Nanobody against african swine fever virus k205r protein and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a nano antibody against African swine fever virus K205R protein and application. The application provides a nano antibody KR-6 against African swine fever virus K205R protein, wherein the nano antibody KR-6 is obtained through construction and screening of a phage display nano antibody library, is a heavy chain antibody variable region fragment, and has an amino acid sequence shown in SEQ ID NO. 1. The nano antibody has high affinity to African swine fever virus K205R protein and African swine fever virus, can specifically recognize African swine fever virus, and can be used for diagnosis, prevention or treatment of African swine fever.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a nanobody against African swine fever virus K205R protein and application thereof. BACKGROUND

[0002] African swine fever (ASF) is a severe infectious disease of pigs caused by infection with African swine fever virus (ASFV). The World Organization for Animal Health lists African swine fever as a legal report animal epidemic, and China lists it as a class animal epidemic. The incubation period of African swine fever is generally 4-19 days, and according to its clinical manifestations, it can be divided into five types, including the most acute type, acute type, subacute type, chronic type and subclinical type. The disease is mainly transmitted by direct contact, and can also be transmitted by vectors. Soft ticks are its reservoir hosts and transmission vectors, and the virus is the only known DNA virus that can be transmitted by biological vectors soft ticks.

[0003] African swine fever virus is a nuclear large DNA virus, which can be divided into 24 genotypes and is easy to mutate. Due to different genotypes, the length of the genome ranges from 171,235bp to 193,886bp, and contains 151-167 open reading frames (ORF). At present, the complexity of epidemic strains has brought new challenges to the prevention and control of African swine fever in China. The detection of African swine fever virus specific nucleic acid has played a very important role in the early prevention and control in China; but due to the appearance of the weak strain, the time of virus shedding is delayed and the amount of virus shedding is relatively low, so the detection of African swine fever virus nucleic acid may not be detected sometimes; and the detection of African swine fever virus specific antibody is very important for the screening of weak virulent strain infection and resistant pigs.

[0004] The variable region of the heavy chain of HCAb in camelid serum is a monomer domain, i.e., a single-domain antibody (VHH), also known as a nanobody (Nb), which has a diameter of 2.5 nm and a length of 4 nm. The molecular weight of Nb is about 15 kDa, which is the smallest antibody that can currently bind to target antigens. Nb has the advantages of small volume, small molecular weight, easy modification, strong penetration, low immunogenicity, fast metabolism, etc. Compared with traditional Ig antibodies (about 150 kDa), Nb is easier to modify; it is easier to recognize and bind to antigen epitopes in hidden places such as folds. Nb has high water solubility and strong affinity. Due to the absence of L chains, VH cannot bind to VL through interaction, which exposes the hydrophilic amino acid residues of Nb, increases its solubility in water, and enables it to bind to water-soluble haptens. In addition, Nb has longer CDR1 and CDR3 amino acid sequences, and a higher affinity for antigens than traditional polyclonal antibodies and monoclonal antibodies. Nb can be expressed at a high level in prokaryotic expression systems, mammalian cell expression systems, and plant cell expression systems. Due to its small molecular weight and simple structure, the production and purification process is simpler, and batch production can be achieved. The unique molecular properties of nanobodies make them have good application prospects in the diagnosis and treatment of various diseases and detection. SUMMARY

[0005] To solve the above technical problems, the present application provides an anti-African swine fever virus K205R protein nanobody KR-6 and a preparation method and application thereof. The nanobody has high affinity for African swine fever virus K205R protein and African swine fever virus, can specifically recognize and bind to African swine fever virus, and can be used for the diagnosis, prevention or treatment of African swine fever. Specifically, the following content is included.

[0006] In a first aspect, the present application provides an anti-African swine fever virus K205R protein nanobody KR-6, wherein the amino acid sequence of the nanobody KR-6 is shown as SEQ ID NO. 1.

[0007] In a second aspect, the present application provides a nucleic acid encoding the nanobody KR-6 of the first aspect.

[0008] Preferably, the nucleic acid sequence is shown as SQE ID NO. 2.

[0009] In a third aspect, the present application provides an expression vector containing the nucleic acid sequence of the second aspect.

[0010] In a fourth aspect, the present application provides an immunoconjugate, which comprises:

[0011] (i) the nanobody KR-6 of the first aspect;

[0012] (ii) and a coupling moiety selected from the group consisting of a detectable label, a drug, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0013] In a fifth aspect, the present application provides use of the Nanobody KR-6 of the first aspect or the immunoconjugate of the fourth aspect in the preparation of an African swine fever virus detection reagent or kit.

[0014] In a sixth aspect, the present application provides an African swine fever virus detection reagent or kit comprising the Nanobody KR-6 of the first aspect.

[0015] In a seventh aspect, the present application provides a composition comprising the Nanobody KR-6 of the first aspect.

[0016] In an eighth aspect, the present application provides a method for preparing the Nanobody KR-6 of the first aspect, the method comprising:

[0017] (1) synthesizing a nucleotide sequence encoding the Nanobody KR-6 of the first aspect;

[0018] (2) connecting the nucleotide sequence of step (1) with a vector / plasmid to construct an expression vector of the Nanobody KR-6;

[0019] (3) transforming the expression vector of the Nanobody KR-6 into bacteria to construct a recombinant cell, inducing expression and purifying to obtain the Nanobody KR-6.

[0020] Preferably, the nucleotide sequence of the Nanobody KR-6 is as shown in SEQ ID NO. 2.

[0021] In a ninth aspect, the present application provides a primer for amplifying the nucleotide sequence encoding the Nanobody KR-6 of the first aspect, characterized in that the primer is:

[0022] the upstream primer F: ccgGAATTCcggATGCATGTGCAGCTGGTGGAG (as shown in SEQ ID NO. 3);

[0023] the downstream primer R: cccAAGCTTgggTGAGGAGACGGTGACCTGGGT (as shown in SEQ ID NO. 4).

[0024] The beneficial effects of the present application are: the present application provides the anti-African swine fever virus K205R protein nanobody KR-6, which is obtained by construction and screening of a phage display nanobody library, is a heavy chain antibody variable region fragment, and has the amino acid sequence shown in SEQ ID NO. 1; the nanobody has high affinity with the African swine fever virus K205R protein and the African swine fever virus, can specifically recognize and combine the African swine fever virus, and can be used for diagnosis, prevention or treatment of African swine fever; and the nanobody is expressed by an E. coli expression system, and the preparation method is simple. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 PCR identification results of recombinant bacteria liquid; wherein M is a molecular weight standard, and 1-9 are recombinant bacteria;

[0026] Figure 2 SDS-PAGE gel electrophoresis results of nanobody KR-6 induced expression; wherein M is a molecular weight standard, 1 is before induction, and 2 is after induction of the recombinant bacteria liquid;

[0027] Figure 3 Nanobody KR-6 induced expression and solubility analysis results; wherein M is a molecular weight standard, 1 is before induction, 2 is after induction, 3 is ultrasonic supernatant, and 4 is ultrasonic sedimentation;

[0028] Figure 4 Purification results of nanobody KR-6; wherein M is a molecular weight standard, 1 is before induction, 2 is before ultrasonic, 3 is ultrasonic sedimentation, 4 is flow-through liquid, 5 is wash 1, 6 is wash 2, 7 is wash 3, 8 is elution 1, 9 is elution 2, 10 is elution 3, and 11 is elution 4;

[0029] Figure 5 ELISA identification of the reactivity of nanobody KR-6 with African swine fever virus K205R recombinant protein results;

[0030] Figure 6 ELISA identification of the reactivity of nanobody KR-6 with African swine fever virus results;

[0031] Figure 7 Western blot identification of the reactivity of nanobody KR-6 with African swine fever virus results. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] After extensive and in-depth research, namely a large amount of screening, the inventors of this application unexpectedly discovered for the first time a nanoantibody KR-6 targeting the K205R protein of African swine fever virus. The experimental results show that the nanoantibody of the present invention can specifically recognize and bind to African swine fever virus, and can be used for the diagnosis, prevention or treatment of African swine fever.

[0034] Specifically, the present invention uses phage display technology to screen a nanobody library (camel heavy chain antibody phage display library) immune to the African swine fever virus K205R protein, thereby obtaining the nanobody KR-6 gene capable of recognizing the African swine fever virus K205R protein. This gene is then transferred into Escherichia coli cells and expressed to obtain the nanobody. The nanobody is then identified by methods such as ELISA to determine its ability to specifically recognize and bind to the African swine fever virus.

[0035] Among them, the nanobody targeting the African swine fever virus K205R protein is represented by KR-6 (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2).

[0036] The experimental viruses described in the following examples were obtained from: African swine fever virus CN / SC / 2019 strain and African swine fever virus antibody positive and negative sera were provided by the National African Swine Fever Regional Laboratory (Lanzhou); the prokaryotic expression vector pET-32a(+) was provided by this laboratory; the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; Q5 Hot start High-Fidelity 2× Master Mix, restriction endonucleases EcoRI, Hind III, and T4 DNA ligase were purchased from NEB; the gel recovery kit and plasmid extraction kit were purchased from OMEGA; IPTG was purchased and synthesized by Sangon Biotech (Shanghai) Co., Ltd.; Rosetta-gami2 (DE3) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; and HRP-labeled rabbit anti-6×His tag antibody was purchased from Abcam (Shanghai) Trading Co., Ltd.

[0037] Unless otherwise specified, other reagents in the experiment are common commercially available reagents; unless otherwise specified, the operations in the experiment are all methods known in the art.

[0038] Example 1 Construction of phage-displayed nanobody library

[0039] The Bactrian camels were immunized with the recombinant protein of African swine fever virus K205R, and the immunization was performed five times. The venous blood was collected before the first immunization and after each immunization, and the serum antibody titer was detected by indirect ELISA. The venous anticoagulant blood was collected after the antibody titer reached the expected immunization effect. The specific animal immunization program and antigen inoculation dose are shown in Table 1.

[0040] Table 1: Bactrian camel immunization program and antigen dose

[0041]

[0042] After the antibody titer reached the requirement, 100 ml of camel anticoagulant blood was collected from the jugular vein. Peripheral blood lymphocytes (PBMCs) were separated, total RNA was extracted, cDNA was obtained by reverse transcription, and VHH fragments were obtained by twice nested PCR amplification using the cDNA as a template. The target band was cut and recovered.

[0043] The gel recovery product of the second round of PCR amplification was cloned into the PHEN2 phage vector, and the recombinant phage was transformed into E. coli TG1. The 2×YT medium was added for recovery, a nanobody library against the K205R protein of African swine fever virus was constructed, the library capacity and correct insertion rate were determined, and the helper phage M13K07 was used to superinfect the host bacteria TG1. After overnight culture, the supernatant was collected to obtain the phage display nanobody library against the K205R protein of African swine fever virus.

[0044] Example 2: Screening of phage display nanobody library

[0045] 1. Coat K205R protein on an enzyme-labeled plate with sodium carbonate-sodium bicarbonate buffer (0.05 mol / L, pH 9.6), and add the phage display nanobody library for screening. High-affinity and specific nanobodies are screened by reducing the antigen coating concentration and increasing the Tween 20 concentration. The screening procedure is as follows: 100 μL of 10 μg / ml K205R protein is used for overnight coating, PBST is used for washing 3 times, 5% skimmed milk powder is added to PBST solution for blocking for 1 hour, the blocking solution is discarded, PBST is used for washing once, the phage display nanobody library is added, incubation is performed at room temperature for 1 hour, PBST containing 0.1% Tween 20 is used for washing 9 times, and then trypsin digestion is performed to obtain the first round of enriched phage. The phage is further amplified for the next round of screening, and a total of three rounds of screening are performed. In the second and third rounds of screening, 5 μg / ml and 2 μg / ml of K205R protein are used to coat the enzyme-labeled plate, respectively, and PBST containing 0.2% and 0.3% Tween 20 is used for washing, respectively. After three rounds of screening, high-affinity and specific phage clones are obtained.

[0046] 2. Coat K205R protein on the enzyme-labeled plate, take the blank enzyme-labeled plate as the control; add 100 μL of the positive phage clone screened and amplified in step 1, and incubate at room temperature for 2 hours, wash 5 times with PBST, add HRP-labeled M13 antibody, and incubate at 37°C for 1 hour; wash 5 times with PBST, add TMB substrate color developing liquid, and place at room temperature in the dark for 20 min, add stop solution, and measure the absorbance. The P / N value (i.e. the OD value of the positive well / the OD value of the control well) is greater than or equal to 2.1 as positive. The positive clone liquid is subjected to sequence determination, and the nucleotide sequence of the nanobody KR-6 specific to the K205R protein is obtained.

[0047] The amino acid sequence of the nanobody KR-6 is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0048] Example 3 Construction of the expression vector of the nanobody KR-6 against the K205R protein of African swine fever virus

[0049] 1. Gene amplification and purification of the nanobody KR-6 against the K205R protein of African swine fever virus

[0050] The gene sequence of the nanobody KR-6 against the K205R protein of African swine fever virus is amplified from the recombinant phage, and the gene sequence is shown in SEQ ID NO. 2. At the same time, EcoR I and Hind III enzyme cutting sites are introduced at both ends of the gene.

[0051] The sequences of the amplification primers used are as follows:

[0052] The upstream primer F: ccgGAATTCcggATGCATGTGCAGCTGGTGGAG (shown in SEQ ID NO. 3);

[0053] The downstream primer R: cccAAGCTTgggTGAGGAGACGGTGACCTGGGT (shown in SEQ ID NO. 4).

[0054] The amplification system is as follows: Q5 Hot start High-Fidelity 2xMaster Mix, 25 μL; upstream primer F, 2.5 μL; downstream primer R, 2.5 μL; template DNA, 2 μL; ddH2O, 18 μL.

[0055] The PCR amplified product was added to a nucleic acid gel well, 120V, 30min, and the target band was cut off under ultraviolet light and transferred to a 2mL centrifuge tube, 6 times the volume of ADB Buffer was added, the centrifuge tube was placed in a 55℃ metal bath for 15min until it melted, and vortexed to mix; the mixture was transferred to a DNA adsorption column, centrifuged at 1000rpm for 1min at room temperature, and the waste liquid was discarded; 600μL of DNA Wash Buffer was added to the DNA adsorption column, centrifuged at 1000rpm for 1min at room temperature, and the waste liquid was discarded; the previous step was repeated, centrifuged at 12000rpm for 2min at room temperature, and the DNA adsorption column was dried; the DNA adsorption column was fitted into a new 1.5mL centrifuge tube, 25μL of Elution Buffer was added to the center of the binding membrane of the DNA adsorption column, and it was placed at room temperature for 2min, centrifuged at 12000rpm for 1min; finally, the eluent in the centrifuge tube was re-added to the center of the binding membrane of the DNA adsorption column, and it was placed at room temperature for 2min, centrifuged at 12000rpm for 2min, and the purified target gene fragment was obtained.

[0056] 2. Construction of recombinant expression vector

[0057] The target gene recovered from the gel in 1 above and the pET-32a vector were double-digested with EcoR I and Hind III, respectively, using pET-32a as the vector. The digested products were purified, and then the target gene and the vector were mixed at a molar ratio of 3:1, connected overnight at 16℃, and the ligation product was transformed into Rosetta-gami2(DE3) competent cells. The cells were cultured in LB medium without resistance at 37℃ and 220rpm for 1h, centrifuged at 3500rpm, the supernatant was discarded, and 100μL of the culture medium was used to resuspend the bacterial liquid, which was evenly spread on an LB culture dish containing ampicillin resistance and incubated at 37℃ for 12-16h. The next day, 9 single colonies were selected for expansion culture, and then bacterial liquid PCR identification was performed.

[0058] The identification results are shown in Table 1. Figure 1 As shown in Table 1, three colony bands met the expected size, indicating that the recombinant vector pET-32a-KR-6 was successfully constructed. The bacterial liquid with the correct size of the target band was selected for sequencing, and the positive clone with correct sequencing was expanded and cultured, and stored at -80℃ with the addition of glycerol at a final concentration of 20%.

[0059] Example 4 Expression and purification of African swine fever virus K205R protein nanobody KR-6

[0060] 1. Expression of African swine fever virus K205R protein nanobody KR-6

[0061] The correct sequencing bacteria liquid was reactivated by shaking, inoculated into 10 mL of TB liquid medium containing ampicillin at a ratio of 1:100, and cultured at 37°C until the OD 600 When the OD value was 0.6-0.8, IPTG (isopropyl thiogalactoside) was added to the medium at a final concentration of 0.2 mM, and the expression was induced at 37°C for 8 h before SDS-PAGE gel electrophoresis analysis.

[0062] The electrophoresis results are shown in Figure 2 The target band size is about 15 KDa, which is consistent with the estimated size, proving that the nanobody can be successfully expressed.

[0063] 2. Nanobody KR-6 induction expression and solubility analysis

[0064] According to the procedure described in 1, the culture was incubated at 37°C until the OD 600 When the OD value was 0.6-0.8, IPTG was added, and the expression was induced at 16°C for 8 h. The bacterial liquid was centrifuged to discard the supernatant, the bacterial body was resuspended in 1 mL of PBS, and was placed on ice for ultrasonic disruption. After centrifugation, the supernatant and precipitate were separated, the precipitate was resuspended with 100 μL of PBS, and then SDS-PAGE gel electrophoresis was performed.

[0065] The electrophoresis results are shown in Figure 3 Under the induction condition of 16°C, the nanobody KR-6 was expressed in the form of inclusion bodies.

[0066] 3. Purification of nanobody KR-6

[0067] After collecting the inclusion body precipitate after ultrasonic crushing, the precipitate was washed three times with 1xPBS, and the inclusion body precipitate was collected by centrifugation at 12000 rpm at 4°C for 10 min; the inclusion body precipitate was resuspended with Binding buffer (20 mM Tris-HCl PH 7.9, 5 mM imidazole, 0.5 M NaCl, 8 M urea), and ice bathed for 1 h to try to fully dissolve the inclusion body; equilibrate the column: add nickel column filler to the chromatography column, let ethanol slowly flow out under the action of gravity, then add 5 times the column volume of deionized water to the column to rinse the ethanol, and then equilibrate the column with 8 times the column volume of Binding buffer; centrifuge the dissolved inclusion body at 12000 rpm at 4°C for 10 min to collect the supernatant, and bind at 4°C overnight; open the column valve to collect the flow-through liquid, add 4 times the column volume of Binding buffer to wash the impurities, mix by blowing, then open the column valve, and collect the liquid flowing out, which is repeated three times, add 3 times the column volume of Binding buffer to wash the impurities, mix by blowing, then open the column valve, and collect the liquid flowing out, which is repeated three times; add 1 mL of Elution Buffer (20 mM Tris-HCl PH 7.9, 500 mM imidazole, 0.5 M NaCl, 8 M urea) to elute the target protein, mix by blowing, then open the column valve, and collect the liquid flowing out; finally, perform SDS-PAGE gel electrophoresis to analyze the protein purification.

[0068] The results, as shown in Figure 4 Figure 2, successfully purified the nanobody KR-6.

[0069] Example 5 Identification of the reactivity of nanobody KR-6 with African swine fever virus K205R recombinant protein

[0070] The laboratory-purified K205R recombinant protein was added to the enzyme-labeled plate at 0.05 μg / well, and coated overnight at 4°C; the enzyme-labeled plate was washed with PBST solution, 300 μL / well each time, a total of three times, and then patted dry; 1% BSA blocking solution was added to the enzyme-labeled plate at 200 μL / well, and blocked at 37°C for 1 h; the enzyme-labeled plate was washed with PBST solution, 300 μL / well each time, and washed once, then patted dry; the purified nanobody KR-6 against African swine fever virus K205R protein was added to the enzyme-labeled plate at a concentration of 10 μg / mL, 100 μL / well, and incubated at 37°C for 1 h; the plate washing steps were the same as above; the MonoRab TMRabbit Anti-Camelid VHH Antibody [HRP] 1 : 5000 dilution, 100 μL / well added to the enzyme-labeled plate, 37°C incubation for 1 h; plate washing steps are the same as above; 100 μL / well of TMB substrate solution was added, 37°C color development for 10 min; 100 μL / well of 0.3 mol / L H2SO4 solution was added to terminate the reaction; OD was measured using a microplate reader 450 Values.

[0071] Results, as shown in Figure 5 Figure 2, the nanobody KR-6 has good reactivity with the African swine fever virus K205R recombinant protein.

[0072] Example 6 Identification of the reactivity of nanobody KR-6 with the natural antigen of African swine fever virus

[0073] 1. ELISA identification of the reactivity of nanobody KR-6 with the natural antigen of African swine fever virus

[0074] The natural antigen of African swine fever virus was diluted 1:2 with phosphate buffer, 100 μL / well added to the enzyme-labeled plate, and coated at 4°C overnight; the enzyme-labeled plate was washed with PBST solution, 300 μL / well each time, a total of three times, and then patted dry; 1% BSA blocking solution, 200 μL / well, was added to the enzyme-labeled plate, and blocked at 37°C for 1 h; the enzyme-labeled plate was washed with PBST solution, 300 μL / well each time, and washed once, and then patted dry; the nanobody KR-6 was diluted at a concentration of 10 μg / mL, 100 μL / well added to the enzyme-labeled plate, and incubated at 37°C for 1 h; the plate washing steps were the same as above; MonoRab TM Rabbit Anti-Camelid VHH Antibody [HRP] 1 : 5000 dilution, 100 μL / well added to the enzyme-labeled plate, 37°C incubation for 1 h; plate washing steps are the same as above; 100 μL / well of TMB substrate solution was added, 37°C color development for 10 min; 100 μL / well of 0.3 mol / L H2SO4 solution was added to terminate the reaction; OD was measured using a microplate reader 450 Values.

[0075] Results, as shown in Figure 6 Figure 2, the nanobody KR-6 has good reactivity with the natural antigen of African swine fever virus.

[0076] 2. Western blot identification of the reactivity of nanobody KR-6 with the natural antigen of African swine fever virus

[0077] Experimental process:

[0078] (1) Sample preparation: Take 20 μL of African swine fever virus antigen and add 20 μL of 2x Loading Buffer. Boil in a metal bath at 100°C for 15 min to denature the protein.

[0079] (2) Protein electrophoresis: After denaturation, add the protein to the protein gel well. Perform electrophoresis at a constant voltage of 80V for 30 min, and then adjust the voltage to 120V for electrophoresis for 50 min.

[0080] (3) Membrane transfer: Cut a piece of PVDF membrane of equal size and soak in methanol for 1 min. Soak filter paper of equal size in the membrane transfer solution. Place the filter paper, membrane, gel and filter paper in the order of filter paper→membrane→gel→filter paper. Remove the bubbles, cover the lid, connect the power supply, and transfer the membrane at a constant voltage of 100V for 60 min.

[0081] (4) Blocking: After membrane transfer, remove the PVDF membrane and place it in 5% skim milk at room temperature for 1 h.

[0082] (5) Incubation of the first antibody: Wash the membrane with PBST for 5 times, each time adding 10 mL, and shaking for 5 min. Wash away the blocking solution and incubate the PVDF membrane with 1:100 diluted nanobody KR-6. Place it in a shaking incubator at 4°C overnight.

[0083] (6) Incubation of the second antibody: Wash the membrane as in step (5) and incubate the PVDF membrane with 1:1000 diluted MonoRab TM Rabbit Anti-CamelidVHH Antibody[HRP] antibody. Incubate at room temperature for 2 h.

[0084] (7) Color development: Wash the membrane as in step (5). Add 1 mL of HRP chemiluminescent substrate A and B to the dark box, respectively, and mix thoroughly for 30 s. Place the PVDF membrane in the dark box and scan it on the film scanner.

[0085] The results are shown in Figure 7 The nanobody KR-6 can recognize the African swine fever virus K205R antigen, and there is a single obvious band at the expected band, indicating that the nanobody KR-6 has good reactivity.

[0086] The embodiments described in the present application are only the results of the implementation of the present application and are not intended to limit the further development of the present application. Any modifications, equivalent replacements, improvements, etc. based on the technology, spirit, principles of the present application and any modifications, equivalent replacements and improvements, etc. developed based on the present application shall be included in the protection scope of the present application.

Claims

1. A nanobody KR-6 against African swine fever virus K205R protein, the amino acid sequence of the nanobody KR-6 is shown in SEQ ID NO.

1.

2. A nucleic acid encoding the Nanobody KR-6 according to claim 1.

3. The nucleic acid according to claim 2, wherein The nucleic acid sequence is shown in SQE ID NO.

2.

4. An expression vector, characterized in that The expression vector contains the nucleic acid sequence according to claim 3.

5. Use of the nanobody KR-6 according to claim 1 in the preparation of an African swine fever virus detection reagent or kit.

6. An African swine fever virus detection reagent or kit, characterized in that: The reagent or kit comprises the Nanobody KR-6 according to claim 1.

7. A composition, characterized in that The composition comprises the Nanobody KR-6 according to claim 1.

8. The method for preparing the Nanobody KR-6 according to claim 1, wherein The method comprises: (1) Amplifying the nucleotide sequence encoding the Nanobody KR-6 according to claim 1; (2) connecting the nucleotide sequence described in step (1) to a vector / plasmid to construct an expression vector for nanobody KR-6; (3) The expression vector of nanobody KR-6 was transformed into bacteria to construct recombinant cells, which were induced to express and purified to obtain nanobody KR-6.

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