African swine fever virus pC129R protein monoclonal antibody, hybridoma cell, quantum dot test strip and application thereof

Abstract

The invention relates to an African swine fever virus pC129R protein monoclonal antibody, a hybridoma cell, a quantum dot test strip and application of the African swine fever virus pC129R protein monoclonal antibody, the hybridoma cell and the quantum dot test strip. After being extracted and purified, the expressed African swine fever virus recombinant pC129R protein is used as an antigen to immunize mice, and the monoclonal antibody of the African swine fever virus pC129R protein is screened out. Meanwhile, according to the quantum dot test strip for detecting the African swine fever virus antibody, the detection line is coated with the staphylococcus aureus protein A, the quality control line is coated with the anti-African swine fever virus pC129R protein monoclonal antibody, the quantum dot test strip has good specificity and sensitivity, the detection time is greatly shortened, and convenience is provided for field detection.

Description

Technical Field

[0001] The present invention application relates to the field of biological immunotechnology, and specifically to monoclonal antibodies against the pC129R protein of African swine fever virus, hybridoma cells, quantum dot test strips and their applications. Background Art

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by African swine fever virus (ASFV). ASF is currently the disease that causes the greatest losses to my country's pig farming industry. The virus has a large genome, complex structure and many immune escape mechanisms. With the emergence of low-virulence natural mutants of African swine fever virus (ASFV) in my country, African swine fever (ASF) has shown the characteristics of persistent, non-lethal, subacute or chronic disease. African swine fever virus (ASFV) infection can cause a strong humoral immune response in pigs and last for a long time. It is difficult to detect the virus in the blood of recovered pigs infected with African swine fever virus (ASFV), but the blood contains a high level of antibodies.

[0003] At present, no effective vaccine or drug has been developed for African swine fever (ASF), so rapid and accurate diagnosis of the disease is particularly important for the prevention, control and purification of African swine fever (ASF). Because the genome of African swine fever virus (ASFV) is extremely complex and large, there is currently no specific therapeutic drug or commercial vaccine for the virus, so accurate and effective detection and early diagnosis are extremely important for the control of the epidemic.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] The pC129R protein is one of the structural proteins with a higher content in African swine fever virus particles. In Vero cells (African green monkey kidney cells) and WSL-HP (wild boar lung cells) infected with African swine fever virus, the pC129R protein is also one of the most abundant proteins. The previous studies by the inventors have shown that the pC129R protein has strong immunogenicity and can stimulate the body to produce a large number of specific antibodies. Therefore, it may become a potential target for the detection of African swine fever virus.

[0006] Based on the above research, a monoclonal antibody against the African swine fever virus pC129R protein was screened, whose heavy chain variable region includes CDR H1, CDR H2, and CDRH3, and whose amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0007] The second aspect disclosed in the present application provides a gene encoding the monoclonal antibody, and the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.7.

[0008] The third aspect disclosed in the present application provides a hybridoma cell which secretes a monoclonal antibody containing the heavy chain variable region described above.

[0009] In a fourth aspect disclosed in the present application, the monoclonal antibody or the hybridoma cell is used in the preparation of an immunological detection reagent.

[0010] According to the fifth aspect disclosed in the present application, the monoclonal antibody or the hybridoma cell is used in the preparation of antibodies or vaccines against African swine fever virus.

[0011] Staphylococcus aureus protein A is a 40-60 kDa surface protein, consisting of 4 or 5 homologous immunoglobulin binding domains composed of 56-61 residues. This protein is a protein component isolated from the cell wall of Staphylococcus aureus. Studies have found that this protein can bind to the Fc segment of IgG molecules of various mammals, but does not affect the binding of IgG to antigens, and can be applied to the field of immunology. In the field of test strips, the protein itself can be used to bind to the Fc segment of IgG molecules in the serum of various mammals, present antibodies to the test strips, and detect antibodies by reacting with the antigens marked on the test strips.

[0012] Based on this, the sixth aspect disclosed in the present application is to provide a quantum dot test strip for detecting antibodies to the African swine fever virus pC129R protein, comprising a conjugation pad, a sample pad, and a base membrane provided with a detection line and a quality control line, the conjugation pad being bound to a quantum dot-labeled African swine fever virus pC129R recombinant protein, the detection line being coated with Staphylococcus aureus protein A, and the quality control line being coated with a monoclonal antibody to the above-mentioned African swine fever virus pC129R protein.

[0013] The seventh aspect disclosed in the present application provides a method for preparing the above-mentioned quantum dot test strip, comprising the following steps: (1) Preparation of binding pad: Cut glass wool into long strips and fix them, spray buffer evenly on them, and then dry them at 40-45°C for 45-50 min. Then, spray 4-5 ml of quantum dot-labeled African swine fever virus pC129R recombinant protein on the glass wool and dry them at 40-45°C for 45-50 min. Finally, dry and seal the glass wool at room temperature for storage. The buffer is 18-22 mmol / L Na2B4O7 buffer containing 0.8-1.2% BSA, 0.08-0.12% Tween 20, 2.5-3.5% sucrose and 0.08-0.12% NaN3. (2) Sample pad preparation: Cut the glass cellulose membrane into long strips, soak it in a buffer solution for 4-5 min, then dry it at 40-45 °C for 3.5-4.5 h, and finally dry it and seal it for storage at room temperature; the buffer solution is 0.01-0.02 mol / L PBS solution containing 0.3-0.7% Triton-100 and 0.08-0.12% sodium azide; (3) Preparation of absorbent pad: Cut the absorbent filter paper into long strips and store them in a sealed container at room temperature for later use; (4) Preparation of nitrocellulose membrane: 0.3-0.5 mg / mL of African swine fever virus pC129R protein monoclonal antibody and 0.8-1.0 mg / mL of Staphylococcus aureus protein A were sprayed on the nitrocellulose membrane as T line and C line, respectively. After spraying, the nitrocellulose membrane was dried at 35-40°C for 3.5-4.5 h; (5) Assemble the pretreated binding pad, sample pad, absorbent pad and nitrocellulose membrane in sequence along the length direction of the plastic base plate, with the edges of each part overlapping by 1.5-2 mm. Finally, cut the assembled card into test strips with a width of 2.5-3 mm. Place the test strips in a dry tin foil bag and store them dry at room temperature.

[0014] In some embodiments of the present disclosure, the preparation process of the quantum dot-labeled African swine fever virus pC129R recombinant protein is as follows: first, the quantum dots are mixed with the EDC solution, and then the reaction is carried out in the dark at 20~30°C and 220rpm for 25~35min; then, the pC129R recombinant protein and BBS buffer are added respectively, and the reaction is carried out in the dark at 20~30°C and 220rpm for 2~3h; finally, after centrifugation at 3500~4500rpm for 4~6min, the supernatant is taken to obtain the coupling product, and it is stored at 2~4°C for future use.

[0015] In some embodiments of the present disclosure, the concentration of the EDC solution is 0.8~1.2 mg / mL; the concentration of the pC129R recombinant protein is 0.1~0.3 mg / mL; the concentration of the BBS buffer is 0.01~0.02 mol / L; the volume ratio of the quantum dots, the EDC solution, the pC129R recombinant protein, and the BBS buffer is 1~1.5:4.5~5.5:3.5~4.5:9~11.

[0016] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. The monoclonal antibody provided in the present application is prepared by synthesizing the C129R gene of the African swine fever virus and then constructing an expression vector, which is then transformed into competent cells and then induced for expression. The expressed recombinant pC129R protein of the African swine fever virus is extracted and purified and then used as an antigen to immunize mice and screen out monoclonal antibodies against the pC129R protein of the African swine fever virus. The monoclonal antibody can specifically recognize and bind to the pC129R protein, which has important guiding significance for the development of vaccines based on the African swine fever virus.

[0017] 2. This application uses a prokaryotic expression system to express and purify a recombinant protein that reacts well with the positive standard serum of African swine fever virus antibodies, and establishes a detection method for detecting African swine fever virus antibodies based on quantum dot visualization. The method is coated with Staphylococcus aureus protein A on the detection line and anti-African swine fever virus pC129R protein monoclonal antibody on the quality control line. It has good specificity and sensitivity, greatly shortens the detection time, provides convenience for on-site detection, and provides a new idea for the construction of rapid visualization detection methods.

[0018] 3. The sensitivity of the test strips in this application can reach 1:10 7 It has good specificity, no cross reaction with porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, classical swine fever virus, pseudorabies virus, and porcine parvovirus, and has good stability.

[0019] 4. Based on the heavy chain variable region sequence and light chain variable region sequence of the monoclonal antibody disclosed in the present application, one or more amino acids can be added, deleted, replaced, etc. through conventional genetic engineering and protein engineering to obtain its active fragments or conservative variants, but it can still specifically bind to the African swine fever virus pC129R protein, laying the foundation for further modifying its antibody variable region sequence to prepare genetically engineered antibodies in different combinations, so as to further enhance the specificity of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the result of PCR electrophoresis identification of the African swine fever virus C129R gene in one embodiment of the present application.

[0021] Figure 2 This is the SDS-PAGE analysis of the induction and purification of the recombinant protein in one embodiment of the present application, wherein: A: M: protein marker; 1: whole bacteria without pC129R induction; 2: supernatant after pC129R induction; 3: precipitate after pC129R induction; B: M: protein marker; 1: before pC129R purification; 2: after pC129R purification.

[0022] FIG3 shows the principle and judgment criteria of a quantum dot test strip in one embodiment of the present application; A: the principle of the test strip; B: the judgment criteria of the test strip.

[0023] Figure 4 This is the sensitivity detection of the quantum dot test strip in one embodiment of the present application.

[0024] Figure 5 This is the specific detection of the quantum dot test strip in one embodiment of the present application. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described below in conjunction with examples. However, the following examples are only used to illustrate the present invention in detail and are not intended to limit the scope of the present invention in any way.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and materials involved are all commercially available conventional products unless otherwise specified; the tests and detection methods involved are all conventional methods unless otherwise specified.

[0027] Example 1. Expression and purification of African swine fever virus pC129R recombinant protein 1. Amplification of C129R gene: Referring to the coding sequence of the ASFV (Pig / HLJ / 2018 strain) gene in GenBank, upstream and downstream primers were designed to amplify the target gene. The C129R gene is shown in SEQ ID NO.1, the upstream primer of the C129R gene is shown in SEQ ID NO.2, and the downstream primer of the C129R gene is shown in SEQ ID NO.3. The composition of the amplification reaction system is: 1 μL template DNA, 2 μL of upstream and downstream primers, 25 μL of Tap DNA polymerase mix, and 20 μL of ddH2O; the PCR amplification program is: 95°C for 5 min, 95°C for 30 s; 58°C for 60 s; 72°C for 30 s; 30 cycles, 72°C for 5 min.

[0028] 2. Purify and recover PCR products: After amplification, use 1% nucleic acid gel electrophoresis to identify the products ( Figure 1 ), and finally, the PCR product with a band close to the target band was selected for purification and recovery using a DNA purification and recovery kit. The concentration of the recovered DNA solution was determined using NanoDrop 2000c and stored at -20°C for later use.

[0029] 3. Double enzyme digestion of target gene and vector: C129R gene and pET-32a vector were double enzyme digested in a 37℃ thermostat. The enzyme digestion reaction system consisted of: 2μg of vector and template gene, 1μL of EcoR I enzyme, 1μL of Xho I enzyme, 5μL of 10×CutSmart Buffer, and ddH2O, which was made up to 50μL. The ligation reaction system consisted of: 0.5μL of T4 ligase and 1μL of T4 buffer. The target fragment and vector were added in a molar ratio of 1:6 and made up to 10μL with ddH2O.

[0030] 4. Transform the target fragment into DH5α competent cells: Add the ligation product to the DH5α competent cells and mix gently, place on ice for 30 minutes, heat shock at 42℃ for 90 seconds, and place on ice for 5 minutes; add 900μL LB medium, place in a 37℃ incubator for 40 minutes (200rpm / min), centrifuge and take 100μL of bacterial solution to spread on an ampicillin-resistant plate, place in a 37℃ incubator for overnight incubation; pick a single colony to LB medium containing ampicillin resistance and place in a 37℃ incubator (200rpm / min) for 6 hours; take 1μL of bacterial solution as a template, and use the designed primers to perform PCR to identify the positive single clones screened out.

[0031] The strain with the correct sequence was preserved and expanded, and then the plasmid was extracted using a plasmid extraction kit and transformed into BL21 (DE3) competent cells. The successfully constructed Escherichia coli recombinant expression strain was named pET-32a-pC129R-BL21 (DE3) and stored in a -80°C refrigerator.

[0032] 5. Preliminary induced expression and identification of ASFV pC129R protein (1) Take out the pET-32a-pC129R-BL21 (DE3) expression bacteria from the -80℃ refrigerator and inoculate them into LB liquid culture medium (ampicillin resistance) at a ratio of 1:1000. Place them in a shaker and culture them at 37℃, 220 rpm overnight.

[0033] (2) Take out the bacterial solution from the above step from the shaker, inoculate it into LB liquid culture medium (ampicillin resistance) at a ratio of 1:100, place it in a shaker, and culture it at 37°C and 220 rpm for about 2 hours.

[0034] (3) When the OD600 value of the bacterial solution reaches between 0.6 and 0.8, add IPTG to a final concentration of 0.1 mmol / L, then place the culture in a shaker at 25°C and 220 rpm for 12 h.

[0035] (4) The next day, centrifuge at 12,000 rpm for 3 min, discard the supernatant, and collect the bacterial cells.

[0036] (5) Add 1 mL of Binding buffer for every 0.1 g of bacterial cells and resuspend the cells.

[0037] (6) The bacterial solution in the above step is subjected to ultrasonic disruption. To prevent overheating and denaturation of the protein, it needs to be placed in an ice-water mixture throughout the process and ultrasonicated until the bacterial solution is clear.

[0038] (7) After the ultrasound, precool the centrifuge at 4°C and centrifuge at 12,000 rpm for 10 min to separate the supernatant from the precipitate, and resuspend the precipitate with an equal volume of Binding buffer.

[0039] The SDS-PAGE identification steps are as follows: (1) The samples include the bacterial solution before induction, the supernatant after ultrasonic disruption, and the precipitate resuspension of pET-32a-pC129R-BL21 (DE3); the supernatant after ultrasonic disruption of pET-32a empty expression bacteria. Take 40 µL of each sample, add 10 µL of 5× loading buffer, mix evenly, boil in water for 10 min, then centrifuge at 12,000 rpm for 1 min, and take the supernatant.

[0040] (2) Add protein marker to the first well, then take 20 µL of the supernatant in step (1) for sample loading. After sample loading, connect the power supply, adjust the voltage to 180 V and run electrophoresis for 45 min.

[0041] (3) Use Coomassie Brilliant Blue R-250 staining solution to stain the protein gel until the protein gel turns dark blue, and then use destaining solution to destain until the background transparent bands are clear.

[0042] (4) Use a gel imager to scan the protein gel and take photos.

[0043] 6. Extraction and purification of African swine fever virus pC129R recombinant protein: centrifuge the cultured bacterial solution at 4°C, 12000rpm, discard the supernatant, resuspend the bacterial solution by adding 20mM Tris-HCl 150mM NaCl in a ratio of 20mM Tris-HCl 150mM NaCl to bacterial solution of 1:10, ultrasonicate on ice for 30min, disrupt by ultrasonication, centrifuge at 4°C, 12000rpm, take the supernatant, and purify the recombinant protein by nickel column affinity chromatography.

[0044] The specific steps of purifying recombinant protein by nickel column affinity chromatography are as follows: (1) Resuspend the cells at a ratio of 0.1 g of cells per 1 mL of 20 mM Tris-HCl 150 mM NaCl and then disrupt by ultrasonication. (2) Wash the nickel column with 10 to 20 column volumes of deionized water; (3) Equilibrate the column with 20 mM Tris-HCl 150 mM NaCl until the color of the protein indicator no longer becomes lighter; (4) Filter the supernatant after ultrasonic disruption with a 0.45 μm filter, then slowly add it to the column and perform column hanging multiple times (2 to 3 times); (5) Use 20 mM Tris-HCl 150 mM NaCl to wash away the proteins that are not bound to the nickel column until the color of the protein indicator no longer becomes lighter; (6) Use 20 mM Tris-HCl 150 mM NaCl (pH 8.25) containing 20, 40, 80, 160, 300, and 500 mM imidazole to gradually wash the nickel column and collect it in a 1.5 mL EP tube. When each gradient is washed until the color of the protein indicator no longer becomes lighter, change to the next gradient; (7) After purification, the nickel column was washed twice with deionized water, and then stored in 20% ethanol and placed at 4°C; (8) Use a dialysis bag to dialyze the eluted target protein in 10 volumes of dialysis buffer (20 mM Tris-HCl 150 mM NaCl, pH 8.25) at 4°C. After dialysis, measure the protein concentration and store it in aliquots at -80°C.

[0045] (9) SDS-PAGE identification: Take the ultrasonic supernatant before purification and the protein eluate after purification for sample processing. The specific method refers to the SDS-PAGE identification step in 5 (initial induction expression and identification of ASFV pC129R protein) above. Figure 2 ).

[0046] Example 2: Preparation of monoclonal antibodies against African swine fever virus pC129R protein 1. Immunization of BALB / c mice (1) An antigen composed of the recombinant protein of African swine fever virus pC129R was added to Freund's complete adjuvant and Freund's incomplete adjuvant respectively for emulsification to prepare Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen, wherein the volume ratio of the recombinant protein of African swine fever virus pC129R (25 μg / animal) to Freund's complete adjuvant and Freund's incomplete adjuvant was 1:1; (2) Two female BALB / c mice aged 6 to 8 weeks were immunized with Freund's complete adjuvant immunogen by multiple subcutaneous injections on the back, approximately 100 μL per mouse; (3) BALB / c mice were boosted with Freund's incomplete adjuvant immunogen using the same method and dose 14 and 28 days after the first immunization; (4) 14 days after booster immunization, blood was collected from the tail to measure the serum titer of mice; (5) 3-5 days before cell fusion, select mice with the highest serum titer and use the non-adjuvanted African swine fever virus pC129R recombinant protein to super-immunize BALB / c mice by intraperitoneal injection. The immunization dose is 50 μg / mouse 2. Determination of the titer of mouse serum The titer of mouse serum was determined by ELISA method. The specific steps are as follows: (1) Dilute the purified African swine fever virus pC129R recombinant protein to 2 μg / mL with coating solution, add 50 μL of coating solution to each well, incubate at 37°C for 2 h, discard the coating solution, and wash three times with PBST; (2) Block with 200 μL blocking solution (5% skim milk powder + PBST) at 4°C overnight; (3) Add 50 μL of the serum to be tested diluted 2-fold with dilution buffer (PBST) to each well (initial dilution factor is 1:400), incubate at 37°C for 1 h, discard the supernatant, and wash 6 times with PBST; (4) Add 50 μL of HRP-labeled goat anti-mouse IgG diluted 1:5000 in dilution buffer to each well, incubate at 37°C for 1 h, discard the supernatant, and wash 6 times with PBST washing solution; (5) Add 50 μL of TMB colorimetric solution to the wells, incubate at room temperature in the dark for 6-8 min, then add 50 μL of 2M H2SO4 stop solution to terminate the reaction; (6) Measure OD using an ELISA reader 450nm value.

[0047] The test results showed that the ELISA titer of mouse No. 1 could reach up to 1:512000, and mouse No. 1 was selected for cell fusion to prepare monoclonal antibodies.

[0048] 3. Cell fusion and culture (1) Preparation of spleen cells: Take No. 1 BALB / c mice that have been superimmunized for 5 days and kill them by cervical scissor lift. Disinfect the body surface with 75% alcohol and remove the mouse spleen aseptically. Wash it twice with GNK solution preheated at 37°C. Add a small amount of HAT culture medium and chop the spleen with small scissors on a sterile 120-mesh nylon gauze. Filter the spleen cells into a sterile beaker and transfer them to a sterile cell centrifuge tube. Centrifuge at 1000 r / min for 10 min. Wash the cells 1 to 2 times with GNK solution for later use.

[0049] (2) Cell fusion and culture of fused cells: Cell fusion was performed using the polyethylene glycol method. The spleen cells of immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 10:1. The fused cells were gently suspended in HAT selection medium and dispersed into a 96-well cell culture plate at 200 μL / well. The plates were cultured in a 37°C, 5% CO2 incubator. Small cell clusters could be observed under a microscope after 3 to 4 days of culture. HT medium was used for half-volume medium replacement 7 days after fusion. On the 10th day, 25 μL of cell culture supernatant was aspirated and initially screened using ELISA.

[0050] 4. Screening, identification and subcloning of hybridoma cells (1) ELISA screening: In the first round of ELISA screening, African swine fever virus pC129R recombinant protein (diluted with CBS 1:100) was used as the coating agent, and 22 ELISA plates were coated to perform preliminary screening of hybridoma cell culture supernatants; the 6 hybridoma cell lines initially screened were transferred to a 24-well cell plate for further culture.

[0051] In the second round of ELISA screening, African swine fever virus pC129R recombinant protein (diluted with CBS 1:100) and pET-32a-pC129R-BL21 ultrasonic supernatant (diluted with CBS 1:1000) were used as coating sources. After excluding false positives by His-tag recombinant protein detection, a total of 3 positive hybridoma cell lines were screened out.

[0052] The third round of ELISA screening was performed on the positive hybridoma cells in the same manner as the second round of screening. As a result, a total of 1 positive hybridoma cell line was screened out.

[0053] (2) Subcloning of positive hybridoma cells: Subclone the positive hybridoma cells by limiting dilution method: dilute the positive hybridoma cells to about 8 cel1s / ml with 1640 complete medium, add 100 μL per well to a 96-well plate pre-coated with 100 μL feeder layer cells, and culture in a 37°C, 5% CO2 incubator for 6-8 days before ELISA screening and identification. Subsequently, transfer the positive monoclonal cell line to a 24-well cell culture plate for expansion culture. If necessary, perform 2-3 subclones until a hybridoma cell line that stably secretes monoclonal antibodies against the African swine fever virus pC129R protein is obtained. The target hybridoma cell 3F4 can be obtained, and the positive monoclonal clone obtained by screening is expanded for culture.

[0054] 5. Preparation and purification of monoclonal antibodies against African swine fever virus pC129R protein The monoclonal hybridoma cell line 3F4 was expanded and cultured, and the titer of the culture supernatant was measured by ELISA to ensure the stability of the monoclonal cell line. The hybridoma cell line 3F4 was collected for mass preparation of monoclonal antibodies. The specific steps are as follows: (1) Select multiparous female BALB / c mice and inject 500 μL of sterilized paraffin intraperitoneally to stimulate immune cells to promote the proliferation of hybridoma cells; (2) Observe the condition of the mice and, after 7 to 10 days, add approximately 1×10 7 The monoclonal positive hybridoma cells prepared in advance were injected at a volume of 2x10 cells, and the mouse status was observed in time; (3) After 10 days, the ascites was extracted and centrifuged at 8000r / min and 4°C for 20 minutes to remove the fat and cell precipitate, and the ascites supernatant was collected and stored at -80°C for later use; (4) One week later, the monoclonal positive hybridoma cells obtained were injected intraperitoneally again, with an injection volume of 2x10 5 cells; (5) One week later, the abdomen of the mouse swells and the ascites is extracted. The supernatant is collected after centrifugation and the ascites is crudely extracted using the ammonium sulfate method; (6) Mouse IgG is purified using a protein A column to obtain high-purity 3F4 IgG.

[0055] 6. Determination of the variable region sequence of monoclonal antibodies: Extract RNA from monoclonal positive hybridoma cell lines and reverse transcribe it into cDNA. Use PCR to amplify the heavy chain variable region sequence and light chain variable region sequence of the monoclonal antibody.

[0056] According to the sequence characteristics of mouse monoclonal antibodies, the heavy chain variable region primer sequences were designed: P1: 5'-CAGGAGTCAGGACCTGAGCT-3'; P2: 5'-TGAGGAGACGGTGACCGTGG-3'.

[0057] Design light chain variable region primer sequences: P3: 5'-TCAGACACACTGCTGTTAT-3'; P4: 5'-GGATGTGGGAAGATGGATACAGT-3'.

[0058] The variable region sequences of monoclonal antibody 3F4 were prepared by molecular cloning technology and sent to General Bio (Anhui) Co., Ltd. for sequencing. The heavy chain variable region of monoclonal antibody 3F4 was determined to be shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain variable region of 3F4 deduced therefrom was shown in SEQ ID NO.5.

[0059] Example 3: Preparation of quantum dot-labeled African swine fever virus pC129R recombinant protein (1) Mix 1.25 μL of quantum dots with 4.8 μL of EDC solution (1 mg / mL) and incubate at 25°C in a shaker at 220 rpm in the dark for 30 min.

[0060] (2) Take 3.9 μL of pC129R recombinant protein (protein concentration is 0.2 mg / mL) and add it to a centrifuge tube. Then add 10.05 μL of BBS buffer (0.01 mol / L) and continue the reaction at 220 rpm in a shaker at 25°C for 3 h in the dark.

[0061] (3) After centrifugation at 4000 rpm for 5 min, the supernatant was collected to obtain the coupling product, which was stored at 4°C for later use.

[0062] Example 4. Preparation of quantum dot test strips for detecting antibodies to the pC129R protein of African swine fever virus The preparation process of the quantum dot test strip for detecting antibodies to the pC129R protein of African swine fever virus is as follows: The structure of the fluorescent immunochromatographic test paper prepared in this example is as follows Figure 3A As shown, the specific assembly process is as follows: (1) Preparation of the binding pad: Cut glass wool (commercial glass fiber wool, size 30 cm × 25 cm. The membrane surface should be clean, smooth and free of foreign matter) into 0.75 × 30 cm 2 The strips were placed on the XYZ 3060 sprayer platform and fixed with a layering strip; the binding pad buffer was evenly sprayed on the glass fiber cotton, and the mixture was placed in a 42°C drying oven for 50 minutes and taken out for use; 4.5 ml of quantum dot-labeled African swine fever virus pC129R recombinant protein was taken and the quantum dot-labeled recombinant protein solution was sprayed on the glass wool using AirjetQuanti 3060 at 8 l / cm; the mixture was placed in a 42°C drying oven for 50 minutes; finally, the binding pad was sealed and stored at room temperature (15-25°C) with a desiccant for later use.

[0063] The binding pad buffer was 20 mmol / L Na2B4O7 buffer containing 1% BSA, 0.1% Tween 20 (v / v), 3% (w / v) sucrose and 0.1% NaN3.

[0064] Preparation method of binding pad buffer: weigh 30g sucrose and 7.628g Na2B4O7 and dissolve them in 800ml distilled water, add 10g BSA and 1g NaN3, and after fully dissolving, measure 1ml Tween-20 and mix thoroughly, then make up to 1L with distilled water, filter and sterilize with a 0.22m filter, and store at 2~8℃.

[0065] (2) Preparation of sample pad: Cut the glass cellulose membrane into 1.5×30 cm 2 The strips were placed in the sample pad buffer and soaked for 5 minutes; dried in a 42°C drying oven for 4 hours; the sample pads were placed in a plastic bag, sealed with desiccant and stored at room temperature (15-25°C) for later use.

[0066] The sample pad buffer was a PBS (0.01 mol / L, pH 7.2) solution containing 0.5% Triton-100 (v / v) and 0.1% (w / v) sodium azide.

[0067] Preparation method of sample pad buffer: weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve in 800ml distilled water, adjust the pH value of the solution to 7.4 with HCl, then add 5ml Triton-100 and 1g sodium azide, and after fully dissolved, make up to 1L with distilled water, filter and sterilize with a 0.22m filter, and store at 2~8℃.

[0068] (3) Preparation of absorbent pad: Cut the absorbent filter paper with a size of 300 × 200 mm into 1.9 × 30 cm 2 Add desiccant and store in sealed container at room temperature (15~25℃) for later use.

[0069] (4) Use the BioDot spraying platform to spray 0.5 mg / mL purified monoclonal antibody against pC129R protein of African swine fever virus and SPA (1 mg / mL) onto the nitrocellulose membrane as T line and C line, respectively. After spraying, place the membrane in a drying oven and dry it at 37°C for 4 h.

[0070] (5) Assemble the pretreated conjugate pad, sample pad, absorbent pad and NC membrane eye in the plastic base plate in the length direction, with the edges of each part overlapping by about 2 mm. Finally, use the BioDot strip cutter to cut the assembled card into test strips with a width of 2.8 mm. Place the test strips in a tin foil bag containing desiccant and store them dry at room temperature.

[0071] Example 5. Determination of the results of quantum dot test strips for detecting antibodies to the pC129R protein of African swine fever virus Add the diluted serum to the sample tank and determine the test results after 10 minutes ( Figure 3B ). If the quantum dot-labeled African swine fever virus pC129R recombinant protein does not bind to the African swine fever virus pC129R protein monoclonal antibody on the quality control line (C line) on the test strip, then no band will appear on the C line, indicating that the test strip is invalid and cannot be effectively detected. If the quantum dot-labeled African swine fever virus pC129R recombinant protein can bind to the monoclonal antibody on the C line, a band will appear on the C line, indicating that the test strip is valid. However, if there are no African swine fever virus pC129R protein antibodies in the serum, these antibodies will not bind to the quantum dot-labeled African swine fever virus pC129R recombinant protein, so no band will appear on the test line T (T line), and the result is judged to be negative; if the serum contains African swine fever virus pC129R protein antibodies, these antibodies will bind to the quantum dot-labeled African swine fever virus pC129R recombinant protein, and then the quantum dot-labeled African swine fever virus pC129R recombinant protein bound to the antibody will bind to SPA on the test line T, causing the T line to develop color, and the result is judged to be positive.

[0072] Example 6. Performance measurement of quantum dot test strips for detecting antibodies to the pC129R protein of African swine fever virus 1. Sensitivity test: The prepared quantum dot test strips were used to test the sensitivity of the positive standard serum for African swine fever virus antibodies. The positive serum was diluted with water at a ratio of 1:10. 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 and 1:10 8 The results were as follows: Figure 4 As shown, serum was diluted to 1:10 7 The test strip can still see the T line at 1:10 8 When the serum is diluted by 100%, the T line band is so faint that it is almost invisible to the naked eye. This shows that the test strip has a high sensitivity and can detect the target antibody even at extremely high dilutions.

[0073] 2. Specificity detection: The quantum dot test strip prepared in Example 4 was used to detect positive serum of African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine parvovirus (PPV), so as to evaluate the specificity of the quantum dot test strip. The results are as follows Figure 5 As shown, the C line of the test strip has bands, proving that the test strip is effective; the T line of the African swine fever virus (ASFV) positive serum has bands, while the T lines of the other sera have no bands, and there is no non-specific situation, indicating that the test strip has good specificity.

[0074] Although some preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations to the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A monoclonal antibody against African swine fever virus pC129R protein, characterized in that: The heavy chain variable region includes CDRH1, CDR H2, and CDR H3, and the amino acid sequences thereof are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. A gene encoding the monoclonal antibody according to claim 1, characterized in that: The DNA sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.

7.

3. A hybridoma cell, characterized in that: It secretes a monoclonal antibody containing the heavy chain variable region described in claim 1.

4. Use of the monoclonal antibody according to claim 1 or the hybridoma cell according to claim 3 in the preparation of immunological detection reagents.

5. Use of the monoclonal antibody according to claim 1 or the hybridoma cell according to claim 3 in the preparation of antibodies or vaccines against African swine fever virus.

6. A quantum dot test strip for detecting antibodies to the pC129R protein of African swine fever virus, comprising a conjugate pad, a sample pad, and a base membrane with a detection line and a quality control line, characterized in that: The conjugate pad is bound to a quantum dot-labeled African swine fever virus pC129R recombinant protein, the detection line is coated with Staphylococcus aureus protein A, and the quality control line is coated with a monoclonal antibody against the African swine fever virus pC129R protein according to claim 1.

7. A method for preparing the quantum dot test strip according to claim 6, characterized in that: The steps include: (1) Preparation of binding pad: Cut glass wool into long strips and fix them, spray buffer on them, and then dry them at 40-45°C for 45-50 min. Then, spray 4-5 ml of quantum dot-labeled African swine fever virus pC129R recombinant protein on the glass wool and dry them at 40-45°C for 45-50 min. Finally, dry and seal the glass wool at room temperature for storage. The buffer is 18-22 mmol / L Na2B4O7 buffer containing 0.8-1.2% BSA, 0.08-0.12% Tween 20, 2.5-3.5% sucrose and 0.08-0.12% NaN3. (2) Sample pad preparation: Cut the glass cellulose membrane into long strips, soak it in a buffer solution for 4-5 min, then dry it at 40-45 °C for 3.5-4.5 h, and finally dry it and seal it for storage at room temperature; the buffer solution is 0.01-0.02 mol / L PBS solution containing 0.3-0.7% Triton-100 and 0.08-0.12% sodium azide; (3) Preparation of absorbent pad: Cut the absorbent filter paper into long strips and store them in a sealed container at room temperature for later use; (4) Preparation of nitrocellulose membrane: 0.3-0.5 mg / mL of African swine fever virus pC129R protein monoclonal antibody and 0.8-1.0 mg / mL of Staphylococcus aureus protein A were sprayed on the nitrocellulose membrane as T line and C line, respectively. After spraying, the nitrocellulose membrane was dried at 35-40°C for 3.5-4.5 h; (5) Assemble the pretreated binding pad, sample pad, absorbent pad and nitrocellulose membrane in sequence along the length direction of the plastic base plate, with the edges of each part overlapping by 1.5-2 mm. Finally, cut the assembled card into test strips with a width of 2.5-3 mm. Place the test strips in a dry tin foil bag and store them dry at room temperature.

8. The preparation method according to claim 7, characterized in that: In the step (1), the preparation method of the quantum dot-labeled African swine fever virus pC129R recombinant protein is as follows: first, the quantum dots and the EDC solution are mixed, and then the mixture is reacted at 20-30°C and 220 rpm in the dark for 25-35 minutes; then, the pC129R recombinant protein and the BBS buffer are added respectively, and the mixture is reacted at 20-30°C and 220 rpm in the dark for 2-3 hours; finally, the mixture is centrifuged at 3500-4500 rpm for 4-6 minutes, and the supernatant is taken to obtain the coupling product, which is stored at 2-4°C for later use.

9. The preparation method according to claim 8, characterized in that: The concentration of the EDC solution is 0.8~1.2 mg / mL; the concentration of the pC129R recombinant protein is 0.1~0.3 mg / mL; the concentration of the BBS buffer is 0.01~0.02 mol / L; the volume ratio of the quantum dots, the EDC solution, the pC129R recombinant protein, and the BBS buffer is 1~1.5:4.5~5.5:3.5~4.5:9~11.

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