CN117126270B

CN117126270B - Type 2 human bocavirus type specific antibody and application thereof

Info

Publication number: CN117126270B
Application number: CN202311394500.3A
Authority: CN
Inventors: 赵林清; 德日; 张可祥
Original assignee: Capital Institute of Pediatrics
Current assignee: Capital Institute of Pediatrics
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-02-13
Anticipated expiration: 2043-10-25
Also published as: CN117126270A

Abstract

The invention relates to a type-specific monoclonal antibody or an antigen binding fragment thereof aiming at type 2 human bocavirus, a related product thereof, a preparation method and application thereof. The monoclonal antibody or antigen binding fragment thereof can specifically bind to VP3 protein of human bocavirus type 2 (HBoV 2), but does not bind to VP3 protein of human bocavirus type 1 (HBoV 1), so that the monoclonal antibody can be used for specific detection or diagnosis of HBoV2 or for differential diagnosis between HBoV1 and HBoV 2; in addition, the binding affinity of the kit and the HBoV2 VP3 protein is higher, so that lower detection line can be provided for the detection of the HBoV2, and the sensitivity of the detection or diagnosis of the HBoV2 is improved.

Description

Type 2 human bocavirus type specific antibody and application thereof

Technical Field

The invention relates to the technical fields of immunology and molecular virology, in particular to a type-specific monoclonal antibody or an antigen binding fragment thereof aiming at type 2 human bocavirus, a preparation method and application thereof.

Background

Since the first time in 2005 swedish scientist alander et al found Human bocavirus (HBoV) in nasopharynx aspirates of infants suffering from respiratory tract infections, researchers in various countries have successively found three different genotypes of HBoV; so far, HBoV has four genotypes, designated HBoV1, 2, 3, 4, respectively. More and more clinical evidence supports that HBoV1 is the causative virus of lower respiratory tract infection, while HBoV 2-4 is detected mainly in stool specimens of children suffering from diarrhea, and the detection rate of HBoVs, especially HBoV2, in the stool specimens of infected intestinal tracts is higher than that of other genotypes, suggesting that the HBoVs may be related to acute gastroenteritis of children.

HBoV belongs to the subfamily Paramyxoviridae, the genus Bokavirus, and is the second member of the family Paramyxoviridae that is pathogenic to humans following parvovirus B19. HBoV is a single negative strand non-enveloped DNA virus, approximately 25 nm in diameter, icosahedral symmetric, and approximately 5kb in genome overall length.

HBoV1 has a hairpin structure specific to parvoviridae, a rabbit-ear hairpin at the 3 '(LEH) end and a classical hairpin at the 5' end (REH). HBoV1 is transcribed by a left end promoter P5 of a genome, and mature mRNA with different lengths is formed through RNA splicing and processing, and then viral protein expression is carried out. The genome of HBoV1 contains three major open reading frame ORFs, wherein the ORFs at the 3' end encode the nonstructural proteins NS1, NS2, NS3, NS4; the middle ORF encodes the nonstructural protein NP1 (which is characteristic of bocavirus); the ORF at the 5' end encodes the capsid proteins VP1, VP2, VP3, wherein the VP2, VP3 gene coding region starts inside the VP1 gene, is a typical overlapping gene, and the encoded proteins have a common carboxy-terminus. Capsid protein VP1: VP2: VP3 is expressed in a ratio of 1:1:10, VP3 is the most main protein for forming icosahedral capsids, and the capsid protein VP3 can be independently expressed to form virus-like particles in a self-assembled mode, so that the virus-like particles have strong immunogenicity and can induce strong humoral and cellular immune responses.

Because of the high homology between VP3 protein sequences of HBoV1 and HBoV2 and the cross-reactive OAS phenomenon in serology research, the preparation of high-affinity type specific antibodies specific to HBoV1 or HBoV2 is a key for specifically recognizing two types of human bocaviruses and performing differential diagnosis on the two types of human bocaviruses.

Disclosure of Invention

Object of the Invention

Aiming at the problems or needs in the prior art, the invention aims to provide a type-specific monoclonal antibody or an antigen binding fragment thereof with higher affinity for human bocavirus type 2, and a preparation method and application thereof.

Solution scheme

In order to achieve the above object, the present invention provides a type-specific monoclonal antibody against HBoV2, which is capable of specifically binding to VP3 protein of HBoV2 with high affinity, but not to VP3 protein of HBoV1, thereby enabling specific detection of HBoV2 virus infection, through a large number of screening.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a type-specific monoclonal antibody, or antigen-binding fragment thereof, directed against human bocavirus type 2 comprising a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region comprises:

three complementarity determining regions HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 37, respectively;

and/or, the light chain variable region comprises:

the amino acid sequences are shown as three complementarity determining regions LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 38, LAS and SEQ ID NO: 39, respectively.

Preferably, the heavy chain variable region further comprises framework regions H-FR1, H-FR2, H-FR3 and H-FR4 of amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, respectively;

and/or the light chain variable region further comprises framework regions L-FR1, L-FR2, L-FR3 and L-FR4 of amino acid sequences shown in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

Further preferably, the heavy chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 48 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 48;

and/or the light chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 49 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 49.

In a preferred embodiment, the monoclonal antibody or antigen binding fragment thereof comprises:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 48; and, a step of, in the first embodiment,

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 49.

In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof further has a leader sequence at the N-terminus of its heavy chain variable region and/or light chain variable region; preferably, the leader sequence of the heavy chain variable region has the amino acid sequence shown as SEQ ID NO. 65, further preferably, the leader sequence is encoded by the nucleotide sequence shown as SEQ ID NO. 66; preferably, the leader sequence of the light chain variable region has the amino acid sequence shown as SEQ ID NO. 67, and more preferably, the leader sequence is encoded by the nucleotide sequence shown as SEQ ID NO. 68.

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 group consisting of: constant regions of IgG, igA, or IgM antibodies.

In preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof 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 shown as SEQ ID NO. 50; also, preferably, the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 51.

In some preferred embodiments, the monoclonal antibody or antigen binding fragment thereof comprises:

a heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID No. 52 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 52; and, a step of, in the first embodiment,

a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID No. 53 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 53.

Further preferred, the monoclonal antibody or antigen binding fragment thereof comprises:

a heavy chain with an amino acid sequence shown as SEQ ID NO. 52; and, a step of, in the first embodiment,

the amino acid sequence of the light chain is shown as SEQ ID NO. 53.

In some possible embodiments, the antigen binding fragment of the monoclonal antibody is selected from the group consisting of Fab, fab ', F (ab') ₂ Fd, fv, dAb, complementarity determining region fragments, single chain antibodies, human antibodies, chimeric antibodies or bispecific or multispecific antibodies.

In a second aspect, the present invention provides a polynucleotide encoding a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above. The polynucleotide is not limited to the method of its production and may be obtained using genetic engineering recombinant techniques or chemical synthesis methods.

In a possible embodiment, the polynucleotides are a set of polynucleotides.

In some preferred embodiments, the set of polynucleotides comprises:

(I) A first polynucleotide encoding the heavy chain variable regions of the monoclonal antibodies or antigen binding fragments thereof of the invention, preferably, the first polynucleotide is a DNA molecule comprising the nucleotide sequences shown as SEQ ID NOs 54, 55, 56 (which may encode the amino acid sequences shown as SEQ ID NOs 35, 36 and 37, respectively, HCDR1, HCDR2 and HCDR3, respectively) or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(II) a second polynucleotide encoding the light chain variable region LCDR1, LCDR2 and LCDR3 of the monoclonal antibodies or antigen-binding fragments thereof of the invention, preferably said second polynucleotide is a DNA molecule comprising the nucleotide sequences set forth in SEQ ID NO 57, CTTGCATCC, SEQ ID NO 58 (which may encode the amino acid sequences set forth in SEQ ID NO 38, LAS and SEQ ID NO 39, respectively, LCDR1, LCDR2 and LCDR 3) or a corresponding mRNA molecule thereof.

Further preferably, the polynucleotide group comprises:

(I) A first polynucleotide encoding the heavy chain variable region of a monoclonal antibody or antigen binding fragment thereof of the invention, preferably said first polynucleotide is a DNA molecule comprising a nucleotide sequence shown as SEQ ID No. 59 (which may encode a heavy chain variable region having the amino acid sequence shown as SEQ ID No. 48) and optionally a nucleotide sequence shown as SEQ ID No. 66 (which encodes a leader sequence at the N-terminus of a VH chain) or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(II) a second polynucleotide encoding the light chain variable region of a monoclonal antibody or antigen binding fragment thereof of the invention, preferably said second polynucleotide is a DNA molecule comprising a nucleotide sequence as set forth in SEQ ID No. 60 (which may encode a light chain variable region having the amino acid sequence set forth in SEQ ID No. 49) and optionally a nucleotide sequence as set forth in SEQ ID No. 68 (which encodes a leader sequence at the N-terminus of the VL chain) or a corresponding mRNA molecule thereof;

preferably, the polynucleotide set further comprises:

(III) a third polynucleotide encoding the heavy chain constant region of the monoclonal antibody or antigen binding fragment thereof of the invention, preferably, the third polynucleotide is a DNA molecule comprising the nucleotide sequence shown as SEQ ID No. 61 (which may encode a heavy chain constant region having the amino acid sequence shown as SEQ ID No. 50) or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(IV) a fourth polynucleotide encoding the light chain constant region of the monoclonal antibody or antigen binding fragment thereof of the invention, preferably, the fourth polynucleotide is a DNA molecule comprising the nucleotide sequence shown as SEQ ID No. 62 (which may encode the light chain constant region of the amino acid sequence shown as SEQ ID No. 51) or a corresponding mRNA molecule thereof.

In a most preferred embodiment, the set of polynucleotides comprises:

(I) Preferably, the first polynucleotide encoding the heavy chain of the monoclonal antibody or antigen binding fragment thereof of the present invention is a DNA molecule having a nucleotide sequence shown as SEQ ID NO. 63 or a corresponding mRNA molecule thereof, which encodes the heavy chain having an amino acid sequence shown as SEQ ID NO. 52; and, a step of, in the first embodiment,

(II) A second polynucleotide encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof of the invention, preferably, the second polynucleotide is a DNA molecule having a nucleotide sequence shown as SEQ ID NO. 64 or a corresponding mRNA molecule thereof, which encodes the light chain having an amino acid sequence shown as SEQ ID NO. 53.

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

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

The recombinant vector of the present invention may be a cloning vector or an expression vector, and may be, for example, a plasmid, a cosmid, a phage, or the like.

In some preferred embodiments, the recombinant vector is an expression vector, preferably a eukaryotic expression vector.

In a fifth aspect, the present invention provides a transformed host cell into which a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect or an expression vector as described in the fourth aspect has been transformed;

such host cells include, but are not limited to: prokaryotic cells, such as E.coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.

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

In a sixth aspect, the present invention provides a reagent or kit for detecting human bocavirus type 2, comprising a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above and/or a transformed host cell as described in the fifth aspect above.

In certain preferred embodiments, the kit is a detection or diagnostic kit, wherein the monoclonal antibodies or antigen-binding fragments thereof of the invention comprised therein further comprise a detectable label; in certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody of the 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, radioisotopes, fluorescent materials, luminescent materials, colored materials, enzymes (e.g., horseradish peroxidase), and the like.

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

In an eighth aspect, the present invention provides the use of a monoclonal antibody or antigen binding fragment thereof according to the first aspect, a polynucleotide according to the second aspect, a nucleic acid construct according to the third aspect, an expression vector according to the fourth aspect, a transformed host cell according to the fifth aspect, a pharmaceutical composition according to the sixth aspect and/or a kit according to the seventh aspect for the preparation of a product for detecting the presence or level of human bocavirus type 2 in a sample, for diagnosing human bocavirus type 2 infection, or for differential diagnosis between human bocavirus type 1 and human bocavirus type 2.

In a possible embodiment, the sample is a biological sample of a subject; in particular embodiments, the sample is a respiratory tract sample from a subject, preferably a nasopharyngeal swab, sputum and/or alveolar lavage.

In a ninth aspect, the present invention provides a method of detecting the presence or level of human bocavirus type 2 in a sample, or diagnosing human bocavirus type 2 infection, or performing a differential diagnosis between human bocavirus type 1 and human bocavirus type 2, the method comprising using a monoclonal antibody or antigen binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, an expression vector as described in the fourth aspect, a transformed host cell as described in the fifth aspect and/or a reagent or kit as described in the sixth aspect.

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

In other preferred embodiments of the method, the method further comprises: the monoclonal antibodies or antigen binding fragments thereof of the invention are detected using a secondary antibody carrying a detectable label.

In a possible embodiment, the sample includes, but is not limited to, a nasopharyngeal swab, sputum, alveolar lavage, etc. respiratory tract sample from a subject.

The method may be used for diagnostic purposes (e.g., the sample is a sample from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

The general methods for detecting the presence or level of a virus or antigen of interest in a sample using monoclonal antibodies or antigen binding fragments thereof are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method, and the like.

Advantageous effects

The monoclonal antibody can be specifically combined with VP3 protein of HBoV2, but is not combined with VP3 protein of HBoV1, so that the monoclonal antibody has extremely high potential to be used for specific detection or diagnosis of type 2 human bocavirus or for differential diagnosis between type 1 human bocavirus and type 2 human bocavirus; in addition, the binding affinity of the kit and the HBoV2 VP3 protein is higher, and lower detection offline can be provided for the detection of the HBoV2, so that the sensitivity of the detection or diagnosis of the HBoV2 is improved.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a diagram showing the SDS-PAGE identification of HBoV1 VP3 and HBoV2 VP3 proteins expressed by HEK293 cells as described in example 2.

FIG. 2 is a SDS-PAGE identification of monoclonal antibodies Mab-HBoV1 DR2, mab-HBoV2 DR 2.

FIG. 3 shows the binding kinetics of monoclonal antibodies Mab-HBoV1 DR2, mab-HBoV2 DR2 to antigenic polypeptides HBoV1 DR2, HBoV2 DR2, respectively; wherein A is the binding kinetics curve of the Mab-HBoV1 DR2 and the HBoV1 DR2, B is the binding kinetics curve of the Mab-HBoV2 DR2 and the HBoV1 DR2, C is the binding kinetics curve of the Mab-HBoV2 DR2 and the HBoV2 DR2, and D is the binding kinetics curve of the Mab-HBoV1 DR2 and the HBoV2 DR 2; and wherein in each figure, seven curves represent seven dilutions, respectively.

FIG. 4 shows the binding of monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 to antigen proteins HBoV1 VP3 and HBoV2 VP3, respectively, detected by Western blot.

FIG. 5 shows ELISA assays for detecting binding of monoclonal antibodies Mab-HBoV1 DR2, mab-HBoV2 DR2 to antigenic polypeptides HBoV1 DR2 (panel A) and HBoV2 DR2 (panel B), respectively.

FIG. 6 shows the binding of the monoclonal antibodies Mab-HBoV1 DR2 (panel A) and Mab-HBoV2 DR2 (panel B) to the antigen polypeptides HBoV1 DR2 and HBoV2 DR2, respectively, detected by the IFA assay.

FIG. 7 shows the clinical diagnostic effectiveness of the monoclonal antibody Mab-HBoV1 DR2 for HBoV1 infection.

FIG. 8 shows the results of cross-reactivity verification of monoclonal antibodies Mab-HBoV1 DR2 (panel A), mab-HBoV1 DR2 (panel B) with other respiratory viruses.

Detailed Description

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

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

In order that the invention may be more readily understood, certain technical and scientific terms are defined as follows. Unless otherwise defined herein, 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. For definitions and terms in the art, the expert may refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including the claims), the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ± 5%, ±2%, ±1% and ± 0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be construed as inclusive, i.e., including at least one of the list of elements or amounts, but also including more than one, and optionally, additional unlisted items. To the extent that only one term is explicitly recited, such as "only one" or "exactly one" or "consisting of" is used in the claims, it will refer to only one number listed or an element of a list.

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 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 alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

"antigen binding fragment" refers to antigen binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen binding or variable regions of a parent antibody, e.g., one or more CDRs. Fragments of the antibodies retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include peptides selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, CDRs comprising peptides, and the like.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains.

The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

"Fab ' fragments" contain a portion of one light chain and one heavy chain comprising a portion of the VH domain, CH1 domain and constant region between the CH1 and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and portions of two heavy chains comprising portions of the VH domain, CH1 domain, and constant region between CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"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. In general, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is so short that it is not possible to pair between two domains of the same strand, the domains pair with complementary domains of the other strand and form two antigen binding sites.

When referring to ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to determining the presence or absence of binding reaction of a protein, such as a monoclonal antibody of the invention, with 2019-nCoV RBD protein in a heterogeneous population of proteins and/or other biological agents. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein. The term "non-binding" protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e. the binding protein or cell has a KD of 1.0X10 ^-6 M or higher, more preferably 1.0X10 ^-5 M or higher, more preferably 1.0X10 ^-4 M or higher, 1.0X10 ^-3 M or higher, more preferably 1.0X10 ^-2 M or higher.

The term "high affinity" for IgG antibodies refers to a KD of 1.0X10 for antigen ^-6 M or less, preferably 5.0X10 ^-8 M or less, more preferably 1.0X10 ^-8 M or less, 5.0X10 s ^-9 M or less, more preferably 1.0X10 ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtype refers to KD of 10 ^-6 M or less, preferably 10 ^-7 M or less, more preferably 10 ^-8 M or lower.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8,278,036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes8:91-98 (1994)).

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The information of the partial sequences according to the present invention is shown in table 1 below.

TABLE 1 sequence information according to the invention

Sequence description	SEQ ID NO:	Sequence information
			HBoV1 -HCDR1	1	GFTFSNHG
HBoV1 -HCDR2	2	INPDGGFT
			HBoV1 -HCDR3	3	TRDRGMIKDRDALFAY
HBoV1 -LCDR1	4	QTIVHSNGNTY
			HBoV1 -LCDR2	-	KVS
HBoV1 -LCDR3	5	FQGSHVPPT
			HBoV1 -H-FR1	6	EVHLVESGGDLVLPGGSLKLSCAVS
HBoV1 -H-FR2	7	MCWVRQTPDKSLELVAN
			HBoV1 -H-FR3	8	YYPDNVKGRFTISRDSAKNTLYLQMTSLRSEDTALYYC
HBoV1 -H-FR4	9	WGQGTLVTVSA
			HBoV1 -L-FR1	10	DILMTQTPLFLPVSLGDQASISCRSS
HBoV1 -L-FR2	11	LEWYLQKPGQSPRLLIY
			HBoV1 -L-FR3	12	NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
HBoV1 -L-FR4	13	FGGGTKLEIK
			HBoV1 -VH	14	EVHLVESGGDLVLPGGSLKLSCAVSGFTFSNHGMCWVRQ TPDKSLELVANINPDGGFTYYPDNVKGRFTISRDSAKNT LYLQMTSLRSEDTALYYCTRDRGMIKDRDALFAYWGQGT LVTVSA
HBoV1 -VL	15	DILMTQTPLFLPVSLGDQASISCRSSQTIVHSNGNTYLE WYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK
			HBoV1 -CH	16	AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTV TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSE TVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSV FIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP IMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNH HTEKSLSHSPGK
HBoV1 -CL	17	RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE YERHNSYTCEATHKTSTSPIVKSFNRNEC
			HBoV 1-H chain	18	EVHLVESGGDLVLPGGSLKLSCAVSGFTFSNHGMCWVRQ TPDKSLELVANINPDGGFTYYPDNVKGRFTISRDSAKNT LYLQMTSLRSEDTALYYCTRDRGMIKDRDALFAYWGQGT LVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPS STWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTV PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEV QFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQD WLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAEN YKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK
HBoV 1-L chain	19	DILMTQTPLFLPVSLGDQASISCRSSQTIVHSNGNTYLE WYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIKRADAA PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN SYTCEATHKTSTSPIVKSFNRNEC
			HBoV 1-HCDR 1 encoding Sequence(s)	20	GGATTCACTTTCAGTAACCATGGC
HBoV 1-HCDR 2 encoding Sequence(s)	21	ATTAATCCTGATGGTGGTTTCACC
			HBoV 1-HCDR 3 encoding Sequence(s)	22	ACAAGAGATCGGGGTATGATTAAGGATAGGGACGCCCTA TTTGCTTAC
HBoV 1-LCDR 1 encoding Sequence(s)	23	CAGACCATTGTCCATAGTAATGGAAACACCTAT
			HBoV 1-LCDR 2 encoding Sequence(s)	-	AAAGTTTCC
HBoV 1-LCDR 3 encoding Sequence(s)	24	TTTCAAGGTTCACATGTTCCTCCGACG
			HBoV 1-VH gene	25	GAAGTACATCTGGTGGAGTCTGGGGGAGACTTAGTGCTG CCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGA TTCACTTTCAGTAACCATGGCATGTGTTGGGTTCGCCAG ACTCCAGACAAGAGCCTGGAGTTGGTCGCAAACATTAAT CCTGATGGTGGTTTCACCTATTATCCGGACAATGTGAAG GGCCGATTCACCATCTCCAGAGACTCTGCCAAGAACACC CTGTACCTGCAAATGACCAGTCTGAGGTCTGAGGACACA GCCTTGTATTACTGTACAAGAGATCGGGGTATGATTAAG GATAGGGACGCCCTATTTGCTTACTGGGGCCAAGGGACT CTGGTCACTGTCTCTGCA
HBoV 1-VL gene	26	GATATTTTGATGACCCAAACTCCACTCTTCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCTAGT CAGACCATTGTCCATAGTAATGGAAACACCTATTTAGAA TGGTACCTGCAGAAACCAGGCCAGTCTCCAAGGCTCCTG ATCTACAAAGTTTCCAACCGCTTTTCTGGGGTCCCAGAC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTAT TACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGT GGGGGCACGAAGCTGGAAATCAAA
			HBoV 1-CH gene	27	GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCT GGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGA TGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTG ACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACC TTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGC AGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAG ACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACC AAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGT AAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTC TTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATT ACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATC AGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTA GATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGG GAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAA CTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAG TTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCC ATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAG ATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACA GACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGG AATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCC ATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAG CTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACT TTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCAC CATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA
	28	CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA TCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTG TGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTC AAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTC CTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACC TACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAG TATGAACGACATAACAGCTATACCTGTGAGGCCACTCAC AAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGG AATGAGTGT
			HBoV 1-H chain gene	29	GAAGTACATCTGGTGGAGTCTGGGGGAGACTTAGTGCTG CCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGA TTCACTTTCAGTAACCATGGCATGTGTTGGGTTCGCCAG ACTCCAGACAAGAGCCTGGAGTTGGTCGCAAACATTAAT CCTGATGGTGGTTTCACCTATTATCCGGACAATGTGAAG GGCCGATTCACCATCTCCAGAGACTCTGCCAAGAACACC CTGTACCTGCAAATGACCAGTCTGAGGTCTGAGGACACA GCCTTGTATTACTGTACAAGAGATCGGGGTATGATTAAG GATAGGGACGCCCTATTTGCTTACTGGGGCCAAGGGACT CTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCT GTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAAC TCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC CCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTG TCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCC AGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCC CACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTG CCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTC CCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCC AAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACG TGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTC CAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACA GCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACT TTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGAC TGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGT GCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAA ACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATT CCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATT ACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAAC TACAAGAACACTCAGCCCATCATGGACACAGATGGCTCT TACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAAC TGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCC CACTCTCCTGGTAAA
HBoV 1-L chain gene	30	GATATTTTGATGACCCAAACTCCACTCTTCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCTAGT CAGACCATTGTCCATAGTAATGGAAACACCTATTTAGAA TGGTACCTGCAGAAACCAGGCCAGTCTCCAAGGCTCCTG ATCTACAAAGTTTCCAACCGCTTTTCTGGGGTCCCAGAC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTAT TACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGT GGGGGCACGAAGCTGGAAATCAAACGGGCTGATGCTGCA CCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTA ACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAAC TTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGAT GGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACT GATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC ACCCTCACGTTGACCAAGGACGAGTATGAACGACATAAC AGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCA CCCATTGTCAAGAGCTTCAACAGGAATGAGTGT
			HBoV 1-H chain leader Column of	31	MNLGLSFIFLALILKGVQC
HBoV 1-H chain leader Column coding gene	32	ATGAACTTAGGGCTCAGCTTCATTTTCCTTGCCCTCATT TTAAAAGGTGTCCAGTGT
			HBoV 1-L chain leader Column of	33	MKLPVRLLVLMFWIPVSSS
HBoV 1-L chain leader Column coding gene	34	ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGG ATTCCTGTTTCCAGCAGT
			HBoV2 -HCDR1	35	GFSLSTSGMG
HBoV2 -HCDR2	36	IWWDDVK
			HBoV2 -HCDR3	37	ARIVGYYASRDALDY
HBoV2 -LCDR1	38	KSVSTSAYSY
			HBoV2 -LCDR2	-	LAS
HBoV2 -LCDR3	39	QHSRELPWT
			HBoV2 -H-FR1	40	QVTLKESGPGILQPSQTLSLTCSFS
HBoV2 -H-FR2	41	VGWIRQPSGKGLEWLTH
			HBoV2 -H-FR3	42	RYNPALKSRLTISKDTSSSQVFLKIASVDTADTAAYYC
HBoV2-H-FR4	43	WGQGTSVTVSS
			HBoV2 -L-FR1	44	DIVLTQSPASLAVSLGQRATISCRAS
HBoV2 -L-FR2	45	MHWYQQRPGQPPKLLIY
			HBoV2 -L-FR3	46	NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
HBoV2 -L-FR4	47	FGGGTKLEIK
			HBoV2 -VH	48	QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWI RQPSGKGLEWLTHIWWDDVKRYNPALKSRLTISKDTSSS QVFLKIASVDTADTAAYYCARIVGYYASRDALDYWGQGT SVTVSS
HBoV2 -VL	49	DIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHW YQQRPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLN IHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK
			HBoV2 -CH	50	AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTV TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSE TVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSV FIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP IMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNH HTEKSLSHSPGK
HBoV2 -CL	51	RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE YERHNSYTCEATHKTSTSPIVKSFNRNEC
			HBoV 2-H chain	52	QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWI RQPSGKGLEWLTHIWWDDVKRYNPALKSRLTISKDTSSS QVFLKIASVDTADTAAYYCARIVGYYASRDALDYWGQGT SVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPS STWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTV PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEV QFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQD WLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAEN YKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK
HBoV 2-L chain	53	DIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHW YQQRPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLN IHPVEEEDAATYYCQHSRELPWTFGGGTKLEIKRADAAP TVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS YTCEATHKTSTSPIVKSFNRNEC
			HBoV 2-HCDR 1 encoding Sequence(s)	54	GGGTTTTCACTGAGCACTTCTGGTATGGGT
HBoV 2-HCDR 2 encoding Sequence(s)	55	ATTTGGTGGGATGATGTCAAG
			HBoV 2-HCDR 3 encoding Sequence(s)	56	GCTCGAATAGTGGGATACTACGCTAGTAGGGATGCTTTG GACTAC
HBoV 2-LCDR 1 encoding Sequence(s)	57	AAAAGTGTCAGTACATCTGCCTATAGTTAT
			HBoV 2-LCDR 2 encoding Sequence(s)	-	CTTGCATCC
HBoV 2-LCDR 3 encoding Sequence(s)	58	CAGCACAGTAGGGAGCTTCCGTGGACG
			HBoV 2-VH gene	59	CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAG CCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGG TTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATT CGTCAGCCATCAGGGAAGGGTCTGGAGTGGCTGACACAC ATTTGGTGGGATGATGTCAAGCGCTATAACCCAGCCCTG AAGAGCCGACTGACTATCTCCAAGGATACCTCCAGCAGC CAGGTATTCCTCAAGATCGCCAGTGTGGACACTGCAGAT ACTGCCGCATACTACTGTGCTCGAATAGTGGGATACTAC GCTAGTAGGGATGCTTTGGACTACTGGGGTCAAGGAACC TCAGTCACCGTCTCCTCA
HBoV 2-VL gene	60	GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTA TCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGC AAAAGTGTCAGTACATCTGCCTATAGTTATATGCACTGG TACCAACAGAGACCAGGACAGCCACCCAAACTCCTCATC TATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAAC ATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTAC TGTCAGCACAGTAGGGAGCTTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAA
			HBoV 2-CH gene	61	GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCT GGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGA TGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTG ACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACC TTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGC AGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAG ACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACC AAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGT AAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTC TTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATT ACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATC AGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTA GATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGG GAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAA CTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAG TTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCC ATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAG GCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAG ATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACA GACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGG AATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCC ATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAG CTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACT TTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCAC CATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA
HBoV 2-CL gene	62	CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA TCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTG TGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTC AAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTC CTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACC TACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAG TATGAACGACATAACAGCTATACCTGTGAGGCCACTCAC AAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGG AATGAGTGT
			HBoV 2-H chain gene	63	CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAG CCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGG TTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATT CGTCAGCCATCAGGGAAGGGTCTGGAGTGGCTGACACAC ATTTGGTGGGATGATGTCAAGCGCTATAACCCAGCCCTG AAGAGCCGACTGACTATCTCCAAGGATACCTCCAGCAGC CAGGTATTCCTCAAGATCGCCAGTGTGGACACTGCAGAT ACTGCCGCATACTACTGTGCTCGAATAGTGGGATACTAC GCTAGTAGGGATGCTTTGGACTACTGGGGTCAAGGAACC TCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCT GTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAAC TCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC CCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTG TCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCT GACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCC AGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCC CACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTG CCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTC CCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCC AAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACG TGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTC CAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACA GCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACT TTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGAC TGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGT GCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAA ACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATT CCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGT CTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATT ACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAAC TACAAGAACACTCAGCCCATCATGGACACAGATGGCTCT TACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAAC TGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCC CACTCTCCTGGTAAA
HBoV 2-L chain gene	64	GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTA TCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGC AAAAGTGTCAGTACATCTGCCTATAGTTATATGCACTGG TACCAACAGAGACCAGGACAGCCACCCAAACTCCTCATC TATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAAC ATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTAC TGTCAGCACAGTAGGGAGCTTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCA ACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACA TCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTC TACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGC AGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGAT CAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACC CTCACGTTGACCAAGGACGAGTATGAACGACATAACAGC TATACCTGTGAGGCCACTCACAAGACATCAACTTCACCC ATTGTCAAGAGCTTCAACAGGAATGAGTGT
			HBoV 2-H chain leader Column of	65	MGRLTSSFLLLIVPAYVLS
HBoV 2-H chain leader Column coding gene	66	ATGGGCAGGCTTACTTCTTCATTCCTGCTACTGATTGTC CCTGCATATGTCCTGTCC
			HBoV 2-L chain leader Column of	67	METDTLLLWVLLLWVPGSTG
HBoV 2-L chain leader Column coding gene	68	ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTC TGGGTTCCAGGTTCCACTGGT
			HBoV1 DR2 antigen peptide	69	IENELADLDGNAAGGNATEKALLYQM
HBoV2 DR2 antigen peptide	70	VIHELAEMEDANAVEKAIALQI
			HBoV1 VP3 Gene PCR amplification primer VP3F1	71	AAGCAGATGCCTCCA
HBoV1 VP3 Gene PCR amplification primer VP3R1	72	CACCCTTCACTTTCCGC
			HBoV1 VP3 Gene PCR amplification primer VP3F2	73	CCCAAGCTTCTGTCTGACACTGACATTCAA
HBoV1 VP3 Gene PCR amplification primer VP3R2	74	CCCTCGAGTTACAACACTTTATTGATGTTTG
			HBoV2 VP3 gene PCR amplification primer VP3F1	75	ATAACGAGCCTAAACCAG
HBoV2 VP3 gene PCR amplification primer VP3R1	76	AATGTATGCTCTTTCGTT
			HBoV2 VP3 gene PCR amplification primer VP3F2	77	GAAGACGCAAATGCTGTA
HBoV2 VP3 gene PCR amplification primer VP3R2	78	CTGTGTTTCCGTGCTGTC
			HBoV2 VP3 gene PCR amplification primer VP3F3	79	GACAGCACGGAAACACAG
HBoV2 VP3 gene PCR amplification primer VP3R3	80	ATGCCTGACGCAGTACA

Example 1: screening and determining HBoV1 and HBoV2 type specific antigen polypeptides

Downloading the uploaded HBoV1 and HBoV2 VP3 amino acid sequences in different national regions in the global scope in a GenBank database, and analyzing the amino acid sequences; the kit comprises HBoV1 VP3 sequences (GenBank: ABK 32030.1) which are detected and uploaded in a nasopharyngeal secretion specimen of an infant suffering from acute respiratory infection and received by the laboratory in 2007, wherein the total is 542aa; HBoV2 VP3 sequences (GenBank: AFW 98869.1), together 538aa, were detected and uploaded in the acute diarrhea infant nasal stool specimens received by the virus institute of the capital pediatric institute in 2012. The HBoV1 VP3 sequence (GenBank: ABK 32030.1) uploaded by the laboratory and the HBoV2 VP3 sequence (GenBank: AFW 98869.1) uploaded by the capital pediatric institute virus laboratory are used as representative amino acid sequences of the HBoV1 VP3 and the HBoV2 VP3 to calculate an antigen index score, and the residue information exposed on the surfaces of the HBoV1 and the HBoV2 VP3 sequences is predicted by using a ProtScale biological prediction website (https:// web. Expasy. Org/protscan /), so as to obtain HBoV1 and HBoV2 type specific antigen polypeptides which are named as HBoV1 DR2 and HBoV2 DR2 respectively, wherein the amino acid sequences are as follows:

HBoV1 DR2： ¹⁹⁵ IENELADLDGNAAGGNATEKALLYQM ²²⁰ （SEQ ID NO: 69）

HBoV2 DR2： ¹⁹⁵ VIHELAEMEDANAVEKAIALQI ²¹⁶ （SEQ ID NO: 70）。

The HBoV1 and HBoV2 DR2 polypeptide is obtained through artificial synthesis, and the purity is 99.9%.

Example 2: expression and purification of HBoV1, HBoV2 VP3 proteins

Design of coding region of HBoV1 and HBoV2 VP3 genes:

the 3 'end of the HBoV1 VP3 (ABK 32030.1) and HBoV2 VP3 (AFW 98869.1) nucleic acid coding sequences are connected with a TwainStrep tag nucleic acid coding sequence, and the 5' end of the HBoV1 VP3 and HBoV2 VP3 gene expression region is formed by connecting a Flag tag nucleic acid coding sequence; nheI restriction sites are added upstream and XhoI restriction sites are added downstream of the expression regions of the HBoV1 and HBoV2 VP3 genes, respectively, so that the expression plasmids can be constructed by NheI/XhoI double restriction.

Primer design for amplifying HBoV1 and HBoV2 VP3 gene coding region:

respectively taking an HBoV1 VP3 nucleotide sequence (ABK 32030.1) and an HBoV2 VP3 nucleotide sequence (AFW 98869.1) as templates, designing primers for amplifying HBoV1 and HBoV2 VP3 genes, and introducing two enzyme cutting sites of NheI and XhoI to be added at two ends of the amplified genes when designing the primers; specifically, an NheI cleavage site is added upstream of the gene and an XhoI cleavage site is added downstream. The primer sequence for PCR amplification of the HBoV1 VP3 gene is shown as SEQ ID NO. 71-74, and the primer sequence for PCR amplification of the HBoV2 VP3 gene is shown as SEQ ID NO. 75-80.

Expression of HBoV1 VP3 protein and HBoV2 VP3 protein:

1) Respectively using an HBoV1 VP3 nucleotide sequence (ABK 32030.1) and an HBoV2 VP3 nucleotide sequence (AFW 98869.1) as templates, and adopting the primers designed above to generate HBoV1 and HBoV2 VP3 gene expression fragments with the enzyme cutting sites and the labels through PCR amplification;

2) Agarose gel electrophoresis is carried out on the PCR amplified product, and gel strips with the size of 1700 and bp are cut under a blue light lamp for recovery;

3) UsingNheI andXhoi, respectively carrying out double enzyme digestion on the recovered gene expression fragment and the pcDNA3.1 empty vector; the enzyme digestion system is as follows:Nhei andXho1 mu L of each fast cutting enzyme, 4 mu L of 10 Xenzyme cutting Buffer, 34 mu L of to-be-cut fragments or carriers, and the cutting conditions are as follows: 37 ℃ water bath 2 h;

4) Using T4 DNA ligase to connect the gene expression fragment subjected to double enzyme digestion with a vector pcDNA3.1;

5) Transforming DH101Bac competent cells (purchased from Tiangen) with the ligation product, and extracting plasmid DNA expressing HBoV1 VP3 protein and HBoV2 VP3 protein;

6) The extracted expression plasmids of HBoV1 VP3 protein and HBoV2 VP3 protein were transfected into HEK293 cells (purchased from Invitrogen), respectively, and expression of HBoV1 VP3 protein and HBoV2 VP3 protein was performed.

Purification of HBoV1 VP3 protein and HBoV2 VP3 protein:

the cell culture solution containing the target protein is purified by nickel ion affinity chromatography (TwainStrep (GE)) and gel filtration chromatography (Superose TM 6 include 10/300GL (GE)) to obtain purer target protein, and the target protein has the size of 70KD (see figure 1) identified by SDS-PAGE, and accords with the expected sizes of HBoV1 VP3 protein and HBoV2 VP3 protein.

Example 3: preparation of monoclonal antibodies, type identification and sequencing

Step one: HBoV1 DR2, HBoV2 DR2 immunized mice:

the HBoV1 DR2, HBoV2 DR2 polypeptides (obtained in example 1) were emulsified separately with equal amounts of freund's complete adjuvant as immunogens; BALB/C female mice (3 mice per group) around 6 weeks old were subjected to subcutaneous multipoint cervical back immunization with immunogen at an inoculum size of 100 μl/mouse; three weeks apart, the HBoV1 DR2 and HBoV2 DR2 polypeptides were emulsified separately with equal amounts of Freund's incomplete adjuvant and immunized a second time at the same dose of 100. Mu.L/mouse; after a third interval of three weeks, a third immunization is performed, and the steps are the same as the second immunization; after the three-phase injection, 14. 14 d, the tail vein blood sampling is carried out to measure the antibody titer in serum, and one mouse with the highest antibody titer is selected to carry out pre-fusion over-injection (namely, 100 mu L of HBoV1 DR2 and HBoV2 DR2 polypeptide without adding adjuvant is injected into the abdominal cavity).

Step two: cell fusion to prepare hybridoma cells:

after aseptically taking out the spleen from the exposed abdominal cavity after the neck-breaking sacrifice of the mouse, the spleen was crushed by shearing after removing connective tissue around the spleen, and the spleen cells of the mouse were prepared into a single cell suspension (about 1×10) ⁸ ) The method comprises the steps of carrying out a first treatment on the surface of the At the same time, the well-grown mouse myeloma cells (about 1X 10) ⁷ ) 5:1, after centrifugation, the supernatant was discarded and quickly transferred to a 37 ℃ water bath. The cells were uniformly dispersed by blowing and cultured at 37℃for 10 days.

Step three: preparation of monoclonal antibodies, type identification and sequencing:

after about 10 days after cell fusion, when hybridoma cell fusion can be observed under a microscope to form clone and occupy the bottom of a 96-well plate, the hybridoma cell plate is preliminarily screened by adopting IPMA, the preliminarily selected positive clone is transferred to a plate which is pre-paved with feeder cells for continuous culture and rechecking, and the strong positive hole is subcloned by adopting a limiting dilution method. After two weeks, positive monoclonal was selected to prepare ascites. Hybridoma cells were collected (1×10) ⁶ ) Is injected into the abdominal cavity of a mouse, and is taken after one week for the patient with abdominal cavity enlargementCollecting ascites, centrifuging the collected ascites, and collecting and preserving supernatant; meanwhile, 2 cell strains which are stably passaged and produce monoclonal antibodies are selected, the subtype of the monoclonal antibodies is verified according to the operation of a Mouse monoclonal antibody subtype identification kit instruction book of Proteintech company, and sequencing is carried out.

Through the steps, and through proper screening, the HBoV1 monoclonal antibody Mab-HBoV1 DR2 and the HBoV2 monoclonal antibody Mab-HBoV2 DR2 are obtained, and the Mab-HBoV1 DR2 and the Mab-HBoV2 DR2 are both of an IgG1 subtype.

The sequence information of the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 are shown in Table 1.

The monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 were subjected to SDS-PAGE gel electrophoresis, stained with Coomassie brilliant blue overnight after the electrophoresis was completed, and after the staining was completed, the membrane was washed with pure water for 1 hour, and then the protein bands were observed. The results are shown in FIG. 2, and FIG. 2 shows that both monoclonal antibodies show distinct two bands, which are the heavy and light chains of the antibody, respectively, as judged by their molecular weight positions.

Example 4: determination of binding Capacity of HBoV1 monoclonal antibody to antigen polypeptide

In this example, the monoclonal antibody obtained in example 3 was tested for its binding capacity to an antigen by a biofilm layer interference experiment, and the specific procedure was as follows:

(1) Starting up a molecular interaction analyzer (Gator, supplied by Gator Bio company), and opening software to automatically initialize and preheat for 30 minutes; the antigens HBoV1 DR2, HBoV2 DR2 and monoclonal antibodies Mab-HBoV1 DR2, mab-HBoV2 DR2 were diluted separately using a K Buffer (available from Gator Bio Inc., cat# 120011) and serial two-fold gradient dilutions of the antibodies were performed for a total of 7 dilutions;

(2) Adding various buffers, samples and Anti-His probes according to the layout, and operating a Kinetics module; pre-wetting the probe in K Buffer for 10 minutes (shaking speed of the shaker, except 400 rpm when binding antigen, all 1000 rpm); equilibrated in Baseline wells for 120 seconds; binding protein in Loading well to interference wavelength shift about 0.8 nm; again equilibrated in K Buffer wells for 60 seconds; binding the antibody in the binding well; dissociation in the dissociation well for 600 seconds when the curve is relatively stationary;

(3) The equilibrium dissociation constant was calculated using Gator system software.

Results:

the binding kinetics of the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 to the HBoV1 DR2 and HBoV2 DR2 antigens are shown in FIG. 3, and the binding kinetics constants are shown in tables 2 and 3 below.

TABLE 2 binding kinetics constant of Mab-HBoV1 DR2 to antigen polypeptide HBoV1 DR2, HBoV2 DR2

。

Table 2 shows that the monoclonal antibody Mab-HBoV1 DR2 binds to the antigen polypeptide HBoV1 DR2 with Kd, kon and Koff values of 3.62X10, respectively ^-11 (M)、1.92×10 ⁶ (1/Ms) and 4.25X10 ^-5 (1/s), prompt: the binding force of the antibody and the specific antigen thereof is stronger; gator systems are unable to calculate Kd, kon and Koff values for binding of Mab-HBoV1 DR2 to HBoV2 DR2, suggesting that Mab-HBoV1 DR2 is not bound to HBoV2 DR 2.

TABLE 3 binding kinetics constant of Mab-HBoV2 DR2 to antigen polypeptide HBoV1 DR2, HBoV2 DR2

。

Table 3 shows that the monoclonal antibody Mab-HBoV2 DR2 binds to the antigen polypeptide HBoV2 DR2 with Kd, kon and Koff values of 2.42×10, respectively ^-10 （M）、1.76×10 ⁶ (1/Ms) and 3.67X10 ^-4 (1/s), prompt: the binding force of the antibody and the specific antigen thereof is stronger; gator systems are unable to calculate Kd, kon and Koff values for binding of Mab-HBoV2 DR2 to HBoV1 DR2, suggesting that Mab-HBoV2 DR2 is not bound to HBoV1 DR 2.

The results show that the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 have higher binding capacity with their respective specific antigen polypeptides, and have no binding with another antigen polypeptide of a different type, respectively, which indicates that: the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 have higher antigen binding specificity and higher binding affinity.

Example 5: type-specific validation of monoclonal antibodies

In this example, binding of monoclonal antibodies Mab-HBoV1 DR2, mab-HBoV2 DR2 to antigen was detected by Western blot, ELISA, IFA experiments with HEK293 eukaryotic expressed HBoV1 VP3 and HBoV2 VP3 proteins (prepared in example 2) as antigens, or with HBoV1 DR2, HBoV2 DR2 polypeptides (prepared in example 1) as antigens, to determine their type specificity.

Western blot experiment:

and (3) detecting the binding condition of antibodies mAb-HBoV1 DR2 and mAb-HBoV2 DR2 by adopting a Western blot (i.e. Western blot) experiment and taking eukaryotic expressed HBoV1 VP3 and HBoV2 VP3 proteins as analysis objects so as to detect the type specificity of the antibodies.

The results are shown in FIG. 4; as can be seen from fig. 4, the monoclonal antibody Mab-HBoV1 DR2 only binds to HBoV1 VP3 protein, but does not bind to HBoV2 VP3 protein at all, i.e., does not cross react; the monoclonal antibody Mab-HBoV2 DR2 can only bind to the HBoV2 VP3 protein, but does not bind to the HBoV1 VP3 protein at all; this illustrates: the Mab-HBoV1 DR2 and the Mab-HBoV2 DR2 are both type-specific.

ELISA experiments:

and detecting the binding condition of antibodies mAb-HBoV1 DR2 and mAb-HBoV2 DR2 and antigen polypeptides HBoV1 DR2 and HBoV2 DR2 by adopting an enzyme-linked immunosorbent assay (ELISA) experiment so as to detect the type specificity of the antibodies. Specifically, the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 are subjected to double-ratio dilution from 1:200 respectively, and the monoclonal antibodies with a series of dilutions are respectively incubated with HBoV1 DR2 and HBoV2 DR2 polypeptides to perform antigen-antibody reaction; after the reaction, the OD450 (mean ± standard deviation) of the reaction mixture was measured, and the binding difference between the antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 and the antigen polypeptides HBoV1 DR2 and HBoV2 DR2 was analyzed.

The results are shown in FIG. 5; as can be seen from FIG. 5, the average of the reactions of Mab-HBoV1 DR2 with the titres 1:25600 with HBoV1 DR2 proteinThe OD450 (1.346 ± 0.659) is significantly higher than its average OD450 (0.055±0032) for the HBoV2 DR2 reaction (t= 40.397,P＜0.001 A) is provided; the OD450 (1.209±0.824) of Mab-HBoV2 DR2 with a titre of 1:25600 reacted with HBoV2 DR2 protein was significantly higher than its OD450 (0.081±0.116) of HBoV1 DR2 (t= 26.000,P＜0.001）。

the results show that: the Mab-HBoV1 DR2 can react with the HBoV1 DR2 polypeptide with little antigen-antibody reaction with the HBoV2 DR2 polypeptide; mab-HBoV2 DR2 is capable of antigen-antibody reaction with HBoV2 DR2 polypeptide, while it hardly reacts with HBoV1 DR2 polypeptide; this illustrates: the antibodies MAB-HBoV1 DR2 and MAb-HBoV2 DR2 are each type-specific.

Indirect Immunofluorescence (IFA) experiments:

HEK293 cells which over express HBoV1 VP3 and HBoV2 VP3 proteins are taken as detection objects, and the binding condition of an antibody Mab-HBoV1 DR2 and an expressed antigen is detected so as to detect the type specificity of the antibody. Specifically, HBoV1 VP3 and HBoV2 VP3 proteins are respectively expressed in a eukaryotic manner in HEK293 cells, after the cells are fixed by paraformaldehyde, the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV2 DR2 are used for diluting and incubating the cells at a ratio of 1:100, then the cells are incubated with FITC goat anti-mouse labeled secondary antibodies, and then the cells are observed and photographed under a fluorescence microscope.

The results are shown in FIG. 6; FIG. 6 shows that in cells expressing antigen HBoV1 VP3, specific green fluorescent cells appeared after incubation with antibody Mab-HBoV1 DR2 dilution, and no specific green fluorescent cells appeared after incubation with antibody Mab-HBoV2 DR2 dilution; for cells expressing antigen HBoV2 VP3, specific green fluorescent cells appear after incubation with antibody Mab-HBoV2 DR2 dilution, and no specific green fluorescent cells appear after incubation with antibody Mab-HBoV1 DR2 dilution; this illustrates: antibody Mab-HBoV1 DR2 reacted with antigen HBoV1 VP3, but not with antigen HBoV2 VP 3; antibody Mab-HBoV2 DR2 reacted with antigen HBoV2 VP3, but not with antigen HBoV1 VP 3. The above results indicate that both Mab-HBoV1 DR2 and Mab-HBoV2 DR2 are type specific.

Example 6: clinical diagnostic validation of HBoV 1-specific monoclonal antibodies

Pediatric patients with acute respiratory infections, including pharyngeal swabs (TS), nasopharyngeal swabs (NPS), nasopharyngeal aspirates (NPA) and Bronchoalveolar Lavage (BLF), were selected from 1 st 2017 to 12 nd 2021, with a pediatric study attached to a pediatric hospital visit, receipts. Adding the group-entering specimen into 2.5 ml virus transfer culture medium in a class II biosafety cabinet, centrifuging at 500 g for 10 minutes, and then using the supernatant for extracting nucleic acid, and preserving the remainder at-80 ℃ for later use; and (3) depositing the smear, fixing the smear with acetone, and preserving the smear at the temperature of minus 20 ℃ for standby. Nucleic acid detection of HBoV1 positive specimen, taking cell sediment to re-suspend and sample on a glass slide, fixing with acetone, preserving at-20deg.C, balancing to room temperature, adding 5 μl of PBS diluted monoclonal antibodies (1:20, 1:40, 1:80) of the present invention onto the glass slide, and incubating in a 37 ℃ wet box for 30 min; washing; FITC-conjugated goat anti-mouse IgG antibody 5. Mu.L diluted in 1:5000 PBS was added to the slide and incubated in a 37℃wet box for 30 min; washing; soaking in trypan blue liquid containing 0.04% for 5 min; the slide was blow-dried and the cells with bright green specific fluorescence were determined to be positive for HBoV1 antigen as observed under a fluorescence microscope.

The results are shown in FIG. 7; fig. 7 shows: in IFA, mab-HBoV1 DR2 has specific green fluorescent cells present; prompting: in clinical respiratory tract specimen antigen detection, the monoclonal antibody Mab-HBoV1 DR2 can effectively detect HBoV1 infection.

Slides of RSV, fluA and B, HAdV, PIV 1, 2 and 3 infected cells in D3 Ultra ™ DFA respiratory virus screening kit (Diagnostic Hybrids inc., athens, OH, USA) were used as other common pathogens for children to verify the diagnostic specificity of the two monoclonal antibodies.

The detection results of the antibodies Mab-HBoV1 DR2 and Mab-HBoV1 DR2 are shown in FIG. 8A and FIG. 8B, respectively; fig. 8A and 8B show: antibodies Mab-HBoV1 DR2 and Mab-HBoV1 DR2 reacted with positive cells infected with RSV, influenza A and B, ADV and PIV 1-3 without the appearance of specific green fluorescent cells; description: the monoclonal antibodies Mab-HBoV1 DR2 and Mab-HBoV1 DR2 have no cross-reactivity with other respiratory viruses.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type-specific monoclonal antibody or antigen-binding fragment thereof directed against human bocavirus type 2 comprising a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises:

and, the light chain variable region comprises:

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region further comprises framework regions H-FR1, H-FR2, H-FR3 and H-FR4 having amino acid sequences shown as SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43, respectively;

and, the light chain variable region further comprises framework regions L-FR1, L-FR2, L-FR3 and L-FR4 of amino acid sequences shown in SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 48;

and, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 49.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region further comprises a leader sequence at the N-terminus of the heavy chain variable region, the leader sequence of the heavy chain variable region having the amino acid sequence shown as SEQ ID No. 65;

and/or, a leader sequence is further arranged at the N end of the light chain variable region, and the leader sequence of the light chain variable region has an amino acid sequence shown as SEQ ID NO. 67.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

a heavy chain comprising the amino acid sequence shown as SEQ ID NO. 52; and, a step of, in the first embodiment,

a light chain comprising the amino acid sequence shown as SEQ ID NO. 53.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

the amino acid sequence of the light chain is shown as SEQ ID NO. 53.

8. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') ₂ Fv, complementarity determining region fragment, single chain antibody, human antibody。

9. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8.

10. The polynucleotide of claim 9, wherein the polynucleotide is a set of polynucleotides comprising:

(I) A first polynucleotide comprising a DNA molecule of the nucleotide sequences shown as SEQ ID NOs 54, 55 and 56 or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(II) a second polynucleotide comprising a DNA molecule of the nucleotide sequence shown as SEQ ID NO. 57, CTTGCATCC and SEQ ID NO. 58 or a corresponding mRNA molecule thereof.

11. The polynucleotide of claim 10, wherein the set of polynucleotides comprises:

(I) A first polynucleotide comprising a DNA molecule having the nucleotide sequence shown as SEQ ID NO. 59 and optionally a nucleotide sequence shown as SEQ ID NO. 66 or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(II) a second polynucleotide comprising a DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO. 60 and optionally a nucleotide sequence set forth in SEQ ID NO. 68 or a corresponding mRNA molecule thereof;

optionally, the polynucleotide set further comprises:

(III) a third polynucleotide comprising a DNA molecule having the nucleotide sequence shown as SEQ ID NO. 61 or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(IV) a fourth polynucleotide comprising a DNA molecule having the nucleotide sequence shown as SEQ ID NO. 62 or a corresponding mRNA molecule thereof.

12. The polynucleotide of claim 11, wherein the set of polynucleotides comprises:

(I) A first polynucleotide which is a DNA molecule having a nucleotide sequence shown as SEQ ID NO. 63 or a corresponding mRNA molecule thereof; and, a step of, in the first embodiment,

(II) the second polynucleotide, which is a DNA molecule having a nucleotide sequence shown as SEQ ID NO. 64 or a corresponding mRNA molecule thereof.

13. A nucleic acid construct comprising the polynucleotide of any one of claims 9-12, and, optionally, at least one expression regulatory element operably linked to the polynucleotide.

14. A recombinant vector comprising the polynucleotide of any one of claims 9-12, or the nucleic acid construct of claim 13.

15. A transformed host cell transformed with the polynucleotide of any one of claims 9-12, the nucleic acid construct of claim 13, or the recombinant vector of claim 14.

16. A reagent or kit for detecting human bocavirus type 2 comprising the monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-8 and/or the transformed host cell according to claim 15.

17. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, the method comprising: allowing the transformed host cell of claim 15 to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.

18. Use of a monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-8, a polynucleotide according to any one of claims 9-12, a nucleic acid construct according to claim 13, a recombinant vector according to claim 14, a transformed host cell according to claim 15 and/or an agent according to claim 16 for the preparation of a product for detecting the presence or level of human bocavirus type 2 in a sample, for diagnosing human bocavirus type 2 infection, or for differential diagnosis between human bocavirus type 1 and human bocavirus type 2.

19. The use of claim 18, wherein the sample is a biological sample of a subject.