Nano antibody for resisting African swine fever virus K205R protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nano antibody for resisting African swine fever virus K205R protein and application. The invention provides a nano-antibody KR-6 for resisting African swine fever virus K205R protein, and the nano-antibody KR-6 is obtained by constructing and screening a phage display nano-antibody library, is a heavy-chain antibody variable region fragment, and has an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a preparation method of the nano-antibody KR-6 for resisting the African swine fever virus K205R protein. The nano antibody has high affinity with the African swine fever virus K205R protein and the African swine fever virus, can specifically recognize the African swine fever virus, and can be used for diagnosing, preventing or treating the African swine fever.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a nano antibody against African swine fever virus K205R protein and an application thereof. Background Art

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by infection with the African swine fever virus (ASFV). The World Organization for Animal Health lists African swine fever as a notifiable animal disease, and my country lists it as a Class I animal disease. The incubation period of African swine fever is generally 4 to 19 days. 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 through direct contact and can also be transmitted through vectors. Soft ticks are its storage host and transmission vector. The virus is also the only known DNA virus that can be transmitted by biological vectors, soft ticks.

[0003] African swine fever virus is a large nucleocytoplasmic DNA virus that can be divided into 24 genotypes and is prone to mutation. Due to different genotypes, the genome length ranges from 171,235bp to 193,886bp, containing 151 to 167 open reading frames (ORFs). In 2018, the first outbreak of African swine fever occurred in Shenyang, Liaoning Province, my country. The African swine fever virus strain was identified as genotype II by sequencing. Since then, African swine fever has spread rapidly, and my country has gradually monitored natural variants of genotype II and gene-deficient strains. In 2021, a weak genotype I strain was reported in China for the first time. Since then, African swine fever virus genotype I and genotype II strains have co-circulated in the fields of my country; subsequently, recombinant strains of genotype I and genotype II were reported. At present, the complexity of the prevalent strains has brought new challenges to the prevention and control of African swine fever in my country. The detection of African swine fever virus-specific nucleic acid has played a very important role in my country's early prevention and control; however, due to the emergence of weak strains, the detoxification time is delayed and the detoxification amount is relatively low, and the detection of African swine fever virus nucleic acid may sometimes fail to detect it; and the detection of African swine fever virus-specific antibodies is very important for the screening of weak virulence strain infections and resistant pigs.

[0004] The variable region of the heavy chain of HCAb in camelid serum is a monomeric domain, namely a single-domain antibody (VHH), also known as a nanobody (Nb), with a diameter of 2.5nm and a length of 4nm. The molecular weight of Nb is about 15kDa, which is the smallest antibody that can bind to the target antigen at present. Nb has the advantages of small size, small molecular weight, easy modification, strong penetrability, low immunogenicity, and fast metabolism. Compared with traditional large molecular weight Ig antibodies (about 150kDa), Nb is easier to modify; it is easier to recognize and bind to antigenic epitopes in hidden places such as tortuous depressions. Nb has high water solubility and strong affinity. Due to the absence of L chain, VH cannot bind to VL through interaction, exposing the hydrophilic amino acid residues of Nb, increasing its solubility in water, and enabling it to bind to water-soluble haptens. In addition, Nb has longer CDR1 and CDR3 amino acid sequences, and its affinity with antigens is higher than that of traditional polyclonal antibodies and monoclonal antibodies. Nb can be expressed at high levels in prokaryotic expression systems, mammalian cell expression systems, and plant cell expression systems. Due to its small molecular weight and simple structure, its production and purification process is simpler and can be prepared in batches. The unique molecular properties of nanobodies make them show good application prospects in the diagnosis and treatment of various diseases and detection. Summary of the invention

[0005] In view of the above technical problems, the present invention provides an anti-African swine fever virus K205R protein nanobody KR-6 and its preparation method and application, wherein the nanobody has a high affinity to both the African swine fever virus K205R protein and the African swine fever virus, can specifically recognize and bind to the African swine fever virus, and can be used for the diagnosis, prevention or treatment of African swine fever. Specifically, it includes the following contents:

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

[0007] In a second aspect, the present invention provides a nucleic acid encoding the Nanobody KR-6 described in the first aspect above.

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

[0009] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid sequence described in the second aspect.

[0010] In a fourth aspect, the present invention provides an immunoconjugate, comprising:

[0011] (i) Nanobody KR-6 as described in the first aspect above;

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

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

[0014] In a sixth aspect, the present invention provides an African swine fever virus detection reagent or kit, wherein the reagent or kit comprises the nanoantibody KR-6 described in the first aspect above.

[0015] In a seventh aspect, the present invention provides a composition comprising the Nanobody KR-6 described in the first aspect above.

[0016] In an eighth aspect, the present invention provides a method for preparing the Nanobody KR-6 described in the first aspect above, the method comprising:

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

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

[0019] (3) The expression vector of nanobody KR-6 is transformed into bacteria to construct recombinant cells, which are then induced to express and purified to obtain nanobody KR-6.

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

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

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

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

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

[0025] Figure 1 PCR identification results of recombinant bacterial solution; where M is the molecular weight standard, and 1-9 are recombinant bacteria;

[0026] Figure 2 SDS-PAGE gel electrophoresis results of the induced expression of nanoantibody KR-6; where M is the molecular weight standard, 1 is before induction, and 2 is after induction of the recombinant bacterial solution;

[0027] Figure 3 The results of the induced expression and solubility analysis of nanoantibody KR-6; where M is the molecular weight standard, 1 is before induction, 2 is after induction, 3 is the ultrasonic supernatant, and 4 is the ultrasonic precipitation;

[0028] Figure 4 The purification results of nanobody KR-6; wherein M is the molecular weight standard, 1 is before induction, 2 is before ultrasound, 3 is ultrasound precipitation, 4 is flow-through, 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 identified the reactivity of nanoantibody KR-6 with African swine fever virus K205R recombinant protein;

[0030] Figure 6 ELISA identified the reactivity of nanoantibody KR-6 with African swine fever virus;

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

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 the present application unexpectedly discovered for the first time a nanoantibody KR-6 that targets the K205R protein of the African swine fever virus. The experimental results show that the nanoantibody of the present invention can specifically recognize and bind to the 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 the African swine fever virus K205R protein immune nanoantibody library (camel heavy chain antibody phage display library), thereby obtaining the nanoantibody KR-6 gene that can recognize the African swine fever virus K205R protein. This gene is then transferred to Escherichia coli cells and expressed to obtain the nanoantibody. Then, the ELISA method and other methods are used to identify that the nanoantibody can 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 virus sources described in the following examples are as follows: 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-Fidelity2×Master Mix, restriction endonucleases EcoRⅠ, Hind III and T4 DNA ligase were purchased from NEB; gel recovery kit and plasmid extraction kit were purchased from OMEGA; IPTG was purchased and synthesized from Sangon Biotech (Shanghai) Co., Ltd.; Rosetta-gami2 (DE3) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; 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 display nanoantibody library

[0039] The African swine fever virus K205R recombinant protein was used to immunize Bactrian camels for five times. Venous blood was collected before the first immunization and after each immunization. The serum antibody titer was tested by indirect ELISA. Venous anticoagulated blood was collected after the antibody titer reached the expected immune effect. The specific animal immunization program and antigen vaccination dose are shown in Table 1.

[0040] Table 1 Immunization schedule and antigen dosage of Bactrian camels

[0041]

[0042] After the antibody titer reaches the required level, 100 ml of camel anticoagulated blood is collected from the jugular vein. Peripheral blood lymphocytes (PBMCs) are separated, total RNA is extracted, and reverse transcription is performed to obtain cDNA, which is used as a template for two nested PCR amplifications to obtain VHH fragments, and the target bands are cut and recovered.

[0043] The gel-recovered products of the second round of PCR amplification were cloned into the PHEN2 phage vector, and the recombinant phage was transformed into Escherichia coli TG1 and revived by adding 2×YT medium. A nanoantibody library targeting the K205R protein of African swine fever virus was constructed, and the library capacity and correct insertion rate were determined. The helper phage M13K07 was used to superinfect the host bacteria TG1, and the supernatant was collected after overnight culture to obtain a phage-displayed nanoantibody library targeting the K205R protein of African swine fever virus.

[0044] Example 2 Screening of phage-displayed nanoantibody library

[0045] 1. K205R protein was coated on an ELISA plate with sodium carbonate-sodium bicarbonate buffer (0.05 mol / L, pH 9.6), and a phage display nanoantibody library was added for screening. Nanoantibodies with high affinity and good specificity were screened by reducing the antigen coating concentration and increasing the concentration of Tween 20. The screening procedure was as follows: 100 μL of 10 μg / ml K205R protein was coated overnight, washed 3 times with PBST, blocked for 1 hour with a PBST solution containing 5% skim milk powder, the blocking solution was discarded, washed once with PBST, added with a phage display nanoantibody library, incubated at room temperature for 1 hour, washed 9 times with PBST containing 0.1% Tween 20, and then digested with trypsin to obtain the phages of the first round of enrichment screening. The phages were further amplified and the next round of screening was performed, and a total of three rounds of screening were performed. In the second and third rounds of screening, 5 μg / ml and 2 μg / ml of K205R protein were used to coat the ELISA plate, respectively, and washed with PBST containing 0.2% and 0.3% Tween 20. After three rounds of screening, phage clones with high affinity and strong specificity were obtained.

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

[0047] Among them, 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 African swine fever virus K205R protein nanobody KR-6

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

[0050] The gene sequence of the nanoantibody KR-6 against the K205R protein of African swine fever virus was 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 restriction sites were introduced at both ends of the gene.

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

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

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

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

[0055] The product obtained by PCR amplification was added to the nucleic acid gel well, 120V, 30min, the target band was cut off in the UV instrument, 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 mixed solution was transferred to the DNA adsorption column, centrifuged at 1000rpm at room temperature for 1min, and the waste liquid was discarded; 600μL of DNA Wash Buffer was added to the DNA adsorption column, centrifuged at 1000rpm at room temperature for 1min, and the waste liquid was discarded; the previous step was repeated, centrifuged at 12000rpm at room temperature for 2min, and the DNA adsorption column was dried; the DNA adsorption column was put 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, left at room temperature for 2min, and centrifuged at 12000rpm for 1min; finally, the eluate in the centrifuge tube was added back to the center of the binding membrane of the DNA adsorption column, left at room temperature for 2min, and centrifuged at 12000rpm for 2min to obtain the purified target gene fragment.

[0056] 2. Construction of recombinant expression vector

[0057] Using pET-32a as the vector, the target gene and pET-32a vector recovered from the gel in 1 were double-digested with EcoR I and HindⅢ, respectively. The digestion product was purified, and then the target gene and the vector were mixed at a molar ratio of 3:1, connected at 16°C overnight, and the connection product was transformed into Rosetta-gami2 (DE3) competent cells, cultured at 37°C and 220rpm for 1h in LB medium without resistance, centrifuged at 3500rpm, discarded the upper culture medium, and left 100μL of culture medium to resuspend the bacterial solution, and evenly coated on the LB culture dish containing ampicillin resistance, and cultured at 37°C for 12-16h. The next day, 9 single colonies were picked for expansion culture, and then PCR identification of the bacterial solution was performed.

[0058] The identification results are as follows Figure 1 As shown, there are 3 colony bands that meet the expected size, indicating that the recombinant vector pET-32a-KR-6 was successfully constructed. The bacterial solution with the correct target band size was selected for sequencing, and the positive clones with the correct sequencing were expanded and cultured, and glycerol with a final concentration of 20% was added and stored at -80°C.

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

[0060] 1. Expression of Nanobody KR-6 against African Swine Fever Virus K205R Protein

[0061] The sequenced bacterial suspension was re-activated by shaking, and inoculated into 10 mL of TB liquid medium containing ampicillin at a ratio of 1:100, and cultured at 37°C in a shaking incubator until OD 600 When the value was 0.6-0.8, IPTG (isopropylthiogalactoside) was added to the culture 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 as follows Figure 2 As shown, the target band size is around 15 KDa, which is consistent with the estimated size, proving that the nanobody can be successfully expressed.

[0063] 2. Inducible expression and solubility analysis of nanobody KR-6

[0064] Follow the steps in 1 and culture at 37°C in a shaking incubator until OD 600 When the value was 0.6-0.8, IPTG was added and the expression was induced at 16°C for 8 h. The bacterial solution was centrifuged and the supernatant was discarded. The bacteria were resuspended in 1 mL of PBS and placed on ice for ultrasonic disruption. The supernatant and precipitate were separated by centrifugation. The sediment was resuspended in 100 μL of PBS and then subjected to SDS-PAGE gel electrophoresis.

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

[0066] 3. Purification of Nanobody KR-6

[0067] Collect the inclusion body precipitate after ultrasonic disruption, wash the precipitate three times with 1×PBS, and then centrifuge at 12000rpm at 4℃ for 10min to collect the inclusion body precipitate; resuspend the inclusion body precipitate with Binding buffer (20mM Tris-HCl PH7.9, 5mM imidazole, 0.5M NaCl, 8M urea), ice bath for 1h, and try to fully dissolve the inclusion body; balance the column: add the nickel column filler to the chromatography column, let the ethanol flow out slowly under the action of gravity, then add 5 column volumes of deionized water to the column to rinse the ethanol, and then balance the column with 8 column volumes of Binding buffer; centrifuge the dissolved inclusion bodies at 12000rpm at 4℃ for 10min to collect the supernatant, and combine at 4℃ overnight; open the column valve to collect the flow-through liquid, add 4 column volumes of Binding buffer to wash the impurities, blow and mix well, open the column valve, collect the outflowing liquid, repeat this step three times, add 3 column volumes of Binding The impurities were washed with buffer, and the column valve was opened after mixing by pipetting, and the liquid flowing out was collected. This step was repeated three times. 1 mL of Elution Buffer (20 mM Tris-HCl PH7.9, 500 mM imidazole, 0.5 M NaCl, 8 M urea) was added to elute the target protein, and the column valve was opened after mixing by pipetting, and the liquid flowing out was collected. Finally, SDS-PAGE gel electrophoresis was performed to analyze the protein purification.

[0068] The results are as follows Figure 4 As shown, nanobody KR-6 was successfully purified.

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

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

[0071] The results are as follows Figure 5 As shown, nanobody KR-6 has good reactivity with African swine fever virus K205R recombinant protein.

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

[0073] 1. ELISA to identify the reactivity of nanoantibody KR-6 with natural antigens of African swine fever virus

[0074] The natural antigen of African swine fever virus was diluted with phosphate buffer at a ratio of 1:2, and 100 μL / well was added to the ELISA plate for overnight coating at 4°C; the ELISA plate was washed with PBST solution, 300 μL / well each time, washed three times in total, and patted dry; 1% BSA blocking solution was added to the ELISA plate at 200 μL / well, and blocked at 37°C for 1h; the ELISA plate was washed with PBST solution, 300 μL / well each time, washed once, and patted dry; the nano antibody KR-6 was diluted at a concentration of 10 μg / mL, 100 μL / well was added to the ELISA plate, and incubated at 37°C for 1h; the plate washing steps were the same as above; MonoRab TM Rabbit Anti-Camelid VHH Antibody [HRP] 1: 5000-fold dilution, 100 μL / well added to the ELISA plate, incubated at 37°C for 1 hour; washing steps are the same as above; 100 μL / well added TMB substrate solution, 37°C color development for 10 minutes; 100 μL / well added 0.3 mol / L H2SO4 solution to terminate the reaction; use an ELISA reader to measure OD 450 value.

[0075] The results are as follows Figure 6 As shown, nanoantibody KR-6 has good reactivity with the natural antigen of African swine fever virus.

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

[0077] Experimental process:

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

[0079] (2) Protein electrophoresis: Add the denatured protein into the wells of the protein gel and run the electrophoresis at a constant voltage of 80 V for 30 min, then adjust the voltage to 120 V for 50 min;

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

[0081] (4) Blocking: After the transfer is completed, take out the PVDF membrane, place it in 5% skim milk, and block it at room temperature for 1 h;

[0082] (5) Incubation with primary antibody: Wash the membrane with PBST five times, adding 10 mL each time, and wash on a shaker for 5 min. Wash off the blocking solution, and incubate the PVDF membrane with 1:100 diluted nanoantibody KR-6, and incubate overnight on a shaker at 4 degrees;

[0083] (6) Incubate with secondary antibody: Wash the membrane in the same way as (5) with 1:1000 dilution of MonoRab TM Rabbit Anti-CamelidVHH Antibody [HRP] antibody was incubated on the PVDF membrane and placed on a shaker at room temperature for 2 hours;

[0084] (7) Color development: The membrane washing steps are the same as (5). 1 mL of HRP chemiluminescent substrate solution A and solution B are added into the dark box respectively. The mixture is allowed to fully combine for 30 s. The PVDF membrane is placed into the dark box and scanned on a membrane scanner.

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

[0086] What is described in this application is only the embodiment result of the present invention and is not intended to limit the in-depth development of the present invention. All the techniques, spirits, principles of the present invention and any modifications, equivalent substitutions and improvements based on the development of the present invention should be included in the protection scope of the present invention.

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, It is characterized in that The nucleic acid sequence is shown in SQE ID NO.

2.

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

5. An immunoconjugate, It is characterized in that The immunoconjugate comprises: (i) Nanobody KR-6 as described in claim 1; (ii) and a conjugated moiety selected from the group consisting of a detectable label, a drug, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

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

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

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

9. The method for preparing the Nanobody KR-6 according to claim 1, It is characterized in that 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 is transformed into bacteria to construct recombinant cells, which are then induced to express and purified to obtain nanobody KR-6.

10. A primer for amplifying the nucleotide sequence encoding the Nanobody KR-6 according to claim 1, It is characterized in that The primers are: Upstream primer F: ccgGAATTCcggATGCATGTGCAGCTGGTGGAG; Downstream primer R: cccAAGCTTgggTGAGGAGACGGTGACCTGGGT.

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