Nano antibody N033 for resisting chikungunya virus E protein and application of nano antibody N033

Abstract

The invention discloses an anti-chikungunya virus E protein nano antibody N033 and application thereof, the anti-chikungunya virus E protein nano antibody has a unique CDR region, the fusion protein of N033 and human Fc has excellent binding activity, high affinity and strong neutralizing activity with chikungunya virus, in a mouse challenge experiment, 200 micrograms of N033-Fc antibody is given before challenge, the binding activity of the N033 and human Fc is improved, and the binding activity of the N033 and human Fc is improved. The prevention and protection rate reaches 100% after 2 * 10 < 3 > TCID50 is challenged, and the protection rate reaches 100% after 100 micrograms of N033-Fc antibody treatment is performed after 1 * 10 < 3 > TCID50 is challenged. The application prospect of the nano antibody provided by the invention in preparation of drugs for treating and / or preventing chikungunya virus diseases is shown, and the application prospect of the nano antibody in preparation of chikungunya virus detection kits is also shown due to excellent binding activity and affinity.

Description

Technical Field

[0001] The invention discloses a nano antibody, belonging to the technical field of polypeptide and immunology. Background Art

[0002] Chikungunya fever (CHIKF) is a disease caused by Chikungunya virus (CHIKV) that was first discovered in Africa. Its clinical manifestations include fever, rash, and body pain. It is one of the fastest-spreading mosquito-borne infectious diseases in the world 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 Chikungunya virus as one of the three sub-hazardous pathogens that may cause severe outbreaks. Due to the limitations of existing diagnostic technologies, CHIKV has not been fully studied and may cause a large-scale epidemic in the future. At present, there are no specific drugs available for the treatment of CHIKV. Although antibody therapy has achieved certain results in preclinical studies, the further implementation of such therapy still faces challenges. Therefore, it is urgent to develop a new generation of neutralizing antibody drugs that can provide protective activity.

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

[0004] The purpose of the present invention is to use phage library technology to obtain nano antibodies against CHIKV with excellent performance in terms of affinity, binding activity and neutralization activity against viruses, and further provide its application in the preparation of CHIKF prevention and treatment drugs. Summary of the invention

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

[0006] The nanobody described in the present invention is an antibody with only a heavy chain, wherein the heavy chain includes a variable region and a constant region, wherein the variable region has three complementary determining regions (CDRs): CDR1, CDR2 and CDR3, which are highly variable and diverse. The sequence diversity of the CDR region determines the specificity and affinity of the antibody, because they recognize and bind to 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 Chikungunya virus E protein is 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 anti-Chikungunya virus E protein Nanobody. In the present invention, the polynucleotide whose sequence is shown in SEQ ID NO.2 is a polynucleotide encoding the amino acid sequence of the Nanobody N033 heavy chain variable region shown in SEQ ID NO.1 according to a specific embodiment. Based on the triplet code rule, any polynucleotide encoding the amino acid sequence of the Nanobody N033 heavy chain variable region shown in SEQ ID NO.1 belongs to the polynucleotide encoding the above-mentioned anti-Chikungunya virus E protein Nanobody defined in the present invention.

[0009] Third, the present invention provides a vector containing the above-mentioned polynucleotide encoding the anti-Chikungunya virus E protein Nanobody. The vector is used to clone and / or express the coding gene of the anti-Chikungunya virus E protein Nanobody. In a specific embodiment of the present invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, especially eukaryotic cell expression vectors, can also be used for 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 anti-Chikungunya virus E protein Nanobody. The host cell is used to express the above-mentioned anti-Chikungunya virus E protein Nanobody. In a specific embodiment of the present invention, the host cell is an 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 nano antibody against Chikungunya virus E protein. Based on different application requirements such as extending the half-life of the antibody in vivo, crossing cells or tissue structures, targeting specific areas, exercising cell effects, dimer or multimer formation, and purification processes, the nano antibody provided by the present invention can be fused with other proteins to form a fusion protein, in which the nano antibody provided by the present invention can exert its unique characteristics of specifically targeting and binding to the 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 shown in SEQ ID NO.3, and preferably, the nucleic acid encoding sequence thereof is shown in SEQ ID NO.4.

[0013] Sixth, the present invention provides the use of the above-mentioned anti-Chikungunya virus E protein nanobody or the fusion protein containing the nanobody in the preparation of a drug for treating and / or preventing Chikungunya virus disease. The nanobody provided by the present invention has excellent binding activity with the Chikungunya virus E protein and can specifically target the Chikungunya virus E protein. Therefore, these properties of the nanobody are used as a specific inhibition of the binding of the Chikungunya virus to the host cell, or the therapeutic drug is specifically targeted to the infection lesion or pathogen to play a role in clinical treatment or prevention of infection. Therefore, the present invention provides the use of the above-mentioned anti-Chikungunya virus E protein nanobody in the preparation of a drug for treating and / or preventing Chikungunya virus disease. In a specific embodiment of the present invention, the fusion protein also contains a human Fc segment protein.

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

[0015] The present invention screened the nano antibody N033 against the E protein of the Chikungunya virus through alpaca immunization and phage antibody library. The nano antibodies all have unique heavy chain variable region CDR regions. The fusion protein N033-Fc of N033 and human Fc has excellent binding activity with the Chikungunya virus. 50 The half-maximal effect concentration is 5.1 ng / mL, and the affinity KD for Chikungunya virus is 3.55 nM. N033-Fc has strong neutralizing activity against CHIΚV, and has a dose-effect relationship. IC 50 The half maximum lethal concentration was 2.9 μg / mL. In the mouse challenge experiment, 200 μg of N033-Fc antibody was given before the challenge, and 2×10 3 TCID 50 The prevention and protection rate after the attack reached 100%, and the virus attack rate was 1×10 3 TCID 50 The protection rate of 100 μg N033-Fc antibody treatment was also 100%, which shows the application prospect of the nano antibody provided by the present invention in the preparation of drugs for treating and / or preventing chikungunya virus disease, and the excellent binding activity and affinity also show its application in the preparation of chikungunya virus detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . SDS-PAGE test results of CHIΚV E protein (separation gel concentration 12%); Figure 2 . SDS-PAGE test results of N033-Fc (separation gel concentration 12%); Figure 3 . IMGT query sequence annotation information of N033; Figure 4 . ELISA binding curve of N033-Fc and CHIKV-E protein; Figure 5 . Binding kinetic curve of N033-Fc and CHIKV-E protein; Figure 6 . Neutralization activity of N033-Fc against CHIΚV virus; Figure 7 . Evaluation of the protective activity of N033-Fc in the preventive administration mode in animals; Figure 8 . Evaluation of the protective activity of N033-Fc in the therapeutic administration model in animals. DETAILED DESCRIPTION

[0017] 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 as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0018] Example 1: Expression and purification of CHIKVE protein The present invention uses an insect cell-baculovirus system to express CHIΚV-E protein. First, the CHIΚV-E protein gene sequence (GenBank: MH670649.1) is optimized according to the insect cell preference for codons, and 6 histidine tags are added to the N-terminus. Then, the gene fragment is synthesized by an artificial method and cloned into the pFastBac1 vector (insect cell baculovirus expression vector) by a seamless cloning method. The recombinant plasmid is transformed into DH10Bac competent cells, and after blue-white screening and PCR identification, the recombinant baculovirus bacmid (Bacmind) is obtained. The recombinant baculovirus bacmid is extracted by isopropanol precipitation, and then Bacmind is transfected with a transfection reagent (ExpiFectamine TM Sf Transfection Reagent, A38915) was transfected into Expi Sf21 insect cells and cultured at 27°C and 120 rpm for 96 hours. The culture supernatant was collected, centrifuged, and stored at -80°C to obtain CHIΚV-E protein P0 virus. Subsequently, the P0 virus was transferred to Expi Sf9 cells to amplify the virus. After 4 days of continuous culture, the supernatant was collected and purified by nickel ion affinity chromatography (HisTrap TM After purification by excel, CHIKV-E protein was obtained. Its expression and purity were identified by SDS-PAGE. Figure 1 As shown, the results show that the molecular weight of the purified CHIKV-E protein is about 100 kDa, which is in line with expectations and the purity meets the requirements.

[0019] Example 2: Alpaca immunization and establishment of VHH nanobody library 1 mg of CHIKV-E protein with 6 histidine tags prepared earlier was diluted to a final volume of 1 mL with PBS, emulsified with 1 mL of Freund's complete adjuvant, and mixed and injected subcutaneously at multiple points for the first immunization of alpacas. Immunization was then performed every three weeks. After the fifth immunization, serum titers were tested using ELISA. Serum titers >10 6 , then 100 mL of blood was collected to separate peripheral blood mononuclear cells (PBMCs), and total RNA was extracted from the isolated PBMCs according to the instructions of QIAGEN's Neasy® Plus MiniRNA Extraction Kit. Using the extracted total RNA as a template, cDNA reverse transcription was performed according to the instructions of Invitrogen's Super Script®III First-Strand Synthesis System Extraction Kit. The reverse transcribed cDNA library was used for gene template amplification, and the first round of PCR reaction was performed using primers CALL001 and CALL002 (primer sequences are shown in Table 1), and the target band was recovered by running gel. The recovered DNA was used as a template for secondary amplification, and VHH-FOR and VHH-REV primers (primer sequences are shown in Table 1) were used to run gel to recover the corresponding band of VHH. Subsequently, the recovered fragments and pcomb3X vector were digested by Sfi Ⅰ enzyme, and the fragments were directly purified by gel recovery purification column. The linear vector of pcomb3X vector was recovered by gel recovery kit. The fragments were mixed with pcomb3X and connected with T4 ligase overnight at 4°C. The purified ligation product was mixed with XL1-Blue competent state in an ice bath, and 80 μL / cup was dispensed into an electric transfer cup, and electroporated at 1800V. After electroporation, the mixture was transferred to 2YT-ATG medium containing ampicillin and tetracycline, and the volume was fixed to 200 mL with 2YT-ATG. The library after fixed volume was shaken and cultured for 1 hour at 37°C and 250rpm, which was the antibody bacterial liquid library.

[0020] Table 1: Reaction primers

[0021] Example 3: Phage display technology to screen specific nanobodies After the library bacterial solution in Example 2 was further cultured at 37°C and 250 rpm for 1 hour, VCSM13 helper phage was added and allowed to infect for half an hour, and then further cultured at 37°C and 200 rpm for 1 hour. Finally, the bacterial precipitate was collected by centrifugation and refloated in 100 mL 2YT-ATK medium (containing ampicillin, tetracycline and kanamycin), and amplified overnight at 30°C and 225 rpm. The supernatant was collected the next day, and the bacterial precipitate was obtained by ice bath and centrifugation using 4% PEG8000 and 3% NaCl. The obtained phage precipitate was resuspended and dissolved in PBS (pH7.4) and sterilized by filtration using a 0.2 μm syringe filter. 10 μL of the filtered library was aspirated and diluted 10 times using the 2YT medium 100-fold dilution method (10 μL+990 μL). 8 After 10 times the concentration, take 10 μL of the dilution to infect 100 μL of XL1-blue. After 30 minutes, apply it to 2YT-ATG semi-solid culture medium plates. The next day, count the number of monoclonal colonies to calculate the concentration of phages in the amplified library. Add 7% DMSO to the filtered library and freeze it at -80℃.

[0022] High-affinity ELISA plates were coated with antigen (antigen: CHIKV-E) overnight, washed with 0.1% PBST, and blocked with 3% skim milk powder at room temperature for 1 hour, then the blocking solution was discarded and the plates were washed for later use. 12 CFU, incubate with antigen for 2 h at 37°C. Wash the plate with 0.1% PBST, add 100 µL of freshly prepared Glycine-HC1 elution binding solution to each well, let stand at room temperature for 10 min, aspirate the eluate and quickly neutralize with an equal volume of 1 M Tris-HCl (pH7.4). Then take the eluate and mix it with Escherichia coli XL1-BIue, incubate at 37°C for 30 minutes, and 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 sequence. 96 monoclonal clones were randomly picked from the bacterial plate after the second round of screening. After inducing VHH antibody expression with IPTG, soluble recombinant VHH antibody crude extracts were prepared for ELISA detection to preliminarily identify the reactivity of VHH antibody clones with CHIKV-E protein. When OD 450 The value was more than 3 times greater than that of the PBS control and was considered positive. A portion of the bacterial suspension of the positive clone was sequenced to obtain the core coding sequence of N033. Log in to https: / / www.imgt.org / IMGT_vquest to obtain Figure 3The sequence annotation information of N033 shown in the figure shows that the homology between V-GENE and allele identification is 88.77%, the homology between J-GENE and allele identification is 79.17%, the amino acid lengths of FR1, FR2, FR3 and FR4 are 25, 17, 38, 11 respectively. The amino acid lengths of CDR1, CD2 and CDR3 are 8, 7, 8 respectively. The amino acid JUNTION sequence is: CNTLPISRDW.

[0023] Example 4: Expression and purification of antibody N033-Fc Based on Example 3, the core coding sequence of N033 was obtained, and the human IgG1 Fc sequence was added to its C-terminus. It was then constructed into the pcDNA3.4 vector through the restriction enzyme sites Eco RI and Eco RV. 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. Expi293F cells were transfected and cultured for 5 days. The supernatant was collected, centrifuged at 3000g for 30 min, filtered through a 0.22 um filter membrane, and purified on a Protein A column to obtain a relatively pure target protein. Its expression and purity were detected by SDS-PAGE. The results are shown in Figure 2. Figure 2 As shown, the purified nanobody N033-Fc was obtained. The non-reduced lane showed a molecular weight of 80 kDa, and the reduced lane showed a molecular weight of 40 kDa, which was in line with expectations.

[0024] Example 5. Identification of the binding activity of antibody N033-Fc The ELISA method was used to analyze the binding activity of bispecific antibodies and monoclonal antibodies to CHIΚ-E. The experimental steps are described as follows: 1. Coating: One day before the experiment, dilute the CHIKV-E antigen to a concentration of 2 μg / mL with coating solution in a 96-well ELISA plate, coat the plate with 100 μL per well, and coat overnight at 4°C; 2. Blocking: Wash three times with a plate washer (BIO-TEΚ, 405_LS), add 100 µL of blocking solution to each well, and incubate at 37°C for 1 h; 3. Sample incubation: Wash the plate 3 times, add 100 μL of diluent to each well except the first well, dilute the antibody to 1 μg / mL in the first well, 3-fold gradient dilution, 100 μL / well, set three replicate wells for each antibody, and incubate at 37°C for 1 h; 4. Secondary antibody incubation: Wash the plate three times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) with diluent at 1:10000, add 100 µL per well to the corresponding wells of the ELISA plate, and incubate at 37°C for 1 h; 5. Color development: Wash the plate 3 times, add 100 µL of TMB single-component color development solution to each well, develop for 6 min at room temperature and avoid light, then add 50 µL of stop solution to each well to terminate the reaction; 6. Detect the OD value at 450-630 nm on an ELISA instrument, draw the curve using the Logistic four-parameter fitting method, and calculate the EC value of the antibody. 50 value.

[0025] ELISA test results Figure 4 The results showed that the nanobody N033-Fc had strong binding activity with CHIKV-E and had a dose-effect relationship. 50 (half-maximal effect concentration) was 5.1 ng / mL.

[0026] Example 6: Affinity identification of antibody N033-Fc The affinity of the nanobody N033-Fc binding to CHIKV-E was detected using a Biacore T200 instrument (Cytiva). The experiment was performed at a flow rate of 30 μL / min and 25°C using 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. First, the antibody N033-Fc was diluted to 1 μg / mL and captured with a protein A chip at a flow rate of 10 μL / min. Then, purified CHIKV-E protein was injected 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 passed through the protein A chip of the capture antibody at a flow rate of 30 μL / min for 120 seconds to determine the association rate (Ka), and then dissociated for 600 seconds to determine the dissociation rate (Kd). The chip surface was regenerated using 10 mM glycine at pH 1.5. The sensorgrams of the association and dissociation phases were fitted to a 1:1 binding model using Biacore Insight Evaluation software (Cytiva). Finally, the affinity of the antibody was calculated based on the ratio of Kd and Ka.

[0027] Test results such as Figure 5The results show 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.

[0028] Table 2. Binding kinetic data of N033-Fc and CHIKV-E protein

[0029] Example 7: Analysis of virus neutralization activity of antibody N033-Fc 1. One day before the experiment, dilute BHK-21 cells to 1.5×10 5 cells / mL concentration, inoculated into 96-well cell culture plates, inoculated with a volume of 200 μL / well, and cultured in a 37°C, 5% CO2 cell culture incubator; 2. On the day of the experiment, the nanobody N033-Fc and the control antibody were diluted to the initial concentration (100 μg / mL) with culture medium (DMEM+2% FBS), diluted 4 times, and added to a 96-well culture plate with a volume of 100 μL / well; then 100 μL of Chikungunya virus suspension (diluted 1:1000 with DMEM+2% FBS) was added to each well, mixed thoroughly, and incubated in a cell culture incubator for 1 hour; 3. Discard the cell culture supernatant in the 96-well plate, add 200 μL of the virus-antibody mixed suspension after co-incubation to each well; set up a survival control (no virus and antibody) and a death control (only virus), and continue to culture in a 37℃ 5% CO2 cell culture incubator for 72 hours; 4. After 72 h, discard the cell culture supernatant and add 50 μL / well crystal violet staining solution (50 mg crystal violet, 20 mL anhydrous ethanol, add water to 100 mL). Let stand at room temperature for 30 min, discard the staining solution, add 200 μL / well pure water, repeat washing 5 times, pat dry the 96-well plate, and take photos for record. 5. Discard the washing solution, add 100 μL / well decolorizing solution (50 mL of anhydrous ethanol, 0.1 mL of acetic acid, add water to 100 mL) to fully dissolve, and measure the OD 630 As a reference, measure OD with a microplate reader 570 Values; cell viability was calculated using (OD sample well - OD death control) / (OD survival control - OD death control). Cell viability and antibody concentration were fitted using GraphPad Prism 8 to calculate antibody IC 50 value.

[0030] Test results such as Figure 6 The results showed that the antibody N033-Fc had strong neutralizing activity against CHIKV and had a dose-effect relationship. 50 (half maximum lethal concentration) was 2.9 μg / mL.

[0031] Example 8: Analysis of the preventive and protective activity of antibody N033-Fc in animals 1. Take 6 IFNARI - / - The mice were divided into two groups (control group and experimental group), with 3 mice in each group; 2. In the clean bench, mice in the control group were injected with 200 μg of isotype control antibody through the tail vein, and mice in the experimental group were injected with 200 μg of N033-Fc antibody through the tail vein; 3. 24 h after antibody administration, CHIKV LR2006 attenuated vaccine strain (Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus. JCIInsight. 2017 Mar 23;2(6):e83527. doi: 10.1172 / jci.insight.83527. ) was diluted to 2×10 3 TCID 50 / 100 μL, each mouse was intraperitoneally injected with 100 μL CHIKV LR2006; 4. After infection, the survival of mice was monitored and recorded for 14 consecutive days.

[0032] Test results such as Figure 7 Compared with the control antibody, the virus 2×10 3 TCID 50 The protective rate of 200 μg N033-Fc antibody reached 100%.

[0033] Example 9: Analysis of the therapeutic protective activity of antibody N033-Fc in animals 1. Take 6 IFNARI - / - The mice were divided into two groups (control group and experimental group), with 3 mice in each group; 2. In a clean bench, dilute the CHIKV LR2006 attenuated vaccine strain to 1×10 3 TCID 50 / 100 μL, each mouse was intraperitoneally injected with 100 μL CHIKV LR2006; 3. 24 h after challenge, mice in the control group were injected with 100 μg of isotype control antibody through the tail vein, and mice in the experimental group were injected with 100 μg of N033-Fc antibody through the tail vein; 4. After antibody treatment, the survival of mice was continuously monitored and recorded for 14 days.

[0034] Test results such as Figure 8 Compared with the control antibody, the virus 1×10 3 TCID 50 , the protection rate of 100 μg 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.

7. The fusion protein according to claim 6, characterized in that The fusion protein also contains human Fc protein.

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

3.

9. Use of the nanobody against Chikungunya virus E protein or a fusion protein containing the nanobody according to claim 1 or 2 in the preparation of a medicament for treating and / or preventing Chikungunya virus disease.

10. Use of the nanobody against Chikungunya virus E protein according to claim 1 or 2 or a fusion protein containing the nanobody in the preparation of a Chikungunya virus detection kit.

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