Antibody capable of specifically recognizing porcine pseudorabies virus gE (52-260aa) 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 gE (52-260aa) 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 can realize specific recognition on gE protein and is used for detecting a target antigen in a sample. The antibody can be used as a core reagent in an immunodetection system, is suitable for laboratory detection and on-site rapid screening of porcine pseudorabies, is beneficial to improving the monitoring efficiency of basic-level and large-scale breeding links, and provides a technical guarantee for early discovery and accurate prevention and control of epidemic situations. Meanwhile, the antibody can also be used as a research tool to be applied to research on gE protein related biological functions and effects of the gE protein in a virus pathopoiesis process, and important support is provided for clarification of virus variation characteristics and optimization of subsequent prevention and control and diagnosis strategies.

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 52-260 of the porcine pseudorabies virus gE 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] gE glycoprotein is an important structural protein of PRV, composed of approximately 579 amino acids. Its N-terminal 403 amino acids constitute the extracellular region, the middle 47 amino acids form the transmembrane region, and the C-terminal 123 amino acids are located in the intracellular region. The extracellular region carries the major antigenic epitopes and is a key marker for distinguishing wild-type virus infection from vaccine immunization. It can form a heterodimer with gI through non-covalent bonds, participating in viral membrane fusion and intercellular transmission, especially playing a crucial role in infection of the nervous system. However, it is not essential for viral replication and does not affect immunogenicity.

[0005] In recent years, PRV variants emerging in my country have shown mutations in the gE gene sequence, further increasing the difficulty and necessity for accurate differential diagnosis. Therefore, the development of specific monoclonal antibodies against the gE protein is of direct and significant importance for establishing domestically produced, efficient differential diagnostic methods. These antibodies can not only serve as core diagnostic reagents for large-scale quarantine at the grassroots level, providing technical support for epidemic control in my country's complex aquaculture environments, but also be crucial tools for in-depth research into gE protein function, revealing viral mutations and pathogenic mechanisms, and laying the foundation for the design of subsequent prevention and control strategies. Summary of the Invention

[0006] The present invention aims to provide an antibody or antigen-binding fragment thereof that specifically recognizes porcine pseudorabies virus gE (52-260 aa) and its application, in order to overcome the shortcomings of the prior art.

[0007] 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 52-260 of the porcine pseudorabies virus gE 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.

[0008] Furthermore, the antibody is a monoclonal antibody.

[0009] Furthermore, the antibody is of the IgG1 type and has a kappa light chain.

[0010] Secondly, the present invention provides a cell line capable of producing the antibodies described above.

[0011] 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.

[0012] Fourthly, a porcine pseudorabies virus detection reagent comprises the antibody or its antigen-binding fragment provided by the present invention.

[0013] 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.

[0014] Sixthly, the application of the antibodies or antigen-binding fragments provided by the present invention in the study of the biological function or pathogenic mechanism of porcine pseudorabies virus gE protein for non-diagnostic purposes.

[0015] The present invention has at least the following beneficial effects: This invention relates to an antibody or its antigen-binding fragment targeting amino acid segments 52–260 of the porcine pseudorabies virus (PRV) gE protein. This antibody or its antigen-binding fragment exhibits good specificity for the PRV gE protein, enabling specific recognition of the gE protein and detection of the target antigen in samples. This antibody can serve as a core reagent in immunoassay systems, suitable for laboratory detection and rapid field screening of porcine pseudorabies, improving monitoring efficiency in grassroots and large-scale farming operations and providing technical support for early detection and precise control of outbreaks. Furthermore, this antibody can also be used as a research tool to study the biological functions of the gE protein and its role in viral pathogenesis, providing crucial support for elucidating viral mutation characteristics and optimizing subsequent prevention and diagnostic strategies. Attached Figure Description

[0016] Figure 1 This is an SDS-PAGE electrophoresis image of the expression and purification of the PRV gE (52-260 aa) recombinant protein in Example 1; where M is the protein molecular weight standard (Marker), 1 is the uninduced bacterial protein, 2 is the induced bacterial protein, 3 is the gE (52-260 aa) recombinant protein sample eluted with 250 mM imidazole, 4 is the sample after enterokinase digestion of gE (52-260 aa) recombinant protein, and 5 is the purified sample after digestion. 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 gE (52-42) in Example 3; Figure 4 This is the result of SDS-PAGE analysis of the purified gE(52-42) monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker), and 1-3 are the purified gE(52-42) monoclonal antibody; Figure 5 This is a graph showing the results of the gE(52-42) monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] Example 1: Obtaining recombinant protein of porcine pseudorabies virus gE (52-260 aa) In this embodiment, gE (52-260 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 gE gene sequence of the prevalent porcine pseudorabies virus variant HN1201 (GenBank accession number: KP722022.1). According to existing literature reports and bioinformatics analysis, the full-length structure of the gE protein can be divided into the following functional domains: the N-terminal 1-21 amino acids (aa) form the signal peptide; the extracellular region is 1-430 aa; the transmembrane region is 431-453 aa; and the intracellular region is 454-579 aa. Based on this, this study precisely defined the truncated variant within the 52-260 aa region. The design strategy is based on the following considerations: firstly, to fully preserve the known major antigenic epitope clusters (52-238 aa); and secondly, to remove the N-terminal signal peptide (1-21 aa) to avoid its potential interference with the prokaryotic expression system, while avoiding the C-terminal transmembrane structure (431-453 aa) and intracellular region. This significantly enhances the solubility and stability of the recombinant protein in the *E. coli* expression system, providing a reliable basis for the subsequent efficient preparation of immunogens. The gene sequence corresponding to this truncated variant was chemically synthesized by Anshengda Biotechnology Co., Ltd., and successfully cloned into the pET-32a(+) vector. After enzyme digestion and sequencing verification, the pET-32a-gE (52-260 aa) recombinant expression plasmid was finally constructed.

[0020] 2. Expression and purification of gE (52-260 aa) protein After amplification and culture of the identified positive BL21(DE3) clone containing the pET-32a-gE (52-260 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 22 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.

[0021] The target protein (gE (52-260 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 supernatant containing the lysate of the gE (52-260 aa) recombinant protein was added to the column, and the permeate was collected and loaded again. Unbound proteins were washed away with binding buffer, followed by washing with 20 mL of washing buffer and elution with 250 mM imidazole elution buffer. The purified recombinant protein was digested with enterokinase to remove the fusion tag. Nickel column affinity chromatography was performed again to purify the recombinant protein. SDS-PAGE electrophoresis was used to detect the digestion and purification efficiency. Figure 1 As shown, the gE (52-260 aa) recombinant protein obtained by affinity chromatography had high purity. The concentration of the purified gE (52-260 aa) recombinant protein was determined by BCA method.

[0022] Example 2: Establishment of a cell line containing gE (52-260 aa) recombinant protein monoclonal antibody This embodiment prepared a gE (52-260 aa) recombinant protein monoclonal antibody cell line. The specific preparation method includes the following steps: 1. Mouse immunization The recombinant gE (52-260 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.

[0023] 2. Mouse serum titer detection (1) Antigen coating: The gE (52-260 aa) recombinant protein obtained in Example 1 was adjusted to 1 μg / mL as the coating agent. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0024] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0025] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37℃ for 1 h.

[0026] (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.

[0027] (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.

[0028] (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.

[0029] (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 2As shown, the reaction of recombinant gE (52-260 aa) protein with immune serum was identified by indirect ELISA, and the titer of mouse serum was 1:64000.

[0030] 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 at 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.

[0031] 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.

[0032] 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 96-well plates at 100 μL per well, 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. If the positive rate reached 100%, the culture was expanded, and the cell line was preserved and labeled as gE(52-42).

[0033] Example 3: Preparation and identification of monoclonal antibodies against gE (52-260 aa) protein In this embodiment, a monoclonal antibody against the gE (52-260 aa) protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation and titer determination of ascites fluid containing gE (52-260 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 gE (52-42) 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, 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.

[0034] 2. Purification of monoclonal antibodies Ascites fluid was collected, and gE(52-42) 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 impurities. 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 gE(52-42) monoclonal antibodies were obtained.

[0035] 3. Identification of Monoclonal Antibody Types and Subclasses The mouse subtype identification kit was used for the experimental procedure. The gE(52-42) monoclonal antibody was identified as IgG1 and kappa light chain.

[0036] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody (i.e., the gE(52-42) monoclonal antibody purified in step 2 of Example 3) produced by the cell line gE(52-42) preserved in Example 2. The results are as follows: The nucleotide sequence (SEQ ID NO: 1) of the heavy chain variable region of the gE(52-42) monoclonal antibody is: 5'-GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGGCATGTCTTGGATTCGCCAGACTCCAGACAAGAGGCTGGAATGGGTCGCAACCGTTAGTACTGATGGTTATTATATCTACTATTCAGACACTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGTCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGGCATTTATTACTGTTCAAGACATGGACGTAACTACGCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3', The corresponding amino acid sequence (SEQ ID NO: 2) is: EVQLVESGGDLVKPGGSLKLSCAASGFTFSTYGMSWIRQTPDKRLEWVATVSTDGYYIYYSDTVKGRFTISRDNVKNTLYLQMSSLRSEDTGIYYCSRHGRNYAFDYWGQGTTLTVSS; The nucleotide sequence (SEQ ID NO: 3) of the light chain variable region is: 5'-GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAACATCAGGTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCTAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAACAGAGTGGAGGCTGAGGATGTGGGTGTTTATTATTGTGCTCAAAATCTAGAACTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3', The corresponding amino acid sequence (SEQ ID NO: 4) is: DIVMTQAAFSNPVTLGTSGSISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRINRVEAEDVGVYYCAQNLELWTFGGGTKLEIK.

[0037] 4. Specificity detection of monoclonal antibodies The following proteins were used as antigens: porcine pseudorabies virus gE (52-260 aa) protein (gE-52-260), porcine pseudorabies virus gB (59-337 aa) protein (gB-59-337), porcine pseudorabies virus gB (507-734 aa) protein (gB-507-734), 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.

[0038] The ELISA plate was coated under the same conditions, using the purified gE (52-42) 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. 450 The 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 gE(52-42) preserved in Example 2 (i.e. the gE(52-42) 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 gE protein in the sample to be tested.

Claims

1. An antibody or its antigen-binding fragment that specifically recognizes amino acid fragments 52-260 of the porcine pseudorabies virus gE protein, 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 according to any one of claims 1 to 3, or the detection reagent according to claim 5 or 6, in the preparation of articles for diagnosing porcine pseudorabies.

8. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3 in the study of the biological function or pathogenic mechanism of porcine pseudorabies virus gE protein for non-diagnostic purposes.

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