A hybridoma cell strain secreting anti-swine herpes virus type 1 gE monoclonal antibody, monoclonal antibody thereof and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a hybridoma cell strain secreting a monoclonal antibody against gE protein of swine herpes virus 1 (SuHV-1), the monoclonal antibody and application thereof. The hybridoma cell strain secreting the monoclonal antibody against gE protein of swine herpes virus 1 (SuHV-1) is characterized in that the hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 46719. The application utilizes the monoclonal antibody secreted by the hybridoma cell strain secreting the monoclonal antibody against gE protein of swine herpes virus 1 (SuHV-1), and provides a full-automatic magnetic microparticle chemiluminescence detection kit, solves the problems existing in the prior art, and realizes full-automatic rapid detection of pseudorabies virus gE antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hybridoma cell line that secretes a monoclonal antibody against porcine herpesvirus type 1 (SuHV-1) gE protein, its monoclonal antibody, and its application. Background Technology

[0002] Pseudorabies (PR) is an acute infectious disease caused by porcine herpesvirus 1 (SuHV-1), also known as pseudorabies virus (PRV). SuHV-1 belongs to the Herpesviridae family, Alphaherpesvirinae subfamily. The gE gene is a virulence-related gene of SuHV-1, encoding a glycoprotein E (gE). This glycoprotein is non-essential; the deletion of the gE gene does not affect viral replication or immunogenicity, but it significantly weakens viral virulence. Therefore, the gE glycoprotein can serve as a marker protein to distinguish between vaccine-immunized pigs and wild-type infected pigs. Since 2011, PR has experienced explosive outbreaks in pig farms in my country, causing severe economic losses to the pig industry. Therefore, establishing rapid and effective diagnostic and detection methods is imperative.

[0003] Existing hybridoma cell lines have low antibody yield and poor passage stability. Furthermore, the monoclonal antibodies secreted by these cells have varying binding abilities to different subtypes of porcine herpesvirus type 1 due to differences in immunogens, resulting in reduced sensitivity and a risk of missed detection.

[0004] Common methods for detecting porcine herpesvirus type 1 gE antibody include enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and indirect immunofluorescence assay (IFA). ELISA carries risks of cross-reactivity, has limited sensitivity, and is time-consuming (typically 1-2 hours). WB is cumbersome, time-consuming (3-6 hours), has low throughput, and requires highly skilled operators. IFA relies on fluorescence microscopy, has high equipment costs, limits its application at the grassroots level, and is prone to subjective interpretation and human error. Furthermore, all of these methods depend on manual operation and cannot achieve fully automated detection. Summary of the Invention

[0005] The present invention aims to provide a hybridoma cell line that can stably secrete high-yield, high-affinity anti-swine herpesvirus type 1 gE monoclonal antibody, overcoming the technical defects of low antibody yield and poor passage stability of existing hybridoma cell lines, and also overcoming the technical defects of large differences in the binding ability of monoclonal antibodies secreted by existing hybridoma cell lines to different subtypes of swine herpesvirus type 1 and reduced sensitivity.

[0006] To solve the above problems,

[0007] In a first aspect, the present invention provides a hybridoma cell line capable of secreting a mouse monoclonal antibody against porcine herpesvirus type 1 gE protein, which is classified and named hybridoma cell line 2C4.

[0008] The hybridoma cell line was deposited on November 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46719.

[0009] Secondly, the present invention provides a mouse anti-porcine herpesvirus type 1 gE monoclonal antibody, which is secreted by the above-mentioned hybridoma cell line.

[0010] Thirdly, the present invention provides the application of the above-mentioned hybridoma cell line monoclonal antibody in the preparation of products for diagnosing porcine pseudorabies.

[0011] Fourthly, the present invention provides a kit for detecting porcine herpesvirus type 1 gE antibody, which comprises the above-mentioned monoclonal antibody.

[0012] Furthermore, the kit is an immunoassay kit based on the principle of competition.

[0013] Furthermore, the kit is a competition-based enzyme-linked immunosorbent assay (ELISA) kit, comprising:

[0014] (a) A solid-phase carrier coated with porcine herpesvirus type 1 gE protein or its antigenic epitopes;

[0015] (b) Enzyme-labeled porcine herpesvirus type 1 gE protein or its antigenic epitopes;

[0016] (c) Substrate solution used for colorimetric reaction.

[0017] Furthermore, the kit is a magnetic particle chemiluminescent immunoassay kit, which comprises:

[0018] (a) Magnetic microparticles coated or coupled with streptavidin;

[0019] (b) Biotin-labeled monoclonal antibodies;

[0020] (c) Chemiluminescent material-labeled porcine herpesvirus type 1 gE protein or its antigenic epitope.

[0021] Fifthly, the present invention provides a method for detecting porcine herpesvirus type 1 gE antibody in a sample, which uses the above-mentioned monoclonal antibody or the above-mentioned kit for detection.

[0022] The beneficial effects of this invention are:

[0023] (1) The hybridoma cell line of the present invention has high antibody yield.

[0024] By optimizing the nucleotide sequence corresponding to the recombinant protein using E. coli preferred codons, the expression level of the recombinant protein is greatly improved. Furthermore, by employing optimized single-cell sorting and stimulation-binding techniques, hybridoma cell clones with the highest monoclonal antibody yield are screened. Under conventional culture conditions, the antibody yield of the hybridoma cell line of this invention can reach 50-100 mg / L, and after optimization, it can reach more than 150 mg / L, which is significantly higher than the antibody yield of similar hybridoma cell lines in the prior art (usually 10-30 mg / L).

[0025] (2) The hybridoma cell line of the present invention has excellent passage stability.

[0026] The hybridoma cell line provided by this invention maintains good growth after 30 consecutive passages, and can stably and efficiently secrete monoclonal antibodies with an antibody titer consistently maintained at 1:10. 6 The above is particularly important. The monoclonal antibodies produced exhibit excellent batch-to-batch and intra-batch consistency, with a batch-to-batch coefficient of variation (CV) of less than 5% and an intra-batch coefficient of variation (CV) of less than 3%. This key advantage significantly solves the technical bottleneck of decreased antibody secretion capacity and unstable yield commonly encountered in existing hybridoma cell lines during long-term passage. Compared to the typical 10%-15% batch-to-batch variation of conventional hybridoma technology products, the batch-to-batch coefficient of variation of the cell line of this invention is reduced by more than 50%, indicating highly stable genetic traits and excellent controllability and reproducibility of the production process. This characteristic ensures that diagnostic kits developed based on this antibody maintain high performance uniformity and reliable test results across different production batches, thus providing a solid technical guarantee for large-scale industrial production and successful commercial application of the product.

[0027] (3) The monoclonal antibody of the present invention has high affinity.

[0028] The heavy chain CDR3 region of the monoclonal antibody of this invention is significantly longer than that of traditional murine antibodies. This longer CDR3 region allows for a more complex three-dimensional structure, enabling a tighter and more precise complementary binding to the antigenic epitope. Therefore, the monoclonal antibody of this invention exhibits extremely high affinity for the SuHV-1 gE antigen, with an equilibrium dissociation constant KD reaching 1 × 10⁻⁶. -11mol / L (10 pM).

[0029] (4) The monoclonal antibody of the present invention has high reproducibility, high purity, high specificity, and no animal-derived components, which can realize rapid screening of monoclonal antibodies.

[0030] This invention utilizes recombinant DNA technology and sequence-defined DNA production to eliminate mutations associated with hybridoma clones, ensuring batch-to-batch consistency. It guarantees consistently high-purity antibody production (≥95%). Produced in a serum-free cell culture system, it is less susceptible to contamination by pathogens or exogenous proteins. Tandem expression of dominant antigenic epitopes enhances the stimulation of the immune system by the target epitope, significantly reducing the difficulty of monoclonal antibody screening. Immunization using the unique multi-epitope fusion protein of SuHV-1 gE focuses the immune response on virus-specific epitopes. The screened antibodies possess longer HCDR3 and unique CDR sequences, forming a highly complementary and precise binding interface. Through rigorous screening and systematic cross-reactivity testing, it ensures that the final monoclonal antibody specifically binds to different subtypes of porcine herpesvirus type 1, exhibiting high specificity.

[0031] (5) The immunoassay method (such as competitive ELISA or magnetic microparticle chemiluminescence assay) based on the mouse anti-swine herpesvirus type 1 gE monoclonal antibody of the present invention shows superior performance in terms of detection sensitivity, specificity and stability compared with existing conventional reagents, realizing fully automated rapid detection of porcine herpesvirus type 1 gE antibody.

[0032] The hybridoma cell line of the present invention, the monoclonal antibody produced therefrom, and the detection kit containing the antibody provide a novel and efficient core tool for the accurate diagnosis and epidemiological surveillance of porcine herpesvirus type 1 infection. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0034] Figure 1 The image of the recombinant plasmid pET28a-SuHV-1-gE in Example 1 of this invention is shown.

[0035] Figure 2 The image of the recombinant plasmid pET28a-SuHV-2-gE in Example 1 of this invention is shown.

[0036] Figure 3 The results of PCR identification of recombinant plasmids pET28a-SuHV-1-gE and pET28a-SuHV-2-gE in Example 1 of this invention;

[0037] Figure 4 This is a diagram showing the SDS-PAGE electrophoresis identification results of Example 1 of the present invention.

[0038] Among them, M1: Trans2K ® DNA Marker; 1: PCR product of empty vector pET28a; 2: PCR product of recombinant plasmid pET28a-SuHV-1-gE; 3: PCR product of recombinant plasmid pET28a-SuHV-2-gE; M2: Protein molecular weight Marker; 4: Protein yield induced by pET28a-SuHV-1-gE expression; 5: Protein yield induced by pET28a-SuHV-2-gE expression. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0040] Based on the embodiments of this invention, all other implementations obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The examples given are for illustrative purposes only and are not intended to limit the scope of this invention.

[0041] The above technical solution will be described in detail below with reference to specific embodiments.

[0042] Example 1: Preparation and Identification of Immunogens

[0043] (1) Preparation of the coding gene: 56 SuHV-1 strains published by NCBI were analyzed, and the highly conserved gE protein (Genbank accession number: MN443975.1) of the representative strain JX18-2 was selected as the target protein region. B-cell epitopes were predicted from the nucleotide sequence of the gE protein using bioinformatics, and linear dominant epitopes were selected as the target gene epitopes for multi-epitope fusion proteins. The nucleotide sequences encoding the dominant epitopes (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3) and the nucleotide sequence encoding the flexible linker peptide were ligated at the DNA level according to the correct reading frame with the dominant epitope sequences 1-2-3 (general formula shown in formula (I)) and 1-3-2 (general formula shown in formula (II)). The nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. The optimized coding gene sequence was obtained according to the codon preference of E. coli. The nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0044] (2) Construction of recombinant plasmids: The optimized coding gene sequences were ligated into the prokaryotic expression vector pET-28a using DNA splicing technology to obtain recombinant plasmids pET28a-SuHV-1-gE and pET28a-SuHV-2-gE, respectively. The recombinant plasmid maps are shown below. Figure 1 and Figure 2 As shown. PCR identification was performed using the universal primers T7 / T7ter for the pET-28a vector, and the results are as follows. Figure 3 As shown.

[0045] (3) Expression of multi-epitope fusion proteins: The successfully ligated recombinant plasmids pET28a-SuHV-1-gE and pET28a-SuHV-2-gE were transformed into BL21(DE3) competent cells. The transformed bacteria were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm for about 4 h until OD was reached. 600 The concentration was 0.6. IPTG was added to a final concentration of 0.2 mmol / L, and the cells were collected after induction at 28℃ and 120 rpm for 12 h.

[0046] (4) Purification of the multi-epitope fusion protein: Crude purification was performed using a nickel ion affinity chromatography column, followed by further purification using a protein G purification column. The purified multi-epitope fusion protein was the porcine herpesvirus type 1 gE protein multi-epitope fusion protein. The amino acid sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. The SDS-PAGE results are shown in... Figure 4 As shown. By Figure 4 It is known that the size of the multi-epitope fusion protein SEQ ID NO.11 expressed by the gene encoding SEQ ID NO.6 is 18 kDa; the size of the multi-epitope fusion protein SEQ ID NO.12 expressed by the gene encoding SEQ ID NO.7 is 18 kDa.

[0047] (5) The purity of the multi-epitope fusion protein was determined by micro-spectrophotometer. The A280 / A260 ratio was 1.8 to 2.0, indicating high protein purity. It can be used for coating, labeling, detection, etc.

[0048] Example 2: Preparation of hybridoma cell lines

[0049] (1) B cell pretreatment:

[0050] Animal Immunization: Three BALB / c mice were immunized four times with purified SuHV-1 gE multi-epitope fusion protein. The immunization dose was 100 μg / mouse, and the route of immunization was intraperitoneal. For the first immunization, the multi-epitope fusion protein was emulsified with an equal volume of Freund's complete adjuvant. For the second and third immunizations, Freund's incomplete adjuvant was used for emulsification. The fourth immunization was a booster immunization three days before fusion, with an immunization dose of 50 μg / mouse, without adjuvant, administered intraperitoneally. After immunization, the spleen of the mice was harvested, ground, and splenic lymphocytes were obtained.

[0051] Spleen cells from immunized mice were first enriched with antigen-specific magnetic beads to obtain antigen-specific B cell populations. Recombinant mouse BAFF (B cell activating factor, final concentration 50 ng / mL) and IL4 (interleukin-4, final concentration 20 ng / mL) were added to the sorting buffer (RPMI-1640 medium containing 10% fetal bovine serum) and pre-cultured at 37°C and 5% CO2 for 12 hours.

[0052] (2) Flow cytometry single-cell sorting

[0053] B cells were labeled with CD19-FITC and antigen-specific B cells were labeled with antigen-Alexa Fluor 647 using a dual-color fluorescent labeling method. Single antigen-specific B cells were sorted into 96-well plates using a BD FACSAria III flow cytometer in "single cell sorting mode" to ensure that only one B cell was in each well. After sorting, BAFF (25 ng / mL) and IL4 (10 ng / mL) were added to the 96-well plates and cultured at 37°C and 5% CO2.

[0054] 24 hours later, pretreated B cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1, and fusion was mediated by PEG4000. The fused cells were then seeded into 96-well plates (containing HAT selection medium). 7-10 days post-fusion, the titer of the multi-epitope fusion protein-specific antibody in the supernatant was detected using indirect ELISA to screen OD cells. 450 Positive wells >1.0.

[0055] (3) Limiting dilution method for subcloning

[0056] First subcloning: Dilute the cells from the initial screening positive wells to a density of 0.5 cells / well in a 96-well plate, culture for 10 days, and then perform ELISA again to record the antibody concentration in each well;

[0057] High-yield strain markers: wells with antibody concentrations >50 mg / L were selected for cell counting and viability detection;

[0058] Second subcloning: The labeled high-yielding cell lines were diluted again at 0.3 cells / well to ensure homozygous monoclonal cells. Finally, three cell lines that stably secreted antibodies with a yield >80 mg / L were selected (named: 2B5, 2C4, and 6D8).

[0059] Stability verification: The selected monoclonal cells were passaged for 30 generations, and the antibody yield was tested at each generation. Cell lines with yield fluctuation of <10% were selected as the final candidate lines.

[0060] Example 3: Identification of hybridoma cell lines

[0061] The obtained hybridoma cell lines were identified as follows:

[0062] Antibody yield assay: Hybridoma cells were prepared at a concentration of 1×10⁻⁶. 5 The cells / mL were seeded in RPMI-1640 medium containing 10% fetal bovine serum. After 5 days of culture, the supernatant was collected, and the antibody was purified by Protein A affinity chromatography. The antibody yield was determined to be 85 mg / L.

[0063] This breakthrough in production volume stems from the following technological innovations:

[0064] By using E. coli-preferred codons to optimize the nucleotide sequence of recombinant proteins, the expression level of recombinant proteins was greatly improved.

[0065] An optimized single-cell sorting and stimulation technique was used, in which BAFF and IL4 were added during the sorting process to promote B cell proliferation and monoclonal antibody secretion. After single-cell sorting stimulation, hybridoma cells with the highest monoclonal antibody production were screened.

[0066] Passage stability test: Hybridoma cells were continuously passaged for 30 generations, and antibody titer was measured at each generation. The results showed that the antibody titer remained at 1:10 after 30 passages. 6 The inter-batch variation coefficient was 3.2%, and the intra-batch variation coefficient was 2.6%.

[0067] This cell line can stably and efficiently secrete monoclonal antibodies against porcine herpesvirus type 1 (SuHV-1) gE protein, overcoming the technical defects of low antibody yield and poor passage stability in existing hybridoma cell lines.

[0068] Affinity assay: The affinity constant of the monoclonal antibody to the SuHV-1 gE antigen was determined using surface plasmon resonance (SPR) technology, with a Ka value of 2.1 × 10⁻⁶. 9 L / moL.

[0069] The most common length of the heavy chain CDR3 (HCDR3) is 8 to 12 amino acids. The heavy chain CDR3 region of the monoclonal antibody of this invention is significantly longer than that of traditional murine antibodies, resulting in a more complex structure. This gives the monoclonal antibody of this invention high affinity, reaching the picomolar level (KD = 10⁻¹² mol / L).

[0070] Specifically, the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody of the present invention are positions 26-35, 51-65, and 96-110 of SEQ ID NO.13, respectively; the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are positions 25-35, 51-57, and 90-98 of SEQ ID NO.14, respectively.

[0071] The monoclonal antibody provided by this invention has a heavy chain variable region CDR3 (HCDR3) with a length of 15 amino acid residues (see positions 96-110 of SEQ ID NO.13), significantly longer than the typical length (8-12 amino acids) of HCDR3 in conventional murine antibodies known in the art. This longer CDR3 region forms a more complex three-dimensional spatial structure, resulting in more complete and stable binding to the antigenic epitope of porcine herpesvirus type 1 gE protein. This directly leads to the antibody of this invention potentially achieving ultra-high affinity at the picomolar level (KD=10⁻¹² mol / L), which is significantly higher than that of conventional murine antibodies (which typically have affinity at the nanomolar level, KD=10⁻¹² mol / L). 9 This is something that cannot be achieved with concentrations of mol / L. Therefore, the structural features of this antibody sequence are key to its unexpected and excellent effects, and are also an important manifestation of the inventiveness of this invention.

[0072] Specificity detection: Western blot and ELISA were used to detect the cross-reactivity of the monoclonal antibody with other porcine viruses. The results showed that there was no cross-reactivity with porcine transmissible gastroenteritis virus, porcine rotavirus, etc., and the cross-reactivity rate was less than 0.1%.

[0073] The above indicators are summarized as follows:

[0074]

[0075] The hybridoma cell lines obtained above were deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46719.

[0076] This hybridoma cell line exhibits excellent passage stability, capable of stable passage for over 30 generations with antibody yields as high as 50-150 mg / L, overcoming the industrialization bottleneck of instability inherent in traditional hybridomas; at its core, its antibody possesses picomolar (Ka10) levels. 11It has an ultra-high affinity (above L / mol), which is 10-10000 times that of traditional technologies.

[0077] Example 4: Preparation of Monoclonal Antibodies

[0078] (1) Preparation of SuHV-1 gE monoclonal antibody: BALB / c mice were pretreated with liquid paraffin via intraperitoneal injection, and then inoculated with hybridoma cells in the logarithmic growth phase 1-2 weeks later. 5-10 days after inoculation, abdominal distension of the mice was observed, and ascites fluid was collected using a 16-gauge needle. Each mouse could be collected 2-3 times consecutively, yielding a total of 5-10 mL of ascites fluid. The ascites fluid was centrifuged (2000 r / min, 5 min) to remove cell debris, preliminarily purified by precipitation with saturated ammonium sulfate, and further purified by ion exchange chromatography to obtain SuHV-1 gE monoclonal antibody with a purity >95%.

[0079] (2) Sequence determination of heavy and light chain variable regions: Hybridoma cells in the logarithmic growth phase were collected and sent to Universal Biotech for sequencing. The amino acid sequences of the heavy chain variable region and light chain variable region of the SuHV-1 gE monoclonal antibody are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0080] Unless otherwise stated, the CDR sequences described in this invention are defined according to the Kabat numbering system.

[0081] The amino acid and nucleotide sequences involved in this invention are used to clearly and completely define the structural characteristics of the monoclonal antibodies necessarily secreted by the hybridoma cell line (CGMCC No. 46719) of this invention, in order to meet the requirements of sufficient disclosure under patent law. The purpose of providing this sequence information is to characterize and identify the biological materials and products of this invention, and it is not itself the subject matter independently claimed in this application.

[0082] Example 5: Biotin labeling of monoclonal antibodies

[0083] Take 1 mg of biotin, bring it to room temperature, add it to 180 μL of ultrapure water, and dissolve and mix well.

[0084] Add 13.5 mg of the prepared SuHV-1 gE monoclonal antibody, mix thoroughly, and let stand at room temperature for 60 min. Add coupling buffer to the ultrafiltration tube, add PBS to the white line at the top of the tube, and centrifuge at 12000 rpm and 4 ℃ for 10 min.

[0085] Discard the waste liquid, wash three times with PBS, and recover the coupling solution.

[0086] Transfer the coupling solution to a clean centrifuge tube, then add an equal volume of glycerol, record the final volume and final concentration, and store at -20 ℃ for later use.

[0087] Example 6: A kit for detecting porcine herpesvirus type 1 gE antibody based on magnetic microparticle chemiluminescence

[0088] In this embodiment, the working principle of the kit for detecting porcine herpesvirus type 1 gE antibody based on magnetic microparticle chemiluminescence is as follows: Utilizing a competitive immunoassay combined with magnetic microparticle separation technology, the sample to be tested is mixed with biotin-labeled monoclonal antibody, acrid ester-labeled multi-epitope fusion protein, and streptavidin magnetic beads and incubated to form an immune complex. After removing unbound impurities by washing with solid-phase carrier magnetic microparticles, a luminescent substrate is added to induce luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is inversely proportional to the porcine herpesvirus type 1 gE antibody titer. The instrument's built-in standard curve outputs RLU = the corresponding porcine herpesvirus type 1 gE antibody titer value.

[0089] In this embodiment, the main components of the kit for detecting porcine herpesvirus type 1 gE antibody based on magnetic microparticle chemiluminescence are: magnetic bead working solution, biotin-labeled antibody working solution, acrid ester-labeled working solution, pre-excitation solution, excitation solution, calibrator 1, and calibrator 2.

[0090] The working solution for magnetic beads was prepared by mixing 225 μL of streptomycin affinity magnetic beads with 4275 μL of PBS-BSA solution (pH=7.4).

[0091] Antibody working solution: prepared from 5.5 μg of biotin-labeled monoclonal antibody and 11000 μL of PBS-BSA solution (pH=7.4).

[0092] Acridinium ester labeled working solution: prepared from 1.1 μg of acridinium ester labeled multi-epitope fusion protein and 11000 μL of PBS-BSA solution (pH=7.4).

[0093] Pre-activation solution: 0.1 mol / L hydrochloric acid solution containing 0.1% hydrogen peroxide

[0094] Activation solution: 0.25 M sodium hydroxide solution containing 2% Triton X-100

[0095] The working process of the kit for detecting porcine herpesvirus type 1 gE antibody based on magnetic particle chemiluminescence includes the following steps:

[0096] Sample (20 μL) + magnetic bead working solution (20 μL) + acrid ester labeled working solution (50 μL) and antibody working solution (50 μL) were reacted at 37°C for 15 min, washed with 0.1 mol / L PBS buffer, and pre-activation solution (100 μL) and activation solution (100 μL) were added and reacted at 37°C for 5 min. The luminescence value was then detected.

[0097] Result determination: S / N value = luminescence value of the sample to be tested / average luminescence value of calibrator 2

[0098] Validity condition: If the average luminescence value of calibrator 1 / the average luminescence value of calibrator 2 < 0.2, the test result is valid; otherwise, the test should be repeated.

[0099] Result interpretation: S / N value > 0.25, judged as negative; S / N value ≤ 0.25, judged as positive.

[0100] Example 7: Kit for detecting porcine herpesvirus type 1 gE antibody based on competitive enzyme-linked immunosorbent assay (ELISA)

[0101] In this embodiment, the detection principle of the kit for detecting porcine herpesvirus type 1 gE antibody based on competitive enzyme-linked immunosorbent assay (ELISA) is as follows: the gE antibody in the sample to be tested competes with the enzyme-labeled antibody to bind to the solid-phase antigen, thereby achieving qualitative analysis of the antibody titer.

[0102] In this embodiment, the main components of the kit for detecting porcine herpesvirus type 1 gE antibody based on competitive enzyme-linked immunosorbent assay (ELISA) are: a microplate coated with SuHV-1 gE multi-epitope fusion protein, horseradish peroxidase (HRP)-labeled anti-porcine IgG monoclonal antibody, substrate solution (such as TMB), stop solution (usually sulfuric acid solution), positive control and negative control.

[0103] The working process of the kit for detecting porcine herpesvirus type 1 gE antibody based on competitive enzyme-linked immunosorbent assay (ELISA) includes the following steps:

[0104] First, add the test sample (e.g., 20 μL) and HRP-labeled antibody working solution (50 μL) simultaneously to the micro-wells coated with the antigen, and incubate at 37°C for 15 minutes to allow the gE antibody in the sample to compete with the enzyme-labeled antibody for binding to the solid-phase antigen. Then, wash with washing buffer (e.g., 0.1 mol / L PBS buffer) to remove unbound material. Add substrate solution (100 μL) and incubate at 37°C in the dark for 5-10 minutes to produce a color reaction. Add stop solution (50 μL) to stop the reaction, and immediately measure the absorbance at 450 nm using a microplate reader. The result is determined based on the S / N value (the ratio of the absorbance of the test sample to the average absorbance of the negative control).

[0105] The validity of the experiment must meet the condition that (average absorbance value of negative control - average absorbance value of positive control) / average absorbance value of negative control ≥ 0.3; if the S / N value > 0.25, it is judged as negative, indicating that there is no gE antibody or the antibody level is low in the sample; if the S / N value ≤ 0.25, it is judged as positive, indicating that SuHV-1 gE antibody is present in the sample.

[0106] The following are the performance tests of the reagent kit:

[0107] Example 8: Sensitivity test

[0108] First, the positive control material for porcine herpesvirus type 1 gE antibody was serially diluted 2-fold using PBST buffer (0.05% Tween-20, pH=7.4) to obtain sensitive control materials diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 times.

[0109] Then, using sensitivity control samples as samples, the sensitivity of a domestic brand (Wuhan Keqian) porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, the SuHV-1 gE competitive ELISA antibody detection kit of this application, and the SuHV-1 gE competitive magnetic particle chemiluminescent antibody detection kit of this application were tested, and the test results were compared and analyzed. The test results are shown in Table 1. The domestic brand could detect 512 times the sensitivity of the control sample, the SuHV-1 gE competitive ELISA antibody detection kit of this application could detect 1024 times the sensitivity of the control sample, and the SuHV-1 gE competitive magnetic particle chemiluminescent antibody detection kit of this application could detect 2048 times the sensitivity of the control sample. The sensitivity of both detection methods of this application is superior to that of the domestic brand.

[0110] Table 1. Sensitivity test comparison results

[0111]

[0112] Example 9: Specificity test

[0113] The SuHV-1 gE competitive ELISA antibody detection method and the SuHV-1 gE competitive magnetic microparticle chemiluminescent antibody detection method constructed in this application were used to simultaneously detect positive sera for SuHV-1 gE, SuHV-1 gB, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and porcine parvovirus (PPV). The results are shown in Table 2. This method showed no cross-reactivity with other susceptible animal viruses and had good specificity.

[0114] Table 2. Specificity test results

[0115]

[0116] Example 10: Repeatability test

[0117] Four swine serum samples with known background (one strongly positive SuHV-1 gE antibody sample, one positive SuHV-1 gE antibody sample, one weakly positive SuHV-1 gE antibody sample, and one negative SuHV-1 gE antibody sample) were selected and subjected to 20 replicate tests each using the competitive ELISA antibody detection method and the competitive magnetic particle chemiluminescence antibody detection method established in this application. The coefficient of variation was calculated based on the absorbance or luminescence values ​​of the serum samples. The results are shown in Table 3. The coefficient of variation (COP) for the SuHV-1 gE antibody competitive ELISA detection method established in this application was 1.92% for strong positive samples, 2.52% for positive samples, 2.91% for weak positive samples, and 2.14% for negative serum samples. The COP for the SuHV-1 gE competitive magnetic microparticle chemiluminescent antibody detection method established in this application was 1.44% for strong positive samples, 1.85% for positive samples, 1.30% for weak positive samples, and 1.73% for negative samples. This indicates that the SuHV-1 gE antibody detection method established in this application has high reproducibility.

[0118] Table 3. Repeatability Test Results

[0119]

[0120] Example 11: Clinical Sample Compliance Test

[0121] This application presents two methods: a competitive ELISA detection method using SuHV-1 gE antibody and a competitive magnetic microparticle chemiluminescence detection method using SuHV-1 gE antibody. Both methods were used to simultaneously detect 112 clinical samples with known backgrounds. The results were compared, and the concordance rate between the two methods was calculated. The results are shown in Table 4. The Kappa value for both methods was 0.93, and the overall concordance rate was 97.32%, indicating a high concordance rate between the two methods.

[0122] Table 4. Results of the comparative test of 112 serum clinical samples

[0123]

[0124] Clinically validated, the immunoassay kit based on the competitive assay principle of this invention exhibits high sensitivity and specificity, making it suitable for rapid diagnosis and epidemiological investigation of SuHV-1. Based on the high affinity and low immunogenicity of the monoclonal antibodies of this invention, they can also serve as candidate molecules for therapeutic antibodies, used for the prevention and treatment of porcine pseudorabies.

[0125] Example 12: Comparative analysis of detection sensitivity for different subtype strains

[0126] Three positive sera from known background genotype I (representative strains: Kaplan strain, KT983811.1; NIA3 strain, KU900059.1; Becker strain, JF797219.1), two positive sera from genotype II (representative strains: Ea strain, KU315430.1; Fa strain, KM189913.1), and four positive sera from genotype II enhanced virulence strains (representative strains: TJ strain, KJ789182.1; JS-2012 strain, KP257591.1; HeN1 strain, KP098534.1; ZJ01 strain, KM061380.1) were analyzed. These were serially diluted 10-fold with SPF porcine serum and detected using the magnetic microparticle chemiluminescent antibody detection kit of this invention. The results demonstrate that the monoclonal antibody and detection kit of this invention are effective at dilutions of 1×10⁻⁶. 4 It still exhibits high specificity and high sensitivity for different genotypes of porcine herpesvirus type 1 (SuHV-1) gE protein, and can cover antibody detection of different genotypes, which is significantly better than a certain domestic brand (Wuhan Keqian) porcine pseudorabies virus gE protein blocking ELISA antibody detection kit.

[0127] The test results are shown in the table below:

[0128] Table 5: Sensitivity detection results of the present invention and other kits against different subtype strains

[0129]

[0130] In summary, the competitive immunoassay method based on the monoclonal antibody constructed in this invention exhibits a significantly higher sensitivity for detecting porcine herpesvirus type 1 gE antibodies compared to existing commercially available kits, and it can uniformly cover different genotypes of the virus. This invention provides a novel solution with stable performance and excellent sensitivity for the accurate diagnosis of porcine pseudorabies.

[0131] Comparative Example 1: Two multi-epitope fusion proteins of the general formula shown in Formula (I) and Formula (II) according to Example 1 of the present invention: preparation and identification of immunogen.

[0132] The multi-epitope fusion protein of the present invention has the general formula shown in formula (I):

[0133]

[0134] As a comparative immunogen, the multi-epitope fusion protein has the general formula shown in formula (II):

[0135]

[0136] The nucleotide sequence of dominant epitope 1 is shown in SEQ ID NO.1, and the corresponding amino acid sequence 1 is shown in SEQ ID NO.8.

[0137] The nucleotide sequence of dominant epitope 2 is shown in SEQ ID NO.2, and the corresponding amino acid sequence 2 is shown in SEQ ID NO.9.

[0138] The nucleotide sequence of dominant epitope 3 is shown in SEQ ID NO.3, and the corresponding amino acid sequence 3 is shown in SEQ ID NO.10.

[0139] AAS1, AAS2, and AAS3 each independently represent the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, and AAS1, AAS2, and AAS3 represent different amino acid sequences; L represents the amino acid sequence GGGGS; each n is independently selected from 1, 2, 3, or 4.

[0140] The solubility, monoclonal antibody yield, and purity of the multi-epitope fusion protein were compared in Table 6. The multi-epitope fusion protein obtained by the general formula (I) used in this invention has better performance in all aspects than the multi-epitope fusion protein obtained by the general formula (II) used in the comparative example, and can significantly improve the detection sensitivity of porcine herpesvirus type 1 gE antibody.

[0141] Table 6: Performance comparison of the multi-epitope fusion protein (Formula I) of this invention with the comparative protein (Formula II)

[0142]

[0143] The multi-epitope fusion protein of formula (I) used in this invention is significantly superior to the comparative protein of formula (II) in terms of solubility, purity and induced antibody yield.

[0144] The protein of formula (I) can be used as an immunogen to induce the production of monoclonal antibodies with higher affinity and broader recognition ability, thereby improving the sensitivity and specificity of the detection kit (Examples 8, 9, 12).

[0145] The optimization of this structural sequence is one of the key innovations of this invention. In other words, one of the core innovations of this invention lies in the discovery that a multi-epitope fusion protein with a specific linkage sequence (Formula (I), corresponding to SEQ ID NO. 11) can be used as an immunogen to induce the production of monoclonal antibodies with unexpectedly superior performance. The monoclonal antibody (with variable region sequences of SEQ ID NO. 13 and SEQ ID NO. 14) secreted by the hybridoma cell line of accession number CGMCC No. 46719, which is the subject of this invention, was obtained by screening using the multi-epitope fusion protein shown in SEQ ID NO. 11 as an immunogen through specific single-cell sorting and stimulation techniques (as described in Example 2).

[0146] Experimental data show that, compared with immunogen of formula (II) (SEQ ID NO.12), immunogen of formula (I) not only significantly improves the solubility, yield, and purity of the immunogen itself, but more importantly, the monoclonal antibodies induced by it achieve significant, non-obvious, improvements in affinity (reaching the picomolar level), broad recognition ability of different genotype strains, and detection sensitivity of the final diagnostic kit. This causal relationship from a specific immunogen structure to a specific high-performance antibody, and the resulting leap in overall technical effect, constitutes the essential feature that distinguishes this invention from the prior art, and provides a novel solution to the technical problems of insufficient sensitivity and incomplete coverage in existing diagnostic technologies.

[0147] Table 7. Sequence information of nucleotide sequences

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] Table 8. Sequence information of amino acid sequences:

[0154] .

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against porcine herpesvirus type 1 gE protein, characterized in that, The hybridoma cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46719.

2. A monoclonal antibody against porcine herpesvirus type 1 gE protein secreted by the hybridoma cell line of claim 1.

3. The use of the hybridoma cell line of claim 1 or the monoclonal antibody of claim 2 in the preparation of a product for diagnosing porcine pseudorabies.

4. A kit for detecting porcine herpesvirus type 1 gE antibody, characterized in that, It includes the monoclonal antibody as described in claim 2.

5. The reagent kit according to claim 4, characterized in that, The kit is an immunoassay kit based on the principle of competition.

6. The reagent kit according to claim 5, characterized in that, The kit is a magnetic particle chemiluminescent immunoassay kit, which contains: (a) Magnetic microparticles coated or coupled with streptavidin; (b) The biotin-labeled monoclonal antibody of claim 2; (c) Chemiluminescent material-labeled porcine herpesvirus type 1 gE protein or its antigenic epitope.

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