A nanobody targeting K205R protein and its application in the detection of African swine fever virus antibodies

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a nano antibody against K205R protein and its application in the detection of African swine fever virus antibodies. The present invention provides a nano antibody KR-1 against the African swine fever virus K205R protein, wherein the nano antibody KR-1 is obtained by the 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 of the present invention has good reactivity and affinity with both the African swine fever virus K205R recombinant protein and the natural antigen, and has broad application prospects in the field of African swine fever virus antibody detection and analysis.

Description

Technical Field

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

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease. The disease is caused by African swine fever virus (ASFV) infecting pigs. The mortality rate caused by infection with highly virulent strains can be as high as 100%, which seriously threatens the world's pig farming industry. The World Organization for Animal Health lists African swine fever as a reportable animal disease, while my country lists it as a Class I animal disease. African swine fever virus belongs to the genus African swine fever virus of the family Asfrinidae. It is a large double-stranded DNA virus with a total length of 170-190kb and encodes more than 200 proteins, including a large number of structural proteins, non-structural proteins, and a series of enzymes related to DNA replication and repair. In 2018, the disease was introduced into my country and spread rapidly, causing huge economic losses to my country's pig farming industry. At present, there is no effective treatment measure or commercial vaccine for the disease. Early, rapid, and accurate diagnosis and effective biosafety measures are the main means of preventing and controlling the disease.

[0003] Nanobodies, the smallest unit known to bind to antigens, are heavy chain antibody variable region fragments that are naturally missing light chains and heavy chain constant region 1 (CH1) from animals such as camelids and sharks. As the smallest antibody fragment that binds to antigens and exerts diagnostic and therapeutic effects, nanobodies retain high antigen affinity and specificity, and have the advantages of low molecular weight, easy preparation, low immunogenicity, and strong tissue penetration. In addition, nanobodies have good solubility and high stability, and are particularly suitable for chemical coupling, and are considered to be the next generation of antibody derivative tools. Currently, nanobodies have been widely used in disease diagnosis, treatment, bioimaging, and basic research.

[0004] The African swine fever virus K205R protein is a non-structural protein encoded by the K205R gene. It is expressed in the early stage of virus infection, starting from 4 hours after virus infection, and diffuses into the cytoplasm 6 hours after infection. The protein has strong immunogenicity and is an ideal target antigen for the detection of African swine fever virus antibodies. The present invention uses the African swine fever virus K205R recombinant protein to immunize healthy Bactrian camels, construct a phage display nano antibody library against the K205R protein, and use phage display technology to screen and obtain nano antibodies specific to the K205R protein, providing an important basis for the development of African swine fever detection technology and the development of therapeutic antibodies. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a nano antibody against K205R protein and its application in the detection of African swine fever virus antibodies. The nano antibody has good reactivity and affinity with both the African swine fever virus K205R recombinant protein and the natural antigen, and has broad application prospects in the field of African swine fever virus antibody detection and analysis. Specifically, it includes the following contents:

[0006] In a first aspect, the present invention provides a nanobody KR-1 against the African swine fever virus K205R protein, and 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-1 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-1 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-1 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-1 described in the first aspect above.

[0015] In a seventh aspect, the present invention provides a composition comprising the Nanobody KR-1 described in the first aspect above.

[0016] In an eighth aspect, the present invention provides a method for preparing the Nanobody KR-1 described in the first aspect above, the method comprising:

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

[0019] (3) The expression vector of nanobody KR-1 is transformed into bacteria to construct recombinant cells, which are then induced to express and purified to obtain nanobody KR-1.

[0020] Preferably, the nucleotide sequence of the Nanobody KR-1 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-1 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: cccAAGCTTgggTGAGGAGACAGTGACCTGGGT (shown in SEQ ID NO.4).

[0024] The beneficial effects of the present invention are as follows: the present invention provides a nano antibody KR-1 against African swine fever virus K205R protein, the nano antibody KR-1 is obtained by 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 of the present invention has good reactivity and affinity with both the African swine fever virus K205R recombinant protein and the natural antigen, and has broad application prospects in the field of African swine fever virus antibody detection and analysis; and the nano antibody 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 KR-1 and pET-32a double digestion results; left: KR-1 double digestion results; right: pET-32a double digestion results, M is the molecular weight standard, 1-7 is the pET-32a vector after digestion, and 8 is the pET-32a without digestion;

[0026] Figure 2 PCR identification results of recombinant bacterial solution; where M is the molecular weight standard, 1-9 are recombinant bacteria;

[0027] Figure 3 SDS-PAGE gel electrophoresis results of nanoantibody KR-1 induced expression; M is the molecular weight standard, 1 is before induction, and 2 is after induction of the recombinant bacterial solution;

[0028] Figure 4The results of the induced expression and solubility analysis of the nanobody KR-1; 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;

[0029] Figure 5 The purification results of nanobody KR-1; 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;

[0030] Figure 6 The reactivity results of nanobody KR-1 and African swine fever virus K205R recombinant protein;

[0031] Figure 7 ELISA identified the reactivity of nanoantibody KR-1 with African swine fever virus;

[0032] Figure 8 Western blot identification of the reactivity of nanoantibody KR-1 with African swine fever virus;

[0033] Fig. 9 The activity results of nanoantibody KR-1 competing with African swine fever virus positive sera of different dilutions for binding to K205R antigen. DETAILED DESCRIPTION

[0034] 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.

[0035] 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 nanobody KR-1 targeting the African swine fever virus K205R protein. The experimental results show that the nanobody of the present invention has good reactivity and affinity with the African swine fever virus K205R recombinant protein and natural antigen, and can be used for the diagnosis, prevention or treatment of African swine fever.

[0036] 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-1 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.

[0037] Among them, the nanobody targeting the African swine fever virus K205R protein is represented by KR-1 (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2).

[0038] 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.

[0039] 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.

[0040] Example 1 Construction of phage display nanoantibody library

[0041] 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.

[0042] Table 1 Immunization schedule and antigen dosage of Bactrian camels

[0043]

[0044] 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.

[0045] 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.

[0046] Example 2 Screening of phage-displayed nanoantibody library

[0047] 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, 5μg / ml and 2μg / ml of K205R protein were used to coat the ELISA plate, respectively, and washed with PBST containing 0.2% and 0.3% Tween 20, respectively. After three rounds of screening, phage clones with high affinity and strong specificity can be obtained.

[0048] 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-1 specific for the K205R protein.

[0049] Wherein, the amino acid sequence of the nanobody KR-1 is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO.2.

[0050] Example 3 Construction of the expression vector of the African swine fever virus K205R protein nanobody KR-1

[0051] 1. Gene amplification and purification of the African swine fever virus K205R protein nanoantibody KR-1

[0052] The gene sequence of the nanoantibody KR-1 against the African swine fever virus K205R protein 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.

[0053] The sequences of the amplification primers used are:

[0054] Upstream primer F: ccgGAATTCcggATGCATGTGCAGCTGGTGGAG (shown in SEQ ID NO.3);

[0055] Downstream primer R: cccAAGCTTgggTGAGGAGACAGTGACCTGGGT (shown in SEQ ID NO.4).

[0056] 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.

[0057] 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.

[0058] 2. Construction of recombinant expression vector

[0059] Using pET-32a as the vector, the target gene and pET-32a vector recovered from the gel in step 1 were double-digested with EcoR I and Hind III, respectively. The results of the double-digestion were as follows: Figure 1 shown.

[0060] The digested 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 connected 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 medium, and left 100μL of medium to resuspend the bacterial solution, and evenly spread 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.

[0061] The identification results are as follows Figure 2 As shown, three colony bands meet the expected size, indicating that the recombinant vector pET-32a-KR-1 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.

[0062] Example 4 Expression and purification of African swine fever virus K205R protein nanobody KR-1

[0063] 1. Expression of Nanobody KR-1 against African Swine Fever Virus K205R Protein

[0064] 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.

[0065] The electrophoresis results are as follows Figure 3 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.

[0066] 2. Inducible expression and solubility analysis of nanobody KR-1 at 16°C and 37°C

[0067] 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 and 37°C for 8 h, respectively. 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.

[0068] The electrophoresis results are as follows Figure 4As shown, under the induction conditions of 16°C and 37°C, nanobody KR-1 was expressed in the form of inclusion bodies.

[0069] 3. Purification of Nanobody KR-1

[0070] 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.

[0071] The results are as follows Figure 5 As shown, nanobody KR-1 was successfully purified.

[0072] Example 5 Identification of the reactivity of nanobody KR-1 with African swine fever virus K205R recombinant protein

[0073] 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-1 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.

[0074] The results are as follows Figure 6 As shown, nanobody KR-1 has good reactivity with African swine fever virus K205R recombinant protein.

[0075] Example 6 Identification of the reactivity of nanobody KR-1 with native antigens of African swine fever virus

[0076] 1. ELISA to identify the reactivity of nanoantibody KR-1 with natural antigens of African swine fever virus

[0077] 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-1 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.

[0078] The results are as follows Figure 7 As shown, nanoantibody KR-1 has good reactivity with the natural antigen of African swine fever virus.

[0079] 2. Western blot identification of the reactivity of nanoantibody KR-1 with natural antigens of African swine fever virus

[0080] Experimental process:

[0081] (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;

[0082] (2) Protein electrophoresis: Add the denatured protein to 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;

[0083] (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.

[0084] (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;

[0085] (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 away the blocking solution, and incubate the PVDF membrane with 1:100 diluted nanoantibody KR-1, and incubate overnight on a shaker at 4 degrees;

[0086] (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;

[0087] (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. Let them fully combine for 30 s. Place the PVDF membrane into the dark box and scan it on a membrane scanner.

[0088] The results are as follows Figure 8 As shown, nanobody KR-1 can recognize the African swine fever virus K205R antigen, with a single obvious band at the expected band, indicating that nanobody KR-1 has good reactivity.

[0089] Example 7 Detection of African swine fever virus antibodies in pig serum samples based on nanobody KR-1

[0090] The K205R protein was added to the ELISA plate at a concentration of 0.05 μg / mL, 100 μL / well, and coated at 4°C overnight; 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; African swine fever virus positive serum was diluted according to the ratio of 1:5, 1:20, 1:40, 1:80, 1:160, 1:320, 100 μL / well was added to the ELISA plate, a negative serum control was set, and incubated at 37°C for 1h; the plate washing steps were the same as above; the nano antibody KR-1 was diluted at 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.

[0091] The results are as follows Fig. 9 As shown, nanoantibody KR-1 has the ability to compete with African swine fever virus-positive sera of different dilutions for binding to K205R protein and can be used to detect African swine fever virus antibodies in pig serum samples.

[0092] 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-1 against African swine fever virus K205R protein, the amino acid sequence of the nanobody KR-1 is shown in SEQ ID NO.

1.

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

3. The nucleic acid according to claim 2, characterized in that The nucleic acid sequence is shown as SEQ 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-1 as claimed in 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-1 according to claim 1.

7. The method for preparing the Nanobody KR-1 according to claim 1, characterized in that: The method comprises: (1) amplifying the nucleotide sequence encoding the Nanobody KR-1 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-1 is transformed into bacteria to construct recombinant cells, which are then induced to express and purified to obtain nanobody KR-1.

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

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