An antibody against African swine fever virus targeting p72 protein

Abstract

The present invention belongs to the technical field of antibody preparation, and specifically relates to an anti-African swine fever virus antibody targeting p72 protein. The antibody belongs to a single-chain antibody that only includes a heavy chain variable region VH and a light chain variable region VL. In the present application, based on the existing mature phage antibody library screening technology, a specific scFv-Fc antibody with high affinity for the African swine fever virus capsid protein p72 protein was screened and obtained. Preliminary experimental results show that the single-chain antibody better retains the affinity activity for the antigen, can specifically recognize and bind to the capsid protein p72; and because of its small molecular weight and strong penetrating power, it is easy to prepare in large quantities through genetic engineering technology with the help of prokaryotic or eukaryotic expression systems, which can lay a certain technical foundation for the development of drugs for the detection, prevention and treatment of African swine fever virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody preparation, and in particular relates to an antibody against African swine fever virus targeting p72 protein. Background Art

[0002] African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease that causes nearly 100% mortality in domestic pigs. Since ASF was first discovered in Kenya in 1921, it has caused great damage to the pig farming industry in various countries.

[0003] Studies have shown that the genome of the African Swine Fever Virus (ASFV), which causes African swine fever, is a double-stranded linear DNA with a size of 170-190 kb, but with a complex structure. Due to limited research on the functions of multiple proteins encoded by the virus and the virus's mutation, there is currently a lack of commercially available vaccines for the prevention of African swine fever. Summary of the invention

[0004] In combination with relevant phage screening technology, the purpose of this application is to provide a specific scFv-Fc (single-chain variable fragment Fc (Fragment Crystallization)) antibody with high affinity for the African swine fever virus capsid protein p72 protein, thereby laying a certain technical foundation for the preparation of related vaccines.

[0005] The technical solution adopted in this application is briefly described as follows.

[0006] An anti-African swine fever virus antibody targeting p72 protein, the antibody is a single-chain antibody (i.e., only includes a heavy chain variable region and a light chain variable region), which includes: a heavy chain variable region VH and a light chain variable region VL;

[0007] The heavy chain variable region VH is 391 bp in length, and its nucleotide sequence (as shown in SEQ ID No. 1) is as follows:

[0008] GAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGG GCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAG;

[0009] The amino acid sequence encoded by the heavy chain hypervariable region VH consists of 130 amino acids, and its amino acid sequence is specifically:

[0010] EEKLVESGGGLVQPGGSLRLSCVGSGFTFSSTYINWVRQAPGKGLQWLAAISTSGGSTYYTDSMEGRFTISKDNSQNTAYLQMNSLRTEDTARYYCATIRGCYSYGASCYRSWASFMDLWGPGVEVVVSS;

[0011] The heavy chain variable region VH contains three hypervariable regions, and the corresponding base sequences are:

[0012] At positions 457-480: GATTCACCTTCAGTATACCTAC (corresponding encoded amino acid sequence: GFTFSSTY);

[0013] At positions 532 to 555: ATTAGTACTAGTGGTGGTAGCACC (the corresponding encoded amino acid sequence is: ISTSGGST);

[0014] Positions 670 to 73: GCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTT

[0015] ATGGATCTC (the corresponding encoded amino acid sequence is: ATIRGCYSYGASCYRSWASFMDL);

[0016] The light chain variable region VL is 321 bp in length, and its nucleotide sequence (as shown in SEQ ID No. 2) is as follows:

[0017] TCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCC CTCAGGGATCCCTGACGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTC;

[0018] The light chain variable region VL consists of 107 amino acids, and its amino acid sequence is specifically as follows:

[0019] SYELTQPSSESVALGSTAKITCSGDLLDEKYTQWYQQKPGQAPLLLIYKDNERPSGIP ERFSGSSSGKTATLTITGAQAEDEADYSCQSGDRINNAIFGGGTHLTVL;

[0020] The light chain variable region VL contains three hypervariable regions, and the corresponding base sequences are:

[0021] Positions 82 to 99: CTGCTGGATGAAAAATAT (corresponding to the encoded amino acid sequence: LLDEKY);

[0022] Positions 151 to 159: AAGGACAAT (corresponding to the encoded amino acid sequence: KDN);

[0023] At positions 268 to 297: CAGTCAGGTGACAGGATTAATAATGCTAT (the corresponding encoded amino acid sequence is: QSGDRINNAI);

[0024] When used, the linker sequence between the heavy chain variable region VH and the light chain variable region VL can be referred to as: GGTGGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCAGGAGGAGGAGGAGGAAGC;

[0025] (The corresponding encoded amino acid sequence is: GGGGGGSGGGGSGGGGGS).

[0026] Based on the above light chain variable region VL and heavy chain variable region VH, combined with the Fc fragment, the present application provides an scFv-Fc antibody against African swine fever p72 protein, the base sequence of which is 1494 bp in length, and the base sequence is shown in SEQ ID NO.3, specifically:

[0027]

[0028] The base sequence corresponds to the encoded amino acid sequence, which is 498 amino acids long. The specific amino acid sequence is:

[0029] MASYELTQPSSESVALGSTAKITCSGDLLDEKYTQWYQQKPGQAPLLLIYKDNERPSGIPERFSGSSSGKTATLTITGAQAEDEADYSCQSGDRINNAIFGGGTHLTVLGGGGGGSGGGGSGGG GGSEEKLVESGGGLVQPGGSLRLSCVGSGFTFSSTYINWVRQAPGKGLQWLAAISTSGGSTYYTDSMEGRFTISKDNSQNTAYLQMNSLRTEDTARYYCATIRGCYSYGASCYRSWASFMDLWGP GVEVVVSSASGQAGLASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0030] The scFv-Fc antibody against African swine fever p72 protein is prepared by the following steps:

[0031] (I) Preparation of recombinant expression vector

[0032] First, obtain the correct antibody nucleotide sequence;

[0033] Subsequently, the above antibody nucleotide sequence is recombined using a eukaryotic expression plasmid (for example, pINFUSE plasmid) as a vector to construct a recombinant eukaryotic expression vector that can translate and express the nucleotide sequence shown in SEQ ID NO.3;

[0034] Taking the plasmid pINFUSE-mIgG2b-Fc2 (product of InvivoGen) as an example, during recombination, the nucleotide sequences of SEQ ID No. 1 and SEQ ID No. 2 can be recombined (if a plasmid vector without an Fc fragment is used, the sequence shown in SEQ ID No. 3 needs to be recombined); the specific recombination operation is as follows:

[0035] The eukaryotic expression vector pINFUSE-mIgG2b-Fc2 is digested with restriction endonucleases EcoRI and NcoI respectively and the digestion products are recovered, and then the recovered digestion products are connected with the nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 using T4 DNA ligase;

[0036] After the connection is completed, the connection product is electrotransformed into TG1 competent cells, and screened and identified to ensure that the plasmid recombination is correct, and the recombinant expression vector plasmid is extracted for use;

[0037] (II) Antibody protein expression

[0038] Referring to the instructions of SinoBiological's SMM 293-TII Expression Medium and Sinofection transfection reagent, the recombinant plasmid vector constructed correctly in step (i) was used to transform 293F cells in good cell condition. After culturing for 5-7 days, the culture containing the scFv-Fc recombinant antibody was collected;

[0039] (III) Purification of scFv-Fc Antibody

[0040] The culture obtained in step (ii) was centrifuged at 4°C and 5000 g for 10 min, the cell precipitate was removed, and the supernatant was retained. The supernatant was the solution containing the single-chain scFv-Fc antibody;

[0041] The supernatant was further concentrated using a Millipore ultrafiltration centrifuge tube with a molecular weight cutoff of 15KD, and then purified using Protein A affinity filler to obtain purified scFv-Fc antibodies.

[0042] In the existing technology, due to the limited research on the innate immunity and adaptive immunity of pigs after infection with African swine fever virus (ASFV), in-depth exploration of the expression levels of various antibody proteins after virus infection and understanding the interactions between these antibody proteins are of great technical significance for the accurate diagnosis of ASFV infection, the mining of key antigenic epitopes and vaccine design.

[0043] In this application, based on the existing mature phage antibody library screening technology, a specific scFv-Fc antibody with high affinity for the African swine fever virus capsid protein p72 protein was screened and obtained. This antibody is a single-chain antibody that only contains the heavy chain variable region and the light chain variable region. This single-chain antibody not only retains the affinity activity for the antigen, but also has the technical advantages of small molecular weight and strong penetration. Therefore, it has good application potential in the diagnosis and treatment of ASFV. Preliminary experimental results show that the single-chain antibody has better retained the affinity activity for the antigen, and can specifically recognize and bind to the capsid protein p72; and because of its small molecular weight and strong penetration, it is easy to prepare in large quantities through genetic engineering technology with the help of prokaryotic or eukaryotic expression systems, which can lay a certain technical foundation for the development of drugs for the detection, prevention and treatment of African swine fever virus, and therefore has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 For the detection of VH and VL gene PCR products by gel electrophoresis, where M: DNA Marker DL5000;

[0045] Figure 2 The results of SOE-PCR amplification of scFv gene were detected by gel electrophoresis, where M: DNA Marker DL2000; 1: scFv spliced ​​by VH-VL (κ); 2: scFv spliced ​​by VH-VL (λ);

[0046] Figure 3 The PCR amplification results of the inserted fragments of the antibody library were detected by gel electrophoresis, where M: DNA Marker DL5000; 1-24: single colonies of the phage antibody library;

[0047] Figure 4 To detect the affinity of recombinant phage antibodies by phage-ELISA;

[0048] Figure 5 The SDS-PAGE was used to detect ASFV-scFv-83-Fc after Protein A purification, where: M, protein marker; 1: blank control 293F cells; 2: eukaryotic expression of specific scFv-83 culture;

[0049] Figure 6 The results of Western blot purification analysis, where: M, protein marker; 1: Western blot results of ASFV-scFv-83-Fc after purification;

[0050] Figure 7Western blotting was used to analyze the reactivity of ASFV-scFv-83 with inactivated ASFV virus-infected PAM cells; 1: blank control group PAM cells; 2: ASFV HLJ / 18-infected PAM cells;

[0051] Figure 8 IFA is used to identify the activity of scFv-p72-83 antibody; p72 is used to identify the activity of scFv-p72-83 antibody; Mock is used to identify the control group; DAPI is used to identify the result of cell nucleus staining; MERGE is used to identify the combined image; DETAILED DESCRIPTION

[0052] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0053] Biomaterials:

[0054] Escherichia coli TG1, pHEN2 phagemid vector, eukaryotic expression vector pFUSE-Fc, proliferation type M13KO7, DH5α competent cells, etc. are all common biological materials in existing biological research and can be obtained from public channels (for example, Qingke Biotechnology Co., Ltd.);

[0055] Main reagents and kits:

[0056] The porcine peripheral blood lymphocyte separation medium kit was purchased from Solebao Biological Products Company;

[0057] DL2000DNAMarker, 6× Loading Buffer, etc. were purchased from Takara Biotech;

[0058] SfiI-HF, EcoRI and NcoI restriction endonucleases were from NEB;

[0059] DNA fragment recovery kit was purchased from Thermo Fisher Scientific;

[0060] Trizol and Protein A affinity chromatography resins were purchased from GenScript Biotechnology Co., Ltd.;

[0061] The plasmid extraction kit is a product of Beijing Quanshijin Company.

[0062] The recombinant p72 protein involved in the following embodiments is a trimeric recombinant p72 protein prepared by the inventors using existing biotechnology to express the ASFV capsid protein p72 and its partner protein pB602L in human embryonic kidney 293 (HEK293) cells, and its amino acid sequence can be referenced: Pig / HLJ / 18GenBank:MK333180.1.

[0063] Example 1

[0064] In combination with relevant phage screening technology, this application screened and obtained a specific porcine scFv-Fc antibody with high affinity for the capsid protein p72 protein of African swine fever virus. Before introducing the specific screening process, it needs to be explained that since the library construction of porcine natural immune single-chain antibody (scFv) is the basis for related antibody screening (in the screening process, the target of the library is the antibody sequence), the relevant screening process is briefly introduced as follows in combination with the library construction process.

[0065] (I) Obtaining the full-length scFv gene by overlapping PCR amplification

[0066] First, obtain the cDNA template of African swine fever antibody-positive pigs. The specific operation is:

[0067] The peripheral blood of African swine fever antibody-positive pigs was used as samples. After isolating the peripheral blood mononuclear cells, the total RNA was extracted and reverse transcribed into cDNA according to the instructions of the relevant kit, and stored at -80°C for future use.

[0068] Secondly, the cDNA prepared as above was used as a template, and the overlapping extension PCR method was used to amplify and prepare the full-length scFv gene. During the specific PCR amplification: a total of three rounds of PCR amplification were performed. During the amplification process: first, PCR amplification was performed to prepare the V(κ), VL and VH genes, and finally the overlapping PCR amplification method was used to obtain the full-length scFv gene. The specific process is briefly described as follows.

[0069] In the first round of PCR amplification:

[0070] For the V(κ) gene, the following primer pairs were designed:

[0071] Forward Primer:

[0072] Vκ-F-1:GCGGCCCAGCCGGCCATGGCCGCCATCCAGCTGACCCAGTCTCC,

[0073] Vκ-F-2:GCGGCCCAGCCGGCCATGGCCGCCATYGTGCTGACCCAGACTCC,

[0074] Vκ-F-3:GCGGCCCAGCCGGCCATGGCCGAAATTGTGCTGACCCAGTCTGC,

[0075] Vκ-F-4:GCGGCCCAGCCGGCCATGGCCGAAACAACASTCACTCAATCTCC,

[0076] Vκ-F-5:GCGGCCCAGCCGGCCATGGCCGCCATGGTGTTGACCCAGAGTGC;

[0077] Reverse Primer:

[0078] Vκ-R-1:CCAGAGCGCCGCCGCCGCCTTTGAKYTCCAGCTTAGTCCCG,

[0079] Vκ-R-2:CCAGAGCCGCCGCCGCCGCCTTTGGGCTCCACTTTGGTCCCCG, Vκ-R-3:CCAGAGCCGCCGCCGCCGCCTTCAATCTCCACGGATGTCCCT,

[0080] Vκ-R-4:CCAGAGCCGCCGCCGCCGCCTTTGAGTTCCAGCTTGGTTCC;

[0081] When PCR amplification of the V(κ) gene was performed, the forward primer and the reverse primer were randomly paired and combined for PCR amplification;

[0082] For VH gene amplification, the following primer pairs were designed:

[0083] Forward Primer:

[0084] VH-F-1: ggttcaggaggaggaggaggaagc GAGGTGAAGCTGGTGGAGTCTGGA,

[0085] VH-F-2: ggttcaggaggaggaggaggaagc GAGGAGAAGCTGGTGGAGTCTGGA;

[0086] Reverse Primer:

[0087] VH-R-1:tgtgcagatgatgaccgcgGCTAGCTGAGGACACGACGACTTCAAC,

[0088] VH-R-2:tgtgcagatgatgaccgcgGCTAGCTGAGGAGACGGTGACCTCGAC,

[0089] VH-R-3:tgtgcagatgatgaccgcgGCTAGCTGAGGAGACGGTGACCAGGA;

[0090] When PCR amplification of the VH gene was performed, the forward primer and the reverse primer were randomly paired and combined for PCR amplification;

[0091] For the VL gene, the following primer pairs were designed:

[0092] Forward Primer:

[0093] VL-F-1:GCGGCCCAGCCGGCCATGGCCCAGACTGTGATCCAGGAGCC,

[0094] VL-F-2:GCGGCCCAGCCGGCCATGGCCTCCTATGAGSTGACTCAGCC,

[0095] VL-F-3:GCGGCCCAGCCGGCCATGGCCCAGGCTGTGCTGACTCAGCCG,

[0096] VL-F-4:GCGGCCCAGCCGGCCATGGCCCAGTCTGCCCTGACTCAGCCC;

[0097] Reverse Primer:

[0098] VL-R-1:CCAGAGCCGCCGCCGCCGCCACCGAGGACGGTCAGATGGGT;

[0099] When PCR amplification of the VL gene was performed, the forward primer and the reverse primer were randomly paired and combined for PCR amplification;

[0100] During PCR amplification, the 25 μL system is designed as follows:

[0101] 2× Taq Master Mix, 12 μL;

[0102] Upstream and downstream primers, 0.5 μL each;

[0103] cDNA template, 1 μL;

[0104] Deionized water, 11.5 μL;

[0105] The amplification program was as follows: 95°C, 5 min, 95°C, 30 s, 56°C, 30 s, 72°C, 20 s, 30 cycles; 72°C, 10 min;

[0106] After PCR amplification, the amplified product was detected by 1% agarose gel, and the target band was extracted, purified and identified.

[0107] During the second round of PCR amplification (since the present application adopts overlapping PCR reaction to prepare the full-length scFv gene fragment, in order to facilitate the use of the second round PCR products in subsequent overlapping PCR reactions, the inventors redesigned the amplification primers by extending the primer length), the specific PCR amplification primer sequences are designed as follows:

[0108] VLκ-F: ACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCAT,

[0109] VLκ-R: TGAACCACCACCACCAGAGCCGCCGCCGCCGCC;

[0110] During PCR amplification, the 25 μL amplification system was designed as follows:

[0111] 2× Taq Master Mix, 12 μL;

[0112] Upstream and downstream primers, 0.5 μL each;

[0113] cDNA template (the product of the first round of amplification), 1 μL;

[0114] Deionized water, 11.5 μL;

[0115] The amplification program was: 95°C, 5 min; 95°C, 30 s, 56°C, 30 s, 72°C, 20 s, 30 cycles; 72°C, 10 min;

[0116] After PCR amplification, the amplified products were detected by 1% agarose gel (the electrophoresis results of some amplified fragments are shown in Figure 1 ), and the target band was extracted, purified and identified.

[0117] During the third round of PCR amplification:

[0118] The second round amplification product was used as a template and the overlap extension PCR splicing method was used to obtain the full-length scFv gene; during PCR amplification, the primer sequence was designed as follows:

[0119] scFv-F:AGGAGGCGCGCATAATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGT T,

[0120] scFv-R:tgtgcagatgatgaccgcgGCTAGCTG;

[0121] During PCR amplification, the 25 μL amplification system was designed as follows:

[0122] 2× Taq Master Mix, 12 μL;

[0123] Upstream and downstream primers, 0.5 μL each;

[0124] cDNA template (second round amplification product), 1 μL;

[0125] Deionized water, 11 μL;

[0126] The amplification program was: 95°C, 5 min; 95°C, 30 s, 56°C, 30 s, 72°C, 20 s, 30 cycles; 72°C, 10 min;

[0127] After PCR amplification, the amplified products were detected by 1% agarose gel (see some amplification results for details). Figure 2 ), the gel was cut under ultraviolet light to recover the target band at a size of about 750 bp, and a gel recovery kit was used to recover the target band at a size of about 750 bp and purify the DNA fragment, and the result was: scFv formed by random splicing of VH gene and VL gene.

[0128] (II) Construction of phage antibody library

[0129] Based on the scFv gene obtained in the above steps, the obtained porcine scFv gene (library) and the phagemid vector pHEN2 vector are respectively digested with SfiI-HF restriction endonuclease, and the digestion products are recovered, and further connected with T4 DNA ligase;

[0130] Then, the ligation product was transformed into E. coli TG1 competent cells by electroporation, and plated for resistance screening. Positive single colonies were randomly selected for colony PCR identification to verify the scFv gene conversion rate (see some test results for details). Figure 3 ), and the diversity of the scFv phage library was analyzed by sequencing to ensure the diversity of the scFv gene sequences in the library to meet the target screening application (in the screening application, the bacterial library containing phage DNA was further packaged into a phage library using M13KO7 helper phage).

[0131] The results of colony counting showed that the capacity of the scFv library constructed in this application was 3.2×10 9 , has the characteristics of good library capacity and diversity, and can better meet the needs of screening applications.

[0132] (III) Phage screening

[0133] Based on the library constructed above, the following is a brief introduction to the implementation steps of phage screening (anti-African swine fever p72 protein antibody).

[0134] The microplate screening method was used to screen the single-chain antibody library constructed above for specific antibodies using the recombinant p72 protein as the target molecule. The screening process is: incubation of antigen with phage antibody library → elution of unbound nonspecific phage → amplification of phage antibodies. After 3 rounds (3 rounds in this example), enriched specific phage antibodies can be obtained. The specific operation is as follows.

[0135] (1) Take the recombinant p72 protein as the target molecule and dilute the protein to the required concentration using coating solution CBS (0.1 M NaHCO3, pH = 9.6) (in the first round of screening, the target molecule concentration of the coating solution is 2 μg / well, and 4-fold dilution is performed in each subsequent round), 100 ul per well; incubate at 4°C overnight;

[0136] On the second day, the liquid in the wells was discarded, and the wells were washed once with 200 μL TBST, and the liquid was discarded; 200 μL blocking solution (TBS solution containing 5% skim milk powder and 0.25% Tween20) was added, and the wells were incubated at 37°C for 2 h;

[0137] Finally, discard the liquid in the wells and wash the well plate once with 200 μL TBS containing 0.25% Tween20 (the Tween concentration can be adjusted according to the situation in the subsequent panning operation);

[0138] (2) After discarding the liquid in the well, add the prepared antibody phage antibody library (1×10 13 / well), incubated at 37°C for 1 h;

[0139] After the incubation, discard the liquid in the wells, shake them clean, wash them 5 times with TBST containing 0.25% Tween20, 5 min each time, discard the liquid by suction, and shake them dry;

[0140] (3) Add 1 mg / ml Trypin (trypsin), 100 μl / well, shake slowly at room temperature for 8 min, and pipette out the supernatant repeatedly;

[0141] Then add 100ul of 4% skim milk-PBS to neutralize (to obtain the eluted product), and directly proceed to the subsequent operation or store at 4°C for future use;

[0142] (4) Add the eluted product in step (3) to the pre-cultured TG1 monoclonal bacterial solution (pick the pre-streaked activated TG1 monoclonal and inoculate it into 20 ml of 2×YT medium and culture it at 37°C and 250 rpm with shaking until the logarithmic phase, OD600 = about 0.5), mix well and let it stand at 37°C for 30 min for infection, then add the resistance component (Amp, final concentration 20 ug / ml), and continue to shake slowly at 37°C and 180 rpm for 60 min for resistance screening;

[0143] (5) Add a corresponding amount of M13KO7 (number of helper phages: number of bacteria ≥ 10:1) to the culture in step (4) and incubate at 37°C for 20 min to assist infection;

[0144] Subsequently, 30 ml of 2×YT medium and Amp (final concentration 20 ug / ml) were added and the culture was shaken slowly at 37°C and 180 rpm for 60 min;

[0145] (6) After the culture was completed, the cells were centrifuged at 5000 rpm for 10 min, the cells were collected, and the cells were resuspended in 50 ml of 2×YT medium (Amp 50 ug / ml + Kan 10 ug / ml), and cultured at 30°C and 220 rpm overnight;

[0146] After the culture, centrifuge at 4°C and 12,000 rpm for 10 min, transfer the supernatant to a new centrifuge tube, and centrifuge the supernatant again at 4°C and 12,000 rpm for 10 min;

[0147] After centrifugation, transfer the supernatant to a new centrifuge tube, add 1 / 4 volume of PEG8000 / NaCl, and precipitate at 4°C for 4 h / ice for 2 h;

[0148] (7) After ice precipitation, centrifuge at 4°C and 12,000 rpm for 20 min, discard the supernatant, resuspend the pellet in 0.5 ml PBS, and centrifuge at 4°C and 12,000 rpm for 10 min (to precipitate the residual cells);

[0149] Finally, the supernatant was transferred to a new centrifuge tube (this is the amplified product) and the titer of the amplified product was measured (titer determination method: inoculate TG1 monoclonal clone in 2×YT antibiotic-free medium, culture at 37°C and 220rpm until OD600≈1.0, take the phage library and dilute it to 10 -10 , take 100ul of the dilution and add it to 200uL of the cultured TG1, let it stand in a 37℃ incubator for 30min, spread it all on an ampicillin-resistant plate, culture it at 37℃ overnight, count the number of clones on the plate the next day, and calculate the titer).

[0150] During the screening process, when the eluted product of the above step (3) is positively identified, PCR is used for identification, and the 20 μl amplification system is designed as follows (the primer sequences are F: TggAATTgTgAgCggATAACAATT, R: ATTCAGATCCTCTTCTGAGAT):

[0151] rTaq, 5ul;

[0152] 10× Buffer, 1ul;

[0153] Template (monoclonal colony), 1ul;

[0154] F primer, 1 μl;

[0155] R primer, 1 μl;

[0156] H2O, add to 10ul;

[0157] PCR conditions: 94°C, 4 min; 94°C, 30 s, 53°C, 30 s, 72°C, 40 s, 25 cycles; 72°C, 2 min, 10°C, 10 s; thus, one round of panning was completed.

[0158] According to the results of the first round of panning, the second and third rounds of panning were carried out to reduce the background and further enrich high-affinity phages by appropriately reducing the concentration of the target molecule coating or increasing the concentration of Tween20 in the washing solution, the number of washes and the time, reducing the binding time and temperature of the target molecule and the phage library, and increasing the panning pressure. During the screening process, 10 monoclones were randomly selected for sequencing analysis after the second and third rounds of panning to analyze the characteristics of the antibody heavy chain variable region sequence and the antibody light chain variable region sequence.

[0159] During the screening process, ELISA test was used to detect the affinity after each round of affinity screening. The specific experimental method is as follows:

[0160] (1) Coating: Dilute the target protein to 3 μg / mL with coating solution, coat the ELISA plate at 100 μL / well, and incubate at 4°C overnight.

[0161] (2) Blocking: Shake off excess coating solution, invert the plate and tap on a clean paper towel to remove residual solution. Wash the plate three times with PBS, add blocking solution, 200 μl / well, and incubate at 37°C for 1 h.

[0162] (3) Sample addition: shake off excess blocking solution, invert the plate and tap on a clean paper towel to remove residual liquid, wash the plate three times with PBS, add phage supernatant, 50 μl / well, and incubate at 37°C for 1 h.

[0163] (4) Secondary antibody: After discarding the excess phage supernatant, invert the plate and tap it on a clean paper towel to remove the residual liquid. Wash the plate three times with PBS, then add 50 μl / well of HRP rabbit anti-M13 diluted 1:1000 with 1% Milk-PBS and incubate at 37°C for 1 h.

[0164] (5) Color development: shake off excess solution, invert the plate and tap on a clean paper towel to remove residual liquid. Wash the plate 4 times with PBS, add TMB color development solution (50 ul / well), and after sufficient reaction and color development, add 2 M H2SO4 to stop color development and detect at 450 nm.

[0165] The results are as follows Figure 4 As shown, it can be seen that scFV-83 can recognize and bind to p72 protein with high specificity.

[0166] The above sequencing analysis results show that scFV-83, as an anti-African swine fever virus antibody targeting p72 protein, is a single-chain antibody (i.e., only includes a heavy chain variable region and a light chain variable region), and the sequence of its heavy chain variable region VH is shown in SEQ ID No. 1, and the sequence of its light chain variable region VL is shown in SEQ ID No. 2, as follows:

[0167] Heavy chain variable region VH:

[0168] GAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGG GCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAG;

[0169] Light chain variable region VL:

[0170] TCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCC CTCAGGGATCCCTGAGCGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTC.

[0171] Based on the above sequence, the base sequence of the scFv-Fc antibody against African swine fever p72 protein (such as SEQ ID No. 3) can be determined as follows:

[0172]

[0173] Example 2

[0174] Based on Example 1, it can be seen that scFV-83 has a strong binding force with p72 protein. In order to further evaluate the application prospects of the protein, the inventor used the pINFUSE-mIgG2b-Fc2 plasmid of InvivoGen (the plasmid has a recombinant CH2-CH3 sequence, and the CH2-CH3 sequence reference is: GenBank: BAE20056.1; it should be emphasized that the use of this plasmid is only based on the convenience of actual experiments, and does not mean that the technical solution of this application must rely on this plasmid. Other universal plasmids can also be used to achieve antibody expression) as a vector, and used 293F cells to express related proteins, and further used Protein A to obtain purified porcine anti-p72 single-chain scFv-Fc antibodies. The specific preparation process is described as follows.

[0175] (I) Recombination of the eukaryotic expression vector pINFUSE-mIgG2b-Fc2

[0176] First, the monoclone corresponding to phage 83 (scFV-83) was inoculated into 1 mL of 2×YT liquid culture medium containing ampicillin (100 μg / mL) and tetracycline resistance (15 μg / mL), and cultured overnight at 37°C and 260 rpm with shaking. The plasmid was extracted from the culture using a plasmid extraction kit (product of TRANS, refer to the instructions for operation) (the plasmid concentration in the extract was measured using a NanoDrop2000 UV spectrophotometer to ensure that it met the needs of subsequent use);

[0177] Subsequently, the extracted plasmid and the eukaryotic expression vector pINFUSE-mIgG2b-Fc2 were digested with restriction endonucleases EcoRI and NcoI, respectively, and the digestion products were subjected to 1% agarose gel electrophoresis, respectively, and then the digestion products were recovered with a gel recovery kit and ligated with T4 DNA ligase.

[0178] During the operation, the enzyme digestion system (20 μL) was designed as follows:

[0179] Extracted plasmid (or eukaryotic expression vector pINFUSE-mIgG2b-Fc2), 2 μg;

[0180] EcoRI and NcoI, 2 μL each;

[0181] 10× CutSmart Buffer, 2 μL;

[0182] Add ddH2O to 20 μL;

[0183] 50℃, enzyme digestion for 2h.

[0184] When using T4 DNA ligase for ligation, the ligation system (10 μL) is designed as follows:

[0185] Extracted plasmid digestion product, 100 ng;

[0186] pFUSE-Fc digestion product, 60 ng;

[0187] T4 DNA ligase, 0.5 μl;

[0188] 10×T4 DNAligase Buffer, 1 μl;

[0189] Add ddH2O to make up to 10 μL;

[0190] 16℃, connect for 3h.

[0191] Then, the ligation product was transformed into TG1 competent cells using the heat shock transformation method. The specific operation reference is as follows:

[0192] Take 10 μL of the ligation product and add it to the TG1 competent medium, place it in an ice bath for 30 minutes, heat shock it at 42°C for 45 seconds, and then incubate it on ice for 5 minutes; add 600 μL of antibiotic-free 2×YT liquid culture medium, resuscitate it at 37°C and 260 rpm for 1 hour, then spread it on a 2×YT plate containing ampicillin (100 μg / mL) and tetracycline resistance (15 μg / mL), and culture it at 37°C overnight.

[0193] Finally, the positive single clones were selected for sequencing verification, and the recombinant plasmid expression vector was named: pINFUSE-scFV-83.

[0194] (II) Expression of scFV-Fc Antibody

[0195] Referring to the instructions of SinoBiological's SMM 293-TII Expression Medium and Sinofection transfection reagent, the recombinant plasmid vector pINFUSE-scFV-83 constructed correctly in the above step (i) was transformed into 293F cells in good cell condition, and cultured in a cell shaker at 37°C, 120 rpm, 5% CO2 for 5-7 days, and the culture containing the scFv-Fc recombinant antibody was collected.

[0196] (III) Purification to obtain single-chain scFV-Fc antibody

[0197] The culture collected in step (ii) was centrifuged at 4°C and 5000 g for 10 min, the cell precipitate was removed and the supernatant was retained. The supernatant was the solution containing the expressed single-chain scFv-Fc antibody.

[0198] Furthermore, the supernatant was concentrated using a Millipore ultrafiltration centrifuge tube with a molecular weight cutoff of 15KD, and purified and eluted using a Protein A affinity filler to obtain a purified scFv-Fc antibody.

[0199] The purified scFv-Fc antibody was identified by SDS-PAGE. Figure 5 As shown in the SDS-PAGE figure, lane 1 is the blank control 293F cells, and lane 2 is the recombinant antibody. The lane shows a single target band, indicating good purity. Western blot analysis was further performed on the purified and eluted target protein, and the results are shown in Figure 6 As shown, it can be seen that the size is in line with expectations, further proving that the recombinant antibody obtained by the present invention has good activity and good purity.

[0200] (IV) Identification

[0201] The inventors further used IFA and Western blot methods to detect the binding of the scFv-Fc antibody obtained above with p72 and inactivated virus, respectively. The specific situation is briefly described as follows.

[0202] (1) Western Blot antibody activity identification

[0203] Using porcine alveolar macrophages (PAMs) as samples, immunoblotting was used to study the reaction of scFV-83 with inactivated ASFV HLJ / 18 strain (provided by Harbin Veterinary Research Institute) infected porcine alveolar macrophages (PAMs) samples.

[0204] During the experiment, PAM cells infected with ASFV HLJ / 18 strain and PAM cells not infected with ASFV in the blank control group were used as samples for SDS-PAGE. All samples were denatured at 95°C for 30 min. The samples were then analyzed using SDS-PAGE (12.5% ​​PAGE gel rapid preparation kit, BioMan), and the proteins were transferred to PVDF membranes using the semi-dry method.

[0205] Then, the PVDF membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) at room temperature for 1 h;

[0206] Then, the collected scFV-83 culture fluid was used as the primary antibody, and after washing with PBST for 5 times, HRP-labeled goat anti-swine IgG (H+L) (Proteintech, China) diluted 1:4000 was added as the secondary antibody, incubated at 37°C for 1 h, and washed with PBST for 5 times;

[0207] Finally, the PVDF membrane was visualized using ECL.

[0208] The results are as follows Figure 7 As shown. It can be seen that this antibody can specifically recognize and bind to PAM cells infected with ASFV HLJ / 18, but does not react with PAM cells in the blank group, proving that scFV-83 is indeed an antibody against the ASFV virus.

[0209] (2) IFA identification of antibody activity

[0210] The test process and operation reference are as follows:

[0211] Referring to the above operation and prior art, the p3xFLAG-CMV-14 expression vector containing the p72 gene (Pig / HLJ / 18GenBank: MK333180.1) constructed and synthesized by Qingke Biotechnology Company was used to transfect PK15 cells, and an empty negative control was set up at the same time; 24 hours after transfection, the culture medium was discarded and the cells were gently rinsed once with PBS;

[0212] Fix with 4% paraformaldehyde for 40 min and wash three times with PBS; permeabilize cells with 0.1% Triton-100 for 10 min and wash three times with PBS;

[0213] After blocking with 5% BSA at room temperature for 40 min, the purified scFv-Fc antibody (1 μg / mL) was added as the primary antibody, incubated at 37°C for 1 h, and washed three times with PBS;

[0214] FITC-Goat Anti-Pig IgG (H+L) (1:100), incubate at 37°C in the dark for 1 h, wash three times with PBS;

[0215] Wash three times with ultrapure water, add DAPI to stain nuclei for 10 min, wash three times with PBS, seal the slides, and observe under a microscope.

[0216] The results are as follows Figure 8 As shown. It can be seen that the antibody prepared in the present application can specifically react with the p72 protein in the cytoplasm, and the fluorescence of scFV-Fc is mainly distributed in the cell nucleus, indicating that scFV-Fc targets the cell nucleus. Overall, the indirect immunofluorescence test also further verifies that the antibody has good affinity and reactivity.

Claims

1. An anti-African swine fever virus antibody targeting p72 protein, characterized in that: The antibody is a single-chain antibody that only includes the heavy chain variable region VH and the light chain variable region VL; The heavy chain variable region VH is 391 bp in length, and its nucleotide sequence is shown in SEQ ID No.1; the light chain variable region VL is 321 bp in length, and its nucleotide sequence is shown in SEQ ID No.

2.

2. The anti-African swine fever virus antibody against p72 protein according to claim 1, characterized in that: A linker is provided between the heavy chain variable region VH and the light chain variable region VL, and the linker sequence is: GGTGGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCAGGAGGAGGAGGAGGAAGC.

3. A scFv-Fc antibody against African swine fever p72 protein, characterized in that: The base sequence length is 1494 bp, and the base sequence is shown in SEQ ID NO.

3.

4. The method for preparing the scFv-Fc antibody against African swine fever p72 protein according to claim 3, characterized in that: The steps include: (I) Preparation of recombinant expression vector First, obtain the correct antibody nucleotide sequence; Subsequently, the above antibody nucleotide sequence is recombined using a eukaryotic expression plasmid as a vector to construct a recombinant eukaryotic expression vector that can translate and express the nucleotide sequence shown in SEQ ID NO.3; (II) Antibody protein expression The correctly constructed recombinant plasmid vector in step (a) is transformed into 293F cells, and after culturing for 5-7 days, the culture containing the scFv-Fc recombinant antibody is collected; (III) Purification to obtain single-chain scFv-Fc antibody The culture obtained in step (ii) is centrifuged to remove the cell precipitate and retain the supernatant, which is the solution containing the single-chain scFv-Fc antibody.

5. The method for preparing the scFv-Fc antibody against African swine fever p72 protein according to claim 4, characterized in that: In step (a), the eukaryotic expression plasmid is pINFUSE-mIgG2b-Fc2 plasmid.

6. The method for preparing the scFv-Fc antibody against African swine fever p72 protein according to claim 4, characterized in that: In step (iii), the obtained supernatant is further concentrated using a Millipore ultrafiltration centrifuge tube with a molecular weight cutoff of 15KD, and then purified using Protein A affinity filler to obtain a purified scFv-Fc antibody.

7. Use of the anti-African swine fever virus antibody against p72 protein as claimed in claim 1 in the preparation of an agent for detecting or preventing African swine fever virus.

8. Use of the scFv-Fc antibody against African swine fever p72 protein according to claim 2 in the preparation of an agent for detecting or preventing African swine fever virus.

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