CHO Cell Expression, Monoclonal Antibody Preparation, Epitope Identification and Application of African Swine Fever Virus D117L Encoded Protein

Abstract

The present invention relates to the expression of a CHO cell against the ASFVD 117L encoded protein, the preparation of monoclonal antibodies, epitope identification and application. By constructing a eukaryotic expression vector pCDNA3.4-D117L-strep, transiently transfecting CHO cells to express recombinant p17 protein, immunizing Balb / c mice after purification, fusing the spleen cells of mice with myeloma SP2 / 0 cells to prepare hybridoma cells after four immunizations, and then using the indirect ELISA method for subcloning to obtain monoclonal antibodies against p17 protein, and identifying the antigen epitope corresponding to the antibody as <supgt;8< / supgt;LLSHNLSTREGIK<supgt;20< / supgt>.

Description

Technical Field

[0001] The present invention relates to the field of biology, and specifically provides the CHO cell expression of a porcine African swine fever virus D117L encoded protein, the preparation of monoclonal antibodies, epitope identification and applications thereof. Background Art

[0002] African swine fever (ASF) is an acute, febrile and highly contagious disease caused by African swine fever virus (ASFV), which often spreads across borders. The disease was first reported in 1921 and is prevalent in sub-Saharan Africa. In 2007, the disease broke out in Georgia and then rapidly spread to Russia. In 2014, the disease was introduced into most countries in Eastern Europe and showed an expanding epidemic trend. The World Organization for Animal Health listed it as a notifiable animal disease. China listed it as a class of animal infectious diseases under key prevention. At present, there is no commercial vaccine for the prevention and control of this disease. On August 3, 2018, the first case in China was reported in Shenyang, and since then the disease has brought heavy losses to the pig industry in China.

[0003] ASFV is an enveloped DNA virus with an icosahedral structure. The genome sizes of different strains vary slightly, about 170 - 190 kb, containing at least 150 open reading frames and encoding at least 200 viral proteins. Among them, proteins related to virus replication, immune escape, pathogenic transmission are of key concern, including p12 (O61R), p17 (D117L), p30 (CP204L), p54 (E183L), p72 (B646L), etc. The ASFV D117L coding sequence is highly conserved among strains. The encoded inner envelope protein p17 plays a crucial role in the step of forming an icosahedron during virus packaging, can affinity with the endoplasmic reticulum of cells, and participates in the formation of virus particles. When the expression of p17 is blocked, it can significantly inhibit the hydrolysis of viral pp220 and pp62, affecting the formation and assembly of virus particles.

[0004] The previous functional studies on the ASFV D117L encoded protein mainly focused on its effects on virus particle formation, virus replication, etc. The reports on its antigenic epitopes are still lacking. The present invention successfully prepared a monoclonal antibody against the ASFV D117L encoded protein by cell fusion and subcloning screening, and identified the antigenic epitope recognized by it, and found that it specifically recognizes the 8 - 20 peptide segment of the ASFV p17 protein ( 8 LLSHNLSTREGIK 20) Software analysis was used to confirm that the peptide sequence is conserved among different ASFV strains. This invention not only provides basic knowledge for vaccine development, but also has the diagnostic potential to identify epitopes. Experiments have confirmed that this monoclonal antibody has strong specificity, laying a good foundation for the research on ASF vaccines and the establishment of highly sensitive detection methods.

[0005] The use of mammalian cell expression systems to express foreign genes has been widely applied in the research and development of various biological products, such as the preparation of monoclonal antibodies, interferons, growth factors, and viral vaccines. Compared with prokaryotic expression systems, the foreign proteins expressed by mammalian cell expression systems have higher biological activities, have efficient and accurate post-translational modification functions, and the recombinant proteins expressed are closest to natural proteins in terms of molecular structure, physical and chemical properties, and biological functions. CHO cells are easy to perform suspension cell culture in serum-free and chemically defined media, with high levels of foreign protein expression, which is conducive to industrial production. This invention uses the CHO cell suspension system to express and purify the ASFV D117L-encoded protein, prepare specific monoclonal antibodies, identify the specific antigen epitopes recognized by them, and establish an indirect ELISA method for ASFV p17 antibodies, providing a basis for subsequent research on the functions of ASFV p17 proteins. Summary of the Invention

[0006] In view of the existing gaps in the prior art, this invention provides a monoclonal antibody against the ASFV D117L-encoded protein, its preparation method and application. The technical solution of this method is: clone the ASFV D117L gene sequence into the pcDNA3.4 vector to construct a eukaryotic expression vector, use the transient transfection CHO mammalian expression system, purify the protein through the downstream strep tag, immunize Balb / c mice with the purified p17 protein as an antigen, take mouse spleen cells and myeloma cells SP2 / 0 cells for fusion to prepare hybridoma cells, coat the ELISA reaction plate with the purified p17 protein, and screen positive hybridoma cells that can specifically recognize the p17 protein through the indirect ELISA method, and identify the antigen epitopes recognized by them.

[0007] The specific invention is: obtaining 1 strain of monoclonal antibody that can specifically recognize the ASFV p17 protein, named 1B4. This monoclonal antibody can be used for IFA and Western blotting detection, and can well recognize the ASFV p17 protein. Subtype identification shows that the heavy chain of this monoclonal antibody is IgG1 and the light chain is the Kappa chain. By expressing the p17 protein in segments through the prokaryotic expression system and synthesizing a series of p17 protein short peptides, it was finally determined that this monoclonal antibody can specifically recognize the 8-20 peptide segment of the p17 protein, and the amino acid sequence it recognizes is 8 LLSHNLSTREGIK 20, as shown in SEQ ID NO.1. Description of the Drawings

[0008] Figure 1 It is the PCR amplification electrophoresis map of the ASFV D117L gene sequence fragment in Example 1 of the present invention. Among them, Lane 1 is the DNA standard DL2000, and Lane 2 is the ASFV D117L gene sequence fragment, indicating that the ASFV D117L fragment with the correct size has been successfully obtained.

[0009] Figure 2 It is the identification of the transient expression of the ASFV D117L-encoded protein in CHO by Coomassie Brilliant Blue staining in Example 2. Among them, Lane M is the protein molecular weight standard, Lane 1 is the untransfected cell control, Lanes 2-4 are the culture supernatant collected 1, 3, and 5 days after transfection, and Lanes 5-7 are the cell ultrasonic supernatant collected 1, 3, and 5 days after transfection. The identification results show that the protein is mainly expressed in the cell ultrasonic supernatant, and the expression level gradually increases with the increase of transfection time. The band is clear and the size is about 17 kDa, which is consistent with the expected size of the p17 protein.

[0010] Figure 3 It is the result of Western blotting identification of the transfected cell sample and the purified protein using the strep-tag antibody in Example 2. Among them, Lane M is the protein molecular weight standard, Lane 1 is the culture supernatant collected 5 days after transfection, Lane 2 is the cell ultrasonic supernatant collected 1 day after transfection, Lane 3 is the cell ultrasonic supernatant collected 3 days after transfection, Lane 4 is the cell ultrasonic supernatant collected 5 days after transfection, and Lane 5 is the purified p17 protein, indicating that the protein can specifically react with the strep tag and is the target protein with the correct size, which can be used as an antigen for immunization.

[0011] Figure 4 It is the IFA identification result of detecting the overexpressed p17 protein in 293T cells with the 1B4 monoclonal antibody in Example 5, indicating that the antibody can specifically recognize the ASFV p17 protein.

[0012] Figure 5 It is the Western blotting identification result of detecting the overexpressed p17 protein in 293T cells with the 1B4 monoclonal antibody in Example 5, indicating that the antibody can specifically recognize the ASFV p17 protein. Among them, Lane M is the protein molecular weight standard, Lane 1 is the 293T cells transfected with the ASFV D117L eukaryotic expression plasmid, and Lane 2 is the 293T cells transfected with the empty vector control.

[0013] Figure 6It shows the detection of the 1B4 monoclonal antibody subtype in Example 5, indicating that the heavy chain of this antibody is IgG1 and the light chain is Kappa chain.

[0014] Figure 7 It is the Western blotting figure for the preliminary screening of the 1B4 monoclonal antibody epitope in Example 6, indicating that the antigen epitopes recognized by this antibody all contain 8 LLSHNLSTREGIK 20 sequence.

[0015] Figure 8 It is the ELISA identification figure for the minimum antigen epitope in Example 6, indicating that the minimum epitope peptide recognized by the 1B4 monoclonal antibody is 8 LLSHNLSTREGIK 20 . Specific implementation manners

[0016] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further illustrates the present invention in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0017] Example 1 Construction of recombinant eukaryotic expression vector pcDNA3.4-D117L-strep

[0018] The inventor first constructed the pCDNA3.4-D117L-strep eukaryotic expression plasmid, and designed the primers as follows:

[0019] D117L-S-F: 5′-ATGGACACAGAAACATCACC-3′(SEQ ID NO.2)

[0020] D117L-S-R: 5′-TTATTTTTCGAACTGCGGGTGGCTCCAAGAATGTGCCAGCTCCGCCA-3′(SEQID NO.3)

[0021] The above primers are all ASFV D117L-specific sequences analyzed by BLAST, and the primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The template was the D117L gene sequence of the ASFV SY18 strain (GenBank: MH766894.2) in NCBI and was ligated into the pcDNA3.4 vector. After sequencing and comparison, it was completely consistent with the ASFV D117L gene sequence and was named pcDNA3.4-D117L-strep. The specific process is as follows:

[0022] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 65°C for 30 s; extension at 72°C for 1 min. There were 35 cycles from denaturation to extension. Final extension was at 72°C for 10 min. After electrophoresis, observation and photography were carried out under ultraviolet light. The synthesized plasmid was identified by PCR using the designed primers. After electrophoresis, observation was carried out under ultraviolet light, and specific bands of approximately 354 bp in size were amplified respectively ( Figure 1 ), and the results were consistent with the expectations.

[0023] Example 2 Transient transfection and protein purification of recombinant eukaryotic expression vector pcDNA3.4-D117L-strep

[0024] One day before transfection, the number of CHO cells was diluted to 3×10 6 cells / mL for inoculation, and their suspension growth was maintained for 24 h. On the day of transfection, the CHO cells were counted, and transfection could continue only when the survival rate was 95 - 99%. The cells were diluted with freshly preheated medium at 37°C to a final density of 6×10 6 cells / mL. The transfection system was prepared in units of 100 mL cell volume. 100 μg plasmid DNA was diluted with 4 mL OptiPRO TM SFM and slowly mixed evenly; 100 mg PEI transfection reagent was diluted with 4 mL OptiPRO TM SFM and slowly mixed evenly. 4 mL of the diluted PEI was added to 4 mL of the diluted plasmid DNA. The tube was slowly inverted for mixing, and the complex was incubated at room temperature for 20 minutes. The incubated complex was slowly added dropwise to 100 mL of the cell suspension, and it was placed in a shaking incubator at 37°C, 8% CO2, and 100 r / min for suspension culture. After 18 - 22 hours, 600 μL of enhancer and 24 mL of excipient were added, and the culture was continued in the shaking incubator for 5 days. After 5 days, the cells were centrifuged, and the culture medium was collected. At the same time, the cells were sonicated and then centrifuged, and the supernatant was collected. The collected samples were subjected to SDS-PAGE gel electrophoresis to identify the protein expression situation ( Figure 2 ). Subsequently, the target protein was purified using the strep tag. The general process was as follows: the collected culture medium, the cell sonicated supernatant, and the pre-treated strep beads were incubated with shaking at 4°C for 3 h. The medium was washed with a PBST washing solution containing 8.8 g NaCl and 20 mL 1M Tris-HCl. Finally, elution was carried out using biotin, and the eluate was collected for SDS-PAGE gel electrophoresis and transferred to an NC membrane for Western blotting identification ( Figure 3 ).

[0025] Example 3 Animal immunization

[0026] Five 4-week-old female Balb / c mice were immunized with purified p17 protein. Another group of five mice was used as a negative control. Before immunization, the concentration of the purified protein was measured. 50 μg of the purified protein was mixed with MnJ(β) colloidal manganese adjuvant and immunized by multi-point injection into muscle, subcutaneous and abdominal cavities. The second immunization was carried out 10 days after the first immunization, and the third and booster immunizations were carried out 7 days apart. The methods and dosages were the same as those in the first immunization.

[0027] Example 4 Preparation of Monoclonal Antibody Against ASFV p17 Protein

[0028] (1) Resuscitation and Culture of SP2 / 0 Cells

[0029] About 5 days before fusion, resuscitate SP2 / 0 cells and adjust the cell density. Specific method: Take out the cryopreserved SP2 / 0 cells from liquid nitrogen, put them into a sealed bag, and quickly put them into a 37 °C water bath and shake to dissolve. After complete dissolution, centrifuge at 1000 rpm for 5 min, aspirate and discard the supernatant in a biosafety cabinet, disperse and mix the cell pellet with 1 mL of medium, spread it evenly in a cell culture dish, add medium to 10 mL, and culture at 37 °C until the cells adhere to the wall, then replace with fresh culture medium.

[0030] (2) Preparation of Feeder Cells

[0031] One day before fusion, collect the serum from the control group mice by eye bleeding as a negative control, and then sacrifice the mice and soak them in 75% alcohol for disinfection. Fix the mice abdomen-up on a sterilized foam board in a biosafety cabinet, use forceps to pick up the abdominal skin and cut a small hole in the skin at the position of the fat layer under the mouse abdomen, then use forceps and scissors to tear the skin from the small hole to expose the abdominal cavity. Inject 5 mL of DMEM culture medium into the abdominal cavity. Gently tap both sides of the peritoneum with forceps to suspend the mouse peritoneal macrophages in the culture medium, and aspirate the culture medium after several taps. Repeat this step, and divide the aspirated culture medium into 96-well plates, 100 μL per well. Culture in an incubator at 37 °C and 5% CO2. The next day, observe the growth state of the feeder cells. If the state is good, fusion can be carried out.

[0032] (3) Preparation of Spleen Cells

[0033] Collect the serum from the immunized group mice by eye bleeding as a positive control, and then sacrifice the mice. Immerse the sacrificed mice in 75% alcohol for 5 min, fix the mice well, gently cut the abdominal skin of the mice with a sterilized scissors, and aseptically remove the spleens of the immunized mice and put them into a culture dish. Place a 40 μm cell sieve on a 50 mL centrifuge tube, grind the spleen with a disposable grinding rod, drip DMEM to wash the spleen, and collect the grinding liquid and centrifuge at 1000 rpm for 5 min.

[0034] (4) Fusion of hybridoma cells

[0035] Mix the SP2 / 0 myeloma cell suspension and the immune spleen cell suspension in a ratio of 1:3 and place them in a 50 mL centrifuge tube. Add DMEM to make up to 30 mL. After thorough mixing, centrifuge the cells at 1000 rpm for 10 min, discard the supernatant, gently tap the bottom of the tube to disperse the cell precipitate into a paste. Place the centrifuge tube containing spleen cells and SP2 / 0 cells in a foam box with warm water at 37 °C. While rotating the centrifuge tube, slowly drip 1 mL of PEG2000 preheated in a 37 °C incubator. After adding it within 1 min, let it stand for 2 min. Then slowly add DMEM basal medium to dilute PEG to terminate the fusion, and centrifuge at 1000 rpm for 10 min. Resuspend the cells with preheated HAT medium containing 20% serum at 37 °C, and add them to a 96-well cell culture plate with feeder cells at a volume of 100 μL / well. Place it in a 37 °C, 5% CO2 incubator for culture. Observe the cell fusion situation 5 days after fusion and appropriately supplement the HAT medium. 10 days after fusion, carefully aspirate 50 μL of cell culture supernatant with a multi-channel pipette, and screen for positive hybridoma cell wells that can secrete antibodies by the indirect ELISA method.

[0036] (5) Screening of positive hybridoma cells

[0037] Use the fusion cell supernatant as the primary antibody, the SP2 / 0 cell supernatant as the negative control, and the mouse serum before fusion as the positive control. Use HRP-labeled goat anti-mouse IgG as the secondary antibody. The specific implementation method is as follows: Coat the ELISA microplate with purified p17 protein, with a coating amount of 200 ng / well, 100 μL per well, and coat overnight at 4 °C. Add 200 μL of 5% skim milk to each well and block at 37 °C for 2 h. After washing 3 times with PBST containing 5‰ Tween-20, take 100 μL of cell supernatant and add it to the microplate coated with the protein and mix well. After incubating in a 37 °C incubator for 1 h, wash 3 times with PBST containing 5‰ Tween-20, 5 min each time. Then add 100 μL of the secondary antibody diluted at a ratio of 1:6000, incubate at 37 °C for 30 min, and wash 3 times with PBST containing 5‰ Tween-20. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 15 min. Finally, add 50 μL of 2M H2SO4 to each well to terminate the reaction, and measure the OD 450 value. Select the positive cell well with the highest OD value for expansion culture, and perform 3 subclonings. Each subcloning is screened by the above indirect ELISA method. Select the hybridoma cell line 1B4 that stably secretes the p17 antibody for expansion culture and prepare ascites.

[0038] (6) Preparation of ascites

[0039] Intraperitoneally inject 8-week-old Balb / c female mice with Freund's incomplete adjuvant 7 days in advance, 0.2 mL for each mouse. One week later, resuspend the well-growing hybridoma cells, transfer them into a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 min. After resuspending the cell pellet with PBS, intraperitoneally inject the mice, 10 6 cells for each mouse. Seven days later, the abdominal cavities of the mice are distended. Collect the ascites, centrifuge it, and aspirate the supernatant, then store it at -80 °C.

[0040] Example 5: Specificity detection and subtype identification of monoclonal antibodies

[0041] To verify the specificity of the monoclonal antibody, the pCDNA3.4 empty vector control and the pCDNA3.4-D117L-strep eukaryotic expression plasmid were transfected into 293T cells respectively for IFA and Western blotting analysis. The IFA results showed that the 1B4 monoclonal antibody could specifically recognize the p17 protein expressed in 293T cells showing red fluorescence, and the strep-tag antibody could specifically show green fluorescence, while there was no fluorescence in the control group ( Figure 4 ). The Western blotting results showed that the 1B4 monoclonal antibody could recognize a specific protein band with a molecular weight of approximately 17 kDa, and the band was clear ( Figure 5 ). Further, the subtype of the antibody was identified according to the Proteinech monoclonal antibody subtype identification kit, and the results showed that the heavy chain of this antibody was IgG1 and the light chain was Kappa chain ( Figure 6 ). Subsequently, the collected ascites was purified using Protein G, and the titer of the purified 1B4 monoclonal antibody was detected by ELISA method to be 1:128000.

[0042] Example 6 Identification of the epitope recognized by the p17 monoclonal antibody

[0043] First, divide p17 (1-354 bp) into two segments (P1: 4-210 bp; P2: 187-354 bp), construct expression plasmids according to the primers in Table 7 (SEQ NO: 4-7), and the schematic diagram of the segmentation is shown in Figure 7A. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 65°C for 30 s; extension at 72°C for 40 s, with 35 cycles set from denaturation to extension; final extension at 72°C for 10 min. The fragments were respectively ligated to the pCold-TF vector, positive clones were selected after transformation with BL21, single colonies were picked for verification, and after correct verification, induction expression was carried out. The bacteria were cultured in a shaker at 37°C until the OD value reached 0.6 - 0.8, then 1 mM IPTG was added for induction expression. After induction at 16°C for 24 h, the bacterial liquid was collected for ultrasonic treatment. The results showed that the epitope of monoclonal 1B4 was located within the P1 region (4 - 210 bp)( Figure 7 B). Then, the P1 fragment was further truncated into three segments (P1-1: 4 - 81 bp; P1-2: 58 - 144 bp; P1-3: 121 - 210 bp), and according to the primers in Table 7 (SEQ NO: 8 - 13), amplification, induction expression and identification were carried out as described above. The results showed that the epitope of the monoclonal antibody 1B4 was located within the P1-1 region (4 - 81 bp)( Figure 7 C).

[0044] To further accurately identify the epitope of the 1B4 monoclonal antibody, 9 different truncated polypeptides (SEQ NO: 14 - 22) were synthesized by Gildo Peptide Biotechnology Co., Ltd., and indirect ELISA identification was carried out by coating the ELISA plates with these 9 polypeptides respectively. The results showed that the minimum epitope recognized by the 1B4 monoclonal antibody for the p17 protein was the amino acid peptide segment at positions 8 - 20, and its sequence was 8 LLSHNLSTREGIK 20 ( Figure 8 ).

[0045] Example 7 Establishment and condition optimization of the p17 indirect ELISA method

[0046] (1) Determination of the optimal coating concentration of the antigen and the optimal dilution degree of the serum

[0047] The p17 protein was diluted to different concentrations for coating, and the ASFV positive serum and negative serum were diluted from 50 - fold to 400 - fold respectively. The results of the checkerboard titration showed that the optimal coating amount of the antigen was 400 ng / well, and the dilution degree of the serum was 1:100. At this time, the OD 450 value difference between the positive serum and the negative serum was the largest (P / N = 25.877).

[0048] Table 1 Determination of the optimal coating concentration of the antigen and the optimal dilution degree of the serum

[0049]

[0050] (2) Optimization of the optimal coating solution, the optimal blocking solution and the blocking time

[0051] Coat an ELISA plate with 400 ng / well using different coating solutions, and block the coated ELISA plate with different blocking solutions for different periods of time. The results show that when the antigen is coated with 0.05 M carbonate coating solution and blocked with 5% BSA at 37 °C for 2 h, the P / N value is the largest (P / N = 22.458), which is the optimal condition.

[0052] Table 2 Determination of the optimal antigen coating solution and blocking conditions

[0053]

[0054] (3) Optimization of coating temperature, time and secondary antibody dilution

[0055] Using 0.05 M carbonate coating solution, coat the antigen under the conditions of 37 °C for 1 h, 37 °C for 2 h, 37 °C for 4 h, and overnight at 4 °C respectively. The experimental results show that the P / N value is the largest when coated overnight at 4 °C; Dilute the enzyme-labeled secondary antibody to 6×10 3 、1×10 4 、2×10 4 、4×10 4 times respectively, incubate at 37 °C for 1 h, and select the optimal dilution multiple of the enzyme-labeled secondary antibody. The test results show that when diluted 2×10 4 times, the P / N value is the highest (P / N = 26.228), so the optimal dilution multiple of the secondary antibody is determined to be 2×10 4 .

[0056] Table 3 Determination of the optimal antigen coating method and secondary antibody dilution

[0057]

[0058] (4) Optimization of the optimal reaction time between serum and enzyme-labeled secondary antibody

[0059] Conduct experiments according to the above determined conditions. Incubate the serum for 20 min, 30 min, 45 min, and 60 min respectively. After washing 3 times, incubate the diluted enzyme-labeled secondary antibody for 20 min, 30 min, 45 min, and 60 min respectively. According to the OD 450 measurement results, calculate the P / N value, and determine that the optimal serum incubation time is 30 min and the optimal secondary antibody incubation time is 45 min.

[0060] Table 4 Determination of the optimal serum reaction time and optimal secondary antibody reaction time

[0061]

[0062]

[0063] (5) Optimization of chromogenic temperature and time

[0064] Under the above conditions, during the final color development, ELISA assays were performed at room temperature and 37 °C, respectively, with color development times of 5 min, 10 min, and 15 min. The analysis of the experimental results showed that the optimal substrate color development temperature and time was 37 °C for 15 min, at which the P / N value could reach 21.746.

[0065] Table 5 Determination of the optimal substrate color development conditions

[0066]

[0067] (6) Establishment of the indirect ELISA cut-off value

[0068] Twenty-seven ASFV-negative serum samples with known backgrounds stored in this laboratory were taken and detected under the optimized indirect ELISA experimental conditions. Statistical analysis was performed on the results, and the calculated average value was 0.083 and the standard deviation was 0.015. Serum with OD 450 ≥ X + 3SD = 0.146 was judged as positive; when OD 450 ≤ X + 2SD = 0.127, the serum was judged as negative, and the sera between the two were suspicious sera.

[0069] Table 6 ELISA test results of 27 porcine negative sera

[0070]

[0071] Table 7 Primer sequences used in this invention patent

[0072]

[0073]

[0074] In summary, the present invention provides the expression of a CHO cell, monoclonal antibody preparation, epitope identification and application against the ASFV D117L-encoded protein. By cloning the ASFV D117L-encoding sequence into the pcDNA3.4 vector, a eukaryotic expression vector carrying a strep tag and capable of expressing the ASFV p17 protein was constructed, transiently transfected into CHO cells, and the ASFV p17 protein was obtained by the method of purification using the strep tag. Using the purified protein as an immunogen to immunize Balb / c mice, monoclonal antibodies against ASFV p17 were obtained through cell fusion and subcloning screening. The identification results showed that the antibody subtype prepared was IgG1 for the heavy chain and Kappa for the light chain. The results of IFA and Western blotting analysis showed that the obtained monoclonal antibodies could specifically recognize the ASFV p17 protein and could be used to detect the ASFV p17 antigen under reducing conditions. The epitope sequence recognized by it is8 LLSHNLSTREGIK 20 , further proving the specificity of the antibody.

[0075] It should be understood that although the content of the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made thereto, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. An antigenic epitope peptide of the porcine ASFV D117L encoded protein, wherein the antigenic epitope peptide is the 8-20 peptide segment of the porcine p17 protein, and its amino acid sequence is LLSHNLSTREGIK.

2. An antigen composition comprising at least one antigenic peptide, wherein the at least one antigenic peptide is an antigenic epitope peptide of a porcine ASFV D117L encoded protein as described in claim 1.

3. Use of an antigenic epitope peptide of a porcine ASFV D117L encoded protein according to claim 1 and the antigen composition according to claim 2 in the preparation of an ELISA antibody detection kit for diagnosing or detecting ASFV.

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