Preparation and application of single-chain antibody of African swine fever virus P30 protein

Abstract

The invention belongs to the technical field of biology, and particularly relates to an African swine fever virus P30 protein single-chain antibody and preparation and application thereof. The invention provides the single-chain antibody 7B9 aiming at the African swine fever virus P30 protein, the single-chain antibody can specifically react with the African swine fever virus P30 protein, and the single-chain antibody has the advantages of good sensitivity, strong antigen binding capacity, strong biological activity and the like; the single-chain antibody 7B9 is applied to establishment of an African swine fever virus blocking ELISA antibody detection method, and an effective detection method is provided for prevention and control of ASF; the single-chain antibody provided by the invention is recombined and modified by a gene engineering method, and is optimized on gene and protein levels, so that the long-term cryopreserved antibody loss of hybridoma cells is avoided, the specificity and affinity of the antibody are improved, and compared with the traditional monoclonal antibody, the scFv-7B9 antibody is lower in preparation cost and higher in specificity. The method is suitable for short-term and rapid large-scale fermentation preparation, and has high cost performance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation of a murine African swine fever virus P30 protein single-chain antibody 7B9 and its application in the detection method of African swine fever virus P30 protein blocking ELISA antibody. Background Technology

[0002] African swine fever (ASF) is an acute, highly contagious, hemorrhagic infectious disease caused by the African swine fever virus (ASFV). It primarily infects domestic and wild pigs, and its transmission routes include direct contact, transmission through contaminated objects, and vector-borne transmission such as soft ticks. Infected domestic pigs exhibit symptoms such as high fever, rapid heartbeat, difficulty breathing, cyanosis of the skin, internal organ hemorrhage, and swollen lymph nodes, with a mortality rate approaching 100%. ASFV is the only member of the African swine fever virus family and is currently the only known arbovirus. It has an enveloped structure, and the virus particle is icosahedral, with a diameter of 175 to 215 nm. The African swine fever virus is a double-stranded DNA virus with a large genome encoding more than 150 proteins, more than 50 of which are packaged into the viral particle and participate in viral replication, host immune evasion, and pathogenicity.

[0003] Due to the complex immune escape mechanism and high variability of ASFV, there are currently no effective commercially available vaccines or treatments worldwide. Therefore, early, rapid, and accurate diagnostic techniques are crucial for ASF prevention and control. Among the many structural proteins of ASFV, p30, p54, and p72 have become major targets for serological diagnosis due to their good immunogenicity and high conservation. The p30 protein, encoded by the CP204L gene, has a molecular weight of approximately 23.6 kDa and is located in the viral inner envelope. It is one of the important proteins expressed in the early stages of ASFV infection. p30 can be detected as early as 2 hours after viral adsorption to host cells and persists throughout the infection cycle. It can induce the body to produce neutralizing antibodies, which can be detected 7–10 days after infection, exhibiting a relatively long duration. Compared with other proteins, p30 has higher antibody detection efficiency and can significantly improve the differentiation between negative and positive sera, thus being considered an ideal target for ASFV antibody detection.

[0004] Blocking ELISA is a detection method based on the competitive binding of antigen and antibody. It works by having an antibody in the sample competitively bind to and immobilize the antigen with an enzyme-labeled antibody, thereby inhibiting signal generation. By detecting changes in the enzyme reaction signal, the presence of virus-specific antibodies in the serum can be determined. This method not only effectively avoids cross-reactions but also detects antibodies in the early stages of infection. scFv (antibody fibroblasts) are composed of variable regions of the antibody heavy and light chains linked by short peptides. They have a small molecular weight, are easily genetically engineered, are low-cost, highly specific, highly reproducible, and widely used, making them widely applicable in diagnostic testing. Using HRP-labeled single-chain antibodies as the enzyme-labeled competitive antibody in blocking ELISA can reduce cross-reactions, improve detection sensitivity and accuracy, and the homogeneity and stability of the single-chain antibody ensures reproducible experimental results and batch-to-batch stability of the test reagents, helping to reduce the preparation cost of the kit. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention has obtained a single-chain antibody 7B9 against African swine fever virus through screening and optimized recombination. This antibody exhibits good biological activity, strong antigenic site blocking effect, and low preparation cost. Using the single-chain antibody 7B9 and coated with recombinant P30 protein, an ELISA antibody detection method for blocking African swine fever virus was established. This method can be used for serum detection of African swine fever virus infection, providing a reference and technical support for the differential diagnosis of African swine fever.

[0006] Specifically, it includes the following:

[0007] In a first aspect, the present invention provides a single-chain antibody against the African swine fever virus P30 protein, the single-chain antibody against the African swine fever virus P30 protein comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; the light chain variable region comprising CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No. 6.

[0008] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0009] Preferably, the heavy chain variable region and the light chain variable region are linked by a linker peptide.

[0010] Preferably, the amino acid sequence of the linker peptide is shown in SEQ ID NO.9.

[0011] Secondly, the present invention provides a nucleic acid molecule that encodes a single-chain antibody against the African swine fever virus P30 protein described in the first aspect above.

[0012] Preferably, the nucleic acid includes the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID No. 10, the nucleotide sequence encoding the light chain variable region shown in SEQ ID No. 11, and the nucleotide sequence encoding the linker peptide shown in SEQ ID No. 12.

[0013] Thirdly, the present invention provides a recombinant vector comprising the nucleic acid described in the second aspect above.

[0014] Fourthly, the present invention provides a recombinant cell, characterized in that the recombinant cell comprises the nucleic acid described in the second aspect above, or the recombinant vector described in the third aspect above.

[0015] Fifthly, the present invention provides a single-chain antibody against the African swine fever virus P30 protein described in the first aspect above, which has any of the following uses:

[0016] (1) Application in the preparation of African swine fever virus detection reagents;

[0017] (2) Application in the detection of African swine fever virus for non-disease diagnosis purposes.

[0018] In a sixth aspect, the present invention provides an African swine fever virus P30 protein blocking ELISA antibody kit, the kit comprising the African swine fever virus P30 protein single-chain antibody described in the first aspect above.

[0019] Preferably, the kit further includes a coated antigen, an enzyme-labeled plate, a blocking solution, a diluent, a washing solution, a chromogenic agent, and a stop solution.

[0020] In a seventh aspect, the present invention provides a method for preparing the African swine fever virus P30 protein single-chain antibody as described in the first aspect above. The method comprises: linking the heavy chain variable region and light chain variable region sequences of the African swine fever virus P30 protein single-chain antibody using a linker peptide and inserting them into an expression vector to obtain a scFv recombinant plasmid; transforming the obtained recombinant expression plasmid into E. coli BL21(DE3) cells, and screening and culturing to obtain the African swine fever virus P30 protein single-chain antibody.

[0021] The beneficial effects of this invention are as follows: This invention uses recombinant ASFV P30 protein expressed in a prokaryotic expression system as an immunogen to immunize mice. Through screening and construction optimization, a single-chain antibody 7B9 targeting the African swine fever virus P30 protein was successfully obtained. The prepared single-chain antibody 7B9 can specifically react with the African swine fever virus P30 protein, and has the advantages of high specificity, sensitivity, and low preparation cost. Through recombinant modification using genetic engineering methods, antibody loss during long-term cryopreservation of hybridoma cells is avoided. At the same time, the antibody is optimized at the gene and protein levels, improving the antibody's specificity and affinity. This provides new materials for the detection of African swine fever virus serum antibodies and also provides new technical support for controlling the spread of African swine fever. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 Results of amplification of the variable region of single-chain antibody 7B9;

[0024] Figure 2 Results of single-chain antibody 7B9 titer assay;

[0025] Figure 3 Affinity identification results for single-chain antibody 7B9;

[0026] Figure 4 Specificity identification results of single-chain antibody 7B9;

[0027] Figure 5 Sensitivity validation results of the ELISA antibody detection method for blocking African swine fever virus P30 protein;

[0028] Figure 6 Specificity validation results of the ELISA antibody detection method for blocking African swine fever virus P30 protein. Detailed Implementation

[0029] The present invention will be described in detail below through specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. Any technical solution that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art shall fall within the scope of protection of the present invention. In addition, for those embodiments where specific technical operation steps or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0030] Light chains are classified into κ chains or λ chains based on their constant regions. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known to those skilled in the art. In this invention, VH represents the variable region of the heavy chain, and VL represents the variable region of the light chain, which is divided into κ and λ types.

[0031] The materials used in the following examples include: BALB / c mice purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; His Tag Mouse Monoclonal Antibody (HRP Conjugated) purchased from Beyotime Biotechnology Co., Ltd.; and HRP Conjugation Kit-Lighting- The reagent kits were purchased from Abcam, His nickel column packing material, Freund's complete and incomplete adjuvants from Sigma, and the mouse monoclonal antibody subtype identification kit from Proteintech. Trizol, the DNA fragment recovery kit, and the plasmid extraction kit were products of OMEGA. Molecular biology reagents were from Sigma-Aldrich, and other biochemical reagents were domestically produced analytical grade. ASFV inactivated serum was provided by the National African Swine Fever Reference Laboratory, O / A type FMDV inactivated serum was provided by the National Foot-and-Mouth Disease Reference Laboratory, and CSFV, PRRSV, and PRV inactivated serums were provided by the State Key Laboratory of Animal Disease Pathogen Biology. These inactivated sera could be obtained by applying to the corresponding laboratories.

[0032] Example 1: Preparation of single-chain antibody against murine African swine fever virus P30 protein

[0033] 1.1 Immunizing mice

[0034] Using recombinant African swine fever virus (ASFV) P30 protein (GenBank: MW828849.1) as the inactivated antigen, the purified ASFV P30 protein was diluted to 200 μg / mL with PBS and emulsified with an equal volume of Freund's complete adjuvant. Six 6-week-old female BALB / c mice were subcutaneously injected with 200 μL of the emulsified antigen at multiple sites on their backs. Two booster immunizations were administered on days 15 and 30 after the initial immunization using Freund's incomplete adjuvant, following the same method. One week after the third immunization, blood was collected from the tail vein to measure antibodies. OD... 450nm Mice with a growth rate >2.0 were given a booster immunization three days prior to fusion by intraperitoneal injection of 0.5 mL of unadjuvanted antigen.

[0035] 1.2 Cell Fusion

[0036] Positive serum was prepared from blood collected from BALB / c mice 3 days after booster immunization. Mice were euthanized, and spleens were aseptically harvested after disinfection, washed, and fat removed. The spleens were minced and ground in incomplete RPMI-1640, and spleen cells were collected. Spleen cells were mixed with Sp2 / 0 cells at a ratio of 1:5 to 1:10, centrifuged, and then PEG 2000 was added to promote fusion. The mixture was then diluted with incomplete RPMI-1640. The fused cells were resuspended in HAT selective medium, seeded in 96-well plates, and cultured at 37°C with 5% CO2.

[0037] 1.3 Screening and subcloning of positive hybridoma cells

[0038] The medium for fusion cells was changed halfway every 2-4 days (discarding 100 μL and adding 100 μL of fresh HAT medium). Observation was performed after 7-8 days, and surviving wells were labeled. When hybridoma cells reached more than 1 / 10 of the bottom of the well, the supernatant was collected and positive wells were screened using ELISA. Cells from positive wells were transferred to 24-well plates, counted after confluence, diluted to 10 cells / mL, and seeded into 96-well plates containing feeder cells. Screening was repeated. A portion of the cells were cryopreserved. Subcloning was performed three times consecutively until highly reactive hybridoma cells were selected. Hybridoma cells with high titers and good reactivity were propagated and cryopreserved. RNA was extracted from hybridoma cells that showed a blocking effect on ASFV P30 protein, and reverse transcription and amplification of the antibody variable region (e.g., Figure 1 (As shown), the amplified products were sent to Qingke Company for sequencing. 1.4 Preparation and Reactivity Identification of Single-Chain Antibodies

[0039] The VH and VL sequences obtained from company sequencing were ligated using the (Gly4Ser)3 linker and a C-terminal 6x-His tag was added. The entire sequence was then inserted into the pET-28a vector to create the scFv construct. The construct was transformed into chemically competent *E. coli* BL21(DE3) cells for expression induction, purification, and validation. A construct that successfully expressed scFv and whose product exhibited blocking activity was obtained and named single-chain antibody 7B9 (scFv-7B9).

[0040] The following protein sequences were retrieved from the NCBI website. After codon optimization, each sequence was inserted into an expression vector, transformed into E. coli BL21(DE3) for induction of expression, and the products were purified and identified for optimization. Tag-free recombinant ASFV P30 protein (GenBank: MW828849.1), SVA VP2 protein (GenBank: MT786457.1), FMDV VP1 protein (GenBank: OR354928.1), and PEDVN protein (GenBank: MW981418.1) were coated into ELISA plates at 100 ng per well, incubated overnight at 4°C, washed with PBST, and blocked overnight with commercial blocking buffer to prepare the microplates.

[0041] Single-chain antibody affinity assay: The concentration of the prepared single-chain antibody 7B9 was determined to be 3 mg / ml. A five-fold serial dilution was performed starting at 1:100, with a blank control retained. Three replicates were performed. The antibody was added to an ELISA plate and incubated at 37°C for 1 hour, followed by 4 washes with PBST. The His-tagged monoclonal antibody-HRP was diluted 1:10000 and added to the ELISA plate, incubated at 37°C for 1 hour, followed by 4 washes with PBST. TMB was added for 15 minutes for color development, then stop solution was added, and the OD was read using an ELISA reader. 450nm The titer of the single-chain antibody 7B9 was determined, and antibody affinity was calculated. The results are as follows: Figure 2 and Figure 3 As shown, the titer of the single-chain antibody 7B9 described in this application is greater than 1:62500, EC50. 50 =177.68ng / ml, indicating good antibody affinity.

[0042] Single-chain antibody specificity assay: The specificity of single-chain antibody 7B9 was detected by indirect ELISA using the aforementioned protein-label-free recombinant ASFV P30 protein, SVAVP2 protein, FMDV VP1 protein, and PEDVN protease plate. Results are as follows: Figure 4 As shown, the single-chain antibody 7B9 described in this application can specifically bind to the African swine fever virus P30 protein and does not react with other proteins. This indicates that the single-chain 7B9 described in this application reacts with the African swine fever virus P30 protein without cross-reactivity and has high specificity.

[0043] Example 2: Sequencing of the variable region of single-chain antibody 7B9

[0044] 2.1 Extraction of total mRNA from positive hybridoma cells

[0045] Resuscitate the aforementioned hybridoma cells, culture for 2-3 generations, and prepare a cell suspension. Add 1 mL of TRizol, mix well, and incubate at 4°C for 5 min. Add 250 μL of chloroform, mix well, incubate at 4°C for 10 min, and centrifuge at 12000 rpm for 15 min. Take 450 μL of the supernatant, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge as before. Discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, and centrifuge for 5 min. Dry the precipitate, dissolve it in 25 μL of RNase-free water, and obtain total mRNA.

[0046] 2.2 cDNA Synthesis and Purification

[0047] Add the components shown in Table 1 to a 0.2 mL PCR amplification tube in sequence, mix well, place the PCR amplification tube in a PCR instrument, and amplify using the reaction program in Table 2. The product obtained is the whole genome cDNA, which should be stored at -20℃ for later use.

[0048] Table 1 Reverse transcription reagent system

[0049]

[0050] Table 2 Reverse Transcription Procedure

[0051]

[0052] 2.3 Sequencing of the variable region of single-chain antibody 7B9

[0053] 2.3.1 Primer Synthesis

[0054] Reference (von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nu ssenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct;11(10):1908-1923. doi:10.1038 / nprot.2016.102. Epub 2016Sep 15.PMID:27658009.) Primers as shown in SEQ ID No. 13-42 were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. Premixed primers were prepared according to the literature, and a PCR amplification program was designed for PCR amplification. The specific amplification primers are as follows:

[0055] First-round amplification primers:

[0056] (1) Primers for amplifying the VH sequence:

[0057] VH-I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 13);

[0058] VH-II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 14);

[0059] VH-III: 5'-TGGCAGCARCAGCTACAGG-3' (SEQ ID No. 15);

[0060] VH-IV: 5'-CTGCCTGGTGACATTCCCA-3' (SEQ ID No. 16);

[0061] VH-V: 5'-CCAAGCTGTGTCCTGTC-3' (SEQ ID No. 17);

[0062] VH-VI: 5'-TTTTAAAAGGTGTCCAGKGT-3' (SEQ ID No.18);

[0063] VH-VII: 5'-CCTGTCAGTAACTRCAGGTGTCC-3' (SEQ ID No.19);

[0064] VH-VIII: 5'-TTTTAAAAGGGGTCCAGTGT-3' (SEQ ID No.20);

[0065] VH-IX: 5'-CGTTCCTGGTATCCTGTCT-3' (SEQ ID No.21);

[0066] VH-X: 5'-ATGAAGTTGTGGYTRAACTGG-3' (SEQ ID No.22);

[0067] VH-XI: 5'-TGTTGGGGCTKAAGTGGG-3' (SEQ ID No.23);

[0068] VH-XII: 5'-AGAAGGTGTGCACACCGCTGGAC-3' (SEQ ID No.24).

[0069] (2) Primers for amplifying VL sequences:

[0070] VL-I: 5'-RGTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID No.25);

[0071] VL-II: 5'-TGGACATGAGGGCYCCTGCTCAGT-3' (SEQ ID No.26);

[0072] VL-III: 5'-CTSTGGTTGTCTGGTGTTGAYGGA-3' (SEQ ID No.27);

[0073] VL-IV: 5'-GTTGCTGCTGCTGTGGCTTACA-3' (SEQ ID No.28);

[0074] VL-V: 5'-GTATCTGGTACCTGTGG-3' (SEQ ID No.29);

[0075] VL-VI: 5'-TGCCTGTTAGGCTGTTGGTGCT-3' (SEQ ID No.3);

[0076] VL-VII: 5'-GCTCAGTTCCTTGGTCTCCTGTTGC-3' (SEQ ID No. 31);

[0077] VL-VIII: 5'-TGGGTGCTGCTGCTCTGGGT-3' (SEQ ID No. 32);

[0078] VL-IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No. 33);

[0079] VL-X: 5'-TGCTCTGGTTATATGGTGCTGATGGG-3' (SEQ ID No. 34);

[0080] VL-XI: 5'-ACTGAGGCACCTCCAGATGTT-3' (SEQ ID No. 35).

[0081] Second round amplification primers:

[0082] 2-I: 5'-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3' (SEQ ID No. 36);

[0083] 2-II: 5'-GCTCAGGGAARTAGCCCTTGAC-3' (SEQ ID No. 37);

[0084] 2-III: 5'-GAYATTGTGMTSACCMCARWCTMCA-3' (SEQ ID No. 38);

[0085] 2-IV: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID No. 39);

[0086] 2-V: 5'-CAGGCTGTTGTGACTCAG-3' (SEQ ID No. 40);

[0087] 2-VI: 5'-CAACTTGTGCTCACTCAG-3' (SEQ ID No. 41);

[0088] 2-VII: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 42).

[0089] 2.3.2 First Round of Amplification

[0090] Add the components listed in Tables 3 and 4 to a 0.2 mL PCR amplification tube in sequence, and perform PCR amplification using the reaction program in Table 5. Store the amplification products at -20℃.

[0091] Table 3 Antibody VH gene PCR amplification system

[0092]

[0093] Table 4 Antibody VL PCR Amplification System

[0094]

[0095] Table 5 Antibody VH / VL Amplification Program

[0096]

[0097] 2.2.3 Second round of amplification

[0098] The VH / VL products from the first round of amplification were amplified again using the amplification system shown in Table 6, following the reaction procedures shown in Table 7. The amplified products were stored at -20℃.

[0099] Table 6 Antibody Second-Round PCR Amplification System

[0100]

[0101] Table 7. Antibody Second-Round PCR Amplification Procedure

[0102]

[0103] 2.3 Comparison of gene sequences

[0104] After amplification, the target fragment was ligated into the pMD-19T vector to construct a sequencing plasmid, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The variable region sequence obtained from sequencing was compared with the mouse antibody heavy chain variable region sequence, light chain Lamda chain, and light chain Kappa chain published on the NCBI website using the NCBI and IMGT gene banks.

[0105] Sequencing results showed that the amplified sequences were the heavy chain variable region and the light chain variable region of the single-chain antibody. The complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region are shown in Table 8, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in Table 9, and the gene sequences encoding the heavy chain variable region and the light chain variable region are shown in Table 10.

[0106] Table 8. Complementation-determining region sequence of antibody variable region

[0107]

[0108] Table 9. Amino acid sequence of antibody variable region

[0109]

[0110] Table 10 Antibody Variable Region Base Sequence

[0111]

[0112] Example 3: Application of single-chain antibody 7B9 in the detection of African swine fever virus P30 protein blocking ELISA antibody 3.1 Establishment of an ELISA antibody detection method for African swine fever virus P30 protein blocking

[0113] 3.1.1 Optimization of working conditions for the detection method

[0114] According to HRP Conjugation Kit-Lighting- The kit procedure involves HRP labeling the purified single-chain antibody 7B9. The working concentration of the enzyme-labeled single-chain antibody 7B9 is fixed at 0.5 μg / mL. The optimal working conditions for the ELISA method are determined using checkerboard titration: 100 ng of capture antibody protein coating per well and a serum dilution ratio of 1:5. Using the optimal working conditions and serum dilution ratio, the working concentration of the enzyme-labeled single-chain antibody 7B9 is determined to be 0.4 μg / mL.

[0115] 3.1.2 Validation of the African swine fever virus P30 protein blocking ELISA antibody detection method

[0116] Sensitivity validation of the ASFV P30 protein blocking ELISA antibody detection method: ASFV-positive serum was serially diluted, and the sensitivity of the method was validated under the aforementioned blocking ELISA method operating conditions. Results are as follows: Figure 5 As shown, the African swine fever virus P30 protein blocking ELISA antibody detection method of the present invention can detect ASFV positive serum at a dilution ratio of 1:128 and has good sensitivity.

[0117] Specificity validation of the ASFV P30 protein blocking ELISA antibody detection method: The specificity of the method was validated by detecting inactivated sera containing ASFV, CSFV, PRRSV, PRV, FMDV type O, and FMDVA under the aforementioned blocking ELISA method operating conditions. Results are as follows: Figure 6 As shown, the African swine fever virus P30 protein blocking ELISA antibody detection method of the present invention can specifically detect ASFV, and has no cross-reactivity with other pathogen sera, thus exhibiting good specificity.

[0118] In summary, this invention provides a single-chain antibody 7B9 (scFv-7B9) against the African swine fever virus (ASFV) P30 protein. This single-chain antibody specifically recognizes and binds to the ASFV P30 protein, exhibiting advantages such as high specificity, sensitivity, and low preparation cost. A specific implementation example demonstrates the application of single-chain antibody 7B9 in an ELISA antibody detection method for blocking ASFV P30 protein serotypes. This establishes an ASFV blocking ELISA antibody detection method, enabling the detection of ASFV-infected serum and providing reference and technical support for the differential diagnosis of ASF. The single-chain antibody sequence provided by this invention is recombinantly modified using conventional genetic engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. The antibody is optimized at the gene and protein levels, improving its specificity and affinity. Compared to traditional monoclonal antibodies, the scFv-7B9 antibody has a lower preparation cost, is suitable for large-scale fermentation preparation, and offers high cost-effectiveness.

[0119] The embodiments described above are only some embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the patent claims of the present invention should be included within the scope of the patent claims of the present invention.

Claims

1. A single-chain antibody against the P30 protein of African swine fever virus, characterized in that, The African swine fever virus P30 protein single-chain antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; the light chain variable region includes CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No.

6.

2. The African swine fever virus P30 protein single-chain antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The African swine fever virus P30 protein single-chain antibody as described in claim 2, characterized in that, The heavy chain variable region and the light chain variable region are linked by a linker peptide.

4. The single-chain antibody as described in claim 3, characterized in that, The amino acid sequence of the linker peptide is shown in SEQ ID NO.

9.

5. A nucleic acid, characterized in that, The nucleic acid molecule encodes a single-chain antibody against the African swine fever virus P30 protein as described in any one of claims 1-4.

6. The nucleic acid as described in claim 5, characterized in that, The nucleic acid includes the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID No. 10, the nucleotide sequence encoding the light chain variable region shown in SEQ ID No. 11, and the nucleotide sequence encoding the linker peptide shown in SEQ ID No.

12.

7. A recombinant vector or recombinant cell, characterized in that, The recombinant vector or recombinant cell comprises the nucleic acid as described in claim 5 or 6.

8. The African swine fever virus P30 protein single-chain antibody according to any one of claims 1-4 has any of the following uses: (1) Application in the preparation of African swine fever virus detection reagents; (2) Application in the detection of African swine fever virus for non-disease diagnosis purposes.

9. An ELISA antibody kit for blocking African swine fever virus P30 protein, characterized in that, The kit includes a single-chain antibody against the African swine fever virus P30 protein as described in any one of claims 1-4.

10. The method for preparing the single-chain antibody against the African swine fever virus P30 protein as described in any one of claims 1-4, characterized in that, The method is as follows: the heavy chain variable region and light chain variable region sequences of the African swine fever virus P30 protein single-chain antibody are linked using a linker peptide and inserted into an expression vector to obtain the scFv recombinant plasmid; the obtained recombinant expression plasmid is transformed into E. coli BL21(DE3) cells, and the African swine fever virus P30 protein single-chain antibody is obtained by screening, culturing and preparing.

Images