Anti-rabies virus antibody and application thereof

Abstract

The present disclosure provides anti-rabies virus antibodies and uses thereof. The invention specifically provides a modified anti-rabies virus antibody, and also provides a coding nucleic acid molecule, an expression vector and a host cell of the antibody disclosed by the invention, a functional row derivative containing the antibody disclosed by the invention, and application of the antibody in antibody drugs for treating or preventing rabies.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-rabies virus antibody and an application thereof. Background Art

[0002] Rabies is a zoonotic viral disease that affects the central nervous system. Once clinical symptoms appear, rabies is almost 100% fatal. Rabies exists in more than 150 countries and regions around the world, causing tens of thousands of deaths each year, mainly in Asia and Africa, 40% of which are children under the age of 15.

[0003] Currently, the prevention methods of rabies mainly include pre-exposure prevention and post-exposure prevention.

[0004] Pre-exposure prophylaxis includes rabies vaccination for dogs and cats and pre-exposure rabies vaccination for humans. Pre-exposure vaccination for humans is mainly targeted at people in certain high-risk occupations (such as laboratory workers who handle rabies and rabies-related live viruses), people whose professional or personal activities may bring them into direct contact with mammals that may be infected with rabies (such as animal disease control personnel and wildlife managers), as well as outdoor travelers and people living in areas where rabies is prevalent but medical resources are limited.

[0005] Post-exposure prophylaxis refers to the immediate treatment of the bite victim after exposure to rabies to prevent the rabies virus from entering the central nervous system and causing death. Post-exposure prophylaxis can include thorough washing with water and soap for at least 15 minutes after suspected exposure and local treatment of the wound as soon as possible; followed by a full vaccination with a potent rabies vaccine that meets WHO standards. If necessary, rabies immunoglobulin or anti-rabies virus antibodies can also be injected into the wound.

[0006] Rabies immunoglobulin or anti-rabies virus antibodies are used in combination with human rabies vaccines to supplement the antibody gap in the active immunization process of human rabies vaccines. They can directly neutralize the rabies virus in the body and play a passive immunization role. Rabies immunoglobulin is the most commonly used rabies passive immunization product in clinical practice in China. It is extracted from plasma donated by plasma donors after they are vaccinated with rabies vaccine and their blood antibodies reach a certain level. The disadvantage is that the supply of plasma raw materials is limited and there is a risk of blood contamination. In contrast, anti-rabies virus antibodies can be mass-produced and standardized in quality, and there is no risk of blood contamination. Therefore, the development of anti-rabies virus antibodies is of great significance for the prevention and treatment of rabies. Summary of the invention

[0007] In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure provides an anti-rabies virus antibody and a fragment thereof, and applications of the anti-rabies virus antibody and a fragment thereof.

[0008] According to a first aspect of the present disclosure, there is provided an anti-rabies virus antibody, the antibody having:

[0009] A heavy chain variable region, whose amino acid sequence has an amino acid mutation at position 5 and / or 117 relative to the amino acid sequence of SEQ ID NO: 1, or has at least 85% sequence identity thereto, or, has an amino acid mutation at position 3 and / or 93 relative to the amino acid sequence of SEQ ID NO: 3, or has at least 85% sequence identity thereto; and,

[0010] A light chain variable region, whose amino acid sequence has an amino acid mutation at position 1 and / or 4 relative to the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having an amino acid mutation at position 13 and / or 106 relative to the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 85% sequence identity thereto.

[0011] In some embodiments, the heavy chain variable region of the antibody has an amino acid sequence in which an amino acid mutation occurs at position 5 and / or 117 relative to the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0012] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:1, the 5th position of the amino acid sequence of the heavy chain variable region of the antibody is an amino acid other than valine (V). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:1, the 5th position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to, for example, but not limited to, glycine (G), alanine (A), leucine (L), isoleucine (I), phenylalanine (F) or proline (P). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:1, the 5th position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to leucine (L). In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0013] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO: 1, the 117th position of the amino acid sequence of the heavy chain variable region of the antibody is an amino acid other than isoleucine (I). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO: 1, the 117th position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to, for example, but not limited to, glycine (G), alanine (A), valine (V), leucine (L), phenylalanine (F) and proline (P). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO: 1, the 117th position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to valine (V). In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0014] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO: 1, the heavy chain variable region of the antibody may have a V5L and / or I117V mutation.

[0015] In some embodiments, the light chain variable region of the antibody has an amino acid sequence in which an amino acid mutation occurs at position 1 and / or position 4 relative to the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0016] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:2, the first position of the amino acid sequence of the light chain variable region of the antibody is an amino acid other than aspartic acid (D). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:2, the first position of the amino acid sequence of the light chain variable region of the antibody can be mutated to, for example, but not limited to, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F) and proline (P). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:2, the first position of the amino acid sequence of the light chain variable region of the antibody can be mutated to alanine (A). In a specific embodiment, the amino acid sequence of the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0017] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:2, the 4th position of the amino acid sequence of the light chain variable region of the antibody is a non-methionine (M) amino acid. In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:2, the 4th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to, for example, but not limited to, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F) and proline (P). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:2, the 4th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to leucine (L). In a specific embodiment, the amino acid sequence of the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0018] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:2, the light chain variable region of the antibody may have a D1A and / or M4L mutation.

[0019] In some embodiments, the heavy chain variable region of the antibody has an amino acid sequence in which an amino acid mutation occurs at position 3 and / or 93 relative to the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0020] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:3, the third position of the amino acid sequence of the heavy chain variable region of the antibody is an amino acid other than arginine (R). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:3, the third position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to, for example, but not limited to, tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), methionine (M), asparagine (N), glutamine (Q) and threonine (T). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:3, the third position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to glutamine (Q). In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0021] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:3, the 93rd position of the amino acid sequence of the heavy chain variable region of the antibody is an amino acid other than isoleucine (I). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:3, the 93rd position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to, for example, but not limited to, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F) and proline (P). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:3, the 93rd position of the amino acid sequence of the heavy chain variable region of the antibody can be mutated to valine (V). In a specific embodiment, the amino acid sequence of the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0022] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:3, the heavy chain variable region of the antibody may have R3Q and / or I93V mutations.

[0023] In some embodiments, the light chain variable region of the antibody has an amino acid sequence in which an amino acid mutation occurs at position 13 and / or position 106 relative to the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0024] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:4, the 13th position of the amino acid sequence of the light chain variable region of the antibody is an amino acid other than threonine (T). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:4, the 13th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to, for example, but not limited to, tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), methionine (M), asparagine (N), glutamine (Q) and threonine (T). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:4, the 13th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to serine (S). In a specific embodiment, the amino acid sequence of the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0025] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:4, the 106th position of the amino acid sequence of the light chain variable region of the antibody is an amino acid other than lysine (K). In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:4, the 106th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to, for example, but not limited to, tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), methionine (M), asparagine (N), glutamine (Q) and threonine (T). In a specific embodiment, relative to the amino acid sequence shown in SEQ ID NO:4, the 106th position of the amino acid sequence of the light chain variable region of the antibody can be mutated to glutamine (Q). In a specific embodiment, the amino acid sequence of the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0026] In some embodiments, relative to the amino acid sequence shown in SEQ ID NO:4, the light chain variable region of the antibody may have T13S and / or K106Q mutations.

[0027] In some embodiments, the heavy chain variable region of the antibody can have an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence or at least 99% identity to the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.

[0028] In some embodiments, the light chain variable region of the antibody can have an amino acid sequence as shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence or at least 99% identity to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0029] In some embodiments, the antibody can have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5 or SEQ ID NO:7, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:5 or SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:8, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:8.

[0030] In some embodiments, the antibody can have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:11, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:11; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:12, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:12.

[0031] In some embodiments, the antibody may have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:5; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:6.

[0032] In some embodiments, the antibody can have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:8.

[0033] In some embodiments, the antibody may have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:9; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO:10.

[0034] In some embodiments, the antibody may have: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO: 11; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO: 12.

[0035] The above specific sequences are shown in Table 1.

[0036] Table 1. Amino acid sequences of heavy and light chains of single-chain antibodies and their variants

[0037]

[0038]

[0039] In some embodiments, the antibody may be an antibody of the IgG and its subclasses, IgM, IgE, IgA, and IgD types.

[0040] In some embodiments, the antibody can be a complete antibody or an antibody fragment, and the antibody fragment can be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), Fv, a single-chain antibody ("scFv"), a double scFv, (scFv)2, a miniantibody, a double antibody, a three-antibody, a four-antibody, a disulfide-stabilized Fv protein ("dsFv"), a single domain antibody (sdAb, nanoantibody), a heavy chain variable region-only antibody (e.g., camel VHH, camel nanoantibody, shark IgNAR), and a portion of a full-length antibody responsible for antigen binding. In some embodiments, the antibody can be a single-chain antibody.

[0041] In some embodiments, the antibodies of the present disclosure have significantly higher antigen (ie, rabies virus) binding activity than the antibody before mutation.

[0042] According to yet another aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the above-mentioned antibody of the present disclosure.

[0043] According to another aspect of the present disclosure, an expression vector is provided, which includes the above-mentioned nucleic acid molecule of the present disclosure.

[0044] According to another aspect of the present disclosure, a host cell is provided, which includes the above-mentioned nucleic acid molecule of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell can be selected from bacteria, yeast cells or mammalian cells. In some embodiments, the host cell can be selected from Escherichia coli, yeast, Chinese hamster ovary (CHO) cells (including CHO-K1, CHO DG44, CHO S, CHOZN and CHODUXB11), human myeloid leukemia cells (including GLYCOEXPRESS), HeLa (human cervical cancer cells), African green monkey kidney fibroblasts (including CV-1, COS-1 and COS-7), African green monkey kidney epithelial cells (including Vero), BHK, MDCK, C127, PC12, human embryonic kidney cells (including HEK-293, HEK-293T, HEK-293F and HEK-293E), PER C6, NS0, WI38, R1610, BALBC / 3T3, HAK, SP2 / O, P3x63-Ag8.653, BFA-1c1BPT, or RAJI cells, etc.

[0045] According to yet another aspect of the present disclosure, a pharmaceutical composition is provided, which includes the antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0046] According to another aspect of the present disclosure, there is provided the use of the antibody of the present disclosure in treating or preventing rabies virus infection-related diseases. In some embodiments, the rabies virus infection-related diseases may include rabies. In some embodiments, the rabies virus (RV) is not limited to any specific rabies virus.

[0047] According to another aspect of the present disclosure, there is provided a method of administering the antibody of the present disclosure for treating or preventing rabies, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present disclosure is not limited to these exemplified administration methods. Preferably, the composition of the present disclosure is administered by intravenous or subcutaneous injection.

[0048] According to yet another aspect of the present disclosure, a detection kit for detecting rabies virus is provided, which comprises the antibody of the present disclosure.

[0049] According to another aspect of the present disclosure, provided is a use of the antibody of the present disclosure in the preparation of a medicament for treating or preventing rabies virus infection or a disease associated with rabies virus infection.

[0050] According to yet another aspect of the present disclosure, provided is a method for treating or preventing rabies virus infection or a disease associated with rabies virus infection, the method comprising administering an effective amount of the antibody of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The anti-rabies virus antibody of the present invention and its grayscale image of SDS-PAGE electrophoresis result are shown.

[0052] Figure 2 The results of the binding activity test of the starting antibody 10H5 and its mutants to the inactivated virus are shown. The horizontal axis is the different concentrations of the purified anti-RV single-chain antibody.

[0053] Figure 3 The results of the binding activity test of the starting antibody 4H3 and its mutants to the inactivated virus are shown. The horizontal axis is the different concentrations of the purified anti-RV single-chain antibody. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0055] Rabies virus is generally classified as a large, bullet-shaped, enveloped, single-stranded RNA virus, and the genome of rabies virus encodes five viral proteins: RNA-dependent RNA polymerase (L); nucleoprotein (N); phosphorylated protein (P); matrix protein (M), which is located on the inside of the viral envelope; and outer surface glycoprotein (G). G protein (62-67 kDa) is a type I glycoprotein composed of 505 amino acids with two to four potential N-glycosylation sites. G protein covers the outer surface of the viral particle envelope and can serve as a target antigen to induce antibodies that neutralize the virus.

[0056] Rabies is a vaccine-preventable zoonotic viral disease that affects the central nervous system. Once clinical signs appear, rabies is almost 100% fatal. Domestic dogs are responsible for transmitting the rabies virus to humans in up to 99% of cases. However, rabies can also infect domestic and wild animals. Rabies is transmitted to humans and animals through bites, scratches, or direct contact with mucous membranes (such as the eyes, mouth, or open wounds), usually through saliva.

[0057] Based on the existing antibodies 4H3 and 10H5, the present invention screened out antibody mutants 4H3_H2L2 and 4H3_H1L1, 10H5_H4L2 and 10H5_H2L1 with significantly improved antigen binding ability through amino acid sequence mutation, laying a foundation for their use as specific antibody drugs for clinical prevention and treatment of rabies virus.

[0058] definition

[0059] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0060] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0061] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0062] "Percentage of sequence identity" or "percent identity" between two amino acid or nucleotide sequences referred to herein refers to the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, a gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides or amino acids are counted, but the nucleotides or amino acids from the reference sequence are not counted, the gaps present in the reference sequence are not counted.

[0063] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0064] The term "antibody" as used herein includes polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term includes genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heterologous conjugate antibodies, multispecific, for example, bispecific antibodies, double antibodies, three antibodies, and four antibodies, tandem two-scFv, tandem three-scFv. Unless otherwise indicated, the term "antibody" should be understood to include its functional antibody fragments. The term also includes complete or full-length antibodies, including antibodies of any type or subtype, including IgG and its subtypes, IgM, IgE, IgA, and IgD. In the present disclosure, the desired biological activity or function refers to the ability of the antibody to specifically bind to rabies virus.

[0065] The term "mutation" as used herein includes amino acid substitution, deletion, insertion or any combination thereof. The "mutation" can generate amino acid mutations using genetic or chemical methods known in the art, including but not limited to methods such as site-directed mutagenesis, PCR, gene synthesis, etc. In some embodiments, the "mutation" of an amino acid can be used interchangeably with the "replacement", "displacement" or "substitution" of an amino acid, and can refer to the substitution of a conservative amino acid, wherein an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0066] In some embodiments, the heavy chain variable region and the light chain variable region in the single-chain antibody can be directly connected or connected through a linker. The term "linker" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. The linker should have a length suitable for connecting two or more monomer domains in this way, and the linker can ensure that the different domains connected to it are correctly folded and appropriately presented, thereby exerting their biologically active functions. In different embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, having glycine, glutamine and / or serine residues. In some embodiments, the linker can be selected from (G n S) m , (G) n 、(EAAAK) n or (XP) n , wherein n and m are each independently selected from integers of 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5. In some embodiments, the linker may further include, for example, GGGGSGGGGSGGGGS (SEQ ID NO: 23) and the like.

[0067] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0068] In this article, the term "recombinant cell" generally refers to a cell that has unique traits of stable inheritance by modifying or recombining the genetic material of a host cell using genetic engineering technology or cell fusion technology. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences disclosed herein, and can be used for the expression and / or secretion of target proteins.

[0069] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active fusion protein with a liquid carrier, a finely divided solid carrier, or both.

[0070] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0071] As used herein, the term "titer" refers to the amount of antigen-antibody (or fragment) reaction to indicate its immune effect. Titer is usually expressed in international units (IU). Herein, titer indicates the titer per mg of protein (IU), expressed in units of "IU / mg", and total titer is the titer per mL of solution, expressed in units of "IU / mL".

[0072] The term "MOI" as used herein refers to the multiplicity of infection, i.e., the ratio of bacteriophage or virus to infected target (e.g., cell). For example, when referring to cells inoculated with infectious viral particles, the multiplicity of infection or MOI is the ratio determined by the number of infectious viral particles precipitated in a well divided by the number of target cells present in the well.

[0073] As used herein, the term "prevent" or "prevent" refers to partially or completely delaying the onset of a disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical manifestations of a specific disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or manifestations of a specific disease, disorder, and / or condition; partially or completely delaying the progression from a specific disease, disorder, and / or condition; and / or reducing the risk of developing pathology associated with a disease, disorder, and / or condition.

[0074] As used herein, the term "treat" refers to, for example, a reduction in the severity of a disease or disorder; a shortening of the duration of a disease course; an improvement or elimination of one or more symptoms associated with a disease or disorder; providing a beneficial effect to a subject suffering from a disease or disorder, but not necessarily curing the disease or disorder. The term also includes the prevention or prophylaxis of a disease or disorder or its symptoms.

[0075] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0076] Example

[0077] Example 1. Single-chain antibody sequence synthesis, expression and purification

[0078] The experimental steps are as follows:

[0079] 1) According to the amino acid sequences of antibodies 10H5_scFv, 10H5_H4L2, 10H5_H2L1, 4H3_scFv, 4H3_H2L2 and 4H3_H1L1 (see Table 2 below), their encoding nucleic acid sequences were synthesized respectively and inserted into pComb 3X expression vector (Genwiss) to obtain expression plasmids of two antibodies respectively.

[0080] Table 2. Amino acid sequences of 10H5_scFv and 4H3_scFv and their variants

[0081]

[0082]

[0083] 2) Transform the scFv expression plasmids into HB2151 competent cells, spread them on 2YT plates (1% glucose, 100 μg / mL ampicillin), and incubate at 30°C overnight;

[0084] 3) Pick a single clone and transfer it to 100 mL 2YT medium (0.2% glucose, 100 μg / mL ampicillin), shake at 37°C, 220 rpm, add 0.5 mM IPTG to a final concentration when the OD value reaches 0.6, and shake at 30°C, 220 rpm overnight;

[0085] 4) 4000g, 10min, centrifuge to collect bacteria. Resuspend the bacteria in 20mL PBS, add 0.5mL 100mg / mL polymyxinb (Aladdin, P105490), shake at 37℃ and 220rpm for half an hour;

[0086] 5) Centrifuge at 4000g for 15 min, take the supernatant, and filter through a 0.45 μm filter membrane. Add imidazole to the supernatant to a final concentration of 10 mM;

[0087] 6) 0.3 mL Ni-NTA (QIAGEN, 30230) was loaded onto the column and balanced with 10 mL binding buffer (PBS, 0.3 M NaCl, 10 mM imidazole), then the bacterial supernatant was added, and after it was drained, 10 mL washing buffer (PBS, 0.3 M NaCl, 20 mM imidazole) was added, and after it was drained, the protein was eluted with 5 mL elution buffer (PBS, 0.3 M NaCl, 200 mM imidazole);

[0088] 7) SDS-PAGE was used to identify the protein; the protein was concentrated using a protein ultrafiltration tube (MilliPore, UFC801008) and the solvent was replaced.

[0089] SDS-PAGE results are as follows Figure 1 As shown, the experiment obtained scFv antibodies of the correct size, and the purified single-chain antibody proteins of 10H5 and its mutants 10H5_H4L2 and 10H5_H2L1, 4H3 and its mutants 4H3_H2L2 and 4H3_H1L1 had high purity (>90%).

[0090] Example 2. ELISA binding activity detection

[0091] The specific experimental steps are as follows:

[0092] 1) Coat an appropriate amount of antigen (100 μL) (RV vaccine (Liaoning Chengda, human rabies vaccine) 1 μg or 3 μg) in an ELISA plate and incubate overnight at 4°C.

[0093] 2) Wash three times with 200 μL PBST, add 5% skim milk powder, and block at 37°C for 1 h.

[0094] 3) Wash three times with 200 μL PBST, add purified anti-RV single-chain antibody of different concentrations, and incubate at 37°C for 1 h.

[0095] 4) Wash three times with 200 μL PBST, add 0.3 μg / mL anti-FLAG HRP (Sigma, A8592), and incubate at 37°C for 1 h.

[0096] 5) Wash three times with 200 μL PBST, add 100 μL TMB colorimetric solution (Biyuntian, P0209) for color development, and then terminate the color development with 2M H2SO4.

[0097] 6) Read the plate at 450 nm using an ELISA reader and record the OD value.

[0098] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 The results showed that after site mutation of the 10H5 antibody, the antibody's virus binding activity was significantly improved. Figure 3 The results showed that after site mutation of the 4H3 antibody, the antibody's virus binding activity was significantly improved.

[0099] Example 3. Rapid fluorescence focus inhibition test method for detecting antibody titer

[0100] This example detects the antibody titer according to the rapid fluorescence focus inhibition test method in the Chinese Pharmacopoeia 2020 Edition. The specific steps are as follows:

[0101] Based on the ability of rabies immunoglobulin in the test sample to neutralize rabies virus, the test sample and standard sample are serially diluted, mixed with rabies virus suspension respectively, and used to infect sensitive cells. The cells are stained with fluorescent antibodies within a specified time and the reduction of fluorescent foci is observed to determine the titer of the test sample.

[0102] 1. Reagents:

[0103] (1) DMEM cell culture medium containing 5% newborn calf serum: Take DMEM cell culture medium containing 5% newborn calf serum, add antibiotics to a final concentration of 100 U / mL antibiotics, and add glutamine to a final concentration of 0.03%. Prepare it before use and add an appropriate amount of NaHCO3 according to the instructions of the DMEM manual to adjust the pH to 7.6.

[0104] (2) DMEM cell culture medium containing 10% newborn calf serum: Take DMEM cell culture medium containing 10% newborn calf serum, add antibiotics to a final concentration of 100 U / mL antibiotics, and add glutamine to a final concentration of 0.03%. Prepare it before use and add an appropriate amount of NaHCO3 according to the instructions of the DMEM manual to adjust the pH to 7.6.

[0105] (3) Phosphate buffer (PBS): Weigh 0.24 g potassium dihydrogen phosphate, 1.44 g disodium hydrogen phosphate (Na2HPO4·12H2O), and 8 g sodium chloride, dissolve in water and dilute to 1000 mL, and adjust the pH to 7.2 with sodium hydroxide. Prepare before use.

[0106] (4) 80% cold acetone: Take 80 mL of acetone, add 20 mL of 0.1 mol / L PBS (pH 7.6), mix well, seal, and store at 4°C.

[0107] (5) 80% glycerol: Measure 80 mL of glycerol, add it to 20 mL of water, mix well, cover and store at 4°C.

[0108] (6) 0.25% trypsin-EDTA.

[0109] (7) Fluorescently labeled rabies virus nucleoprotein antibody (MILLIPORE Rabies DFAReagent): Dilute to working concentration according to the instructions.

[0110] 2. Preparation of virus for neutralization:

[0111] (1) Preparation of virus suspension: Take CVS-11 virus seed (usually freeze-dried virus seed) and make appropriate dilutions, inoculate well-growing BHK-21 golden hamster kidney cells (BSR cells) at an infection dose of 0.1, culture at 37°C and 5% carbon dioxide for 1 day, then transfer to 34°C for further culture. After 2 days, collect the culture supernatant, centrifuge at 4000 rpm at 4°C for 10 minutes to remove cell debris, take the supernatant, add 10% newborn calf serum, mix well, divide into small tubes, and freeze at below -70°C for later use.

[0112] Pre-titration of virus solution: Take one tube of frozen virus suspension, thaw it in running water, and make 5-fold serial dilutions starting from 1:5 on a 24-well culture plate. Take 100 μL of virus solution and add it to 400 μL of DMEM culture medium containing 10% inactivated newborn calf serum. After thorough mixing, take 50 μL of each dilution and transfer it to a 96-well culture plate. Make 2 copies of each dilution in parallel, and add 5×10 6 / mL BSR cell suspension 50μL, cultured at 37℃, 5% carbon dioxide conditions for 24 hours. After the culture, discard the supernatant, wash once with PBS, add 80% cold acetone, 50μL per well, fix at 4℃ for 30 minutes, or -30℃ for 10 minutes, discard acetone, add 50μL working concentration of FITC-labeled rabies virus nucleoprotein antibody to each well after evaporation and drying, incubate at 37℃ for 30 minutes, wash 3 times with PBS, spin dry, add 80% glycerol 50μL per well, observe under a fluorescence microscope; count the number of fluorescent foci in each well, take the wells with less than 30 fluorescent foci in each well, record the number of fluorescent foci in the adjacent 4 wells, take the average value, and calculate as follows:

[0113] Virus titer (FFU / mL) = (average value of fluorescence foci in the well with the highest dilution factor × 5 + average value of fluorescence foci in the adjacent well with a lower dilution factor) / 2 × virus dilution factor in the well with a lower dilution factor × 20

[0114] (2) Preparation of virus solution for neutralization: Take one tube of virus suspension and perform the same procedure as the pre-titration of the virus solution. Count the proportion of fluorescent foci in each well under a fluorescence microscope, and use the virus dilution at which 80% to 95% of the cells are infected by the virus as the virus dilution for the neutralization test.

[0115] Take one tube of frozen virus suspension, thaw it with running water, dilute the virus to the virus dilution for neutralization test with DMEM culture medium containing 5% inactivated newborn calf serum, and place it in an ice bath for later use.

[0116] Preparation of rabies immunoglobulin standard solution: Rabies immunoglobulin standard is provided by the national drug testing agency, or a working standard calibrated by the national drug testing agency is used. The preparation method is carried out according to the instruction manual. The rabies immunoglobulin standard is diluted 3 times in series with DMEM culture medium containing 10% inactivated newborn calf serum, that is, 100 μL of culture medium is added to each well of the 96-well culture plate in advance, and 50 μL of the test sample is added thereto to make a 1:3 dilution. After sufficient mixing, 50 μL is drawn and added to the 100 μL culture medium in the next well to make a 1:9 dilution. Repeat this series dilution for several wells to the appropriate dilution.

[0117] Preparation of test solution: The test sample (inactivated for 30 minutes) was diluted 3-fold in series with DMEM culture medium containing 10% inactivated newborn calf serum, i.e., 100 μL of culture medium was pre-added to each well of a 96-well culture plate, and 50 μL of the test sample was added thereto, resulting in a 1:3 dilution. After thorough mixing, 50 μL was pipetted and added to the 100 μL culture medium in the next well, resulting in a 1:9 dilution. Repeat this series of dilutions for several wells to the appropriate dilution, and 50 μL in the last well was discarded.

[0118] Assay method: Add the neutralization virus to each well of the diluted standard and test sample, 50 μL / well, and set up normal cell control wells (add only 100 / μL DMEM to the well) and neutralization virus control wells (100 μL DMEM containing 5% inactivated newborn calf serum, add 50 μL neutralization virus), mix well and neutralize at 37°C for 1 hour, add 1×10 6 50 μL of BSR cell suspension with a concentration of cells / mL was cultured at 37°C and 5% carbon dioxide for 24 hours. After the culture was completed, the culture medium was aspirated, 100 μL of PBS was added to each well for washing and aspiration, and 50 μL of 80% acetone precooled to 4°C was added to each well, fixed at 4°C for 30 minutes, or -30°C for 10 minutes, and the acetone was discarded. After evaporation and drying, the working concentration of fluorescently labeled rabies virus nucleoprotein antibody was added, 50 μL / well, and incubated at 37°C for 30 minutes. The liquid was discarded, and the plate was washed 2 to 3 times with PBS, dried, and 50 μL of 80% glycerol was added to each well, and observed under a fluorescence microscope. The calculation formula is as follows:

[0119]

[0120] Where A is the dilution of the standard that is less than 50% of the fluorescent focus ratio;

[0121] B is the percentage of fluorescent foci in wells where the standard sample is lower than 50% of the fluorescent foci ratio;

[0122] C is the percentage of fluorescent foci in wells where the standard is higher than 50% of the fluorescent foci ratio;

[0123] n1 is the dilution multiple of the standard.

[0124]

[0125] Where E is the dilution of the test product that produces less than 50% fluorescent focus ratio;

[0126] F is the percentage of fluorescent foci in the wells where the test sample has a ratio of less than 50% fluorescent foci;

[0127] G is the percentage of fluorescent foci in the wells with a ratio of more than 50% fluorescent foci for the test article;

[0128] n2 is the dilution multiple of the test sample.

[0129] Test product potency (IU / ml) = 10 （J-K） ×L

[0130] Where J is the standard lgED50;

[0131] K is the lgED50 of the test product;

[0132] L is the potency of the standard, IU / mL.

[0133] in:

[0134] (1) The virus titer for neutralization should not be less than 10 6 FFU / mL.

[0135] (2) When diluting the virus, it should be done in an ice bath if possible.

[0136] (3) The test is valid only if 80% to 95% of the cells in the virus control wells are fluorescently stained and no fluorescence is present in the cell control wells.

[0137] Table 3. Antibody titer test results

[0138] Sample name Protein concentration (mg / mL) Total titer (IU / mL) Potency (IU / mg) 4H3 0.34 769.07 2294.92 4H3_H1L1 0.45 937.68 2085.35 4H3_H2L2 0.30 769.07 2542.64 10H5 1.52 2861.80 1881.02 10H5_H2L1 2.24 3162.15 1409.56 10H5_H4L2 0.56 1140.88 2021.35

[0139] The antibody titer test results are shown in Table 3. According to the test results, the titers of single-chain antibodies 4H3_H2L2 and 10H5_H4L2 are improved to a certain extent compared with the starting antibodies 4H3 and 10H5. For example, the titer of 4H3_H2L2 is increased by 10.8% compared with the starting antibody 4H3, and 10H5_H4L2 is increased by 7.5% compared with the starting antibody 10H5. The present invention obtains anti-rabies virus single-chain antibodies by mutation, and successfully obtains a series of human new genetically engineered single-chain antibodies with higher binding activity and can specifically neutralize rabies virus.

[0140] Example 4. IgG antibody expression and activity detection

[0141] In this example, IgG antibodies of 4H3_H2L2_1 and 10H5_H4L2_1 were prepared. In brief, nucleic acid sequences encoding IgG antibody amino acid sequences (see the table below) were inserted into expression vectors, transfected into CHO cells to express IgG antibodies 4H3_H2L2_1 and 10H5_H4L2_1, and their activities were detected after purification. The sequences are shown in Table 4. It was found that IgG antibodies of 4H3_H2L2_1 and 10H5_H4L2_1 had improved binding and neutralization activity against rabies virus.

[0142] Table 4. Light and heavy chain sequences of IgG antibodies 4H3_H2L2_1 and 10H5_H4L2_1

[0143]

[0144]

[0145] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An anti-rabies virus antibody, characterized in that: The antibody has: A heavy chain variable region, an amino acid sequence having an amino acid mutation at position 5 and / or 117 relative to the amino acid sequence of SEQ ID NO: 1, or having at least 85% sequence identity thereto, or an amino acid mutation at position 3 and / or 93 relative to the amino acid sequence of SEQ ID NO: 3, or having at least 85% sequence identity thereto; and A light chain variable region, whose amino acid sequence has an amino acid mutation at position 1 and / or 4 relative to the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence with at least 85% sequence identity thereto, or, relative to the amino acid sequence shown in SEQ ID NO:4, has an amino acid mutation at position 13 and / or 106, or an amino acid sequence with at least 85% sequence identity thereto.

2. The antibody according to claim 1, characterized in that The heavy chain variable region of the antibody has: An amino acid sequence having an amino acid mutation at position 5 and / or position 117 relative to the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity thereto; preferably an amino acid other than valine at position 5 and / or an amino acid other than isoleucine at position 117, more preferably a mutation of V5L and / or I117V, or An amino acid sequence having an amino acid mutation at position 3 and / or position 93 relative to the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 85% sequence identity thereto; preferably an amino acid other than arginine at position 3 and / or an amino acid other than isoleucine at position 93, and more preferably having mutations of R3Q and / or I93V.

3. The antibody according to claim 1, characterized in that The light chain variable region of the antibody has: An amino acid sequence having an amino acid mutation at position 1 and / or position 4 relative to the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity thereto; preferably an amino acid other than aspartic acid at position 1 and / or an amino acid other than methionine at position 4, more preferably a mutation of D1A and / or M4L, or An amino acid sequence having an amino acid mutation at position 13 and / or position 106 relative to the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 85% sequence identity thereto; preferably an amino acid other than threonine at position 13 and / or an amino acid other than lysine at position 106, more preferably a mutation of T13S and / or K106Q.

4. The antibody according to claim 1, characterized in that The antibody has: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, or an amino acid sequence having at least 85% identity to the amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and A light chain variable region having an amino acid sequence as shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, or an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:

12.

5. The antibody according to claim 1, characterized in that The antibody has: A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 7, or an amino acid sequence that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 8, or an amino acid sequence that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 8, and / or A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:11, or an amino acid sequence that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:11; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:12, or an amino acid sequence that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:

12.

6. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid molecule according to claim 6. A host cell comprising the nucleic acid molecule according to claim 6 .

9. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

10. A detection kit for detecting rabies virus, comprising the antibody according to any one of claims 1 to 5.

11. Use of the antibody according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing diseases related to rabies virus infection.

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