Monoclonal antibody binding to feline herpesvirus-gd protein and its use as a diagnostic marker

Abstract

The application belongs to the technical field of biology and specifically relates to a cat herpes virus-gD protein combined monoclonal antibody and application thereof as a diagnostic marker. The application provides a variable region sequence specifically combined with a cat herpes virus gD protein, and the variable region sequence is a variable region sequence of a mouse monoclonal antibody 3D1-F5-F8 or a variable region sequence of a mouse monoclonal antibody 7G12. The application of the antibody as the diagnostic marker in the preparation of a detection reagent for quantitatively detecting the expression amount of the cat herpes virus gD protein can improve the accuracy and sensitivity of virus detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to monoclonal antibodies that bind to feline herpesvirus-gD protein and their application as diagnostic markers. Background Technology

[0002] Feline herpesvirus (FHV-1) is a large virus, 100-130 nm in diameter, with a capped and double-stranded DNA. It replicates within the cell nucleus, forming intranuclear inclusion bodies. Also known as viral rhinobronchitis, feline herpesvirus is widespread globally. It replicates in the conjunctival and upper respiratory tract epithelial cells, as well as in neurons; infection with neurons can lead to lifelong latent infection. The virus enters the cat's body through the mouth, nose, and conjunctiva, causing lytic infection of the nasal mucosal epithelial cells, and then spreads to the conjunctiva, pharynx, trachea, bronchi, and small bronchi. Lesions are characterized by localized, multiple necrotic (neutrophilic infiltration) and inflammatory lesions. Transient viremia has been reported, especially in newborns or hypothermic kittens, as feline herpesvirus replicates at lower temperatures. Viral shedding begins 24 hours after infection and continues for 1-3 weeks. Acute symptoms usually subside within 10-14 days, but some cases develop into chronic lesions in the upper respiratory tract and eyes. The virus spreads along sensory nerves and reaches neurons, particularly in the trigeminal ganglion, which is the primary site of latent infection. Almost all infected cats become lifelong carriers. Currently, there is no simple test to detect this latent state because the viral genome resides in the nucleus of infected neurons and does not replicate. Approximately 70% of infected cats can be reactivated and shedding the virus through steroid administration. Other stressors that may induce viral reactivation include lactation (40%) and environmental changes (18%). Conjunctivitis may be accompanied by corneal ulcers, potentially leading to chronic corneal osteonecrosis. Stromal keratitis is a secondary immune-mediated response because the virus is present in the corneal epithelial cells or stroma. If acute cases cause damage to the nasal turbinate bones, it can easily lead to chronic rhinitis in some cats.

[0003] However, current methods for the prevention and diagnosis of FHV-1 remain significantly inadequate. In terms of treatment, clinical practice primarily relies on broad-spectrum antiviral drugs such as famciclovir. These drugs have limited efficacy and potential toxic side effects, failing to meet the clinical demand for highly effective and specific treatment regimens. Regarding diagnosis, although some molecular biological detection methods such as PCR exist, they are complex to operate and require sophisticated equipment, making them unsuitable for point-of-care testing (POCT). Immunological detection methods (such as ELISA and colloidal gold test strips) are ideal choices due to their speed and convenience; however, core issues, such as the lack of high-affinity and high-specificity monoclonal antibodies against key FHV-1 antigens, limit the development and application of related diagnostic products.

[0004] The gD protein plays a crucial role in FHV-1 infection of host cells. This protein mediates the fusion of the viral envelope and cell membrane by specifically recognizing receptors on the host cell surface (such as adhesion molecules like nectin-1), making it a key protein essential for viral invasion. More importantly, the region of the gD protein exposed on the viral surface contains abundant linear and conformational epitopes, exhibiting strong immunogenicity and serving as a primary target for inducing the production of viral neutralizing antibodies in the host. Among all FHV-1 envelope glycoproteins, the gD protein is considered the optimal antigenic target for FHV-1 detection due to its high conservation and low mutation rate. Neutralizing antibodies against the gD protein can directly inhibit viral adsorption and invasion by blocking the binding of gD to host cell receptors. Therefore, the development of high-performance monoclonal antibodies that specifically recognize the gD protein is of significant practical and clinical value for establishing accurate FHV-1 antigen detection methods and exploring novel antibody therapy strategies.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a monoclonal antibody that binds to the feline herpesvirus-gD protein and its application as a diagnostic marker.

[0007] The first aspect of this invention provides a variable region sequence that specifically binds to the feline herpesvirus gD protein. The variable region sequence is the variable region sequence of mouse monoclonal antibody 3D1-F5-F8, including: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region amino acid sequence as shown in SEQ ID NO:4; or the variable region sequence is the variable region sequence of mouse monoclonal antibody 7G12, including: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6 and the light chain variable region amino acid sequence as shown in SEQ ID NO:8.

[0008] A second aspect of the present invention provides an antibody that binds to the feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region amino acid sequence as shown in SEQ ID NO:4.

[0009] A third aspect of the present invention provides an antibody that binds to the feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6 and the light chain variable region amino acid sequence as shown in SEQ ID NO:8.

[0010] Optionally, the antibody mentioned above is a monoclonal antibody or a genetically engineered antibody; the genetically engineered antibody is selected from one of the following: single-chain antibody, single-chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody, modified monoclonal antibody fragment, mouse monoclonal antibody, and mouse monoclonal antibody fragment.

[0011] Optionally, the antibody is a mouse monoclonal antibody 3D1-F5-F8 or 7G12.

[0012] The fourth aspect of this invention proposes the application of the above-mentioned antibody as a diagnostic marker in the preparation of a detection reagent for quantitative detection of feline herpesvirus gD protein expression.

[0013] The fifth aspect of this invention provides a kit for detecting feline herpesvirus, comprising the aforementioned antibody, detection reagent, and FHV-1-gD protein standard.

[0014] Optionally, one of the mouse monoclonal antibodies 3D1-F5-F8 or 7G12 is coated on a microplate, and the other is labeled; the labeling markers include enzymes, fluorescent groups or chemiluminescent groups.

[0015] Optionally, the detection reagent includes a substrate that reacts with the label in color; preferably, the detection reagent includes an enzyme chromogenic reagent, a fluorescent reagent, or a chemiluminescent reagent.

[0016] The sixth aspect of the present invention provides a nucleotide sequence encoding the above-mentioned variable region sequence, the nucleotide sequence comprising the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:3; or the nucleotide sequence comprising the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7.

[0017] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0018] This invention uses FHV-1-gD protein as an immunogen to immunize BALB / c mice. Post-immunization, tail blood titers were evaluated by indirect ELISA, reaching 1:50,000. Mouse spleen cells and mouse myeloma cells SP2 / 0 were fused under PEG conditions. After multiple rounds of screening and confirmation, further expansion culture was conducted to verify positive clones, yielding multiple highly sensitive monoclonal antibodies. Then, pairwise pairing tests were performed on multiple mouse monoclonal antibodies to detect antigen proteins. The antibody pairs with the best pairing performance were obtained, and specificity tests showed that the paired antibodies could effectively detect antigen proteins, viral stock, and viral stock with added lysis buffer. Therefore, the above antibodies, as diagnostic markers, can improve the accuracy and sensitivity of viral detection when used in the preparation of reagents for quantitative detection of feline herpesvirus gD protein expression. Attached Figure Description

[0019] Figure 1 The results of the initial cloning screening;

[0020] Figure 2 This is the result of the second monoclonal antibody screening. Detailed Implementation

[0021] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0022] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0023] As used herein, the term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length, whose amino acid sequence corresponds to the heavy chain amino acid sequence of the monoclonal antibody starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length, whose amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody starting from the N-terminal amino acid of the light chain. Those skilled in the art will readily recognize that, based on the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody specifically disclosed in this invention, one or more amino acids can be added, deleted, or substituted using conventional genetic engineering and protein engineering methods to obtain conserved variants that still maintain specific binding to feline herpesvirus. The monoclonal antibody in this invention also includes its active fragment or conserved variant.

[0024] The term "conservative variant" refers to a variant that essentially retains the characteristics of its parent polypeptide, such as basic immunobiological, structural, regulatory, or biochemical properties. Generally, the amino acid sequence of a conservative variant of a polypeptide differs from that of the parent polypeptide, but the difference is limited, ensuring that the sequence of the parent polypeptide and the conservative variant is generally very similar and identical in many regions. The differences in the amino acid sequence between the conservative variant and the parent polypeptide can be substitutions, additions, and deletions of one or more amino acid residues or any combination thereof. The substituted or inserted amino acid residues may or may not be encoded by the genetic code. Conservative variants of polypeptides can arise naturally or unnaturally. Unnaturally occurring conservative variants of polypeptides can be produced through mutagenesis or direct synthesis.

[0025] In a first aspect, embodiments of the present invention provide a variable region sequence that specifically binds to the feline herpesvirus gD protein. The variable region sequence is the variable region sequence of mouse monoclonal antibody 3D1-F5-F8, comprising: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 or a conserved variant of sequence SEQ ID NO:2 obtained by adding, deleting, substituting or modifying one or more amino acids; and the light chain variable region amino acid sequence as shown in SEQ ID NO:4 or a conserved variant of sequence SEQ ID NO:4 obtained by adding, deleting, substituting or modifying one or more amino acids.

[0026] The variable region sequence may be the variable region sequence of mouse monoclonal antibody 7G12, including: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6 or a conserved variant of sequence SEQ ID NO:6 obtained by adding, deleting, substituting or modifying one or more amino acids; the light chain variable region amino acid sequence as shown in SEQ ID NO:8 or a conserved variant of sequence SEQ ID NO:8 obtained by adding, deleting, substituting or modifying one or more amino acids.

[0027] Secondly, embodiments of the present invention provide an antibody that binds to the feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region amino acid sequence as shown in SEQ ID NO:4. The antibody is a monoclonal antibody or a genetically engineered antibody; the genetically engineered antibody is selected from one of the following: single-chain antibody, single-chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody, modified monoclonal antibody fragment, mouse monoclonal antibody, and mouse monoclonal antibody fragment. Specifically, the antibody is mouse monoclonal antibody 3D1-F5-F8. The ELISA titer detection sensitivity of mouse monoclonal antibody 3D1-F5-F8 exceeds 0.005 μg / mL, and the subtype is IgG1,κ subtype.

[0028] Thirdly, embodiments of the present invention provide an antibody that binds to the feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6 and the light chain variable region amino acid sequence as shown in SEQ ID NO:8. The antibody is a monoclonal antibody or a genetically engineered antibody; the genetically engineered antibody is selected from one of the following: single-chain antibody, single-chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody, modified monoclonal antibody fragment, mouse monoclonal antibody, and mouse monoclonal antibody fragment. Specifically, the antibody is mouse monoclonal antibody 7G12. The ELISA titer detection sensitivity of mouse monoclonal antibody 7G12 reaches 0.5 μg / mL, and the subtype is IgG1,κ subtype.

[0029] The "monoclonal antibody" used in this invention can also be prepared using hybridoma methods. Because the DNA sequence encoding the murine antibody of this invention can be obtained using conventional methods well known to those skilled in the art, such as artificial synthesis or PCR amplification of the amino acid sequence disclosed in this invention, recombinant DNA methods can also be used, and the sequence can be ligated into a suitable expression vector using various methods well known in the art. Finally, under conditions suitable for antibody expression of this invention, the transformed host cells are cultured, and then those skilled in the art purify them using well-known conventional separation and purification methods to obtain the monoclonal antibody of this invention.

[0030] Fourthly, embodiments of the present invention provide the application of the above-mentioned antibody in the preparation of a detection reagent for quantitative detection of feline herpesvirus gD protein expression.

[0031] Fifthly, embodiments of the present invention provide a kit for detecting feline herpesvirus, comprising the aforementioned antibody, detection reagent, and FHV-1-gD protein standard. Optionally, one of the aforementioned mouse monoclonal antibodies 3D1-F5-F8 or 7G12 is coated onto a microplate, and the other is labeled; the label includes an enzyme, a fluorescent group, or a chemiluminescent group. The detection reagent includes a substrate that reacts with the label; preferably, the detection reagent includes an enzyme chromogenic reagent, a fluorescent reagent, or a chemiluminescent reagent. Specificity verification is performed on the antigen protein, stock solution, lysis buffer, and negative control, respectively, to identify the antibody pair 3D1-F5-F8 and Biotin-7G12 with the best virus recognition effect.

[0032] Fifthly, embodiments of the present invention provide a nucleotide sequence encoding a variable region sequence as claimed in claim 1, wherein the nucleotide sequence comprises the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:3; or the nucleotide sequence comprises the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7.

[0033] To make the present invention easier to understand, the present invention will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0034] Example 1

[0035] 1. Immunogen preparation

[0036] The antigen protein FHV-1-gD was mixed with Freund's complete adjuvant and emulsified to prepare immunogen A. The antigen protein was mixed with AD adjuvant to prepare immunogen B, which was then prepared for use.

[0037] 2. Evaluation of mouse immunity and immune response using ELISA

[0038] For the first mouse immunization, three mice were immunized using the immunogen prepared in step 1. The mouse immunization procedure was as follows: for the initial immunization, mice were immunized with immunogen A and immunogen B respectively; for the first booster immunization, immunogen B was used; for the second booster immunization, immunogen B was used; for the third booster immunization, immunogen A and immunogen B were used; and for the fourth booster immunization, immunogen B was used. The immunization sites were the subcutaneous tissue on the back and the muscle on the leg.

[0039] Following immunization, mouse tail blood was collected and indirect ELISA was performed. The plate was coated with antigen protein (1 µg / mL), with 100 µL added to each well and incubated overnight at 4°C. The plate was washed three times with PBS, blocked with 5% milk-PBS at room temperature for 1 hour, and then washed once with PBS. Serially diluted mouse tail blood (1 / 500, 1 / 1000, 1 / 5000, 1 / 10000, and 1 / 50000) were added to the ELISA plate and incubated for 1 hour at room temperature. The plate was then washed three times with PBS, patted dry, and incubated with 1:2000 diluted HRP-labeled goat anti-mouse IgG (Fc) secondary antibody for 1 hour at room temperature. After washing five times with PBS, the plate was patted dry, and TMB was added for color development. The plate was incubated for 20 minutes in the dark at room temperature. Finally, 50 µL of stop solution was added, mixed, and the OD was read using a microplate reader. 450 The tail blood was evaluated using the value. The results are shown in Table 1.

[0040] Table 1: Results of ELISA evaluation of mouse tail blood

[0041]

[0042] As can be seen from the tail blood evaluation results in Table 1, the antibody titer of mouse tail blood in recognizing antigen proteins can reach 1 / 50,000, which can be further used for cell fusion and clone screening.

[0043] 2. Cell fusion and clone screening

[0044] Based on the ELISA evaluation results of the tail blood in step 2, mouse #3 with the highest tail blood antibody detection value was selected for cell fusion. Mouse spleen cells were fused with myeloma SP2 / 0 cells. (Well-grown SP2 / 0 cells were pipetted and centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 30 mL of preheated DMEM medium. After centrifugation at 1500 rpm for 3 min, the supernatant was discarded, and an appropriate amount of preheated DMEM medium was added to resuspend the cells and count them. The appropriate cell amounts were taken at a ratio of 10:1 for mouse spleen cells and SP2 / 0 cells, mixed thoroughly in a 50 mL centrifuge tube, and centrifuged at 1500 rpm for 3 min. The supernatant was discarded. Then, 1 mL of preheated PEG was slowly added, and the mixture was incubated at 37 ℃ for 25 min. 5 mL of preheated DMEM medium containing 10% fetal bovine serum was slowly added dropwise along the tube wall, and then the speed was gradually increased, adding 15 mL of preheated DMEM medium containing 10% fetal bovine serum, finally adding to a total of 40 mL. After centrifugation at 1200 rpm for 5 min, the supernatant was discarded, and 1 mL of preheated DMEM medium containing 10% fetal bovine serum was added.) The suspension was uniformly dissolved in mL of recovery culture medium.

[0045] Then, 0.5 mL of fusion cells were added to 80 mL of DMEM medium containing HAT and 10% fetal bovine serum. After mixing, the mixture was added to four 96-well cell culture plates (1#-4#), with 200 µL of cell suspension added to each well. The plates were incubated at 37°C and 5% CO2 for 8 days. Then, 50 µL of cell culture supernatant was taken, diluted 1 / 2 with 5% milk-PBS, and added to an ELISA plate coated with antigen protein for clonal screening. The experimental results are as follows. Figure 1 As shown in Table 2.

[0046] Table 2: Initial Cloning Screening

[0047]

[0048] Note: H11 wells of each plate are negative controls, diluted with 5% milk-PBS; H12 wells of each plate are positive controls, with mice #3 bled at the end and diluted 1:500.

[0049] From the initial clonal screening results shown in Table 2, 23 clones with high OD values ​​(greater than 3.0) were selected. After adding culture medium again and culturing for 2 days, the cell culture supernatant of positive clones was screened again to confirm whether the first screening was a false positive reaction. The experimental results are shown in Table 3.

[0050] Table 3: Rescreening of positive clones

[0051]

[0052] Note: NC is the negative control, 5% milk-PBS; PC is the positive control, serum was released from mice #3 at the end, 1 / 500.

[0053] Based on the results in Table 3, 13 polyclonal positive cells with an OD value greater than 1.0 were selected after secondary screening. The cell culture supernatant was then confirmed again by ELISA. The experimental results are shown in Table 4.

[0054] Table 4: Confirmation of positive clones

[0055]

[0056] Note: NC is the negative control, 5% milk-PBS; PC is the positive control, serum was released from mice #3 at the end, 1 / 500.

[0057] Based on the results in Table 4, eight polyclonal cell lines (1A10, 1F2, 1F9, 2E4, 3D1, 3H10, 4F3, and 4F12) were selected for the first subcloning of single cells. The experimental results are shown in Table 5.

[0058] Table 5: First Subcloning Screening

[0059]

[0060] Note: The last well of each subclone is a positive control well, containing the original cell culture supernatant.

[0061] Based on the first subcloning results in Tables 2-5, three positive clones were obtained (numbered according to the cell culture plate number, not the detection plate number). Among them, 3H10-H10 were monoclonal cells and were directly used for antibody preparation. The other two, 3D1-F5 and 2E4-H5, were polyclonal positive cell wells and underwent a second subcloning screening. The experimental results are shown in Table 6.

[0062] Table 6: Second Subcloning Screening

[0063]

[0064] Note: The last well of each subclone is a positive control well, containing the original cell culture supernatant.

[0065] Based on the subcloning results in Table 6, two monoclonal antibody cell lines, 3D1-F5-G7 and 3D1-F5-F8, were obtained from 3D1-F5; no strong positive clones were obtained from 2E4, so it was discarded.

[0066] In summary, the first cloning yielded two monoclonal cell lines, 3H10-H10 and 3D1-F5-F8 / G7.

[0067] The other half of the fused cells were added to 30 mL of recovery medium and cultured for 48 hours before being transferred to selective semi-solid selection medium. After 10 days of culture, 564 monoclonal cell lines were selected and transferred to 96-well plates for further culture (6 plates in total, #7-10). After 7 days of culture in the 96-well plates, the supernatant was collected for analysis. Cell selection was performed using an indirect ELISA method (steps as above) on the cell culture supernatant (1:1 dilution) in the 96-well plates. The results are as follows. Figure 2 As shown.

[0068] Table 7-1: Second Cloning Screening

[0069]

[0070] Table 7-2: Second Cloning Screening

[0071]

[0072] Note: Well H11 of each plate is a negative control, diluted with 5% milk-PBS; Well H12 of each plate is a positive control, with mice bled at the end and diluted 1:500.

[0073] The OD450 of the culture supernatant of positive clones was significantly higher than that of the NC control, yielding 14 relatively positive clones. These 14 positive hybridoma cells were transferred and cultured in 48-well plates. After culturing for another 2 days, the cell supernatant (diluted 1:1) was collected for further screening and verification using an indirect ELISA method. The experimental results are shown in Table 8.

[0074] Table 8: Validation of Positive Clones Through Rescreening

[0075]

[0076] Note: NC is the negative control, diluted with 5% milk-PBS; PC is the positive control, with mice bled at the end and diluted 1:500.

[0077] From the rescreening results in Table 2-8, three positive clones with specific recognition of the antigen (OD value greater than 0.5) were obtained: 5B1, 7G12, and 8D2. After expanded culture, the cell culture supernatant of the positive clones was further confirmed. The experimental results are shown in Table 9.

[0078] Table 9: Confirmation of Positive Clones

[0079]

[0080] Note: NC is the negative control, diluted with 5% milk-PBS; PC is the positive control, with mice bled at the end and diluted 1:500.

[0081] As can be seen from the results in Table 9, two monoclonal cell lines, 5B1 and 7G12, were obtained in the second screening.

[0082] Therefore, after two clonal screenings, a total of four monoclonal cell lines were obtained, namely 3H10-H10, 3D1-F5-F8 / G7, 5B1 and 7G12. Next, ascites fluid production and antibody preparation were carried out on the four clones.

[0083] Example 2: Preparation of mouse monoclonal antibodies

[0084] The four positive clones obtained in Example 1 were further prepared into ascites fluid, and approximately 10 μL of each was used to prepare ascites fluid. 7 One cell was injected into the peritoneal cavity of two BALB / c mice that had been pre-injected with IFA adjuvant. Ten days later, ascites fluid was extracted from each positive clone and centrifuged at 4°C and 12,000 rpm for 15 min. The supernatant was collected for further Protein G purification.

[0085] Add 1 mL of G protein-conjugated column material to an empty column. After washing with PBS, dilute an appropriate amount of ascites fluid with PBS and load it onto the column. Then, reload the column with the eluent. Elute with glycine elution buffer (pH 2.7). Collect 1 mL of eluent per tube (pre-mixed with 100 µL of neutralization buffer), for a total of 5 tubes. Then, perform OD analysis on each tube of eluent. 280 Reading, OD 280 The eluent with a concentration greater than 0.5 was mixed, and the OD of the mixture was measured again. 280 Antibody concentration was calculated based on the average extinction coefficient of IgG of 1.4. Antibody concentration = OD 280 / 1.4. Among them, four positive clones yielded purified antibodies, and the antibody information is shown in Table 10.

[0086] Table 10: Information on mouse monoclonal antibodies

[0087]

[0088] The antibodies were evaluated by ELISA, and the results are shown in Table 11.

[0089] Table 11: ELISA evaluation of purified antibodies

[0090]

[0091] Note: NC is the negative control, which is a diluted solution of 5% milk-PBS.

[0092] The test results show that the mouse monoclonal antibody 3D1-F5-F8 has the highest detection sensitivity, exceeding 0.005 μg / mL, followed by 3H10-H10 at 0.05 μg / mL, and 7G12 at 0.5 μg / mL. 5B1 showed a low detection OD value at an antibody concentration of 1 μg / mL, indicating that this antibody strain has too weak an affinity and was therefore discarded.

[0093] Antibody subtypes of the three mouse monoclonal antibodies were detected, and the results are shown in Table 12.

[0094] Table 12: Antibody Subtype Detection

[0095]

[0096] The experimental results show that the three mouse monoclonal antibody subtypes are IgG1,κ subtypes.

[0097] Example 3 Selection of Paired Antibodies

[0098] First, the mouse monoclonal antibodies 3H10-H10 and 7G12 were biotin-labeled. The mouse monoclonal antibodies were first dialyzed in PBS to change the medium, and then biotin was added at a molecular ratio of 20:1. After reacting for 2 hours, the free biotin molecules in the reaction solution were removed using a desalting column to obtain biotin-labeled mouse monoclonal antibodies. The labeled antibodies were detected by ELISA, and the results are shown in Table 13.

[0099] Table 13: Detection of Biotin-Labeled Antibodies

[0100]

[0101] The results show that the biotin-labeled mouse monoclonal antibody can still bind well to the antigen protein, indicating that the biotin labeling was successful and can be used for paired detection.

[0102] Then, three mouse monoclonal antibodies (3H10-H10, 3D1-F5-F8 and 7G12) were used as coating antibodies, and two biotin-labeled mouse monoclonal antibodies (3H10-H10 and 7G12) were used as detection antibodies. The antigen proteins were detected by pairwise pairwise detection, and the results are shown in Table 14.

[0103] Table 14: Antibody Pairing Detection

[0104]

[0105] The results showed that all four pairings could detect the antigen protein at different gradients. The best pairing was 7G12 coating antibody and 3H10-H10 detection antibody; the next best was 3D1-F5-F8 coating antibody and 7G12 detection antibody. Then, the coating antibody concentration of these two pairs was increased to 20 μg / mL, and specificity was verified for the antigen protein, stock solution, lysis buffer, and negative control, respectively. The results are shown in Table 15.

[0106] Table 15: Specificity Detection

[0107]

[0108] The results show that both pairs can specifically detect antigen proteins, viral stock, and viral stock with added lysis buffer, meeting the project requirements. Among them, the antibody pair 3D1-F5-F8 and Biotin-7G12 showed the best virus recognition effect.

[0109] Example 4 Antibody Variable Region

[0110] 1. RNA extraction from hybridoma cells

[0111] 1) Collect 3D1-F5-F8 and 7G12 clone hybridoma cells, approximately 1×10⁻⁶ cells per cell. 7 .

[0112] 2) Add an appropriate amount of RLT buffer and pipette the cells until they are completely dissolved.

[0113] 3) Add an equal volume of 70% ethanol to RLT and mix thoroughly with a pipette tip.

[0114] 4) Place the RNeasy centrifuge column in a 2 mL collection tube, take the maximum amount of 700 µL sample and put it into the nucleic acid purification column, centrifuge at 12000 rpm for 5 min, and discard the waste liquid.

[0115] 5) Add 700 µL of RW1 solution to the nucleic acid purification column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0116] 6) Add 500 µL of RPE solution to the nucleic acid purification column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0117] 7) Repeat step 6 once.

[0118] 8) Place the nucleic acid purification column into a new 2 mL collection tube and centrifuge at 12,000 rpm for 2 min.

[0119] 9) Place the nucleic acid purification column into a new 1.5 mL centrifuge tube, add 30-50 µL of RNase-free water, centrifuge at 12000 rpm for 1 min, discard the purification column, and collect the RNA.

[0120] The reverse transcription of first-strand cDNA synthesis involves the addition of the following components, as shown in Table 16:

[0121] Table 16:

[0122]

[0123] The total reaction volume was 20 µL. After mixing thoroughly, the mixture was placed in a PCR instrument and incubated at 42°C for 30 min and then at 85°C for 5 s.

[0124] 2. Amplify the target fragment and sequence it:

[0125] The following components are added in this step, as shown in Table 17:

[0126] Table 17:

[0127]

[0128] The total reaction volume was 25 µL. After mixing thoroughly, the mixture was placed in a PCR instrument and the PCR program was set as follows: 94℃, 5 min; 94℃, 30 s; 55℃, 30 s; 72℃, 1 min; 35 cycles; 72℃, 10 min.

[0129] 3. Experimental Results:

[0130] 3D1-F5-F8 variable region sequence: The heavy chain base sequence is shown in SEQ ID NO:1, and the heavy chain amino acid sequence is shown in SEQ ID NO:2. The light chain base sequence is shown in SEQ ID NO:3, and the light chain amino acid sequence is shown in SEQ ID NO:4. The sequences of the heavy chain CDR and light chain CDR are shown in Table 18, and the annotations are shown in Table 19.

[0131] 7G12 variable region sequence: The heavy chain base sequence is shown in SEQ ID NO:5, and the heavy chain amino acid sequence is shown in SEQ ID NO:6. The light chain base sequence is shown in SEQ ID NO:7, and the light chain amino acid sequence is shown in SEQ ID NO:8. The sequences of the heavy chain CDR and light chain CDR are shown in Table 18, and the annotations are shown in Table 20.

[0132] Table 18:

[0133]

[0134] Table 19:

[0135]

[0136] Table 20:

[0137]

[0138] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A variable region sequence that specifically binds to the feline herpesvirus gD protein, characterized in that, The variable region sequence is the variable region sequence of mouse monoclonal antibody 3D1-F5-F8, including: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region amino acid sequence as shown in SEQ ID NO:4; Alternatively, the variable region sequence may be the variable region sequence of mouse monoclonal antibody 7G12, including: the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6, and the light chain variable region amino acid sequence as shown in SEQ ID NO:

8.

2. An antibody that binds to feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region amino acid sequence as shown in SEQ ID NO:

4.

3. An antibody that binds to feline herpesvirus gD protein, comprising the heavy chain variable region amino acid sequence as shown in SEQ ID NO:6 and the light chain variable region amino acid sequence as shown in SEQ ID NO:

8.

4. The antibody according to claim 2 or 3, characterized in that, The antibody is a monoclonal antibody or a genetically engineered antibody; the genetically engineered antibody is selected from one of the following: single-chain antibody, single-chain antibody fragment, chimeric monoclonal antibody, chimeric monoclonal antibody fragment, modified monoclonal antibody, modified monoclonal antibody fragment, mouse monoclonal antibody, and mouse monoclonal antibody fragment.

5. The antibody according to claim 4, characterized in that, The antibody is either mouse monoclonal antibody 3D1-F5-F8 or 7G12.

6. The use of an antibody as described in any one of claims 2 to 5 in the preparation of a detection reagent for quantitative detection of feline herpesvirus gD protein expression.

7. A kit for detecting feline herpesvirus, comprising the antibody, detection reagent, and FHV-1-gD protein standard as described in any one of claims 2 to 5.

8. The reagent kit according to claim 7, characterized in that, One of the mouse monoclonal antibodies 3D1-F5-F8 or 7G12 is coated on a microplate, and the other is labeled; the label includes an enzyme, a fluorescent group, or a chemiluminescent group.

9. The reagent kit according to claim 8, characterized in that, The detection reagent includes a substrate that reacts with the marker in a color; preferably, the detection reagent includes an enzyme chromogenic reagent, a fluorescent reagent, or a chemiluminescent reagent.

10. A nucleotide sequence encoding the variable region sequence as described in claim 1, characterized in that, The nucleotide sequences include those shown in SEQ ID NO:1 and SEQ ID NO:3; Or the nucleotide sequence may include the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7.

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