CN115975011A - anti-African swine fever virus antibody aiming at p72 protein - Google Patents

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-African swine fever virus antibody aiming at a p72 protein. The antibody belongs to a single-chain antibody only comprising a heavy chain variable region VH and a light chain variable region VL. In the application, a specific scFv-Fc antibody with high affinity to the African swine fever virus capsid protein p72 protein is obtained by screening based on the existing mature phage antibody library screening technology. Preliminary experiment results show that the single-chain antibody well retains the affinity activity to the antigen and can specifically recognize and combine with capsid protein p72; and because the molecular weight is small and the penetrating power is strong, the preparation method is easy to carry out mass preparation by means of a prokaryotic or eukaryotic expression system through a genetic engineering technical means, and can lay a certain technical foundation for the development of the detection, prevention and treatment medicines of the African swine fever virus.

Description

anti-African swine fever virus antibody aiming at p72 protein

Technical Field

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-African swine fever virus antibody aiming at a p72 protein.

Background

African Swine Fever (ASF) is a highly contagious and acute hemorrhagic viral disease, with mortality rates approaching 100% in domestic pigs after infection. Since the ASF is discovered in Kenya for the first time in 1921, huge damage is caused to the pig breeding industry of various countries.

It has been shown that the genome of African Swine Fever Virus (ASFV), which causes African Swine fever, is double stranded DNA, 170-190kb in size, but complex in structure. Due to limited research on functions of various proteins encoded by viruses and virus variation and the like, a commercial vaccine product which is better for preventing African swine fever is not available at present.

Disclosure of Invention

The application aims to provide a specific scFv-Fc (Fragment variable Fragment Fc) antibody with high affinity for the African swine fever virus capsid protein p72 protein by combining with a related phage screening technology, thereby laying a certain technical foundation for the preparation of related vaccines.

The technical scheme adopted by the application is briefly described as follows.

An anti-african swine fever virus antibody against a p72 protein, which is a single chain antibody (i.e., comprising only a heavy chain variable region and a light chain variable region), comprising: a heavy chain variable region VH and a light chain variable region VL;

the length of the heavy chain variable region VH is 391bp, and the nucleotide sequence (shown as SEQ ID No. 1) is as follows:

GAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGGGCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAG；

the amino acid sequence coded by the heavy chain hypervariable region VH consists of 130 amino acids, and the amino acid sequence is specifically as follows:

EEKLVESGGGLVQPGGSLRLSCVGSGFTFSSTYINWVRQAPGKGLQWLAAISTSGGSTYYTDSMEGRFTISKDNSQNTAYLQMNSLRTEDTARYYCATIRGCYSYGASCYRSWASFMDLWGPGVEVVVSS；

the heavy chain variable region VH comprises three hypervariable regions, and the corresponding base sequences are respectively:

GATTCACCTTCAGTATACCTAC (corresponding to encoded amino acid sequence: GFTFSSTY) at positions 457-480;

at positions 532-555: ATTAGTACTAGTGGTGGTAGCACC (corresponding encoded amino acid sequence: ISTSGST);

at positions 670 to 73: GCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTT

ATGGATCTC (corresponding encoded amino acid sequence: ATIRGCYSYGASCYRSWASFMDL);

the light chain variable region VL has the length of 321bp, and a nucleotide sequence (shown as SEQ ID No. 2) as follows:

TCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTC；

the light chain variable region VL consists of 107 amino acids, and the amino acid sequence of the light chain variable region VL is specifically as follows:

SYELTQPSSESVALGSTAKITCSGDLLDEKYTQWYQQKPGQAPLLLIYKDNERPSGIP ERFSGSSSGKTATLTITGAQAEDEADYSCQSGDRINNAIFGGGTHLTVL；

the light chain variable region VL comprises three hypervariable regions, and the corresponding base sequences are respectively:

at positions 82 to 99: CTGCTGGATGAAAAATAT (the corresponding encoded amino acid sequence is LLDEKY);

at positions 151 to 159: AAGGACAAT (the corresponding encoded amino acid sequence is KDN);

at positions 268 to 297: CAGTCAGGTGACAGGATTAATAATGCTAT (corresponding encoded amino acid sequence: QSGDRINNAI);

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;

(the corresponding encoded amino acid sequence is GGGGGGSGGGGSGGGGGS).

On the basis of the light chain variable region VL and the heavy chain variable region VH, the application provides an scFv-Fc antibody for resisting African swine fever p72 protein, the length of a base sequence of the scFv-Fc antibody is 1494bp, and the base sequence is shown as SEQ ID NO.3, and specifically comprises the following components:

ATGGCCTCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTCGGTGGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCAGGAGGAGGAGGAGGAAGCGAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGGGCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGCTAGTGGCCAGGCCGGCCTGGCATCTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA；

the base sequence corresponds to an encoded amino acid sequence with the length of 498 amino acids, and the amino acid sequence is specifically as follows:

MASYELTQPSSESVALGSTAKITCSGDLLDEKYTQWYQQKPGQAPLLLIYKDNERPSGIPERFSGSSSGKTATLTITGAQAEDEADYSCQSGDRINNAIFGGGTHLTVLGGGGGGSGGGGSGGGGGSEEKLVESGGGLVQPGGSLRLSCVGSGFTFSSTYINWVRQAPGKGLQWLAAISTSGGSTYYTDSMEGRFTISKDNSQNTAYLQMNSLRTEDTARYYCATIRGCYSYGASCYRSWASFMDLWGPGVEVVVSSASGQAGLASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

the scFv-Fc antibody for resisting the African swine fever p72 protein is prepared by the following steps:

(one) preparation of recombinant expression vector

Firstly, obtaining a correct antibody nucleotide sequence;

then, taking an eukaryotic expression plasmid (for example, pINFUSE plasmid) as a vector, recombining the nucleotide sequence of the antibody, and constructing to obtain a recombined eukaryotic expression vector capable of translationally expressing the nucleotide sequence shown in SEQ ID NO. 3;

taking the plasmid pINFUSE-mIgG2b-Fc2 as an example (product of InvivoGen company), the nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 can be recombined during recombination (if a plasmid vector without Fc fragment is adopted, the sequence shown in SEQ ID No.3 needs to be recombined); for specific recombination operations, reference is made to the following:

respectively carrying out enzyme digestion on eukaryotic expression vectors pINFUSE-mIgG2b-Fc2 by using restriction enzymes EcoRI and NcoI, recovering an enzyme digestion product, and connecting the recovered enzyme digestion product with nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 by using T4 DNA ligase;

after connection is finished, electrically transforming the connection product into TG1 competent cells, screening and identifying to ensure correct plasmid recombination, and extracting recombined expression vector plasmids for later use;

(II) performing antibody protein expression

Transforming 293F cells with good cell state by the correct recombinant plasmid vector constructed in the step (I) by using a Sinofection transfection reagent according to the instructions of SinoBiolocal SMM 293-TII Expression Medium (SMM 293-TII Expression Medium) and the Sinofection transfection reagent, culturing for 5-7 days, and collecting a culture containing the scFv-Fc recombinant antibody;

(III) purification to obtain scFv-Fc antibody

Centrifuging the culture obtained in the step (II) at 4 ℃ for 10min at 5000g, removing cell precipitates, and reserving supernatant, wherein the supernatant is the solution containing the single-chain scFv-Fc antibody;

and further concentrating the supernatant by using a Millipore ultrafiltration centrifugal tube with the molecular weight cut-off of 15KD, and purifying the supernatant by using protein A affinity packing to obtain the purified scFv-Fc antibody.

In the prior art, because the research on innate immunity and adaptive immunity of pigs infected with African Swine Fever Virus (ASFV) is limited, the expression levels of various antibody proteins after the virus infection are deeply researched, the interaction condition among the antibody proteins is known, and the method has important technical significance for accurate diagnosis of ASFV infection, excavation of key antigen epitope and vaccine design.

In the application, a specific scFv-Fc antibody with high affinity to the African swine fever virus capsid protein p72 protein is obtained by screening based on the existing mature phage antibody library screening technology. The antibody belongs to a single-chain antibody only containing a heavy chain variable region and a light chain variable region, and the single-chain antibody not only retains the affinity activity to the antigen, but also has the technical advantages of small molecular weight and strong penetrating power, so the antibody has better application potential in the diagnosis and treatment of ASFV. Preliminary experiment results show that the single-chain antibody well retains the affinity activity to the antigen and can specifically recognize and combine with capsid protein p72; and because the molecular weight is small and the penetrating power is strong, the preparation method is easy to carry out mass preparation by means of a prokaryotic or eukaryotic expression system through a genetic engineering technical means, and can lay a certain technical foundation for the development of drugs for detecting, preventing and treating African swine fever viruses, thereby having better application prospect.

Drawings

FIG. 1 is a gel electrophoresis detection of VH and VL gene PCR products, where M: DNA Marker DL5000;

FIG. 2 shows the result of detecting the amplification of scFv gene SOE-PCR by gel electrophoresis, wherein M: DNA Marker DL2000;1: scFv spliced by VH-VL (kappa); 2: scFv spliced by VH-VL (lambda);

FIG. 3 shows the result of PCR amplification of the insert fragment of the antibody library detected by gel electrophoresis, wherein M: DNA Marker DL5000;1-24: a single colony of a phage antibody library;

FIG. 4 shows the detection of the affinity of recombinant phage antibodies by the phase-ELISA;

FIG. 5 shows ASFV-scFv-83-Fc after Protein A purification by SDS-PAGE, in which: m, protein Marker;1: blank control 293F cells; 2: eukaryotic expressed specific scFv-83 cultures;

FIG. 6 shows the result of Western blot purification analysis, in which: m, protein Marker;1: western blot result of the purified ASFV-scFv-83-Fc;

FIG. 7 is a Western blotting method for analyzing the reactivity of ASFV-scFv-83 with inactivated ASFV virus infected PAM cells; 1: a blank control group of PAM cells; 2: ASFV HLJ/18 infected PAM cells;

FIG. 8 identification of scFv-p72-83 antibody activity for IFA; p72 represents Mock for control; DAPI is a nucleus staining result graph; MERGE represents a combinatorial graph;

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Biological material:

escherichia coli TG1, pHEN2 phagemid vector, eukaryotic expression vector pFUSE-Fc, proliferation type M13KO7, DH5 alpha competent cell, etc., are common biological materials in the existing biological research, can be obtained by open channel (for example, scophyta organism company);

main reagents and kits:

porcine peripheral blood lymphocyte isolate Kit, purchased from Solebao Biochemical company;

DL2000 DNArker, 6 × Loading Buffer, etc., available from Baozhi corporation;

SfiI-HF, ecoRI and NcoI restriction enzymes, product of NEB corporation;

DNA fragment recovery kit, purchased from Thermo Fisher Scientific;

trizol, protein A affinity chromatography resin, purchased from Kisry Bio Inc;

the plasmid extraction kit is a product of Beijing Quanzijin company.

The recombinant p72 protein referred to in the following examples, trimeric recombinant p72 protein prepared by the present inventors from ASFV capsid protein p72 and its chaperone protein pB602L expressed in human embryonic kidney 293 (HEK 293) cells by using the existing biotechnology, whose amino acid sequence can be referred to: pig/HLJ/18GenBank MK333180.1.

Example 1

By combining with a related phage screening technology, the specific swine scFv-Fc antibody with high affinity for the African swine fever virus capsid protein p72 is obtained by screening. Before describing a specific screening process, it should be explained that, since the construction of a library of porcine-derived natural immune single chain antibodies (scFv) is the basis of the screening of the relevant antibodies (in the screening process, the target of the library is the antibody sequence), the process of combining the library construction with the relevant screening process is briefly described below.

(I) overlapping PCR amplification method to obtain full-length scFv gene

Firstly, obtaining a cDNA template of an African swine fever antibody positive pig, and specifically operating:

taking peripheral blood of a pig with positive African swine fever antibody as a sample, separating to obtain peripheral blood mononuclear cells, extracting total RNA and synthesizing cDNA through reverse transcription by referring to a related kit instruction, and storing at-80 ℃ for later use.

Secondly, the prepared cDNA is used as a template, an overlap extension PCR method is adopted to amplify and prepare a full-length scFv gene, and during the specific PCR amplification: three rounds of PCR amplification were performed, during which: the V (kappa), VL and VH genes are obtained by PCR amplification respectively, and finally the full-length scFv gene is obtained by an overlapping PCR amplification method, and the specific process is briefly described as follows.

During the first round of PCR amplification:

for the V (kappa) gene, the following primer pairs were designed:

a forward primer:

Vκ-F-1:GCGGCCCAGCCGGCCATGGCCGCCATCCAGCTGACCCAGTCTCC，

Vκ-F-2:GCGGCCCAGCCGGCCATGGCCGCCATYGTGCTGACCCAGACTCC，

Vκ-F-3:GCGGCCCAGCCGGCCATGGCCGAAATTGTGCTGACCCAGTCTGC，

Vκ-F-4:GCGGCCCAGCCGGCCATGGCCGAAACAACASTCACTCAATCTCC，

Vκ-F-5:GCGGCCCAGCCGGCCATGGCCGCCATGGTGTTGACCCAGAGTGC；

reverse primer:

Vκ-R-1:CCAGAGCCGCCGCCGCCGCCTTTGAKYTCCAGCTTAGTCCCG，

Vκ-R-2:CCAGAGCCGCCGCCGCCGCCTTTGGGCTCCACTTTGGTCCCCG，Vκ-R-3:CCAGAGCCGCCGCCGCCGCCTTCAATCTCCACGGATGTCCCT，

Vκ-R-4:CCAGAGCCGCCGCCGCCGCCTTTGAGTTCCAGCTTGGTTCC；

when the V (kappa) gene is amplified by PCR, the forward primer and the reverse primer are randomly matched and combined for PCR amplification;

for VH gene amplification, the following primer pairs were designed:

a forward primer:

VH-F-1：ggttcaggaggaggaggaggaagc GAGGTGAAGCTGGTGGAGTCTGGA，

VH-F-2：ggttcaggaggaggaggaggaagc GAGGAGAAGCTGGTGGAGTCTGGA；

reverse primer:

VH-R-1:tgtgcagatgatgaccgcgGCTAGCTGAGGACACGACGACTTCAAC，

VH-R-2:tgtgcagatgatgaccgcgGCTAGCTGAGGAGACGGTGACCTCGAC，

VH-R-3:tgtgcagatgatgaccgcgGCTAGCTGAGGAGACGGTGACCAGGA；

when the VH gene is amplified by PCR, the forward primer and the reverse primer are randomly matched and combined for PCR amplification;

for the VL gene, the following primer pairs were designed:

a forward primer:

VL-F-1:GCGGCCCAGCCGGCCATGGCCCAGACTGTGATCCAGGAGCC，

VL-F-2:GCGGCCCAGCCGGCCATGGCCTCCTATGAGSTGACTCAGCC，

VL-F-3:GCGGCCCAGCCGGCCATGGCCCAGGCTGTGCTGACTCAGCCG，

VL-F-4:GCGGCCCAGCCGGCCATGGCCCAGTCTGCCCTGACTCAGCCC；

reverse primer:

VL-R-1:CCAGAGCCGCCGCCGCCGCCACCGAGGACGGTCAGATGGGT；

when the VL gene is amplified by PCR, the forward primer and the reverse primer are randomly paired and combined for PCR amplification;

during PCR amplification, the 25. Mu.L system was designed as follows:

2×Taq Master Mix，12μL；

upstream and downstream primers, each 0.5 μ L;

cDNA template, 1. Mu.L;

deionized water, 11.5 μ L;

the amplification procedure was: 95 deg.C, 5min,95 deg.C, 30s,56 deg.C, 30s,72 deg.C, 20s,30 cycles; 72 ℃ for 10min;

after PCR amplification is finished, 1% agarose gel detection is carried out on the amplification product, and the target band is extracted, purified and identified.

In the second round of PCR amplification (since the present application employs the overlap PCR reaction to prepare the full-length scFv gene fragment, for this reason, in order to use the second round of PCR product in the subsequent overlap PCR reaction, the inventors redesign the primers for amplification by extending the length of the primers), the specific sequences of the primers for PCR amplification are designed as follows:

VLκ-F：ACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCAT，

VLκ-R：TGAACCACCACCACCAGAGCCGCCGCCGCCGCC；

during PCR amplification, a 25. Mu.L amplification system was designed as follows:

2×Taq Master Mix，12μL；

upstream and downstream primers, each 0.5. Mu.L;

cDNA template (first round amplification product), 1 μ L;

deionized water, 11.5 μ L;

the amplification procedure was: 95 deg.C for 5min;95 ℃, 30s,56 ℃, 30s,72 ℃, 20s and 30 cycles; 72 ℃ for 10min;

after the PCR amplification is finished, 1% agarose gel detection is carried out on the amplification product (the electrophoresis result of part of the amplification fragment is shown in figure 1), and the target band is extracted, purified and identified.

During the third PCR amplification:

taking the second round amplification product as a template, and adopting an overlap extension PCR splicing method to obtain a full-length scFv gene; during PCR amplification, the primer sequence is designed as follows:

scFv-F:AGGAGGCGCGCATAATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGT T，

scFv-R:tgtgcagatgatgaccgcgGCTAGCTG；

during PCR amplification, a 25. Mu.L amplification system was designed as follows:

2×Taq Master Mix，12μL；

upstream and downstream primers, each 0.5. Mu.L;

cDNA template (second round amplification product), 1 μ L;

deionized water, 11 μ L;

the amplification procedure was: 95 deg.C for 5min;95 ℃, 30s,56 ℃, 30s,72 ℃, 20s and 30 cycles; 72 ℃ for 10min;

after the PCR amplification is finished, carrying out 1% agarose gel detection on the amplification product (partial amplification result is shown in figure 2), cutting the gel under an ultraviolet lamp to recover a target band at the position of about 750bp, and recovering the target band at the position of about 750bp and purifying a DNA fragment by using a gel recovery kit to obtain the DNA fragment: and the scFv is formed by randomly splicing the VH gene and the VL gene.

(II) construction of phage antibody library

On the basis of obtaining the scFv gene in the step, carrying out enzyme digestion on the obtained pig scFv gene (library) and the phagemid vector pHEN2 vector by using SfiI-HF restriction endonuclease respectively, recovering an enzyme digestion product, and further connecting the enzyme digestion product by using T4 DNA ligase;

then, the ligation products are transformed into escherichia coli TG1 competent cells by an electric shock transformation method, plates are coated for resistance screening, positive single colonies are further randomly selected for colony PCR identification to verify scFv gene transformation rate (partial detection results are shown in figure 3), and diversity of scFv phage libraries is analyzed by sequencing to ensure diversity of scFv gene sequences in the libraries to meet target screening application (in screening application, M13KO7 auxiliary phage is further used for packaging bacterial libraries containing phage DNA into phage libraries).

The measurement results by colony calculation show that the scFv library constructed in the application has the capacity of 3.2 × 10 ⁹ The method has the characteristics of good library capacity, good diversity and the like, and can better meet the requirements of screening application.

(III) phage selection

On the basis of the library constructed above, the following steps were carried out for screening of (anti-African swine fever p72 protein antibody) phage as follows.

And (3) carrying out specific antibody panning and screening on the constructed single-chain antibody library by adopting a microplate screening method and taking the recombinant p72 protein as a target molecule. The screening process comprises the following steps: incubating antigen and phage antibody library → eluting unbound non-specific phage → amplifying phage antibody, and screening 3 rounds (3 rounds in this example) to obtain enriched specific phage antibody. Specific operations are referred to as follows.

(1) Taking recombinant p72 protein as target molecule, coating solution CBS (0.1M NaHCO) is used ₃ pH = 9.6) to the desired concentration (coating solution target molecule concentration 2 μ g/well in the first round of screening, followed by 4-fold dilutions in each round), 100ul per well; incubating overnight at 4 ℃;

on the next day, the well was aspirated away, and the wells were washed 1 time with 200 μ L TBST, and the liquid was aspirated away; add 200. Mu.L blocking solution (TBS solution containing 5% skimmed milk powder and 0.25% Tween 20), incubate well for 2h at 37 ℃;

finally, abandon the liquid in the hole, with 200u L containing 0.25% Tween20 TBS washing hole plate 1 times (according to the circumstances, in the subsequent panning operation according to the circumstances to adjust the concentration of Tween);

(2) After discarding the well fluid, add the prepared antibody phage antibody library (1X 10) ¹³ Hole), standing and incubating for 1h at 37 ℃;

after incubation, abandoning liquid in the holes, beating and throwing the holes clean, washing the holes for 5 times by TBST containing 0.25 percent Tween20 for 5min each time, sucking and throwing the liquid, and beating and drying the holes;

(3) Adding 1mg/ml trypsin (100 ul/well), slowly shaking at room temperature for 8min, repeatedly blowing and sucking with pipette, and sucking out supernatant;

adding 100ul of 4% skimmed milk-PBS for neutralization (to obtain eluate), and directly performing subsequent operation or storing at 4 deg.C;

(4) Adding the elution product in the step (3) into a pre-cultured TG1 monoclonal bacterium solution (a monoclonal of TG1 activated by a pre-streaked plate is selected and inoculated into 20ml of 2 XYT culture medium, shaking culture is carried out at 37 ℃ and 250rpm until the logarithmic phase is reached, and OD600= about 0.5), uniformly mixing, standing at 37 ℃ for 30min for infection, then supplementing resistance components (Amp, the final concentration is 20 ug/ml), and slowly shaking at 37 ℃ and 180rpm for 60min for resistance screening;

(5) Adding a corresponding amount of M13KO7 (the number of helper phages: the number of thalli is more than or equal to 10: 1) into the culture of the step (4), and standing for 20min at 37 ℃ to assist infection;

subsequently, 30ml of 2 XYT medium and Amp (final concentration 20 ug/ml) were supplemented, and the mixture was shaken at 37 ℃ and 180rpm for 60min;

(6) After the culture is finished, centrifuging at 5000rpm for 10min, collecting thalli, suspending the thalli in 50ml of 2 XYT culture medium (Amp 50ug/ml + Kan 10 ug/ml), and culturing at 30 ℃ and 220rpm overnight;

after the culture is finished, centrifuging at 4 ℃ and 12000rpm for 10min, transferring the supernatant into a new centrifuge tube, and centrifuging the supernatant again at 4 ℃ and 12000rpm for 10min;

after the centrifugation is finished, transferring the centrifuged supernatant into a new centrifuge tube again, adding 1/4 volume of PEG 8000/NaCl, and precipitating at 4 ℃ for 4 h/ice for 2h;

(7) After the ice precipitation, centrifugation at 12000rpm for 20min at 4 ℃ was performed, the supernatant was discarded, and the precipitate was resuspended in 0.5ml of PBS and centrifuged at 12000rpm for 10min at 4 ℃ (to precipitate the residual cells);

finally, the supernatant was transferred to a new centrifuge tube (this is the amplification product), the titer of the amplification product was determined (titer determination method: inoculating TG1 monoclonal in 2 XYT non-resistant medium, culturing at 37 ℃ and 220rpm until OD600 ≈ 1.0, and gradient dilution of phage library to 10 ≈ g ^-10 100uL of the diluted solution was added to 200uL of the cultured TG1, and the mixture was left to stand in an incubator at 37 ℃ for 30min, and all the diluted solution was spread on an ampicillin-resistant plate, and cultured overnight at 37 ℃, and the number of clones on the plate was counted the next day, and the titer was calculated).

In the screening process, when positive condition identification is carried out on the elution product in the step (3), a PCR mode is adopted for identification, and a 20-mu-l amplification system is designed as follows (the primer sequences are F: tggAATTgTgAgCggATAACAATT and R: ATTCAGATCCTCTTCTGAGAT respectively):

rTaq，5ul；

10×Buffer，1ul；

template (monoclonal colonies), 1ul;

primer F, 1ul;

primer R, 1ul;

H ₂ o, adding to 10ul;

PCR conditions were as follows: 94 ℃ for 4min;94 ℃ and 30s,53 ℃ and 30s,72 ℃ and 40s, and 25 cycles; 72 ℃, 2min,10 ℃ and 10s; thus, a round of panning is completed.

According to the first round of panning results, the concentration of the target molecule coating is properly reduced, or the concentration of Tween20 in the washing liquid is increased, the washing times and time are increased, the binding time and temperature of the target molecule and the phage library are reduced, the panning pressure is increased, and the second and third rounds of panning are carried out to reduce the background and further enrich the high-affinity phage. In the screening process, 10 single clones are randomly selected for sequencing analysis after 2 nd and 3 rd rounds of panning, and the characteristics of the antibody heavy chain variable region sequence and the antibody light chain variable region sequence are analyzed.

In the screening process, ELISA test is adopted to detect the affinity condition after each round of affinity screening. Specific experimental methods are referenced below:

(1) Coating: diluting the target protein to 3 mu g/mL by using a coating solution, coating ELISA plate holes according to the quantity of 100 mu L/hole, and standing overnight at 4 ℃;

(2) And (3) sealing: throwing out the redundant coating solution, inverting the flat plate, beating and throwing on a clean paper towel to remove residual liquid, washing the plate for 3 times by PBS, adding the confining liquid, keeping the plate at 200 ul/hole, and standing for 1h at 37 ℃;

(3) Sample adding: throwing out redundant confining liquid, inverting the flat plate, beating and throwing on a clean paper towel to remove residual liquid, washing the plate for 3 times by PBS, adding the phase supernatant, keeping the plate at 50 ul/hole, and standing for 1h at 37 ℃;

(4) Secondary antibody: after throwing out the excess phase supernatant (phage supernatant), inverting the plate and patting on a clean paper towel to remove the residual liquid, after washing the plate for 3 times with PBS, adding HRP rabbit antibody M13 diluted with 1% Milk-PBS 1;

(5) Color development: throwing out excessive solution, inverting the plate, beating on clean paper towel to remove residual liquid, washing the plate for 4 times with PBS, adding TMB color developing solution (50 ul/hole), reacting completely, and adding 2M H ₂ SO ₄ The color development is stopped, and the detection is carried out at 450 nm.

The results are shown in FIG. 4. As can be seen, scFV-83 can recognize and bind to p72 protein with high specificity.

The above sequencing analysis results show that scFV-83, as an anti-African swine fever virus antibody against p72 protein, belongs to a single chain antibody (i.e. it only comprises heavy chain variable region and light chain variable region), whose heavy chain variable region VH sequence is shown in SEQ ID No.1 and light chain variable region VL is shown in SEQ ID No.2, specifically as follows:

heavy chain variable region VH:

GAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGGGCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAG；

light chain variable region VL:

TCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTC。

on the basis of the above sequence, the base sequence (as shown in SEQ ID No. 3) of the scFv-Fc antibody against African swine fever p72 protein can be clearly determined as follows:

ATGGCCTCCTATGAGCTGACTCAGCCGTCTTCAGAGTCAGTGGCCTTGGGAAGTACGGCCAAGATCACCTGCTCCGGGGATCTGCTGGATGAAAAATATACGCAATGGTACCAGCAGAAGCCAGGCCAGGCCCCCCTGCTGCTCATTTATAAGGACAATGAGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGGTCCAGCTCAGGGAAAACGGCCACCCTGACCATCACTGGGGCCCAGGCTGAGGACGAGGCCGACTACTCGTGTCAGTCAGGTGACAGGATTAATAATGCTATTTTCGGCGGTGGGACCCATCTGACCGTCCTCGGTGGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCAGGAGGAGGAGGAGGAAGCGAGGAGAAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGTAGTACCTACATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAGTGGCTTGCAGCTATTAGTACTAGTGGTGGTAGCACCTACTACACAGACTCTATGGAGGGCCGATTCACCATCTCCAAAGACAACTCCCAGAACACGGCCTATCTGCAAATGAACAGCCTGAGAACAGAAGACACGGCCCGCTATTACTGTGCAACCATCCGGGGTTGCTATAGCTATGGTGCTAGTTGCTATCGTTCCTGGGCGTCCTTTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGCTAGTGGCCAGGCCGGCCTGGCATCTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA。

example 2

Based on example 1, it can be seen that scFV-83 has stronger binding force with p72 Protein, and in order to further evaluate the application prospect of the Protein, the inventor uses pINFUSE-mIgG2b-Fc2 plasmid (the plasmid is recombined with CH2-CH3 sequence, and the CH2-CH3 sequence is referred to as GenBank: BAE20056.1; it should be emphasized that the plasmid is only used for convenience based on practical experiments, and does not show that the technical scheme of the application needs to depend on the plasmid, and other universal plasmids are used for realizing antibody expression) as a vector, and uses 293F cell for related Protein expression, and further uses Protein A to obtain a purified pig-derived single-chain scFv-Fc antibody against p 72. The specific preparation process is described as follows.

(I) recombining eukaryotic expression vector pINFUSE-mIgG2b-Fc2

Firstly, inoculating a monoclonal corresponding to phage No. 83 (scFV-83) into 1mL of 2 XYT liquid medium containing ampicillin (100. Mu.g/mL) and tetracycline resistance (15. Mu.g/mL), performing shaking culture at 37 ℃ and 260rpm overnight, and performing plasmid extraction on the culture by using a plasmid miniprep kit (TRANS products, the reference instruction is used for operation) (the concentration of the plasmid in the extract is measured by a NanoDrop2000 ultraviolet spectrophotometer to ensure that the requirement of subsequent use is met);

subsequently, the extracted plasmid and the eukaryotic expression vector pINFUSE-mIgG2b-Fc2 were digested with restriction enzymes EcoRI and NcoI, respectively, and the digested products were subjected to 1% agarose gel electrophoresis, respectively, followed by recovery of the digested products with a gel recovery kit and ligation of the digested products with T4 DNA ligase.

During the operation, the enzyme digestion system (20. Mu.L) is designed as follows:

the extracted plasmid (or eukaryotic expression vector pINFUSE-mIgG2b-Fc 2) is 2 mug;

EcoRI and NcoI, 2. Mu.L each;

10×CutSmart Buffer，2μL；

ddH2O to 20 μ L;

the enzyme digestion is carried out for 2h at 50 ℃.

For T4 DNA ligase ligation, the ligation system (10. Mu.L) was designed as follows:

100ng of the extracted plasmid enzyme digestion product;

pFUSE-Fc enzyme digestion product, 60ng;

T4 DNA ligase，0.5μl；

10×T4 DNAligase Buffer，1μl；

ddH2O to 10 μ L;

ligation was carried out for 3h at 16 ℃.

Then, the ligation product was transformed into TG1 competent cells by a heat shock transformation method, which is specifically referred to as:

adding 10 μ L of the ligation product into TG1 competence, performing ice bath for 30min, performing heat shock at 42 ℃ for 45s, and then performing incubation on ice for 5min; add 600 u L non-resistant 2 XYT liquid medium, 37 degrees C, 260rpm recovery 1h after plating containing ampicillin (100 u g/mL) and tetracycline resistance (15 u g/mL) 2 XYT plate, 37 degrees C overnight culture.

And finally, selecting positive single clone for sequencing verification, and naming the recombinant plasmid expression vector as: pINFUSE-scFV-83.

(II) performing scFV-Fc antibody expression

The correct recombinant plasmid vector pINFUSE-scFV-83 constructed in the above step (one) was transformed into 293F cells with good cell status by reference to SinoBiolocal SMM 293-TII Expression Medium and SinoFection transfection reagent instructions, and placed in a shaker at 37 deg.C and 120rpm,5% CO ₂ After 5-7 days of culture, cultures containing scFv-Fc recombinant antibodies were collected.

(III) purifying to obtain the single-chain scFV-Fc antibody

And (d) centrifuging the culture collected in the step (II) at 4 ℃ for 10min at 5000g, removing cell precipitates and reserving a supernatant, wherein the supernatant is the solution containing the single-chain scFv-Fc antibody after expression.

Further, the supernatant was concentrated by a Millipore ultrafiltration tube with a cut-off of 15KD, and purified and eluted by ProteinA affinity packing, to obtain the purified scFv-Fc antibody.

The obtained purified scFv-Fc antibody was identified by SDS-PAGE, and the results are shown in FIG. 5. Lane 1 of the SDS-PAGE shows the blank control 293F cells, lane 2 shows the recombinant antibody, lane shows a single band of interest, indicating better purity. Western blot analysis is further carried out on the purified and eluted target protein, and the result is shown in FIG. 6, which shows that: the size of the recombinant antibody accords with expectation, and the recombinant antibody obtained by the invention is further proved to have good activity and good purity.

(IV) identification

The inventors further examined the binding of the scFv-Fc antibody obtained above to p72 and inactivated virus by the IFA and Western blot methods. The specific case is briefly described as follows.

(1) Western Blot antibody activity identification

The reaction of scFV-83 with inactivated infected ASFV HLJ/18 strain (provided by the Harbin veterinary institute) Porcine Alveolar Macrophages (PAMs) samples were studied by immunoblotting using Porcine Alveolar Macrophages (PAMs) as samples.

In the experimental process, PAM cells infected by ASFV HLJ/18 strain and PAM cells not infected by ASFV in a blank control group are taken as SDS-PAGE samples, and all the samples are denatured for 30min at 95 ℃; the samples were then resolved by SDS-PAGE (12.5% PAGE gel Rapid preparation kit, bioMan), proteins were transferred to PVDF membrane by semidry method;

then, the PVDF membrane was blocked with 5% skim milk in Phosphate Buffered Saline (PBS) for 1h at room temperature;

then, after 5 times of PBST washing with the collected scFV-83 culture stock as a primary antibody, HRP-labeled goat anti-porcine IgG (H + L) (Proteintech, china) diluted 1;

finally, the PVDF membrane was visualized using ECL.

The results are shown in FIG. 7. It can be seen that: the antibody can specifically recognize and bind to ASFV HLJ/18 infected PAM cells, but does not react with blank PAM cells, and proves that the scFV-83 is indeed the antibody aiming at ASFV virus.

(2) IFA identification of antibody Activity

The test procedures and operations are referred to as follows:

with reference to the above operation and the prior art, a synthetic p3xFLAG-CMV-14 expression vector containing a p72 gene (Pig/HLJ/18GenBank; removing the culture medium 24h after transfection, and gently washing the cells with PBS;

fixing with 4% paraformaldehyde for 40min, washing with PBS for three times; 0.1% Triton-100 permeabilized cells for 10min, washed three times with PBS;

blocking with 5% BSA at room temperature for 40min, adding purified scFv-Fc antibody (1. Mu.g/mL) as primary antibody, incubating at 37 deg.C for 1h, and washing with PBS for three times;

FITC-Goat Anti-Pig IgG (H + L) (1, 100), incubated at 37 ℃ in the dark for 1h, washed three times with PBS;

washing with ultrapure water for 3 times, adding DAPI staining for 10min, washing with PBS for three times, sealing, and observing with a microscope.

The results are shown in FIG. 8. It can be seen that the antibody prepared by the application can specifically react with p72 protein in cytoplasm, and the fluorescence of the scFV-Fc is mainly distributed in nucleus, which indicates that the scFV-Fc targets to nucleus. Overall, indirect immunofluorescence assays also further confirmed that the antibody affinity and reactivity were good.

Claims (8)

1. An anti-african swine fever virus antibody against the p72 protein, which is a single chain antibody comprising only a heavy chain variable region VH and a light chain variable region VL;

the length of the heavy chain variable region VH is 391bp, and the nucleotide sequence is shown as SEQ ID No. 1; the light chain variable region VL has the length of 321bp, and the nucleotide sequence of the light chain variable region VL is shown as SEQ ID No. 2.

2. An anti-african swine fever virus antibody according to claim 1, against the p72 protein, wherein a linker is provided between the heavy chain variable region VH and the light chain variable region VL, the linker sequence being:

GGTGGCGGCGGCGGCGGCTCTGGTGGTGGTGGTTCAGGAGGAGGAGGAGGAAGC。

3. an scFv-Fc antibody for resisting African swine fever p72 protein is characterized in that the length of the base sequence is 1494bp, and the base sequence is shown as SEQ ID NO. 3.

4. The method for preparing the scFv-Fc antibody against African swine fever p72 protein according to claim 3, comprising the steps of:

(one) preparation of recombinant expression vector

Firstly, obtaining a correct antibody nucleotide sequence;

then, the eukaryotic expression plasmid is taken as a vector to recombine the nucleotide sequence of the antibody, and a recombined eukaryotic expression vector capable of translating and expressing the nucleotide sequence shown in SEQ ID NO.3 is constructed;

(II) performing antibody protein expression

Transforming 293F cells by the correct recombinant plasmid vector constructed in the step (one), culturing for 5-7 days, and collecting a culture containing the scFv-Fc recombinant antibody;

(III) purifying to obtain the single-chain scFv-Fc antibody

And (d) centrifuging the culture obtained in the step (II) to remove cell precipitates, and reserving a supernatant, wherein the supernatant 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, wherein in step (one), the eukaryotic expression plasmid is pINFUSE-mIgG2b-Fc2 plasmid.

6. The method according to claim 4, wherein in the step (III), the obtained supernatant is further concentrated by a Millipore ultrafiltration centrifugal tube with a molecular weight cut-off of 15KD, and then purified by ProteinA affinity filler to obtain the purified scFv-Fc antibody.

7. Use of an anti-african swine fever virus antibody against the p72 protein according to claim 1 for the detection or prevention of african swine fever virus.

8. Use of the scFv-Fc antibody against the african swine fever p72 protein according to claim 2 for the detection or prevention of african swine fever virus.

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