Anti-porcine pseudorabies virus glycoprotein gB antibody and application thereof

Abstract

The invention belongs to the technical field of immunological detection, and particularly relates to an antibody for resisting porcine pseudorabies virus glycoprotein gB and application of the antibody. The antibody is a first antibody or a second antibody, amino acid sequences of light chains CDR1-3 of the first antibody are respectively shown as SEQ ID NO.3-5, and amino acid sequences of heavy chains CDR1-3 of the first antibody are respectively shown as SEQ ID NO.8-10; the amino acid sequences of light chains CDR1-3 of the second antibody are respectively as shown in SEQ ID NO.13-15, and the amino acid sequences of heavy chains CDR1-3 of the second antibody are respectively as shown in SEQ ID NO.18-20. The antibody and the PRV glycoprotein gB have good reactivity, recognition specificity and binding sensitivity, the detection sensitivity and accuracy of the gB protein or the anti-gB protein antibody are improved, and an antibody tool with excellent performance is provided for qualitatively or quantitatively evaluating the immune effect of swine herd vaccines and detecting the content of porcine pseudorabies virus antigens in a sample.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly to antibodies against glycoprotein gB of porcine pseudorabies virus and their applications. Background Art

[0002] Pseudorabies (PR) is an acute infectious disease caused by Pseudorabies virus (PRV) in various animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, wild boars, minks, bears, and foxes, with main clinical symptoms including fever, severe itching (except in pigs), and encephalomyelitis. Pseudorabies virus is also known as porcine herpesvirus type I, infectious bulbar paralysis virus, pruritus virus, and Oiezkij virus. It belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae of the Herpesviridae family, is a linear double-stranded DNA virus, with a full-length gene of approximately 150 kb, containing 77 open reading frames, and an average G+C content as high as 73.6%. Porcine pseudorabies exists widely. After pigs are infected with PRV, it mainly causes abortion, stillbirth, mummified fetuses in pregnant sows and infertility in breeding pigs, high mortality in piglets after showing loss of appetite, intractable diarrhea, and neurological symptoms, as well as respiratory symptoms in fattening pigs. Moreover, PRV infection is prone to form latent infection, resulting in lifelong virus carriage and intermittent virus excretion; in addition, PRV is easily activated under stress conditions, which can cause recurrent infection and virus dissemination. This latent-activation cycle mechanism determines the high incidence and explosive epidemic of pseudorabies virus in pig populations, and it is one of the primary diseases endangering pig populations, causing huge economic losses to the global pig industry.

[0003] Vaccination is an important measure for the prevention and control of infectious diseases. Before and after immunizing with a pseudorabies vaccine, it is necessary to timely detect the neutralizing antibody level in the sera of pig populations as a basis for the effectiveness of vaccination, to understand whether the vaccine has achieved the expected immunization effect and reasonably arrange the time for supplementary vaccination. On the other hand, neutralizing antibody detection can also monitor the wild virus infection status or the situation of disease treatment, which is of great significance for the prevention and control of diseases.

[0004] Glycoprotein gB is the main immunogen of the PRV virus and plays an important role in the invasion stage of PRV replication. It can help the fusion of the cell membrane and the viral envelope, thus successfully completing viral invasion. It can also mediate the membrane fusion between infected and uninfected cells and help the virus spread between cells. The development of vaccines targeting the gB protein has been very successful. The gB protein can stimulate the body to produce complement-dependent and independent neutralizing antibodies. Therefore, using the PRV gB protein as a coating antigen and competing and blocking the binding of serum neutralizing antibodies to the gB protein through anti-gB protein antibodies can achieve efficient evaluation of the immune antibody level, which is a key link in the prevention and control of porcine PRV infection, the evaluation of vaccine immune effects, and the purification of porcine pseudorabies. At the same time, the gB protein is also an important target of many current PRV virus detection kits. Therefore, it is of great significance to specifically prepare monoclonal antibodies targeting glycoprotein gB. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the present invention provides two antibodies with good reactivity, recognition specificity and binding sensitivity to porcine pseudorabies virus glycoprotein gB and the antibody pair composed thereof, and provides the encoding genes and expression vectors of the antibodies. The present invention further provides the application of the aforementioned antibody or antibody pair in the preparation of a kit for detecting porcine pseudorabies virus glycoprotein gB or anti-porcine pseudorabies virus glycoprotein gB antibody, or the application of the aforementioned antibody in the preparation of anti-porcine pseudorabies virus drugs. The present invention is specifically realized through the following technical solutions:

[0006] In the first aspect of the present invention, an antibody against porcine pseudorabies virus glycoprotein gB is provided, selected from the first antibody or the second antibody. The antibody includes a light chain variable region and a heavy chain variable region, wherein: the amino acid sequences of complementarity-determining regions CDR1-3 on the light chain variable region of the first antibody are respectively as shown in SEQ ID NO.3-5, and the amino acid sequences of complementarity-determining regions CDR1-3 on the heavy chain variable region are respectively as shown in SEQ ID NO.8-10; the amino acid sequences of complementarity-determining regions CDR1-3 on the light chain variable region of the second antibody are respectively as shown in SEQ ID NO.13-15, and the amino acid sequences of complementarity-determining regions CDR1-3 on the heavy chain variable region are respectively as shown in SEQ ID NO.18-20.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the first antibody is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the second antibody is as shown in SEQ IDNO.12, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.17.

[0008] Further, the amino acid sequence of the light chain of the first antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.6; the amino acid sequence of the light chain of the second antibody is as shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.16.

[0009] Further, the first antibody or the second antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and sc(Fv)2 fragment.

[0010] In the second aspect of the present invention, a nucleic acid molecule or a recombinant vector is provided. The recombinant vector contains the nucleic acid molecule, and the nucleic acid molecule encodes the antibody against porcine pseudorabies virus glycoprotein gB as described above.

[0011] In the third aspect of the present invention, an antibody conjugate is provided, which includes the antibody against porcine pseudorabies virus glycoprotein gB as described above and a detection label connected to the antibody.

[0012] In the fourth aspect of the present invention, an antibody pair against porcine pseudorabies virus glycoprotein gB is provided, which is composed of the first antibody and the second antibody as described above.

[0013] In the fifth aspect of the present invention, the application of the antibody against porcine pseudorabies virus glycoprotein gB, the antibody conjugate or the antibody pair as described above in the preparation of a kit for detecting porcine pseudorabies virus glycoprotein gB is provided.

[0014] In the sixth aspect of the present invention, the application of the antibody against porcine pseudorabies virus glycoprotein gB, the antibody conjugate or the antibody pair as described above in the preparation of a kit for detecting the antibody against porcine pseudorabies virus glycoprotein gB is provided.

[0015] In the seventh aspect of the present invention, the application of the antibody against porcine pseudorabies virus glycoprotein gB as described above in the preparation of a drug against porcine pseudorabies virus is provided.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0017] 1. The antibody provided by the present invention has good reactivity, recognition specificity and binding sensitivity with porcine pseudorabies virus glycoprotein gB. The immunoassay system constructed with this antibody for detecting the neutralizing antibody of gB protein in porcine pseudorabies positive serum has the advantages of good specificity, no cross-reaction with other common viruses, and good linear relationship and wide linear range of the detection results, which is beneficial to improving the detection sensitivity and accuracy of the antibody against gB protein, and provides an antibody tool with excellent performance for qualitatively or quantitatively evaluating the vaccine immunization effect of pig herds.

[0018] 2. The antibodies provided by the present invention recognize different antigenic epitopes of the gB protein and can be used as paired antibodies. By pairing the first antibody and the second antibody to construct a double-antibody sandwich immunoassay system, the quantitative detection of the gB protein has the characteristics of good specificity, wide linear range, and high sensitivity. It has good application value in the fields of detecting the content of PVR virus with the gB protein as the detection target, identifying pigs infected with pseudorabies virus, and assisting in the purification of PRV.

[0019] 3. The antibodies provided by the present invention have the function of neutralizing PRV virus and blocking virus infection of cells by binding to the gB protein, which is beneficial to providing new means for the prevention and treatment of porcine pseudorabies and shows good clinical potential in the research and development field of porcine pseudorabies antibody drugs. Detailed implementation manners

[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] Based on the information contained in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for schematically illustrating specific aspects of the present invention. In fact, various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are all covered within the scope of the appended claims.

[0022] In order to better understand the present invention rather than limit its scope, all numbers representing amounts, percentages and other numerical values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired properties. Each numerical parameter should be regarded as obtained at least according to the reported significant figures and by the conventional rounding method.

[0023] In addition, it should be noted that unless otherwise defined, the scientific and technical terms used in the context of the present invention should have the meanings commonly understood by those of ordinary skill in the art.

[0024] The meanings of terms such as "comprising", "including", "containing", "having" and the like are non-restrictive, that is, other steps and other components can be added without affecting the results.

[0025] The term "and / or" shall be regarded as a specific disclosure of each of two designated features or components with or without the other. For example, "A and / or B" is regarded as including the following cases: (i) A, (ii) B, and (iii) A and B.

[0026] Similar terms such as "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody", and "mAb" have the same meaning. Unless otherwise specified, they all refer to antibodies that specifically bind to the glycoprotein gB of porcine pseudorabies virus. Terms such as "glycoprotein gB", "gB protein", and "gB" have the same meaning. The modifier "rabbit" indicates that the complementarity-determining region (CDR) of the antibody is derived from rabbit immunoglobulin sequences.

[0027] An antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity. An antibody fragment can be one or more parts or fragments of a full-length antibody, retaining the ability of the antibody to specifically bind to the target antigen.

[0028] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). The light chains can be divided into two types, namely κ chains and λ chains; the heavy chains can be classified into five types, namely μ, δ, γ, α, and ε chains, and the antibodies are defined as IgM, IgD, IgG, IgA, and IgE respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions with relatively large amino acid sequence variations near the N-terminus in the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity-determining regions (CDR), which are loop structures. The CDRs of the heavy chain and the CDRs of the light chain are closely juxtaposed and cooperate with each other through the FR regions to jointly form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the specificity of the antibody and being the site where the antibody recognizes and binds to the antigen. The FR regions are the more conserved parts of VH and VL. They generally have a β-sheet configuration and are connected by three CDRs that form connecting loops. Each VH and VL usually consists of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0029] The CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the cumulative of the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition or any CDR determination method well-known in the art.

[0030] The constant region of the light chain (CL) and the constant region of the heavy chain (CH) do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The lengths of the CLs of different Ig types (κ or λ) are basically the same, but the lengths of the CHs of different Ig classes are different. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the constant regions of the heavy and light chains of the antibody are well-known in the art and can be obtained by querying the IMGT database.

[0031] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "intact antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0032] Antibody fragments are one or more parts or segments of a full-length antibody, which substantially retain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR regions as the full-length antibody, and more preferably have the same variable regions, thereby retaining a complete antigen recognition and binding site, and being able to bind to the same antigen as the full-length antibody, especially binding to the same epitope. In typical examples, antibody fragments include: Fab, F(ab)2, Fab’, F(ab’)2, Fv, (Fv)2, scFv, sc(Fv)2, and these antibody fragments can be obtained by conventional techniques in the art.

[0033] (i) Fab: The antigen-binding fragment (Fab) is a monovalent fragment composed of a complete light chain (variable region and constant region) and a partial heavy chain (variable region and the first constant region). By protease digestion of the full-length antibody, fragments such as Fab, F(ab’)2, and Fab’ can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab’)2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab’ fragments. Since Fab has an antigen-binding region and a partial constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration ability as scFv, but also has a more stable structure.

[0034] (ii) F(ab)2: A bivalent fragment containing two Fabs linked by a disulfide bridge in the hinge region.

[0035] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment and only contains the variable regions, and is composed of the variable regions of a light chain and a heavy chain. It is a non-covalently bound dimer of a VH and a VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, and have the ability to recognize and bind antigens, although the affinity is lower than that of the full-length antibody.

[0036] (iv) (Fv)2: Composed of two Fv fragments covalently linked together.

[0037] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment consisting of a single polypeptide chain, formed by linking a variable heavy chain region (VH) and a variable light chain region (VL) through a flexible linker (usually composed of 10 - 25 amino acids). It retains the antigen-binding specificity of the original antibody. In the present invention, the linker is not particularly limited as long as it does not interfere with the expression of the antibody variable regions connected at both ends. Compared with full-length antibodies, scFv has the characteristics of a small molecular weight, and thus has higher penetrability and lower immune side reactions.

[0038] (vi) An sc(Fv)2 fragment is formed by connecting two variable heavy chain regions and two variable light chain regions through a linker or the like.

[0039] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include complementarity-determining regions (CDRs) and framework regions (FRs) from rabbit immunoglobulin sequences. In other embodiments, the antibody may contain amino acid residues encoded by non-rabbit immunoglobulin sequences, such as types like porcine antibodies and chimeric antibodies, to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which a part is derived from a specific source or species, while the remaining part is derived from a different source or species. For example, a porcine-rabbit chimeric antibody is formed by combining the variable region of a rabbit antibody with the constant region of a porcine antibody. The term "porcine antibody" is a chimeric antibody of a non-porcine antibody such as a rabbit antibody's CDR region and a porcine FR region. This framework sequence can be derived from the FR sequences of a single or multiple other porcine antibody variable regions. In some cases, a porcine antibody can also be formed by combining the CDR region of a rabbit antibody with the FR region and constant region derived from a porcine antibody sequence. In the present invention, the CDR region in the chimeric antibody or porcine antibody is derived from the rabbit CDR region.

[0040] Terms such as "monoclonal antibody" or "mAb" can be used interchangeably and refer to a homogeneous group of antibodies, that is, except for a small number of possible natural mutations and / or post-translational modifications (such as isomerization, amidation), each antibody constituting the population is the same. "Monoclonal antibodies" are highly specific and show a single binding specificity and affinity for the same or substantially the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to the hybridoma method, phage display method, yeast display method, recombinant DNA method, single-cell screening or single-cell sequencing method.

[0041] The term "specific binding" is a well-known term in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a specific target antigen or epitope than with other target antigens or epitopes. "Specific binding", also known as "preferential binding", does not necessarily require (although it can include) exclusive binding.

[0042] To make the objectives, features, and advantages of the present invention more obvious and understandable, the following provides a detailed description of the specific embodiments of the present invention.

[0043] The envelope glycoprotein gB is an essential glycoprotein for the replication of pseudorabies virus (PRV). Both live vaccines and wild strains contain the gB protein, and both live vaccine immunization and wild virus infection can stimulate the body to produce gB antibodies. Therefore, collecting blood samples from immunized pigs, separating the serum, and detecting the serum antibody level specific to the gB protein are important means for monitoring the immunization effect of pseudorabies vaccines and the virus infection situation, and are of great significance for formulating immunization programs and monitoring the epidemic status of pseudorabies. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used method in PRV serological detection. A blocking gB-ELISA commercial kit is often used to evaluate the PRV antibody level to supervise the vaccine immunization effect in pig farms and to warn of the presence of PRV wild virus infection. Currently, the relationship between the detection value of the commonly used ELISA method and the level of immune protection is not clear, resulting in the failure to timely detect pigs with a low level of immune protection. When the number of these pigs reaches a certain value, the pig herd may be infected, leading to an increase in the virus-carrying rate. Moreover, the serum antibody detection values of some kits are very close, making it inconvenient to analyze their corresponding relationship with neutralizing antibodies, and thus unable to accurately evaluate the vaccine immunization effect of the pig herd. Developing novel monoclonal antibodies targeting glycoprotein gB to solve the aforementioned problems is of great significance.

[0044] An embodiment of the present invention provides an antibody against porcine pseudorabies virus glycoprotein gB, which is a first antibody or a second antibody. The antibody includes a light chain variable region and a heavy chain variable region. Both the light chain variable region and the heavy chain variable region include 3 complementary determining regions (CDRs), named CDR1, CDR2, and CDR3 respectively. Among them: the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the first antibody are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20 respectively.

[0045] The present invention immunizes New Zealand white rabbits with a live vaccine of porcine pseudorabies virus (PRV), and screens 2 rabbit-derived monoclonal antibodies with good reactivity, recognition specificity, and binding sensitivity to PRV glycoprotein gB. The immunoassay systems constructed by conjugating the aforementioned antibodies with detection labels, such as blocking ELISA and chemiluminescence detection systems, have the advantages of good specificity for detecting gB protein neutralizing antibodies in porcine pseudorabies positive sera, no cross-reaction with positive sera of other common porcine viral diseases, and a good linear relationship between the detection results and the neutralizing antibody concentration. The linear range is better than that of commercially available kits of the same type, and it can better differentiate the detection of gB protein serum antibody levels, which is beneficial to improving the detection sensitivity and accuracy of gB protein antibody levels and realizing the accurate evaluation of the vaccine immunization effect of the pig population. Moreover, based on the different epitopes of the gB protein targeted by the antibodies of the present invention, the 2 antibodies can be used as paired antibodies to construct a double-antibody sandwich immunoassay system, which has the characteristics of good specificity, wide linear range, and high sensitivity when quantitatively detecting the gB protein, and has great application value in the fields of PVR virus content targeting the gB protein, differentiating pseudorabies virus-infected pigs, and assisting in PRV purification. In addition, the antibodies of the present invention also have the function of neutralizing PRV virus and blocking virus infection of cells by binding to the gB protein, and can be used for antiviral treatment, and also show good clinical potential in the research and development field of porcine pseudorabies antibody drugs. In short, the antibodies against PRV glycoprotein gB provided by the present invention have broad application prospects and important public health significance in the fields of PRV infection diagnosis, clinical treatment, and immune evaluation.

[0046] Optionally, both the light chain variable region and the heavy chain variable region include 4 framework regions (FRs), and the 4 FRs and 3 CDRs are arranged alternately in sequence to form the variable region. The amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7. The amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.17.

[0047] Optionally, the antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH). CL and VL form the light chain, and CH and VH form the heavy chain. The constant regions of the antibody are usually obtained by querying the IMGT online database. For example, through the IMGT online database (www.imgt.org), CH is obtained by searching for rabbit IgG gamma C reign, and CL is obtained by searching for rabbit IgG Kappa C reign.

[0048] Specifically, the amino acid sequence of the light chain (FL) of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.6. The amino acid sequence of the light chain (FL) of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.16.

[0049] It should be noted that the antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR regions as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site and being able to bind to the same antigen as the full-length antibody, especially binding to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab’, F(ab’)2, Fv, (Fv)2, scFv, and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0050] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule, and the nucleic acid molecule is used to encode the antibody against pseudorabies virus glycoprotein gB as described above.

[0051] The nucleic acid molecule can be in the form of DNA (such as cDNA, genomic DNA, or synthetic DNA) or RNA (such as mRNA or synthetic RNA). The DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand.

[0052] The sequence of the nucleic acid molecule can be obtained by derivation through conventional means such as codon coding rules based on the antibody AA sequence. The full-length sequence or fragments of the nucleic acid molecule can usually be obtained by PCR amplification, recombination, or artificial synthesis methods.

[0053] The original vectors for constructing recombinant vectors are various conventional vectors in the art, as long as they can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1, and M13), cosmids, and minichromosomes. The vector can be a cloning vector (i.e., used to transfer the nucleic acid molecule into a host and multiply it in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in the host cell). Insert the nucleic acid molecule into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, then introduce it into the host cell and culture it under specific conditions to express and obtain the antibody. This is a well-known technology in the art and will not be introduced in detail here.

[0054] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two vectors respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form the antibody. In some other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into one vector, and each nucleic acid sequence is linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably linked to different promoters, or the nucleic acid sequences encoding the heavy chain and the light chain can be operably linked to a single promoter such that both the heavy chain and the light chain can be expressed by the same promoter. The choice of the expression vector / promoter depends on the type of host cell used to produce the antibody.

[0055] The transfection or transformation of the recombinant vector into the host cell is carried out by conventional techniques. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with the CaCl2 method or MgCl2; it can also be through microinjection, electroporation, or liposome packaging, etc. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, microinjection method, electroporation method, liposome packaging, or gene gun bombardment, etc.

[0056] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (such as DH5α, JM109, BL21, W3110), Bacillus spp. (such as Bacillus subtilis, Bacillus thuringiensis), Enterobacteriaceae strains (such as Salmonella typhimurium, Serratia marcescens), and Pseudomonas spp. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining the host cell transfected or transformed with the recombinant vector as described above, culturing it under suitable conditions can express the antibody, and then separating it to obtain the purified monoclonal antibody.

[0057] Another embodiment of the present invention provides an antibody conjugate, comprising the antibody against pseudorabies virus glycoprotein gB as described above and a detection label linked to the antibody.

[0058] It should be emphasized that the monoclonal antibody of the present invention can be used alone or linked to a detection label (covalently or non-covalently conjugated) to form an antibody conjugate. The detection label is used to directly or indirectly generate a recognizable signal change to identify the antibody of the present invention according to the signal change, and then qualitatively or quantitatively detect the analyte through the specific reaction of antigen-antibody, including but not limited to gB protein, PRV virus, and antibody against gB protein. In some embodiments, the antibody of the present invention is used as an antigen-binding (or capturing) antibody, which specifically recognizes and binds the gB protein in the test sample, and then the analyte is qualitatively or quantitatively detected by analyzing the signal of the detection label linked to it. In other embodiments, the antibody against gB protein (as the primary antibody or capturing antibody) is not labeled, but the detection label is conjugated to a secondary antibody (as the detection antibody) or other molecules that can bind to the primary antibody. For example, if the antibody against gB protein is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. Thus, the qualitative or quantitative detection of the analyte is achieved by analyzing the signal change of the detection label generated after the secondary antibody specifically binds to the antibody of the present invention. The antibodies of the present invention can also be used in pairs. When used in pairs, one of the two antibodies is used as the primary antibody or capturing antibody, and the other is used as the secondary antibody or detection antibody.

[0059] The detection markers include, but are not limited to: biotin, fluorescent dyes (such as acridinium ester, umbelliferone, fluorescein, cyanine, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP, and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0060] Another embodiment of the present invention provides an antibody pair against glycoprotein gB of porcine pseudorabies virus, which is composed of the first antibody and the second antibody as described above.

[0061] Based on the fact that the antibodies of the present invention recognize different gB protein epitopes, the first antibody and the second antibody of the present invention can be used as paired antibodies in a double-antibody sandwich. Using this antibody pair to construct a double-antibody sandwich immunoassay method such as a chemiluminescence detection system can specifically and sensitively detect the gB protein, providing an effective way for the efficient and accurate detection of the gB protein and the detection of PRV virus targeting the gB protein.

[0062] The embodiments of the present invention also provide the applications of the antibody against glycoprotein gB of porcine pseudorabies virus as described above, its antibody conjugate, or the antibody pair composed thereof. Specifically, the present invention provides the following applications:

[0063] 1) The application of the antibody against glycoprotein gB of porcine pseudorabies virus, its antibody conjugate, or the antibody pair as described above in the preparation of a kit for detecting glycoprotein gB of porcine pseudorabies virus;

[0064] 2) The application of the antibody against glycoprotein gB of porcine pseudorabies virus, its antibody conjugate, or the antibody pair as described above in the preparation of a kit for detecting the antibody against glycoprotein gB of porcine pseudorabies virus;

[0065] 3) The application of the antibody against glycoprotein gB of porcine pseudorabies virus as described above in the preparation of a drug against porcine pseudorabies virus.

[0066] The above-mentioned detection method uses conventional immunoassay methods. The detection kits include, but are not limited to: enzyme-linked immunosorbent assay (ELISA) kits, enzyme-linked immunospot (ELISPOT) kits, immunohistochemistry (IHC) kits, immunofluorescence (IF) kits, immunoblotting (WB) kits, flow cytometry (FC) kits, chemiluminescence immunoassay kits, etc. The detection samples include, but are not limited to: serum, plasma, urine, cell culture medium, and tissue homogenate.

[0067] When detecting the gB protein, the gB protein should be understood in the broadest sense, which includes not only the pure gB protein, but also mixtures containing the gB protein or organisms containing the gB protein such as the PRV virus. When detecting the gB protein, the antibody of the present invention is used to bind to the gB protein, and then the detection of the gB protein is achieved by detecting the antibody of the present invention. Specifically, the test substance such as the gB protein can be coated on a solid-phase carrier, and then the antibody of the present invention is used to specifically recognize and bind to the solid-phase gB protein. For example, in a direct ELISA system, the antibody of the present invention is conjugated with a detection label, and the gB protein can be qualitatively or quantitatively detected by analyzing the signal of the detection label connected thereto; in an indirect ELISA system, the antibody of the present invention (as the primary antibody) is not labeled, but the detection label is conjugated with a secondary antibody that can bind to the antibody of the present invention, and qualitative or quantitative detection is achieved by analyzing the detection label signal generated by the secondary antibody, such as the principle shown by the indirect ELISA detection system established in Example 1 below of the present invention for measuring the gB antibody titer. Of course, in some other embodiments, the test substance may not be coated. For example, in a double-antibody sandwich ELISA system, one of the antibodies of the present invention is coated on a solid-phase carrier, and then the test substance is used to bind to the antibody, and then another antibody conjugated with a detection label is used to bind to the test substance to achieve detection, such as the principle shown by the double-antibody sandwich ELISA detection system established in Example 5 below of the present invention for measuring the gB protein; in a competitive ELISA system, the antibody of the present invention is coated on a solid-phase carrier, and then the test substance conjugated with a detection label is used to bind to the antibody to achieve detection, which is a conventional method in the art and will not be elaborated in the present invention.

[0068] When detecting the gB protein antibody, the antibody of the present invention is used to competitively bind to the gB protein antigen with the gB protein neutralizing antibody (produced by vaccine immunization) in the test substance, so as to detect the content of the gB protein neutralizing antibody. Specifically, a quantitative gB protein can be coated on a solid-phase carrier, and then the antibody of the present invention conjugated with a detection label and the test substance are simultaneously used to competitively bind to the coated antigen. The signal intensity generated by the detection label is inversely proportional to the level of the gB protein neutralizing antibody in the test substance. Therefore, the content of the gB protein antibody can be calculated according to the change in the signal intensity, and then the immune effect can be evaluated; such as the principle shown by the blocking ELISA established in Example 3 below of the present invention and the magnetic particle chemiluminescence immunoassay system established in Example 4 for measuring the gB neutralizing antibody.

[0069] When preparing a drug against porcine pseudorabies virus, the antibody of the present invention is used as a drug active ingredient to neutralize porcine pseudorabies virus and achieve antiviral treatment.

[0070] The present invention will be further described below in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" published by Cold Spring Harbor Laboratory, or usually according to the conditions recommended by the manufacturer.

[0071] Example 1 Preparation of Monoclonal Antibodies Against Porcine Pseudorabies Virus Glycoprotein gB

[0072] In this example, New Zealand white rabbits were immunized with a commercially available live vaccine of porcine pseudorabies virus (PRV). Subsequently, based on single B lymphocyte labeling and sorting technology, B lymphocytes that could recognize gB protein were enriched and screened from the spleens of immunized rabbits. The isolated B lymphocytes were cultured in single cell form to obtain secreted monoclonal antibodies. Finally, through genetic engineering recombinant expression technology, the genes of the naturally paired antibody light chain (VL) and heavy chain variable region (VH) were first amplified by PCR from the B lymphocytes secreting monoclonal antibodies, and were respectively inserted into the expression vector in tandem with the light chain (CL) and heavy chain constant region (CH). After co-transfection of host cells, culture, purification, and screening, rabbit-derived monoclonal antibodies 17F12 and 20F3 against gB protein were obtained. The antibody sequencing work was completed by Wuhan Kingcare Biotechnology Co., Ltd. The amino acid (AA) sequences of the antibodies are shown in Table 1, where LCDR1-3 represent the light chain complementarity determining regions CDR1-3, and HCDR1-3 represent the heavy chain complementarity determining regions CDR1-3 respectively.

[0073] Table 1 Sequence Information of Monoclonal Antibodies 17F12 and 20F3 and Immunogen in this Example

[0074]

[0075]

[0076] 1.1 Animal Immunization: The immunogen used was a heat-stable live vaccine against porcine pseudorabies (Keqian Biology; HB2000 strain). Each New Zealand white rabbit was initially immunized with 0.4 doses by multiple subcutaneous injections in the inguinal and back regions. Booster immunizations were performed every 2 weeks after the first immunization with a dose of 0.2 doses, for a total of four booster immunizations. Ten days after the fifth immunization, blood was collected from the marginal ear vein to isolate serum. The titer of PRV gB neutralizing antibodies in the serum was determined by indirect enzyme-linked immunosorbent assay (ELISA) coated with gB protein (amino acid sequence shown in SEQ ID NO.21). Rabbits with high serum titers were selected for one additional booster immunization. Five days later, the animals were sacrificed and spleens were removed to isolate splenocytes.

[0077] Preparation of gB protein: According to the PRV-gB sequence shown by the GeneBank accession number (MT949537.1), the gB gene was synthesized, with restriction enzyme sites HindⅢ, XhoⅠ and His tag introduced at the upstream and downstream. Then the synthesized gB gene fragment was ligated to the vector pcDNA3.1. The ligation product was transformed into DH5α competent cells. After the plasmid was extracted and sequenced to be correct, the recombinant plasmid pcDNA3.1-gB was obtained. This recombinant plasmid was transfected into CHO cells. When the cell viability ≤ 60%, the cell culture medium was harvested. After the cells were lysed, the cell debris was removed by centrifugation at 12000×g for 20 min. An appropriate amount of the supernatant was taken for SDS-polyacrylamide gel electrophoresis. At the same time, the centrifuged culture supernatant was filtered through a 0.45 μm filter and purified with a Ni column. After dialysis and buffer exchange, the PRV gB protein was obtained.

[0078] The steps for determining the immune serum titer by indirect ELISA method are as follows: (1) Coating: Prepare a 0.5 μg / mL PRV gB protein solution with carbonate buffer (pH 9.6), add 100 μL per well to the enzyme-linked immunosorbent assay (ELISA) plate, and coat overnight at 4°C; (2) Blocking: Wash with washing buffer PBST at 300 μL per well, and then add blocking buffer at 200 μL per well and block at 37°C for 1 h; (3) Serial dilution of the serum to be tested and sample addition: Repeat the washing process of step (2) to wash the plate, and then serially dilute the serum to be tested, starting from a dilution factor of 1:1000, with a three-fold serial dilution. Add the serum dilution at 100 μL per well to the plate and incubate at room temperature for 1 h; (4) Secondary antibody incubation: Repeat the washing process of step (2) to wash the plate, and then add horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG diluted 1:5000 (purchased from ThermoFisher, catalog number 31460) at 100 μL per well and incubate at room temperature in the dark for 1 h; (5) Termination of reaction and color development: Repeat the washing process of step (2) to wash the plate, and then add TMB chromogenic solution at 100 μL per well and react in the dark at 25°C for 15 min. Finally, add termination buffer at 100 μL per well to terminate the reaction, and measure the absorbance at 450 nm. The pre-immune rabbit serum was used as the negative control, and the detection system without adding immune serum was used as the blank control (NC). The formulations of each buffer are described in detail in Example 3.

[0079] The results of serum titer detection are shown in Table 1. It can be seen that the titers of all three rabbits reached the requirements after the fifth immunization, and a strong immune response was generated in the rabbits, which can be used for subsequent monoclonal antibody isolation.

[0080] Table 1 Detection results of the titers of anti-gB protein neutralizing antibodies in the sera of three rabbits after the fifth immunization (OD 450 )

[0081] Serum dilution Negative Rabbit No. 1 Rabbit No. 2 Rabbit No. 3 1:1000 0.314 1.679 1.712 1.698 1:3000 0.057 1.676 1.701 1.553 1:9000 0.036 1.5 1.582 1.317 1:27000 0.038 1.112 1.287 1.002 1:81000 0.03 0.585 0.697 0.463 1:243000 0.03 0.246 0.28 0.208 1:729000 0.029 0.092 0.106 0.071 1:2187000 0.029 0.045 0.066 0.038

[0082] 1.2 Isolation of B lymphocytes in the spleen and sorting and culture of antigen-specific B lymphocytes: For relevant methods, refer to the publicly disclosed patents "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: July 16, 2019)" and "An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: August 11, 2020)".

[0083] The supernatant of the cultured B lymphocytes was identified by indirect ELISA coated with gB antigen to identify positive clones that could recognize and bind to the PRV gB protein. A total of 1152 cell supernatants were detected. Among them, the OD450 values of 7 strains, namely 7A1, 2H5, 3G4, 8D8, 20F3, 17F12, and 5D5, were greater than 1.0, and they were positive clones. The results are shown in Table 2.

[0084] Table 2 Screening results of antigen-specific B lymphocytes

[0085] Clone number 16E3 14B7 15H11 11A2 7H11 1A7 12H6 13A5 4E6 3F6 6B1 <![CDATA[OD 450 > 0.323 0.355 0.357 0.369 0.369 0.372 0.372 0.384 0.401 0.404 0.524 Clone number 8H2 10C12 1G7 7A1 2H5 3G4 8D8 20F3 17F12 5D5 / <![CDATA[OD 450 > 0.622 0.705 0.718 1.192 1.27 1.332 1.36 1.61 1.701 1.715 /

[0086] 1.3 Cloning of genes encoding rabbit monoclonal antibodies: Collect the B lymphocytes corresponding to the positive clones, lyse them, extract RNA, and reverse transcribe it into cDNA. Using the aforementioned cDNA as a template, the genes of the light chain variable region (VL) and heavy chain variable region (VH) of the naturally paired rabbit antibodies were amplified by the PCR method. The PCR reaction system includes: 4 μL of cDNA, 1 μL of forward primer (10 mM), 1 μL of reverse primer (10 mM), 12.5 μL of 2×Gloria HiFi (from Abclonal), and 6.5 μL of H2O; the PCR amplification program includes: pre-denaturation at 98 °C for 30 s, and then 40 cycles under the conditions of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 40 s, and finally hold at 72 °C for 5 min.

[0087] The amplified DNA products were sequenced, and then the sequences of the constant regions were obtained by querying the IMGT online database (www.imgt.org) to obtain the antibody gene sequences of the complete light chain (FL) and complete heavy chain (FH).

[0088] 1.4 Expression and large-scale production of antibodies: Rabbit monoclonal antibodies that recognize the PRV gB protein were produced on a large scale through genetic engineering recombinant expression technology. The heavy chain gene and light chain gene of the rabbit monoclonal antibody positive clone strain selected in the above steps were respectively loaded downstream of the signal peptide of the expression vector pcDNA3.1 by homologous recombination, and the successful construction of the recombinant plasmid was verified by sequencing. The above process was entrusted to Wuhan Abclonal Biotechnology Co., Ltd. to complete.

[0089] The light and heavy chain recombinant expression vectors were co-transfected into 293F cells. After 72 - 96 hours of transfection, the cell supernatant was collected to obtain a rabbit monoclonal antibody that recognizes the gB protein recombinantly. The target antibody was purified from the cultured cell supernatant using protein A affinity gel resin (purchased from Tiandi Renhe; product number: SA023100), and the purification protocol was operated according to the instruction manual of the protein A affinity gel resin. Subsequently, the antibody was collected by dialysis and buffer exchange, and the purity of the antibody was verified to be ≥95% using 12% polyacrylamide gel electrophoresis (SDS-PAGE). After passing the verification, it was aliquoted and stored at -20 °C for future use.

[0090] Example 2 Neutralization experiment of monoclonal antibodies 17F12 and 20F3 against porcine pseudorabies virus

[0091] The neutralization ability of antibodies 7A1, 2H5, 3G4, 8D8, 20F3, 17F12, and 5D5 against porcine pseudorabies virus (PRV) was verified using the method of fixed virus - diluted antibody. The steps are as follows: (1) The purified rabbit monoclonal antibody was serially diluted 2-fold with cell maintenance medium, with the initial antibody concentration of 1 μg / mL and dilution factors of 1:2, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256; (2) 50 μL of the monoclonal antibody solution at different dilution factors was mixed with 50 μL of standard porcine pseudorabies virus (containing 100 median tissue culture infective doses (TCID 50 )) and incubated in a 37 °C carbon dioxide incubator for 1 h; (3) 100 μL of the incubated monoclonal antibody and virus mixture was transferred to a 96-well cell culture plate in which porcine kidney cells (PK-15) had grown into a monolayer. Four wells of cells were inoculated for each dilution factor, and at the same time, 4 wells of normal cell controls and 4 wells of 100 TCID 50 virus controls were set up. Additionally, a virus titer control group was set up, that is, the virus was serially diluted 10-fold and then added to a 96-well cell culture plate in which the cells had grown into a monolayer. Four wells were inoculated for each dilution factor, and 100 μL was added to each well; (4) The 96-well cell culture plate was placed in a 37 °C, 5% CO2 cell culture incubator for culture, and the cytopathic effect (CPE) was observed daily; (6) Calculation of the 50% neutralization endpoint: According to the degree of cytopathic effect, the 50% neutralization endpoint was calculated using the Reed-Muench method, which is defined as: the antibody dilution factor at which 50% of the cells do not produce a cytopathic effect (CPE).

[0092] The results of the neutralization ability of the antibodies of the present invention against PRV are shown in Table 3. The results indicate that the screened antibodies 17F12 and 20F3 have good neutralizing effects on porcine pseudorabies virus, with neutralizing antibody titers of 90.51 and 44.32 respectively, and can effectively neutralize porcine pseudorabies virus.

[0093] Table 3 Test results of the neutralization ability of the antibodies screened in the present invention against PRV virus

[0094]

[0095] Example 3: Analysis of the Effect of Establishing a Blocking Enzyme-Linked Immunosorbent Assay (ELISA) for Detecting gB Neutralizing Antibodies Based on Monoclonal Antibody 17F12

[0096] In this example, the gB protein was coated and fixed on a solid-phase carrier, and then the serum to be tested and the enzyme-labeled rabbit antibody 17F12 were added. The rabbit antibody 17F12 and the gB neutralizing antibody in the serum to be tested competitively bound to the solid-phase antigen. Therefore, the amount of labeled antibody 17F12 bound to the solid phase was inversely proportional to the amount of gB neutralizing antibody in the serum to be tested. Finally, the substrate was added for color development to detect the concentration of neutralizing antibody in the serum tested.

[0097] The blocking ELISA system includes the following components: an enzyme-labeled plate coated with gB protein, washing buffer, dilution buffer (for diluting the sample to be tested), blocking buffer, enzyme-labeled monoclonal antibody 17F12, substrate solution, termination solution, and negative control and positive control for quality control. The washing buffer is PBS buffer containing 1% Tween-20 (PBST buffer), usually prepared as a 10-fold concentrated type. The dilution buffer and blocking buffer are phosphate buffer containing 0.5% (wt) casein (pH 7.4 ± 0.1, concentration 0.2 M). The substrate solution is 0.3 g / L TMB chromogenic solution, and the termination solution is 2 M H2SO4 solution. The negative control (NC) serum is diluted specific pathogen free (SPF)-level experimental pig serum (purchased from Tianhang Biology, product number 60011-8615), and its OD value is about 1.30. The positive control (PC) serum is diluted pig serum containing pseudorabies virus gB neutralizing antibody, and its OD value is about 0.20.

[0098] The preparation method of the enzyme-labeled plate coated with gB protein includes: adding 0.25 μg / mL gB protein to a polystyrene microplate (96-well ELISA detection plate) at 100 μL / well, placing it at 4°C overnight, then washing once with PBST at 150 μL / well, and then adding the blocking buffer, placing it at 4°C overnight to block the unbound sites, and then centrifuging to dry and placing it in a drying chamber for drying. Preferably, it is sealed and stored in a package containing a desiccant.

[0099] The enzyme-labeled monoclonal antibody 17F12 was prepared using an HRP rapid labeling kit (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., product number HRP-L-100). In actual use, it was appropriately diluted according to the titer of each batch of enzyme-labeled monoclonal antibody. The diluent used for dilution was Tris HCl buffer (pH 7.6) containing 1% (wt) bovine serum albumin (BSA).

[0100] The blocking ELISA method includes the following steps: (1) Serum sample treatment: Dilute the serum sample to be tested with an equal volume of dilution buffer, i.e., add 60 μL of dilution buffer to 60 μL of the serum sample to be tested, and mix well; (2) Serum sample addition: Add the diluted serum sample to the ELISA plate coated with gB protein at 100 μL / well, incubate at 37 °C for 30 min, and set up 2 wells for positive control serum and 2 wells for negative control serum respectively; (3) Plate washing: Discard the liquid in the wells, add 300 μL of washing buffer to each well, wash 4 times, and pat dry; (4) Addition of enzyme-labeled monoclonal antibody: Add 100 μL of enzyme-labeled monoclonal antibody to each well, incubate at 37 °C for 30 min; (5) Repeat step (3) for plate washing; (6) Color development: Add 100 μL of TMB color development solution to each well, incubate at 37 °C in the dark for 10 min, and then add 50 μL of stop solution to each well to terminate the color development; Measure the optical density (OD) value at 450 nm.

[0101] Judgment criteria for the validity of the test: If the average value of the positive control (PC) < 0.30 and the average value of the negative control (NC) > 0.80, the experiment is valid. Calculation of the S / N value: S / N = average OD value of the sample to be tested / average OD value of the negative control; If S / N ≤ 0.4, the sample is determined to be positive for neutralizing antibodies, and if the S / N value > 0.4, the sample is determined to be negative for neutralizing antibodies.

[0102] Verify the linear range of the blocking ELISA for detecting gB protein neutralizing antibodies. Dilute the positive serum of porcine pseudorabies virus by 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold respectively, and use the above detection system and IDEXX porcine pseudorabies virus gB protein antibody detection kit (product number 99-09732) for detection respectively. The detection results are shown in Table 4. It can be seen that the linear range of the present invention for detecting positive serum containing gB protein neutralizing antibodies is between 16-fold and 256-fold dilution, which is equivalent to the linear range of the commercially available kit, providing an important antibody tool for the detection of gB protein neutralizing antibodies and the evaluation of the immune effect of positive serum containing such neutralizing antibodies.

[0103] Table 4 Linear range of the blocking enzyme-linked immunosorbent assay for detecting gB neutralizing antibodies established based on monoclonal antibody 17F12

[0104] Positive serum dilution multiple Antibody 17F12 ELISA system (S / N) IDEXX detection system (S / N) 2 0.0689(0.047) 0.0478(0.049) 4 0.0715(0.049) 0.0584(0.060) 8 0.0734(0.050) 0.0647(0.066) 16 0.0739(0.051) 0.0722(0.074) 32 0.183(0.126) 0.2113(0.217) 64 0.2162(0.149) 0.3056(0.313) 128 0.415(0.285) 0.3791(0.389) 256 0.6938(0.477) 0.5824(0.597) Positive control mean value 1.4557 0.9754 Negative control mean value 0.108 0.1634

[0105] Verify the specificity of the blocking ELISA for detecting neutralizing antibodies against gB protein. The coated antigen is gB protein, and the positive and negative sera of classical swine fever, porcine reproductive and respiratory syndrome, porcine circovirus disease, porcine parvovirus disease, porcine foot-and-mouth disease type O, and pseudorabies virus are detected respectively. The negative sera are confirmed to be negative for pseudorabies virus by the IDEXX ELISA kit. The test results are shown in Table 5. The results show that except for the S / N value of the positive porcine serum of pseudorabies virus ≤ 0.4 (judged as positive), the S / N values of the other sera are between 0.7 - 0.9, and all are judged as negative, indicating that the method established by the present invention has good specificity and no cross-reaction with the positive sera of other pathogens.

[0106] Table 5 Specificity of the blocking ELISA for detecting neutralizing antibodies against gB protein

[0107]

[0108]

[0109] Example 4 Analysis of the effect of establishing a chemiluminescence immunoassay based on monoclonal antibody 17F12 for detecting gB neutralizing antibodies

[0110] In this example, gB protein is coated on the surface of magnetic particles, and then the serum to be tested and acridinium ester-labeled antibody 17F12 are added. The rabbit-derived antibody 17F12 and the gB neutralizing antibody in the serum to be tested competitively bind to the solid-phase antigen to form a solid-phase coated antigen-labeled antibody complex. The amount of this complex is inversely proportional to the amount of gB neutralizing antibody in the serum to be tested. Finally, the pre-excitation solution and the excitation solution are added. The acridinium ester absorbs the chemical energy in the chemical reaction and emits light, which can be detected by the optical system of the chemiluminescence analyzer to detect the concentration of neutralizing antibodies in the tested serum.

[0111] The chemiluminescence immunoassay system based on magnetic particles includes the following components: magnetic beads coated with gB protein, washing buffer, acridinium ester-labeled monoclonal antibody 17F12, excitation solution, pre-excitation solution, positive and negative calibrators for calibration, and quality control products 1 and 2 for quality control. The washing buffer is an aqueous solution containing 25 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.2). The pre-excitation solution is an aqueous solution containing 0.1% H2O2 and 0.1 M HCl. The excitation solution is an aqueous solution containing 0.2 M NaOH and 2% Triton X-100. The negative calibrator and quality control product 1 are fetal bovine serum (purchased from Solarbio, product number S9030). The positive calibrator and quality control product 2 are 6 μg / mL gB monoclonal antibody 17F12.

[0112] The preparation method of gB protein-coated magnetic beads includes: taking carboxyl magnetic beads (purchased from JSR, product number MS160) into a centrifuge tube, performing magnetic separation on a magnetic stand, washing the magnetic beads 3 times with 100 mM MES buffer (pH 5.0), adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to a final concentration of 4 mg / mL, reacting at room temperature for 30 min, then washing the magnetic beads 3 times, adding gB protein (the mass ratio of protein to magnetic beads is 1:20), mixing evenly and reacting at room temperature for 2 h; finally, washing the magnetic beads 3 times, adding a preservation solution, and storing at 2-8 °C.

[0113] The preparation method of acridinium ester-labeled monoclonal antibody includes: taking 50 μL of antibody 17F12 (concentration 2 mg / mL) solution into a brown centrifuge tube, adding 35 μL of 0.02 M PBS, mixing evenly, then adding 15 μL of 5 mM acridinium ester solution dissolved in dimethyl sulfoxide (DMSO), mixing evenly and centrifuging briefly, labeling at 1500 rpm at room temperature in the dark for 2 h. After briefly centrifuging the acridinium ester-labeled antibody, load it into a dialysis bag, use 0.02 M PBS as the dialysis buffer, and change the dialysis solution every 2 h until the RLU of the peripheral dialysis solution is less than 10000.

[0114] The magnetic particle chemiluminescence method includes the following steps: adding 20 μL of calibrator or quality control product or test serum and 30 μL of gB protein-coated magnetic beads to a reaction cup, mixing evenly, incubating at 37 °C for 20 min, washing away unbound substances with a washing buffer, then adding 30 μL of acridinium ester-labeled antibody 17F12, mixing evenly, incubating at 37 °C for 10 min, washing away unbound substances with a washing buffer, adding 50 μL of pre-excitation solution and 50 μL of excitation solution, reacting for 64 s, and measuring the luminescence value (RLU) of the sample with an automatic chemiluminescence analyzer.

[0115] The judgment standard for the test to be valid: the S / Co of quality control product C1 is between 0.9 and 1.1, and the S / Co of quality control product C2 is between 0.04 and 0.06, then the test is judged to be valid. Calculation of the S / Co value: S / Co = sample luminescence value / Cutoff value; for the test sample, if S / Co ≤ 0.7, it is judged as positive, if S / Co ≥ 0.75, it is judged as negative, and if 0.7 < S / Co < 0.75, it is judged as suspicious.

[0116] Verify the linear range of the magnetic particle chemiluminescence method for detecting gB protein neutralizing antibodies. Dilute the positive serum of porcine pseudorabies virus 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, and 256-fold respectively, and use the above detection system and IDEXX porcine pseudorabies virus gB protein antibody detection kit for detection respectively. The detection results are shown in Table 6. It can be seen that the antibodies of the present invention can show a good antibody titer change trend when the positive serum is diluted 4-258 times, while the imported ELISA kit does not show the titer change of the serum before the positive serum is diluted 16 times. The method established by the antibodies of the present invention has a wider linear range.

[0117] Table 6 Linear range of magnetic particle chemiluminescence method for detecting gB neutralizing antibodies based on monoclonal antibody 17F12

[0118] Standard positive serum dilution multiple Antibody 17F12 magnetic particle chemiluminescence system (S / Co) IDEXX detection system (S / N) 2 25624(0.031) 0.0478(0.049) 4 46397(0.056) 0.0584(0.060) 8 101818(0.123) 0.0647(0.066) 16 179529(0.217) 0.0722(0.074) 32 280856(0.339) 0.2113(0.217) 64 311524(0.376) 0.3056(0.313) 128 466428(0.563) 0.3791(0.389) 256 547886(0.661) 0.5824(0.597) Quality control product 1 533205 0.9754 Quality control product 2 828469 0.1634

[0119] Verify the specificity of the magnetic particle chemiluminescence method for detecting gB protein neutralizing antibodies. Detect the positive and negative sera of classical swine fever, porcine reproductive and respiratory syndrome, porcine circovirus disease, porcine parvovirus disease, porcine foot-and-mouth disease type O, and porcine pseudorabies virus respectively. The negative sera are confirmed to be negative for porcine pseudorabies virus by IDEXX ELISA kit. The detection results are shown in Table 7. The results show that except for the S / Co of the positive serum of porcine pseudorabies <0.7, the S / Co of the measured values of the other sera are all >0.75, and they are all judged to be negative, indicating that the detection system of the present invention has good specificity and no cross-reaction with the positive sera of other pathogen antibodies.

[0120] Table 7 Specificity of magnetic particle chemiluminescence method for detecting gB protein neutralizing antibodies

[0121]

[0122] Example 5 Analysis of the effect of establishing a magnetic particle chemiluminescence method for detecting the content of gB protein based on antibodies 17F12 and 20F3

[0123] In this example, the double antibody sandwich method is used. Antibody 20F3 is coated on the surface of magnetic particles, and then the sample to be detected and acridinium ester-labeled antibody 17F12 are added in sequence to form an antibody 20F3-antigen-antibody 17F12 complex. Finally, the pre-excitation solution and the excitation solution are added. The acridinium ester absorbs the chemical energy in the chemical reaction and emits light. The content of PRV gB protein in the sample is positively correlated with the luminescence value.

[0124] The magnetic particle chemiluminescence system includes the following components: magnetic beads coated with monoclonal antibody 20F3, washing buffer, acridinium ester-labeled monoclonal antibody 17F12, excitation solution, pre-excitation solution, positive calibration product and negative calibration product for calibration, and quality control product 1 and quality control product 2 for quality control. The negative calibration product and quality control product 1 are 0.01M PBS, and the positive calibration product and quality control product 2 are gB protein.

[0125] The preparation method of magnetic beads coated with monoclonal antibody 20F3 and the method of acridinium ester labeling monoclonal antibody 17F12 are shown in Example 4.

[0126] The magnetic particle chemiluminescence method of double antibody sandwich includes the following steps: Add 20 μL of calibrator or quality control product or sample to be tested and 30 μL of magnetic beads coated with monoclonal antibody 20F3 into the reaction cup, mix well, incubate at 37 °C for 20 min, wash away the unbound substances with washing buffer, then add 30 μL of acridinium ester-labeled antibody 17F12, mix well, incubate at 37 °C for 10 min, wash away the unbound substances with washing buffer, then add 50 μL of pre-excitation solution and 50 μL of excitation solution, react for 64 s, and immediately measure the luminescence value of the sample with an automatic chemiluminescence analyzer. The sample to be tested contains serially diluted gB protein, and the gB protein concentrations are: 100000, 20000, 4000, 800, 160, 32, and 6.4 pg / mL in sequence.

[0127] Table 8 shows the luminescence values of detecting the content of gB protein with different concentrations by the magnetic particle chemiluminescence method based on the antibody pair of the present invention. The standard curve obtained by linear fitting is: Y = 8.7677X + 6599, where X is the content of gB protein and Y is the luminescence value, R 2 = 0.998. The results show that the antibodies 17F12 and 20F3 of the present invention can be used as paired antibodies for immunoassay. The established detection method has good dilution linearity and sensitivity, and the content of gB protein and the content of PRV virus containing gB protein can be calibrated according to this method.

[0128] Table 8 Standard curve of detecting gB protein by magnetic particle chemiluminescence method established based on monoclonal antibodies 17F12 and 20F3

[0129] gB protein concentration (unit: pg / mL) Luminescence value 100000 878942 20000 205264 4000 36198 800 6923 160 1082 32 1069 6.4 1154 0 1125

[0130] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antibody against porcine pseudorabies virus glycoprotein gB, characterized in that, Selected from the first antibody or the second antibody, the antibody comprising a light chain variable region and a heavy chain variable region, wherein: The amino acid sequences of complementarity determining regions CDR1, CDR2, and CDR3 on the light chain variable region of the first antibody are shown in SEQ ID NO.3-5 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8-10 respectively; The amino acid sequences of complementarity determining regions CDR1, CDR2, and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO.13-15 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.18-20 respectively.

2. The antibody against pseudorabies virus glycoprotein gB according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

17.

3. The antibody against pseudorabies virus glycoprotein gB according to claim 2, characterized in that, The amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; The amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

16.

4. The antibody against pseudorabies virus glycoprotein gB according to claim 1, characterized in that, The first antibody or the second antibody is a full-length antibody or an antigen-binding region of the full-length antibody; the antigen-binding region is selected from Fab, F(ab)2, Fab’, F(ab’)2, Fv, (Fv)2, scFv or sc(Fv)2.

5. A nucleic acid molecule or recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule, and the nucleic acid molecule encodes an antibody against pseudorabies virus glycoprotein gB as described in any one of claims 1-4.

6. An antibody conjugate, characterized in that, Comprising an antibody against pseudorabies virus glycoprotein gB as described in any one of claims 1-4 and a detection label linked to the antibody.

7. An antibody pair against porcine pseudorabies virus glycoprotein gB, characterized in that, Composed of the first antibody and the second antibody as described in any one of claims 1-4.

8. Use of the antibody against pseudorabies virus glycoprotein gB as described in any one of claims 1-4, the antibody conjugate as described in claim 6, or the antibody pair against pseudorabies virus glycoprotein gB as described in claim 7 in the preparation of a kit for detecting pseudorabies virus glycoprotein gB.

9. Use of the antibody against pseudorabies virus glycoprotein gB as described in any one of claims 1-4, the antibody conjugate as described in claim 6, or the antibody pair against pseudorabies virus glycoprotein gB as described in claim 7 in the preparation of a kit for detecting an antibody against pseudorabies virus glycoprotein gB.

10. Use of the antibody against pseudorabies virus glycoprotein gB as described in any one of claims 1-4 in the preparation of a drug against pseudorabies virus.

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