Single-domain antibody against african swine fever virus e165r protein and screening method and application thereof

Abstract

The application discloses a single-domain antibody against African swine fever virus E165R protein and a screening method and application thereof, relates to the technical field of bioengineering, and provides the single-domain antibody against African swine fever virus E165R protein and a specific amino acid sequence, a gene coding the single-domain antibody, an expression vector comprising the gene and a bacteriophage expressing the gene, and applies the single-domain antibody against African swine fever virus E165R protein to immunological detection of African swine fever virus. The single-domain antibody provided by the application has strong specificity, high sensitivity and strong neutralization activity, and can be used in daily immunological detection of epidemic diseases and future system construction for prevention and control of epidemic diseases.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a single-domain antibody against the E165R protein of African swine fever virus, its screening method, and its application. Background Technology

[0002] African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars, causing enormous economic losses to the global livestock industry. Currently, there is no effective vaccine for ASF worldwide, and control measures primarily focus on preventing its introduction and timely culling. Given the difficulties in vaccine development (long development time, technical challenges, an incomplete vaccine evaluation system, high investment, and the need for specialized personnel) and the severe ASF epidemic, immunological methods based on antigen-antibody interactions and the advantages of single-domain antibodies can contribute to disease control. The core of this method lies in preparing single-domain antibodies with high specificity, stability, and strong neutralizing activity. Most reported antibodies are traditional, with few reports on single-domain antibodies; therefore, developing single-domain antibodies against ASF is of great significance for the detection of the ASF virus and the control of the disease. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a single-domain antibody against the African swine fever virus E165R protein, along with its screening method and application. The provided single-domain antibody exhibits strong specificity, high sensitivity, and strong neutralizing activity, and can be used for routine immunological detection of diseases and the construction of future disease prevention and control systems.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a single-domain antibody against African swine fever virus E165R protein is provided, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The present invention provides a gene encoding a single-domain antibody against the above-mentioned African swine fever virus E165R protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The present invention also provides an expression vector comprising the gene encoding the single-domain antibody against the African swine fever virus E165R protein described above.

[0007] The present invention also provides a bacteriophage that expresses the gene encoding the single-domain antibody against the African swine fever virus E165R protein.

[0008] This invention also provides the application of the above-mentioned single-domain antibody against African swine fever virus E165R protein in the immunological detection of African swine fever virus.

[0009] This invention also provides the application of the above-mentioned single-domain antibody against African swine fever virus E165R protein in the preparation of immunological detection reagents for African swine fever virus.

[0010] The present invention also provides a detection kit comprising a single-domain antibody against the above-mentioned African swine fever virus E165R protein.

[0011] This invention also provides a method for screening single-domain antibodies against the E165R protein of African swine fever virus, comprising the following steps:

[0012] (1) Mice expressing heavy chain antibodies were immunized with African swine fever virus E165R protein;

[0013] (2) RNA was extracted from samples immunized with African swine fever virus E165R protein antigen, and then the heavy chain variable region gene was obtained by PCR amplification.

[0014] (3) The heavy chain variable region gene obtained in step (2) is cloned into a vector and then transformed into Escherichia coli ER2738. After being rescued by helper phage M13KO7, an anti-African swine fever virus E165R protein phage library is obtained. Then, single-domain antibodies against African swine fever virus E165R protein are obtained by screening for African swine fever virus E165R protein.

[0015] Furthermore, in step (3), the vector is the phage vector pComb3xss.

[0016] Furthermore, in step (3), the elution buffer used during screening is a pure African swine fever virus E165R protein with a concentration of 5 μg / mL.

[0017] The present invention has the following beneficial effects:

[0018] 1. This invention utilizes antibody gene engineering technology and phage display technology to construct a mouse-derived immune phage single-domain antibody library of African swine fever virus E165R protein, and screens out single-domain antibodies of African swine fever virus E165R protein from it, providing a theoretical basis and material foundation for the subsequent establishment of a rapid immunological detection method for E165R protein.

[0019] 2. This invention uses electroporation to transform the recombinant vector into Escherichia coli ER2738. At the same time, in order to ensure the high activity of competent cells, competent cells are prepared in advance before transformation and can be used for transformation immediately after preparation, so as to maximize the transformation efficiency.

[0020] 3. In the subsequent screening process, the present invention uses high concentrations of pure E165R protein for elution in order to reduce non-specific binding of single-domain antibodies and improve the efficiency and accuracy of screening. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the expression levels of antigen-specific antibodies in serum.

[0022] Figure 2 This is a schematic diagram of the amplification results;

[0023] Figure 3 This is a schematic diagram showing the library positivity rate results;

[0024] Figure 4 This is a schematic diagram of the identification results;

[0025] Figure 5 This is a schematic diagram showing the expression results of the anti-E165R single-domain antibody.

[0026] Figure 6 This is a schematic diagram showing the sensitivity and specificity analysis results of the anti-E165R single-domain antibody.

[0027] Figure 7 This is a schematic diagram showing the sensitivity and specificity detection results of a single-domain antibody.

[0028] Figure 8 This is a schematic diagram showing the results of the neutralizing activity assay for the anti-E165R single-domain antibody. Detailed Implementation

[0029] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0030] Example 1

[0031] 1. Immunization of mice that produce heavy chain antibodies

[0032] Antigen emulsification: Dilute the antigen to 2 μg / μL with sterile PBS and draw it into a syringe for later use. Take another syringe and draw an equal volume of adjuvant. Connect the two syringes together with a syringe tube and quickly push the adjuvant into the antigen. Mix well by pushing and drawing back and forth several times. After about 40 minutes, draw 10 μL and drop it onto the surface of water. If it does not spread, it means that the emulsification is complete.

[0033] Mouse immunization: The immunization procedure consisted of one primary immunization and four booster immunizations, with a 14-day interval between the two immunizations. The primary immunization was administered subcutaneously at a dose of 100 μg / 50 μL; the booster immunizations were administered intraperitoneally at a dose of 100 μg / 50 μL. Blood samples were collected from the orbital rim three days before the primary immunization and three days after each immunization, and serum was separated for subsequent ELISA experiments.

[0034] 2. Immunization effect detection

[0035] 1. Dilute the antigen to a concentration of 2 μg / mL using coating buffer, vortex to mix, and add 100 μL to each well. Coat at 37°C for 1 hour or at 4°C overnight.

[0036] 2. Shake off the coating solution, wash the plate 5 times with a plate washer, and pat it dry on paper;

[0037] 3. Add 200 μL of blocking solution to each well and block at room temperature for 1 hour or at 4°C overnight;

[0038] 4. Shake off the sealing liquid, wash the plate 5 times with a plate washer, and pat it dry on paper;

[0039] 5. Dilute the serum to an appropriate concentration using sample diluent, vortex mix, and add 100 μL to each well. At the same time, dilute the standard serum proportionally, vortex mix, and add 100 μL to each well. Perform two technical replicates for each sample, and allow it to bind at room temperature for 1 hour.

[0040] 6. Remove the sample, wash the plate 5 times with a plate washer, and pat it dry on paper;

[0041] 7. Dilute the HRP-labeled antibody to the target concentration using sample dilution buffer, vortex to mix, and add 100 μL to each well. Incubate at room temperature for 1 hour.

[0042] 8. Remove the antibodies, wash the plate 5 times with a plate washer, and pat it dry on paper;

[0043] 9. Mix equal volumes of TMB solutions A and B, add 100 μL to each well, and incubate for 20 min. Then add 100 μL of stop solution to each well and read the absorbance at 560 nm using an ELISA reader to detect the expression level of antigen-specific antibodies in serum.

[0044] The result is as follows Figure 1 As shown. Among them, Figure 1 The study showed the response trend of specific antibodies in the serum of mice immunized against African swine fever. Each color represents a different mouse; a total of 5 mice were used.

[0045] Depend on Figure 1It can be seen from the overall absorbance values ​​that the mice produced a high immune response to the E165R protein.

[0046] 3. Extracting RNA from mouse spleen

[0047] RNA was extracted from two mice with good immunization response. Following the steps outlined in the Magen Tissue Cell Total RNA Extraction Kit, the extraction was performed as follows:

[0048] 1. Place the tissue in liquid nitrogen for quick freezing, break it into small pieces as much as possible, put it into 1 mL of Trizol, add steel balls, homogenize in a tissue homogenizer at 50 Hz for 5-10 min, and after the tissue is completely broken, put it in an ice bath for 2 min.

[0049] 2. Add 200 μL of chloroform, quickly invert and mix 8-10 times, let stand for 2 min, then centrifuge at 12000 rpm and 4℃ for 10 min;

[0050] 3. Carefully aspirate approximately 500 μL of the upper aqueous phase and mix it with 250 μL of anhydrous ethanol;

[0051] 4. Place the RNA binding column into the collection tube, add the mixture from step 3, and centrifuge at 12000 rpm for 1 min;

[0052] 5. Discard the filtrate, reassemble the column into the collection tube, add 500 μL of RW1, and centrifuge at 12000 rpm for 1 min;

[0053] 6. Discard the filtrate, reassemble the column into the collection tube, mix 10 μL of DNase and 100 μL of DNase Buffer, add to the center of the column, and digest at room temperature for 30 min.

[0054] 7. Add 500 μL of RW1 and centrifuge at 12000 rpm for 1 min;

[0055] 8. Discard the filtrate, reassemble the column into the collection tube, add 500 μL of RW2 (diluted with anhydrous ethanol); centrifuge at 12000 rpm for 1 min;

[0056] 9. Repeat step 8 once;

[0057] 10. Discard the filtrate, reassemble the column into the collection tube, and run it at 12000 rpm for 2 minutes.

[0058] 11. Place the column into a new centrifuge tube, add 50 μL of DEPC water to the center of the column, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min. To improve elution efficiency, the eluted RNA can be added to the center of the column and eluted again.

[0059] 12. The concentration and purity of RNA were detected using Nano 3000, and its integrity was checked by agarose gel electrophoresis. The RNA was then stored at -80°C.

[0060] 4. Reverse transcription

[0061] 2 μg of tissue or cell RNA was used for reverse transcription experiments using the Abm G490 reverse transcription kit; the system and amplification program are shown in Tables 1 and 2.

[0062] Table 1 G490 reverse transcription system

[0063]

[0064] After mixing all samples according to the system shown in Table 1, briefly centrifuge and then perform reverse transcription according to the procedure shown in Table 2.

[0065] Table 2 G490 Reaction Procedure

[0066]

[0067] The cDNA after reverse transcription was cooled on ice and then stored at -20°C.

[0068] 5. Amplification of mouse antibody variable region library

[0069] In the phage display experiment, the variable region library was constructed using nested PCR amplification. Spleen cDNA from immunized mice was used as a template. Enzyme restriction sites and protective bases were added to the 5' ends of the second-round primers. However, to ensure library diversity, the products from the first round (12 tubes) were used as templates for amplification with the five primer pairs from the second round. After the reaction, agarose gel electrophoresis was performed to detect the amplification of the target bands, and the gel was excised and purified. The recovered products were stored at -20°C.

[0070] The procedures for the first round of amplification of variable region libraries and the procedures for the second round of amplification of variable region libraries are shown in Table 3-4, respectively:

[0071] Table 3. One-round expansion procedure for variable region libraries

[0072]

[0073] Table 4. Second-round expansion procedure for variable region libraries

[0074]

[0075] Amplification results as follows Figure 2 As shown.

[0076] 6. Amplify single-domain antibody libraries

[0077] Preparation of ER2738 competent cells:

[0078] 1. Activate the bacterial cells: Dip the pipette tip into a small amount of ER2738 bacterial solution and streak it on a culture plate containing tetracycline resistance, then place it in a 37℃ incubator for 12 hours to activate the cells;

[0079] 2. Small-scale culture: Dip the pipette tip into 5 mL of 2YT medium containing tetracycline resistance and incubate overnight at 37°C and 220 rpm;

[0080] 3. Large-scale inoculum formation: Transfer the above bacterial suspension to 500 mL of tetracycline-resistant 2YT medium at a ratio of 1:100, and activate at 37°C and 220 rpm for 3 hours until OD500 reaches zero. 600 =0.5-0.7;

[0081] 4. Transfer the bacterial culture to a pre-cooled collection bottle and let it stand on ice for 30 minutes to allow it to cool completely;

[0082] 5. Centrifuge at 4500 rpm and 4℃ for 10 min, discard the supernatant, add a small amount of pre-cooled 10% glycerol to wash the bottle wall, discard the supernatant, add 500 mL of pre-cooled 10% glycerol to resuspend the bacterial cells on ice, and incubate on ice for 10 min.

[0083] Centrifuge at 4500 rpm and 4℃ for 10 min, discard the supernatant, add 250 mL of pre-cooled 10% glycerol to resuspend the bacterial cells on ice, and incubate on ice for 10 min;

[0084] Centrifuge at 4500 rpm and 4℃ for 10 min, discard the supernatant, add 100 mL of pre-cooled 10% glycerol to resuspend the bacterial cells on ice, and incubate on ice for 10 min;

[0085] Centrifuge at 4500 rpm and 4℃ for 10 min, discard the supernatant, add 2 mL of pre-chilled 10% glycerol to resuspend the bacterial cells, and then aliquot 200 μL into each pre-chilled EP tube and store on ice.

[0086] Variable region library electrotransfer ER2738 competent state

[0087] 1. Take 3-4 μg of the variable region fragment and ligate it at a fragment to vector molar ratio of 5:1. Then, perform column purification of the ligation product to remove salt ions in the ligation product to avoid affecting electroporation.

[0088] 2. Mix 10 μL of ligation product with 200 μL of competent cells, then add the mixture to a pre-cooled electrode cup. Adjust the parameters to 2.5 kV / 125 Ω for electroporation. Immediately after the electroporation, add 1 mL of preheated SOC medium to the electrode cup, and quickly pipette three times while avoiding foaming. Aspirate this 1 mL of bacterial solution and place it in 10 mL of SOC medium. Repeat the electroporation reaction 10 times. Then, incubate approximately 20 mL of bacterial solution at 37°C and 220 rpm for 1 h.

[0089] 3. Spread the activated bacterial solution at a rate of 2 mL per plate onto 2YT solid culture plates containing tetracycline-resistant, ampicillin-resistant, and 1% glucose solutions. Simultaneously, take 20 μL of the bacterial solution and serially dilute it (10⁻⁶ dilutions). 4 times, 10 5 times, 10 6 Double 10 7 After dilution (by a factor of 10), the cells are spread onto LB agar plates containing ampicillin-resistant bacteria and incubated upside down overnight at 37°C to calculate the volume of the culture medium. The volume of the culture medium is calculated as: volume = colony count × dilution factor. Generally, a dilution ratio of 10 is required. 8 above;

[0090] 4. After overnight incubation, scrape the large culture plate with 30 mL of 2YT blank medium, add 20 mL of 50% glycerol, mix well, and then dispense into 1.5 mL centrifuge tubes. Store at -80℃. This is the primary storage.

[0091] Construction of phage libraries

[0092] 1. Add 1 mL of the primary culture medium to 50 mL of 2YT medium containing ampicillin resistance, tetracycline resistance, and 500 μL of 1M glucose. Incubate at 37°C and 220 rpm for 1-2 hours until OD500 reaches 50%. 600 =0.8-1.0;

[0093] 2. Add 1 mL of M13KO7 helper phage (titer greater than 10) at a ratio of MOI = 20. 11 (cfu / mL), incubate at 37℃ for 30 min, then incubate at 37℃ and 220 rpm for 1 h;

[0094] Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the cells in 100 mL of 2YT medium containing tetracycline resistance, ampicillin resistance and kanamycin resistance, and incubate overnight at 30°C and 220 rpm.

[0095] 4. Centrifuge at 10,000 rpm and 4°C for 20 min, then transfer the supernatant to another centrifuge tube;

[0096] 5. Add 1 / 4 volume of pre-cooled PEG 8000 / NaCl, mix by inversion, and precipitate on ice for 5-8 hours;

[0097] 6. Centrifuge at 10,000 rpm and 4°C for 20 min, discard the supernatant, turn the precipitate side of the collection tube outwards from the rotor, centrifuge again at 10,000 rpm and 4°C for 10 min, and pipette to discard the supernatant.

[0098] 7. Resuspend the precipitate in 1 mL of PBS buffer, filter the phage suspension through a 0.45 μm filter, take 10 μL to test the titer, and store the remainder at 4 °C. The titer of the initial phage library is generally 1 × 10⁻⁶. 9 cfu / mL;

[0099] 8. Titer (i.e., the number of clones obtained per milliliter of phage infection of the host bacteria) detection method: Mix 10 μL of phage library with 990 μL of PBS, and perform 10 tests according to this ratio. 2 10 4 ...10 10 Dilute 100 μL of the diluted phage library and 200 μL of ER2738 bacterial culture, mix well, incubate overnight at 37°C with the culture inverted position, and then spread on LB agar plates containing ampicillin. The next day, calculate the number of clones and randomly select single clones for colony PCR to detect the positive rate.

[0100] The phage plasmid used in this experiment was pComb3xss. The variable region library and plasmid were digested with SfiI restriction endonuclease and ligated at a 3:1 molar ratio, followed by electroporation into ER2738 competent cells. Generally, the volume of the bacterial library after electroporation should reach 10⁻⁶ cells. 8 Only at or above these levels can diversity and coverage be guaranteed. The library size of the antibody library constructed in this experiment is shown in Table 5, and the results of the accuracy of colony PCR detection of the library are as follows: Figure 3 As shown.

[0101] Table 5 Library Capacity

[0102]

[0103] From Table 5 and Figure 3 It can be seen that the library capacity meets the experimental requirements, and the positive rate of the library is above 83%, which meets the requirements and subsequent experiments can be carried out.

[0104] Example 2

[0105] Screening and identification of anti-E165R single-domain antibodies

[0106] filter:

[0107] 1. Antigen coating: The antigen coating amounts for the first, second, and third rounds of screening were 10 μg / mL, 5 μg / mL, and 2.5 μg / mL, respectively. Eight enzyme-labeled wells were coated with 100 μL of antigen per well, and the coating was carried out overnight at 4°C.

[0108] 2. Discard the coating solution, wash the plate 3 times with 250 μL of 0.1% PBST, pat dry, add 200 μL of 3% skim milk powder, and incubate at room temperature for 2 hours;

[0109] 3. Discard the blocking solution, wash the plate three times with 250 μL of 0.1% PBST, pat dry, add 100 μL of phage library to each well, and incubate at room temperature for 1 hour;

[0110] 4. Discard the sample solution, wash the plate 10 times with 250 μL of 0.1% PBST, pat dry, and use 100 μL of Gly-HCl for polar elution in the first and second rounds of screening. Incubate at 37°C for 6-8 min, gently pipette and collect the elution into centrifuge tubes. Add 30 μL of neutralization solution to control the pH at 7-8. In the third round of screening, use 100 μL of 5 μg / mL pure antigen for competitive elution on a shaker at room temperature for 1 h. Then collect the supernatant into centrifuge tubes, take 10 μL to detect the titer, and amplify the remaining product for subsequent screening.

[0111] The screening results are shown in Table 6.

[0112] Table 6. Screening of E165R-specific single-domain antibodies using phage display technology.

[0113]

[0114] Identification:

[0115] Phage ELISA:

[0116] 1. Select positive clones and place them in 1 mL of 2YT medium containing tetracycline and ampicillin resistance. Incubate at 37°C and 220 rpm until OD500. 600 =0.6-0.8;

[0117] 2. Add 10 μL of M13KO7, let stand at 37℃ for 30 min, then shake at 220 rpm for 2 h;

[0118] 3. Add kanamycin at a volume ratio of 1:2000, incubate overnight at 30°C and 220 rpm;

[0119] 4. Centrifuge at 12000 rpm and 4℃ for 15 min, and collect the supernatant for subsequent detection experiments;

[0120] 5. The subsequent experimental steps are similar to those for the detection of serum antigen-specific antibodies, but during the sample loading process, 50 μL of diluted serum and 50 μL of sample diluent are added to the sample wells, and 50 μL of diluted serum and 50 μL of 4 μg / mL antigen diluent are added to the competition wells.

[0121] The results of the identification are as follows Figure 4 As shown.

[0122] Depend on Figure 4 It can be seen that the phage ELISA screening product has good binding affinity to the African swine fever antigen E165R.

[0123] Example 3

[0124] Expression of anti-E165R single-domain antibody

[0125] Prokaryotic expression and purification method for African swine fever E165R specific single-domain antibodies: pComb3xss vector was used as the expression plasmid, and BL21 was used as the expression strain. Four monoclonal single-domain antibodies were expressed and purified in prokaryotes using an induction temperature of 37℃ and an induction concentration of 1 mM IPTG.

[0126] The specific process is as follows:

[0127] Sample preparation

[0128] 1. Pick positive clones and add them to 5 mL of LB medium containing the target antibiotic, and incubate overnight at 37°C and 220 rpm;

[0129] 2. Transfer the above 5 mL bacterial culture to 500 mL of LB medium containing the target resistance at a ratio of 1:100, and activate it at 37°C and 220 rpm for 3 hours until OD500 reaches the target level. 600 It reaches between 0.6 and 1.0;

[0130] 3. Add 100mM IPTG stock solution at a volume ratio of 1:100 to a final concentration of 1mM, and induce overnight at an induction temperature of 30℃ or 37℃;

[0131] Centrifuge at 4,500 rpm and 4°C for 10 min, discard the supernatant, and collect the bacterial cells;

[0132] 5. Resuspend the bacterial cells in a small amount of pre-cooled PBS buffer, centrifuge at 5000 rpm and 4°C for 10 min, discard the supernatant, and collect the bacterial cells;

[0133] 6. Resuspend the bacterial cells in 30 mL of pre-cooled PBS buffer, add PMSF and Tritan-100 at a ratio of 1:100, and sonicate on ice to break the bacteria.

[0134] Centrifuge at 12000 rpm and 4℃ for 20 min, aspirate the supernatant, and purify by filtration through a 0.45 μm filter;

[0135] Protein purification

[0136] 1. Add 5 mL of nickel packing material to the purification column and wash the nickel column with 3-5 column volumes of Milli-Q water to thoroughly remove the nickel packing buffer.

[0137] 2. Add 5-10 column volumes of PBS buffer to equilibrate the column;

[0138] 3. Add the filtered supernatant of induced bacterial cells to the purification column and spin-bind at 4°C for 1 hour on a mixer;

[0139] 4. Collect the percolation solution. To increase the loading of the target protein, the percolation solution can be loaded onto the column again. Repeat this step twice and collect a portion of the percolation solution for subsequent detection.

[0140] 5. Add 25-50 column volumes of 20 mM imidazole wash buffer, 5 column volumes of 50 mM imidazole wash buffer, and 2 column volumes of 100 mM imidazole wash buffer to wash the column. Take about 50 μL of the wash buffer and mix it with 500 μL of Bradford solution. The washing can be stopped when the color no longer changes significantly. Collect a portion of the wash buffer for subsequent detection.

[0141] 6. Add 1 mL of Elution Buffer sequentially for elution. Take about 50 μL of the eluent and mix it with 500 μL of Bradford solution. The elution can be stopped when the color no longer changes significantly. Collect the eluent for subsequent Coomassie Brilliant Blue staining and Western Blot experiments.

[0142] 7. Add 10 mL of Elution Buffer to the purification column and store at 4°C. The purification column can be reused 2-3 times.

[0143] Sample dialysis

[0144] Based on the results of Coomassie Brilliant Blue staining and Western Blot, high-purity and high-concentration eluent was selected and placed into a dialysis tube. The dialysis tube was placed in a beaker containing 2 L of PBS buffer using a float, and dialysis was accelerated on a magnetic stirrer at 4 °C. The buffer was changed every 4 hours. The volume of PBS buffer used for dialysis was at least 1000 times that of the eluent, that is, 1 mL of eluent required 1 L of PBS buffer for dialysis. After dialysis, the eluent was aspirated, and a small amount was taken to determine the protein concentration using the BCA protein concentration assay method. The eluent was then stored at 4 °C.

[0145] The expression results of the anti-E165R single-domain antibody are as follows: Figure 5 As shown. Among them, Figure 5 The images, from top to bottom, show the results of Coomassie brilliant blue staining and the results of specific detection of the target protein expression using a tag antibody (anti-His 6x histidine).

[0146] Depend on Figure 5 It can be seen that a large amount of the target protein was successfully eluted using Elution buffer, with relatively few other proteins.

[0147] Example 4

[0148] Sensitivity and specificity analysis of anti-E165R single-domain antibody

[0149] The antigen affinity of the purified, dialyzed E165R-specific single-domain antibody was detected using biomembrane interferometry. The specific procedure is as follows:

[0150] 1. Affinity detection of single-domain antibodies

[0151] Testing steps:

[0152] The biosensor used in this experiment carries streptavidin, so it is necessary to biotin label the antibody or antigen to be tested. This experiment mainly focuses on biotin labeling the antigen.

[0153] Antigen biotinylation:

[0154] 1. The molar ratio of biotin to antigen is usually controlled between 1:1 and 3:1. Take an appropriate volume of biotin stock solution and antigen according to the following formula, mix them well, and let them stand at room temperature for 30 minutes or at 4°C for 1 hour to bind.

[0155] 2. Required volume of 10mM biotin stock solution (μL) = molar ratio × antigen volume (μL) × 0.1 × antigen concentration (mg / mL) / antigen molecular weight (kDa);

[0156] Desalting of biotinylated antigens:

[0157] 1. Remove the bottom cap of the desalting column, loosen the top cap (do not open), place the desalting column in the EP tube, detach it briefly at 1500 rpm, and discard the waste liquid;

[0158] 2. Mark the position of the desalting column slant, add biotinylated antigen to it, and point the column slant towards the outside of the centrifuge rotor to increase the binding area between the antigen and the desalting column. Collect the protein by instantaneous centrifugation at 1500 rpm.

[0159] Biomembrane Layer Interference (BLI) Experiment

[0160] 1. Dilute the single-domain antibody and biotinylated antigen to be tested to an appropriate concentration using BLI sample buffer. Add 200 μL to each well of a black flat-bottomed 96-well plate. Add 200 μL of sample buffer to the baseline well. Add one blank well as a reference well for each test sample.

[0161] 2. Take the corresponding number of biosensors and pre-wet them in the sample buffer for 10-20 minutes;

[0162] 3. Set the program according to the following steps: reference well 1 (180s) - antigen solidification (Loading) (600s) - reference well 2 (120s) - association (500s) - dissociation (500s). The reference well 2 and dissociation well are the same for each sample. The measurement temperature is set to 30℃ and the sample plate rotation speed is 1000rpm.

[0163] 4. After the experiment, Fortebio analysis software was used for data analysis. The last 10 seconds of reference well 2 was selected as the start time. For data where the binding and dissociation curves were not connected, "Align to disassociation" was selected for data processing. After processing the curves, a 1:1 model was used to integrate the data of different concentrations of a single sample for fitting analysis to obtain the binding and dissociation constants of each sample. Generally, the closer the Full R^2 value is to 1, the smaller the Full X^2 ​​value (less than 3), and the higher the Kon / Kon Error ratio is to 10, the higher the reliability of the data.

[0164] Sensitivity and specificity analysis results are as follows Figure 6 As shown in Table 7, the antigen affinity test results for antigen-specific single-domain antibodies are presented. In Table 7, K... D K is the dissociation constant. off K is the dissociation rate constant. on For the binding rate constant, FullR 2 The closer the value is to 1, the more Full X 2 The smaller the value, the higher the reliability of the data.

[0165] Table 7 shows the antigen affinity test results for E165R antigen-specific single-domain antibodies.

[0166]

[0167] Depend on Figure 6 As shown in Table 7, the single-domain antibodies obtained through screening have an affinity for antigens in the nanomolar range, indicating high affinity.

[0168] 2. Sensitivity and specificity detection of single-domain antibodies

[0169] The specificity of the single-domain antibody was verified using dot blot hybridization. The E1 monoclonal single-domain antibody can detect pg-level antigens and does not cross-react with P54 and OVA.

[0170] The specific process is as follows:

[0171] 1. Dilute the antigen to be tested to an appropriate concentration using PBS buffer, take 2 μL and spot it onto a nylon membrane, then let it air dry at room temperature;

[0172] 2. Place the nylon film in 5% skim milk powder and seal at room temperature for 1-2 hours;

[0173] Wash the membrane once with 3.0.1% PBST, dilute the purified single-domain antibody to the appropriate concentration using antibody dilution buffer, and incubate at room temperature for 2 hours or overnight at 4°C;

[0174] Wash the membrane three times with 4.01% TBST, 10 minutes each time;

[0175] 5. Dilute the HRP-labeled secondary antibody to an appropriate concentration using antibody dilution buffer and incubate at room temperature for 1 hour;

[0176] Wash the membrane three times with 6.0.1% TBST, 10 minutes each time;

[0177] 7. Mix equal volumes of solutions A and B of the chemiluminescence developer and add them to the membrane for development in a chemiluminescence imager.

[0178] The result is as follows Figure 7 As shown.

[0179] Depend on Figure 7 It is known that E1 monoclonal single-domain antibodies can detect pg-level antigens and do not cross-react with P54 and OVA.

[0180] Example 5

[0181] Neutralization activity assay of anti-E165R single-domain antibody

[0182] The specific process is as follows:

[0183] 1. The selected single-domain antibodies were used to perform African swine fever virus neutralization experiments, with an infection dose MOI of 2;

[0184] 2. Incubate the virus with antibodies diluted to different concentrations or PBS (negative control) at 37°C for 1 hour;

[0185] 3. Porcine alveolar macrophages were cultured into 6-well cell culture plates that had grown into a monolayer. Then, the antibody-virus mixture was added to the cells and incubated at 37°C for 1 hour.

[0186] 4. Then replace with a new culture medium and incubate at 37°C for 72 hours;

[0187] 5. Collect cells to extract RNA, perform quantitative real-time PCR detection, and convert the CT value to viral copy number according to the following formula;

[0188] 6. Viral copy number Y = 10^[(X-36.272) / (-3.3652)], where X is the CT value, comparing the host infection with the virus under antibody-positive and antibody-negative conditions.

[0189] The result is as follows Figure 8 As shown.

[0190] Depend on Figure 8 It can be seen that, compared with the PBS-treated control group, the viral copy number of porcine primary alveolar macrophages (PAM) cells treated with E-1 single-domain antibody was significantly reduced, indicating that the single-domain antibody has a strong virus neutralizing ability.

[0191] In summary, this invention provides a single-domain antibody against the African swine fever virus (ASV) E165R protein. Sequencing results show that the full-length gene of this single-domain antibody is 465 bp. Soluble-expressing recombinant single-domain antibody protein was successfully obtained by inducing expression in strain ER2738 after infection with a strongly positive phage against the E165R antigen. Identification showed that it exhibits good binding activity to E165R. Specificity testing results showed that the E1 single-domain antibody can detect pg-level antigens and does not cross-react with P54 and OVA. This single-domain antibody provides a theoretical basis and material foundation for establishing a rapid immunological detection method for E165R.

[0192] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against the E165R protein of African swine fever virus, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding a single-domain antibody against the African swine fever virus E165R protein as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The gene comprising the single-domain antibody encoding the E165R protein against African swine fever virus as described in claim 2.

4. A bacteriophage, characterized in that, The gene expressing the single-domain antibody encoding the anti-African swine fever virus E165R protein as described in claim 2.

5. The use of the single-domain antibody against African swine fever virus E165R protein as described in claim 1 in the preparation of an immunological detection reagent for African swine fever virus.

6. A test kit, characterized in that, The single-domain antibody against the African swine fever virus E165R protein as described in claim 1.

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