Antibody capable of specifically recognizing porcine pseudorabies virus gB (507-734 aa) or antigen binding fragment thereof and application

Abstract

The invention relates to the technical field of antibodies, and discloses an antibody for specifically recognizing porcine pseudorabies virus gB (507-734 aa) or an antigen binding fragment thereof and application. The antibody or the antigen binding fragment thereof comprises a heavy chain and a light chain; the antibody or the antigen binding fragment thereof can be used for detecting the existence or the level of the porcine pseudorabies virus gB protein in a sample, and has key significance on optimization of a porcine pseudorabies virus detection method, improvement of diagnosis accuracy and realization of effective prevention and control of epidemic situations.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an antibody or its antigen-binding fragment that specifically recognizes amino acid fragments 507-734 of the porcine pseudorabies virus gB protein and its applications. Background Technology

[0002] Pseudorabies (PR) is a highly dangerous acute viral infectious disease caused by the pseudorabies virus (PRV). While the virus has a broad host spectrum, pigs are the only natural host and primary source of infection. The virus can spread rapidly within pig herds through direct contact and vertical transmission, posing a serious risk of large-scale outbreaks. Clinical symptoms vary significantly among pigs at different growth stages: pregnant sows often experience reproductive disorders such as abortion, stillbirth, and mummified fetuses; newborn piglets frequently exhibit acute and fatal neurological symptoms; and finishing pigs are characterized primarily by respiratory symptoms and growth retardation. These complex and diverse clinical manifestations result in continuous economic losses for the pig farming industry. Currently, there is no specific drug that can directly eliminate the virus; clinical interventions mainly focus on alleviating symptoms, reducing mortality, and controlling secondary infections. PRV infection often occurs in combination with other pathogens and is prone to secondary bacterial infections, leading to complex and nonspecific symptoms, making accurate diagnosis difficult based solely on clinical observation. Therefore, establishing a sensitive and specific PRV diagnostic method is an important foundation for achieving pig farm purification and implementing effective prevention and control strategies.

[0003] Laboratory diagnosis of porcine pseudorabies primarily employs two technical systems: etiological and serological. In etiological detection, while traditional virus isolation is considered the gold standard, its cumbersome operation and long cycle make it unsuitable for rapid clinical diagnosis. Immunofluorescence technology requires highly specialized equipment and personnel, limiting its application. While PCR methods excel in sensitivity and specificity, they still require specialized laboratory platforms and are not suitable for rapid on-site screening. In serological diagnosis, serum neutralization tests struggle to distinguish antibody responses from vaccine immunization and wild-type virus infection. Immunochromatography, though simple to operate, is susceptible to sample matrix interference. Fluorescent microsphere detection, while possessing potential for multiplex analysis and differential diagnosis, is limited by instrument costs. Enzyme-linked immunosorbent assay (ELISA), recommended by the World Organisation for Animal Health (OIE), offers significant advantages in throughput and ease of operation; however, its diagnostic accuracy is closely related to the quality of the antigens and antibodies used. Reagents from different sources often vary in specificity and sensitivity, affecting the reliability and comparability of test results.

[0004] Among the various glycoproteins encoded by porcine pseudorabies virus (PRV), the gB glycoprotein plays a central role in viral infection due to its highly conserved structure and essential requirement for viral replication. This protein is encoded by the UL region gene of the viral genome and has a molecular weight of approximately 92 kDa. Structurally, the gB protein consists of 913 amino acid residues: the first 799 amino acid residues at the N-terminus form the extracellular region, the middle 23 amino acid residues form the transmembrane region, and the 91 amino acid residues at the C-terminus form the intracellular region. Studies have confirmed that the gB protein mediates the initial binding of the virus to host cells by forming a complex with gC and plays a core role in membrane fusion and viral invasion. Simultaneously, gB is also a major antigenic target for inducing the production of neutralizing antibodies, and its antigenic epitopes remain highly conserved among different circulating strains, demonstrating good genetic stability. In practical applications, antibody detection targeting the gB protein has multiple significances: in immune surveillance, it can objectively assess the level of protective antibodies after vaccination; in diagnostic differentiation, combined with gE antibody detection, it can effectively distinguish between wild-type virus infection and vaccine immunization; in the eradication phase, continuous monitoring of gB antibody dynamics helps to scientifically formulate immunization strategies, and its negative antibody status is one of the key indicators for achieving disease eradication. Therefore, the gB protein is not only a key molecule in the viral invasion mechanism, but also a core target for establishing a highly sensitive and specific serological diagnostic system. Obtaining high-titer gB antibodies with accurate identification capabilities is crucial for optimizing detection methods, improving diagnostic accuracy, and achieving effective prevention and control. Summary of the Invention

[0005] This invention provides an antibody or its antigen-binding fragment that specifically recognizes the amino acid fragment at positions 507-734 of the porcine pseudorabies virus gB protein, thereby overcoming the shortcomings of the prior art.

[0006] Firstly, to achieve the above objectives, the present invention adopts the following technical solution: an antibody or its antigen-binding fragment that specifically recognizes amino acid fragments 507-734 of the porcine pseudorabies virus gB protein, comprising: (a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and / or (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 4.

[0007] Furthermore, the antibody is a monoclonal antibody.

[0008] Furthermore, the antibody is of the IgG1 type and has a kappa light chain.

[0009] Secondly, the present invention provides a cell line capable of producing the antibodies described above.

[0010] Thirdly, the application of the antibody or its antigen-binding fragment provided by the present invention in the preparation of a reagent for detecting porcine pseudorabies virus.

[0011] Fourthly, a porcine pseudorabies virus detection reagent comprises the antibody or its antigen-binding fragment provided by the present invention.

[0012] Fifthly, the use of the antibody or its antigen-binding fragment provided by the present invention, or the detection reagent described herein, in the preparation of products for diagnosing porcine pseudorabies.

[0013] The present invention has at least the following beneficial effects: This invention relates to an antibody or its antigen-binding fragment targeting amino acid fragments 507-734 of the porcine pseudorabies virus gB protein. The antibody or its antigen-binding fragment exhibits good specificity for the porcine pseudorabies virus gB protein and can be used to detect the presence or level of the porcine pseudorabies virus gB protein in a sample. This invention is of key significance for optimizing porcine pseudorabies virus detection methods, improving diagnostic accuracy, and achieving effective epidemic prevention and control. Attached Figure Description

[0014] Figure 1 The image shows the SDS-PAGE results of imidazole-eluted samples collected at different times in Example 1; where M is the protein molecular weight standard (Marker), 1 is the gB (507-734 aa) recombinant protein sample obtained by elution with 250 mM imidazole, 2 is the sample after thrombin digestion, and 3-6 are the samples after enzyme digestion and purification. Figure 2 This is a graph showing the results of serum titer detection in mice after immunization in Example 2; Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of cell line gB (507-1) in Example 3; Figure 4 This is the result of SDS-PAGE analysis of the purified gB(507-1) monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker), and 1-3 are the purified gB(507-1) monoclonal antibody; Figure 5 This is a graph showing the results of the gB(507-1) monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0015] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0016] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0017] Example 1: Obtaining recombinant protein of porcine pseudorabies virus gB (507-734 aa) In this embodiment, gB (507-734 aa) recombinant protein was prepared. The specific preparation method includes the following steps: 1. Construction of recombinant expression vectors This study was designed based on the gB sequence of the prevalent variant strain HN1201 of porcine pseudorabies virus (GenBank accession number: KP722022.1). Combining literature reports and bioinformatics analysis, three confirmed core antigenic epitope regions (59-129 aa, 214-289 aa, and 507-734 aa) were identified within the extracellular region (1-799 aa) of the gB protein. This study selected the 507-734 aa epitope fragment, whose gene sequence was synthesized by Anshengda Biotechnology Co., Ltd., and successfully cloned into the pET-32a vector, thus constructing the pET-32a-gB (507-734 aa) recombinant expression vector.

[0018] 2. Expression and purification of gB (507-734 aa) protein After amplification and culture of the identified positive BL21(DE3) clone containing the pET-32a-gB (507-734 aa) recombinant expression vector, plasmid was extracted, transformed using standard methods, and single colonies were picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600 When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16℃. The bacterial cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated on ice for 3 min with Binding buffer at 30 W power. The sonication was repeated for 2 s with a 2 s interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 min for purification.

[0019] The target protein (gB(507-734 aa) recombinant protein) was purified by nickel-column affinity chromatography: A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The lysate containing the gB(507-734 aa) recombinant protein was added to the column, and the permeate was collected and re-loaded. Unbound contaminants were washed away with binding buffer, followed by washing with 20 mL of washing buffer and eluting with 250 mM imidazole elution buffer. The purified recombinant protein was cleaved using thrombin to remove the fusion tag. Nickel-column affinity chromatography was used again to purify the recombinant protein. SDS-PAGE electrophoresis was used to detect the cleavage and purification efficiency. Figure 1 As shown, protein concentration was determined using the BCA method.

[0020] Example 2: Establishment of a cell line containing gB (507-734 aa) recombinant protein monoclonal antibody This embodiment prepared a gB(507-734 aa) recombinant protein monoclonal antibody cell line. The specific preparation method includes the following steps: 1. Mouse immunization The recombinant gB (507-734 aa) protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, administered subcutaneously at five sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg per mouse, following the same method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing. If the antibody titer was greater than 1:10000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10000, a fourth immunization was performed on day 43, following the same method. Three days before cell fusion, an intraperitoneal pulse immunization was performed, with 100 μg of immunogen injected directly into the peritoneum without adjuvant.

[0021] 2. Mouse serum titer detection (1) Antigen coating: The coating buffer was adjusted to 1 μg / mL with the recombinant gB (507-734 aa) protein obtained in Example 1 as the coating antigen. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0022] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0023] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37℃ for 1 h.

[0024] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0025] (5) Add primary antibody: Use blocking solution to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37℃ for 1 h, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0026] (6) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0027] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2 M sulfuric acid to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) is ≥2.1 is taken as the serum titer. Mice with a dilution greater than 1:100,000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction of recombinant gB (507-734 aa) protein with immune serum was identified by indirect ELISA, and the titer of mouse serum was 1:64000.

[0028] 3. Cell fusion Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible. The mixture was allowed to stand for 1 min. 20 mL of preheated serum-free DMEM culture medium (37°C) was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated.

[0029] 4. Screening of positive hybridoma cells Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0030] 5. Subcloning of positive hybridoma cells Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in 96-well plates, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was measured using an indirect ELISA method. The positive rate reached 100%. The culture was expanded, and the cell line was preserved and labeled as gB(507-1).

[0031] Example 3: Preparation and Identification of Monoclonal Antibodies against gB (507-734 aa) Protein In this embodiment, a monoclonal antibody against the gB (507-734 aa) protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation and titer determination of ascites fluid from gB (507-734 aa) protein monoclonal antibody. 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of a cell suspension (5 × 10⁻⁶) of the gB(507-1) cell line preserved in Example 2. 5 (Each mouse was injected with the cell suspension, and the day of injection was counted as day 1). On day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, and the supernatant was collected and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0032] 2. Purification of monoclonal antibodies Ascites fluid was collected, and gB(507-1) monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then allowed to stand at 4°C for 1 h to allow for complete precipitation of contaminating proteins. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 4 As shown, high-purity gB(507-1) monoclonal antibody was obtained.

[0033] 3. Identification of Monoclonal Antibody Types and Subclasses The mouse subtype identification kit was used to perform the experimental procedure. The gB(507-1) monoclonal antibody was identified as IgG1 and kappa light chain.

[0034] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody produced by the cell line gB (507-1) preserved in Example 2 (i.e., the gB (507-1) monoclonal antibody purified in step 2 of Example 3). The results are as follows: The nucleotide sequence of the heavy chain variable region of gB(507-1) monoclonal antibody (SEQ ID NO: 1) is: 5'-GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCTACGCCATGAACTGGGTTCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAACTAAAAGTAATAA TTATGGAACATATTATGCCGATTCAGTGAAGGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGGTCTATCTGCAAATGCACAACTTGAAAACTGAGGACACAGGCATGTATTACTGTGTGAATCCGTTTGCTGACTGGGGCCAAGGGACTCTGGTCATTGTCTCTGCA-3' ; The corresponding amino acid sequence (SEQ ID NO: 2) is: EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRTKSNNYGTYYADSVKDRFTISRDDSQSMVYLQMHNLKTEDTGMYYCVNPFADWGQGTLVIVSA; Nucleotide sequence of the light chain variable region (SEQ ID NO: 3) For: 5'-GACTTTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTATTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTCTTGATCTACTG GGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTGTCAGAATGATTATAGGTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3' ; The corresponding amino acid sequence (SEQ ID NO: 4) is: DFVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYRYPWTFGGGTKLEIK.

[0035] 4. Specificity detection of monoclonal antibodies The following proteins were used as antigens: porcine pseudorabies virus gB (507-734 aa) protein (gB-507-734), porcine pseudorabies virus gB (59-337 aa) protein (gB-59-337), porcine pseudorabies virus gE (52-260 aa) protein (gE-52-260), porcine reproductive and respiratory syndrome virus GP5 protein (GP5), porcine reproductive and respiratory syndrome virus N protein (N), porcine reproductive and respiratory syndrome virus Nsp protein (NSP), porcine foot-and-mouth disease virus 3ABC protein (3ABC), African swine fever virus I226R protein (I226R), African swine fever virus CD2v protein (CD2v), African swine fever virus p30 protein (p30), and African swine fever virus p72 protein (p72). All the antigens used in the tests were obtained by constructing expression vectors in the laboratory and inducing prokaryotic expression.

[0036] The ELISA plate was coated under the same conditions, using the purified gB(507-1) monoclonal antibody from step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5 As shown, the monoclonal antibody produced by the cell line gB(507-1) preserved in Example 2 (i.e. the gB(507-1) monoclonal antibody purified in step 2 of Example 3) has good specificity and can be used for qualitative or quantitative detection of porcine pseudorabies virus gB protein in the sample to be tested.

Claims

1. An antibody or its antigen-binding fragment that specifically recognizes amino acid fragments 507-734 of the gB protein of porcine pseudorabies virus, characterized in that, Include: (a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and / or (b) Light chain variable region containing the amino acid sequence shown in SEQ ID NO:

4.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody is of the IgG1 type and has a kappa light chain.

4. A cell line, characterized in that, The cell line is capable of producing the antibody as described in any one of claims 1 to 3.

5. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3 in the preparation of a reagent for detecting porcine pseudorabies virus.

6. A reagent for detecting porcine pseudorabies virus, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

7. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or the detection reagent as described in claim 5 or 6, in the preparation of articles for the diagnosis of porcine pseudorabies.

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