Hybridoma cell strain secreting PRRS GP2 protein monoclonal antibody and application

Abstract

The invention belongs to the technical field of biology, and discloses a hybridoma cell strain capable of secreting a PRRS GP2 protein monoclonal antibody and application of the hybridoma cell strain. The preservation number of the hybridoma cell strain for secreting the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody is CCTCC (China Center For Type Culture Collection) NO: C202551. A good tool is provided for structure and function research of the PRRSV GP2 protein and establishment of a diagnosis method, and a foundation is laid for PRRSV diagnosis and biological research.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hybridoma cell line secreting a PRRS GP2 protein monoclonal antibody and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive dysfunction in sows and respiratory dysfunction in piglets. PRRS is one of the most important diseases in the pig industry and has been prevalent for many years. PRRS has caused great economic losses to the pig industry worldwide. At present, according to the serotype of PRRSV, PRRSV can be roughly divided into two types: the European type represented by LV (lelysted virus) and the American type represented by VR2332. The genetic similarity between the two is 50%-70%. The popular strains are classic strains, HP-PRRSV strains, NADC30-like strains and NADC34-like strains. In recent years, European strains have also been discovered, and the genetic variability of PRRSV is very strong, which makes the detection and control of PRRSV more difficult.

[0003] PRRSV is a single-stranded positive-strand RNA virus with a genome size of about 15 kilobase pairs (kb) and a diameter of about 50-65 nanometers. It encodes at least 10 open reading frames (ORFs). The GP2a protein is encoded by ORF2a, with a molecular weight of about 29kDa-30kDa. GP2a and GP2 are the same protein, but the descriptions in different articles are different. GP2 contains a predicted N-terminal signal sequence, an exodomain of 168 amino acids, a transmembrane helix, and an intracellular domain of 20 amino acids. GP2, GP3, and GP4 in the virus particle will form a heterotrimer that can cause viral infection. It is the main determinant for arteritis virus to enter cultured cells and plays a key role in the virus attachment process. GP2 may be involved in inducing the host's immune response. During the infection process, GP2 may act as part of the immunogen in the antibody response produced by the host to stimulate the host's B cell and T cell response. At present, the structure and antigenic characteristics of the PRRSV GP2 protein are not very clear. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a hybridoma cell line secreting PRRS GP2 protein monoclonal antibody and its application.

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

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

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

[0008] In a second aspect, 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] The present invention uses the PRRSV HuN4-F112 strain sequence as a template to construct an Escherichia coli recombinant expression plasmid pET-30a-GP2 to express and prepare recombinant GP2 protein. After the recombinant protein is purified using a nickel ion metal affinity chromatography column, 6-week-old female BALB / c mice are immunized, and positive hybridoma cells are screened by indirect ELISA and indirect immunofluorescence methods, and the specificity of the monoclonal antibody is identified by protein immunoblotting. The epitope region recognized by the monoclonal antibody is identified by constructing a truncation of the GP2 protein gene. A hybridoma cell line (2G10C3) that stably secretes GP2 protein antibodies was successfully screened. IFA results showed that the monoclonal antibody can specifically react with PRRSV HuN4-F112 and Marc-145 GP2 cell lines. The antigenic epitope of the monoclonal antibody was identified as 133 EATLSRI 139 Through amino acid comparison analysis, it was found that it is relatively conservative in American strains, and through modeling and visualization analysis, it was found that it exists on the surface of the protein structure in the form of α helix. The IFA results showed that in the reactivity identification with different genetic subtypes of PRRSV, 2G10C3 reacted with NADC30, HuN4, CH-1a and VR2332 strains. The monoclonal antibody obtained by the present invention provides a good tool for the structural and functional research of PRRSV GP2 protein and the establishment of diagnostic methods.

[0010] As a preferred embodiment of the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody of the present invention, the antigenic epitope recognized by the monoclonal antibody is 133 EATLSRI139 .

[0011] In a third aspect, the present invention provides a porcine reproductive and respiratory syndrome virus detection reagent, chip, test paper or kit, including the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody.

[0012] In a fourth aspect, the present invention provides a method for preparing a monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus, comprising culturing the hybridoma cell line; and isolating and purifying the monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus from the cell culture species.

[0013] In a fifth aspect, the present invention provides an in vitro non-diagnostic method for detecting porcine reproductive and respiratory syndrome virus, wherein a sample to be tested is contacted with the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody.

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

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

[0016] In an eighth aspect, the present invention uses the hybridoma cell line, the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody, the detection reagent, chip, test paper or kit in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus.

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

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The invention amplifies the ORF2 gene according to the sequence of the PRRSV HuN4-F112 strain, successfully prepares the recombinant GP2 protein through a prokaryotic expression system, uses the recombinant GP2 protein as an immunogen to immunize BALB / c mice, prepares a GP2 monoclonal antibody, and identifies an antigenic epitope. The invention provides a good tool for the study of the structure and function of the PRRSV GP2 protein and the establishment of a diagnostic method, and lays a foundation for the diagnosis and biological research of PRRSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Purification and identification of PRRSV recombinant GP2 protein; Figure 1 In the figure, A represents recombinant protein expression; B represents inclusion body purification; C represents Western blot identification; M represents protein relative molecular mass standard, 1 represents the whole bacteria containing an empty vector, 2 represents the 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 It is the serum antibody titer of mice after three immunizations.

[0022] Figure 3 Western blot was used to identify the reactivity of GP2 protein monoclonal antibody with recombinant GP2 protein; Figure 3 In the figure, M is the protein relative molecular mass standard, 1 is the recombinant GP2 protein, and 2 is the empty-load induced whole bacterial protein.

[0023] Figure 4 To identify the IFA reaction characteristics of GP2 protein monoclonal antibodies with Marc-145 GP2 cell line and PRRSV HuN4-F112 strain.

[0024] Figure 5 To construct strategies for PRRSV GP2 protein truncations and identify antigenic epitopes; Figure 4 In the figure, A is the first GP2 protein truncation and monoclonal antibody recognition region, B is the second GP2 protein truncation and monoclonal antibody recognition region, C is the third GP2 protein truncation and monoclonal antibody recognition region, and D is the fourth GP2 protein truncation and monoclonal antibody recognition region.

[0025] Figure 6 To analyze the conservation of PRRSV GP2 protein antigenic epitopes and identify the epitope antigenicity based on the 3D structural model of GP2 protein.

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

[0027] Figure 8 This is the sensitivity test result of monoclonal antibody. DETAILED DESCRIPTION

[0028] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

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

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

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

[0032] The ORF2 gene sequence was amplified by PCR using the full-length HuN4-F112 of PRRSV as a template 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 through the lentiviral packaging system and transduced into Marc-145 cells. The cells were screened using a culture medium containing puromycin. The cells after the initial screening were cloned using the limiting dilution method to eventually obtain a cell line. The stable expression of GP2 protein in the cell line was verified by PCR and Western blot experiments.

[0037] Example 1: Construction of recombinant expression plasmid

[0038] Add the homology arm of the vector sequence to the primer for amplifying the target gene GP2. The primer sequence is:

[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 GP2 gene was amplified with a size of 456 bp.

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

[0045] To construct the GP2 gene recombinant plasmid, the target fragment and pET-30a vector were linearized by double restriction digestion with BamH I and Hind III and then connected. The ligation product was transformed into DH5α cloning competent cells, cultured at 37°C for 4 hours, and then sent to Qingke Biotechnology Company for sequencing after PCR identification of the bacterial solution. The plasmid with the correct sequencing result was selected for expansion and culture, and the recombinant plasmid pET-30a-GP2 was stored at -20°C for future use.

[0046] In order to analyze the antigenic epitope of GP2 protein, a series of truncated primers were designed and a BamH I restriction site was introduced at the 5′ end of the primer, and a Hind III restriction site was introduced at the 3′ end. The primer sequences are shown in the table (Table 1) and sent to Shanghai Shenggong Company for synthesis. Using the recombinant plasmid pET-30a-GP2 as a template, 13 GP2 protein gene truncations were amplified by PCR, and the truncations and pET-30a vector were double-digested and connected to construct 13 GP2 protein truncated recombinant plasmids.

[0047] Table 1 GP2 protein truncated PCR primer sequences

[0048]

[0049]

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

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

[0052] The GP2 protein gene PCR amplification product was cloned into the pET-30a vector and transformed into E. coli after correct identification. After IPTG induction, SDS-PAGE identification showed that the target protein size was 22.6 kDa, which was consistent with the expected protein size (see Figure 1 A), after the inclusion bodies were purified and concentrated, the Western blot results showed a single band, indicating that the purification effect was good (see Figure 1 (B, C).

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

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

[0055] The recombinant protein purified in Example 2 and complete Freund's adjuvant were mixed and emulsified in a volume ratio of 1:1, and then 6-8 week old female BALB / c mice (100 μg / mouse) were immunized by multiple subcutaneous injections in the back of the neck. After emulsification with incomplete Freund's adjuvant, the second immunization (100 μg / mouse) and the third immunization (100 μg / mouse) were performed in the same immunization route as the first immunization, and the immunization interval was 2 weeks. Blood was collected two weeks after the third immunization, and the purified recombinant GP2 protein was subjected to WB specific analysis using mouse serum as the primary antibody; the purified recombinant GP2 protein was used as an antigen to coat the ELISA plate, and the mouse serum antibodies were detected by the indirect ELISA method. When the serum titer reached the fusion requirement, shock immunization was performed, and the immunization method was intraperitoneal injection (100 μg / mouse).

[0056] (2) Cell fusion and hybridoma cell screening

[0057] Prepare feeder layer cells one day in advance and culture them in 96-well plates for later use. Digest and centrifuge the SP2 / 0 cells in good growth state and fuse them with spleen cells at a cell number ratio of 1:5 under the action of PEG, and culture them in medium supplemented with HAT. Observe cell colonies after three days, replace medium containing HT on the seventh day, and screen positive cell wells by indirect ELISA and IFA methods on the tenth day. Subclone the initially screened cell lines three times by limiting dilution method until hybridoma cells proliferated from a single cell population that can stably secrete anti-GP2 protein antibodies are screened, and then subculture and expand the culture to T75 cell bottles, freeze them, and store them in liquid nitrogen.

[0058] (3) Preparation of monoclonal antibody ascites

[0059] Three 8-week-old female BALB / c mice were used for ascites preparation. First, 0.5 mL of paraffin oil was given to each experimental mouse through the peritoneal route for sensitization pretreatment. After a 7-day sensitization period, hybridoma cell lines in the logarithmic proliferation phase were selected, and the concentration of the cell suspension was adjusted to 1-2×10^6 cells / mL. Subsequently, 0.5 mL of PBS mixed solution containing cells at this concentration was injected into the peritoneal cavity of each experimental mouse to complete the immune cell implantation operation. After 7 days, the state of the mice was observed, and the ascites was collected when their abdominal circumference became larger, they had difficulty eating, and they had difficulty walking. The collected ascites was centrifuged at room temperature at 3000r / min for 10 minutes, and the colorless and transparent middle layer was aspirated as the ascites, and the ascites titer was detected by Elisa method.

[0060] The results showed that the serum titer of mice after immunization twice could reach 1:12800 (see Figure 2). Cell fusion can be performed 3 days after shock immunization. Through ELISA screening and combined with subclone screening technology, a positive hybridoma cell line that can continuously produce GP2 antigen was successfully screened out. The cell line was named: 2G10C3 (hybridoma cell line PRRSV-GP2-2G10C3), and its Latin name was classified as Hybridoma cell line. It was deposited in the China Center for Type Culture Collection on January 20, 2025, with the deposit number CCTCC NO: C202551. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a zip code of 430072 and a contact number of 027-68752319.

[0061] The culture supernatant of hybridoma cells cultured to the fifteenth generation was collected. The results in Table 2 showed that the titer was basically stable. The titer of mouse ascites showed >1×10 6 .

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

[0063]

[0064] Example 4: Identification of biological properties of monoclonal antibodies

[0065] (1) Stability of antibody secretion from hybridoma cell lines

[0066] The positive hybridoma cells obtained in Example 3 were serially passaged, and the monoclonal hybridoma cell supernatants of the 5th, 10th, and 15th passages were collected to detect the antibody titer using the indirect ELISA method to determine the stability of antibody secretion by the hybridoma cell line.

[0067] (2) Western blot specificity identification

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

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

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

[0071] Marc-145 cells were subcultured to six-well plates. When the cell density reached 80%, the medium was discarded, the cells were washed twice with PBS, and replaced with serum-free medium. The cells were infected with PRRSV HuN4-F112. When the cells showed pathological changes but did not fall off in large quantities, the cells were fixed with ice methanol, incubated at 4°C for 10 minutes, washed 3 times with PBS, and inverted at -20°C. Marc-145 cells stored in the laboratory were GP2 cell line cells were plated and fixed with ice methanol after they were fully grown, incubated at 4°C for 10 min, washed three times with PBS, and both samples were blocked with 5% skim milk solution at 37°C for 2 h, and washed three times with PBS; the monoclonal cell supernatant was added to the culture plate as the primary antibody, 500 μL / well, incubated at 37°C for 1 h, and washed three times with PBS; goat anti-mouse fluorescent secondary antibody (1:2000 dilution) was incubated at 37°C for 1 h, washed three times with PBS, incubated with DAPI stain at room temperature in the dark for 10 min, washed three times with PBS, and the results were observed under a fluorescence microscope and photographed.

[0072] The results are as follows Figure 4 As shown: 2G10C3 mAb can produce specific fluorescence with Marc-145 GP2 cell line and Marc-145 cells infected with PRRSV HuN4-F112.

[0073] (4) Identification of monoclonal antibody subtypes

[0074] According to the instructions for monoclonal antibody subtype identification, the monoclonal antibody cell supernatant was diluted at 1:200, and the sample to be tested was added to the strip, 50 μL per well; 1× goat anti-mouse IgM+IgG-HRP (diluted at 1:100) was added, 50 μL per well, and mixed; incubated at room temperature for 1 hour; the liquid in the well was discarded, and the plate was washed 3 times with PBST; the color development solution was added, and the color was developed at room temperature in the dark for 10-20 minutes, and the stop solution was added, and the OD of each well was measured. 450 The well with the highest value is the corresponding mAb subtype.

[0075] The results showed that the heavy chain of 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) Monoclonal Antibody Epitope Identification

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

[0080] Results Figure 5 By constructing a series of GP2 protein truncations, western blot results showed that the prokaryotic expression proteins of the 13 GP2 protein gene truncations recombinant plasmids all reacted with His mouse monoclonal antibody, indicating that the recombinant plasmid was expressed correctly. The preliminary identification results of the antigen epitope recognized by the mAb reacted with F1, F2 and F3, and recognized 131-148aa ( Figure 5 Similarly, Figure 5 The results in B show that it only reacts with F5 and F7 and recognizes 131-142aa. Further verification results show that ( Figure 5 In C), it reacts with F8 and F11 and recognizes 132-140aa. In order to further refine the minimum recognition region, truncated expressions of F12 and F13 were constructed and combined with adjacent recognition regions. The results showed that it reacted with both F12 and F13. Finally, it was inferred that the antigen epitope recognized by 2G10C3 monoclonal antibody is 133 EATLSRI 139 ( Figure 5 (middle D).

[0081] (6) Comparative analysis of amino acid sites of GP2 monoclonal antibody epitopes

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

[0083] The amino acid analysis of GP2 protein of four major epidemic lineages of PRRSV-1 and 2 (Lineage 1 3 58) was performed using Megalign software. Figure 6 Middle A, 133 EATLSRI 139 In the European strain137 SRI 139 There are large differences, but they are more conservative among the four lineages of American strains.

[0084] The three-dimensional structure of PRRSV GP2 was constructed using the SWISS-MODEL server, and the spatial location of the newly identified linear epitopes was visualized using PyMOL software. 133 EATLSRI 139 It exists in the form of an α-helical epitope. This epitope is exposed on the surface of the structure, which is conducive to the identification of the antigenic epitope. These findings suggest that this antigenic epitope may be an important linear B cell epitope in PRRSV GP2 (see Figure 6 (middle 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, had a good reaction, and could be used for the detection of the above strains.

[0088] (8) Sensitivity test of monoclonal antibodies

[0089] The expressed and purified PRRSV GP2 protein was coated on the ELISA coated plate at a concentration of 100 ng / well (the protein was diluted with a coating buffer of pH 9.6), 100 μl per well, and incubated at 4°C overnight; 100 μl of 5% skim milk was added to each well, blocked at 37°C for 2 h, the blocking solution in the plate was discarded, and the plate was washed 3 times with 200 μl PBST, the liquid was discarded for the last time, and the remaining liquid was gently patted dry on absorbent paper; the hybridoma cell supernatant was diluted in a gradient of 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, and 100 μl / well.

[0090] After the wells were added to the 96-well plate, they were incubated at 37°C for 1 hour, the liquid in the plate was discarded, 200 μl PBST was added to each well, washed 3 times, and the remaining liquid was gently patted dry on absorbent paper; HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was diluted 1:5000 and reacted at 37°C for 1 hour, the liquid in the plate was discarded, 200 μl PBST was added to each well, washed 3 times, and the remaining liquid was gently patted dry on absorbent paper; 100 μl TMB color development solution was added, and the color was developed for 15 minutes at room temperature in the dark; 50 μl 2M H2SO4 was added to terminate the reaction, and OD 450nm reading. The result is Figure 8 As shown, the OD value was linearly related to the antibody concentration, and it also had a good reaction with GP2 protein at a dilution ratio of 1:6400, indicating that the antibody had good sensitivity.

[0091] (9) Repeatability testing of monoclonal antibodies

[0092] The GP2 protein prepared in this study was used to coat the ELISA plate at 2 μg / ml, and the cell supernatant collected at the same time was used as the primary antibody to perform intra-batch repeatability tests according to the ELISA detection method used in this case; the cell supernatant collected at different times was used as the primary antibody to perform inter-batch repeatability tests. The results are shown in Tables 4 and 5. The coefficients of variation of intra-batch repeats and inter-batch repeats were both less than 3%, indicating that the antibody had good repeatability.

[0093] Table 4 Intra-batch repeatability test

[0094]

[0095] Table 5 Batch-to-batch repeatability test

[0096]

[0097] In summary, the present invention fused and expressed the GP2 protein of PRRSV HuN4-F112, and used it as an antigen to immunize mice to screen mAbs. A hybridoma cell line that can stably secrete monoclonal antibodies against the GP2 protein was successfully obtained, and the specific antigenic epitope recognized by the mAb was identified, and the conservation and visualization of the antigenic epitope were analyzed. This provides new insights into the antigenicity of the GP2 protein.

[0098] Visualization of protein expression in PRRSV-infected cells is essential for studying the biological properties of GP2 protein. The present invention uses the monoclonal antibody to detect the expression of GP2 protein in PRRSV-infected cells and Marc-145 GP2 cell lines. The IFA test results show that the monoclonal antibodies that recognize the antigenic epitope are able to detect the expression of GP2 protein, and the expressed protein is mainly distributed around the cell nucleus. In the monoclonal antibody detection and reaction experiments with different PRRSV-2 strains, it reacts with NADC30, HuN4, CH-1a and VR2332 strains, has good reactivity, and can be used for the detection of the above strains. In summary, the preparation of GP2 monoclonal antibodies provides new materials for PRRSV serological detection and lays a foundation for further exploring the structure and function of PRRSV GP2 protein.

[0099] Virus protein antigen epitope analysis plays an important role in clarifying protein structure and antigenic characteristics. The present invention constructs and expresses GP2 protein gene truncations and uses developed monoclonal antibodies to identify a B cell linear epitope as 133 EATLSRI 139 , which is different from previous studies and the antigen epitope is more accurately located. By analyzing the differences in GP2 protein amino acids between 21 European and American strains, the results showed that the epitope is well conserved among American strains and has great differences with European strains. The spatial structure of GP2 was analyzed by PyMOL (version 2.5.0) and the epitope was found 133 EATLSRI 139 The part that forms the α-helix has a stable structure and is exposed on the surface of the protein, which is conducive to antibody recognition.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against porcine reproductive and respiratory syndrome virus GP2 protein, characterized in that: Its deposit number is CCTCC NO: C202551.

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

3. The porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody according to claim 2, characterized in that: The antigenic epitope it recognizes is 133 EATLSRI 139 .

4. A porcine reproductive and respiratory syndrome virus detection reagent, chip, test paper or kit, characterized in that: It comprises the monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus as described in claim 2 or 3.

5. A method for preparing a monoclonal antibody against the GP2 protein of porcine reproductive and respiratory syndrome virus, characterized in that: Cultivate the hybridoma cell line described in claim 1; and separate and purify the porcine reproductive and respiratory syndrome virus GP2 protein monoclonal antibody from the cell culture species.

6. A method for non-diagnostic in vitro detection of porcine reproductive and respiratory syndrome virus, characterized in that: The sample to be tested is contacted with the monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus according to claim 2 or 3.

7. Use of the hybridoma cell line according to claim 1, the monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus according to claim 2 or 3, and the detection reagent, chip, test paper or kit according to claim 4 in in vitro non-diagnostic detection of porcine reproductive and respiratory syndrome virus.

8. Use of the hybridoma cell line according to claim 1, the monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus according to claim 2 or 3, and the detection reagent, chip, test paper or kit according to claim 4 in the preparation of a detection product for detecting porcine reproductive and respiratory syndrome virus.

9. Use of the hybridoma cell line according to claim 1, the monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus according to claim 2 or 3, and the detection reagent, chip, test paper or kit according to claim 4 in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus.

10. Use of the hybridoma cell line according to claim 1, the monoclonal antibody against GP2 protein of porcine reproductive and respiratory syndrome virus according to claim 2 or 3, and the detection reagent, chip, test paper or kit according to claim 4 in the preparation of a pharmaceutical preparation for preventing or treating diseases caused by porcine reproductive and respiratory syndrome virus infection.

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