Single-domain antibody for resisting African swine fever virus E165R protein as well as screening method and application of single-domain antibody

Abstract

The invention discloses a single-domain antibody for resisting African swine fever virus E165R protein as well as a screening method and application of the single-domain antibody, and relates to the technical field of bioengineering. The invention provides a single-domain antibody for resisting African swine fever virus E165R protein and a specific amino acid sequence, and also provides a gene for coding the single-domain antibody, an expression vector comprising the gene and a phage for expressing the gene, and the single-domain antibody for resisting the African swine fever virus E165R protein is applied to African swine fever virus immunological detection. The single-domain antibody provided by the invention has relatively strong specificity, high sensitivity and strong neutralizing activity, and can be used for immunological detection of daily epidemic diseases and system construction of future epidemic disease prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a single-domain antibody against African swine fever virus E165R protein and a screening method and application thereof. Background Art

[0002] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars, causing huge economic losses to the world's animal husbandry. Since there is no effective vaccine for the prevention of African swine fever in the world, the current prevention and control measures for African swine fever are mainly to prevent introduction and timely culling. In view of the difficulties in the current vaccine development process (long time, technical difficulties, imperfect vaccine evaluation system, high investment, and the need for professionals) and the very serious African swine fever epidemic, immunological methods based on antigen-antibody interactions and the application advantages of single-domain antibodies can help prevent and control the disease. The core of this method is to prepare single-domain antibodies with high specificity, stability and strong neutralizing activity. Most of the reports are traditional antibodies, and there are few reports on single-domain antibodies. Therefore, the development of single-domain antibodies against African swine fever is of great significance for the detection of African swine fever virus and the prevention and control of the disease. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a single-domain antibody against African swine fever virus E165R protein and its screening method and application. The provided single-domain antibody has strong specificity, high sensitivity and strong neutralizing activity, and can be used for daily immunological detection of epidemic diseases and the construction of future disease prevention and control systems.

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

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

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

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

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

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

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

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

[0012] (1) Immunizing mice expressing heavy chain antibodies with African swine fever virus E165R protein;

[0013] (2) extracting RNA from samples immunized with African swine fever virus E165R protein antigen, and then amplifying by PCR to obtain the heavy chain variable region genes;

[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 rescue by helper phage M13KO7, a phage library against African swine fever virus E165R protein is obtained, and then a single domain antibody against African swine fever virus E165R protein is obtained by screening the African swine fever virus E165R protein.

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

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

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

[0018] 1. The present 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, from which single-domain antibodies of African swine fever virus E165R protein are screened, providing a theoretical basis and material foundation for the later establishment of an immunological rapid detection method for E165R protein.

[0019] 2. The present invention uses electroporation to transform the recombinant vector into Escherichia coli ER2738; at the same time, in order to ensure the high activity of the competent state, the competent state is prepared in advance before transformation in the present invention and can be used for transformation immediately after preparation, thereby ensuring the high efficiency of transformation to the greatest extent.

[0020] 3. In the subsequent screening process, the present invention uses a high concentration of pure E165R protein for elution in order to reduce the nonspecific binding of the single domain antibody and improve the efficiency and accuracy of the screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the expression level of antigen-specific antibodies in serum;

[0022] Figure 2 Schematic diagram of amplification results;

[0023] Figure 3 It is a schematic diagram of the library positive rate results;

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

[0025] Figure 5 It is a schematic diagram of the expression results of the anti-E165R single domain antibody;

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

[0027] Figure 7 It is a schematic diagram of the sensitivity and specificity detection results of single domain antibodies;

[0028] Figure 8 Schematic diagram of the neutralization activity test results of anti-E165R single domain antibody. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] Example 1

[0031] 1. Immunization of mice producing heavy chain antibodies

[0032] Antigen emulsification: dilute the antigen to 2μg / μL with sterile PBS, suck it into a syringe for later use, use another syringe to draw an equal volume of adjuvant, use a syringe to connect the two syringes together, quickly push the adjuvant into the antigen, push and draw back and forth several times to mix, and after about 40 minutes, draw 10μL and drop it on the water surface. If it does not spread, it means that the emulsification is complete.

[0033] Mouse immunization: The immunization process is divided into 1 primary immunization and 4 booster immunizations, with an interval of 14 days between the two immunizations. The primary immunization is injected subcutaneously with an immunization dose of 100μg / 50μL; the booster immunization is injected intraperitoneally with an immunization dose of 100μg / 50μL. Blood is collected from the eye sockets three days before the primary immunization and three days after each immunization, and the serum is separated for subsequent ELISA experiments.

[0034] 2. Immune effect detection

[0035] 1. Use coating buffer to dilute the antigen to a concentration of 2 μg / mL, vortex and mix, then add 100 μL per well to the enzyme-labeled wells, and coat at 37°C for 1 hour or at 4°C overnight;

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

[0039] 5. Use sample diluent to dilute the serum to an appropriate concentration, vortex mix and add 100 μL per well to the enzyme-labeled wells. At the same time, dilute the standard serum in proportion, vortex mix and add 100 μL per well to the enzyme-labeled wells. Perform 2 technical replicates for each sample and combine at room temperature for 1 hour.

[0040] 6. Shake off the sample, wash the plate 5 times using a plate washer, and pat dry on paper;

[0041] 7. Use sample diluent to dilute the HRP-labeled antibody to the target concentration, vortex and mix, then add 100 μL per well to the enzyme-labeled wells and allow to bind for 1 hour at room temperature;

[0042] 8. Shake off the antibody, wash the plate 5 times using a plate washer, and pat dry on paper;

[0043] 9. Mix equal volumes of TMB solution A and solution B, add 100 μL to each well into the enzyme-labeled well, allow to develop color for 20 minutes, then add 100 μL of stop solution to each well, place in an enzyme-labeled instrument and read the absorbance at 560 nm to detect the expression level of antigen-specific antibodies in the serum.

[0044] The results are as follows Figure 1 As shown. Among them, Figure 1 Figure 2. Response trend of specific antibodies in the serum of mice after African swine fever immunization. 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 value that the mice produced a higher immune response to the E165R protein.

[0046] 3. Extract RNA from mouse spleen

[0047] Extract RNA from two mice with good immune effects, refer to the Magen tissue cell total RNA extraction kit, the steps are as follows:

[0048] 1. Quickly freeze the tissue in liquid nitrogen, break it into small pieces as much as possible, put it into 1 mL Trizol, add steel beads, homogenize it in a tissue homogenizer at 50 Hz for 5-10 minutes, and put it in an ice bath for 2 minutes after the tissue is completely broken;

[0049] 2. Add 200 μL chloroform, quickly invert up and down to mix 8-10 times, let stand for 2 minutes, and then centrifuge at 12000 rpm, 4°C for 10 minutes;

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

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

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

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

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

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

[0056] 9. Repeat step 8 once;

[0057] 10. Discard the filtrate, reinstall the column into the collection tube, and run at 12000 rpm for 2 min;

[0058] 11. Place the column in a new centrifuge tube, add 50 μL of DEPC water to the center of the column, let stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes. To improve the elution efficiency, add the eluted RNA to the center of the column and elute again;

[0059] 12. Use Nano 3000 to detect the concentration and purity of RNA, and perform agarose gel electrophoresis to detect its integrity, and then store it at -80℃.

[0060] 4. Reverse transcription

[0061] Take 2 μg of tissue or cell RNA and use Abm G490 reverse transcription kit for reverse transcription experiment; the system and amplification procedure are shown in Table 1 and Table 2:

[0062] Table 1 G490 reverse transcription system

[0063]

[0064] After mixing each sample according to the system shown in Table 1, centrifuge briefly, and then perform reverse transcription according to the procedure shown in Table 2;

[0065] Table 2G490 reaction procedure

[0066]

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

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

[0069] The construction strategy of the variable region library in the phage display experiment was carried out by nested PCR amplification, with spleen cDNA of immunized mice as the template, and restriction sites and protective bases added to the 5' end of the second round primers. The difference was that in order to ensure the diversity of the library, the 12 tubes of products in the first round were used as templates for amplification using 5 pairs of primers in the second round. After the reaction, agarose gel electrophoresis was performed to detect the amplification of the target band, and the gel was cut and recovered for purification, and the recovered product was stored at -20℃.

[0070] The variable region library first round amplification program and the variable region library second round amplification program are shown in Table 3-4 respectively:

[0071] Table 3 Variable region library first round amplification program

[0072]

[0073] Table 4 Variable region library second round amplification program

[0074]

[0075] The amplification results are as follows Figure 2 shown.

[0076] 6. Amplification of single domain antibody library

[0077] Preparation of ER2738 competent cells:

[0078] 1. Activate the bacteria: Use the tip of the pipette to take a small amount of ER2738 bacterial solution and streak it on a culture plate containing tetracycline resistance, then place it in a 37°C incubator for activation and culture for 12 hours;

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

[0080] 3. Large-scale shaking: Take the above bacterial solution and transfer it into 500 mL of 2YT medium containing tetracycline resistance at a ratio of 1:100, and activate it at 37°C, 220 rpm for 3 hours until the OD 600 =0.5-0.7;

[0081] 4. Transfer the bacterial solution to a pre-cooled bacterial collection bottle and place it in ice for 30 minutes to cool it thoroughly;

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

[0083] 6. Centrifuge at 4500 rpm, 4°C for 10 min, discard the supernatant, add 250 mL of pre-cooled 10% glycerol to resuspend the cells on ice, and place on ice for 10 min;

[0084] 7. Centrifuge at 4500 rpm, 4°C for 10 min, discard the supernatant, add 100 mL of pre-cooled 10% glycerol to resuspend the cells on ice, and place on ice for 10 min;

[0085] 8. Centrifuge at 4500rpm and 4℃ for 10min, discard the supernatant, add 2mL of pre-cooled 10% glycerol to resuspend the bacteria, then dispense into pre-cooled EP tubes at 200μL per tube and store on ice.

[0086] Variable region library electroporated into ER2738 competent cells

[0087] 1. Take 3-4 μg of variable region fragments and connect them at a molar ratio of 5:1 between the fragment and the vector. Then, the connection product is purified by column recovery to remove salt ions in the connection product to avoid affecting the electrotransfer.

[0088] 2. Take 10 μL of ligation product and 200 μL of competent cells and mix them, then add them to the pre-cooled electrode cup, adjust the parameters to 2.5 kV / 125Ω for electric shock, and immediately add 1 mL of preheated SOC medium to the electrode cup after the end, blow quickly 3 times to avoid foaming, aspirate this 1 mL of bacterial solution and place it in 10 mL of SOC medium, repeat the electroporation reaction 10 times, and then resuscitate about 20 mL of bacterial solution at 37°C, 220 rpm for 1 hour;

[0089] 3. Spread 2 mL of the activated bacterial solution on a 2YT solid culture plate containing tetracycline resistance, ampicillin resistance and 1% glucose, and take 20 μL of the bacterial solution for gradient dilution (10 dilutions per plate). 4 times, 10 5 times, 10 6 Times and 10 7 After spreading onto a small LB culture plate containing ampicillin resistance, invert and culture overnight in a 37°C incubator to calculate the reservoir capacity, which is the number of colonies × dilution factor. Generally, it should reach 10 8 above;

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

[0091] Construction of phage library

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

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

[0094] 3. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the cells in 100 mL 2YT medium containing tetracycline resistance, ampicillin resistance, and kanamycin resistance, and culture overnight at 30°C and 220 rpm;

[0095] 4. Centrifuge at 10,000 rpm, 4°C for 20 min and 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, 4°C for 20 min, discard the supernatant, place the collection tube with the sediment side facing out of the rotor, centrifuge again at 10,000 rpm, 4°C for 10 min, and discard the supernatant with a pipette;

[0098] 7. Use 1mL PBS buffer to suspend the precipitate, filter the phage suspension with a 0.45μm filter, take 10μL to test the titer, and store the rest at 4℃. Generally, the titer of the initial phage library is 1×10 9 cfu / mL;

[0099] 8. Titer (i.e. the number of clones obtained per ml of phage infecting the host bacteria) detection method: Take 10 μL of phage library and 990 μL of PBS and mix them evenly. 2 , 10 4 ……10 10 Dilution, take 100 μL of the diluted phage library and 200 μL of ER2738 bacterial solution, mix them, invert and culture them at 37°C overnight, then spread them on LB plates containing ampicillin, calculate the number of clones the next day and randomly pick single clones for colony PCR detection of positive rate.

[0100] The phagemid used in this experiment was pComb3xss. The variable region library and plasmid were digested with SfiⅠ restriction endonuclease and ligated at a molar ratio of 3:1, and then electroporated into ER2738 competent cells. Generally speaking, the library capacity after electroporation should reach 10 8 The diversity and coverage can be guaranteed only when the library capacity of the antibody library constructed in this experiment is shown in Table 5, and the results of the colony PCR detection library accuracy are shown in Figure 3 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 can be used for subsequent experiments.

[0104] Example 2

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

[0106] filter:

[0107] 1. Antigen coating: The antigen coating amount for the first, second and third rounds of screening is 10μg / mL, 5μg / mL and 2.5μg / mL respectively, coating 8 enzyme-labeled wells, 100μL per well, and coating at 4℃ overnight;

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

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

[0110] 4. Throw out the sample solution, wash the plate 10 times with 250μL 0.1% PBST, pat dry, use 100μL Gly-HCl for polar elution in the first and second rounds of screening, let stand at 37℃ for 6-8min, gently blow and collect into a centrifuge tube, add 30μL neutralizing solution to control the pH at 7-8; in the third round of screening, use 100μL 5μg / mL pure antigen for competitive elution, compete on a shaker at room temperature for 1h, then collect the supernatant into a centrifuge tube, take 10μL to test the titer, and expand 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. Pick the positive clones and place them in 1 mL of 2YT medium containing tetracycline and ampicillin resistance, and activate and culture at 37°C, 220 rpm until OD 600 =0.6-0.8;

[0117] 2. Add 10 μL M13KO7, incubate at 37°C for 30 min, and then shake at 220 rpm for 2 h;

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

[0119] 4. Centrifuge at 12000 rpm, 4°C 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 addition process, 50 μL of diluted serum and 50 μL of sample diluent are added to the sample well, and 50 μL of diluted serum and 50 μL of 4 μg / mL antigen diluent are added to the competition well.

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

[0122] Depend on Figure 4 It can be seen that the phage ELISA detection and screening products have a good binding affinity with the African swine fever antigen E165R.

[0123] Example 3

[0124] Expression of anti-E165R single domain antibody

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

[0126] The specific process is as follows:

[0127] Sample preparation

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

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

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

[0131] 4. Centrifuge at 5000 rpm, 4°C for 10 min, discard the supernatant and collect the bacteria;

[0132] 5. Use a small amount of pre-cooled PBS buffer to resuspend the bacteria, centrifuge at 5000rpm, 4℃ for 10min, discard the supernatant, and collect the bacteria;

[0133] 6. Resuspend the cells in 30 mL of pre-cooled PBS buffer, add PMSF and Tritan-100 at a volume ratio of 1:100, and disrupt the cells by ultrasonication on ice;

[0134] 7. Centrifuge at 12000 rpm, 4°C for 20 min, remove the supernatant, and purify it after filtering with a 0.45 μm filter;

[0135] Protein purification

[0136] 1. Take 5 mL of nickel filler into the purification column and wash the nickel column with 3-5 column volumes of Milli-Q water to fully remove the nickel filler buffer;

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

[0138] 3. Add the filtered supernatant of the induced bacteria to the purification column and rotate on a mixer at 4°C for 1 hour;

[0139] 4. Collect the column liquid. In order to increase the loading capacity of the target protein, the column liquid can be loaded onto the column again. Repeat this step twice and collect part of the column liquid for later detection.

[0140] 5. Add 25-50 column volumes of 20mM imidazole washing solution, 5 column volumes of 50mM imidazole washing solution and 2 column volumes of 100mM imidazole washing solution to wash the column. Take about 50μL of washing solution and 500μL Bradford and mix them evenly. When the color no longer changes significantly, the washing can be stopped and part of the washing solution can be collected for later detection;

[0141] 6. Add 1 mL Elution Buffer for elution, take about 50 μL of elution buffer and mix with 500 μL Bradford, and stop elution when the color no longer changes significantly. Collect the elution buffer for subsequent Coomassie Brilliant Blue staining and Western Blot experiments;

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

[0143] Sample dialysis

[0144] Combined with the results of Coomassie Brilliant Blue staining and Western Blot, the eluate with higher purity and concentration was selected and placed in the dialysis tube. The dialysis tube was placed in a beaker containing 2L PBS buffer using a float. The dialysis was accelerated on a 4°C magnetic stirrer. The buffer was changed every 4 hours. The volume of PBS buffer used for dialysis was at least 1000 times that of the eluate, that is, 1mL of the eluate needed to be dialyzed with 1L of PBS buffer. After the dialysis, the eluate was aspirated, and a small amount was taken to detect the protein concentration using the BCA protein concentration determination method, and then stored at 4°C.

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

[0146] Depend on Figure 5 It can be seen that a large amount of target protein was successfully eluted using Elution buffer, and there were fewer impurity proteins.

[0147] Example 4

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

[0149] The antigen affinity of the purified and dialyzed E165R-specific single domain antibody was tested using biofilm interferometry. The specific process is as follows:

[0150] 1. Affinity testing of single domain antibodies

[0151] Testing steps:

[0152] The biosensor used in this experiment carries streptavidin, so the antibody or antigen to be tested needs to be labeled with biotin. This experiment mainly labels the antigen with biotin.

[0153] Antigen biotinylation:

[0154] 1. The molar ratio of biotin to antigen is usually controlled at 1:1-3:1. Pipette an appropriate volume of biotin stock solution and antigen according to the following formula, mix well, and place at room temperature for 30 minutes or at 4°C for 1 hour to combine;

[0155] 2. Required volume of 10 mM 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 cover of the desalting column, loosen the top cover (do not open), place the desalting column in the EP tube, centrifuge at 1500rpm, and discard the waste liquid;

[0158] 2. Mark the position of the slope of the desalting column bed, add biotinylated antigen into it, and point the slope of the column bed toward the outside of the centrifuge rotor to increase the binding area between the antigen and the desalting column. Centrifuge at 1500 rpm to collect the protein;

[0159] Biolayer Interferometry (BLI) experiments

[0160] 1. Use BLI sample buffer to dilute the single domain antibody and biotinylated antigen to the appropriate concentration, add 200 μL per well to a black flat-bottom 96-well plate, add 200 μL sample buffer to the baseline well, and add a blank well as a reference well for each measured sample;

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

[0162] 3. Set up the program according to reference well 1 (180s) - antigen immobilization (Loading) (600s) - reference well 2 (120s) - association (Association) (500s) - dissociation (Disassociation) (500s). The reference well 2 and dissociation well of each sample are the same. The measurement temperature is set to 30°C and the sample plate speed is 1000rpm.

[0163] 4. After the experiment, use Fortebio analysis software for data analysis, select the last 10s of reference well 2 as the starting time, and select "Align to disassociation" for data processing when the binding curve and dissociation curve are not connected. After processing the curve, use the 1:1 model to integrate the data of different concentrations of a 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 is (less than 3), and the Kon / Kon Error ratio is greater than 10, the higher the credibility of the data.

[0164] The results of sensitivity and specificity analysis are as follows Figure 6 The antigen affinity test results of the antigen-specific single domain antibody are shown in Table 7. In Table 7, K D is the dissociation constant, K off is the dissociation rate constant, K on is 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 credibility of the data.

[0165] Table 7 Antigen affinity test results of E165R antigen-specific single domain antibodies

[0166]

[0167] Depend on Figure 6 As shown in Table 7, the affinity of the screened single domain antibodies to the antigen is in the nanomolar range, which has a relatively high affinity.

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

[0169] The specificity of the single-domain antibody was verified by dot blot assay. The E1 monoclonal single-domain antibody could detect pg-level antigens and did not cross-react with P54 and OVA.

[0170] The specific process is as follows:

[0171] 1. Use PBS buffer to dilute the antigen to be tested to an appropriate concentration, apply 2 μL onto the nylon membrane, and dry at room temperature;

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

[0173] 3. Wash the membrane once with 0.1% PBST, dilute the purified single domain antibody to an appropriate concentration using antibody diluent, and incubate at room temperature for 2 hours, or at 4°C overnight;

[0174] 4. Wash the membrane with 0.1% TBST 3 times, 10 min each time;

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

[0176] 6. Wash the membrane with 0.1% TBST 3 times, 10 min each time;

[0177] 7. Mix equal volumes of chemiluminescent developer A and B and add them to the membrane, and develop in a chemiluminescent imager.

[0178] The results are as follows Figure 7 shown.

[0179] Depend on Figure 7 It can be seen that E1 monoclonal single domain antibody can detect antigens at the pg level and does not cross-react with P54 and OVA.

[0180] Example 5

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

[0182] The specific process is as follows:

[0183] 1. The screened single domain antibodies were used for African swine fever virus neutralization experiments, with an inoculation dose of MOI = 2;

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

[0185] 3. Culture porcine alveolar macrophages into a 6-well cell culture plate that has grown into a monolayer, then add the antibody-virus mixture to the cells and incubate at 37°C for 1 hour;

[0186] 4. Then replace the new culture medium and culture in a 37°C incubator for 72 hours;

[0187] 5. Collect cells to extract RNA, perform fluorescence quantitative PCR detection, and convert the CT value into the number of viral copies according to the following formula;

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

[0189] The results are as follows Figure 8 shown.

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

[0191] In summary, the present invention provides a single-domain antibody against the E165R protein of African swine fever virus. Sequencing results show that the full-length size of the single-domain antibody gene is 465bp. After the strong positive phage against the E165R antigen was infected into the expression strain ER2738 and induced to express, a soluble expressed single-domain antibody recombinant protein was successfully obtained, which was identified to have good binding activity with E165R. Specific identification results show that the E1 single-domain antibody can detect pg-level antigens and does not cross-react with P54 and OVA; the single-domain antibody can provide a theoretical basis and material basis for establishing an immunological rapid 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

1.

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

2.

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

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

5. Use of the single-domain antibody against African swine fever virus E165R protein according to claim 1 in immunological detection of African swine fever virus.

6. Use of the single-domain antibody against African swine fever virus E165R protein according to claim 1 in the preparation of an African swine fever virus immunological detection reagent.

7. A detection kit, characterized in that: Comprising the single domain antibody against African swine fever virus E165R protein as described in claim 1.

8. A method for screening single domain antibodies against African swine fever virus E165R protein according to claim 1, characterized in that: The following steps are involved: (1) Immunizing mice expressing heavy chain antibodies with African swine fever virus E165R protein; (2) extracting RNA from samples immunized with African swine fever virus E165R protein antigen, and then amplifying by PCR to obtain the heavy chain variable region genes; (3) The heavy chain variable region gene obtained in step (2) is cloned into a vector, and then transformed into Escherichia coli ER2738. After rescue by helper phage M13KO7, a phage library against African swine fever virus E165R protein is obtained, and then a single domain antibody against African swine fever virus E165R protein is obtained by screening the African swine fever virus E165R protein.

9. The method for screening single domain antibodies against African swine fever virus E165R protein according to claim 8, characterized in that: In step (3), the vector is a phagemid vector pComb3xss.

10. The method for screening single domain antibodies against African swine fever virus E165R protein according to claim 8, characterized in that: In step (3), the eluent used during screening is a pure product of African swine fever virus E165R protein at a concentration of 5 μg / mL.

Images