A hybridoma cell line secreting a monoclonal antibody against PRRS GP2 protein and its application

Abstract

This invention belongs to the field of biotechnology and discloses a hybridoma cell line that secretes a monoclonal antibody against the PRRS GP2 protein and its applications. The hybridoma cell line secreting the monoclonal antibody against the porcine reproductive and respiratory syndrome virus (PRRSV) GP2 protein is registered under CCTCC NO: C202551. This invention provides a valuable tool for studying the structure and function of the PRRSV GP2 protein and establishing diagnostic methods, laying the foundation for PRRSV diagnosis and biological research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a hybridoma cell line that secretes a monoclonal antibody against the PRRS GP2 protein and its applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), characterized primarily by reproductive dysfunction in sows and respiratory dysfunction in piglets. PRRS is one of the most important diseases in the swine industry, having been prevalent for many years and causing significant economic losses to the global swine industry. Currently, based on serotypes, PRRSV can be broadly classified into two types: the European type, represented by the LV (lelysted virus) strain, and the American type, represented by the VR2332 strain, with a genetic similarity of 50%-70%. Common strains include classic strains, HP-PRRSV strains, NADC30-like strains, and NADC34-like strains. In recent years, European strains have also been discovered, and the high genetic variability of PRRSV makes its detection and control even more challenging.

[0003] PRRSV is a single-stranded positive-sense RNA virus with a genome size of approximately 15 kilobase pairs (kb) and a diameter of approximately 50-65 nanometers. It encodes at least 10 open reading frames (ORFs). The GP2a protein, encoded by ORF2a, has a molecular weight of approximately 29-30 kDa. GP2a and GP2 are the same protein, differing only in description in different publications. GP2 contains a predicted N-terminal signal sequence, a 168-amino acid outer cellular domain, a transmembrane helix, and a 20-amino acid inner cellular domain. GP2, GP3, and GP4 within the viral particle together form a heterotrimer that induces viral infection, a key determinant of PRRSV entry into cultured cells, and plays a crucial role in viral attachment. GP2 may be involved in inducing the host's immune response. During infection, GP2 may act as part of the immunogen in the host's antibody response, stimulating B and T cell responses. Currently, the structure and antigenic properties of the PRRSV GP2 protein are not fully understood. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a hybridoma cell line that secretes a monoclonal antibody against PRRS GP2 protein and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a hybridoma cell line that secretes a monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus, the preservation number of which is CCTCC NO: C202551.

[0007] The hybridoma cell line PRRSV-GP2-2G10C3 of this invention, classified as Hybridoma cell line in Latin, was deposited on January 20, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202551. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The contact number is 027-68752319.

[0008] Secondly, the present invention provides a monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus, which is secreted by the hybridoma cell line.

[0009] This invention uses the PRRSV HuN4-F112 strain sequence as a template to construct the E. coli recombinant expression plasmid pET-30a-GP2, express and prepare recombinant GP2 protein. The recombinant protein was purified using a nickel-ion metal affinity chromatography column and immunized with 6-week-old female BALB / c mice. Positive hybridoma cells were screened by indirect ELISA and indirect immunofluorescence, and the specificity of the monoclonal antibody was identified using Western blotting. The epitope region recognized by the monoclonal antibody was identified by constructing a truncated GP2 protein gene. A stable hybridoma cell line (2G10C3) secreting GP2 protein antibody was successfully screened. IFA results showed that this monoclonal antibody specifically reacted with PRRSV HuN4-F112 and Marc-145 GP2 cell lines. The antigenic epitope of this monoclonal antibody was identified as... 133 EATLSRI 139 Amino acid alignment analysis revealed that the protein GP2 protein is relatively conserved in American strains, and modeling and visualization analysis showed that it exists on the protein surface in the form of an α-helix. IFA results showed that in the reactivity identification with different PRRSV genotypes, 2G10C3 reacted with NADC30, HuN4, CH-1a, and VR2332 strains. The monoclonal antibody obtained by this invention provides a good tool for the structural and functional study of the PRRSV GP2 protein and the establishment of diagnostic methods.

[0010] In a preferred embodiment of the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody described in this invention, the antigenic epitope it recognizes is 133 EATLSRI139 .

[0011] Thirdly, the present invention provides a detection reagent, chip, test strip or kit for porcine reproductive and respiratory syndrome virus (PRRSV), including a monoclonal antibody against the PRRSV GP2 protein.

[0012] Fourthly, the present invention provides a method for preparing a monoclonal antibody against porcine reproductive and respiratory syndrome virus (PRRSV) GP2 protein, comprising culturing the hybridoma cell line and isolating and purifying the monoclonal antibody against PRRSV GP2 protein from the cell culture species.

[0013] Fifthly, the present invention provides a method for in vitro non-diagnostic detection of porcine reproductive and respiratory syndrome virus (PRRSV), wherein the sample to be tested is contacted with the PRRSV GP2 protein monoclonal antibody.

[0014] In a sixth aspect, the present invention applies the hybridoma cell line, the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody, the detection reagent, chip, test strip or kit to the in vitro non-diagnostic detection of porcine reproductive and respiratory syndrome virus.

[0015] In a seventh aspect, the present invention applies the hybridoma cell line, the monoclonal antibody against porcine reproductive and respiratory syndrome virus GP2 protein, the detection reagent, chip, test strip, or kit to the preparation of a detection product for detecting porcine reproductive and respiratory syndrome virus.

[0016] Eighthly, the present invention utilizes the hybridoma cell line, the monoclonal antibody against the porcine reproductive and respiratory syndrome virus GP2 protein, the detection reagent, chip, test strip, or kit in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus.

[0017] In a ninth aspect, the present invention utilizes the hybridoma cell line, the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody, the detection reagent, chip, test strip, or kit in the preparation of pharmaceutical formulations for the prevention or treatment of diseases caused by porcine reproductive and respiratory syndrome virus infection.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention amplifies the ORF2 gene based on the PRRSV HuN4-F112 strain sequence, successfully prepares recombinant GP2 protein using a prokaryotic expression system, and uses it as an immunogen to immunize BALB / c mice, producing a GP2 monoclonal antibody and identifying one antigenic epitope. This invention provides a valuable tool for studying the structure and function of the PRRSV GP2 protein and establishing diagnostic methods, laying the foundation for PRRSV diagnosis and biological research. Attached Figure Description

[0020] Figure 1 Purification and identification of PRRSV recombinant GP2 protein; Figure 1 In the table, A represents recombinant protein expression; B represents inclusion body purification; C represents Western blot identification; M represents the relative molecular mass standard of the protein; 1 represents whole bacteria with empty vector; 2 represents whole bacteria without induced expression; 3 represents the supernatant of induced expression; 4 represents the precipitate of induced expression; and 5 represents the purified recombinant GP2 protein.

[0021] Figure 2 The serum antibody titer after three immunizations in mice is shown.

[0022] Figure 3 Western blot was used to identify the reactivity of GP2 protein monoclonal antibodies with recombinant GP2 protein; Figure 3 In this context, M represents the relative molecular mass of the protein, 1 represents the recombinant GP2 protein, and 2 represents the empty vector-induced whole bacterial protein.

[0023] Figure 4 The IFA reaction characteristics of GP2 protein monoclonal antibody with Marc-145 GP2 cell line and PRRSV HuN4-F112 strain were identified.

[0024] Figure 5 Construction strategy and identification of antigenic epitopes for PRRSV GP2 protein truncated variant; Figure 4 In the diagram, A represents the first GP2 protein truncation and monoclonal antibody recognition region, B represents the second GP2 protein truncation and monoclonal antibody recognition region, C represents the third GP2 protein truncation and monoclonal antibody recognition region, and D represents the fourth GP2 protein truncation and monoclonal antibody recognition region.

[0025] Figure 6 This study aimed to analyze the conservation of PRRSV GP2 protein epitopes and identify epitope antigenicity based on a 3D structural model of the GP2 protein.

[0026] Figure 7 To identify the IFA reaction characteristics of GP2 protein monoclonal antibodies with different PRRSV subtypes.

[0027] Figure 8 This is the result of a sensitivity test for monoclonal antibodies. Detailed Implementation

[0028] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] Unless otherwise specified, the test methods used in the examples are conventional methods; the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0030] The PRRSV strain HuN4-F112, SP2 / 0 cells, Escherichia coli E. coli DH5α, and BL-21 competent cells involved in the examples are from the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences; 6-8-week-old female BALB / c mice are purchased from Suzhou Speyford Biotechnology Co., Ltd., license: SCXK (Su) 2022-0006; BamH I and Hind III restriction enzymes, BL21(DE3) competent cells are purchased from TaKaRa; the plasmid miniprep kit, Ni-NTA (His tag protein purification medium) are purchased from Beyotime; Escherichia coli competent cell DH5α is purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; the gel extraction kit, 2×Taq Master Mix are purchased from Nanjing Novoprotein Scientific Co., Ltd.; the BCA protein quantification kit, affinity chromatography column, SDS-PAGE protein loading buffer (5×) are purchased from Shanghai Beyotime Biotechnology Co., Ltd.; protein marker is purchased from Thermo; 1640 and DMEM cell culture media are purchased from Gibco; isopropyl β-D-thiogalactoside (IPTG), PEG1450, 50×HAT, and 50×HT are purchased from Sigma, USA; the mouse monoclonal antibody subtype identification kit is purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0031] The preparation method of the Marc-145 GP2 cell line involved in the examples is as follows:

[0032] Using the full-length PRRSV HuN4-F112 as a template, the ORF2 gene sequence was amplified by PCR and cloned into a lentiviral vector to obtain the recombinant plasmid pLV-EF1a-EGFP-2A-GP2.

[0033] The primer sequences used are:

[0034] ORF2-F: 5’-ATGAAATGGGGTCTATGCAAAGCC-3’;

[0035] ORF2-R: 5’-CCACGAGTTCAAAAGAAAAATTGCC-3’.

[0036] Recombinant lentiviral particles were obtained using a lentiviral packaging system and transduced into Marc-145 cells. Cells were screened using a medium containing puromycin, and the cells after initial screening were cloned using a limiting dilution method to obtain cell lines. The stable expression of GP2 protein in the cell lines was verified by PCR and Western blot experiments.

[0037] Example 1: Construction of recombinant expression plasmid

[0038] Homologous arms of the vector sequence were added to the primers for amplifying the target gene GP2. The primer sequences are as follows:

[0039] pET-30a-PRRSV-GP2-F:

[0040] 5'-GGCTGATATCGGATCCCCGCGGTACTCCGTGCGC-3';

[0041] pET-30a-PRRSV-GP2-R:

[0042] 5'-GTGCGGCCGCAAGCTTTCATTGCTGAAAATCGTGAAGCTTTGGC-3'.

[0043] The target fragment of the GP2 gene was amplified, with a size of 456 bp.

[0044] The PCR reaction conditions were: 95℃ pre-denaturation for 2 min; 98℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and 72℃ extension for 10 min.

[0045] To construct the GP2 gene recombinant plasmid, the target fragment and the pET-30a vector were linearized by double digestion with BamHI and HindIII, followed by ligation. The ligation product was transformed into DH5α competent cells, cultured at 37°C for 4 hours, and then identified by bacterial PCR before being sent to Qingke Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from the correctly sequenced cells and expanded. The recombinant plasmid pET-30a-GP2 was stored at -20°C for later use.

[0046] To analyze the GP2 protein antigenic epitopes, a series of truncated primers were designed, with a BamHI restriction site introduced at the 5′ end and a HindIII restriction site introduced at the 3′ end. The primer sequences are shown in Table 1, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Using the recombinant plasmid pET-30a-GP2 as a template, 13 truncated GP2 protein genes were amplified by PCR. The truncated variants and the pET-30a vector were double-digested with enzymes and then ligated to construct 13 recombinant plasmids of truncated GP2 proteins.

[0047] Table 1. Primer sequences for GP2 protein truncated variant PCR

[0048]

[0049]

[0050] Example 2: Expression and purification of recombinant proteins

[0051] The recombinant plasmid pET-30a-GP2 prepared in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells, plated on solid LB medium containing 50 mg / L kanamycin, and cultured at 37°C for 12 h. Single colonies were picked and inoculated into liquid LB medium containing 50 mg / L kanamycin for expansion culture. After incubation at 37°C and 220 rpm for 2 h-4 h, when the bacterial OD of the culture was... 600 When the pH reached 0.4-0.6, expression was induced by 0.1 mmol / L IPTG at 37℃ for 6 h, followed by centrifugation at 8000 r / min for 10 min. The bacterial cell pellet was collected, washed twice with PBS, resuspended in PBS, and sonicated to disrupt the cells. The supernatant and pellet were then separated by high-speed centrifugation to prepare the sample. The sample was identified and analyzed by SDS-PAGE, stained with Coomassie brilliant blue, and purified using Ni-NTA affinity chromatography. The purified recombinant protein was desalted and concentrated by dialysis. The concentration of the recombinant protein was determined using the BCA method. The reactivity of the recombinant protein with the His-Tag antibody was identified by Western blot.

[0052] The PCR amplification product of the GP2 protein gene was cloned into the pET-30a vector. After successful identification, it was transformed into E. coli. IPTG-induced expression was followed by SDS-PAGE analysis, and the target protein size was 22.6 kDa, consistent with the expected protein size (see [link to original text]). Figure 1 (A) After purification and concentration of inclusion bodies, the Western blot results showed a single band, indicating good purification effect (see [reference]). Figure 1 (B, C)

[0053] Example 3: Preparation of recombinant protein monoclonal antibodies

[0054] (1) Immunization of BALB / c mice

[0055] The purified recombinant protein from Example 2 was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and then immunized with 100 μg / mouse in 6-8 week old female BALB / c mice via subcutaneous injection at multiple sites on the back of the neck. After emulsification with incomplete Freund's adjuvant, a second immunization (100 μg / mouse) and a third immunization (100 μg / mouse) were performed via the same route as the first immunization, with an interval of 2 weeks between each immunization. Two weeks after the third immunization, blood samples were collected, and the purified recombinant GP2 protein was analyzed by Western blotting using mouse serum as the primary antibody. The purified recombinant GP2 protein was used as the antigen to coat an enzyme-linked immunosorbent assay (ELISA) plate, and mouse serum antibodies were detected by indirect ELISA. When the serum titer reached the fusion requirement, a shock immunization was performed via intraperitoneal injection (100 μg / mouse).

[0056] (2) Cell fusion and screening of hybridoma cells

[0057] Prepare feeder cells in 96-well plates one day in advance for later use. After digesting and centrifuging SP2 / 0 cells in good growth condition, fuse them with spleen cells at a cell ratio of 1:5 using PEG. Culture in medium supplemented with HAT. Observe cell colonies after three days. Replace with medium containing HT on the seventh day. On the tenth day, screen positive cell wells using indirect ELISA and IFA. Perform three subclonings on the initially screened cell lines using limiting dilution until hybridoma cells derived from a single cell population that can stably secrete anti-GP2 protein antibodies are selected. Then passage and expand the culture to T75 cell flasks, freeze and store in liquid nitrogen.

[0058] (3) Preparation of monoclonal antibody ascites

[0059] Three 8-week-old female BALB / c mice were used to prepare ascites fluid. First, each mouse was pre-sensitized with 0.5 mL of paraffin oil via intraperitoneal injection. After a 7-day sensitization period, hybridoma cell lines in the logarithmic growth phase were selected, and the cell suspension concentration was adjusted to 1-2 × 10^6 cells / mL. Subsequently, 0.5 mL of PBS containing this cell concentration was injected intraperitoneally into each mouse to complete the immunization cell implantation. Seven days later, the mice were observed, and ascites fluid was collected when their abdominal girth increased, they experienced difficulty eating, and they had difficulty walking. The collected ascites fluid was centrifuged at 3000 rpm for 10 min at room temperature, and the colorless, transparent middle layer was collected as ascites fluid. The ascites fluid titer was determined using ELISA.

[0060] The results showed that the serum titer of mice could reach 1:12800 after two immunizations (see...). Figure 2Cell fusion can be performed 3 days after shock immunization. Using ELISA combined with subclonal screening, a positive hybridoma cell line continuously producing the GP2 antigen was successfully screened. This cell line was named 2G10C3 (hybridoma cell line PRRSV-GP2-2G10C3), and its Latin classification is Hybridoma cell line. It was deposited on January 20, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: C202551, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The contact number is 027-68752319.

[0061] Culture supernatants from hybridoma cells passaged to the fifteenth generation were collected, and the results in Table 2 show that the titer remained basically stable. The titer in mouse ascites fluid was >1×10⁻⁶. 6 .

[0062] Table 2. ELISA antibody titer determination of hybridoma cell culture supernatant and mouse ascites fluid.

[0063]

[0064] Example 4: Identification of the biological characteristics of monoclonal antibodies

[0065] (1) Antibody secretion stability of hybridoma cell lines

[0066] The positive hybridoma cells obtained in Example 3 were passaged continuously, and the supernatant of monoclonal hybridoma cells from passages 5, 10, and 15 was used to detect antibody titers using an indirect ELISA method to determine the stability of antibody secretion by the hybridoma cell line.

[0067] (2) Western blot specificity identification

[0068] The prepared purified GP2 protein sample was subjected to SDS-PAGE electrophoresis and transferred to an NC membrane. It was blocked with 5% skim milk powder solution at room temperature for 2 hours, washed three times with TBST, and incubated overnight at 4°C with the supernatant of the prepared monoclonal antibody as the primary antibody. It was washed three times with TBST, and incubated at room temperature for 1 hour with goat anti-mouse IgG (1:10000 dilution) as the secondary antibody. It was washed three times with TBST, and an appropriate amount of chromogenic solution was added to the front side of the NC membrane for observation in a luminescence imaging system.

[0069] The results showed that 2G10C3 mAb specifically reacted with the recombinant GP2 protein expressed in the prokaryotic system, but did not react with the pET-30a empty vector (see [link to study]). Figure 3 ).

[0070] (3) Specificity identification by indirect immunofluorescence assay (IFA)

[0071] Marc-145 cells were passaged into six-well plates and cultured until the cell density reached 80%. The culture medium was discarded, the cells were washed twice with PBS, and the medium was replaced with serum-free medium. Cells were infected with PRRSV HuN4-F112. When cell pathogenesis was observed but no large amount of cells detached, the cells were fixed with ice-cold methanol, incubated at 4°C for 10 min, washed three times with PBS, and inverted at -20°C. Marc-145 cells stored in the laboratory were then... GP2 cell line cells were seeded and, after confluence, fixed with ice-cold methanol and incubated at 4°C for 10 min. The cells were then washed three times with PBS. Both samples were simultaneously blocked with 5% skim milk powder solution at 37°C for 2 h and washed three times with PBS. The supernatant of monoclonal cells was added to the culture plate as primary antibody at 500 μL / well and incubated at 37°C for 1 h. The cells were then washed three times with PBS. Goat anti-mouse fluorescent secondary antibody (1:2000 dilution) was incubated at 37°C for 1 h and washed three times with PBS. The cells were then incubated with DAPI staining solution at room temperature in the dark for 10 min and washed three times with PBS. The results were observed and photographed under a fluorescence microscope.

[0072] The results are as follows Figure 4 As shown, 2G10C3 mAb exhibits specific fluorescence in Marc-145 GP2 cell lines and Marc-145 cells infected with PRRSV HuN4-F112.

[0073] (4) Identification of monoclonal antibody subtypes

[0074] Following the instructions for monoclonal antibody subtype identification, the monoclonal antibody cell supernatant was diluted 1:200. 50 μL of the sample to be tested was added to each well of the strip. 50 μL of 1× goat anti-mouse IgM + IgG-HRP (diluted 1:100) was added to each well, and the mixture was stirred. The plate was incubated at room temperature for 1 hour. The liquid in the wells was discarded, and the plate was washed three times with PBST. The chromogenic solution was added, and the plate was incubated at room temperature in the dark for 10-20 minutes. The stop solution was added, and the OD values ​​of each well were recorded. 450 The well with the highest value corresponds to the monoclonal antibody subtype.

[0075] The results showed that the heavy chain of the 2G10C3 monoclonal antibody belonged to the IgG1 subclass, and the light chain belonged to the Kappa type (see Table 3).

[0076] Table 3. Monoclonal antibody subtype identification (OD) 450 nm)

[0077]

[0078] (5) Identification of monoclonal antibody epitopes

[0079] The truncated recombinant plasmid of the GP2 protein gene was transformed into the expression strain BL-21, and expression was induced by IPTG. After SDS-PAGE, the protein was transferred onto an NC membrane. The antigenic epitopes recognized by the monoclonal antibody were identified by Western blot using the monoclonal antibody supernatant and His tag antibody as primary antibodies.

[0080] See results Figure 5 Western blot analysis of a series of constructed GP2 protein truncated variants showed that the prokaryotic expression proteins of all 13 GP2 protein gene truncated recombinant plasmids reacted with His mouse monoclonal antibody, indicating correct expression of the recombinant plasmids. Preliminary identification of the antigenic epitopes recognized by this mAb showed that it reacted with F1, F2, and F3 antibodies, recognizing 131-148 amino acids (amino acids). Figure 5 (A) Similarly, such as Figure 5 The results for B showed that it only reacted with F5 and F7, recognizing 131-142aa. Further validation results indicated that ( Figure 5 The 2G10C3 monoclonal antibody (C) reacts with F8 and F11, recognizing 132-140 amino acids. To further refine the minimum recognition region, F12 and F13 truncated expression sequences were constructed and combined with adjacent recognition regions. The results showed that it reacted with both F12 and F13, ultimately leading to the conclusion that the antigenic epitope recognized by the 2G10C3 monoclonal antibody is... 133 EATLSRI 139 ( Figure 5 (D).

[0081] (6) Comparative analysis of the antigenic epitope amino acid sites of GP2 monoclonal antibodies

[0082] A total of 21 PRRSV-1 strains and PRRSV-2 strains from different lineages circulating in China, along with reference strain sequences (BB0907, SY0608, HuN4, JXA1, WUH4, GD1404, CH-1a, IngelvacATP, QYYZ, GM2, VR2332, BF4, S1, MN184C, NADC31, NADC30, FJ1402), were selected. The conservation of the antigenic epitope sequences in each strain was analyzed using Megalign software. The spatial structure of the PRRSV GP2 protein was modeled using SWISS-MODEL, and the antigenic epitopes were visualized and analyzed in the spatial structure of the GP2 protein using PyMOL (version 2.5.0).

[0083] The amino acid composition of GP2 protein was analyzed using Megalign software for representative strains of PRRSV-1 and type 2 from four major circulating lineages (Lineage 1, 3, 58). The results are shown in [Figure 1]. Figure 6 China A, 133 EATLSRI 139 European strains137 SRI 139 The differences are significant, but the conservation is relatively good among the four lineages of the American type.

[0084] A three-dimensional structure of PRRSV GP2 was constructed using the SWISS-MODEL server, and the spatial locations of the newly identified linear epitopes were visualized using PyMOL software. The results show that the epitopes... 133 EATLSRI 139 It exists in the form of an α-helical epitope. This epitope is exposed on the structural surface, which facilitates its identification. These findings suggest that this epitope may be an important linear B-cell epitope in PRRSV GP2 (see [link to study].) Figure 6 (B)

[0085] (7) Reactivity of monoclonal antibodies with different strains

[0086] To evaluate the application of this monoclonal antibody in the detection of different PRRSV strains, Marc-145 cells were infected with four different PRRSV-2 strains, namely NADC30, HuN4, CH-1a and VR2332.

[0087] Figure 7 The results showed that the monoclonal antibody produced specific fluorescence with four PRRSV-2 strains and had a good reaction, making it suitable for the detection of these strains.

[0088] (8) Sensitivity detection of monoclonal antibodies

[0089] The purified PRRSV GP2 protein was coated onto an ELISA plate at a concentration of 100 ng / well (the protein was diluted with coating buffer at pH 9.6), 100 μl per well, and incubated overnight at 4°C. 100 μl of 5% skim milk was added to each well, and the plate was blocked at 37°C for 2 h. The blocking solution was discarded, and the plate was washed three times with 200 μl of PBST. The last wash was discarded, and the remaining liquid was gently patted dry on absorbent paper. The hybridoma cell supernatant was serially diluted at 100, 200, 400, 800, 1600, 3200, 6400, 12800, and 25600, and then coated at a concentration of 100 μl / well.

[0090] After adding the contents to each well of a 96-well plate, incubate at 37°C for 1 hour. Discard the liquid in the plate, add 200 μl of PBST to each well, wash three times, and gently pat dry on absorbent paper. Dilute HRP-labeled goat anti-mouse enzyme-labeled secondary antibody 1:5000 and react at 37°C for 1 hour. Discard the liquid in the plate, add 200 μl of PBST to each well, wash three times, and gently pat dry on absorbent paper. Add 100 μl of TMB chromogenic solution and incubate at room temperature in the dark for 15 minutes. Stop the reaction by adding 50 μl of 2M H2SO4. OD 450nm reading. Results as follows Figure 8 As shown, the OD value is linearly related to the antibody concentration, and it also reacts well with GP2 protein at a dilution ratio of 1:6400, indicating that the antibody has good sensitivity.

[0091] (9) Repeatability testing of monoclonal antibodies

[0092] The GP2 protein prepared in this study was used to coat ELISA plates at a concentration of 2 μg / ml. Intra-assay reproducibility tests were performed using cell supernatants collected at the same time as primary antibodies, following the ELISA detection method used in this study. Inter-assay reproducibility tests were performed using cell supernatants collected at different times as primary antibodies. The results are shown in Tables 4 and 5. The coefficients of variation for both intra-assay and inter-assay reproducibility were less than 3%, indicating good reproducibility of the antibody.

[0093] Table 4. Intra-batch repeatability testing

[0094]

[0095] Table 5 Inter-batch repeatability testing

[0096]

[0097] In summary, this invention fuses and expresses the GP2 protein of PRRSV HuN4-F112, and uses it as an antigen to immunize mice for mAb screening. A hybridoma cell line that stably secretes a monoclonal antibody against the GP2 protein was successfully obtained. The specific antigenic epitope recognized by this mAb was also identified, and its conservation and visualization were analyzed. This provides new insights into the antigenicity of the GP2 protein.

[0098] Visualization of protein expression in PRRSV-infected cells is crucial for studying the biological characteristics of the GP2 protein. This invention uses a monoclonal antibody to detect GP2 protein expression in PRRSV-infected cells and the Marc-145 GP2 cell line. IFA assay results showed that all monoclonal antibodies recognizing this epitope could detect GP2 protein expression, with the expressed protein mainly distributed around the cell nucleus. In experiments detecting the reaction of this monoclonal antibody with different PRRSV-2 strains, it showed good reactivity with NADC30, HuN4, CH-1a, and VR2332 strains, and can be used for the detection of these strains. In summary, the preparation of the GP2 monoclonal antibody provides new material for PRRSV serological detection and lays the foundation for further investigation of the structure and function of the PRRSV GP2 protein.

[0099] Viral protein epitope analysis plays a crucial role in clarifying protein structure and antigenic characteristics. This invention utilizes a truncated version of the GP2 protein gene for expression and, using a developed monoclonal antibody, identifies one linear B-cell epitope. 133 EATLSRI 139 This study differs from previous studies and demonstrates a more precise localization of the antigenic epitope. Analysis of the amino acid differences in GP2 protein among 21 European and American strains revealed that the epitope is well-conserved among American strains but significantly different from European strains. Spatial structure analysis of GP2 using PyMOL (version 2.5.0) further revealed the epitope. 133 EATLSRI 139 The portion that forms the α-helix is ​​structurally stable and exposed on the protein surface, which is beneficial for antibody recognition.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A hybridoma cell strain secreting a monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus, characterized in that, The preservation number is CCTCC NO: C202551.

2. A porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody, characterized in that, Secreted by the hybridoma cell strain of claim 1.

3. A detection reagent for porcine reproductive and respiratory syndrome virus, characterized by comprising an antibody which binds to a polypeptide of SEQ ID NO:

1. The pig reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of claim 2.

4. A detection chip for porcine reproductive and respiratory syndrome virus, characterized by, The pig reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of claim 2.

5. A test strip for porcine reproductive and respiratory syndrome virus, characterized by, The pig reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of claim 2.

6. A detection kit for porcine reproductive and respiratory syndrome virus, characterized by, The pig reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of claim 2.

7. The use of the hybridoma cell strain of claim 1, the pig reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of claim 2 in the preparation of a detection product for detecting the pig reproductive and respiratory syndrome virus.

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