Nanobody N033 against chikungunya virus E protein and its application

Abstract

The present invention discloses a nanobody N033 against Chikungunya virus E protein and its application. The nanobody against Chikungunya virus E protein has a unique CDR region. The fusion protein of N033 and human Fc has excellent binding activity, high affinity and strong neutralizing activity against Chikungunya virus. In the mouse challenge experiment, 200 μg of N033-Fc antibody was administered before challenge with 2×10 3 TCID 50 , and the preventive protection rate reached 100%. When 100 μg of N033-Fc antibody was administered after challenge with 1×10 3 TCID 50 , the protection rate also reached 100%. It shows the application prospect of the nanobody provided by the present invention in the preparation of drugs for the treatment and / or prevention of Chikungunya virus disease. At the same time, the excellent binding activity and affinity also show its application prospect in the preparation of Chikungunya virus detection kits.

Description

Technical Field

[0001] The present invention discloses a nanobody, belonging to the technical fields of polypeptides and immunology. Background Art

[0002] Chikungunya fever (CHIKF) is a disease first discovered in Africa and caused by the Chikungunya virus (CHIKV). Its clinical manifestations include fever, rash, and body pain, etc. It is one of the fastest-spreading mosquito-borne infectious diseases globally after malaria and dengue fever. Since its discovery in 1953, CHIKV has spread from Africa to more than 100 countries and regions in the world, infecting millions of people. In 2015, the World Health Organization identified the Chikungunya virus as one of the three sub-dangerous pathogens that may cause severe epidemic outbreaks. Due to the limitations of existing diagnostic technologies, CHIKV has not been fully studied at present and may trigger large-scale epidemics in the future. At present, there is no specific drug available for the treatment of CHIKV. Although antibody therapy has achieved certain results in preclinical studies, the further implementation of such therapies still faces challenges. Therefore, there is an urgent need to develop a new generation of neutralizing antibody drugs that can provide protective activity.

[0003] Nanobodies (Nb) were first reported in 1993. It was found that there is a "heavy-chain antibody" lacking the light chain in the blood of camelids. The variable region ((variable domain of heavy chain of heavy-chain antibody, VHH) of this antibody retains all the antigen-binding ability and is called a single-domain antibody or nanobody (nanobody, Nb), with a molecular weight of only 15 kDa. Compared with ordinary antibodies, nanobodies have a small molecular weight and are easy to penetrate into dense tissues or even the blood-brain barrier; they have a simple structure and are easy to genetically modify, showing broad application prospects in the diagnosis and treatment of diseases. One of the most important characteristics of VHH is its weak immunogenicity and good biocompatibility, with nanomolar-level affinity. In addition, the sequences of antibody VH and nanobody VHH are highly homologous. By appropriately modifying the VHH region, a humanized nanobody can be obtained, and the hypervariable region CDR3 of VHH is longer, making it easier to specifically bind to antigens and more tightly and stably. Compared with single-chain antibodies, there are disulfide bonds inside nanobodies, greatly improving their stability under harsh conditions (such as proteases, extreme pH, etc.), and they can be used for the treatment of diseases by inhalation administration. These unique characteristics provide many advantages for VHH and its recombinant fragments that cannot be replaced by traditional antibodies, making them a powerful tool in the development of immunotherapy and immunodiagnosis.

[0004] The object of the present invention is to obtain nanobodies against CHIKV by phage library technology, which have excellent properties in terms of affinity, binding activity and neutralizing activity against the virus, and further provide their application in the preparation of drugs for the prevention and treatment of CHIKV. Summary of the Invention

[0005] Based on the above object, the present invention firstly provides a nanobody against the E protein of Chikungunya virus. The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the nanobody against the E protein of Chikungunya virus are respectively shown at positions 26-33, 51-57 and 96-103 of SEQ ID NO.1.

[0006] The nanobody of the present invention is an antibody with only a heavy chain. The heavy chain includes a variable region and a constant region. The variable region has three Complementarity Determining Regions (CDRs): CDR1, CDR2 and CDR3, which have high variability and diversity. The sequence diversity of the CDR region determines the specificity and affinity of the antibody because they recognize and bind specific antigenic determinants by interacting with the antigen.

[0007] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the nanobody against the E protein of Chikungunya virus is as shown in SEQ ID NO.1. In the present invention, the nanobody having this variable region is named "N033".

[0008] Secondly, the present invention provides a polynucleotide encoding the above-mentioned nanobody against the E protein of Chikungunya virus. In the present invention, the polynucleotide shown in SEQ ID NO.2 is a polynucleotide encoding the heavy chain variable region of the nanobody N033 with the amino acid sequence shown in SEQ ID NO.1 according to a specific embodiment. Based on the triplet codon rule, any polynucleotide encoding the heavy chain variable region of the nanobody N033 with the amino acid sequence shown in SEQ ID NO.1 belongs to the polynucleotide encoding the above-mentioned nanobody against the E protein of Chikungunya virus defined by the present invention.

[0009] Thirdly, the present invention provides a vector containing the above-mentioned polynucleotide encoding the nanobody against the E protein of Chikungunya virus. The vector is used for cloning and / or expressing the coding gene of the nanobody against the E protein of Chikungunya virus. In a specific embodiment of the present invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, especially eukaryotic expression vectors, can also be used for the cloning and expression of the coding gene of the present invention.

[0010] Fourth, the present invention provides a host cell containing a vector of a polynucleotide encoding the above-mentioned nanobody against Chikungunya virus E protein. The host cell is used to express and obtain the above-mentioned nanobody against Chikungunya virus E protein. In a specific embodiment of the present invention, the host cell is Expi293F cell. Other host cells known to those skilled in the art, especially eukaryotic host cells, can also be used for the expression of the nanobody of the present invention.

[0011] Fifth, the present invention provides a fusion protein containing the above-mentioned nanobody against Chikungunya virus E protein. Based on different application requirements such as prolonging the half-life of the antibody in vivo, crossing cells or tissue structures, targeting specific regions, exerting cell effects, dimer or multimer formation, and purification processes, the nanobody provided by the present invention can be fused with other proteins to form a fusion protein. In the fusion protein, the nanobody provided by the present invention can exert its unique property of specifically targeting and binding to Chikungunya virus.

[0012] In a preferred embodiment, the fusion protein further contains a human Fc segment protein. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.3, and preferably, its nucleic acid coding sequence is as shown in SEQ ID NO.4.

[0013] Sixth, the present invention provides the application of the above-mentioned nanobody against Chikungunya virus E protein or the fusion protein containing the nanobody in the preparation of drugs for treating and / or preventing Chikungunya virus disease. The nanobody provided by the present invention has excellent binding activity with Chikungunya virus E protein and can specifically target Chikungunya virus E protein. Therefore, by utilizing these properties of the nanobody, it can specifically inhibit the binding of Chikungunya virus to host cells, or specifically target the infected lesion or pathogen with a therapeutic drug to play a role in clinical treatment or prevention of infection. Therefore, the present invention provides the application of the above-mentioned nanobody against Chikungunya virus E protein in the preparation of drugs for treating and / or preventing Chikungunya virus disease. In a specific embodiment of the present invention, the fusion protein further contains a human Fc segment protein.

[0014] Finally, the present invention also provides a detection kit containing the above-mentioned nanobody against Chikungunya virus E protein or a fusion protein containing the nanobody. Based on the excellent affinity between the nanobody provided by the present invention and Chikungunya virus E protein, it can be used to detect Chikungunya virus particles with E protein that may exist in a sample. The detection antigen is the detection of a single antibody, that is, the nanobody binds specifically to the pathogen as the primary antibody, and then the binding is detected with a secondary antibody; it can also be a double antibody sandwich combination detection. In a specific embodiment of the present invention, the fusion protein further contains a human Fc segment protein.

[0015] The present invention screened the nanobody N033 against Chikungunya virus E protein through alpaca immunization and phage antibody library screening. The nanobodies all have unique heavy chain variable region CDR regions. The fusion protein N033-Fc of N033 and human Fc has excellent binding activity with Chikungunya virus, and the EC 50 (half maximal effective concentration) is 5.1 ng / mL, the affinity KD with Chikungunya virus is 3.55 nM, N033-Fc has strong neutralizing activity against CHIKV, and there is a dose-effect relationship. The IC 50 (half maximal lethal concentration) is 2.9 μg / mL. In the mouse challenge experiment, 200 μg of N033-Fc antibody was given before challenge. After challenging with 2×10 3 TCID 50 , the preventive protection rate reached 100%. After challenging with 1×10 3 TCID 50 , the protection rate of giving 100 μg of N033-Fc antibody for treatment was also 100%. It shows the application prospect of the nanobody provided by the present invention in the preparation of drugs for the treatment and / or prevention of Chikungunya virus disease. At the same time, the excellent binding activity and affinity also show its application in the preparation of Chikungunya virus detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . SDS-PAGE detection results of CHIKV E protein (concentration of separating gel is 12%);

[0017] Figure 2 . SDS-PAGE detection results of N033-Fc (concentration of separating gel is 12%);

[0018] Figure 3 . Sequence annotation information of N033 queried by IMGT;

[0019] Figure 4 . ELISA binding curve of N033-Fc and CHIKV-E protein;

[0020] Figure 5 . Binding kinetic curve of N033-Fc and CHIKV-E protein;

[0021] Figure 6 . Neutralizing activity of N033-Fc against CHIKV virus;

[0022] Figure 7 . Evaluation of protective activity of N033-Fc in prophylactic administration mode in animals;

[0023] Figure 8 . Evaluation of protective activity of N033-Fc in therapeutic administration mode in animals. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0025] Example 1: Expression and purification of CHIKV E protein

[0026] The present invention uses an insect cell-baculovirus system to express CHIKV-E protein. First, the CHIKV-E protein gene sequence (GenBank: MH670649.1) is optimized according to the codon preference of insect cells, and 6 histidine tags are added to the N-terminus. Then, the gene fragment is synthesized by artificial methods and cloned into the pFastBac1 vector (insect cell baculovirus expression vector) by seamless cloning method. The recombinant plasmid is transformed into DH10Bac competent cells. After blue-white screening and PCR identification, the recombinant baculovirus bacmid (Bacmind) is obtained. The recombinant baculovirus bacmid is extracted by isopropanol precipitation method, and then Bacmind is transfected into Expi Sf21 insect cells using a transfection reagent (ExpiFectamine TM Sf Transfection Reagent, A38915). After culturing in a shaker at 27°C and 120 rpm for 96 hours, the culture supernatant is collected, centrifuged, aliquoted and stored at -80°C to obtain the CHIKV-E protein P0 virus. Subsequently, the P0 virus is transferred to Expi Sf9 cells to amplify the virus. After continuing to culture for 4 days, the supernatant is collected and purified by nickel ion affinity chromatography (HisTrap TM excel) to obtain the CHIKV-E protein. Its expression and purity are identified by SDS-PAGE. As Figure 1 shown, the results show that the purified CHIKV-E protein has a molecular weight of about 100 kDa, which meets the expectation and the purity meets the requirements.

[0027] Example 2: Alpaca Immunization and Establishment of VHH Nanobody Library

[0028] Using 1 mg of CHIΚV-E protein with a 6-histidine tag prepared previously, dilute it with PBS to a final volume of 1 mL, add 1 mL of Freund's complete adjuvant for emulsification, and mix for multi-point subcutaneous injection for the first immunization of the alpaca. Immunization is carried out every three weeks thereafter. After the fifth immunization, ELISA is used to detect the serum titer, and the serum titer > 10 6 , then collect 100 mL of blood, isolate peripheral blood mononuclear cells (PBMC), and extract total RNA from the isolated PBMC according to the instruction manual of QIAGEN's Neasy® Plus MiniRNA Extraction Kit. Using the extracted total RNA as a template, cDNA reverse transcription is carried out according to the instruction manual of Invitrogen's Super Script®III First-Strand Synthesis System Extraction Kit. Gene template amplification is carried out with the reverse transcription cDNA library, using primers CALL001 and CALL002 (primer sequences are shown in Table 1), and the first round of PCR reaction is carried out, and the target band is recovered by gel electrophoresis. The recovered DNA is used as a template for secondary amplification, using VHH-FOR and VHH-REV primers (primer sequences are shown in Table 1), and the VHH corresponding band is recovered by gel electrophoresis. Subsequently, the recovered fragment and the pcomb3X vector are digested with Sfi Ⅰ enzyme respectively. After digestion, the fragment is directly purified using a gel recovery purification column, and the pcomb3X vector is recovered as a linear vector using a gel recovery kit. The fragment and pcomb3X are mixed and then ligated overnight at 4°C using T4 ligase. The purified ligation product is mixed with XL1-Blue competent cells on ice, aliquoted 80 μL / cup into an electroporation cuvette, and electroporated at 1800 V. After electroporation, the mixture is transferred into 2YT-ATG medium containing ampicillin and tetracycline, and made up to 200 mL with 2YT-ATG. The aliquoted library is cultured and resuscitated by shaking at 37°C and 250 rpm for 1 hour, and this is the antibody bacterial liquid library.

[0029] Table 1: Reaction Primers

[0030]

[0031] Example 3: Screening of Specific Nanobodies by Phage Display Technology

[0032] After culturing the library bacterial solution in Example 2 at 37 °C and 250 rpm for 1 hour, add VCSM13 helper phage and let it stand for half an hour for infection. Then further culture at 37 °C and 200 rpm for 1 hour. Finally, centrifuge to collect the bacterial pellet and resuspend it in 100 mL of 2YT-ATK medium (containing ampicillin, tetracycline, and kanamycin), and express and amplify overnight at 30 °C and 225 rpm. The next day, collect the supernatant, use 4% PEG8000 and 3% NaCl for ice bath, and centrifuge to obtain the bacterial pellet. The obtained phage pellet is resuspended and dissolved with PBS (pH 7.4), filtered through a 0.2 μm needle filter to remove bacteria, and 10 μL of the filtered library is taken and diluted 100-fold with 2YT medium (10 μL + 990 μL) dilution method. 8 After dilution by 10 times, take 10 μL of the diluted solution to infect 100 μL of XL1-blue, coat a 2YT-ATG semi-solid medium plate after 30 minutes, and count the number of monoclonal colonies the next day to calculate the concentration of the amplified library phage. The filtered library is added with 7% DMSO by volume and stored at -80 °C.

[0033] Coat the high-affinity enzyme-linked immunosorbent assay (ELISA) plate with antigen (antigen: CHIKV-E) overnight, wash the plate with 0.1% PBST, and then block it with 3% skim milk powder at room temperature for 1 h. Discard the blocking solution and wash the plate for later use. Add about 5×10 12 CFU to the above-mentioned blocked antigen plate, and incubate with the antigen at 37 °C for 2 h. Wash the plate with 0.1% PBST, add 100 µL of freshly prepared Glycine-HC1 elution binding solution to each well, let it stand at room temperature for 10 min, and quickly neutralize the eluate with an equal volume of 1 M Tris-HCl (pH 7.4). Then mix the eluate with Escherichia coli XL1-Blue, incubate at 37 °C for 30 min and then shake culture. After rescuing the phage, shake culture overnight at 30 °C to obtain the amplified antibody 1st library. Repeat the above process to obtain the 2nd library in turn. Randomly pick 96 monoclonal colonies from the bacterial plate after the second round of screening. After inducing the expression of VHH antibody by IPTG, prepare a crude extract of soluble recombinant VHH antibody for ELISA detection to preliminarily identify the reactivity of the VHH antibody clone with the CHIΚV-E protein. When the OD 450 value is more than 3 times that of the PBS control, it is judged as positive. Take a part of the bacterial solution of the positive clone for sequencing to obtain the core coding sequence of N033, and log in to https: / / www.imgt.org / IMGT_vquest to obtain Figure 3The sequence annotation information of N033 shown has a homology of 88.77% for V-GENE and allele identification, a homology of 79.17% for J-GENE and allele identification, and the amino acid lengths of FR1, FR2, FR3, and FR4 regions are 25, 17, 38, and 11 respectively. The amino acid lengths of CDR1, CD2, and CDR3 regions are 8, 7, and 8 respectively. The amino acid JUNTION sequence is: CNTLPISRDW.

[0034] Example 4: Expression and Purification of Antibody N033-Fc

[0035] Based on the core coding sequence of N033 obtained in Example 3, the human IgG1 Fc sequence was added to its C-terminus, and through the restriction enzyme sites Eco RI and Eco RV, it was constructed into the pcDNA3.4 vector. The amino acid sequence of the constructed N033-Fc fusion protein is shown in SEQ ID NO.3, and the nucleic acid coding sequence is shown in SEQ ID NO.4. After transfection of Expi293F cells and culturing for 5 days, the supernatant was collected. After centrifugation at 3000g for 30 min, it was filtered through a 0.22 μm filter membrane and then purified by a Protein A column to obtain a relatively pure target protein. Its expression and purity were detected by SDS-PAGE, and the results are as Figure 2 shown. The purified nanobody N033-Fc was obtained. The non-reduced (Non reduced) lane showed a molecular weight of 80 kDa, and the reduced lane showed a molecular weight of about 40 kDa, which was in line with expectations.

[0036] Example 5. Identification of the Binding Activity of Antibody N033-Fc

[0037] The ELISA method was used to analyze the binding activities of bispecific antibodies and monoclonal antibodies to CHIΚ-E. The experimental steps are described as follows:

[0038] 1. Coating: One day before the experiment, a 96-well ELISA plate was taken. The CHIΚV-E antigen was diluted to a concentration of 2 μg / mL with the coating solution and used to coat the ELISA plate, 100 μL per well, and coated overnight at 4°C;

[0039] 2. Blocking: Washed 3 times with a plate washer (BIO-TEΚ, 405_LS), 100 μL of blocking solution was added to each well, and incubated at 37°C for 1 h;

[0040] 3. Sample Incubation: Washed the plate 3 times. Except for the first well, 100 μL of dilution solution was added to each well. The antibody was diluted to 1 μg / mL in the first well and diluted in a 3-fold gradient, 100 μL / well. Three replicates were set for each antibody and incubated at 37°C for 1 h;

[0041] 4. Secondary antibody incubation: Wash the plate 3 times. Dilute the goat anti-human IgG secondary antibody labeled with HPR (Abcam, ab97225) 1:10,000 with the diluent and add 100 µL to the corresponding wells of the ELISA plate. Incubate at 37 °C for 1 h;

[0042] 5. Color development: Wash the plate 3 times. Add 100 µL of TMB single-component color development solution to each well, develop color for 6 min at room temperature in the dark, and then add 50 µL of stop solution to each well to terminate the reaction;

[0043] 6. Detect the OD value at 450 - 630 nm using an enzyme-linked immunosorbent assay (ELISA) reader, use Logistic four-parameter fitting to plot the curve, and calculate the EC 50 value.

[0044] The ELISA test results are as Figure 4 shown. The nanobody N033-Fc has strong binding activity with CHIKV-E and shows a dose-response relationship. The EC 50 (half maximal effective concentration) is 5.1 ng / mL.

[0045] Example 6: Affinity identification of antibody N033-Fc

[0046] Use a Biacore T200 instrument (Cytiva) to detect the affinity of the nanobody N033-Fc for binding to CHIKV-E. The experiment uses HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, pH 7.4) as the running buffer and is carried out at a flow rate of 30 μL / min and 25 °C. First, dilute the antibody N033-Fc to 1 μg / mL and capture it with a protein A chip at a flow rate of 10 μL / min. Then, inject the purified CHIKV-E protein at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM, and bind it to the protein A chip capturing the antibody at a flow rate of 30 μL / min for 120 s to determine the association rate (Ka), and then dissociate for 600 s to determine the dissociation rate (Kd). Regenerate the chip surface with 10 mM glycine at pH 1.5. Use the Biacore Insight Evaluation software (Cytiva) to fit the sensorgrams in the association and dissociation phases to a 1:1 binding model. Finally, calculate the affinity of the antibody based on the ratio of Kd and Ka.

[0047] The detection results are as follows Figure 5 shown that the affinity constant KD value of the nanobody N033-Fc is 3.55 nM, which has a good affinity with the CHIKV-E antigen, making it possible to develop it into a specific drug for chikungunya fever. The detailed kinetic parameters are shown in Table 2 below.

[0048] Table 2. Binding kinetic data of N033-Fc and CHIKV-E protein

[0049]

[0050] Example 7: Analysis of the virus neutralizing activity of antibody N033-Fc

[0051] 1. One day before the experiment, dilute BHK-21 cells with the medium (DMEM + 10% FBS) to a concentration of 1.5×10 5 cells / mL, inoculate into a 96-well cell culture plate, with an inoculation volume of 200 μL / well, and place it in a cell culture incubator at 37 °C and 5% CO2 for culture;

[0052] 2. On the day of the experiment, dilute the nanobody N033-Fc and the control antibody with the medium (DMEM + 2% FBS) to the initial concentration (100 μg / mL), dilute them 4-fold, add them to the 96-well culture plate, with a volume of 100 μL / well; then add 100 μL of chikungunya virus suspension (diluted 1:1000 with DMEM + 2% FBS) to each well, mix well, and place it in the cell culture incubator for co-incubation for 1 h;

[0053] 3. Discard the cell culture supernatant in the 96-well plate, add 200 μL of the co-incubated virus-antibody mixed suspension to each well; additionally set up a survival control (without virus and antibody) and a death control (only add virus), and continue to culture in a cell culture incubator at 37 °C and 5% CO2 for 72 h;

[0054] 4. After 72 h, discard the cell culture supernatant, add 50 μL / well of crystal violet staining solution (50 mg of crystal violet, 20 mL of absolute ethanol, add water to 100 mL), let it stand at room temperature for 30 min, discard the staining solution, add 200 μL / well of pure water, repeat the washing 5 times, pat dry the 96-well plate, and take pictures for record keeping;

[0055] 5. Discard all the washing solutions, add 100 μL / well of decolorizing solution (50 mL of absolute ethanol, 0.1 mL of acetic acid, add water to 100 mL) to dissolve completely, taking OD 630 as a reference, measure OD 570Value; calculate the cell viability using (OD sample well - OD dead control) / (OD live control - OD dead control), and fit the curves of cell viability and antibody concentration with GraphPad Prism 8 to calculate the antibody IC 50 value.

[0056] The detection results are as Figure 6 shown. The antibody N033-Fc has strong neutralizing activity against CHIKV, and there is a dose-effect relationship. The IC 50 (half-maximal inhibitory concentration) is 2.9 μg / mL.

[0057] Example 8: Analysis of the prophylactic protective activity of antibody N033-Fc in animals

[0058] 1. Take 6 IFNARI - / - mice and divide them into two groups (control group and experimental group), with 3 mice in each group;

[0059] 2. In the laminar flow hood, inject 200 μg of isotype control antibody into the tail vein of the mice in the control group, and inject 200 μg of N033-Fc antibody into the tail vein of the experimental group;

[0060] 3. 24 h after administration of the antibody, in the laminar flow hood, dilute the CHIKV LR2006 attenuated vaccine strain (Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus. JCI Insight. 2017 Mar 23;2(6):e83527. doi: 10.1172 / jci.insight.83527.) to 2×10 3 TCID 50 / 100 μL, and inject 100 μL of CHIKV LR2006 into the peritoneal cavity of each mouse;

[0061] 4. After challenge, continuously monitor the survival of the mice for 14 days and record.

[0062] The detection results are as Figure 7 shown. Compared with the control antibody, after challenge with 2×10 3 TCID 50 , the prophylactic protection rate of 200 μg of N033-Fc antibody reaches 100%.

[0063] Example 9: Analysis of the therapeutic protective activity of antibody N033-Fc in animals

[0064] 1. Take 6 IFNARI - / -The mice were divided into two groups (control group and experimental group), with 3 mice in each group;

[0065] 2. In the laminar flow hood, dilute the CHIKV LR2006 attenuated vaccine strain to 1×10 3 TCID 50 / 100 μL, and intraperitoneally inject 100 μL of CHIKV LR2006 into each mouse;

[0066] 3. 24 h after virus challenge, inject 100 μg of isotype control antibody into the tail vein of the control group mice, and inject 100 μg of N033-Fc antibody into the tail vein of the experimental group;

[0067] 4. After antibody treatment, continuously monitor the survival of the mice for 14 days and record.

[0068] The test results are as Figure 8 shown. Compared with the control antibody, after virus challenge with 1×10 3 TCID 50 , the protection rate of 100 μg of N033-Fc antibody treatment reached 100%.

Claims

1. A nanobody against Chikungunya virus E protein, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-Chikungunya virus E protein nanobody are shown in positions 26-33, 51-57 and 96-103 of SEQ ID NO.1, respectively.

2. The nanobody against Chikungunya virus E protein according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the nanobody against Chikungunya virus E protein is shown in SEQ ID NO.

1.

3. A polynucleotide encoding the nanobody against Chikungunya virus E protein according to claim 1 or 2, characterized in that: The sequence of the polynucleotide encoding the heavy chain variable region of the nanobody against Chikungunya virus E protein is shown in SEQ ID NO.

2.

4. A vector containing the polynucleotide encoding the anti-Chikungunya virus E protein nanobody according to claim 3.

5. A host cell containing a vector of a polynucleotide encoding the anti-Chikungunya virus E protein nanobody according to claim 4.

6. A fusion protein containing the nanobody against Chikungunya virus E protein according to claim 1 or 2, characterized in that: The fusion protein also contains human Fc protein.

7. The fusion protein according to claim 6, characterized in that The amino acid sequence of the human Fc protein is shown in SEQ ID NO.

3.

8. Use of the fusion protein containing the nanobody according to claim 6 in the preparation of a medicament for treating and / or preventing Chikungunya virus disease.

9. Use of the fusion protein containing the nanobody according to claim 6 in the preparation of a Chikungunya virus detection kit.

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