Nanobodies against rabies virus g protein and uses thereof

Abstract

This invention discloses nanobodies against rabies virus G protein and their applications, belonging to the field of nanobody technology. The nanobodies are antibody 11G6, antibody 9F7, or antibody 9A3; the amino acid sequence of antibody 11G6 is shown in SEQ ID NO. 8; the amino acid sequence of antibody 9F7 is shown in SEQ ID NO. 13; and the amino acid sequence of antibody 9A3 is shown in SEQ ID NO. 18. This invention uses phage display technology to screen for nanobodies with high affinity and high neutralizing activity. Based on this, this invention utilizes the advantage of the small size of nanobodies and verifies their unique ability to cross the blood-brain barrier (BBB) ​​through Transwell experiments, overcoming the limitation that large molecules such as antibodies and drugs cannot cross the BBB to enter the brain. This invention provides a new drug option for the treatment of rabies virus and has significant clinical implications.

Description

Technical Field

[0001] This invention relates to the field of nanobody technology, and in particular to nanobodies against rabies virus G protein and their applications. Background Technology

[0002] Rabies is an acute infectious disease caused by the rabies virus (RABV). Currently, the main methods of rabies prevention are post-exposure prevention (PEP) and pre-exposure prophylaxis (PrEP). Since the late 19th century, PrEP has primarily relied on rabies vaccination, which provides complete protection in the short term (6 months to 1 year after vaccination) but does not induce lifelong immunity. In humans, vaccine-induced neutralizing antibody levels typically decline 1 to 5 years after vaccination; therefore, regular revaccination is necessary to maintain neutralizing antibody titers. The main methods of PEP include thorough wound cleaning and administration of rabies immunoglobulin (RIG).

[0003] Rabies virus (RABV) belongs to the family Rabieviridae and the genus Rabievirus. It is a segmentless, negative-sense RNA virus. The entire RABV genome primarily encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). The rabies virus glycoprotein (RABV-G) is the only protein on the virus exposed on its surface and has multiple antigenic epitopes. RABV is a neurotropic virus, and large molecular drugs such as neutralizing antibodies cannot cross the blood-brain barrier to enter the central nervous system. RABV is easily transmitted through the bite of infected animals (most commonly dogs) and can infect and replicate in the central nervous system, subsequently leading to severe neurological disorders. The clinical features of RABV infection typically include multiple neuronal dysfunctions, which almost inevitably lead to death. Therefore, research on antibody therapy for brain invasion after RABV infection is essential.

[0004] Researchers have been searching for ways to overcome this limitation. However, to date, no strategy has shown satisfactory efficacy. On the one hand, direct drug delivery to the brain carries significant risks and is highly localized; on the other hand, modifying molecules to increase their diffusion across the barrier is only applicable to a limited number of small-molecule drugs, and the structure of the drug itself can easily be altered. Studies have shown that nanomaterials can encapsulate monoclonal antibodies and enable them to cross the blood-brain barrier, providing a new means for efficient antibody delivery. However, due to the stability of nanoparticles, the release of monoclonal antibodies and the metabolic pathways of nanoparticles in the brain microenvironment require further investigation.

[0005] Therefore, developing nanobodies targeting RABV-G could provide innovative drug options for the treatment of rabies virus and has significant clinical implications. Summary of the Invention

[0006] The purpose of this invention is to provide nanobodies against the G protein of rabies virus and their applications, thereby addressing the problems existing in the prior art. This invention utilizes phage display technology to screen for nanobodies with high affinity and high neutralization activity. Furthermore, this invention leverages the small size of nanobodies, verifying their unique ability to cross the blood-brain barrier (BBB) ​​through Transwell assays, overcoming the limitation that large molecules such as antibodies and drugs cannot cross the BBB to enter the brain. This invention provides a new drug option for the treatment of rabies virus and has significant clinical implications.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a nanobody against rabies virus G protein, wherein the nanobody is antibody 11G6, antibody 9F7 or antibody 9A3;

[0009] The antibody 11G6 comprises CDR1 with the amino acid sequence shown in SEQ ID NO. 9, CDR2 with the amino acid sequence shown in SEQ ID NO. 10, and CDR3 with the amino acid sequence shown in SEQ ID NO. 11;

[0010] The antibody 9F7 comprises CDR1 with the amino acid sequence shown in SEQ ID NO.14, CDR2 with the amino acid sequence shown in SEQ ID NO.15, and CDR3 with the amino acid sequence shown in SEQ ID NO.16;

[0011] The antibody 9A3 comprises CDR1 with the amino acid sequence shown in SEQ ID NO.19, CDR2 with the amino acid sequence shown in SEQ ID NO.20, and CDR3 with the amino acid sequence shown in SEQ ID NO.21.

[0012] Optionally, the amino acid sequence of antibody 11G6 is shown in SEQ ID NO.8;

[0013] The amino acid sequence of the antibody 9F7 is shown in SEQ ID NO.13;

[0014] The amino acid sequence of antibody 9A3 is shown in SEQ ID NO.18.

[0015] The present invention also provides a nucleotide sequence encoding the nanobody, wherein the nucleotide sequence encoding the antibody 11G6 is shown in SEQ ID NO.7;

[0016] The nucleotide sequence encoding the antibody 9F7 is shown in SEQ ID NO.12;

[0017] The nucleotide sequence encoding antibody 9A3 is shown in SEQ ID NO.17.

[0018] The present invention also provides an expression vector containing the nucleotide sequence.

[0019] The present invention also provides a host cell containing the expression vector.

[0020] The present invention also provides the use of the nanobody, the nucleotide sequence, the expression vector, or the host cell in the preparation of products for the prevention and / or treatment of rabies virus.

[0021] Optionally, the product may include a drug.

[0022] The present invention also provides a product for the prevention and / or treatment of rabies virus, the product comprising the nanobody or the nucleotide sequence or the expression vector or the host cell.

[0023] Optionally, the product may also contain pharmaceutically acceptable excipients.

[0024] Optionally, the product may include a drug.

[0025] The present invention discloses the following technical effects:

[0026] This invention screened for high-affinity, high-neutralization-activity antibodies that can target and bind to the alpaca RABV-G protein by immunizing it. It also demonstrated the ability of nanobodies to cross the body's blood-brain barrier (BBB). These nanobodies have significant potential for future applications in RABV detection and treatment, particularly in rabies research.

[0027] This invention utilizes phage display technology to screen for nanobodies with high affinity and high neutralizing activity. Building upon this, the invention leverages the small size of nanobodies, verifying their unique ability to cross the blood-brain barrier (BBB) ​​using Transwell assays, thus overcoming the limitation of large molecules such as antibodies and drugs being unable to cross the BBB to reach the brain. This invention provides a new drug option for the treatment of rabies virus and has significant clinical implications. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The results show the affinity test between antibody 11G6 and RABV-G protein antigen.

[0030] Figure 2 The results show the affinity test between antibody 9F7 and the RABV-G protein antigen.

[0031] Figure 3 The results show the affinity test between antibody 9A3 and the RABV-G protein antigen.

[0032] Figure 4 The graph shows the neutralizing antibody titers of the three antibodies.

[0033] Figure 5 A schematic diagram of an experiment to establish the blood-brain barrier in vitro;

[0034] Figure 6 The graph shows the results of nanobody crossing the BBB efficiency. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example

[0041] 1. Alpaca immunization injection

[0042] Two mg of RABV-G protein was divided into four equal parts (0.5 mg each), and alpacas were subcutaneously immunized four times on days 1, 11, 21, and 31. Approximately 200 mL of peripheral venous blood was collected from alpacas before the fourth immunization (day 30) and on day 14 after immunization (day 45).

[0043] Compared to traditional immunization protocols involving mice and rabbits, the method of this invention provides a richer source for subsequent screening of highly diverse nanobodies by collecting a large amount of peripheral blood from alpacas.

[0044] 2. Construction of an alpaca nanobody library

[0045] (1) Isolate lymphocytes from blood by density gradient centrifugation;

[0046] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA;

[0047] (3) Using cDNA as a template, primer 1 and primers 2-5 mixed in equal moles were used to amplify the VH-CH1-CH2 fragment of IgG1 and the VHH-CH2 fragment of IgG2 and IgG3 by PCR.

[0048] (4) The VHH-CH2 fragment was recovered as a template, and primer 6 was mixed with an equimolar amount of primer 2-5 for further PCR amplification to obtain the VHH gene fragments of IgG2 and IgG3.

[0049] PCR amplification reaction system: 1 μL VHH-CH2 fragment, 0.5 μL upstream primer (equal molar mixture of primers 2-5), 0.5 μL downstream primer (primer 6), 0.5 μL rtaq enzyme (Vazyme, P505-d1-AA), 5 μL 2×PCR Buffer (Vazyme, P505-d1-AB), 1 μL dNTP (Vazyme, P505-d1-AC), and 1.5 μL sterile water.

[0050] PCR amplification reaction program: 95℃ pre-denaturation for 2-5 min; 95℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 40 s, 25 cycles; 72℃ final extension for 10 min (to ensure complete extension of all fragments).

[0051] The nucleotide sequences of primers 1-6 used to construct the nanobody library are shown below:

[0052] Primer 1: 5'-CGCCATCAAGGTACCAGTTGA-3' (SEQ ID NO.1);

[0053] Primer 2: 5'-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG-3' (SEQ ID NO. 2);

[0054] Primer 3: 5'-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTCTGG-3' (SEQ ID NO. 3);

[0055] Primer 4: 5'-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGG-3' (SEQ ID NO. 4);

[0056] Primer 5: 5'-CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG-3' (SEQ ID NO.5);

[0057] Primer 6: 5'-CCACGATTCTGCGGCCGCTGAGGAGACRGTGAACCTGGGTCC-3' (SEQ ID NO. 6).

[0058] (5) The recovered VHH gene fragment was treated with restriction endonucleases SfiI (NEB, Cat.#R0123L) and NotI (NEB, Cat.#R0189L) at its 5' and 3' ends, respectively; at the same time, the phage vector plasmid pHen1 (self-prepared) was linearized using the same restriction enzyme digestion system.

[0059] (6) The digested VHH fragment was ligated with the linearized pHen1 vector under the catalysis of T4 DNA ligase (NEB, Cat.#M0202S) to construct a VHH-pIII fusion protein expression plasmid library. pIII is a flagellin protein on the surface of bacteriophage, and its encoding gene is located downstream of the NotI restriction site of the pHen1 vector.

[0060] (7) The above-mentioned ligation product was introduced into TG1 competent bacteria by electroporation. After appropriate culture, all colonies were collected to obtain the alpaca nanobody library.

[0061] 3. Displaying and screening specific nanobodies on the surface of bacteriophages

[0062] (1) After inoculating and culturing the frozen nanobody library, helper phage M13KO7 (NEB, Cat. #N0315S) was added for amplification.

[0063] (2) Enrich phage particles from culture supernatant using the PEG-NaCl method.

[0064] (3) The obtained phage was co-incubated with the IL-18 antigen embedded in Maxisorp immunoassay tubes (ThermoFisher Scientific) to allow them to fully bind.

[0065] (4) Screening: Discard unbound phages and wash repeatedly with PBS buffer to remove non-specifically bound phages.

[0066] (5) Elution: Add glycine solution to dissociate the specifically bound phage from the antigen and collect the eluent.

[0067] At this point, a phage pool expressing specific nanobodies has been obtained, which can then be used for two subsequent processes: constructing and enriching nanobodies libraries and preparing single-clone colonies.

[0068] (6) Constructing an enriched nanobody library: The eluted phages are re-infected with Escherichia coli in the logarithmic growth phase (without helper phages), and all infected bacteria are collected to obtain an antigen-specific nanobody library. This library can be used directly for the next round of screening (return to (1) of “3. Displaying screening-specific nanobodies on the surface of phages”).

[0069] (7) Preparation of monoclonal colonies: Take a small amount (about 0.5%) of eluted bacteriophage to infect Escherichia coli, spread it on solid culture medium, and incubate overnight at 37°C to obtain monoclonal colonies for subsequent positive clone identification.

[0070] 4. Identification of positive monoclonal nanobodies

[0071] (1) Select single colonies and inoculate them into microplates for culture, and add IPTG to induce the expression of VHH-pIII fusion protein (i.e. nanobody-pIII protein).

[0072] (2) Collect the bacterial culture supernatant containing nanobodies and add it to a Maxisorp clear 96-well plate (ThermoFisher Scientific) coated with RABV-G antigen and incubate with shaking at room temperature.

[0073] (3) The binding of nanobodies to antigens was detected by enzyme-linked immunosorbent assay (ELISA), and the steps are as follows:

[0074] I. Add the supernatant of the induced VHH-pIII protein to the wells and incubate with shaking at room temperature;

[0075] II. Add c-myc-tagged mouse monoclonal antibody (Genscript, Cat. #A00863), and incubate at room temperature in the dark with shaking.

[0076] III. Add HRP substrate ABTS (Sigma-Aldrich, Cat. #A9941) and incubate at room temperature in the dark with shaking.

[0077] IV. OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 405 nm Absorbance.

[0078] (4) Screen out monoclonal nanobody colonies that can bind to the antigen, expand the culture, extract DNA plasmids and sequence them to obtain the nucleotide and amino acid sequences of the nanobody.

[0079] 5. Recombinant Expression and Purification of Small-Batch Monoclonal Nanobodies

[0080] (1) The above “4. Identification of positive monoclonal nanobodies” yielded monoclonal nanobodies that could specifically recognize and bind to RABV-G. The DNA plasmid encoding the nanobodies was transformed into BL21(DE3) competent cells, and the monoclonal nanobodies were expressed and purified in small batches using the E. coli expression system.

[0081] (2) Using the ELISA method, nanobodies of different concentrations were incubated, and the affinity between the nanobodies and the antigen was measured based on the binding ability of the nanobodies to RABV-G.

[0082] Three groups of monoclonal colony-corresponding antibodies (11G6, 9F7, and 9A3) were obtained. The results of the affinity tests for the RABV-G protein antigen are as follows: Figures 1-3 As shown.

[0083] Depend on Figures 1-3 It can be seen that 11G6 has the best affinity, K D The value is 241.5 nM; 9F7 has moderate affinity, K D The value is 642.4 nM; K of 9A3 D The value is slightly worse, K D The value is 1467 nM.

[0084] SEQ ID NO.7 (11G6 nucleotide sequence):

[0085] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGATCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCACCATCGATGGCATGGCCTGGTTCCCCCAGACTCCGGGGAAGGAGCGCGACTTCGTCGCACGTATTAGTAGTACTGGCAGCACAAACTATGC AAACTCCGTGAAGGGCCGATTCACCATCCCAGAGACGATGCCAAGAACACGGAGTATCTGCAAATGAACGGCCTGAAAACCTGAGGACACGGCCGGCTATTACTGTAATGCAGATCGTGTAAAGATGCATTGGGGGCCCGACTACTGGGGCCAGGGGGACCCACGGCACTGTCTCCTCA;

[0086] SEQ ID NO.8 (11G6 amino acid sequence):

[0087] EVQLVESGGGLVQAGGSLRLSCAASGSIFTIDGMAWFPQTPGKERDFVARISSTGSTNYANSVKGRFTISRDDAKNTEYLQMNGLKPEDTAGYYCNADRVKMHWGPDYWGQGTHGTVSS;

[0088] Among them, amino acid sequence 1-25 is FR1, amino acid sequence 26-30 is CDR1 (GSIFT, SEQ ID NO. 9), amino acid sequence 31-50 is FR2, amino acid sequence 51-57 is CDR2 (ISSTGST, SEQ ID NO. 10), amino acid sequence 58-95 is FR3, amino acid sequence 96-108 is CDR3 (NADRVKMHWGPDY, SEQ ID NO. 11), and amino acid sequence 109-119 is FR4.

[0089] SEQ ID NO.12 (9F7 nucleotide sequence):

[0090] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCACCAGGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTGGGTCGCGTCTATTAGTAGTGGTGGTAAAACATACTATGTAGACTCCGTGAAGGGCCG ATTCACCATTTCAAGAGACACACCAGGACACGATTAATTTGGAAATGAACAGCCTGACACCGGAGGACACGGCCGTTTATTACTGTGCAGCAGCCGGAATGGAGCGAATCTATACTTTTGCTAACTACCGATGGGCGTCTGAATATGACTACTGGGGCCAGGGGGACCCAGGTCACTGTCTCCTCA;

[0091] SEQ ID NO.13 (9F7 amino acid sequence):

[0092] EVQLVESGGGLVQAGGSLRLSCAASGRTTFTRAWFRQAPGKEREWVASISSGGKTYYVDSVKGRFTISRDNTKDTINLEMNSLTPEDTAVYYCAAAGMERIYTFANYRWASEYDYWGQGTQVTVSS;

[0093] Among them, amino acid sequence 1-25 is FR1, amino acid sequence 26-30 is CDR1 (GRTFT, SEQ ID NO. 14), amino acid sequence 31-47 is FR2, amino acid sequence 48-54 is CDR2 (ISSGGKT, SEQ ID NO. 15), amino acid sequence 55-92 is FR3, amino acid sequence 93-114 is CDR3 (AAAGMERIYTFANYRWASEYDY, SEQ ID NO. 16), and amino acid sequence 115-125 is FR4.

[0094] SEQ ID NO.17 (9A3 nucleotide sequence):

[0095] CAGGGTGCAGCTGGTGCAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGCCACGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTGGGTCGCAGCTATTAGCGGTAGTGGTAGGACATACTATGCAGACTCCGTGAAGGGCCG ATTCACCGGCTCAAGAGACACACCAGAACACGGTGTATTTGCAAATGAACAGCCTGACACCAGAGGACACGGCCGATTACTACTGTGCAGCAGCCGGAATGGAGCGAGTCTATACTTTTGCTAACTACCGATGGGCTTCTGAATATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA;

[0096] SEQ ID NO.18 (9A3 amino acid sequence):

[0097] QVQLVQSGGGLVQAGGSLRLSCAASGRTFATAWFRQAPGKEREWVAAISGSGRTYYADSVKGRFTGSRDNTKNTVYLQMNSLTPEDTADYYCAAAGMERVYTFANYRWASEYDYWGQGTQVTVSS;

[0098] Among them, amino acid sequence 1-25 is FR1, amino acid sequence 26-30 is CDR1 (GRTFA, SEQ ID NO. 19), amino acid sequence 31-47 is FR2, amino acid sequence 48-54 is CDR2 (ISGSGRT, SEQ ID NO. 20), amino acid sequence 55-92 is FR3, amino acid sequence 93-114 is CDR3 (AAAGMERVYTFANYRWASEYDY, SEQ ID NO. 21), and amino acid sequence 115-125 is FR4.

[0099] 6. Neutralization activity detection

[0100] Three types of antibodies were incubated with fluorescently labeled rabies virus (CVS-11 strain, 100 FFU) at 37°C for 1 hour to allow the nanobodies to bind to the virus. This mixture was then seeded into pre-formed monolayer BSR cells and cultured for 48 hours. Finally, the cells were observed under a fluorescence microscope. By statistically analyzing the proportion of uninfected (i.e., non-fluorescent) cell wells at each dilution, the highest serum dilution that inhibited infection in 50% of cell wells was calculated; this was the titer of the neutralizing antibody. Figure 4 As shown.

[0101] Depend on Figure 4 It can be seen that the neutralizing activities of the three nanobodies are similar, with 11G6 having the highest IC50. 50 IC with a capacity of 21.52 nM and 9F7 50 A 9A3 IC with a capacity of 12.84 nM. 50 It is 14.82 nM.

[0102] 7. Nanobody Cross-BB Detection

[0103] An in vitro blood-brain barrier (BBB) ​​model was established using human brain microvascular endothelial cells (hCMEC / D3, Procell, CL-0843). Figure 5 As shown. Cells were packed at 6 × 10⁻⁶. 4 Cells were seeded at a density of 1 / cm² in 0.4 μm pore size Transwell chambers (24 mm diameter; Corning) and cultured for 48–72 hours until confluence. Subsequently, nanobodies 9F7 dissolved in fresh culture medium were added to the upper chamber. Samples were taken from the lower chamber at 0.5, 1, 1.5, 2, and 3 hours after drug addition. The antibody concentration in the samples was quantitatively determined using ELISA. The efficiency of the nanobodies across the BBB was as follows: Figure 6 As shown.

[0104] Depend on Figure 6 It can be seen that the efficiency of nanobodies in crossing the BBB is significantly higher than that of full-length antibodies, with a transport efficiency of up to 50% in 3 hours.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nanobody against rabies virus G protein, characterized in that, The nanobody is antibody 11G6, antibody 9F7, or antibody 9A3; The antibody 11G6 comprises CDR1 with the amino acid sequence shown in SEQ ID NO. 9, CDR2 with the amino acid sequence shown in SEQ ID NO. 10, and CDR3 with the amino acid sequence shown in SEQ ID NO. 11; The antibody 9F7 comprises CDR1 with the amino acid sequence shown in SEQ ID NO.14, CDR2 with the amino acid sequence shown in SEQ ID NO.15, and CDR3 with the amino acid sequence shown in SEQ ID NO.16; The antibody 9A3 comprises CDR1 with the amino acid sequence shown in SEQ ID NO.19, CDR2 with the amino acid sequence shown in SEQ ID NO.20, and CDR3 with the amino acid sequence shown in SEQ ID NO.

21.

2. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the antibody 11G6 is shown in SEQ ID NO. 8; The amino acid sequence of the antibody 9F7 is shown in SEQ ID NO.13; The amino acid sequence of antibody 9A3 is shown in SEQ ID NO.

18.

3. A nucleic acid encoding the nanobody of claim 1, characterized in that, The nucleic acid sequence encoding the antibody 11G6 is shown in SEQ ID NO.7; The nucleic acid sequence encoding the antibody 9F7 is shown in SEQ ID NO.12; The nucleic acid sequence encoding the antibody 9A3 is shown in SEQ ID NO.

17.

4. An expression vector containing the nucleic acid of claim 3.

5. A host cell containing the expression vector of claim 4.

6. The use of the nanobody as described in claim 1 or 2, the nucleic acid as described in claim 3, the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of products for the prevention and / or treatment of rabies virus infection.

7. The application as described in claim 6, characterized in that, The product is a medicine.

8. A product for the prevention and / or treatment of rabies virus infection, characterized in that, The product comprises the nanobody of claim 1 or 2, the nucleic acid of claim 3, the expression vector of claim 4, or the host cell of claim 5.

9. The product as described in claim 8, characterized in that, The product also contains pharmaceutically acceptable excipients.

10. The product as described in claim 8, characterized in that, The product in question is a medicine.

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