Fully human monoclonal antibody targeting rabies virus G protein epitope and application thereof

Abstract

The present invention provides a rabies virus G protein antigen epitope targeting completely human monoclonal antibody and applications thereof, the completely human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementary determining region amino acid sequences: GDISSSCFY, IHYSGST and ARHRRGYCYDSEKGGTNWFDP, and the light chain variable region has three complementary determining region amino acid sequences: GDISSSCFY, IHYSGST and ARHRRGYCYDSEKGGTNWFDP. The amino acid sequences of the three complementary determining regions of the light chain variable region are respectively as follows: QGISND, ATS and LQDYEFPLT. The fully human monoclonal antibody has efficient and broad-spectrum anti-rabies virus neutralizing activity, is high in expression, fully human-derived and good in stability, and can be used for preparing rabies virus detection products or drugs for preventing and treating rabies.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and immunology, and relates to a fully human monoclonal antibody that targets the G protein antigenic epitope of rabies virus and its application. Background Technology

[0002] Rabies is an acute zoonotic infectious disease caused by the rabies virus (RABV), and it is one of the deadliest diseases (nearly 100%). The disease is primarily transmitted through bites or scratches from infected animals. After the virus invades the central nervous system, it causes encephalitis, clinically manifesting as hydrophobia, aerophobia, pharyngeal muscle spasms, and progressive paralysis, hence the name "hydrophobia." The rabies virus belongs to the family Rhabdoviridae and the genus Lyssavirus. The virus particle is bullet-shaped, approximately 75 nm in diameter and 180 nm in length. Its core is a single-stranded negative-sense RNA genome, approximately 12 kb in length, encoding five structural proteins (N, P, M, G, L). It is enclosed in a helical symmetrical nucleocapsid and a lipid envelope, with glycoprotein (G protein) spikes embedded on its surface. The G protein is the main antigen, determining the virus's virulence and immunogenicity. Rabies virus is sensitive to ultraviolet light, high temperature (56°C for 30 minutes), and strong acids and alkalis, but can survive for weeks to months at low temperatures or in glycerol.

[0003] Dogs are the primary source of rabies virus infection (accounting for 99% of human cases), while bats are a major host in the Americas. Wild animals such as foxes, raccoons, and wolves can also carry the virus. The main route of transmission for rabies virus is through bites or scratches from animals carrying the rabies virus (such as dogs, cats, and bats). The virus enters the wound through saliva. There are also very rare reports of infection through contact with viral contaminants on mucous membranes (such as the conjunctiva of the eye), organ transplantation, or aerosol inhalation (most commonly found in bat caves).

[0004] The pathological process of rabies virus invasion can be divided into the following stages: 1. Local replication: The virus initially replicates in the muscle tissue of the wound, with an incubation period of 1-3 months (from a few days to several years). 2. Nerve invasion: The virus is transported to the spinal cord via the axons of motor nerve endings, eventually spreading to the entire brain and causing encephalomyelitis. 3. Central nervous system infection: The virus replicates extensively within neurons, leading to nerve cell apoptosis and inflammatory responses, clinically presenting as an excitation phase (mania, hydrophobia) and a paralytic phase (paralysis, coma). 4. Peripheral spread: The virus migrates from the central nervous system to peripheral organs, especially invading the salivary glands, promoting transmission.

[0005] The clinical stages of viral infection can be divided into the following phases: 1. Prodromal phase (2-10 days): low-grade fever, numbness or tingling around the wound. 2. Acute neurological phase: divided into manic (80%) and paralytic (20%) types. The former is characterized by hydrophobia, aerophobia, and hallucinations, while the latter is mainly characterized by limb paralysis. 3. Coma and death: usually occurring within 7-10 days after symptom onset due to respiratory failure. Viral RNA can be detected by PCR in saliva or cerebrospinal fluid, or by antigen detection via skin biopsy.

[0006] Without intervention, the fatality rate of rabies infection is close to 100%. Post-exposure prophylaxis (PEP) is currently the most effective rabies prevention and control strategy, and passive immunization is a crucial measure in PEP. It primarily provides immediate immune protection during the window period between rabies vaccine injection and the production of sufficient antibodies. Current passive immunization agents mainly include human rabies immunoglobulin and equine anti-rabies serum. These agents are expensive, have limited supply, and pose risks such as serum infection and allergic reactions. Monoclonal antibodies, due to their higher safety, lower production cost, and stable source, have become the WHO-recommended alternative to immunoglobulins; however, there is currently a severe global shortage of rabies virus monoclonal antibodies.

[0007] Therefore, there is an urgent need to develop a fully human monoclonal therapeutic antibody that provides good protection against rabies. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a fully human monoclonal antibody targeting the G protein antigenic epitope of rabies virus and its application. This fully human monoclonal therapeutic antibody exhibits good protective efficacy against rabies.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, a fully human monoclonal antibody targeting the G protein antigenic epitope of rabies virus is provided. The fully human monoclonal antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequences of the three complementarity-determining regions of the heavy chain variable region are: GDSISSSCFY, IHYSGST, and ARHRRGYCYDSEKGGTNWFDP, respectively. The amino acid sequences of the three complementarity-determining regions of the light chain variable region are: QGISND, ATS, and LQDYEFPLT, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.

[0011] Furthermore, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 7.

[0012] Furthermore, the monoclonal antibody also includes: The monoclonal antibody is modified by substituting, deleting, and / or adding one or more amino acids to obtain an antibody with the same function. Alternatively, it may include a heavy chain variable region having an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region. Alternatively, an antibody obtained by attaching a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0013] In some other implementations, V H and / or V L The amino acid sequence can be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the above sequence. It has a V-shaped structure similar to the above sequence. H and V L V regions with high (i.e., 80% or higher) homology H and V L The antibody for the region can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NO: 1 and / or SEQ ID NO: 3, followed by detection of the retained function of the encoded altered antibody using the functional assays described herein.

[0014] In other embodiments, the variable region gene can be converted into the scFv gene, once the encoding V is obtained. H and V L These DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes.

[0015] In these operations, the encoding V L or V H The DNA fragment is effectively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used herein, "effective linking" means that two DNA fragments are linked together such that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, the nucleic acid molecule comprising a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0017] Furthermore, the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0018] Furthermore, the polynucleotide sequences encoding the heavy chain constant region of the monoclonal antibody are all as shown in SEQ ID NO: 6, and the polynucleotide sequences encoding the light chain constant region are all as shown in SEQ ID NO: 8.

[0019] In a third aspect of the invention, an expression vector comprising the nucleic acid is provided, the expression vector being capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0020] The vector can specifically be a plasmid vector, a bacteriophage vector, a viral vector, or a mammalian expression vector. This invention specifically uses a mammalian expression vector.

[0021] In a fourth aspect of the invention, an engineered bacterium or eukaryotic host cell comprising the expression vector described above is provided.

[0022] In a fifth aspect of the invention, the use of the monoclonal antibody described herein is provided in the preparation of rabies treatment drugs or virus detection products.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The fully human monoclonal antibody targeting the G protein epitope of rabies virus provided by this invention exhibits good neutralizing and protective effects against rabies virus-infected cells. The results of this invention show that the antibody has broad application prospects in the preparation of rabies treatment drugs. Specifically, the monoclonal antibody disclosed in this invention also has the following technical advantages: (1) All human origin, no humanization modification is required in clinical application.

[0024] (2) High activity, in a cell model of rabies virus infection, the half-maximal effective concentration (EC50) of WHU-R10 against rabies virus strain CVS-11 was 5.48 ng / mL.

[0025] (3) Broad spectrum: WHU-R10 has good neutralizing activity against all 10 rabies virus pseudoviruses detected.

[0026] (4) Good stability: Because the heavy chain and light chain genes of monoclonal antibodies come from the same cell in the human body and are naturally paired, the half-life of IgG1 antibodies in the human body is known to be 21 to 28 days. Theoretically, publicly available monoclonal antibodies have a consistent half-life in the human body. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a flow cytometry image showing single-cell sorting. Figure 2 The image is a pattern detected by an automated nucleic acid electrophoresis instrument for amplifying the variable region gene of a monoclonal antibody. Figure 3 The EC50 assay curve of the antibody in a pseudoviral cell model; Figure 4 The EC50 assay curve of the antibody in a true viral cell model; Figure 5 This is a diagram showing the protective effect of the antibody on a mouse model of a real virus.

[0029] Figure 6 The image shows the protective effect of the antibody of the present invention in a mouse model of rabies virus street strain infection, comparing the survival rates of mice in the treatment group and the control group. Detailed Implementation

[0030] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0031] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all 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 pertains. In the event of any conflict, this specification shall prevail.

[0032] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0033] The following will provide a detailed description of the monoclonal antibody, its preparation method, and its application effects, in conjunction with examples and experimental data.

[0034] Example 1: Screening and preparation of human anti-rabies virus monoclonal antibodies 1. Prepare a 96-well plate. Add 20 μL of RNase-free water and 20 U of RNase inhibitor to each well, cover with sealing film, and incubate at 4°C until use.

[0035] 2. Prepare samples (1) Cell thawing: The frozen PBMC cells isolated from the peripheral blood of vaccine recipients were taken out from -80℃ and quickly placed in warm water at 37℃. After the cells thawed, they were centrifuged at 800 rpm for 5 min and the supernatant was discarded. The cells were resuspended in 1 mL PBS into a flow cytometry tube, balanced, centrifuged at 800 rpm for 5 min and the supernatant was discarded. The cells were then resuspended in 1 mL PBS, centrifuged, and the supernatant was discarded. Finally, 100 μL of PBS was added for resuscitation, and 2 μL of the solution was diluted 10-fold for cell counting.

[0036] (2) Single-color tubes: 7 tubes (FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, CD20-BV421, CD38-FITC), 1×10 per tube 6 For each cell, the dye was added to a flow cytometry tube containing cells according to the antibody concentration recommended in the instructions, and the reaction volume was made up to 50 μL with PBS.

[0037] (3) Nude cell control: 1 tube, 1×10 6 One cell was added to a final volume of 50 μL with PBS.

[0038] (4) Cells used for sorting: 1 tube, first determine the number of cells, final concentration is 100 μL system (1×10 6 The cells were treated with FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, and G-His fluorescent dyes.

[0039] (5) Incubate the sample at 4°C in the dark for 1 h.

[0040] (6) Add 1 mL of PBS to each tube, centrifuge at 800 rpm for 5 min at 4℃, discard the supernatant, and repeat the washing process twice.

[0041] (7) After resuspending with 400 μL PBS, remove cell clusters with a 40 μm cell sieve and store at 4°C in the dark for sorting.

[0042] 3. Flow sorting Select CD3 - CD4 - CD8 - CD19 + IgD - G + The cells were sorted. The results of the flow cytometry sorting are shown in [the figure]. Figure 1First, select lymphocytes, then select unattached single cells, then select live cells, and finally select CD3 cells. － CD4 － CD8 － CD19 + B cells, and then IgD － Mature B cells were selected, and finally G cells were chosen. + Mature B cells are our target cells.

[0043] 4. Amplification of the variable region gene of the fully human monoclonal antibody using single-cell PCR technology. 4.1 Reverse Transcription PCR The reverse transcription PCR reference manual (QIAGEN, 210212) provides a simplified procedure as follows: After sorting cells by flow cytometry, add all of the following specific primers (primer sequences are shown in Table 1) to each reaction system simultaneously for each subtype of the heavy chain (H), kappa light chain (κ), and Lamda light chain (λ).

[0044] The PCR reaction system was prepared according to the QIAGEN instructions. The PCR reaction conditions were: reverse transcription at 50℃ for 30 min; followed by pre-denaturation at 95℃ for 15 min, 95℃ for 40 s, 55℃ for 30 s, 72℃ for 1 min, for 40 cycles, and finally extension at 72℃ for 10 min.

[0045] Table 1 - Primers for Reverse Transcription PCR

[0046] 4.2 Nested PCR Using 1 μL of the reverse transcription product as a template, PCR was performed to amplify the variable regions of H, κ, and λ. The primers for amplifying the variable regions of the heavy chain, kappa light chain, and λ light chain are shown in Table 2.

[0047] The PCR reaction system contains: 12.5 μL of DNA polymerase mixture (Kangwei Century Biotechnology Co., Ltd., CW2849), primers as above, 1 μL of reverse transcription product as template, and water to a final volume of 25 μL.

[0048] The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by 40 cycles of 94℃ for 30 s, 57℃ for 30 s, 72℃ for 45 s, and a final extension at 72℃ for 10 min.

[0049] The products after the reaction are used for nucleic acid electrophoresis identification.

[0050] Table 2. Nested PCR primers

[0051] 4.3 Detection was performed using an automated nucleic acid electrophoresis instrument. Some results of automated nucleic acid electrophoresis detection are shown below. Figure 2 The figure shows the heavy chain and light chain variable zone bands of WHU-R10.

[0052] 5. Synthesis of polynucleotide sequences with variable sequencing Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the variable region DNA sequence of the antibody based on the above polynucleotide sequence. A signal peptide sequence and restriction enzyme sites were added before the variable regions of the antibody heavy chain and light chain, and restriction enzyme sites were added at the tail.

[0053] 6. Construction of plasmid expression vectors Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize antibody heavy chain constant region DNA sequences and light chain constant region DNA sequences by adding restriction enzyme sites before and after the heavy chain and light chain constant region DNA sequences, based on the antibody heavy chain constant region DNA sequence and light chain constant region DNA sequence (heavy chain constant region sequence shown in SEQ ID NO: 5, DNA coding sequence shown in SEQ ID NO: 6; Kappa type light chain constant region sequence shown in SEQ ID NO: 7, DNA coding sequence shown in SEQ ID NO: 8). The pCAGGS empty vector plasmid (purchased from Shanghai Yuanmu Biotechnology Co., Ltd., catalog number P0165) and the antibody heavy chain constant region DNA sequence were digested using restriction endonucleases Nhe I and Sac I. The digested plasmid and antibody constant region DNA fragment were ligated using T4 DNA ligase to obtain a plasmid vector containing the antibody heavy chain constant region. The pCAGGS empty vector plasmid and the antibody light chain constant region DNA sequence were digested with restriction endonucleases Nhe I and Sac I. The digested plasmid and antibody constant region DNA fragments were then ligated using T4 DNA ligase to obtain a plasmid vector containing the antibody light chain constant region.

[0054] The constructed plasmid vector containing the constant region of the antibody heavy chain and the variable region of the antibody heavy chain synthesized in step 5 were digested with restriction endonucleases EcoRI and KpnI. The digestion products were then ligated to obtain a eukaryotic expression vector for expressing the WHU-R10 antibody heavy chain. Similarly, the constructed plasmid vector containing the constant region of the antibody light chain and the variable region of the antibody light chain synthesized in step 5 were digested with restriction endonucleases EcoRI and KpnI. The digestion products were then ligated using T4 DNA ligase to obtain a eukaryotic expression vector for expressing the WHU-R10 antibody light chain. Both vectors can then be used for antibody expression via transfection into eukaryotic cells.

[0055] The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of monoclonal antibody WHU-R10 are shown in SEQ ID NO: 1, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 3, as detailed in Table 3.

[0056] Table 3. Antibody CDR Sequences

[0057] 7. Transient expression and affinity chromatography purification of monoclonal antibodies Using the Expi 293 expression system, 15 μg of heavy chain and 15 μg of light chain were mixed and transfected into Expi 293F cells according to the instructions of the transfection reagent (Shanghai Liji Biotechnology Co., Ltd., EZ Trans Plus, AC04L011). After 5-6 days, the culture medium was harvested, and about 50 mL of supernatant was centrifuged. A 5 mL pre-packed Protein A affinity chromatography column was used, equilibrated with 20 mM PBS before loading. After the conductivity reached the baseline, the column was injected. After loading, the column was washed with 20 mM PBS until the baseline was stable. The target protein was eluted with 0.1 M pH 3.0 glycine buffer. When the OD280 was close to the baseline, the collection was stopped. The column was washed with at least 3 column volumes of 20 mM PBS until the baseline was stable. The column was then washed with 20% ethanol to purify and obtain the monoclonal antibody WHU-R10, which was then aliquoted and frozen.

[0058] Example 2: Neutralization activity analysis of rabies virus pseudovirus cell model 1. Pseudovirus Packaging: Different pseudoviruses were packaged based on the spike protein (G protein) of wild-type rabies virus and rabies virus mutant strains. Plasmids expressing different G proteins were transfected into Vero-E6 cells using Lipofectamine 2000 (Biosharp, BL623B). After 24 h, VSV-dG-GFP (1×10⁻⁶) diluted with DMEM was used. 6Transfected cells were inoculated with TCID50 / mL. After 24 hours, the supernatant containing the rabies virus pseudovirus was collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and stored at -80°C. The TCID50 of the pseudovirus was determined by serial dilution of BHK-21 cells and calculated according to the Reed-Muench method.

[0059] 2. Diluted antibodies: Rabies virus pseudovirus (3×10⁻⁶) 5 TCID50 / well) and diluted antibody were incubated on 96-well white plates at room temperature for 30 min, then inoculated with trypsin-treated BHK-21 cells at 2 × 10⁻⁶. 4 / Density mixing of pores.

[0060] 3. Detection and analysis of fluorescence readings: After 16 h of culture, the culture medium for infected cells was removed, and fluorescence readings were performed using a multi-functional microplate imaging system to calculate the neutralization percentage. The titer of a 50% neutralizing dilution (NT50) was calculated using GraphPad Prism software, and a nonlinear regression curve was fitted.

[0061] See results Figure 3 (The horizontal axis represents antibody concentration, and the vertical axis represents the neutralization effect (%) relative to the negative control group.) In a pseudovirus model of rabies virus, WHU-R10 was able to effectively neutralize 10 wild-type rabies virus pseudoviruses detected.

[0062] Example 3: Neutralization activity analysis in a rabies virus-infected cell model 1. Preparation and titration of rabies virus: CVS-11 virus stock was appropriately diluted and used to infect well-grown BHK-21 cells (MOI=0.1). After culturing at 37℃ and 5% CO2 for 2 days, the culture supernatant was collected. Cell debris was removed by centrifugation at 5000 rpm for 5 minutes, and the supernatant was aliquoted and stored at -80℃. One vial of the frozen virus solution was thawed rapidly with running water and serially diluted 5-fold. 50 μL of each dilution was transferred to a 96-well plate, with two replicates per well. 5 × 10⁵ mmol / L of the virus solution was added to each well. 5 50 μL of BHK-21 cell suspension (FFU / mL) was added and cultured at 37°C and 5% CO2 for 24 hours. After culture, the supernatant was discarded, the cells were washed once with PBS, and 50 μL of 80% cold acetone was added to each well. The cells were fixed at 4°C for 30 minutes, the acetone was discarded, and after evaporation and drying, 50 μL of FITC-labeled rabies virus nucleoprotein antibody at the working concentration was added to each well for staining. The cells were incubated at 37°C for 30 minutes, washed three times with PBS, and dried. The cells were read using a multi-well microplate imaging system. The number of fluorescent foci in each well was counted. For wells with fewer than 30 fluorescent foci, the number of fluorescent foci in four adjacent wells was recorded, and the average value was taken to calculate the virus titer (FFU / mL).

[0063] 2. Preparation of rabies virus solution for neutralization: Take one vial of virus suspension and proceed with the titration method for virus solution. Count the proportion of fluorescent foci in each well under fluorescence. The virus dilution used for the neutralization test is the one at which 80%–95% of the cells are infected by the virus.

[0064] 3. Neutralization Assay: Take one vial of frozen virus suspension, thaw it under running water, and dilute the virus to the neutralization assay concentration using DMEM culture medium containing 5% inactivated newborn calf serum. Keep the solution on ice. After serially diluting the antibody to be tested, mix it with the virus and incubate at 37°C for 1 hour. Add 5 × 10⁻⁶ antibodies to each well. 5 50 μL of BHK-21 cell suspension ( / mL) was added and cultured at 37°C and 5% CO2 for 24 hours. After culture, the culture medium was aspirated, and 100 μL of PBS was added to each well for washing and aspiration. Then, 50 μL of 80% acetone pre-cooled to 4°C was added to each well. The cells were fixed at 4°C for 30 minutes or at -30°C for 10 minutes. The acetone was discarded, and after evaporation and drying, 50 μL of FITC-labeled rabies virus nucleoprotein antibody at the working concentration was added to each well for staining. The cells were incubated at 37°C for 30 minutes. The liquid was discarded, and the cells were washed 2-3 times with PBS. The cells were then dried and the values ​​were read using a multi-well microplate imaging system to calculate the antibody neutralization percentage.

[0065] Result: See Figure 4 (The horizontal axis represents antibody concentration, and the vertical axis represents the neutralization percentage relative to the negative control group.) The half-maximal effective concentration (EC50) of WHU-R10 against rabies virus strain CVS-11. 50 The concentration was 5.48 ng / mL.

[0066] Example 4: Analysis of the protective ability of rabies virus in a mouse model 1. Antibody protective efficacy test in a mouse model of rabies virus fixed strain infection. Rabies virus strain CVS-11 was appropriately diluted and injected intramuscularly into BALB / c mice via the leg, with an infectious dose of 20 LD50 per mouse. One day post-infection, the test monoclonal antibody and control antibody were injected intraperitoneally at a dose of 20 mg / kg. The mice's condition was observed, and the disease progression was recorded. The experimental endpoint was set at 30 days post-infection. Mice that died during the experiment were recorded at the time of death.

[0067] See results Figure 5 Mice injected with WHU-R10 one day after infection had 100% survival, while all mice in the control group died within 13 days.

[0068] 2. Antibody protective efficacy test in a mouse model of rabies virus street strain infection. A street strain of rabies virus was appropriately diluted and injected intramuscularly into the leg of BALB / c mice at an infectious dose of 20 LD50 / mouse. One day post-infection, the test monoclonal antibody and control antibody were injected intraperitoneally at a dose of 20 mg / kg. The mice's condition was observed, and the onset of disease was recorded. The experimental endpoint was set at 20 days post-infection. Mice that died during the experiment were recorded at the time of death.

[0069] See results Figure 6 Mice that were injected with WHU-R10 one day after infection had 100% survival, while all mice in the control group died within 9 days.

[0070] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A fully human monoclonal antibody targeting an epitope of the rabies virus G protein antigen, characterized in that, The full human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region has complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 9-SEQ ID NO: 11, respectively; The light chain variable region has complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 12-SEQ ID NO: 14, respectively.

2. The fully human monoclonal antibody targeting the epitope of the G protein of rabies virus according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

3. 3.The fully human monoclonal antibody targeting the epitope of the G protein of rabies virus according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:

7.

4. The fully human monoclonal antibody targeting the epitope of the G protein of rabies virus according to claim 1, characterized in that, The monoclonal antibody further comprises: The amino acid sequence of the monoclonal antibody is obtained by substituting, deleting and / or adding one or more amino acids to obtain an antibody with the same function; Or comprises a heavy chain variable region with an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region with an amino acid sequence having at least 80% homology with the light chain variable region; Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

5. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-4. The nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

6. The nucleic acid molecule of claim 5, wherein The polynucleotide sequence encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

7. The nucleic acid molecule of claim 5, wherein The polynucleotide sequence encoding the heavy chain constant region of the monoclonal antibody is as shown in SEQ ID NO: 6, and the polynucleotide sequence encoding the light chain constant region is as shown in SEQ ID NO:

8.

8. An expression vector comprising the nucleic acid molecule of any one of claims 5-7. The expression vector can express the nucleic acid in prokaryotic or eukaryotic host cells.

9. An engineered bacterium or eukaryotic host cell comprising the expression vector of claim 8.

10. Use of the monoclonal antibody of any one of claims 1-4 in the preparation of a rabies treatment drug or a rabies virus detection product.

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