High-affinity nanoantibody targeting african swine fever virus deoxyuridine triphosphatase, and preparation method therefor and use thereof

Abstract

Provided are a high-affinity nanoantibody targeting African swine fever virus deoxyuridine triphosphatase, and a preparation method therefor and a use thereof. The nanoantibody targeting African swine fever virus deoxyuridine triphosphatase has three complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is positions 26-38 of SEQ ID No. 2, the amino acid sequence of CDR2 is positions 55-64 of SEQ ID No. 2, and the amino acid sequence of CDR3 is positions 100-113 of SEQ ID No. 2. Upon verification by ELISA, the nanoantibody can specifically bind to African swine fever virus deoxyuridine triphosphatase (dUTPase), and has relatively high affinity.

Description

High-affinity nanoantibodies targeting African swine fever virus deoxyuridine pyrophosphatase and their preparation and application Technical Field

[0001] The present invention relates to a high-affinity nanobody targeting African swine fever virus pyrophosphatase, and a preparation method and application thereof. Background Art

[0002] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease of pigs caused by the African swine fever virus (ASFV). It is highly contagious and lethal. Symptoms of ASFV infection are very similar to those of common swine fever, making them difficult to distinguish. Both present with high fever and red spots on the skin, but the morbidity and mortality rates of ASFV-infected pigs can reach 100%. As African swine fever spreads across the globe, no effective biological treatment has yet been developed, resulting in the culling of pigs to control its spread, causing severe losses to the economy and the swine industry.

[0003] The function of deoxyuridine triphosphatase (dUTPase, also known as E165R) is to increase the fidelity of DNA replication and improve the accuracy of DNA replication. It has played an important role as a drug target in Mycobacterium tuberculosis and Plasmodium falciparum. The dUTPase present in ASFV can also effectively ensure the fidelity of ASFV DNA replication, enabling ASFV to replicate smoothly and accurately in the host body. Therefore, it can also be used as a detection target and drug target for ASFV. The non-classical two-subunit active center of ASFV dUTPase is also a potential antiviral target. Viral inhibitors can be designed based on the active site differences, hydrophobicity distribution and electrostatic distribution between the enzyme and the host dUTPase (Ariza ME, Cox B, Martinez B, et al. Viral dUTPases: Modulators of Innate Immunity. Biomolecules, 2022, 12(2): 227.). Mouse monoclonal antibody studies have shown that ASFV dUTPase has three main antigenic regions, located between amino acids 100-120, 120-140, and 140-165, respectively. Antibodies screened based on these epitopes have the potential for integrated diagnosis and treatment (Chen CC, Lai HR, Liang HK, et al. A new method for detection African Swine Fever Virus: time-resolved fluorescence immunoassay. Journal of Fluorescence, 2021, 31(5): 1291-1296.).

[0004] Currently, ASFV diagnostic techniques can be divided into three categories: virus (intact particle) detection methods, nucleic acid detection methods, and immunoassay methods. Petrovan et al. prepared a panel of ASFV-p30-specific monoclonal antibodies and used three of these monoclonal antibodies for immunofluorescence detection of Vero cells and porcine macrophages infected with ASFV type I and type II. The results showed that the p30 monoclonal antibody could recognize both ASFV genotypes (Sanchez-Vizcaino JM, Mur L. African Swine Fever Diagnosis Update. Developments in Biologicals, 2013, 135: 159-165). Feng Chunyan et al. immunized mice with recombinant protein p72 to prepare eight monoclonal antibodies. They paired HRP-labeled and unlabeled antibodies to screen for the best antibody pairing. Finally, they established a double-antibody sandwich ELISA method using 5G8 as the capture antibody and 12F6-HRP as the detection antibody. At present, colloidal gold immunochromatography technology is used to detect ASFV. The antibodies used are generally monoclonal or polyclonal antibodies obtained by animal immunization. Zhang Xinyu et al. used ASFV-p54 and ASFV-p72 as targets and established a colloidal gold test strip method for p54 polyclonal antibodies and p72 monoclonal antibodies.

[0005] Antibody drugs play a crucial role in antiviral therapy. However, monoclonal antibodies suffer from poor permeability and poor binding to sterically hindered epitopes. Therefore, miniaturization of antibody molecules has become a research hotspot. In 1993, the Hamers group at the Free University of Brussels first discovered heavy-chain antibodies (HcAbs) in camel serum that naturally lack light chains and consist only of heavy chain dimers. These single-domain antigen-binding fragments, also known as the heavy chain variable region, constitute single-domain antibodies (VHHs) or nanobodies (Nb) (Hamers-Casterman C, et al. Naturally occurring antibodies devoid of light chains. Nature. 1993, 363(6428):446-8. DOI:10.1038 / 363446a0. PMID:8502296).

[0006] Nanobodies (VHHs) are composed of four conserved regions (Frameworks, FRs) and three complementarity determining regions (CDRs). They have inherent advantages such as low molecular weight, good permeability, easy passage through the blood-brain barrier, easy binding to antigenic epitopes in sterically hindered locations such as narrow slits, and easier prokaryotic expression and purification. Furthermore, the amino acids in the FR2 region of VHHs are more hydrophilic, reducing the aggregation of nanobodies. Compared to conventional full-length antibodies, VHHs offer significant advantages in stability, tissue permeability, antigen binding ability, and immunogenicity.

[0007] Invention Disclosure

[0008] The main problem to be solved by the present invention is to develop a nanoantibody that can bind to deoxyuridine pyrophosphatase in ASFV and play a role in the diagnosis or treatment of ASFV.

[0009] In order to solve the above problems, the present invention provides a nanobody targeting African swine fever virus deoxyuridine pyrophosphatase or an antigen-binding fragment containing the nanobody.

[0010] The nanobody targeting African swine fever virus deoxyuridine pyrophosphatase provided by the present invention or an antigen-binding fragment containing the nanobody comprises a complementary determining region CDR of a VHH chain, and has three complementary determining clusters CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is positions 26-38 of SEQ ID No.2 or a sequence having more than 80% identity thereto, the amino acid sequence of the CDR2 is positions 55-64 of SEQ ID No.2 or a sequence having more than 80% identity thereto, and the amino acid sequence of the CDR3 is positions 100-113 of SEQ ID No.2 or a sequence having more than 80% identity thereto.

[0011] In the present invention, the above 80% identity may be 80%, 85%, 90% or 95% identity or more.

[0012] The 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0013] The nanobodies described herein generally include a VHH consisting of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment comprises at least a portion of the nanobody that is sufficient to confer upon the fragment the ability to specifically bind to the dUTPase of African swine fever virus.

[0014] In the present invention, the Nanobody may be the following A1) or A2):

[0015] A1) a Nanobody with the amino acid sequence of SEQ ID No. 2 or a Nanobody with an amino acid sequence that is more than 80% identical to SEQ ID No. 2 and has the same function;

[0016] A2) Nanobodies obtained by linking a protein tag to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID No. 2.

[0017] A protein tag is a polypeptide or protein that is fused with a target protein using in vitro DNA recombination techniques to facilitate expression, detection, tracing, and / or purification of the target protein. Examples of protein tags include His tags, FLAG tags, MBP tags, HA tags, myc tags, GST tags, and / or SUMO tags.

[0018] In a specific embodiment of the present invention, the nanobody is specifically obtained by connecting a FLAG tag to the C-terminus of the amino acid sequence of SEQ ID No. 2.

[0019] In the above-mentioned nanobody, the nanobody consists of the complementary determining cluster region and the framework region.

[0020] The term "antibody" as used herein refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, with the number of disulfide bonds varying between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain opposes the first constant region of the heavy chain, and the variable region of the light chain opposes the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0021] In the present invention, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning, referring to a single-domain antibody (VHH) naturally present in camelids consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function.

[0022] The above-mentioned antigen-binding fragment can be a complete antibody, a fusion antibody, an antibody-drug conjugate, a Fab fragment, a Fv fragment, a Fab′ fragment, a F(ab′)2 fragment, a single-chain antibody (ScFv) or a minimal recognition unit (MRU) containing the nanobody.

[0023] The term "Fab fragment" refers to a heterodimer formed by disulfide bonds between a heavy chain Fd and a complete light chain. The heavy chain Fd refers to approximately half of the H chain in Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0024] The term "Fv fragment" refers to the construction of vectors containing VH and VL genes separately, co-transfection into cells, expression of each gene, and assembly into a functional Fv antibody. Alternatively, a stop codon can be set between VH and VL in the vector to express two small molecule protein fragments separately, which are then combined through non-covalent bonds to form an Fv antibody (Fv fragment).

[0025] The term "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CH1 domain as well as the region between the CH1 and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0026] The term "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0027] The term "variable" as used herein refers to the fact that certain portions of the variable region of an antibody differ in sequence, which contribute to the binding and specificity of each particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of both the light and heavy chains. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which generally have a β-pleated structure and are connected by three CDRs that form a connecting loop, which in some cases may form a partial β-pleated structure. The CDRs in each chain are closely aligned together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit various effector functions, such as involvement in the antibody's antibody-dependent cytotoxicity.

[0028] In some embodiments, the Nanobodies described herein may be truncated at the N- or C-terminus so that they comprise only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as they substantially retain antigen binding and specificity.

[0029] In the present invention, the nanobody is named ASFV4F1.

[0030] The present invention also provides a biomaterial related to the aforementioned nanobody, which can be any of the following:

[0031] B1) a nucleic acid molecule encoding a Nanobody as described above or an antigen-binding fragment of said Nanobody;

[0032] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0033] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0034] B4) a recombinant vector containing the expression cassette described in B2);

[0035] B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0036] B6) a recombinant microorganism containing the expression cassette described in B2);

[0037] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0038] B8) a recombinant microorganism containing the recombinant vector described in B4);

[0039] B9) a recombinant cell line containing the nucleic acid molecule of B1) or a recombinant cell line containing the expression cassette of B2);

[0040] B10) a transgenic animal cell containing the nucleic acid molecule described in B1), or a transgenic animal cell containing the expression cassette described in B2);

[0041] B11) transgenic animal tissue containing the nucleic acid molecule described in B1) or transgenic animal tissue containing the expression cassette described in B2);

[0042] B12) A transgenic animal organ containing the nucleic acid molecule described in B1) or a transgenic animal organ containing the expression cassette described in B2).

[0043] In the above-mentioned biological materials, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0044] In the above-mentioned biological material, the expression cassette B2) refers to a DNA capable of expressing the Nanobody in a host cell, which DNA may include not only a promoter for initiating transcription of the Nanobody-encoding gene, but also a terminator for terminating transcription of the Nanobody-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0045] Existing expression vectors can be used to construct a recombinant vector containing the expression cassette.

[0046] In the above biological materials, the vector may be a plasmid, cosmid, phage or viral vector.

[0047] In the above-mentioned biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule described in B1) into a pET-22b(+) vector.

[0048] In one embodiment of the present invention, the recombinant vector B3) is a recombinant vector obtained by introducing the Nanobody encoding gene (SEQ ID No. 1) into the pET-22b(+) vector. The recombinant vector pET-22b(+)-ASFV4F1-FLAG expresses the Nanobody ASFV4F1 of SEQ ID No. 2.

[0049] In the above-mentioned biological materials, the microorganisms may be bacteria (such as Escherichia coli), yeast, algae or fungi.

[0050] In the above-mentioned biological materials, the animal cells may not include germ cells, fertilized eggs and human embryonic stem cells, and may be somatic cells or cell lines. The animal organs may not include embryos.

[0051] The transgenic animals are animals obtained through biological methods such as recombinant DNA technology of genetic engineering.

[0052] In the above-mentioned biological material, the gene coding sequence of the CDR1 of the Nanobody is SEQ ID No. 1 from 76 to 114 or a sequence having 80% or more identity thereto, the gene coding sequence of the CDR2 of the Nanobody is SEQ ID No. 1 from 163 to 192 or a sequence having 80% or more identity thereto, and the gene coding sequence of the CDR3 of the Nanobody is SEQ ID No. 1 from 298 to 339 or a sequence having 80% or more identity thereto.

[0053] In the above-mentioned biological material, the nucleic acid molecule in B1) can be any of the following:

[0054] C1) a DNA molecule having a nucleotide sequence of SEQ ID No. 1;

[0055] C2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in C1) and encodes said Nanobody;

[0056] C3) A DNA molecule that is 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identical to a DNA sequence as defined in any one of C1) or C2) and encodes said Nanobody.

[0057] Wherein, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 2×SSC and 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 1×SSC and 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC and 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC and 0.1% SDS; and hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC and 0.1% SDS. Hybridize in a mixed solution of SDS, 0.5M Na3PO4 and 1mM EDTA, and rinse in 0.1×SSC, 0.1% SDS at 65°C; or hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0058] Those skilled in the art can readily mutate the nucleotide sequence of the nanocarrier ASFV4F1 described in B1) of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotides that are 75% or more identical to the nucleotide sequence of ASFV4F1 described in B1) of the present invention, as long as they encode the ASFV4F1 and have ASFV4F1 activity, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0059] The term "identity" as used herein refers to sequence similarity to a nucleotide sequence. Identity can be evaluated by the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0060] The aforementioned 75% or greater identity may be 75%, 80%, 85%, 90% or 95% or greater identity.

[0061] The present invention also provides a method for preparing the above-mentioned nanoantibody, which may include the following steps: introducing a nucleic acid molecule encoding the nanoantibody into a recipient cell to obtain a transgenic cell expressing the nanoantibody, and culturing the transgenic cell to obtain the nanoantibody.

[0062] The recipient cell may be a microorganism, a plant, or a non-human animal.

[0063] Furthermore, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0064] The nucleotide sequence of the nucleic acid molecule encoding the nanobody is specifically SEQ ID No. 1 in the sequence table.

[0065] Furthermore, the recipient cell may be a microbial cell, such as bacteria (eg, Escherichia coli), yeast, algae, or fungi.

[0066] In a specific embodiment of the present invention, the recipient cell is specifically Escherichia coli.

[0067] The present invention also provides the use of the nanobody, the biomaterial, and the preparation method in preparing a reagent for detecting African swine fever virus deoxyuridine pyrophosphatase.

[0068] The present invention also provides an ELISA detection kit targeting African swine fever virus deoxyuridine pyrophosphatase, wherein the kit comprises the nanoantibody or the biomaterial described above.

[0069] The present invention also claims protection for any of the following applications:

[0070] E1) Use of the Nanobody in the preparation of a product for detecting the expression of African swine fever virus deoxyuridine pyrophosphatase fusion protein;

[0071] E2) Use of the biological material in the preparation of a product for detecting the expression of African swine fever virus deoxyuridine pyrophosphatase fusion protein;

[0072] E3) Application of the preparation method in the preparation of a product for detecting the expression of African swine fever virus deoxyuridine pyrophosphatase fusion protein;

[0073] E4) Use of the kit in the preparation of a product for detecting the expression of African swine fever virus deoxyuridine pyrophosphatase fusion protein;

[0074] E5) Use of the Nanobody in the preparation of a product that binds to deoxyuridine pyrophosphatase;

[0075] E6) Use of the biomaterial in the preparation of a product combined with deoxyuridine pyrophosphatase;

[0076] E7) Use of the preparation method in preparing a product combined with deoxyuridine pyrophosphatase;

[0077] E8) Use of the kit in the preparation of a product combined with deoxyuridine pyrophosphatase;

[0078] E9) Use of the Nanobody in the preparation of a product for detecting deoxyuridine pyrophosphatase;

[0079] E10) Use of the biological material in the preparation of a product for detecting deoxyuridine pyrophosphatase;

[0080] E11) Use of the method in the preparation of a product for detecting deoxyuridine pyrophosphatase;

[0081] E12) Use of the kit in the preparation of a product for detecting deoxyuridine pyrophosphatase.

[0082] In E1)-E4), the expression of the nanobody and the deoxyuridine pyrophosphatase fusion protein can be studied by ELISA.

[0083] The above-mentioned product may be a medicine.

[0084] The present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Therefore, the present invention also encompasses polypeptides such as fragments, derivatives, and analogs of the Nanobodies that retain the same biological function or activity as the antibodies of the present invention.

[0085] The present invention also provides a polypeptide molecule containing the aforementioned nanobody structure, which can be any of the following:

[0086] D1) a single-chain antibody containing said Nanobody;

[0087] D2) a Fab containing said Nanobody;

[0088] D3) intact antibodies containing said Nanobodies;

[0089] D4) a fusion antibody containing said Nanobody;

[0090] D5) an antibody drug conjugate containing said Nanobody.

[0091] As known to those skilled in the art, the conjugates and fusion antibody expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules bound to the antibodies of the invention or their fragments. The present invention also includes cell surface markers or antigens bound to the Nanobodies or their fragments.

[0092] The present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical or at least 90% identical to the heavy chain variable region of an antibody of the present invention, preferably at least 95% identical.

[0093] The antigen described in the present invention is African swine fever virus deoxyuridine pyrophosphatase.

[0094] The nucleic acid molecule encoding the African swine fever virus deoxyuridine pyrophosphatase may be F1) or F2) or F3):

[0095] F1) a DNA molecule having a nucleotide sequence of SEQ ID No. 5;

[0096] F2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in F1) and encodes the deoxyuridine pyrophosphatase;

[0097] F3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined in any one of F1) or F2) and encodes the deoxyuridine pyrophosphatase.

[0098] The present invention also provides a method for detecting African swine fever virus, comprising contacting the aforementioned nanoantibody with a sample from a subject to detect whether the sample contains African swine fever virus or the content of African swine fever virus in the sample.

[0099] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human or a non-human primate, such as a monkey or chimpanzee), a non-primate (e.g., a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, or whale), or a bird (e.g., a duck or goose). BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is a schematic diagram of the structure of VHH.

[0101] Figure 2 shows the nanobody VHH library displayed on the phage surface. A: Nanobody VHH library diluted 10 8 B: Nanobody VHH library diluted 10 9 Colonies on the plate after multiple dilution; C: Nanobody VHH library diluted 10 10 Colonies on the plate after multiple dilution; D: Nanobody VHH library diluted 10 11 After multiples, spread the colonies on the plate.

[0102] Figure 3 shows the SDS-PAGE analysis of the purified antigen (African swine fever virus deoxyuridine pyrophosphatase dUTPase). M: Protein molecular weight standard, kDa; 0: Protein eluted with 20 mM Tris-HCl, pH 8.0, containing 0 mM imidazole; 5: Protein eluted with 20 mM Tris-HCl, pH 8.0, containing 5 mM imidazole; 150: Protein eluted with 20 mM Tris-HCl, pH 8.0, containing 150 mM imidazole.

[0103] Figure 4 is the SDS-PAGE detection of the purified nanoantibody ASFV4F1.

[0104] Figure 5 shows the ELISA assay for the binding of ASFV4F1 to African swine fever virus deoxyuridine pyrophosphatase dUTPase.

[0105] Best Mode for Carrying Out the Invention

[0106] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0107] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0108] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0109] Example 1. Construction and screening of a phage-displayed VHH library of African swine fever virus deoxyuridine pyrophosphatase-specific nanobodies

[0110] The camel nanobody VHH was selected as the framework (Figure 1), with its amino acid sequence as SEQ ID No. 4 and its encoding gene sequence as SEQ ID No. 3. The three CDR region sequences of VHH are: the gene sequence encoding CDR1 is from positions 76 to 99 of SEQ ID No. 3, and the amino acid sequence is from positions 26 to 33 of SEQ ID No. 4; the gene sequence encoding CDR2 is from positions 148 to 177 of SEQ ID No. 3, and the amino acid sequence is from positions 50 to 59 of SEQ ID No. 4; the gene sequence encoding CDR3 is from positions 283 to 342 of SEQ ID No. 3, and the amino acid sequence is from positions 95 to 114 of SEQ ID No. 4.

[0111] Camelid nanobody VHH (nucleotide sequence is SEQ ID No. 3, amino acid sequence is SEQ ID No. 4) was used as the backbone and the method was carried out according to the existing literature (Yau KY, et al., J Immunol Methods, 2005, 297(1-2): 213-224; E, et al., Methods Mol Biol, 2018, 1701: 169-187; Caucheteur D, et al., Methods Mol Biol. 2018, 1827: 93-108; Bobkov V, et al., Methods Mol Biol, 2018, 1827: 129-144; ) Primers were designed for the CDR region and a phage surface display library was constructed (AD in Figure 2), which was applied to the antibody screening of African swine fever virus deoxyuridine pyrophosphatase (dUTPase).

[0112] The primers for constructing the phage surface displayed nanobody library are as follows:

[0113] FR1_F: 5'-GAGGCGGGGCCCAGGCGGCCCAGGTTCAGCTGCAGGAAAG-3' (SEQ ID No. 9);

[0114] FR1_R: 5'-GCTTGCTGCACATGACAGAC-3' (SEQ ID No. 10);

[0115] CDR1_F: 5'-GTCTGTCATGTGCAGCAAGCNNNN-3' (SEQ ID No.11);

[0116] CDR1_R: 5'-GCCTGACGAAACCAACCCATNNNN-3' (SEQ ID No.12);

[0117] FR2_F: 5'-ATGGGTTGGTTTCGTCAGGC-3' (SEQ ID No.13);

[0118] FR2_R: 5'-TGCCACAAATTCACGTTCTT-3' (SEQ ID No.14);

[0119] CDR2_F: 5'-AAGAACGTGAATTTGTGGCANNNN-3' (SEQ ID No.15);

[0120] CDR2_R: 5'-CCTTTAACACTATCGGTATANNNN-3' (SEQ ID No.16);

[0121] FR3_F: 5'-TATACCGATAGTGTTAAAGG-3' (SEQ ID No.17);

[0122] FR3_R: 5'-ATAAACGGCGGTATCATCAG-3' (SEQ ID No.18);

[0123] CDR3_F: 5'-CTGATGATACCGCCGTTTATNNNN-3' (SEQ ID No.19);

[0124] CDR3_R: 5'-GGTCACCTGGGTACCCTGACCCCAATAATCNN-3' (SEQ ID No.20);

[0125] FR4_R: 5'-GGCCGGCCTGGCCGCTGCCAGATGACACGGTCACCTGGGTACC-3' (SEQ ID No.21).

[0126] The steps for constructing the nanobody library are as follows:

[0127] 1) Using primer pairs (FR1_F and FR1_R), (FR2_F and FR2_R), (FR3_F and FR3_R), and VHH nucleotide (SEQ ID No. 3) as templates, PCR amplified FR1, FR2, and FR3 gene fragments, respectively; using primer pairs (CDR1_F and CDR1_R), (CDR2_F and CDR2_R), (CDR3_F and CDR3_R), and VHH nucleotide (SEQ ID No. 3) as templates, PCR amplified CDR1, CDR2, and CDR3 gene mutation fragments, respectively, using GeneMorph II-Random Mutagenesis Kits (Aglient, Catalog No.: 200550) (refer to the kit manual for the operating steps). The PCR amplified products were recovered using a PCR product kit (QIAGEN, Catalog No.: 28006).

[0128] The FR1-CDR1 gene segment was amplified using FR1 and CDR1 as templates and FR1_F and CDR1_R as primers; the FR2-CDR2 gene segment was amplified using FR2 and CDR2 as templates and FR2_F and CDR2_R as primers; the FR3-CDR3 gene segment was amplified using FR3 and CDR3 as templates and FR3_F and CDR3_R as primers. The PCR products were recovered using a PCR product kit (QIAGEN, Cat. No. 28006).

[0129] 2) Using FR1-CDR1 and FR2-CDR2 as templates and FR1_F and CDR2_R as primers, respectively, the FR1-CDR1-FR2-CDR2 gene fragment was amplified; using FR3-CDR3 as a template and FR3_F and FR4_R as primers, the FR3-CDR3-FR4 gene fragment was amplified. The PCR products were recovered using a PCR product kit (QIAGEN, Cat. No. 28006).

[0130] 3) Using FR1-CDR1-FR2-CDR2 and FR3-CDR3-FR4 as templates and FR1_F and FR4_R as primers, the complete full-length mutant fragment of the nanobody VHH gene was amplified. After digestion with SfiI restriction endonuclease (New England Biolabs, Catalog No.: R0123L), it was ligated into the phagemid vector pComb3XSS (Addgene, Catalog No.: 63890) that had also been digested with SfiI to construct the pComb3XSS-VHH nanobody library recombinant vector, which was then electroporated into TG1 Escherichia coli competent cells (Agilent, Catalog No.: 200123) to obtain a phage surface-displayed nanobody library.

[0131] The purified African swine fever virus deoxyuridine pyrophosphatase (dUTPase) antigen was coated on a 96-well plate and incubated overnight at 4°C. The phage surface-displayed nanoantibody library was used for panning to obtain candidate phage antibody clones that specifically bound to the antigen. After four rounds of screening, a nanoantibody clone was enriched and named antibody ASFV4F1.

[0132] Sequencing revealed the complementary determinant clusters CDR1, CDR2, and CDR3 of ASFV4F1. The amino acid sequence of CDR1 is shown in SEQ ID No. 2, positions 26-38, and the gene sequence encoding CDR1 is shown in SEQ ID No. 1, positions 76-114. The amino acid sequence of CDR2 is shown in SEQ ID No. 2, positions 55-64, and the gene sequence encoding CDR2 is shown in SEQ ID No. 1, positions 163-192. The amino acid sequence of CDR3 is shown in SEQ ID No. 2, positions 100-113, and the gene sequence encoding CDR3 is shown in SEQ ID No. 1, positions 298-339. The full-length gene sequence of ASFV4F1 is shown in SEQ ID No. 1, and the full-length amino acid sequence is shown in SEQ ID No. 2. CDR1, CDR2, and CDR3 are numbered according to their amino acid sequences.

[0133] The encoding nucleotide sequence of antibody ASFV4F1 is SEQ ID No.1:

[0134] The amino acid sequence of ASFV4F1 is SEQ ID No. 2:

[0135] Example 2: Expression and purification of African swine fever virus deoxyuridine pyrophosphatase dUTPase

[0136] Codon optimization was performed according to the amino acid sequence of African swine fever virus deoxyuridine pyrophosphatase (dUTPase) (NCBI accession: NP_042823.1), and the base sequence after codon optimization was SEQ ID No. 5; the codon-optimized base sequence was inserted between NcoI and XhoI in the multiple cloning site of the vector pET-22b(+) (Novagen, catalog number: 69744) to construct a prokaryotic expression vector pET-22b(+)-dUTPase.

[0137] The structure of the vector pET-22b(+)-dUTPase is described as follows: a recombinant vector capable of expressing a histidine-tagged dUTPase protein was obtained by replacing the small fragment between the NcoI and XhoI restriction enzyme cleavage sites in pET-22b(+) with a codon-optimized dUTPase fragment (nucleotide sequence is SEQ ID No. 5).

[0138] The nucleotide sequence (i.e., coding sequence) of pET-22b(+)-dUTPase is the codon-optimized nucleotide sequence of dUTPase of SEQ ID No. 5 in the sequence listing, and the expressed amino acid sequence is the fusion protein of SEQ ID No. 6 in the sequence listing with a histidine tag at the C-terminus.

[0139] 20 ng of the recombinant vector pET-22b(+)-dUTPase was transformed into Escherichia coli BL21(DE3) competent cells (NEW ENGLAND Biolabs, Cat. No. C2527H). Positive clones were screened and verified by PCR, 2% agarose gel electrophoresis, or sequencing. The successfully verified positive clone recombinant strain was named recombinant strain BL21-pET-22b(+)-dUTPase.

[0140] The recombinant strain BL21-pET-22b(+)-dUTPase was inoculated into LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) containing ampicillin at a final concentration of 100 μg / mL, and the OD 600 When the p-value reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mmol / L and induce at 16°C, 220 rpm for 16-20 hours. Collect the induced cells by centrifugation at 6000 rpm for 10 minutes at 4°C, and resuspend the pellet in PBS buffer. Disrupt the cells by ultrasonication in an ice-water bath, collect the supernatant by centrifugation, and purify by imidazole gradient elution over Ni-NTA medium. The imidazole is then removed by dialysis.

[0141] The purity was verified by SDS-PAGE. As shown in Figure 3, there was a clear target protein size band in the purified eluted protein from the supernatant after the recombinant strain BL21-pET-22b(+)-dUTPase bacterial solution was disrupted, indicating that the African swine fever virus deoxyuridine pyrophosphatase dUTPase protein containing a His tag at the C-terminus was obtained.

[0142] Example 3. Expression and purification of antibody ASFV4F1

[0143] The antibody ASFV4F1 gene was fused with the FLAG tag gene (Merck) and connected between the NcoI and XhoI of the multiple cloning site of the vector pET-22b(+) (Novagen, Cat. No.: 69744) to construct the expression vector pET-22b(+)-ASFV4F1-FLAG.

[0144] The structure of the expression vector pET-22b(+)-ASFV4F1-FLAG is described as follows: a DNA molecule containing an ASFV4F1 nucleotide fragment (nucleotide sequence of SEQ ID No. 1 and SEQ ID No. 7 connected) carrying a FLAG tag replaces the small fragment between the NcoI and XhoI restriction endonuclease cleavage sites in pET-22b(+), while keeping the other nucleotides of pET-22b(+) unchanged, resulting in a recombinant expression vector carrying a FLAG tag. The expression vector pET-22b(+)-ASFV4F1-FLAG contains a recombinant ASFV4F1-FLAG protein gene composed of the ASFV4F1 gene and the FLAG tag connected, with a nucleotide sequence (i.e., coding sequence) of SEQ ID No. 1, and expresses a fusion protein with an amino acid sequence of SEQ ID No. 2 and SEQ ID No. 8.

[0145] Transform 20 ng of the expression vector pET-22b(+)-ASFV4F1-FLAG into competent E. coli BL21(DE3) cells. Screen for positive clones and verify them by PCR, 2% agarose gel electrophoresis, or sequencing. Name the recombinant strain BL21-pET-22b(+)-ASFV4F1-FLAG.

[0146] The recombinant strain BL21-pET-22b(+)-ASFV4F1-FLAG was inoculated into LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) containing ampicillin at a final concentration of 100 μg / mL, and the OD 600 When the p-value reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mmol / L and induce at 16°C, 220 rpm for 16-20 hours. Collect the induced cells by centrifugation at 6000 rpm for 10 minutes at 4°C, and resuspend the pellet in PBS buffer. Disrupt the cells by ultrasonication in an ice-water bath, collect the supernatant by centrifugation, and purify the protein by Ni-NTA media and dialyze to remove imidazole. Verify protein purity by SDS-PAGE.

[0147] As can be seen from Figure 4, there is a clear target protein size band in the purified eluted protein from the supernatant after the bacterial solution of the recombinant strain BL21-pET-22b(+)-ASFV4F1-FLAG was broken, and the purity of the protein is very high; the molecular weight is about 15 kDa, which is consistent with the theoretical molecular weight, that is, the ASFV4F1 antibody fusion protein containing a FLAG tag at the C-terminus was expressed and purified.

[0148] Example 4: Evaluation of the binding ability of ASFV4F1 to African swine fever virus deoxyuridine pyrophosphatase dUTPase by ELISA

[0149] The His-tagged African swine fever virus deoxyuridine pyrophosphatase (dUTPase) protein (10 μg / mL) obtained in Example 2 was coated onto the wells of a 96-well plate and incubated overnight at 4°C. The plate was then blocked with 1% (w / v) BSA at 37°C for 1 hour. The FLAG-tagged ASFV4F1 antibody protein obtained in Example 3 was added at serial dilutions of 0, 50, 100, 150, and 200 nM. The plate was incubated at 37°C for 2 hours, followed by four washes with PBS + 0.05% (v / v) Tween 20. Horseradish peroxidase (HRP)-labeled anti-FLAG tag mouse monoclonal antibody (Sigma, Catalog No. A8592) was then added and incubated at 37°C for 1 hour. The plate was then washed four times with PBS + 0.05% (v / v) Tween 20. A framework VHH was used as a negative control. The backbone VHH is a protein having an amino acid sequence of SEQ ID No. 4, i.e., the camel nanobody VHH in Example 1, obtained by prokaryotic expression and purification. The preparation of the camel nanobody VHH is as follows:

[0150] The camel nanobody VHH nucleotide sequence (SEQ ID No. 3) was concatenated with the FLAG tag gene (Merck) and synthesized by Beijing Qingke Biotechnology Co., Ltd. The expression vector pET-22b(+)-VHH-FLAG was constructed by ligating the vector between NcoI and XhoI in the multiple cloning site of the pET-22b(+) (Novagen, Cat. No. 69744).

[0151] The structure of the pET-22b(+)-VHH-FLAG vector is described as follows: a small fragment between the NcoI and XhoI restriction endonuclease cleavage sites in pET-22b(+) is replaced with a DNA molecule containing a FLAG-tagged VHH nucleotide fragment (nucleotide sequence of SEQ ID No. 3 and SEQ ID No. 7 connected together), while the other nucleotides in pET-22b(+) remain unchanged. This results in a recombinant vector expressing a FLAG-tagged protein. The recombinant pET-22b(+)-VHH-FLAG vector contains the nucleotide sequence (i.e., coding sequence) of SEQ ID No. 3 in the sequence listing, which is a recombinant VHH-FLAG protein gene formed by connecting the VHH gene and the FLAG tag. The expressed amino acid sequence is the fusion protein VHH-FLAG of SEQ ID No. 4 and SEQ ID No. 8 in the sequence listing.

[0152] Transform 20 ng of the plasmid (pET-22b(+)-VHH-FLAG) into competent E. coli BL21(DE3) cells. Screen for positive clones and verify by PCR, 2% agarose gel electrophoresis, or sequencing. Name the recombinant strain BL21-pET-22b(+)-VHH-FLAG.

[0153] The recombinant strain BL21-pET-22b(+)-VHH-FLAG was inoculated into LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) containing ampicillin at a final concentration of 100 μg / mL, and the OD 600 When the p-value reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mmol / L and induce at 16°C, 220 rpm for 16-20 hours. Collect the induced cells by centrifugation at 6000 rpm for 10 minutes at 4°C, and resuspend the pellet in PBS buffer. Disrupt the cells by ultrasonication in an ice-water bath, collect the supernatant by centrifugation, and purify the protein by Ni-NTA media and dialyze to remove imidazole. Verify protein purity by SDS-PAGE.

[0154] The results are shown in Figure 5. The EC of nanobody ASFV4F1 binding to African swine fever virus deoxyuridine pyrophosphatase dUTPase protein 50 The concentration of VHH was 45 nM, while VHH could not interact with dUTPase.

[0155] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

[0156] Industrial Applications

[0157] The present invention uses the African swine fever virus deoxyuridine pyrophosphatase (dUTPase) as an antigen from a polypeptide and protein library with VHH as the skeleton to screen and obtain high-affinity nano antibodies targeting the African swine fever virus deoxyuridine pyrophosphatase (dUTPase). The present invention provides a preparation scheme for the above-mentioned ASFV4F1 nano antibody, optimizes the Escherichia coli prokaryotic expression system, and expresses and purifies the ASFV4F1 nano antibody in large quantities, effectively reducing the development and production costs of the ASFV4F1 antibody. The nano antibody was verified by ELISA to be able to specifically bind to the African swine fever virus deoxyuridine pyrophosphatase (dUTPase) with a high affinity. The ASFV4F1 nano antibody provides essential experimental materials for the study of the pathogenic mechanism of ASFV, has the potential function of treating African swine fever virus clinically, and can be used in combination with other antibody drugs to achieve an ideal therapeutic effect.

Claims

1. A nanobody targeting African swine fever virus deoxyuridine pyrophosphatase or an antigen-binding fragment containing the nanobody, characterized in that: The nanobody has three complementary determining clusters CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is positions 26-38 of SEQ ID No.2 or a sequence with more than 80% identity thereto, the amino acid sequence of the CDR2 is positions 55-64 of SEQ ID No.2 or a sequence with more than 80% identity thereto, and the amino acid sequence of the CDR3 is positions 100-113 of SEQ ID No.2 or a sequence with more than 80% identity thereto.

2. The Nanobody or antigen-binding fragment according to claim 1, characterized in that: The Nanobody is the following A1) or A2): A1) a Nanobody with the amino acid sequence of SEQ ID No. 2 or a Nanobody with an amino acid sequence that is more than 80% identical to SEQ ID No. 2 and has the same function; A2) Nanobodies obtained by attaching a protein tag to the N-terminus and / or C-terminus of SEQ ID No.

2.

3. Biomaterial, wherein the biomaterial is any of the following: B1) a nucleic acid molecule encoding the Nanobody or antigen-binding fragment of claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) a recombinant microorganism containing the recombinant vector described in B4); B9) a recombinant cell line containing the nucleic acid molecule of B1) or a recombinant cell line containing the expression cassette of B2); B10) a transgenic animal cell containing the nucleic acid molecule described in B1), or a transgenic animal cell containing the expression cassette described in B2); B11) transgenic animal tissue containing the nucleic acid molecule described in B1) or transgenic animal tissue containing the expression cassette described in B2); B12) A transgenic animal organ containing the nucleic acid molecule described in B1) or a transgenic animal organ containing the expression cassette described in B2).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a nucleic acid molecule encoding the Nanobody according to claim 1 or 2, in which the gene encoding the CDR1 is nucleotides 76-114 of SEQ ID No. 1; the gene encoding the CDR2 is nucleotides 163-192 of SEQ ID No. 1; and the gene encoding the CDR3 is nucleotides 298-339 of SEQ ID No.

1.

5. The biomaterial according to claim 3 or 4, characterized in that: B1) The nucleic acid molecule is any one of the following: C1) a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 1; C2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in C1) and encodes said Nanobody; C3) A DNA molecule that has more than 80% identity with the DNA sequence defined in any one of C1) or C2) and encodes said Nanobody.

6. A method for preparing the Nanobody according to claim 1 or 2, comprising the following steps: The nucleic acid molecule according to any one of claims 3 to 5 is introduced into a recipient cell to obtain a transgenic cell expressing the nanobody, and the transgenic cell is cultured to obtain the nanobody.

7. An ELISA detection kit targeting African swine fever virus deoxyuridine pyrophosphatase, characterized by: The kit comprises the Nanobody according to claim 1 or 2 or the biomaterial according to any one of claims 3-5.

8. Any of the following applications: E1) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a product for detecting African swine fever virus deoxyuridine pyrophosphatase; E2) Use of the biological material according to any one of claims 3 or 4 in the preparation of a product for detecting African swine fever virus deoxyuridine pyrophosphatase; E3) Use of the preparation method according to claim 6 in the preparation of a product for detecting African swine fever virus deoxyuridine pyrophosphatase; E4) Use of the kit according to claim 7 in the preparation of a product for detecting African swine fever virus deoxyuridine pyrophosphatase; E5) Use of the Nanobody of claim 1 or 2 in the preparation of a product that binds to deoxyuridine pyrophosphatase; E6) Use of the biomaterial according to any one of claims 3 or 4 in the preparation of a product combined with deoxyuridine pyrophosphatase; E7) Use of the preparation method according to claim 6 in the preparation of a product combined with deoxyuridine pyrophosphatase; E8) Use of the kit according to claim 7 in the preparation of a product combined with deoxyuridine pyrophosphatase.

9. Any of the following applications: F1) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of an African swine fever virus detection reagent; F2) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a diagnostic reagent for African swine fever; F3) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a drug for preventing and / or treating African swine fever.

10. A method for detecting African swine fever virus, comprising contacting the Nanobody according to claim 1 or 2 with a sample from a subject to detect whether the sample contains African swine fever virus or the content of African swine fever virus in the sample.

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