Akaban virus nanobody Nb2 and its application

Abstract

This invention discloses an akabanvirus nanobody Nb2 and its applications, belonging to the field of nanobody technology. The purpose of this invention is to provide an efficient detection method for akabanvirus. This invention provides a nanobody for detecting akabanvirus. The amino acid sequence of the CDR1 variable region of the akabanvirus-detecting nanobody is shown in SEQ ID NO.11, the amino acid sequence of the CDR2 variable region of the akabanvirus-detecting nanobody is shown in SEQ ID NO.12, and the amino acid sequence of the CDR3 variable region of the akabanvirus-detecting nanobody is shown in SEQ ID NO.13. A competitive ELISA kit is constructed for the detection of akabanvirus.

Description

Technical Field

[0001] This invention belongs to the field of nanobody technology, specifically relating to an akaban virus nanobody Nb2 and its applications. Background Technology

[0002] Akabane disease is a polymorphic infectious disease of cattle, sheep, and goats caused by Akabanevirus (AKAV), a member of the Szymborovirus group of the Bunyavirus genus. It is characterized by abortion, premature birth, stillbirth, congenital joint deformities, and Arthrogryposis-Hydraencephaly (AH syndrome). Akabane disease is primarily transmitted by arthropods such as mosquitoes, midges, and mites. Classified as a Category II infectious disease, Akabane virus causes severe economic losses to livestock farming and has become a key quarantine target in international animal trade. Currently, most research on ELISA methods for Akabane virus is still in the laboratory research stage. There are currently no commercially available ELISA kits for detecting Akabane antigen. Antibody detection for Akabane virus mainly relies on classic neutralization assays or imported ELISA kits, which poses challenges to the rapid diagnosis of Akabane disease. Summary of the Invention

[0003] The purpose of this invention is to provide an efficient detection method for akaban virus.

[0004] The present invention provides a nanobody for detecting akaban virus. The amino acid sequence of CDR1 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.11, the amino acid sequence of CDR2 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.12, and the amino acid sequence of CDR3 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.13.

[0005] Further specifying, the amino acid sequence of FR1 of the nanobody for detecting akaban virus is shown in SEQ ID NO.14, the amino acid sequence of FR2 of the nanobody for detecting akaban virus is shown in SEQ ID NO.15, the amino acid sequence of FR3 of the nanobody for detecting akaban virus is shown in SEQ ID NO.16, and the amino acid sequence of FR4 of the nanobody for detecting akaban virus is shown in SEQ ID NO.17.

[0006] This invention provides a nanobody for detecting Akaban virus, the amino acid sequence of which is shown in SEQ ID NO. 10.

[0007] The present invention provides a nucleotide sequence encoding the above-mentioned nanobody.

[0008] The present invention provides a recombinant vector containing the above-mentioned nucleotide sequence.

[0009] The present invention provides a recombinant microbial cell containing the above-mentioned nucleotide sequence.

[0010] This invention provides the application of the above-mentioned nanobody for detecting akaban virus, the above-mentioned nucleotide sequence, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in the preparation of a kit for detecting akaban virus.

[0011] This invention provides a nanobody kit for detecting akaban virus, the kit comprising the aforementioned nanobody for detecting akaban virus.

[0012] Further specifying, HRP is fused with the aforementioned nanobody for detecting Akaban virus to obtain a nanobody fused with HRP.

[0013] To further specify, the kit is a competing ELISA kit.

[0014] Beneficial effects: The akaban virus competitive ELISA method based on nanobodies has high sensitivity, strong specificity, good repeatability, and a detection time of ≤1.5h, which can meet the needs of rapid detection of clinical samples. Attached Figure Description

[0015] Figure 1 Figure showing the purification and identification results of Akaban virus;

[0016] Figure 2 The image shows the results of serum titer testing for immunized alpaca.

[0017] Figure 3 Figure showing the results of PCR identification of the nanobody library;

[0018] Figure 4 This is a graph showing the results of the fourth round of indirect ELISA screening.

[0019] Figure 5 The image shows the prokaryotic expression results of the nanoantibody strain;

[0020] Figure 6 The image shows the results of indirect immunofluorescence identification of nanobodies;

[0021] Figure 7 This is a diagram illustrating the expression pattern of nanobody-HRP fusion.

[0022] Figure 8 The image shows the results of identifying nanobody-HRP using an indirect ELISA method.

[0023] Figure 9Figure showing experimental results for the specificity of the competing ELISA method. Detailed Implementation

[0024] Example 1. Purification of Akaban virus and immunization of alpacas

[0025] I. Purification of Akaban virus

[0026] Iodixanol is a preferred medium for virus purification due to its low osmotic pressure, low toxicity, and high stability. Akabanovirus (CGMCC NO.45970, genotype Ia) cell culture medium was repeatedly freeze-thawed to release the virus. Cell debris and other impurities were removed by low-speed centrifugation. Protease inhibitors were added to prevent degradation. The mixture was then loaded into a continuous iodixanol density gradient of 10%-40% and centrifuged at 35,000-40,000 rpm at 4°C for 2-4 h. One tube was collected per milliliter from top to bottom, for a total of 11 tubes. The TCID values ​​at different gradients were measured. 50 The peak positions of complete viral particles were determined, and the results are as follows: Figure 1 As shown, viral nucleic acid was measured using quantitative real-time PCR, and viral protein was detected by Western blot. The results showed that the viral content was highest in layer 11. Figure 1 The 11th layer, after being centrifuged at 30,000 rpm to remove alcohol, can be used as an immunogen, aliquoted and frozen, with low-temperature operation throughout to avoid virus inactivation.

[0027] II. Immunizing alpacas:

[0028] Healthy alpacas weighing 400-500 kg and aged 3-5 years were selected. They were immunized with akabanvirus antigen. The initial immunization dose was 100 μg / alpacia, emulsified with an equal volume of Freund's complete adjuvant and akabanvirus antigen, and administered subcutaneously at multiple sites, including the neck and back, with 0.5 mL injected at each site. A second immunization was administered 2 weeks later, with the same dose as the initial immunization, emulsified with an equal volume of Freund's incomplete adjuvant and akabanvirus antigen, and administered subcutaneously at multiple sites. Booster immunizations were then administered every 2 weeks for a total of 3-4 booster immunizations, with a booster dose of 50 μg / alpacia, administered subcutaneously with a mixture of physiological saline and akabanvirus antigen. Seven days after each immunization, venous blood was collected from the alpacas, serum was separated, and the titer of anti-akabanvirus antibodies in the serum was detected using an indirect ELISA method. Figure 2 When the antibody titer reaches 1:10000 or higher, a final booster immunization is performed. Seven days after the final booster immunization, peripheral blood lymphocytes are collected from each alpaca, with 100 mL of peripheral blood collected from each alpaca, and lymphocytes are separated.

[0029] Example 2. Construction and screening of nanobody libraries

[0030] I. Construction of Nanobody Library:

[0031] Total RNA was extracted from lymphocytes. Primers VHH-F1 and VHH-R1 were designed based on the conserved sequences at both ends of VHH. A first round of PCR amplification was performed using cDNA as a template, followed by a second round of nested PCR amplification using VHH-FR1 and VHH-R1 to obtain the VHH gene fragment of the nanobody. Specific primer sequences are shown in Table 1. The PCR-amplified VHH gene fragment clone pComb3 was electroporated into TG1 competent cells to construct a library with a capacity of 4 × 10⁻⁶ cells. 7 The AKAV nanobody phage display library was CFU / mL, and the recombination rate of the library was verified to be 98% by colony PCR. Figure 3 ).

[0032] Table 1. Primer List

[0033]

[0034] Note: W = A or T, R = A or G, Y = C or T, M = A or C.

[0035] II. Screening for specific nanobodies:

[0036] The akabanvirus nanobody library was screened using indirect ELISA. First, akabanvirus antigen (the akabanvirus obtained in step one) was diluted with coating buffer (0.05 mol / L carbonate buffer, pH 9.6) and coated onto the ELISA plate at 100 μL per well, incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST buffer for 3 min each time. Blocking buffer (PBST buffer containing 5% skim milk powder) was added at 200 μL per well, and the plate was blocked at 37°C for 1 h. The blocking buffer was then discarded, and the plate was washed three times with PBST buffer for 3 min each time. Phage-displayed nanobody library was added at 100 μL per well, and the plate was incubated at 37°C for 1 h. Discard any unbound phages and wash three times with PBST buffer for 3 min each time to remove non-specifically bound phages. Add 100 μL of elution buffer (0.2 mol / L glycine-HCl buffer, pH 2.2) to each well and incubate at room temperature for 15 min to elute bound phages. Immediately add 50 μL of neutralization buffer (1 mol / L Tris-HCl buffer, pH 9.1) to each well to neutralize the pH of the elution buffer. The eluted phage was used to infect competent TG1 cells. The specific procedure was as follows: 100 μL of eluted phage was added to 100 μL of competent TG1 cells and incubated at 37°C for 30 min; 5 mL of LB liquid medium was added and cultured at 37°C and 200 rpm for 1 h; 100 μL of the culture was plated onto LB solid medium containing ampicillin and incubated overnight at 37°C for amplification culture to obtain enriched phage, which was then detected by indirect ELISA. The above "adsorption-elution-amplification" screening process was repeated four times. Figure 4 The screening pressure was gradually increased by progressively decreasing the coating concentration of akabanvirus antigen (from 10 μg / mL to 1 μg / mL) and shortening the incubation time (from 2 h to 1 h), resulting in a phage enrichment factor of 10. 3 The results showed that 52 positive clones specifically binding to the akaban virus antigen were obtained. Sequencing analysis revealed that they encompassed five unique VHH sequences, named Nb1, Nb2, Nb3, Nb4, and Nb5.

[0037] Table 2. Statistics of input-output ratio for screening

[0038]

[0039] III. Prokaryotic Expression and Purification of Nanobodies

[0040] Five nanobody genes were amplified by PCR using Pcold-VHH-F and Pcold-VHH-R, and then cloned into the prokaryotic expression vector pCold to construct a recombinant expression vector. The recombinant expression vector was transformed into BL21 (DE3) competent cells. Single colonies were picked and inoculated into LB liquid medium containing kanamycin. The cells were cultured at 37°C and 200 rpm until the OD600 value reached 0.6. IPTG was then added to a final concentration of 1 mmol / L, and the cells were induced at 16°C for 10 h. The induced bacterial culture was collected, centrifuged at 4°C and 8000 rpm for 10 min, and the cells were collected. The cells were resuspended in lysis buffer (50 mmol / L Tris-HCl, pH 8.0, 100 mmol / L NaCl, 1 mmol / L EDTA), and the cells were sonicated. The supernatant was collected and the protein expression was detected by SDS-PAGE electrophoresis. Figure 5 ). Use Ni 2+ -NTA affinity chromatography column is used to purify nanobodies in the supernatant.

[0041] IV. Indirect Immunofluorescence (IFA) Identification

[0042] First, BHK21 cells were seeded into 96-well plates and cultured for 48 hours. Then, AKAV cells were seeded and fixed with pre-cooled anhydrous ethanol for 10 minutes. The ethanol was discarded, and the cells were washed with PBS to remove any fixative residue. Purified nanobodies were added, and the cells were incubated at 37°C for 1 hour. The cells were then washed with PBS, and FITC-tagged anti-his monoclonal antibody was added. After washing, the fluorescence signal was observed under a fluorescence microscope. The results are shown in (…). Figure 6 All five nanobodies reacted with Akaban virus, exhibiting specific green fluorescence, but did not react with BHK21 cells, showing no specific fluorescence.

[0043] Example 3. Akaban nanobody fusion with HRP expression

[0044] I. Construction of eukaryotic expression vectors

[0045] The eukaryotic expression vector pCAGGS was selected, containing a strong CMV promoter, an Igκ signal peptide (to promote protein secretion), an HRP gene, and a C-terminal His tag (for easy purification). Using the validated nanobody gene as a template, amplification was performed using pCAGGS-F and pCAGGS-R primers. The amplified product was then ligated to the linearized vector via homologous recombination via fusion PCR. Figure 7The cells were transformed into Escherichia coli DH5α competent cells. Positive clones were selected for ampicillin resistance, verified by colony PCR and sequencing, and named pCAGGS-Nb1-HRP, pCAGGS-Nb2-HRP, pCAGGS-Nb3-HRP, pCAGGS-Nb4-HRP and pCAGGS-Nb5-HRP, respectively. The recombinant plasmids were extracted and purified for later use.

[0046] II. Cell Transfection and Expression:

[0047] After resuscitating HEK293F cells, they were incubated in serum-free CD293 medium at 37°C and 5%... Suspension culture at 120 rpm until cell density reaches... When cells / mL and viability ≥95%, PEI transfection was used: plasmid (μg): PEI (μL) was mixed at a ratio of 1:3, incubated at room temperature for 20 min to form a complex, and slowly added to the cell culture medium. Fetal bovine serum was then added to a final concentration of 0.1%, and the mixture was cultured for another 48–72 h. After culture, the mixture was centrifuged at 8000×g for 15 min at 4°C, and the supernatant was collected and filtered through a 0.22 μm filter to remove cell debris and impurities.

[0048] III. Protein purification:

[0049] A Ni-NTA affinity chromatography column was used. After equilibrating the column with PBS buffer (pH 7.4) containing 20 mM imidazole, the filtered cell supernatant was slowly loaded. Impurities were eluted with 50–80 mM imidazole buffer, followed by elution with 250 mM imidazole buffer. The elution peak was collected, and the imidazole was removed by overnight dialysis with PBS. High-purity samples were obtained by concentration using a 10 kDa ultrafiltration centrifuge tube and named Nb1-HRP, Nb2-HRP, Nb3-HRP, Nb4-HRP, and Nb5-HRP, respectively. The five purified nanobody-HRP strains were validated using an indirect ELISA method. The results ( Figure 8 It can be seen that all five nanobody-HRP strains can specifically react with Akaban virus and can be used to establish detection methods.

[0050] Nb2 gene (SEQ ID NO.9):

[0051] CAGGTGCAGCTGCAGGAATCTGGGGGAGGACTGGTGCGGCCGGGGGGCAGCCTGCGGCTGAGCTGCGCTGCTTCTGGGTTCACCTTCAGCCGGTATGGCATGTCTTGGGTGCGGCAGGCTCCCGGGAAGGAACTTGAATGGGTGAGCACCCTTAGCGTGGGGGGGGGGGGCACCACCTATGCTGACTCTGTGAAGGGCCGGTTCAGCATCAGCCGGGACAACGCTCGGAACACCTTGTACCTGCAGATGAACTCTCTGAAGCCCGAAGACACCGCTGTGTATTATTGCGAAGACGGTGTGCCGACCCAGTTCCCCCGGGGCCAGGGGACCCAGGTGACCGTGAGCTCT;

[0052] Nb2 Amino acids (SEQ ID NO.10):

[0053] QVQLQESGGGLVRPGGSLRLSCAASGFTFSRYGMSWVRQAPGKELEWVSTLSVGGGGTTYADSVKGRFSISRDNARNTLYLQMNSLKPEDTAVYYCEDGVPTQFPRGQGTQVTVSS;

[0054] Nb2 CDR1 Amino acids (SEQ ID NO.11):

[0055] FTFSRYGMS;

[0056] Nb2 CDR2 Amino acids (SEQ ID NO.12):

[0057] STLSVGGGGTT;

[0058] Nb2 CDR3 Amino acids (SEQ ID NO.13):

[0059] EDGVPTQFPR;

[0060] Nb2 FR1 Amino acids: (SEQ ID NO.14):

[0061] QVQLQESGGGLVRPGGSLRLSCAASG;

[0062] Nb2 FR2 Amino acids: (SEQ ID NO.15):

[0063] WVRQAPGKELEWV;

[0064] Nb2 FR3 amino acids: (SEQ ID NO.16):

[0065] YADSVKGRFSISRDNARNTLYLQMNSLKPEDTAVYYC;

[0066] Nb2 FR4 amino acid: (SEQ ID NO.17):

[0067] GQGTQVTVSS;

[0068] Nb2 CDR1 nucleotide sequence (SEQ ID NO.18):

[0069] TTCACCTTCAGCCGGTATGGCATGTCT;

[0070] Nb2 CDR2 nucleotide sequence (SEQ ID NO.19):

[0071] AGCACCCTTAGCGTGGGGGGGGGGGGCACCACC;

[0072] Nb2 CDR3 nucleotide sequence (SEQ ID NO.20):

[0073] GAAGACGGTGTGCCGACCCAGTTCCCCCGG;

[0074] Nb2 FR1 nucleotide sequence (SEQ ID NO.21):

[0075] CAGGGTGCAGCTGCAGGAATCTGGGGGAGGACTGGTGCGGCCGGGGGGCAGCCTGCGGCTGAGCTGCGCTGCTTCTGGG;

[0076] Nb2 FR2 nucleotide sequence (SEQ ID NO.22):

[0077] TGGGTGCGGCAGGCTCCCGGGAAGGAACTTGAATGGGTG;

[0078] Nb2 FR3 nucleotide sequence (SEQ ID NO.23):

[0079] TATGCTGACTCTGTGAAGGGCCGGTTCAGCATCAGCCGGGACAACGCTCGGAACACCTTGTACCTGCAGATGAACTCTCTGAAGCCCGAAGACACCGCTGTGTATTATTGC;

[0080] FR4 nucleotide sequence (SEQ ID NO.24):

[0081] GGCCAGGGGACCCAGGTGACCGTGAGCTCT.

[0082] Example 3. Akabanvirus competitive ELISA detection method

[0083] I. Competitive Identification of Nanobodies

[0084] This study used a competitive ELISA method to competitively identify purified nanobodies. The experimental principle is as follows: if a nanobodies can competitively bind to the same epitope of the akaban virus antigen with known akaban virus positive serum, the amount of antigen bound to the positive serum will decrease when the two are incubated together, and the absorbance (OD value) detected by the ELISA reader will also decrease accordingly. The specific operating steps are as follows: First, dilute the akaban virus antigen (the akaban virus purified in step one of Example 1) to 10 μg / mL with carbonate buffer (pH 9.6), and coat the microplate with 100 μL / well, incubating overnight at 4°C; the next day, block with PBST buffer containing 5% skim milk, incubate at 37°C for 1 h, and after washing, add equal volumes of akaban virus positive serum (ID.Vet MRI-AKA) and nanobody (working concentration 1 μg / mL), with a negative serum (Gibco fetal bovine serum) control, and incubate at 37°C for 1 h; discard the mixture and wash the plate, incubate at 37°C for 30 min, wash again, add TMB substrate solution for color development, and finally use 2 mol / L... The reaction was terminated, and the OD values ​​of each well were measured at a wavelength of 450 nm. Wells containing negative serum were used as negative controls. The competitive inhibition rate of each nanobody was calculated using the formula: Inhibition rate = (Negative control OD value - Sample OD value) / Control OD value × 100%. The results showed that Nb2-HRP had an inhibition rate exceeding 50% among the five nanobodies, with Nb2-HRP achieving an inhibition rate of 94.7%, indicating its strongest ability to competitively bind to the akaban virus antigen against positive antibodies. The other four nanobodies (Nb1-HRP, Nb3-HRP, Nb4-HRP, and Nb5-HRP) had inhibition rates between 59.3% and 72.2% (Table 3), all of which were determined to be highly competitive nanobodies. Nb2-HRP was subsequently selected as the core material to establish a competitive ELISA method.

[0085] Table 3.5 Competitive Identification of Nanobody Strains

[0086]

[0087] II. Establishing a Competitive ELISA Method

[0088] A competitive ELISA method was established using the nanobody Nb2-HRP as the detection antibody. The reaction conditions of the competitive ELISA were systematically optimized using the checkerboard titration method to determine the optimal experimental parameters. First, the antigen coating concentration and the working concentration of the nanobody were optimized: Akaban virus was used as the antigen and coated onto the ELISA plate at concentrations of 0.25, 0.5, 1, 2, and 10 μg / mL, respectively. Simultaneously, NbA2-HRP was serially diluted at concentrations of 0.1, 0.25, 0.5, 1, and 2 μg / mL. The OD values ​​of each combination were detected by competitive ELISA. It was ultimately determined that when the antigen coating concentration was 2 μg / mL and the working concentration of the nanobody was 1 μg / mL, the difference in OD values ​​between the negative and positive controls was the largest, and the background value was the lowest. Subsequently, the incubation conditions were optimized, comparing the reaction effects of incubation at 37℃ for 30 min, 1 h, 2 h, and overnight at 4℃. The results showed that incubation at 37℃ for 1 h resulted in the highest reaction efficiency and the most stable results. In addition, the types of blocking solutions (5% skim milk, 1% BSA, 1% gelatin, 10% rabbit serum) and substrate color development times (10 min, 15 min, 20 min) were screened, and 5% skim milk as the blocking solution and a color development time of 15 min at room temperature were determined to be the optimal conditions. Results were interpreted as follows: Inhibition rate = (OD value of negative control - OD value of sample) / OD value of negative control × 100%. An inhibition rate ≥ 50% was considered positive, and < 50% was considered negative.

[0089] III. Evaluation of Competitive ELISA Methods

[0090] 1. Sensitivity

[0091] To verify the sensitivity of the established akabanvirus competitive ELISA method, a concordance test was conducted using the classic virus neutralization assay (VN assay) as a reference. Sixty bovine serum samples clinically suspected of akabanvirus infection were selected (including 20 known positive, 20 known negative, and 20 unknown samples). All samples were free from hemolysis, turbidity, or other interference and were stored at -20°C. The competitive ELISA assay was performed strictly according to the previously optimized conditions.

[0092] The virus neutralization assay employed the fixed virus dilution serum method, where samples were serially diluted twofold (1:4 to 1:512) in 96-well cell culture plates, with 100 μL of serum added to each well. Akaban virus solution was incubated at 37°C for 1 hour, and then sensitive cell suspension was added. The cells were cultured for another 5-7 days, and cytopathic effects (CPE) were observed. The highest serum dilution that could completely inhibit CPE was used as the neutralizing titer. A titer ≥1:4 was considered positive, and <1:4 was considered negative.

[0093] The experimental results showed that out of 60 samples, 19 samples were both positive (true positives) and both were negative (true negatives) as determined by the competitive ELISA and neutralization test. The concordance rate was calculated as follows: Overall concordance rate = (true positives + true negatives) / total number of samples × 100% = (19 + 40) / 60 × 100% = 98.3% (Table 4). This indicates that the competitive ELISA and the neutralization test, which is considered the "gold standard," showed good consistency. This result suggests that the competitive ELISA can be used as a rapid detection method to replace the neutralization test for the batch screening of clinical samples for akabanvirus.

[0094] Table 4. Comparison of Competitive ELISA and Neutralization Test

[0095]

[0096] 2. Specificity

[0097] To evaluate the specificity of the akaban virus competitive ELISA method, five bovine disease-positive sera (bovine coronavirus, bluetongue virus, epidemic hemorrhagic disease virus, bovine viral diarrhea virus, and bovine ephemeral fever virus) were selected. Akaban virus positive (virus with accession number CGMCC NO.45970) and negative control (PBS) were also set up. Each sample was tested in triplicate and detected by the competitive ELISA method.

[0098] The results showed that the inhibition rate of Akaban virus positive serum was 98%, while the inhibition rates of negative control and other pathogen-positive serum were between 20% and 40%, all below 50%. Figure 9 This indicates that the nanobody Nb2-HRP can specifically compete with akabanvirus-positive serum without interference from antibodies of irrelevant pathogens. This competitive ELISA method has extremely high specificity and can accurately distinguish akabanvirus-positive serum from serum of other pathogens, and can be used for the differential diagnosis of akabanvirus.

[0099] 3. Repeatability

[0100] Repeatability tests included intra-assay and inter-assay replicates. Intra-assay replicates: Three positive and three negative sera were selected, and six replicates were set up for each concentration in the same experiment. Detection was performed using a competitive ELISA method, and the coefficient of variation (CV) of the OD values ​​for each concentration replicate was calculated. Inter-assay replicates: The above six sera were selected, and three different batches of the nanobody Nb2-HRP were used for three independent assays, with three replicates for each concentration. The inter-assay CV value was calculated. The results showed that the CV in the intra-assay replicates was less than 5%, and the CV in the inter-assay replicates was less than 7%. This indicates that the method has good stability and excellent repeatability under different experimental conditions and batches, and can be used for batch detection of akaban virus.

[0101] The above experimental results show that the akaban virus competitive ELISA method based on nanobodies has high sensitivity, strong specificity, good repeatability, and a detection time of ≤1.5h, which can meet the needs of rapid detection of clinical samples.

Claims

1. A nanobody for detecting an akabane virus, characterized in that, The amino acid sequence of CDR1 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.11, the amino acid sequence of CDR2 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.12, and the amino acid sequence of CDR3 of the variable region of the nanobody for detecting akaban virus is shown in SEQ ID NO.

13.

2. Nanobody according to claim 1, characterized in that, The amino acid sequence of FR1 of the nanobody for detecting akaban virus is shown in SEQ ID NO.14, the amino acid sequence of FR2 of the nanobody for detecting akaban virus is shown in SEQ ID NO.15, the amino acid sequence of FR3 of the nanobody for detecting akaban virus is shown in SEQ ID NO.16, and the amino acid sequence of FR4 of the nanobody for detecting akaban virus is shown in SEQ ID NO.

17.

3. The gene encoding the nanobody of claim 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 3.

5. A recombinant microbial cell, characterized in that, The recombinant microbial cell contains the gene described in claim 3.

6. The use of the nanobody for detecting akaban virus as described in claim 1 or 2, the gene as described in claim 3, the recombinant vector as described in claim 4, or the recombinant microbial cell as described in claim 5 in the preparation of a kit for detecting akaban virus.

7. A nanobody kit for detecting an akabane virus, characterized in that, The kit shown includes the nanobody for detecting akaban virus as described in claim 1 or 2.

8. The reagent kit according to claim 7, characterized in that, HRP is fused with the nanobody for detecting akaban virus as described in claim 1 or 2 to obtain a nanobody fused with HRP.

9. The reagent kit according to claim 7, characterized in that, The kit is a competing ELISA kit.

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