A dual epitope llama-derived nanobody and uses thereof

Abstract

The application discloses a dual-epitope llama-derived nanobody and application thereof, and belongs to the field of biological medicine. The application is aimed at different epitopes of MERS-CoV, and two strains of antibodies 2-77 and 4-58 aiming at different binding sites of MERS-CoV RBD are obtained by immunizing a llama, performing specific screening on a ribosome display nanobody library, performing a pseudovirus neutralization experiment, performing a competitive BLI experiment, identifying VHH epitopes and molecular docking by using a phage random peptide library, and constructing a dual-epitope llama-derived nanobody 2F4 with good neutralization activity, high expression and stability by using the two strains of nanobodies. It is proved by a lesion formation test and a mouse in-vivo experiment that the dual-epitope llama-derived nanobody 2F4 constructed by the application can effectively neutralize MERS-CoV and effectively protect animals, and the application provides a train of thought and technical support for exploring new antigen epitopes of MERS-CoV and designing a therapeutic antibody drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a dual epitope llama-derived nanobody and application thereof. BACKGROUND

[0002] Middle East Respiratory Syndrome (MERS) is caused by infection with Middle East Respiratory Syndrome coronavirus (MERS-CoV) and is a highly pathogenic zoonosis. MERS-CoV has the highest mortality rate among the known coronaviruses that can infect humans, with a mortality rate of about 36%. After human infection with MERS-CoV, the main performance is acute respiratory symptoms, and the incubation period is about 6-10 days; infected persons often have fever, cough, joint and muscle pain and are accompanied by systemic chills in the early stage, and gastrointestinal symptoms such as vomiting, diarrhea, abdominal pain and other clinical symptoms appear. With the progression of the disease, it rapidly develops into pneumonia, and severe cases can develop respiratory failure and even endanger life.

[0003] The main immunogen of MERS-CoV is the spike glycoprotein (Spike, S), which is anchored on the membrane in the form of a trimer in the natural state and is the core effector molecule for viral invasion of cells; when MERS-CoV infects cells, the receptor binding domain (Receptor binding domin, RBD) on the S1 subunit recognizes and binds to the host cell surface receptor dipeptidyl peptidase 4 (DPP4) to complete virus adsorption, and the S2 subunit undergoes a conformational change after RBD binds to DPP4, allowing the fusion peptide to enter the target cell membrane. Therefore, the development of anti-RBD antibody drugs to block the interaction between RBD and DPP4 is of great significance for the prevention and control of MERS-CoV.

[0004] Currently, there is neither a marketed vaccine nor an approved antibody therapeutic drug for the prevention and control of MERS-CoV. The MERS spike protein has become a target protein for antibody screening, vaccine development, and pathogen detection. Neutralizing antibodies have precise pathogen targeting ability and a key role in blocking viral infection, and have become one of the specific drugs for the prevention and control of infectious diseases.

[0005] However, the conventional antibody has a molecular weight of 150 KDa, and its stability is greatly affected by temperature, and it cannot pass through the blood-brain barrier. Nanobody (Nb) prepared based on the Variable domain of Heavy chain of Heavy-chain antibody (VHH) of Camelidae has the advantages of small molecular weight, strong penetration, high temperature resistance, etc., and has been increasingly applied to drug research and development and structure and function research in recent years. Nanobody has unique epitope binding properties that traditional whole molecule antibodies do not have due to its structural characteristics, and it is easy to construct multivalent nanobody clusters, fuse with Fc fragments, and construct bispecific antibodies (BsAb) due to its small molecular weight. Bispecific antibodies are antibody structures that can bind to different epitopes on the same or different antigens, and have the advantages of blocking escape mutations and enhancing neutralization effects for different epitopes of the same antigen.

[0006] Therefore, the present application develops bispecific nanobodies for different epitopes of MERS-CoV in order to provide ideas and technical support for exploring new antigen epitopes and therapeutic antibody drug design for MERS-CoV. SUMMARY

[0007] One of the purposes of the present application is to provide a dual epitope nanobody of camelid origin, which comprises two nanobodies 2-77 and 4-58 that recognize different antigen epitopes of MERS-CoV, respectively, wherein the amino acid sequence of nanobody 2-77 is shown in SEQ ID No: 1, and the amino acid sequence of nanobody 4-58 is shown in SEQ ID No: 2.

[0008] Further, the dual epitope nanobody of camelid origin described above is obtained by adding a human IgG1 FC sequence to the C-terminal sequence of nanobody 2-77, and simultaneously adding a linker to the C-terminal sequence of the human IgG1 FC sequence and connecting it to the sequence of nanobody 4-58, and is named antibody 2F4.

[0009] Further, the amino acid sequence of IgG1 FC is shown in SEQ ID No: 3.

[0010] Further, the amino acid sequence of the linker is shown in SEQ ID No: 4.

[0011] The second purpose of the present application is to provide the use of the dual epitope nanobody of camelid origin described above in the preparation of a preparation for preventing or treating Middle East respiratory syndrome coronavirus.

[0012] The third purpose of the present application is to provide a preparation for preventing or treating Middle East respiratory syndrome coronavirus, comprising the dual epitope nanobody of camelid origin described above.

[0013] The fourth objective of this invention is to provide a nucleotide molecule that encodes the aforementioned biepisode alpaca-derived nanobody.

[0014] The fifth objective of this invention is to provide an expression vector comprising the aforementioned nucleotide molecules.

[0015] The sixth objective of this invention is to provide a host cell containing the above-mentioned expression vector or nucleotide molecules integrated into the genome.

[0016] Compared with the prior art, the technical effects of the present invention are as follows:

[0017] This invention targets different epitopes of MERS-CoV. By immunizing alpacas, a ribosome-displayed nanobody library was used for specific screening, and a pseudovirus neutralization experiment was used for preliminary neutralization identification, yielding six antibodies with neutralizing activity. Furthermore, through competitive BLI experiments, phage random peptide library identification of VHH epitopes, and molecular docking, two antibodies, 2-77 and 4-58, targeting different binding sites of the MERS-CoV RBD were obtained. These two nanobodies recognizing different antigenic epitopes were used to construct three bispecific nanobodies using different construction strategies. A pseudovirus neutralization experiment was used to screen for the bispecific alpaca-derived nanobody 2F4, which exhibited good neutralizing activity, high expression levels, and stability. Lesion formation assays and in vivo mouse experiments demonstrated that the bispecific alpaca-derived nanobody 2F4 constructed in this invention can effectively neutralize MERS-CoV, providing effective protection to animals and offering insights and technical support for the discovery of novel antigenic epitopes and the design of therapeutic antibody drugs for MERS-CoV. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 The results of SDS-PAGE identification of six nanobody purification strains;

[0020] Figure 2 Results of ELISA detection of the binding activity of 6 nanobodies;

[0021] Figure 3 Results of a pseudovirus neutralization experiment on six nanoantibodies;

[0022] Figure 4 Results of competition among 6 nanobody strains;

[0023] Figure 5The effect of DPP4 on the binding of antibodies 2-77 and 4-58 to RBD;

[0024] Figure 6 The results show the docking of GD27, m336, 6516, and VHH9 antibodies with 2-77 and 4-58 antibody molecules.

[0025] Figure 7 The construction strategy for three bispecific antibodies;

[0026] Figure 8 The results of neutralizing activity detection of three bispecific antibodies for pseudovirus neutralization;

[0027] Figure 9 To detect the neutralizing effect of 2F4 antibody on live virus using the FFA method;

[0028] Figure 10 To assess the protective efficacy of the bispecific nanobody 2F4 in treating and preventing MERS-CoV infection. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0031] Example 1: Screening of Nanobodies

[0032] This invention utilizes immunization of alpacas, employs ribosome-displayed nanobody libraries for specific screening, and employs a pseudovirus neutralization assay for preliminary neutralization identification, revealing six nanobody strains (numbered 2-77, 2-150, 4-21, 4-23, 4-58, and 4-94) to possess neutralizing activity. The specific methods are as follows.

[0033] 1. Transfection

[0034] By immunizing alpacas, a nanobody library was specifically screened using ribosome-displayed nanobody libraries. The nanobodies were then transfected into ExpiCHO cells for high-level antibody expression. ExpiCHO-S cells were cultured at 37°C, 8% CO2, and 125 rpm for several hours until a cell density of 7-10 × 10⁶ cells was reached. 6When the concentration is 1 / ml, place it in a 60mL culture flask. Follow the instructions for ExpiFectamine. TM Transfection was performed according to the instructions for the CHO transfection kit.

[0035] 2. Purification of nanobodies

[0036] ExpiCHO cells transfected with the recombinant plasmid were centrifuged at 15,000g for 20 min at 4°C. The cell culture supernatant was transferred to a new centrifuge tube and filtered twice through a 0.22 μM filter. VHH monomers were purified using a HISTRAP HP purification column, and hFCs were purified using a HiTrapMabSelect SuRe column.

[0037] The specific procedure for purifying VHH monomer using a HISTRAP HP purification column is as follows:

[0038] (1) Connect the HISTRAP HP purification column and constant flow pump to the computer UV chromatograph at a flow rate of 2 mL / min and equilibrate the purification column with 20 mM imidazole for 20 min.

[0039] (2) The cell supernatant of the nanobody monomer was purified twice through a purification column;

[0040] (3) Wash away contaminating proteins with 20 mM imidazole;

[0041] (4) The target protein was eluted with 100 / 300mM imidazole, respectively;

[0042] (5) Elute impurities with high concentration imidazole for 10 min, then pass through a 20 mM imidazole equilibration column for 20 min;

[0043] (6) Store the HISTRAP HP purification column in 20% anhydrous ethanol.

[0044] The purification steps for hFC using a HiTrap MabSelect SuRe column are the same as those for purifying VHH monomers, except that the equilibration buffer is replaced with PBS, elution is performed with glycine solution at pH 2.7, and the pH is adjusted to neutral.

[0045] The results of SDS-PAGE identification of the purified 6 nanobody strains are as follows: Figure 1 As shown in the figure. The results indicate that the nanobodies exhibit a single band at 15 kDa, consistent with the expected size, and six nanobodies were successfully prepared.

[0046] 3. ELISA detection of nanobody binding activity

[0047] Using MERS-CoV RBD protein as the coating antigen, the binding activity of six nanobodies was detected by indirect ELISA. The specific method is as follows:

[0048] (1) Dilute MERS-CoV RBD protein to 1 μg / mL with coating buffer, 100 μL / well, and coat overnight at 4℃;

[0049] (2) Wash the plate with PBST buffer, 150 μL / well, 3 times / 5 min;

[0050] (3) The blocking solution was 1% BSA solution, 150 μL / well, and incubated at 37°C for 1 h;

[0051] (4) Wash the plate 3 times with PBST, add 100 μL of the protein to be tested (1 μg / mL) per well, and incubate at 37°C for 1 h;

[0052] (5) Wash the plate 3 times with PBST, add 100 μL of HRP-labeled Rabbit Anti-Camelid VHH Antibody (1:100000) antibody to each well, and incubate at 37°C for 1 h.

[0053] (6) Wash the plate 3 times with PBST, add 100 μL of TMB colorimetric solution per well, and incubate in the dark for 5 min.

[0054] (7) Add 50 μL of ELISA stop solution per well and read the OD value using a microplate reader. 450nm Numerical value.

[0055] The results of ELISA detection of the binding activity of 6 nanobodies are as follows: Figure 2 As shown. The results indicate that the six nanobodies exhibited good binding activity to MERS-CoV RBD, EC... 50 Between 0.068 nM and 1.310 nM.

[0056] 4. Counterfeit virus packaging and neutralizing antibody detection

[0057] MERS-CoV pseudoviruses were packaged using a human immunodeficiency virus lentiviral system. The pNL4-3.LUC.RE backbone plasmid and the pCDNA3.1-MERS-CoV S plasmid encoding the MERS-CoV / KOR / KNIH / 002_05_2015 strain (GenBank: KT029139.1) were co-transfected into 293T cells. The specific method is as follows:

[0058] (1) Pass 293T cells into 6-well plates and start transfection when the cell density is 80%-90%;

[0059] (2) Take 8 μL of Lipofectamine 3000 liposomes and add them to 100 μL of Opti-MEM cell culture medium. After gently mixing thoroughly, incubate at room temperature for 5 min.

[0060] (3) Add 3 μg each of the backbone plasmid and the rescue virus plasmid to 100 μL of Opti-MEM cell culture medium, add 12 μL of P3000 reagent, gently mix thoroughly, and incubate at room temperature for 5 min;

[0061] (5) Gently mix the diluted liposome transfection reagent with plasmid DNA at a volume ratio of 1:1, and incubate the thoroughly mixed complex at room temperature for 20 min.

[0062] (6) During incubation, remove the cell culture dish, discard the original culture medium, add the DNA-liposome complex dropwise to the cell culture dish, and incubate at 37°C and 5% CO2 cell culture incubator for 4-6 hours.

[0063] (7) Discard the cell culture medium, add DMEM (2% FBS) cell culture medium, and continue to culture in a 37℃, 5% CO2 cell culture incubator for 48h;

[0064] (8) Harvest the cell supernatant and centrifuge at 3000 rpm / min and 4℃ for 10 min to remove cell debris;

[0065] (9) The serum to be tested was started with a 30-fold dilution, serially diluted 3-fold, and cell control and virus control were set up;

[0066] (10) Add 50 μL of diluted pseudovirus to each well, incubate at 37°C for 1 h, add Huh-7 cells, 50 μL per well, cell density is 2×105 cells / mL, and continue to culture at 37°C for 48 h.

[0067] (11) Discard 100 μL of supernatant from each well, add 100 μL of luciferase substrate, lyse for 1-3 min, and then detect the relative luciferase unit (RLU) activity of each well using a multi-functional microplate reader.

[0068] The results of the pseudovirus neutralization experiment on 6 nanobodies are as follows: Figure 3 As shown in the figure. The results showed that all six nanobodies had good virus neutralizing activity against the MERS-CoV pseudovirus (KOR / KNIH / 002-05-2015strain), with IC50 values ​​ranging from 7.38 nM to 82.13 nM.

[0069] Example 2: Competitive BLI Experiment

[0070] Competitive bio-layer interference (BLI) was used to identify whether six antibodies (2-77, 2-150, 4-21, 4-23, 4-58, and 4-94) recognize the same or different antigenic epitopes. An Octet RH16 molecular interaction analyzer (SA chip) was used. After opening the software, the machine was preheated for 30 minutes. The specific operating steps are as follows:

[0071] (1) Dilute MERS-CoV RBD protein to 5 μg / mL with HBS-P buffer and fix for 180 s;

[0072] (2) Dilute the six antibodies 2-77, 2-150, 4-21, 4-23, 4-58 and 4-94 to 5 μg / mL, bind for 900s until saturation, and then incubate with competitive antibodies;

[0073] (3) Calculate the inhibition rate between antibodies. The criteria are: <20% is a self-reaction signal or complete competition, >60% is no competition between antibodies, and 20%-60% is partial competition between antibodies.

[0074] (4) Analyze the results using Data Analysis 12.0 software.

[0075] Experimental results are as follows Figure 4 As shown, from Figure 4 As can be seen in Figure A, all antibodies begin to incubate other antibodies 900 seconds after incubation. If there is competition for epitopes between antibodies, no signal will be generated. Conversely, if the signal value increases, it indicates that there is no competition between antibodies and that the antigen epitopes they bind to are different.

[0076] The antibodies were divided into two groups: group 1 consisted of 2-77 and 2-150 antibodies, and group 2 consisted of 4-21, 4-23, 4.58, and 4-94 antibodies. Experimental results showed that antibodies did not compete between groups; however, they did compete within each group. Figure 4 As shown in Figure B, the two antibodies VHH2-77 and 4-58 with the best neutralizing activity in each group were selected for further identification and evaluation.

[0077] Example 3: Identification of VHH epitopes using a random phage peptide library

[0078] To further determine whether antibodies 2-77 (amino acid sequence as shown in SEQ ID No:1) and 4-58 (amino acid sequence as shown in SEQ ID No:2) bind to different antigenic epitopes, a random 12-peptide library was subjected to three rounds of affinity panning using phage display technology. Specifically, the Ph.D.-12Phage Display Peptide Library Kit was used for VHH antigenic epitope screening, and the steps are as follows:

[0079] (1) Escherichia coli ER2738 was streaked onto an LB-Tet plate and incubated overnight at 37°C; a single colony was picked and placed into 5 mL of LB-Tet culture medium and incubated at 37°C and 200 rpm / min until the OD600 was around 0.5; and the phage library was determined.

[0080] (2) Dilute the antibody to 100 μg / mL with NaHCO3 solution. The antibody concentration and TBST concentration are shown in Table 1. Add 200 μL / well to a 96-well plate and coat overnight at 4℃. Discard the coating solution, add 150 μg / mL TBST to wash the plate 3 times, and then add 3% BSA-TBS to block for 2h.

[0081] (3) Wash the plate 10 times with TBST, add 2×10¹¹ pfu of phage solution, incubate at room temperature for 1 h, discard the unbound phage solution (2.2 pfu), wash the plate 10 times with TBST, add elution buffer (0.2 mol / L glycine, pH=2.2) and incubate at room temperature for 1 h; then add neutralization solution (1 mol / L Tris-HCl, pH=9.1), determine the titer of the eluted phage and perform a new round of screening;

[0082] (4) After the third round of screening, blue bacterial colonies were randomly picked from the titer plate, and ER2738 bacterial culture was amplified and DNA was extracted for sequencing. The sequencing primers were 5'-CCCTCATAGTTAGCGTAACG-3'. The randomly selected peptides were analyzed.

[0083] Table 1 Screening antibody concentrations

[0084]

[0085] The results of three rounds of affinity screening are shown in Table 1. Twenty-four clones were randomly selected from each antibody strain for sequencing and analysis. The results are shown in Table 2. The enriched sequence for antibody 2-77 was “HLSWFSYAWSTV”, accounting for 83.3% of the total sequence, while the enriched sequence for antibody 4-58 was “YSHAKCCYSVNS”, accounting for 58.3% of the total sequence. These results are consistent with the results of the competitive BLI assay, further indicating that 2-77 and 4-58 bind to different antigenic epitopes in the RBD region.

[0086] Table 2 Antibody enrichment sequences

[0087]

[0088]

[0089] To investigate the binding sites of antibodies 2-77 and 4-58 with RBD (PDB number: 4kqz), the spatial structures of 2-77 and 4-58 were predicted and modeled using AlphaFold 3 software, and molecular docking was performed using PyMOL software. To further explore the binding sites of bispecific antibodies, published nanobodies GD27, m336 (PDB number: 4XAK), VHH9 (PDB number: 8IDI), and 6516 (PDB number: 7V3L) were molecularly docked with antibodies 2-77 and 4-58 in the three-dimensional structure visualization software PyMOL to analyze the binding sites between the antibodies and MERS-CoV RBD.

[0090] By modeling the antibodies and performing molecular docking with RBD, the results are as follows: Figure 5 As shown: Figure 5 In Figure A, the docking result of 2-77 with RBD is shown. MERS-CoV RBD is green, and 2-77 is purple. Ser416, Phe418, Ser419, and Asn421 in RBD play crucial roles in binding to 2-77. The hydroxyl group in Ser416 forms hydrogen bonds with the oxygen atom in the carboxylic acid and the hydrogen atom in the amino group of Trp101, respectively. The carbonyl oxygen atom in Phe418 forms a hydrogen bond with the amino hydrogen atom in Arg45. The hydroxyl group in Ser419 forms hydrogen bonds with the carbonyl and amino groups in Gln39, respectively; the carboxylic acid oxygen atom in Asn421 forms a hydrogen bond with the amino hydrogen atom in Gln39. Figure 5 Table B shows the docking results of 4-58 with RBD. Gln522, Tyr523, Pro525, and Val527 in RBD play crucial roles in the binding of 4-58. The amino group in Gln522 forms hydrogen bonds with the oxygen atom in the carbonyl group of Tyr113, and the carbonyl oxygen atom in Tyr523 forms hydrogen bonds with the amino hydrogen atom in Trp116. The hydroxyl group in Pro525 forms hydrogen bonds with the amino hydrogen atom in the Arg45 residue; the carboxylic acid oxygen atom in Val527 forms hydrogen bonds with the amino hydrogen atom in Arg45. The docking results show that the binding sites of 2-77 and 4-58 with RBD are different from the known binding sites (Table 3).

[0091] Table 3. Known MERS-CoV RBD binding sites

[0092]

[0093]

[0094] Further molecular docking was performed in pymol with three fully human antibodies against MERS-CoV (GD27, m336, and 6516) and the nanobody VHH9, as well as antibodies with predicted structures 2-77 and 4-58. The results are as follows: Figure 6 As shown, the MERS-CoV RBD is green, 2-77 is purple, antibody 6516 is gray, VHH9 is yellow, 4-58 is blue, GD27 is orange, and m336 is grayish-purple. It can be seen that 2-77, 6516, and VHH9 bind to one side of the RBD, while 4-58, GD27, and m336 bind to the other side of the RBD. Molecular docking further indicates that 2-77 and 4-58 bind to different regions of the RBD.

[0095] Example 3: Construction of Bispecific Nanobodies

[0096] After identification, nanobodies 2-77 and 4-58 recognized different antigenic epitopes. The two nanobodies were then constructed into a bispecific antibody. The specific construction strategy is as follows: Figure 7 As shown, Figure 7 In the middle, A is 2-77 and 4-58 linked by a flexible linker sequence ((G4S)3) (e.g., SEQ ID No:4), and a TEV site (sequence ENLYFQG) and an HIS tag (sequence HHHHHH) are added sequentially to the C-terminus of the antibody sequence, named 24H. Figure 7 The sequences 2-77 and 4-58 in B are expressed by linker ((G4S)3) (e.g., SEQ ID No:4) and then fused with the human IgG1 FC sequence (e.g., SEQ ID No:3) at the C-terminus of the 4-58 sequence, and named 24F. Figure 7 The C in the sequence is formed by adding the human IgG1 FC sequence to the C-terminus of the 2-77 sequence, and adding a linker (such as SEQ ID No:4) to the C-terminus of the FC sequence to link with the 4-58 sequence, named 2F4.

[0097] The neutralizing activity of three bispecific antibodies was detected using a neutralization assay with a MERS-CoV pseudovirus (KOR / KNIH / 002-05-2015strain). Among them, 2F4 showed good neutralizing activity (e.g., ...). Figure 8 As shown in the figure, the expression level is high and stable. We then selected the 2F4 bispecific antibody for live virus neutralization and broad-spectrum evaluation, as well as for animal protection evaluation.

[0098] Example 4: Detection of neutralization of live virus with 2F4 antibody

[0099] The neutralizing effect of 2F4 antibody on live virus was detected using the Focus Forming Assay (FFA). FFA utilizes immunostaining technology to specifically label viral antigens with fluorescent antibodies to detect infected host cells and infectious viruses. The simplified steps are as follows:

[0100] (1) Pre-catch Vero E6 cells in 96-well plates and incubate overnight at 37°C;

[0101] (2) Dilute the 2F4 antibody to 100 μg / mL and perform a 3-fold serial dilution. Add live MERS-CoV virus and incubate at 37°C for 1 h.

[0102] (3) Add 125 μL of carboxymethylcellulose (CMC) to each well to cover the virus plaque with the semi-solid culture medium and incubate at 37°C for 24 h;

[0103] (4) Add 4% paraformaldehyde and 2% Triton X-100, add MRES-CoV specific antibody and HRP-labeled secondary antibody;

[0104] (5) The lesions were visualized using TrueBlue peroxidase substrate and counted using an ELISPOT dot image analyzer.

[0105] Experimental results are as follows Figure 9 As shown, 2F4 exhibits high neutralizing activity against representative MERS-CoV strains EMC / 2012, ChinaGD01, and Nigeria / NV1657 (from the Biosafety Level 3 Laboratory of Guangzhou Medical University), with IC50 values ​​of 3.95 nM, 4.0 nM, and 9.33 nM, respectively.

[0106] Example 5: Evaluation of Animal Protection Experiments

[0107] To further investigate the therapeutic and preventive effects of the bispecific nanobody 2F4, an Ad5-hDPP4 mouse model was used for evaluation. A prevention group, a treatment group, and a control group were established, with 10 female Ad5-hDPP4 mice aged 5-6 weeks in each group. The prevention group received 20 mg / kg / mouse of 2F4 antibody 24 hours before live virus infection; the treatment group received 20 mg / kg / mouse of 2F4 antibody 24 hours after live virus infection. Changes in mouse body weight were measured, and 6 mice from each group were randomly selected 5 days after virus infection for viral load determination.

[0108] Evaluation results, such as Figure 10 As shown, the mice in the prevention group did not experience a significant decrease in body weight, and their body weight remained stable; the mice in the treatment group experienced a transient decrease in body weight, which then returned to normal levels. Figure 10In mice challenged with MERS-CoV for 5 days, no MERS-CoV was detected in either the treatment or prevention groups, indicating that 2F4 can effectively neutralize MERS-CoV in animals 5 days after challenge. Figure 10 B.

[0109] The embodiments described above are merely specific implementations of this application, used to illustrate the technical solutions of this application, and not to limit them. The protection scope of this application is not limited thereto. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in this application, or make equivalent substitutions for some of the specific technologies; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application. All should be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the protection scope of the claims.

Claims

1. A dual-epitope alpaca-derived nanobody, characterized in that, The invention contains two nanobodies, 2-77 and 4-58, which respectively recognize different antigenic epitopes of MERS-CoV. The amino acid sequence of nanobodies 2-77 is shown in SEQ ID No:1, and the amino acid sequence of nanobodies 4-58 is shown in SEQ ID NO:

2.

2. The dual-epitope alpaca-derived nanobody as described in claim 1, characterized in that, The nanobody 2F4 was obtained by adding a human IgG1 FC sequence to the C-terminus of the nanobody 2-77 sequence and simultaneously adding a linker to the C-terminus of the human IgG1 FC sequence and linking it to the nanobody 4-58 sequence.

3. The dual-epitope alpaca-derived nanobody as described in claim 2, characterized in that, The amino acid sequence of IgG1 FC is shown in SEQ ID NO:

3.

4. The dual-epitope alpaca-derived nanobody as described in claim 2, characterized in that, The amino acid sequence of the linker is shown in SEQ ID NO:

4.

5. The use of the biepisode alpaca-derived nanobody as described in any one of claims 1-4 in the preparation of a preventive or therapeutic formulation of Middle East Respiratory Syndrome Coronavirus.

6. A preventive or therapeutic agent for Middle East Respiratory Syndrome Coronavirus, characterized in that, It contains a biepisode alpaca-derived nanobody as described in any one of claims 1-4.

7. A nucleic acid molecule encoding a biepisode alpaca-derived nanobody as described in claim 1.

8. An expression vector comprising the nucleotide molecule as described in claim 7.

9. A host cell, characterized in that, It contains the expression vector as described in claim 8 or the genome in which the nucleic acid molecule as described in claim 7 is integrated.

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