed by methods well known to those skilled in the art. These methods include, but are not limited to, recombinant DNA technology, DNA synthesis technology, etc. The DNA encoding the antibody can be effectively ligated to a multiple cloning site in the vector to direct mRNA synthesis and thus express the protein, or for homologous recombination.

[0028] The fifth aspect of the present application provides a recombinant host cell comprising the expression vector of the fourth aspect of the present application.

[0029] Further, the recombinant host cell includes prokaryotic cells, eukaryotic cells.

[0030] In the present application, the term "host cell" refers to a cell that can be used for introducing a vector, which includes but is not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or mammalian cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0031] In some embodiments, the nucleic acid molecule or the expression vector of the present application as described above can be introduced into a host cell by various suitable means, which are not limited to the methods listed in the present application, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposome), or viral vector-mediated transfection (such as lentivirus infection, retrovirus infection, adenovirus infection), and other physical, chemical or biological means for transferring into cells, such as transposon technology, CRISPR-Cas9 technology, etc.

[0032] The sixth aspect of the present application provides any one of the following products, which comprises: 1) an antibody conjugate, which is a complex formed by directly or indirectly conjugating the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application to a detectable marker.

[0033] 2) a detection reagent, which comprises the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, or the antibody conjugate.

[0034] 3) a detection product, which comprises the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the antibody conjugate, or the detection reagent.

[0035] 4) a pharmaceutical composition, which comprises the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application.

[0036] 5) a biological agent, which comprises the pharmaceutical composition.

[0037] Further, the detectable marker comprises a chemiluminescent marker, a chemiluminescent catalyst, a fluorescent dye, avidin, a paramagnetic atom, a radioisotope, an enzyme marker, colloidal gold.

[0038] In some embodiments, the chemiluminescent label is luminol and its derivatives, isoluminol and its derivatives, acridinium ester, acridinium ester derivatives, adamantane, rare earth elements or a ruthenium complex of dipyridyl. The chemiluminescent catalyst is horseradish peroxidase or alkaline phosphatase. The fluorescent pigment includes fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, peridinin-chlorophyll protein. The avidin includes biotin, avidin, streptavidin, ovomucoid, affililin. The radioisotope includes radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt. The enzyme label includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, lysozyme, malate dehydrogenase.

[0039] Further, the detection product includes a kit, a chip, a test strip.

[0040] In some embodiments, the biological agent can further comprise a pharmaceutically acceptable carrier and / or excipient, which is described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995), and the present application does not have a particular limitation on the pharmaceutically acceptable carrier and / or excipient contained in the biological agent.

[0041] The seventh aspect of the present application provides any one of the following methods, which comprises: 1) A method for preparing the recombinant host cell of the fifth aspect of the present application, which comprises: introducing the expression vector of the fourth aspect of the present application into a host cell to obtain the recombinant host cell of the fifth aspect of the present application.

[0042] 2) A method for producing the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application, which comprises: culturing the recombinant host cell of the fifth aspect of the present application, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell.

[0043] 3) A method for detecting metapneumovirus M protein in a sample to be tested for non-diagnostic and non-therapeutic purposes, which comprises: contacting the sample to be tested with the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the antibody conjugate of the sixth aspect of the present application, the detection reagent or the detection product, and detecting the formation of an immune complex of metapneumovirus M protein and the antibody.

[0044] 4) A method for inhibiting the activity of metapneumovirus M protein in a sample in vitro, which comprises: contacting the sample with the antibody of the first aspect of the present application or the bispecific antibody of the second aspect of the present application.

[0045] In some embodiments, the sample / subject sample includes, but is not limited to, tissue, blood, cells, lymph fluid, synovial fluid, exosomes, cell extracts, fecal, urine, saliva, sputum, joint cavity fluid, pleural effusion, peritoneal effusion, lymph fluid, cerebrospinal fluid, uterine cavity fluid, digestive fluid, bile, alveolar bronchial lavage fluid, organs, and any combination thereof, derived from a subject.

[0046] In the present application, the subject includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles.

[0047] The eighth aspect of the present application provides any one of the following uses, which comprises: 1) use of the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, or the recombinant host cell of the fifth aspect of the present application in the preparation of an antibody conjugate for detecting metapneumovirus M protein.

[0048] 2) use of the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, or the antibody conjugate of the sixth aspect of the present application in the preparation of a detection reagent or a detection product for detecting metapneumovirus M protein.

[0049] 3) use of the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application, the detection reagent, or the detection product in the detection of metapneumovirus M protein for non-diagnostic, non-therapeutic purposes.

[0050] 4) use of the antibody of the first aspect of the present application, the bispecific antibody of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application, the detection reagent, or the detection product in the preparation of a diagnostic product for diagnosing or aiding in the diagnosis of a disease associated with metapneumovirus M protein infection.

[0051] 5) The antibody of the first aspect of the application, the bispecific antibody of the second aspect of the application, the nucleic acid molecule of the third aspect of the application, the expression vector of the fourth aspect of the application or the recombinant host cell of the fifth aspect of the application for use in the preparation of a medicament for the treatment and / or prevention of a metapneumovirus M protein infection related disease.

[0052] Further, the metapneumovirus M protein infection related disease includes bronchiolitis, pneumonia, rhinitis, cold.

[0053] In some embodiments, the metapneumovirus M protein infection related disease is collectively referred to, and is not limited to the specific disease types listed above, and any disease related to metapneumovirus M protein infection will fall within the protection scope of the present application.

[0054] Advantages and beneficial effects of the present application: The heavy chain and light chain variable region complementarity determining region amino acid sequences of the metapneumovirus M protein antibody provided by the present application are clear, as shown in SEQ ID NO: 1-8, and have high affinity and specificity for metapneumovirus M protein. The metapneumovirus M protein antibody of the present application can effectively avoid cross-reaction, significantly improve the accuracy and sensitivity of detection, and is suitable for early antigen detection of metapneumovirus infection, providing a reliable technical means for screening and disease prevention and control of metapneumovirus, and having a broad clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 Electrophoretogram of antibody 7G1.

[0056] Figure 2 HPLC chromatogram of antibody 7G1.

[0057] Figure 3 ELISA binding curve of antibody 7G1. DETAILED DESCRIPTION

[0058] The present application will be further described below in conjunction with specific examples, which are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0059] The materials and equipment used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions are typically performed under conventional conditions or as recommended by the manufacturer. Specifically, the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0060] Example 1: Preparation and detection of antibody 7G1 I. Experimental Methods 1. Preparation of antibody 7G1 1) Synthesize the metapneumovirus M protein sequence and construct it into the pCDNA3.1 vector; extract the plasmid for transfection; transfect it into HEK293 cells and culture the cells for 7 days; harvest the supernatant, purify it with a Ni column, and obtain the recombinant metapneumovirus M protein after concentration and replacement buffer.

[0061] The amino acid sequence information of the recombinant metapneumovirus M protein is as follows: MESYLVDTYQGIPYTAAVQVDLVEKDLPASLTIWFPLFQANTPPAVLLDQLKTLTITTLYAASQSGPILKVNASAQGAAMSVLPKKFEVNATVALDEYSKLEFDKLTVCEVKTVYLTTMKPYGMVSKFVSS AKPVGKKTHDLIALCDFMDLEKNTPVTIPAFIKSVSIKESESATVEAAISSEADQALTQAKIAPYAGLIMIMTMNNPKGIFKKLGAGTQVIVELGAYVQAESISKICKTWSHQGTRYVLKSRHHHHHH* (SEQ ID NO:9) 2) Immunization of mice: The first immunization was administered with Freund's complete adjuvant, 100 μg per mouse, via intraperitoneal injection, with a total dose of 0.5 ml / mouse. The second immunization was administered 3 weeks later. From the second immunization onwards, Freund's incomplete adjuvant was administered at a dose of 50 μg / 0.5 ml / mouse. The third immunization was administered 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0062] SP2 / 0 fusion: Take feeder cells, and mix at 10... 5 / Hole application, lay 10 plates the day before fusion. 5 100 μl / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μl / well.

[0063] Screening: positive well screening was performed by ELISA detection method, recombinant metapneumovirus M protein was plated overnight; plate was washed, blocked with skimmed milk powder, 37°C, 1h; plate was washed, 100 μl 96 well culture supernatant was added, 37°C, incubated for 1 h; plate was washed, HRP labeled goat anti-mouse secondary antibody was added, 37°C, incubated for 30 min; plate was washed, color developing solution was added, color developed for 10 min, stop solution was added, OD value was read 450 Screening: positive well screening was performed by ELISA detection method, recombinant metapneumovirus M protein was plated overnight; plate was washed, blocked with skimmed milk powder, 37°C, 1h; plate was washed, 100 μl 96 well culture supernatant was added, 37°C, incubated for 1 h; plate was washed, HRP labeled goat anti-mouse secondary antibody was added, 37°C, incubated for 30 min; plate was washed, color developing solution was added, color developed for 10 min, stop solution was added, OD value was read

[0064] 3) Sequence fishing: collect cells, extract RNA, reverse transcription, design primers, PCR, transformation, pick clones, and send for sequencing. Metapneumovirus M protein antibody targets M protein. Recombinant metapneumovirus M protein antibody with clone number 7G1 was obtained.

[0065] 4) The amino acid sequence of antibody 7G1 is shown in Table 1.

[0066] Table 1 Amino acid sequence of antibody 7G1

[0067] 5) Take the amino acid sequence of 7G1 protein as the core template, and systematically optimize based on the codon usage bias of human cells: first search the human genome codon frequency database, replace low frequency codons in the sequence with human high frequency synonymous codons corresponding to the amino acid; at the same time, avoid poly (A) signal, cryptic splice site and other redundant elements affecting transcription, and adjust the GC content to the conventional 40%-60% range of human genes; optimize the codon combination through RNA secondary structure prediction to avoid the formation of stem-loop structure that hinders ribosome binding; strictly retain the key amino acid regions such as active center and functional binding site of 7G1 protein throughout the process to ensure that the protein structure and function are not affected, and finally obtain an optimized coding sequence adapted to the human expression system. The optimized nucleotide sequence is shown in Table 2. The optimized nucleic acid sequence is sent to Kingsway for synthesis and constructed into pcDNA3.4 vector. The plasmid is prepared for standby.

[0068] Table 2 Nucleotide sequence of antibody 7G1

[0069] 2, Antibody 7G1 detection: 1 day before transfection, 293 cells in good condition (adherent cell confluence reached 70-80%, and the density of suspended cells was 3×10 5DNA (cells / mL) was seeded into culture dishes. On the day of transfection, endotoxin-free plasmids were prepared at an HC:LC ratio of 1:1-1.2. The amount of PEI was calculated with N / P = 3:1. DNA and PEI were diluted separately with serum-free medium and mixed. The mixture was incubated at room temperature for 15-20 min to form a complex. The complex was added to the cells and cultured at 37°C with 5% CO2. The medium was changed after 6-12 h. The supernatant was collected after 48-96 h of culture. The antibody concentration and activity were detected by UV 280 nm, ELISA, SDS-PAGE and HPLC.

[0070] II. Experimental Results The results showed that the concentration of antibody 7G1 was 323 mg / L. SDS-PACE electrophoresis and HPLC analysis revealed that the purity of antibody 7G1 was greater than 95%. Figure 1 , Figure 2 ELISA results showed that antibody 7G1 had ECG... 50 The concentration was 0.1050 μg / ml, indicating good affinity. Figure 3 ).

[0071] The specificity test results of antibody 7G1 are shown in Table 3. Only the metapneumovirus M protein showed normal color development, while the other antigens showed no positive reaction. This means that antibody 7G1, which targets the metapneumovirus M protein, can specifically bind to the metapneumovirus M protein and has high specificity.

[0072] Table 3. Specificity detection results of antibody 7G1

[0073] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A metapneumovirus M protein antibody, characterized in that, The heavy chain variable region CDR-H1 of the antibody is shown in SEQ ID NO:1; CDR-H2 is shown in SEQ ID NO:2; CDR-H3 is shown in SEQ ID NO:3; the light chain variable region CDR-L1 of the antibody is shown in SEQ ID NO:5; CDR-L2 is shown in SEQ ID NO:6; CDR-L3 is shown in SEQ ID NO:

7.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% homology with SEQ ID NO:4; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% homology with SEQ ID NO:

8.

3. A bispecific antibody, characterized in that, The bispecific antibody comprises the antibody as described in any one of claims 1 or 2; Preferably, the bispecific antibody further comprises a second antibody that specifically binds to other antigens.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody as described in any one of claims 1 or 2, or the bispecific antibody as described in claim 3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the expression vector according to claim 5; Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells.

7. Any of the following products, characterized in that, The products include: 1) An antibody conjugate, wherein the antibody conjugate is a complex formed by directly or indirectly conjugating the antibody of claim 1 or 2 or the bispecific antibody of claim 3 to a detectable marker; 2) A detection reagent comprising the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, or the antibody conjugate thereof; 3) A detection product comprising the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the antibody-drug conjugate, or the detection reagent; 4) A pharmaceutical composition comprising the antibody as described in any one of claims 1 or 2 or the bispecific antibody as described in claim 3; 5) A biological agent comprising the pharmaceutical composition.

8. The product according to claim 7, characterized in that, The detectable markers include chemiluminescent markers, chemiluminescent catalysts, fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, and colloidal gold; Preferably, the detection product includes a reagent kit, a chip, and a test strip.

9. The following method, characterized in that, The method includes: 1) A method for preparing the recombinant host cell according to claim 6, the method comprising: introducing the expression vector according to claim 5 into a host cell to obtain the recombinant host cell according to claim 6; 2) A method for producing the antibody as described in any one of claims 1 or 2 or the bispecific antibody as described in claim 3, the method comprising: culturing the recombinant host cell as described in claim 6, and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell; 3) A method for detecting metapneumovirus M protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the antibody conjugate as described in claim 7, a detection reagent or a detection product, and detecting the formation of a metapneumovirus M protein immune complex with the antibody; 4) A method for inhibiting the activity of metapneumovirus M protein in a sample in vitro, the method comprising: contacting the sample with the antibody as described in any one of claims 1 or 2 or the bispecific antibody as described in claim 3.

10. The following application, characterized in that, The applications include: 1) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, or the recombinant host cell as described in claim 6 in the preparation of antibody-drug conjugates for detecting metapneumovirus M protein; 2) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, or the antibody conjugate as described in claim 7 in the preparation of a detection reagent or detection product for detecting metapneumovirus M protein; 3) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, the antibody conjugate as described in claim 7, the detection reagent or detection product in the detection of metapneumovirus M protein for non-diagnostic and non-therapeutic purposes; 4) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, the recombinant host cell as described in claim 6, the antibody conjugate as described in claim 7, the detection reagent or detection product in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of diseases related to metapneumovirus M protein infection; 5) The use of the antibody as described in any one of claims 1 or 2, the bispecific antibody as described in claim 3, the nucleic acid molecule as described in claim 4, the expression vector as described in claim 5, or the recombinant host cell as described in claim 6 in the preparation of a medicament for the treatment and / or prevention of diseases related to metapneumovirus M protein infection; Preferably, the diseases associated with metapneumovirus M protein infection include bronchiolitis, pneumonia, rhinitis, and the common cold.

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