Monoclonal antibodies, detection reagents and applications thereof for detecting human metapneumovirus

Abstract

The present invention relates to a monoclonal antibody, a detection reagent and an application thereof for detecting human metapneumovirus. The monoclonal antibody or an antigen-binding fragment thereof of the present invention can specifically bind to the N protein of human metapneumovirus with high affinity, has a broad spectrum of effectiveness against various genotypes, and has no cross-reactivity with other respiratory viruses. Therefore, it has great potential application value in clinical detection of human metapneumovirus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular virology, and in particular to a monoclonal antibody for detecting human metapneumovirus, a detection reagent and application thereof. Background Art

[0002] Human metapneumovirus (hMPV) is a new virus discovered by Dutch scholars in 2001. It belongs to the genus Metapneumovirus in the family Pneumoviridae and is the only member of this genus that can infect humans. hMPV is an important viral pathogen that causes acute respiratory tract infections in children, second only to human respiratory syncytial virus (hRSV) in the genus Orthopneumovirus in the family Pneumoviridae. The clinical symptoms of hMPV infection are difficult to distinguish from those of hRSV, ranging from mild upper respiratory tract infection (URTI) to more severe lower respiratory tract infection (LRTI); hMPV infection can aggravate the inflammatory response and lead to severe bronchiolitis and pneumonia. Therefore, the rapid and accurate identification of human metapneumovirus plays an important role in the prevention and quarantine of the disease. However, there is still a lack of clinical detection and diagnostic kits for the rapid detection of hMPV, and there is no kit for clinical antigen detection of hMPV on the market in China.

[0003] The hMPV genome consists of non-segmented, single-stranded negative-strand RNA with a total length of about 13kb, containing 8 gene open reading frames, encoding a total of 9 proteins, with the coding order: 3'-NPMF-M2-SH-GL-5'. Among them, F, G, and SH proteins are viral surface membrane proteins and are located on the surface of the virus; N protein is the core component of the virus particle. It binds to the viral genome RNA and packages the RNA into a ribonucleoprotein (RNP) complex, which is an important structural protein. In addition to assembly, N protein also plays an important role in viral mRNA transcription and replication, and participates in immune regulation; at the same time, N protein can also induce humoral and cellular immune responses after infection, making it a key target for early rapid diagnosis and vaccine development. hMPV can be divided into two genotypes, A and B, and each genotype can be further divided into at least two genotypes: A1, A2 and B1, B2. G protein has the greatest variability and is the main basis for hMPV genotyping; F protein is also the main gene used for hMPV typing, and N protein can also achieve complete typing of the virus. Therefore, the development of broad-spectrum antigen detection reagents and kits that can cover various genotypes has more practical and extensive application significance in the clinical detection and diagnosis of hMPV infection.

[0004] At present, the diagnostic techniques for hMPV infection include virus culture, nucleic acid detection, antigen detection and serological antibody detection. Virus culture is the gold standard for hMPV detection, but hMPV grows slowly, takes a long time to isolate and culture, and its lesions are not easy to observe, and the isolation positive rate is low. Viral nucleic acid detection is to collect respiratory specimens (nasopharyngeal swabs, sputum, alveolar lavage fluid, etc.) and extract nucleic acids, and use fluorescent quantitative PCR, viral gene sequencing and other methods to detect hMPV nucleic acid. It has high sensitivity and specificity, but is easily interfered by non-infectious residual nucleic acids, which is not conducive to the judgment of the course of disease. Serological antibody detection is a serological technique that uses the specific reaction of antigen and antibody to detect viral antibodies. It has high sensitivity and specificity, but because it takes time for antibodies to be produced, it is not conducive to the diagnosis of the acute stage of infection. Antigen detection is also a technique that directly detects viral antigens using the specific reaction of antigen and antibody. A positive result is an important basis for viral replication and symptomatic infection. Immunofluorescence is the main method for antigen detection and a commonly used method for laboratory pathogen detection. However, this method requires monoclonal antibodies that can specifically recognize and bind to hMPV antigen proteins with high affinity to ensure the accuracy and sensitivity of the detection. Currently, there is an urgent need to develop a highly specific, high-affinity monoclonal antibody against hMPV antigen that can be used for immunofluorescence detection of hMPV. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a specific monoclonal antibody with high affinity to human metapneumovirus N protein, its related products, and its preparation method and use.

[0006] The inventors of the present invention have obtained a monoclonal antibody after a large number of experimental screenings, which can specifically recognize and target the human metapneumovirus N protein and show high antigen binding activity. Therefore, the monoclonal antibody of the present invention is particularly suitable for antigen detection of human metapneumovirus.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human metapneumovirus N protein, which comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0009] The heavy chain variable region comprises:

[0010] HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

[0011] The light chain variable region comprises:

[0012] The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; wherein the amino acid sequence shown in SEQ ID NO: 5 is SAS.

[0013] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0014] a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:7; and,

[0015] A light chain variable region comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 8.

[0016] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0017] A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 7; and,

[0018] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0019] In addition, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody.

[0020] In a preferred embodiment, the monoclonal antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9; and, preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10.

[0021] In some preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0022] a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11; and,

[0023] A light chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0024] Further preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0025] A heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 11; and

[0026] The light chain has an amino acid sequence as shown in SEQ ID NO:12.

[0027] In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof further has a leader sequence (e.g., a signal peptide sequence) at the N-terminus of its heavy chain variable region and / or light chain variable region. In a feasible embodiment, the N-terminus of the heavy chain variable region has a leader sequence with an amino acid sequence such as that shown in SEQ ID NO: 13; and / or, the N-terminus of the light chain variable region has a leader sequence with an amino acid sequence such as that shown in SEQ ID NO: 14.

[0028] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, human antibody, chimeric antibody or bispecific or multispecific antibody.

[0029] The monoclonal antibody or antigen-binding fragment thereof of the present invention can bind to the human metapneumovirus N protein with high affinity and specificity, and thus can be used for antigen detection of the human metapneumovirus.

[0030] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0031] In a possible embodiment, the polynucleotide is a set of polynucleotides.

[0032] In some preferred embodiments, the polynucleotide set comprises:

[0033] (I) a first polynucleotide encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the first polynucleotide is a DNA molecule comprising the nucleotide sequences as shown in SEQ ID NOs: 15, 16, 17 (which can encode the amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively) or its corresponding mRNA molecule; and,

[0034] (II) A second polynucleotide encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the second polynucleotide is a DNA molecule comprising the nucleotide sequences as shown in SEQ ID NOs: 18, 19, 20 (which can encode the amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6 for LCDR1, LCDR2 and LCDR3, respectively) or its corresponding mRNA molecule; wherein the nucleotide sequence as shown in SEQ ID NO: 19 is TCGGCATCC.

[0035] Further preferably, the polynucleotide group comprises:

[0036] (I) a first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the first polynucleotide is a DNA molecule comprising the nucleotide sequence as shown in SEQ ID NO: 21 (which can encode the heavy chain variable region of the amino acid sequence as shown in SEQ ID NO: 7) or its corresponding mRNA molecule; and,

[0037] (II) a second polynucleotide encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the second polynucleotide is a DNA molecule comprising the nucleotide sequence as shown in SEQ ID NO: 22 (which can encode the light chain variable region of the amino acid sequence as shown in SEQ ID NO: 8) or its corresponding mRNA molecule;

[0038] In some specific embodiments, the first polynucleotide encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention and / or the second polynucleotide encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a nucleotide sequence encoding a leader sequence (e.g., a signal peptide sequence) at the 5' end; feasibly, the nucleotide sequence encoding the 5' leader sequence of the heavy chain variable region as shown in SEQ ID NO: 13 is as shown in SEQ ID NO: 27, and the nucleotide sequence encoding the 5' leader sequence of the light chain variable region as shown in SEQ ID NO: 14 is as shown in SEQ ID NO: 28.

[0039] Preferably, the above polynucleotide set also includes:

[0040] (III) a third polynucleotide encoding the heavy chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the third polynucleotide comprises a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 23 (which can encode the heavy chain constant region having an amino acid sequence as shown in SEQ ID NO: 9) or its corresponding mRNA molecule; and,

[0041] (IV) a fourth polynucleotide encoding the light chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention. Preferably, the fourth polynucleotide is a DNA molecule comprising the nucleotide sequence as shown in SEQ ID NO: 24 (which can encode the light chain constant region of the amino acid sequence as shown in SEQ ID NO: 10) or its corresponding mRNA molecule.

[0042] In the most preferred embodiment, the set of polynucleotides comprises:

[0043] (I) a first polynucleotide encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention, preferably, the first polynucleotide is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 25 or its corresponding mRNA molecule, which can encode the heavy chain with an amino acid sequence as shown in SEQ ID NO: 11; and,

[0044] (II) a second polynucleotide encoding a light chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention. Preferably, the second polynucleotide is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 26 or its corresponding mRNA molecule, which can encode a light chain having an amino acid sequence as shown in SEQ ID NO: 12.

[0045] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and, optionally, at least one expression control element operably linked to the polynucleotide.

[0046] In a fourth aspect, the present invention provides a vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0047] The vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0048] In some preferred embodiments, the vector is an expression vector, preferably a eukaryotic expression vector.

[0049] In a fifth aspect, the present invention provides a host cell comprising the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect;

[0050] The host cell includes, but is not limited to, prokaryotic cells, such as Escherichia coli cells, eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.

[0051] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0052] In a sixth aspect, the present invention provides a detection reagent for detecting human metapneumovirus, which comprises the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.

[0053] In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in certain preferred embodiments, the kit further comprises a second antibody, which specifically recognizes the monoclonal antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present invention; preferably, the second antibody further comprises a detectable label; such detectable labels are well known to those skilled in the art, and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

[0054] In a seventh aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0055] In an eighth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect and / or the detection reagent as described in the sixth aspect in the preparation of a product for detecting the presence or level of human metapneumovirus in a sample and / or for diagnosing human metapneumovirus infection.

[0056] Optionally, the sample is a biological sample from a subject.

[0057] In the ninth aspect, the present invention provides a method for detecting the presence or level of human metapneumovirus in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above and / or the detection reagent as described in the sixth aspect above.

[0058] In some preferred embodiments of this method, the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable label.

[0059] In some other preferred embodiments of the method, the method further comprises: using a secondary antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0060] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0061] Therefore, in some specific embodiments, the present invention provides a method for diagnosing whether a subject is infected with human metapneumovirus, which comprises: using the monoclonal antibody or antigen-binding fragment thereof of the present invention as described in the first aspect above to detect the presence of human metapneumovirus N protein in a sample from the subject. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in other preferred embodiments, the method further comprises using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present invention. In a feasible embodiment, the subject is a mammal, such as a human.

[0062] The general method of using monoclonal antibodies or antigen-binding fragments thereof to detect the presence or level of target viruses or antigens in samples is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method and similar detection methods.

[0063] Beneficial Effects

[0064] The monoclonal antibody of the present invention can specifically bind to the human metapneumovirus N protein with high affinity, and has broad-spectrum effectiveness against various genotypes of hMPV, and is therefore suitable for the detection of human metapneumovirus infections of all genotypes; and it has no cross-reactivity with other common respiratory viruses, and therefore has extremely high potential to be developed into a specific detection antibody for human metapneumovirus, and may be able to provide a lower detection limit and higher detection sensitivity, and has great clinical application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0066] Figure 1 This is the SDS-PAGE identification result of the monoclonal antibody Mab-hMPV 85D8.

[0067] Figure 2 This is the SDS-PAGE identification result of hMPV M, N, and F proteins expressed by transient transfection in HEK293FT cells.

[0068] Figure 3 The Western blot experiment is shown to detect the binding of monoclonal antibody Mab-hMPV 85D8 to hMPV N protein; wherein, "85D8" represents the monoclonal antibody Mab-hMPV 85D8 detection group, and "DFA" represents the positive control detection group.

[0069] Figure 4 The ELISA experiment is shown to detect the binding of monoclonal antibody Mab-hMPV 85D8 to hMPV M, N, and F proteins respectively.

[0070] Figure 5The IFA detection of the binding of the monoclonal antibody Mab-hMPV 85D8 to the hMPV N protein is shown; wherein, "DFA" represents the positive control detection group, and each concentration refers to the used concentration of the monoclonal antibody Mab-hMPV 85D8.

[0071] Figure 6 The results of IFA experiment on the detection efficiency of monoclonal antibody Mab-hMPV 85D8 for A and B genotype hMPV strains are shown; among them, "85D8-A genotype" and "85D8-B genotype" respectively represent the detection results of monoclonal antibody Mab-hMPV85D8 for A and B genotype hMPV strains, and "DFA-A genotype" and "DFA-B genotype" respectively represent the detection results of A and B genotype hMPV strains using the detection kit of the prior art.

[0072] Figure 7 The data show the clinical diagnostic effectiveness of the monoclonal antibody Mab-hMPV 85D8 for infection with different genotypes of hMPV strains; among them, "85D8-A2" and "85D8-B1" respectively represent the detection results of the monoclonal antibody Mab-hMPV 85D8 for A2 and B1 genotype hMPV strains, and "DFA-A2", "DFA-B1" and "DFA-B2" respectively represent the detection results of A2, B1 and B2 genotype hMPV strains using the detection kits using the prior art.

[0073] Figure 8 The cross-reactivity of the monoclonal antibody Mab-hMPV 85D8 with other respiratory viruses was shown. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] Unless explicitly stated otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0076] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0077] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity.

[0078] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, etc.

[0079] The "Fab" fragment consists of a light chain and the CH1 and variable regions of a heavy chain.

[0080] A "Fab'" fragment contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0081] The "F(ab')2" fragment contains two light chains and portions of two heavy chains including the VH domain, the CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0082] The "Fv" region contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0083] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0084] "Bispecific antibodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0085] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0086] The relevant sequence information of the monoclonal antibody 18_14D described in the following examples is shown in Table 1 below.

[0087] Table 1. Sequence information of monoclonal antibody Mab-hMPV 85D8

[0088] SEQ ID NO: Sequence description Sequence information 1 VH CDR1 GYSITSDYA 2 VH CDR2 ISYSGST 3 VH CDR3 ANADTRIYGSRRYWNFDV 4 VL CDR1 QNVGSN 5 VL CDR2 SAS 6 VL CDR3 QQYNNYPLT 7 VH DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYISYSGSTSYNPSLKSRISITRDPSKNQFFLQLNSVTTEDTATYYCANADTRIYGSRRYWNFDVWGAGTTVTVSS 8 VL DIVMTQSQKFMSTSVGDRVSVTCKASQNVGSNVAWYQQKPGQSPKPLIYSASYRSSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQQYNNYPLTFGVGTKLELK 9 CH AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 10 CL RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 11 Heavy chain DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYISYSGSTSYNPSLKSRISITRDPSKNQFFLQLNSVTTEDTATYYCANADTRIYGSRRYWNF DVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 12 L chain DIVMTQSQKFMSTSVGDRVSVTCKASQNVGSNVAWYQQKPGQSPKPLIYSASYRSSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQQYNNYPLTFGVGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 13 Signal peptide for VH MRVLILLWLFTAFPGILS 14 Signal peptide for VL MESQTQVFVYMLLWLSGVDG 15 VH CDR1 coding sequence GGCTACTCAATCACCAGTGATTATGCC 16 VH CDR2 coding sequence ATAAGCTACAGTGGTAGCACT 17 VH CDR3 coding sequence GCAAACGCAGACACTCGAATCTACGGTAGTAGACGGTACTGGAACTTCGATGTC 18 VL CDR1 coding sequence CAAAATGTGGGTAGTAAT 19 VL CDR2 coding sequence TCGGCATCC 20 VL CDR3 coding sequence CAGCAATATAACAACTATCCTCTCACG 21 VH genes GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGCCTACATAAGCTACAGTGGTAGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACCCATCCAAGAATCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAACGCAGACACTCGAATCTACGGTAGTAGACGGTACTGGAACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA 22 VL gene GACATTGTGATGACCCAGTCGCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAAAATGTGGGTAGTAATGTTGCCTGGTATCAGCAGAAACCAGGGCAATCTCCTAAACCACTGATTTACTCGGCATCCTATCGGTCCAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAGCAATATAACAACTATCCTCTCACGTTCGGTGTTGGGACCAAGCTGGAGCTGAAA 23 CH gene GCCAAAACGACACCCCCATCCGTTTATCCACTGGCCCCTGGAAGCGCCGCACAAACAAACTCAATGGTTACCCTCGGTTGCCTCGTCAAGGGATATTTTCCAGAGCCAGTCACAGTGACGTGGAATTCAGGCAGCTTGTCCTCCGGTGTTCACACATTTCCTGCCGTGCTCCAGTCCGACCTCTACACGCTCAGCTCCAGCGTTACAGTGCCAAGTTCTACTTGGCCAAGCCAGACCGTAACATGTAATGTGGCACACCCAGCTAGTTCAACGAAGGTGGACAAGAAAATAGTGCCCCGAGATTGTGGCTGTAAGCCATGCATCTGTACCGTCCCCGAAGTGTCAAGCGTGTTTATTTTTCCGCCTAAGCCCAAGGATGTCCTTACCATCACGTTGACTCCCAAGGTCACCTGTGTAGTGGTGGACATCTCTAAGGATGACCCCGAGGTACAATTTAGTTGGTTTGTGGATGACGTCGAGGTGCATACCGCCCAAACAAAGCCGAGGGAGGAGCAGATTAACAGCACTTTTCGCAGCGTGTCTGAGTTGCCTATCATGCACCAAGACTGGCTGAACGGGAAAGAATTCAAGTGCAGAGTGAACTCAGCAGCATTTCCGGCCCCAATCGAGAAGACCATCTCCAAGACTAAAGGGAGACCCAAGGCACCCCAGGTGTACACAATTCCCCCTCCGAAAGAACAAATGGCCAAAGACAAGGTCTCTTTGACCTGTATGATCACCAACTTTTTCCCTGAAGATATCACTGTGGAGTGGCAATGGAACGGCCAGCCTGCAGAGAACTACAAGAACACCCAGCCAATCATGGATACCGACGGGAGCTACTTCGTTTATAGTAAGTTGAACGTCCAGAAAAGTAACTGGGAAGCCGGCAACACCTTTACATGCAGCGTCCTTCACGAGGGGCTGCACAACCACCATACAGAGAAAAGCCTGTCCCATAGTCCCGGAAAG 24 CL gene CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 25 Heavy chain gene GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGCCTACATAAGCTACAGTGGTAGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACCCATCCAAGAATCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAACGCAGACACTCGAATCTACGGTAGTAGACGGTACTGGAACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCCGTTTATCCACTGGCCCCTGGAAGCGCCGCACAAACAAACTCAATGGTTACCCTCGGTTGCCTCGTCAAGGGATATTTTCCAGAGCCAGTCACAGTGACGTGGAATTCAGGCAGCTTGTCCTCCGGTGTTCACACATTTCCTGCCGTGCTCCAGTCCGACCTCTACACGCTCAGCTCCAGCGTTACAGTGCCAAGTTCTACTTGGCCAAGCCAGACCGTAACATGTAATGTGGCACACCCAGCTAGTTCAACGAAGGTGGACAAGAAAATAGTGCCCCGAGATTGTGGCTGTAAGCCATGCATCTGTACCGTCCCCGAAGTGTCAAGCGTGTTTATTTTTCCGCCTAAGCCCAAGGATGTCCTTACCATCACGTTGACTCCCAAGGTCACCTGTGTAGTGGTGGACATCTCTAAGGATGACCCCGAGGTACAATTTAGTTGGTTTGTGGATGACGTCGAGGTGCATACCGCCCAAACAAAGCCGAGGGAGGAGCAGATTAACAGCACTTTTCGCAGCGTGTCTGAGTTGCCTATCATGCACCAAGACTGGCTGAACGGGAAAGAATTCAAGTGCAGAGTGAACTCAGCAGCATTTCCGGCCCCAATCGAGAAGACCATCTCCAAGACTAAAGGGAGACCCAAGGCACCCCAGGTGTACACAATTCCCCCTCCGAAAGAACAAATGGCCAAAGACAAGGTCTCTTTGACCTGTATGATCACCAACTTTTTCCCTGAAGATATCACTGTGGAGTGGCAATGGAACGGCCAGCCTGCAGAGAACTACAAGAACACCCAGCCAATCATGGATACCGACGGGAGCTACTTCGTTTATAGTAAGTTGAACGTCCAGAAAAGTAACTGGGAAGCCGGCAACACCTTTACATGCAGCGTCCTTCACGAGGGGCTGCACAACCACCATACAGAGAAAAGCCTGTCCCATAGTCCCGGAAAG 26 Light chain gene GACATTGTGATGACCCAGTCGCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAAAATGTGGGTAGTAATGTTGCCTGGTATCAGCAGAAACCAGGGCAATCTCCTAAACCACTGATTTACTCGGCATCCTATCGGTCCAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAGCAATATAACAACTATCCTCTCACGTTCGGTGTTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 27 Coding sequence of the signal peptide for VH ATGAGAGTGCTGATTCTTTTGGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCT 28 Coding sequence for signal peptide of VL ATGGAGTCACAGACTCAGGTCTTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGA .

[0089] Example 1: Preparation and concentration of inactivated hMPV virus for the preparation of monoclonal antibodies

[0090] Step 1: hMPV virus culture and amplification

[0091] The hMPV 35378 strain was isolated from nasopharyngeal secretion specimens from children with acute respiratory tract infection who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics. The strain sequence has been uploaded to the GenBank database (GenBank: OR338754.1).

[0092] The hMPV 35378 strain was inoculated into the LLC-MK2+Vero E6 mixed culture cell culture system. When about 90% of the cells showed lesions and fell off, a large amount of virus liquid was collected and frozen at -80°C for later use.

[0093] Step 2: hMPV virus inactivation

[0094] After centrifuging the hMPV 35378 virus solution obtained in step 1 at 10,000 rpm and 4°C for 15 minutes, take the supernatant and add β-propiolactone (β-propiolactone working concentration 0.025%) to mix thoroughly. Place the mixed system at 4°C overnight to inactivate the virus, then place it in a 37°C water bath for 2 hours to decompose β-propiolactone to obtain an inactivated virus solution.

[0095] Step 3: Concentration of hMPV virus solution

[0096] The hMPV 35378 inactivated virus solution obtained in step 2 was concentrated by ultrafiltration, and the protein ultrafiltration column (Millipore Ultra-15, 10kD) was activated by 0.1N NaOH at 4°C before use. The inactivated virus solution was operated according to the instructions for use of the ultrafiltration column and centrifuged to obtain a virus concentrate; finally, after washing 3 times with PBS, the concentrated hMPV 35378 inactivated virus solution was collected and frozen at -80°C for future use.

[0097] Example 2: Preparation, screening, typing and sequencing of monoclonal antibodies

[0098] Step 1: Immunization of mice with hMPV

[0099] An equal amount of Freund's complete adjuvant was used to fully emulsify the inactivated hMPV virus (as obtained in Example 1) as an immunogen; the immunogen was used to perform multiple subcutaneous immunizations on the back of the neck of BALB / C female mice about 6 weeks old, with 3 mice in each group and an inoculation volume of 100 μL / mouse; two weeks later, an equal amount of Freund's incomplete adjuvant was used to fully emulsify the inactivated hMPV virus and perform a second immunization, with the same dose of 100 μL / mouse; after another two weeks, a third immunization was performed, with the same steps as the second immunization; 7 days after the third immunization, blood was collected from the tail vein to determine the antibody titer in the serum, and the mouse with the highest antibody titer was selected for fusion.

[0100] Step 2: Cell fusion to produce hybridoma cells

[0101] The mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, and fixed on the dissection board in the clean bench. After the abdominal cavity was exposed and the spleen was aseptically removed, the spleen was cut into pieces after the connective tissue around the spleen was removed. The spleen was transferred to a 3.5 cm plate with a filter to separate the spleen cells. The spleen cells were resuspended in serum-free medium, centrifuged at 800 rpm for 5 minutes at room temperature, and the supernatant was discarded. The mouse spleen cells were prepared into a single cell suspension (about 1×10 8 / mL); at the same time, the growing mouse myeloma cells (about 3×10 7 / mL) in a volume ratio of 3:1, and discard the supernatant after centrifugation. Gently shake the centrifuge tube to loosen and evenly form a paste; use a 1mL pipette to slowly drip 1mL of 50% PEG preheated to 37°C along the tube wall, while rotating while dripping. After the addition is complete, the cells are still for 30 seconds to 1 minute; use a pipette to add incomplete culture medium preheated to 37°C in batches, while rotating and dripping the culture medium, slowly terminate the effect of PEG, 1mL each time for a total of 10 mL; centrifuge at 800 rpm for 5 minutes at room temperature, discard the supernatant; suspend with bovine serum. Add the fused cells to HAT selection medium (containing 20% ​​bovine serum, 1% double antibody, HAT) containing an appropriate amount of feeder layer cells and spread 10 96-well plates.

[0102] Step 3: Preparation and typing of monoclonal antibodies

[0103] About 10 days after cell fusion, cell clusters can be seen forming. The culture medium in the 96-well plate is discarded and replaced with HAT selection medium without feeder cells. The next day, the culture supernatant is taken and ELISA is performed using an enzyme-labeled plate coated with hMPV virus lysate to preliminarily screen the hybridoma cell plate. The initially selected positive clones are transferred to a plate pre-coated with feeder cells for continued culture and re-examination, and the strongly positive wells are subcloned by limiting dilution. Two weeks later, positive monoclonal clones are selected to prepare ascites. Hybridoma cells (0.5×10 6 / mL) was injected into the peritoneal cavity of mice. A total of 5 mice were inoculated. Ascites was collected from mice with swollen abdominal cavity 7-10 days later. The collected ascites was centrifuged and the supernatant was collected and frozen.

[0104] Through the above steps and after appropriate screening, hMPV monoclonal antibody 85D8 (also referred to as Mab-hMPV85D8 in this article) was obtained; the supernatant was detected using the Luoyang Bio-Tong Experimental Materials Center Company Mouse Monoclonal Antibody Ig Class / Subclass Identification Kit (Cat. No.: C060101), and its antibody subtype was rated as IgG1 subtype.

[0105] Step 4: Monoclonal Antibody Sequencing

[0106] The monoclonal antibody hybridoma cell line was cultured at 37°C and 5% CO2 for 3 days until the vigorous growth phase, and 1×10 6 The cells were centrifuged at 900 rpm for 5 min, and the supernatant was removed. The cell pellet was fully lysed using TRIzol (Thermo) to extract total RNA. The RNA was reverse transcribed into cDNA using the SMART RACE method, and the cDNA was amplified by PCR to obtain the antibody heavy chain / light chain gene. The antibody gene was connected to the T vector, and the positive clones were selected and sequenced using the antibody universal primers for first-generation sequencing to obtain the antibody sequence.

[0107] The relevant sequence information of the monoclonal antibody Mab-hMPV 85D8 is shown in Table 1.

[0108] The monoclonal antibody Mab-hMPV 85D8 was subjected to SDS-PAGE gel electrophoresis, and then stained with Coomassie Brilliant Blue overnight. After staining, the monoclonal antibody Mab-hMPV 85D8 was washed with pure water for 1 hour, and then the protein bands were observed.

[0109] The results are as follows Figure 1 As shown, Figure 1 Display: The SDS-PAGE electrophoresis result of the monoclonal antibody Mab-hMPV 85D8 showed two clear bands. Judging from their molecular weight positions, the two bands were the heavy chain and light chain of the antibody, respectively.

[0110] Example 3: Expression and purification of hMPV M, N, and F protein antigens

[0111] In this example, hMPV M, N, and F protein antigens were prepared by the following steps:

[0112] Step 1: Construction of hMPV M, N, and F gene expression vectors

[0113] The F, M and N proteins of hMPV virus were expressed in the prokaryotic expression system. The amino acid sequence of the F protein of hMPV BJ1887 strain (GenBank: DQ843659.1) is GenBank: AAV88364.1, the amino acid sequence of the M protein is GenBank: AAV88366.2, and the amino acid sequence of the N protein is GenBank: AAS88425.1. Beijing Qingke Biotechnology Co., Ltd. was commissioned to design and synthesize the gene sequence according to the Escherichia coli expression system, and cloned into the pRec3.0R expression vector, respectively, to obtain three expression plasmids: pRec3.0R-MPVF, pRec3.0R-MPVM and pRec3.0R-MPVN.

[0114] Step 2: Expression, purification and identification of hMPV M, N and F proteins

[0115] The expression plasmids pRec3.0R-MPVF, pRec3.0R-MPVM and pRec3.0R-MPVN were transformed into BL21 (DE3) respectively, and single colonies were picked and cultured in LB medium shake flasks at 30°C. After 6 hours, IPTG with a final concentration of 1mM was added to induce expression. After 4 hours of continuous culture, the cells were collected by centrifugation; resuspended in 10mM PBS, ultrasonically disrupted, and centrifuged at 10,000 rpm for 10 minutes to collect the supernatant. Metal chelation chromatography was performed using Ni affinity filler for purification. The Ni column was balanced with equilibration solution (20mMTris+0.15M NaCl+20mM imidazole, pH8.0) for 3 column volumes. After loading the supernatant sample, the equilibration solution was used to wash the column for 1 column volume. 13%, 35%, and 100% eluent (20mM Tris+0.15M NaCl+250mM imidazole, pH8.0) were used for gradient elution, and the eluent was collected for SDS-PAGE to detect the purity of the product. The sample with the highest purity was selected and the buffer was replaced with PBS.

[0116] The size of the relatively pure target protein hMPV-F protein was 58KDa, the size of M protein was 28KDa, and the size of N protein was 43KDa. The SDS-PAGE analysis showed that the sizes of hMPV-F, M, and N proteins were consistent with the expected sizes (see the results). Figure 2 ).

[0117] Example 4: Validation of the antigen binding site of hMPV monoclonal antibodies

[0118] In this example, hMPV M, N, and F proteins expressed in Example 3 were used as antigens, and the binding of the monoclonal antibody Mab-hMPV 85D8 to these antigens was detected by ELISA and indirect immunofluorescence assay to preliminarily determine its specific antigen.

[0119] ELISA experiment

[0120] Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of monoclonal antibody Mab-hMPV 85D8 to prokaryotically expressed hMPV M, N, and F proteins, and to detect the binding of antibody Mab-hMPV 85D8 to each antigen to determine the antigen binding site of the antibody.

[0121] Specifically, 293FT cells were cultured at 2×10 4 96-well plates were plated with 10 cells / well. After 24 hours, serum-free medium was replaced and hMPV M, N, and F prokaryotic expression plasmids were transiently transfected at a transfection volume of 0.6 μg / well. After 48 hours, the serum-free medium was discarded and the cells were fixed with paraformaldehyde cell fixative at room temperature for 10-15 minutes. The plate was washed 3 times with PBST, and then 250 μL HSA blocking solution was added for blocking. After incubation at 37°C for 2 hours, 1 μg / mL Mab-hMPV 85D8 primary antibody solution was added to the cell wells, incubated at 37°C for 30-60 minutes, and the reaction wells were washed 3 times with PBST washing solution. A 1:6000 dilution of HRP enzyme-labeled goat anti-mouse secondary antibody was added, incubated at 37°C for 60 minutes, the reaction wells were washed 3 times with PBST washing solution, and the plate was washed 3 times with purified water. Color development process: add 100 μL of single-component color substrate diaminobenzidine (DAB) color development solution, develop at 37°C in the dark for 10 min, then add 100 μL of stop solution to terminate the reaction, detect the OD450 (mean ± standard deviation) of the reactants, and analyze the binding of antibody Mab-hMPV 85D8 to antigen hMPV M, N, and F proteins.

[0122] The results are shown in Table 2.

[0123] Table 2. Binding activity test of Mab-hMPV and hMPV M, N, and F proteins

[0124] Blank cell control M F N Negative control 0.1803 0.2041 0.1600 1.0629 0.1228 0.1841 0.1835 0.1355 1.1901 0.1238 0.1889 0.1446 0.1627 0.4583 0.1254 .

[0125] As shown in Table 2, when the concentration of Mab-hMPV 85D8 was 1 μg / mL, the OD450 of its reaction with hMPV N protein was 0.90±0.39, which was significantly higher than the OD450 of the reaction with the other two antigenic proteins hMPV M and F. At the same time, the reactivity of antigenic proteins hMPV M and F with antibodies was similar to that of the negative control group, with no statistical difference. This result shows that Mab-hMPV 85D8 binds to hMPV N protein, but not to M and F proteins, suggesting that the antigen binding site of Mab-hMPV 85D8 may be located on the hMPV N protein.

[0126] Indirect immunofluorescence (IFA) experiments

[0127] The IFA experiment was used to detect the binding of the monoclonal antibody Mab-hMPV 85D8 to the prokaryotic expressed hMPV N protein to confirm the antigen binding site of the antibody.

[0128] Specifically, 293FT cells were cultured at 1×10 5 12-well plates were plated with cells / well. After 24 hours, the serum-free medium was replaced, and the hMPV N prokaryotic expression plasmid was transiently transfected with a transfection amount of 0.6 μg / well. After 48 hours, the serum-free medium was discarded, and the cells were digested and collected using trypsin. The cells were fixed on the slides at room temperature for 10-15 minutes using paraformaldehyde cell fixative. The slides were washed 3 times with PBS, and Mab-hMPV 85D8 was added for antibody incubation at 4°C overnight, with 5 μL of antibody added to each well. Afterwards, the slides were washed 3 times with PBS, and FITC-labeled sheep anti-mouse fluorescent secondary antibody IgG was added to each well. The antibodies were incubated in a 37°C wet box for 1 hour. The slides were washed 3 times with PBS and observed under a fluorescent microscope. At the same time, a positive detection control group was set up. In the positive detection control group, the hMPV in vitro diagnostic kit D of the prior art was used. 3 DFA Metapneumovirus Identification Kit (REF: 01-030000, DIAGMOSTIC HYBRIDS, INC., Athens, OH, USA) was used for detection. Specifically, the detection reagent was directly added to the slide and the antibody was incubated in a 37°C wet box for 30 min. After that, the slide was washed 3 times with PBS and observed by fluorescence microscopy.

[0129] The results are as follows Figure 3 As shown, Figure 3The results show that in the cell smear expressing hMPV-BJ1816 N protein, more specific green fluorescent cells appeared after incubation with antibody Mab-hMPV 85D8; compared with the positive detection control group (i.e., the "DFA" group in the attached figure), its specificity is outstanding. This indicates that the antibody Mab-hMPV 85D8 has a significant antibody-antigen reaction with the hMPV-BJ1816 N protein, and the antibody binding site of Mab-hMPV 85D8 and hMPV may be located on the N protein.

[0130] Example 5: Potency Verification of Mab-hMPV 85D8 Monoclonal Antibody

[0131] In this example, hMPV M, N, and F proteins expressed in Example 3 were used as antigens, and the binding of monoclonal antibody Mab-hMPV 85D8 to hMPV N protein antigen was detected by ELISA and IFA experiments to preliminarily verify its potency.

[0132] ELISA experiment

[0133] Specifically, a 96-well ELISA plate was taken, and hMPV M, N, and F antigen protein coating solution (100 μL / well) was added, and the plate was coated overnight at 4°C. The protein concentration of the coating solution was 1 μg / well. The negative control was coated with PBS; the positive control group was directly coated with HRP-labeled goat anti-mouse secondary antibody. The plate was washed 3 times with PBST, and then 250 μL of HSA blocking solution was added for blocking. After incubation at 37°C for 2 hours, Mab-hMPV 85D8 primary antibody solution was added to each well, with an initial concentration of 8 μg / mL, followed by 2-fold dilution, incubation at 37°C for 1 hour, and the reaction wells were washed 3 times with PBST washing solution. A 1:6000 diluted HRP enzyme-labeled goat anti-mouse secondary antibody was added, incubated at 37°C for 1 hour, the reaction wells were washed 3 times with PBST washing solution, and the plate was washed 3 times with purified water. Color development process: add 100 μL of single-component color substrate diaminobenzidine (DAB) color development solution, develop at 37°C in the dark for 10 min, then add 100 μL of stop solution to terminate the reaction, detect the OD450 (mean ± standard deviation) of the reactants, and analyze the binding of antibody Mab-hMPV 85D8 to antigen hMPV M, N, and F proteins.

[0134] The results are as follows Figure 4 As shown. Figure 4It can be seen that when the dilution concentration of Mab-hMPV 85D8 is 8μg / mL, the OD450 of its reaction with hMPV N protein is about 2.6, which is significantly higher than the reactivity with other hMPV proteins; when the Mab-hMPV 85D8 antibody is diluted 2-fold, its antibody titer gradually decreases, and the reactivity of the antibody with hMPV N protein shows a dose-dependent characteristic, until the antibody dilution drops to 62.5ng / mL, its reactivity with hMPV N protein is similar to that with hMPV M and F proteins, and it is determined that the antibody activity is no longer. After calculation, the EC50 of the monoclonal antibody Mab-hMPV 85D8 binding to hMPV N protein is 1.02μg / mL.

[0135] At the same time, no matter at what dilution, the average OD450 of the reaction of Mab-hMPV 85D8 binding to hMPV M and F proteins did not change significantly, indicating background interference activity, that is, Mab-hMPV 85D8 did not bind to hMPV M and F.

[0136] The above results show that the binding reactivity of Mab-hMPV 85D8 to hMPV N protein is significant, and the antigen-antibody reactivity shows a clear dose-dependent relationship.

[0137] IFA Experiment

[0138] The IFA experiment was used to detect the binding of the monoclonal antibody Mab-hMPV 85D8 to the antigen in the positive cells infected with the hMPV strain to confirm the titer of the antibody.

[0139] Specifically, the hMPV strain (BJ35378, GenBank: OR338754.1) was isolated, cultured and amplified from clinical specimens using the Vero-E6 & LLC-MK2 mixed culture cell system. At the same time, infected positive cells were collected and cell smears were performed. The cells were fixed with cold acetone for 15 minutes, and then Mab-hMPV 85D8 antibody with a 2-fold dilution was added. The antibody was incubated overnight at 4°C, and 5 μL of antibody was added to each well. After that, the slides were gently washed 3 times with PBS, and FITC-labeled sheep anti-mouse fluorescent secondary antibody IgG was added to each well. The antibodies were incubated in a 37°C wet box for 1 hour, and fluorescent microscopy was performed after gently washing with PBS. In the positive control group, the hMPV in vitro diagnostic kit D was used. 3 DFA Metapneumovirus Identification Kit, add the detection reagent directly to the slide, incubate the antibody in a 37℃ wet box for 30min. Then, wash the slide 3 times with PBS and observe under fluorescence microscope.

[0140] The results are as follows Figure 5 As shown. Figure 5 It can be seen that after Mab-hMPV 85D8 was incubated with hMPV-infected positive cell smears, obvious specific green fluorescent cells appeared; and the fluorescence intensity was proportional to the antibody dose, that is, as the antibody dose decreased, the fluorescence intensity decreased. When the antibody dose reached 1μg / mL, its fluorescence intensity was similar to that of the DFA positive detection control group. This shows that the antibody Mab-hMPV 85D8 had an obvious antibody-antigen reaction with hMPV, and when the antibody concentration reached 1μg / mL, its titer could reach the detection level of the current detection kit.

[0141] Example 6: Verification of the broad-spectrum effectiveness of Mab-hMPV 85D8 monoclonal antibody in detecting hMPV types

[0142] In this example, cells infected with genotype A and B hMPV strains and clinical samples of children were used as antigen specimens. The binding of the monoclonal antibody Mab-hMPV 85D8 to antigens of different genotypes of hMPV strains was detected by IFA experiments to determine whether it is effective for each genotype when used for hMPV detection. At the same time, a prior art kit detection group was set up as a control test to compare the consistency and sensitivity of the detection of the kit of the present invention with that of the prior art kit; in this example, the hMPV in vitro diagnostic kit D was used. 3 DFA Metapneumovirus Identificacion Kit is a kit used as a prior art kit for clinical hMPV detection and identification.

[0143] Cell experiment detection

[0144] Specifically, hMPV strains of genotype A and B (genotype A strain, BJ35378, GenBank: OR338754.1; genotype B strain, BJ29472, GenBank: OR338753.1) were isolated, cultured and amplified from clinical specimens, and infected Vero-E6 & LLC-MK2 mixed culture cells were collected and cell smears were performed. The cells were fixed with cold acetone for 15 minutes, and then 1 μg / mL Mab-hMPV 85D8 was added for antibody incubation at 4°C overnight, and 5 μL of antibody was added to each well. After that, the slides were gently washed with PBS 3 times, and FITC-labeled goat anti-mouse fluorescent secondary antibodies were added to each well, and the antibodies were incubated for 1 hour in a 37°C wet box, and then washed with PBS for fluorescence microscopy observation.

[0145] At the same time, a positive detection control group was set up, in which the hMPV in vitro diagnostic kit D of the prior art was used. 3DFA Metapneumovirus Identification Kit (REF: 01-030000, DIAGMOSTICHYBRIDS, INC., Athens, OH, USA) was used for detection. Specifically, the detection reagent was directly added to the slide and the antibody was incubated in a 37°C wet box for 30 minutes. Afterwards, the slide was washed 3 times with PBS and observed by fluorescence microscopy.

[0146] The results are as follows Figure 6 shown by Figure 6 It can be seen that in the cell smears infected with hMPV-A and B genotype strains, after incubation with 1μg / mL antibody Mab-hMPV 85D8, obvious specific green fluorescent cells appeared; its fluorescence intensity was significantly higher than that of the positive detection control group. This result shows that the antibody Mab-hMPV 85D8 has an antibody-antigen reaction with both A and B genotype hMPV, that is, the immune reaction of Mab-hMPV 85D8 with each genotype of hMPV has a broad spectrum of effectiveness.

[0147] Clinical specimen testing

[0148] Clinical specimens from pediatric patients with acute respiratory tract infection who were admitted to the Children's Hospital of Capital Institute of Pediatrics from January 2010 to December 2014 were collected, including pharyngeal swabs (TS), nasopharyngeal swabs (NPS), nasopharyngeal aspirates (NPA), and bronchoalveolar lavage fluid (BLF). The samples were added with 2.5 ml of virus transport medium in a Class II biosafety cabinet and centrifuged at 500 g for 10 minutes. The supernatant was used for nucleic acid extraction, and the remainder was stored at -80°C for later use; the precipitate was smeared, fixed with acetone, and stored at -20°C for later use. For specimens that were positive for hMPV in nucleic acid detection, the cell pellet was resuspended in PBS and spotted on a slide, fixed in precooled acetone, and stored at -20°C. Before the experiment, the smear was balanced to room temperature, and 5 μL of the monoclonal antibody of the present invention (1:20, 1:40, 1:80) diluted with PBS was added to the slide, and incubated in a 37°C wet box for 20-30 minutes. After gently washing with PBS, 5 μL of FITC-conjugated goat anti-mouse IgG antibody diluted with 1:1000 PBS was added to the slide, incubated in a 37°C wet box for 30 minutes, and then washed with PBS. The smear was placed in a 0.04% trypan blue liquid and stained for 5 minutes. After washing with PBS, the slide was blown dry and the results were observed under a fluorescence microscope. The treatment method of the control test group was the same as that described in the "Cell Experiment Detection" section above.

[0149] The results are as follows Figure 7 shown by Figure 7It can be seen that in the clinical specimen smears infected with hMPV-A and B genotypes (genotypes A2, B1, and B2 respectively according to nucleic acid detection), after incubation with 1 μg / mL antibody Mab-hMPV 85D8, good specific green fluorescent cells appeared; especially in the detection of specimens of children infected with B1 and B2 genotype strains, the fluorescence intensity of the reaction was significantly better than that of the control detection group. The results show that the antibody Mab-hMPV 85D8 has an antibody-antigen reaction with hMPV of genotypes A and B, that is, the reaction of Mab-hMPV 85D8 with each genotype of hMPV has a broad spectrum of effectiveness; and in the detection of clinical specimens infected with different genotypes of hMPV, the detection efficiency of Mab-hMPV 85D8 is equal to or even higher than that of the control detection group using the prior art kit. This suggests that in the detection of antigens in clinical respiratory specimens, the monoclonal antibody Mab-hMPV 85D8 of the present invention can efficiently detect hMPV infection.

[0150] The above results indicate that Mab-hMPV 85D8 is a broad-spectrum and highly effective monoclonal antibody against hMPV.

[0151] Example 7: Validation of clinical diagnostic effectiveness of Mab-hMPV 85D8-specific monoclonal antibody

[0152] Slides of cells infected with HAdV, Flu A and B, RSV, PIV1, 2 and 3 in the D3 Ultra™ DFA Respiratory Virus Screening Kit (Diagnostic Hybrids Inc., Athens, OH, USA) were used as other common pediatric pathogens to verify the diagnostic specificity of Mab-hMPV 85D8 monoclonal antibody. HAdV: Human adenovirus; Flu A, B: Influenza A, B; RSV: Respiratory Syncytial Virus; PIV1, 2 and 3: Parainfluenza Virus, Parainfluenza virus type 1, type 2 and type 3. NC: Negetive Control, negative uninfected cell control.

[0153] Test results such as Figure 8 As shown; Figure 8 The results showed that no specific green fluorescent cells appeared when the antibody Mab-hMPV 85D8 reacted with positive cells infected with ADV, influenza A and B, RSV and PIV1-3, indicating that the monoclonal antibody Mab-hMPV 85D8 had no cross-reactivity with other respiratory viruses.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human metapneumovirus N protein, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; The light chain variable region comprises: The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; wherein the amino acid sequence shown in SEQ ID NO: 5 is SAS.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:7; and, A light chain variable region comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein: The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 7; and, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof further comprises a constant region; the constant region comprises a heavy chain constant region as shown in SEQ ID NO: 9 and a light chain constant region as shown in SEQ ID NO:

10.

5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof comprises: a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11; and, A light chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:

12.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein The antibody or antigen-binding fragment thereof comprises: A heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 11; and The light chain has an amino acid sequence as shown in SEQ ID NO:

12.

7. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The polynucleotide according to claim 7, wherein The polynucleotide is a polynucleotide group, and the polynucleotide group includes: (I) a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 15, 16, 17; and, (II) A DNA molecule comprising the nucleotide sequences shown in SEQ ID NOs: 18, 19, and 20; wherein the nucleotide sequence shown in SEQ ID NO: 19 is TCGGCATCC.

9. The polynucleotide according to claim 8, wherein The polynucleotide set comprises: (I) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 21; and, (II) A DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:

22.

10. The polynucleotide according to claim 9, wherein The polynucleotide set also includes: (III) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 23; and, (IV) A DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:

24.

11. The polynucleotide according to claim 9, wherein The polynucleotide set comprises: (I) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 25; and, (II) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO:

26.

12. A nucleic acid construct comprising the polynucleotide according to any one of claims 7 to 11, and at least one expression control element operably linked to the polynucleotide.

13. A vector comprising the polynucleotide according to any one of claims 7 to 11, or the nucleic acid construct according to claim 12.

14. A host cell transformed with the polynucleotide according to any one of claims 7 to 11, the nucleic acid construct according to claim 12 or the vector according to claim 13.

15. A detection reagent for detecting human metapneumovirus, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

16. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the method comprising: The host cell of claim 14 is allowed to express the monoclonal antibody or the antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or the antigen-binding fragment thereof, and the expressed monoclonal antibody or the antigen-binding fragment thereof is recovered from the culture of the host cell.

17. Use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, the polynucleotide as described in any one of claims 7 to 11, the nucleic acid construct as described in claim 12, the vector as described in claim 13, the host cell as described in claim 14 and / or the detection reagent as described in claim 15 in the preparation of a product for detecting the presence or level of human metapneumovirus in a sample and / or for diagnosing human metapneumovirus infection.

18. The use according to claim 17, characterized in that The sample is a biological sample from a subject.

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