Preparation of monoclonal antibody of p30 protein of african swine fever virus and application thereof

Abstract

The application relates to the technical field of antibodies, in particular to an anti-African swine fever virus p30 protein monoclonal antibody and application thereof. The anti-African swine fever virus p30 protein monoclonal antibody of the application can specifically bind to African swine fever virus p30 protein. The anti-ASFV p30 protein monoclonal antibody is prepared by immunizing BALB / c mice through an immunological method, and the antibody has high specificity, high titer and high affinity, and can be applied to immunological detection or an antibody detection kit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and particularly relates to a monoclonal antibody of an African swine fever virus p30 protein and application thereof. BACKGROUND

[0002] African swine fever is an acute, hemorrhagic, and virulent infectious disease caused by African swine fever virus (ASFV) infection in domestic pigs and various wild pigs. African swine fever was first reported in 1921, and has caused serious economic losses and heavy blows to the animal husbandry industry in the world for more than a hundred years. The World Organization for Animal Health (OIE) lists ASF in the list of animal diseases that must be reported, and China lists it as a class animal disease. The clinical symptoms of African swine fever mainly depend on the difference in virulence of the virus strain, which are divided into acute, subacute and chronic. Acute and subacute generally mainly show that sick pigs have high fever, moderate anorexia, dyspnea, hematochezia, skin cyanosis, and so on, and pregnant sows will abort; chronic generally shows dyspnea, wet cough, emaciation, joint swelling, and so on. The infection of African swine fever can be caused by direct contact with sick pigs or contact with contaminated tools, feed and the like, and its transmission mode is various.

[0003] African swine fever virus is a large, hexagonal, complex icosahedral, double-stranded linear DNA virus with a capsule membrane, a diameter of 172-260 nm, and a full-length genome of 170-194 kb. The virus particle is a spherical particle assembled by more than 30,000 protein subunits and 17,280 proteins, which is the largest virus particle with a near-atomic resolution structure. According to whether the virus has the red blood cell adsorption property, eight serogroups have been identified, and according to the 3' end sequence of the B646L gene encoding the p72 protein, at least 24 genotypes have been identified in the world, among which the European prevalent types I and II.

[0004] The genome of African swine fever virus is large, and can encode 150-200 viral proteins, including 68 structural proteins and more than 100 non-structural proteins. Most of the proteins are capsid proteins, regulatory proteins that help viral genome replication and viral assembly, and enzymes, while the membrane protein is the main structural protein of the virus particle and an important surface antigen, which is closely related to the host cell tropism, pathogenicity and immunogenicity, and plays an important role in the process of viral transmembrane entry.

[0005] The p30 protein is encoded by the CP204L gene, has a relative molecular mass of about 30 kDa, appears in the early stage of viral infection, and is one of the main antigenic structural proteins for the virus to penetrate into host cells. Studies have shown that the expression of the protein can be detected after 2-4 hours of infection, and then continues to be expressed throughout the infection process. The expression of the p30 protein indicates that the virus has penetrated into the cell and uncoated, and some early viral gene expression has begun. The p30 protein has strong immunogenicity and can produce high levels of antibodies during viral infection.

[0006] The African swine fever virus has a large and complex genome, high variability, an immune escape mechanism, and unclear mechanisms of action between the virus and the host, making it difficult to develop an African swine fever vaccine. At present, there is still no specific drug and vaccine for the African swine fever virus, and the main prevention and control strategy relies on the implementation of health measures and the slaughter of infected or exposed animals. Therefore, the detection and early diagnosis of the African swine fever virus are crucial for epidemic confirmation and control.

[0007] Monoclonal antibodies have the characteristics of strong specificity, high stability, and single biological activity, and are often used in biological immunology tests, disease diagnosis, and prevention, etc. At present, the monoclonal antibody technology is developing increasingly perfect, and gradually expands to various fields. There is great application potential in the research of preparation methods, the development of new monoclonal antibodies, and the expansion of application range. In the detection of ASFV, according to the specificity of the monoclonal antibody, the developed kit can detect various ASFV, or detect a specific serotype or genotype of ASFV. The preparation of the monoclonal antibody against the p30 protein provides material support for subsequent serological tests related to ASFV and the biological function research of the p30 protein. SUMMARY

[0008] The present application screens hybridoma cells by immunizing BALB / c mice to obtain a monoclonal antibody against ASFV p30, which lays a foundation for establishing a safe, reliable, and rapid African swine fever diagnosis and detection method. In order to realize the detection of African swine fever virus antigens and antibodies, the present application provides an anti-African swine fever virus p30 protein monoclonal antibody and its application.

[0009] The present application scheme is specifically as follows:

[0010] 1. An anti-African swine fever virus p30 protein monoclonal antibody, wherein the antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) of a heavy chain and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0011] the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 6, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 7, and the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 8,

[0012] the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 3, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 4, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 5.

[0013] 2. The monoclonal antibody of item 1, wherein the antibody comprises a heavy chain variable region, wherein:

[0014] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 2, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2.

[0015] 3. The monoclonal antibody of item 1 or 2, wherein the antibody comprises a light chain variable region, wherein:

[0016] the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 1, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0017] 4. The monoclonal antibody of any one of items 1-3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 2, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 1, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0019] 5. The monoclonal antibody of any one of items 1-4, wherein the antibody is a mouse antibody or a chimeric antibody.

[0020] 6. An isolated nucleic acid comprising a polynucleotide sequence encoding the monoclonal antibody of any one of items 1-5.

[0021] 7. A recombinant vector comprising the nucleic acid of item 6.

[0022] 8. A host cell comprising the nucleic acid of item 6 or the recombinant vector of item 7.

[0023] 9. A kit for detecting African swine fever virus p30 protein, wherein the kit comprises the monoclonal antibody of any one of items 1-5.

[0024] Advantages:

[0025] The anti-African swine fever virus p30 protein monoclonal antibody of the present application can specifically bind to African swine fever virus p30 protein, and the anti-ASFV p30 protein monoclonal antibody is prepared by immunizing BALB / c mice by immunological methods. The antibody has high specificity, high titer and high affinity, and can be applied to immunological detection or antibody detection kits. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is an ELISA assay immune mouse serum titer result graph;

[0027] Figure 2 is an IFA fluorescence graph of hybridoma cell culture supernatant and SP2 / 0 cell culture supernatant;

[0028] Figure 3 is an indirect Elisa assay of monoclonal antibody 6D5 titer graph;

[0029] Figure 4 is an indirect Elisa assay of monoclonal antibody relative affinity graph;

[0030] Figure 5 is a Western Blot detection of monoclonal antibody 6D5 specificity result graph;

[0031] Figure 6 is an IFA fluorescence graph of monoclonal antibody 6D5 reacting with different ASFV;

[0032] Figure 7 is an IFA fluorescence graph of monoclonal antibody 6D5 reacting with other strains. DETAILED DESCRIPTION

[0033] The present application will be further illustrated by the following examples, which should be understood as merely further illustrating and explaining the present application, and not limiting the present application.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described below. However, the materials, methods, and examples are illustrative only and not intended to be limiting. Additional information concerning various aspects of this application is set forth in the following sections.

[0035] The term "anti-African swine fever virus p30 protein monoclonal antibody" as used herein is intended to encompass a variety of antibody structural molecules that bind to African swine fever virus p30 protein, including but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2), linear antibodies, and single-chain antibody molecules (e.g., scFv), and the like, so long as they exhibit the desired binding activity to African swine fever virus p30 protein.

[0036] The modifier "monoclonal" in the term "anti-African swine fever virus p30 protein monoclonal antibody" as used herein refers to the preparation of the antibody obtained from a population of substantially homogeneous antibodies, which contain only minor amounts of naturally occurring mutant or other impurities that arise during the production of the monoclonal antibody. Each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the porcine antigen, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different epitopes. An anti-African swine fever virus p30 protein IL-31 monoclonal antibody is a highly homogeneous antibody produced from a single B-cell clone, which is directed only against a single African swine fever virus epitope. The monoclonal antibodies of the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and the like.

[0037] The sequences herein comply with the IUPAC convention for nucleic acid degenerate code, i.e., "R" comprises adenine or guanine; "Y" comprises cytosine, thymine or uracil; "S" comprises guanine or cytosine; "W" comprises adenine, thymine or uracil; "K" comprises "guanine", thymine or uracil; "M" comprises adenine or cytosine.

[0038] In some embodiments, the antibody of the present application is a full length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is generally a polypeptide that consists of, in N-terminal to C-terminal orientation, a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full length antibody heavy chain" is a polypeptide that consists of, in N-terminal to C-terminal orientation, VH, CH1, HR, CH2, and CH3. A "full length antibody light chain" is generally a polypeptide that consists of, in N-terminal to C-terminal orientation, a light chain variable region (VL), and a light chain constant region (CL), abbreviated as VL-CL.

[0039] In the present application, the light chain constant region (CL) is kappa (k), and the heavy chain constant region is IgG.

[0040] As known to those skilled in the art, each heavy chain variable region can be composed of three complementarity determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementarity determining regions (CDRs) and four framework regions (FRs), the complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) being the regions in the variable region that have the most influence on the affinity and specificity of the antibody. In some embodiments, both the heavy chain variable region and the light chain variable region comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N-terminal to C-terminal.

[0041] The CDR sequences of the heavy chain variable region or the light chain variable region can be identified by reference to any of the numbering systems known in the art, such as the Kabat system (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia and Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains," Dev. Comp. Immunol. 27, 55-77 (2003)).

[0042] In embodiments of the application, the CDR sequences are determined using the IMGT system.

[0043] However, it will be appreciated by those skilled in the art that not all antibodies or antigen-binding fragments thereof comprise a complete heavy chain variable region and a light chain variable region. Also, in many cases, protein molecules comprising only an antibody antigen-binding fragment still have the ability to specifically bind and / or neutralize an antigen. In some embodiments, the antibody can comprise only the VH and / or VL of an antibody, such as a dAb, a single domain antibody containing only a light chain variable region, a nanobody, and the like.

[0044] In the present context, the term "identity" or "identical" is defined as the percentage of identical residues in a variant amino acid or nucleotide sequence after alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. In the present context, reference to an amino acid sequence having a certain percentage of identity to a given amino acid sequence means a sequence having said percentage of identity over the entire length of the given amino acid sequence.

[0045] In the present context, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector which is primarily used for insertion of a DNA or RNA into a cell, a vector which is primarily used for replication of DNA or RNA, and a vector which is primarily used for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide which is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0046] In the present context, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its single-stranded or double-stranded form. Unless specifically limited, the term can encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties to the reference nucleic acid (e.g., show sequence information) and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, the sequence of a nucleic acid can include conservatively modified variants thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences.

[0047] In the present context, "affinity" indicates the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in the present specification indicates the intrinsic binding affinity reflecting 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K D ) of the complex X:Y. The affinity can be determined by common methods known in the art.

[0048] Antibodies

[0049] In one aspect, the present application provides an anti-swine fever virus p30 protein monoclonal antibody, wherein the antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3).

[0050] In some embodiments, the amino acid sequence of CDR-H1 is as set forth in SEQ ID NO: 6 (GYTFTDYN), the amino acid sequence of CDR-H2 is as set forth in SEQ ID NO: 7 (IYPYNGGT), the amino acid sequence of CDR-H3 is as set forth in SEQ ID NO: 8 (AMDGNYGY), the amino acid sequence of CDR-L1 is as set forth in SEQ ID NO: 3 (ESVDNYGISF), the amino acid sequence of CDR-L2 is as set forth in SEQ ID NO: 4 (AAS), and the amino acid sequence of CDR-L3 is as set forth in SEQ ID NO: 5 (QQSKEVPRT).

[0051] In some embodiments, the antibody comprises a heavy chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 2, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2.

[0052] SEQ ID NO: 2:

[0053]

[0054] In some embodiments, the antibody comprises a light chain variable region, wherein: the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 1, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0055] SEQ ID NO: 1:

[0056]

[0057] In some embodiments, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 2, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 1, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0058] In some embodiments, the antibody is a mouse antibody or a chimeric antibody.

[0059] In some embodiments, the heavy chain of the antibody further comprises a heavy chain constant region, and the light chain further comprises a light chain constant region.

[0060] In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO: 9, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above identical to SEQ ID NO: 9.

[0061] SEQ ID NO: 9:

[0062]

[0063] In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO: 9, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above identical to SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 10, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above identical to SEQ ID NO: 10.

[0064] SEQ ID NO: 10:

[0065]

[0066] In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO: 9, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above identical to SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 10, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above identical to SEQ ID NO: 10.

[0067] Nucleic acids, vectors, host cells

[0068] In another aspect, the present application also provides an isolated nucleic acid comprising a polynucleotide sequence encoding any of the foregoing antibodies.

[0069] In some embodiments, the nucleic acid molecule is an RNA or a DNA molecule. In some embodiments, the nucleic acid is single-stranded or double-stranded. In some embodiments, the nucleic acid is linear or circular. The nucleic acid can be synthesized by a cell or chemically synthesized. In some embodiments, the nucleic acid is a chemically modified nucleic acid molecule.

[0070] In another aspect, the present application also provides a vector comprising the nucleic acid described above.

[0071] In some embodiments, the vector is a plasmid.

[0072] In another aspect, the present application also provides a host cell comprising the nucleic acid or the vector described above.

[0073] Example 1 Preparation of hybridoma cell strain of anti-swine fever virus (ASFV) p30 protein monoclonal antibody

[0074] 1. Immunization of animals

[0075] Four SPF female BALB / c mice were selected, and the purified p30 recombinant protein was mixed with an equal volume of Freund's complete adjuvant for emulsification, and the mice were injected subcutaneously with multiple points. The first immunization dose was 60 μg per mouse. The booster immunization was performed every 14 days, and the mice were immunized three times. The p30 recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant for emulsification, and the immunization method was the same as the first immunization. The immunization dose was 30 μg per mouse. Seven days after the third immunization, the tail blood was collected to determine the antibody titer. Three to five days before cell fusion, the mouse with the highest serum titer was selected, and the BALB / c mouse was subjected to superimmunization by intraperitoneal injection of the ASFV p30 protein without adjuvant. The immunization dose was 30 μg per mouse.

[0076] 2. Determination of serum antibody titer of immunized mice

[0077] ELISA titer determination:

[0078] (1) ASFV p30 recombinant protein 2 μg / mL coating, 100 ul per well, 4 degree coating overnight, PBST washing 3 times, and drying;

[0079] (2) 2% skimmed milk powder (diluted with PBST), 200 ul per well, 37 degree blocking for 2 hours, PBST washing 3 times, and drying;

[0080] (3) Dilute the serum with PBST by gradient dilution, the dilution gradient is from 1:200 to 1:102400, dilute 10 gradients, the negative serum dilution is 1:200, 100 ul per well, 37°C incubation for 1 hour, PBST washing 3 times, and drying;

[0081] (4) HRP labeled goat anti-mouse IgG 1:10000 dilution (diluted with PBST), 100 ul per well, 37°C incubation for 1 hour, PBST washing 3 times, and drying;

[0082] (5) TMB color developing solution, 100 ul per well, 37 degree incubation for 10-15 min in the dark, and termination liquid 50 ul per well to terminate the reaction;

[0083] (6) Measure OD450 with microplate reader.

[0084] The results are shown in Table 1. Figure 1 The ELISA results show that the ELISA titer of No. 1 mouse can reach 1:102400 at the highest, and No. 1 mouse is selected for cell fusion to prepare monoclonal antibodies.

[0085] 3. Preparation and screening of hybridoma cells

[0086] The spleen cells of the mouse are collected by conventional method, and the spleen cells are fused with SP2 / 0 cells of mouse myeloma cells at a ratio of 10:1 under the induction of PEG1450 (fusion agent, purchased from Sigma Company). The selective culture solution of HAT (purchased from Sigma Company) is used for culture, and the culture condition is 5% carbon dioxide, 37°C.

[0087] The specific process is as follows:

[0088] (1) The well-conditioned sp2 / 0 cells are gently blown off from the wall of the culture bottle and sucked into a 50ml centrifuge tube.

[0089] (2) The mouse is taken out by enucleation, and then killed by pulling the neck, and soaked in 75% alcohol for 5min.

[0090] (3) A small amount of serum-free IMDM is poured into a flat dish, and the cell screen and the inner core of the syringe are put into the flat dish. The spleen of the mouse is taken out by scissors and forceps and put on the cell screen. The spleen is gently crushed by the inner core of the syringe, and the crushed cells are sucked into the centrifuge tube containing sp2 / 0, and centrifuged at 1500 rad / min for 5min.

[0091] (4) The thymus of the mouse is taken out by scissors and forceps and crushed. The crushed thymus cells are put into a 15ml centrifuge tube, and 2ml of HAT and 1ml of HT are added and placed in an incubator for standby.

[0092] (5) The centrifuged cells are poured, and the supernatant is blown evenly with serum-free IMDM, and centrifuged (1500 rad / min, 5min).

[0093] (6) The supernatant of the centrifuged cells is poured as much as possible. The bottom of the centrifuge tube is tapped to suspend the cells, and the centrifuge tube is placed in warm water at 37°C. 1ml of PEG is slowly added in about 1min, and then placed in warm water for 1min after addition. Then 2ml of serum-free IMDM is slowly added within 2min, followed by slowly adding 8ml of serum-free IMDM within 2min. Centrifuge at 1000 rad / min for 5min.

[0094] (7) Discard the supernatant, add 10 ml of serum, carefully blow the cells, pour into the prepared thymus cells. Add sterilized semi-solid medium to 50 ml, mix well. Then evenly pour into 30 cell culture dishes. Put the cell culture dishes in a humid box, then put them in an incubator for culture. After 12 days of fusion, 10 plates x 93 cell monoclonal, cultured in 96-well cell culture plates.

[0095] Example 2 Screening and identification of hybridoma cell strains secreting anti-ASFV p30 protein monoclonal antibodies

[0096] 1. Positive hybridoma cell strains secreting anti-ASFV p30 were screened by indirect ELISA method, and positive hybridoma cells were subcloned by limiting dilution method, a total of three times. The operation steps of indirect ELISA method are as follows:

[0097] (1) Dilute P30 recombinant protein with coating solution to a final concentration of 2 μg / ml, 100 ul / well, 4℃, overnight, then wash with washing solution for 3 times.

[0098] (2) Blocking solution (2% skim milk powder) blocking, 200ul / well, 37℃ incubation for 2h, then washing with washing solution for 3 times.

[0099] (3) Add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), positive control (positive serum diluted 1000 times with PBS), all 100ul / well, 37℃ incubation for 1h, then washing with washing solution for 3 times.

[0100] (4) Add secondary antibody diluted 20000 times with PBS, 100ul / well, 37℃ incubation for 1h, then remove and wash with washing solution for 3 times.

[0101] (5) Add 100ul / well of color developing solution, color developing time is about 10min.

[0102] (6) Add 50ul / well of stop solution to each well.

[0103] (7) Measure the absorbance at dual wavelengths (450, 630), record and save the data.

[0104] Through three rounds of screening, three hybridoma cell strains capable of stably secreting specific monoclonal antibodies against p30 protein were obtained, named hybridoma cell strains 1E9, 4C7 and 6D5.

[0105] 2. Indirect immunofluorescence (IFA) identification of monoclonal antibodies

[0106] After three subcloning, the cell supernatant secreted by the single cell strain obtained was identified by IFA. The specific operation steps of IFA are as follows:

[0107] 1.25 x 105PAM cells were plated in 96-well cell plates, and after the cells adhered, 1000-fold diluted ASFV was inoculated. 72 h after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with PBS 3 times, then permeabilized with 0.1% Triton X-100, and incubated at room temperature for 20 min, washed with PBS 3 times, then incubated with hybridoma cell supernatant and anti-mouse FITC-labeled goat anti-mouse IgG, and the results were observed under a fluorescence microscope after the reaction was completed. 5

[0108] IFA identification results are shown in Figure 2. Figure 2 As shown in Figure 2, 1E9, 4C7, and 6D5 hybridoma cell supernatants can detect specific green fluorescent signals in ASFV-infected PAM cells, and no fluorescent signals are observed in the negative control.

[0109] 3. Monoclonal antibody subclass identification

[0110] According to the requirements of the mouse monoclonal antibody Ig class / subclass identification kit (ELISA), the type of antibody secreted by the hybridoma cells was identified. The specific operation process is as follows:

[0111] (1) Dilute the subclass coating antibody (Goat Anti-Mouse Ig, Human ads-UNLB) with coating solution to a final concentration of 2 μg / ml, 100 u wells, 4°C, overnight, then wash with washing solution 3 times.

[0112] (2) Block with blocking solution, 200 u1 well, 37°C for 2 h, then wash with washing solution 3 times.

[0113] (3) Add the primary antibody (cell culture supernatant) and the negative control (SP2 / 0 culture supernatant) to 100 u1 wells, 37°C for 1 h, then wash with washing solution 3 times.

[0114] (4) Dilute each type of subclass secondary antibody (Goat Anti-Mouse IgM, IgG1, IgG2a, IgG2b, etc.) with PBS, 100 u / well, and add to the appropriate wells, 37°C for 1 h, then remove and wash with washing solution 3 times.

[0115] (5) Add 100 u / well of color developing solution, and develop for about 10 min.

[0116] (6) Add 50 ul of stop solution to each well.

[0117] (7) Measure the absorbance at two wavelengths (450, 630), and record the data.

[0118] ​The results are shown in Table 1 below. The heavy chain constant regions of the three monoclonal antibodies are IgG1 type, and the light chain constant regions are Kappa type.

[0119] Table 1

[0120]

[0121] Preparation and purification of anti-ASFV p30 protein monoclonal antibody ascites

[0122] Preparation of anti-ASFV p30 protein monoclonal antibody ascites

[0123] The 6D5 monoclonal antibody was prepared in large quantities by in vivo induction of ascites, and the specific steps are as follows:

[0124] Female BALB / c mice in production were injected intraperitoneally with 500ul sterilized paraffin to stimulate immune cells to promote the proliferation of hybridoma cells. The state of the mice was observed, and 14 days later, about 1x10 7 cells were injected into the prepared monoclonal positive cells, and the state of the mice was observed in time; 7 days later, the ascites was extracted, centrifuged at 8000r / min at 4℃ for 20min to remove oil and cell precipitate, and the ascites supernatant was collected and stored at-80℃ for standby; one week later, the obtained monoclonal hybridoma cells were injected intraperitoneally, and the injection amount was 2x10 5 cells; one week later, the ascites was extracted after the abdominal swelling of the mice, and the supernatant was taken after centrifugation. The ascites IgG was coarsely extracted by saturated ammonium sulfate method; and the mouse IgG was purified by protein A column.

[0125] Determination of the sequence of the variable region of the monoclonal antibody

[0126] The RNA of the 6D5 hybridoma cell strain was extracted and reverse transcribed into cDNA, and the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody were amplified by PCR.

[0127] According to the sequence characteristics of the mouse-derived monoclonal antibody, the primer sequence of the light chain variable region is as follows:

[0128] The gene sequence of the forward primer 1 is as shown in SEQ ID NO: 11:

[0129] SEQ ID NO: 11:

[0130] The gene sequence of the forward primer 2 is as shown in SEQ ID NO: 12:

[0131] SEQ ID NO: 12:

[0132] The gene sequence of the reverse primer is as shown in SEQ ID NO: 13:

[0133] SEQ ID NO: 13:​​

[0134] According to the sequence characteristics of the mouse monoclonal antibody, the primer sequence of the heavy chain variable region is designed as follows:

[0135] The gene sequence of the forward primer is as shown in SEQ ID NO: 13:

[0136] SEQ ID NO: 14:

[0137] The gene sequence of the reverse primer is as shown in SEQ ID NO: 15:

[0138] SEQ ID NO: 15:

[0139] The variable region sequence of the monoclonal antibody is obtained by molecular cloning technology, and is sent to Shanghai Shengong Biological Co., Ltd. for sequencing.

[0140] The sequencing results are as follows, the heavy chain variable region and the light chain variable region gene DNA sequences of the 6D5 monoclonal antibody of the application are respectively as shown in SEQ ID NO: 16 and SEQ ID NO: 17:

[0141] SEQ ID NO: 16:

[0142]

[0143]

[0144]

[0145] SEQ ID NO: 17:

[0146]

[0147]

[0148] The corresponding amino acid sequences of the heavy chain variable region and the light chain variable region derived therefrom are respectively as shown in SEQ ID NO: 2 and SEQ ID NO: 1. Further analysis obtains that the amino acid sequences of the CDR of the heavy chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 6-8; the amino acid sequences of the CDR of the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 3-5.

[0149] Example 5 Identification of monoclonal antibody

[0150] 1. Indirect Elisa titer determination of monoclonal antibody

[0151] ​​​​​The antibody titer was detected by indirect Elisa. The purified 6D5 monoclonal antibody was diluted by 2 times series (1:100 to 1:204800) as primary antibody, and the negative control (mouse negative serum) was set, and HRP labeled goat anti-mouse IgG was used as secondary antibody. 100 μL / well TMB color developing liquid was added for incubation at room temperature for 15 min, 100 μL / well stop solution was added, and D450 nm value was read by microplate reader. The specific operation steps were the same as those in Example 2.

[0152] Results: The p30 protein was used as the coating antigen, the monoclonal antibody diluted by 2 times was used as the primary antibody, and the indirect ELISA method was used to determine the titer of the monoclonal antibody. The results are shown in Figure 3 The ratio of OD450 values of the test well to the negative control well (P / N) was calculated. If P / N≥2.1, the test well was determined to be positive. The calculation results showed that the indirect Elisa titer of the monoclonal antibody 1E9 was 1:51200.

[0153] 2. Affinity determination of monoclonal antibody

[0154] The relative affinity of the monoclonal antibody was determined by indirect Elisa. The purified p30 protein was coated at a concentration of 2 μg / ml, 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml, and the diluted monoclonal antibody was added as the primary antibody for indirect Elisa detection. The affinity constant was calculated by the formula K=(n+1) / 2(nAb'-Ab). In the formula, Ab and Ab' are the antibody concentrations that produce half of the absorbance value when the antigen concentrations are Ag and Ag'; n=Ag / Ag'; when n=2, 3 K values can be obtained; when n=4, 2 K values can be obtained; when n=8, 1 K value can be obtained, and the average value is the final result.

[0155] Results: The affinity constant reflects the tightness of the antibody and antigen binding, which is an important indicator for evaluating the performance of the monoclonal antibody. 6D5 was 2.6 mg / mL, and then it was diluted to different concentrations. The antibody concentration at 50% OD value of the antigen-antibody binding platform represented the relative affinity of the monoclonal antibody. The affinity determination results of the 6D5 monoclonal antibody are shown in Figure 4 The affinity constant of the monoclonal antibody 6D5 was calculated to be 1.82×10 7 mol / L by substituting the affinity determination formula.

[0156] 3. Western Blot identification of monoclonal antibody

[0157] The lysate of PAM cells infected with ASFV and the pre-stained protein marker were subjected to SDS-PAGE electrophoresis, and the electrophoresis products were transferred to a PVDF membrane. The protein bands of each lane and the pre-stained protein marker bands were cut off, and then the PVDF membrane bands containing the cell lysate were washed 3 times in TBST for 3-5 min each time; 5% skim milk was blocked at room temperature for 1 h. The blocked PVDF membrane bands were incubated with 1:3000 diluted purified mouse ascites monoclonal antibody at room temperature for 1 h, washed 3 times with TBST, and then incubated with HRP-goat anti-mouse IgG (1:5000) at room temperature for 1 h, washed 3 times with TBST, and then developed.

[0158] The results of Western blot detection are shown in Figure 5 The results show that the monoclonal antibody prepared in Example 3 can specifically react with p30 protein.

[0159] 4. Detection of reactivity of monoclonal antibody

[0160] The specific operation steps of IFA are as follows: 1.25 x 10 5 PAM cells were plated in a 96-well cell plate, and after the cells adhered, 1000-fold diluted ASFV genotype I strain, ASFV genotype II strain, ASFV genotype II double gene deletion strain, ASFV genotype I and genotype II recombinant strain were inoculated. 72 h after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed 3 times with PBS; then permeated with 0.1% Triton X-100, and incubated at room temperature for 20 min, and washed 3 times with PBS; then incubated with the purified ascites monoclonal antibody prepared in Example 3 and anti-mouse FITC-labeled goat anti-mouse IgG, and the results were observed under a fluorescence microscope after the reaction was completed.

[0161] The results of IFA are shown in Figure 6 The results show that the anti-ASFV p30 monoclonal antibody can react with different ASFV-infected PAMs, and fluorescent signals can be seen under a fluorescence microscope, and normal PAM cells do not show fluorescence, indicating that the obtained monoclonal antibody can react with different ASFV, and has strong specificity.

[0162] 5. Identification of specificity of monoclonal antibody

[0163] The identification was carried out by indirect immunofluorescence. PRRSV was inoculated into Marc-145 cells, FMDV was inoculated into BHK-21 cells, PRV and CSFV were inoculated into PK-15 cells, respectively, and 72 h later, the cells were fixed with a mixture of pre-cooled methanol and acetone (1:1) at -20°C for 1 h, and then incubated with the purified monoclonal antibody and anti-mouse FITC-labeled goat anti-mouse IgG, and the results were observed under a fluorescence microscope after the reaction was completed.

[0164] Results: The results are shown in Table 1 Figure 7 Antibodies Nucleic acids, vectors, host cells Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 It is shown that 6D5 monoclonal antibody does not appear specific fluorescence with PRRSV, FMDV, PRV and CSFV, which indicates that the monoclonal antibody does not cross-react with PRRSV, FMDV, PRV and CSFV, and proves that the specificity of 6D5 monoclonal antibody is better.

[0165] Example 6 Preparation of ASFV P30 protein blocking ELISA antibody detection kit

[0166] 1. Preparation of antigen-coated plate

[0167] The P30 protein was diluted with coating solution to 1.5 ug / ml, and 100 ul was added to each well of the enzyme-labeled plate, and incubated at 4°C for 16 h. The solution in the wells was discarded, 200 ul of PBST was added to each well, and the washing was repeated for 3 times, and the last time was dried. 200 ul of blocking solution was added to each well, and incubated at 37°C for 2 h. The blocking solution was discarded, 200 ul of PBST was added to each well, and the washing was repeated for 3 times, and the last time was dried.

[0168] 2. Preparation of negative control

[0169] The negative serum was collected, diluted, filtered to remove bacteria, and quantitatively packaged as a negative control.

[0170] 3. Preparation of positive control

[0171] The positive serum was collected, diluted, filtered to remove bacteria, and quantitatively packaged as a positive control.

[0172] 4. Preparation of enzyme-labeled monoclonal antibody

[0173] The purified monoclonal antibody in Example 3 was labeled by horseradish peroxidase labeling method, and the concentration of ASFV enzyme-labeled monoclonal antibody was adjusted to 1 mg / ml as the stock solution of ASFV enzyme-labeled monoclonal antibody. The stock solution of ASFV enzyme-labeled monoclonal antibody was diluted with commercial HRP conjugate stabilizer / diluent, filtered to remove bacteria, and quantitatively packaged as enzyme-labeled monoclonal antibody.

[0174] 5. Preparation of sample diluent, 20x concentrated washing solution, substrate developing solution, and termination solution.

[0175] 6. Assembly The qualified antigen-coated plate, negative control, positive control, enzyme-labeled monoclonal antibody, sample diluent, 20x concentrated washing solution, substrate developing solution, and termination solution were assembled into an ASFV P30 protein blocking ELISA antibody detection kit.

[0176] Example 7 Identification of ASFV P30 protein blocking ELISA antibody detection kit

[0177] 1. Specific identification

[0178] Four specific quality control serum samples were selected to verify the specificity of the kit. The results of the African swine fever virus blocking ELISA antibody detection kit were all negative, indicating that the kit prepared in Example 6 had good specificity. The detailed results are shown in Table 2.

[0179] Table 2

[0180]

[0181] Note: “—” indicates a negative result

[0182] 2. Identification of African swine fever clinical detection samples

[0183] Thirty-six ASFV positive sera determined by IFA method were selected for detection. The results of the African swine fever virus blocking ELISA antibody detection kit were all positive, consistent with the IFA identification results. Table 3 shows the results of the African swine fever virus blocking ELISA antibody detection kit for clinical sample detection.

[0184] Table 3

[0185]

[0186]

[0187] Note: The determination criteria for the African swine fever virus blocking ELISA antibody detection kit are as follows: calculate the blocking rate. When the blocking rate is ≥27.7%, it is determined to be positive; when the sample blocking rate is ≤18.26%, it is determined to be negative; and when it is between the two values, it is determined to be suspicious, and the suspicious sample is rechecked.

[0188] Formula: Blocking rate = (1-S / N) x 100%, where S represents the OD of the sample 450 , and N represents the OD of the negative sample 450 .

[0189] The above is only a preferred embodiment of the present application, and is not intended to limit the other forms of the present application. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments without departing from the technical content of the present application, and according to the technical essence of the present application, still belongs to the protection scope of the present application.

Claims

1. An anti-African swine fever virus p30 protein monoclonal antibody, wherein, The antibody comprises three heavy chain complementarity determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID NO: 6, the amino acid sequence of CDR-H2 is as shown in SEQ ID NO: 7, the amino acid sequence of CDR-H3 is as shown in SEQ ID NO: 8, the amino acid sequence of CDR-L1 is as shown in SEQ ID NO: 3, the amino acid sequence of CDR-L2 is as shown in SEQ ID NO: 4, the amino acid sequence of CDR-L3 is as shown in SEQ ID NO:

5.

2. The monoclonal antibody of claim 1, wherein, The antibody comprises a heavy chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

2.

3. The monoclonal antibody of claim 1, wherein, The antibody comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO:

2.

4. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region, wherein: the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

1.

5. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO:

1.

6. The monoclonal antibody of claim 1, wherein, The antibody comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

1.

7. The monoclonal antibody of claim 1, wherein, The antibody comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO: 2, the amino acid sequence of the light chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO:

1.

8. The monoclonal antibody of any one of claims 1-7, wherein, The antibody is a mouse antibody or a chimeric antibody.

9. An isolated nucleic acid encoding the monoclonal antibody of any one of claims 1-8.

10. A recombinant vector comprising the nucleic acid of claim 9.

11. A host cell comprising the nucleic acid of claim 9 or the recombinant vector of claim 10.

12. A kit for detecting an African swine fever virus p30 protein, wherein, comprising the monoclonal antibody of any one of claims 1-8.

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