A specific recognition antigen epitope 16 AQTGYAPV 23 Monoclonal antibody D3-mAb against porcine rotavirus and its application

Abstract

This invention discloses a method for specifically recognizing antigen epitopes. 16 AQTGYAPV 23 This invention relates to a monoclonal antibody D3-mAb against porcine rotavirus and its applications, belonging to the field of antibody bioengineering technology. The monoclonal antibody D3-mAb specifically binds to the VP8 protein of porcine rotavirus and exhibits neutralizing activity; its amino acid sequence is shown in SEQ ID NO. 8. This invention screened a monoclonal antibody D3 with neutralizing activity against the VP8 protein, and subsequently identified the neutralizing antigenic epitope. 16 AQTGYAPV 23 Recognized by D3-mAb, sequence comparison analysis of currently circulating PoRVA strains revealed that this antigenic epitope is absolutely conserved. Furthermore... 16 AQTGYAPV 23 The epitope, located on the surface of the VP8 protein, interacts with the antibody's variable region via six hydrogen bonds. This invention provides a novel approach for designing novel epitope vaccines and antiviral strategies, and the provided monoclonal antibody holds promise for development into a biotherapeutic agent and diagnostic agent for porcine rotavirus infection.

Description

Technical Field

[0001] This invention relates to the field of antibody bioengineering technology, and in particular to a method for specifically recognizing antigen epitopes. 16 AQTGYAPV 23 The monoclonal antibody D3-mAb against porcine rotavirus and its application. Background Technology

[0002] In 1976, porcine rotavirus (PoRV) was first isolated from pigs suffering from diarrheal disease. It primarily causes diarrhea in suckling piglets aged 1-4 weeks. Typical clinical manifestations in infected piglets include watery diarrhea, dehydration, and weight loss. Infected piglets are also prone to secondary infections with Escherichia coli or Salmonella, further exacerbating their growth and development. Recent epidemiological surveys show that the infection and morbidity rates of PoRV in Chinese pig herds are rising, posing a significant threat to public health. However, there are currently no effective vaccines or treatments for PoRV control. Therefore, strengthening the research and development of PoRV prevention and control agents is of great importance for PoRV prevention and control.

[0003] PoRV belongs to the genus Rotavirus in the family Reoviridae, and is a non-enveloped double-stranded RNA (dsRNA) virus. The viral genome is approximately 18.5 kb in size, composed of 11 segments encoding six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP5 / NSP6). VP4 (approximately 86 kDa), located in the viral capsid, is a key antigen for inducing neutralizing antibodies and an important target for vaccine development. During viral infection, VP4 can be hydrolyzed by proteases into two proteins: N-terminal VP8 (28 kDa) and C-terminal VP5 (60 kDa). VP8 contains the major antigenic site of VP4, is relatively conserved, and can induce neutralizing antibodies, playing a crucial role in immune protection against rotavirus infection. Studies have found that amino acid segments 26-231 of VP8 can induce higher titers of neutralizing antibodies, providing better protective efficacy.

[0004] Currently, there is no effective treatment for PoRV infection, and vaccination remains the key strategy for controlling the disease. However, while existing commercial vaccines (attenuated vaccines and subunit vaccines) provide some immune protection, they still have limitations. Given the current shortcomings of PoRV vaccines in terms of safety and immunogenicity, the development of novel vaccines and therapeutic drugs with high protective efficacy and good safety is particularly urgent. Compared with vaccines and chemical drugs, specific antibodies have gradually become an important strategy for treating infectious diseases due to their advantages such as fewer side effects, longer half-life, and high specificity. Neutralizing antibodies are specific immunoglobulins secreted by plasma cells that can inhibit pathogen invasion and infection by binding to pathogen surface antigens and blocking their binding to host cells. Currently, various specific neutralizing antibodies against viral infections (such as SARS-CoV-2, Ebola virus, and HIV) have been developed. Monoclonal antibodies have become the mainstream product in the development of neutralizing antibody drugs due to their strong targeting, fewer side effects, and high development success rate.

[0005] Because newborn piglets are highly susceptible to PoRV and suffer the most severe damage, immunization strategies that obtain specific antibodies through breast milk are crucial for their protection. However, existing vaccines have low efficiency in inducing mucosal immunity in sows, and it is difficult to ensure that the colostrum of immunized sows contains high levels of specific antibodies, leading to infection and disease in clinical piglets. Therefore, developing passive immunization and therapeutic antibodies can address the key issues of insufficient passive immunization from breast milk. Summary of the Invention

[0006] The purpose of this invention is to provide a method for specifically recognizing antigen epitopes. 16 AQTGYAPV 23 This invention relates to a monoclonal antibody D3-mAb against porcine rotavirus and its applications, aiming to address the problems existing in the prior art. Based on an insect baculovirus expression system, PoRVA VP8 (26-231 aa) was expressed. Using hybridoma cell fusion technology, a specific monoclonal antibody with neutralizing activity against PoRVA was prepared. Furthermore, the antigenic epitopes recognized by this neutralizing antibody were identified and analyzed, laying the foundation for the development of PoRVA antibody drugs and immunodiagnostic technologies.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a monoclonal antibody D3-mAb, wherein the monoclonal antibody D3-mAb specifically binds to the antigenic epitope of porcine rotavirus VP8 protein. 16 AQTGYAPV 23 It has neutralizing activity and contains a light chain as shown in SEQ ID NO.8 and a heavy chain as shown in SEQ ID NO.9.

[0008] The second technical solution of the present invention is the application of the monoclonal antibody D3-mAb in the preparation of vaccines or drugs, wherein the vaccines or drugs are used to prevent and / or treat porcine rotavirus infection.

[0009] The third technical solution of the present invention is a detection reagent or kit containing the monoclonal antibody D3-mAb.

[0010] The fourth technical solution of the present invention is the application of the monoclonal antibody D3-mAb in the preparation of reagents or kits for detecting porcine rotavirus.

[0011] The fifth technical solution of the present invention is the application of the monoclonal antibody D3-mAb in the preparation of a drug for the prevention or treatment of porcine rotavirus infection.

[0012] The sixth technical solution of the present invention is a drug for preventing and / or treating porcine rotavirus infection, comprising the monoclonal antibody D3-mAb and a pharmaceutically acceptable carrier.

[0013] Based on the above technical solution, the present invention has the following technical effects: This invention successfully expressed the PoRV VP8 protein using an insect baculovirus expression system. The expressed VP8 protein could be released into the supernatant of Sf9 cell culture while retaining its original structure. A monoclonal antibody D3 with neutralizing activity was screened for the VP8 protein, and subsequently, neutralizing epitopes were identified. 16 AQTGYAPV 23 Recognized by D3-mAb, sequence comparison analysis of currently prevalent PoRVA strains revealed that this epitope is absolutely conserved. Furthermore... 16 AQTGYAPV 23 The epitope, located on the surface of the VP8 protein, interacts with the antibody's variable region via six hydrogen bonds. This invention provides a novel approach for designing novel epitope vaccines and antiviral strategies, and the provided monoclonal antibody holds promise for development into a biotherapeutic agent and diagnostic agent for porcine rotavirus infection. Attached Figure Description

[0014] Figure 1 This study focuses on the expression and identification of the PoRV VP8 protein. Lane A represents PCR and restriction enzyme digestion identification of the recombinant plasmid pFastBac HTB-VP8. Lane M represents the DL5000 DNA Marker; lanes 1-2 represent PCR identification of the empty plasmid pFastBac HTB and the recombinant plasmid pFastBac HTB-VP8, respectively; lanes 3-4 represent the expression and identification of the empty plasmid pFastBac HTB and the recombinant plasmid pFastBac HTB-VP8, respectively. BamH I- SalI: Double enzyme digestion identification. B: PCR identification of DH10Bac single colonies. Lane M: DL5000 DNA Marker; Lane 1: PCR results using VP8 specific primers; Lane 2: PCR results using M13 primers. C: SDS-PAGE analysis. Lane M: 10-250kDa protein Marker; Lanes 1 and 2: Culture supernatant and cell lysate of Sf9 cells infected with recombinant baculovirus, respectively; Lanes 3 and 4: Culture supernatant and cell lysate of Sf9 cells, respectively. D: Western blot identification. Lane M: 10-250kDa protein Marker; Lanes 1-2: Culture supernatant and cell lysate of VP8 recombinant baculovirus, respectively; Lanes 3-4: Culture supernatant and cell lysate of Sf9 cells. E: IFA identification. Infect: Sf9 cells infected with recombinant baculovirus; NC: Sf9 cell control.

[0015] Figure 2 This section describes the identification and affinity assay of monoclonal antibodies. A: Western blotting. Lane M: 10-250 kDa protein marker; Lanes 1-2: VP8 protein and PoRVA reaction results with VP8 mouse antiserum (positive control); Lanes 3-4: VP8 protein and PoRVA reaction results with D3 hybridoma cell supernatant; Lanes 5-6: VP8 protein and PoRVA reaction results with SP2 / 0 cell supernatant (negative control). B: IFA. C: Type identification. D: Ascites antibody titer. E: Specificity assay. F: Affinity analysis.

[0016] Figure 3 This study aims to determine the neutralizing activity of monoclonal antibodies. A: IFA detection; B: Analysis of fluorescence intensity using ImageJ; C: qRT-PCR detection; D: TCID. 50 Determination; E: Determination of antibody neutralizing titer.

[0017] Figure 4 Epitopes recognized by D3-mAb and their conservation analysis. A and B: ELISA and Dot-blot identification of antigenic epitopes recognized by D3-mAb; C: Conservation analysis of the amino acid sequences of antigenic epitopes recognized by D3-mAb among common PoRVA genotypes.

[0018] Figure 5 This study aims to predict the locations of synthesized epitopes in the VP8 protein and analyze the interaction between antibodies and antigenic epitopes. In the diagram, A represents the predicted locations of the five synthesized epitopes in the VP8 protein. Gray-blue represents the VP8 protein, and red represents the epitopes. 16 AQTGYAPV 23 Green: Epitope 49FNPPVSYWILLSPFNAGVVVE 69 Yellow: Epitope 77 WLATILIEPNVT 88 Pink: Tablet 128 TQHGPLLSDTKLYGVM 143 Orange: Epitope 172 VNMVSHCDFYIIP 184 B: Antibodies and 16 AQTGYAPV 23 Epitope complex structure prediction. Gold: VH, Blue: VL, Red: Epitope 16 AQTGYAPV 23 Green: Amino acid residues on the antibody that form hydrogen bonds with the epitope.

[0019] Figure 6 Western blot, immunohistochemistry, and immunofluorescence were used to detect specific antigens. A: Western blot detection of expressed VP8 recombinant protein. Lane M: 10-180 kDa protein marker; Lanes 1-2: VP8 protein expressed by baculovirus; Lane 3: Sf9 cell culture supernatant control; Lane 4: Sf9 cell lysate control. B: Immunohistochemical detection of viral antigens in the small intestine tissue of PoRVA (rotavirus type A) infected piglets; NC: Control piglet small intestine. C: Immunofluorescence identification of VP8 recombinant protein expressed in Sf9 cells; NC: Sf9 cell control.

[0020] Figure 7 To identify porcine rotavirus type A isolated from MA-104 cells using D3-mAb. Detailed Implementation

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention provides a monoclonal antibody D3-mAb, which specifically binds to the antigenic epitope of porcine rotavirus VP8 protein. 16 AQTGYAPV 23 It has neutralizing activity and contains a light chain as shown in SEQ ID NO.8 and a heavy chain as shown in SEQ ID NO.9.

[0023] In some specific implementations, the monoclonal antibody D3-mAb is the IgG2b subtype, and the light chain is the κ chain.

[0024] This invention also provides the application of the monoclonal antibody D3-mAb in the preparation of vaccines or drugs for the prevention and / or treatment of porcine rotavirus infection.

[0025] This invention also provides a detection reagent or kit containing the monoclonal antibody D3-mAb.

[0026] This invention also provides the application of the monoclonal antibody D3-mAb in the preparation of reagents or kits for detecting porcine rotavirus.

[0027] This invention also provides the use of the monoclonal antibody D3-mAb in the preparation of a drug for the prevention or treatment of porcine rotavirus infection.

[0028] This invention also provides a medicament for the prevention and / or treatment of porcine rotavirus infection, comprising the monoclonal antibody D3-mAb and a pharmaceutically acceptable carrier.

[0029] PoRV has multiple groups, including A, B, and C, with group A PoRV (PoRVA) being the most prevalent. However, there is currently no ideal immunomodulatory and therapeutic agent. VP4 (which can be hydrolyzed by proteases into C-terminal VP5 and N-terminal VP8) is located on the outer layer of the rotavirus particle and participates in viral adsorption and invasion of host cells, making it a key target for vaccine research and antibody preparation. Neutralizing antibodies are important biological agents for the specific treatment of viral infections and have great development potential. This invention utilizes hybridoma technology to screen and obtain a stable hybridoma cell line (D3) that secretes a monoclonal antibody (mAb) against PoRVA VP8. This monoclonal antibody belongs to the IgG2b subtype, with a κ light chain, and an antibody titer of 10 in mouse ascites fluid. 6 It exhibits no cross-reactivity with other proteins and can neutralize the PoRVA virus. Further analysis of the amino acid sequence of D3-mAb, using indirect ELISA and dot blot hybridization techniques, revealed that D3-mAb specifically recognizes the epitope of the PoRVA-VP8 protein. 16 AQTGYAPV 23 It is conserved among PoRVA strains. Analysis using PyMOL molecular graphics software (v2.6.0) revealed that the epitope recognized by D3-mAb is located on the surface of the VP8 protein, where... 16 A, 19 G, 23 N、 20 Y interacts with multiple amino acids in the heavy chain variable region (VH) and light chain variable region (VL) of D3-mAb, respectively. The results of this invention provide important theoretical basis and candidate materials for the development of PoRV antibody drugs and immunodiagnostic technologies.

[0030] Example 1 1. Materials and Methods 1.1 Cells and Viruses Spodoptera frugiperda Sf9 cells were donated by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. The cell culture medium was Sf-900 III SFM (Womei Biotechnology Co., Ltd., China) containing 1% penicillin-streptomycin. The cells were cultured at 27°C and 120 r / min for later use.

[0031] Myeloma cells (SP2 / 0) and MA-104 cells were preserved in our laboratory. The PoRVA G9P strain was also preserved in our laboratory, and its half-maximal tissue culture infectious dose (TCID) was [not specified]. 50 ) is 10 5.4 / mL.

[0032] 1.2 Laboratory Animals and Animal Welfare 6-8 week Balb / c mice were purchased from Beijing Speford Biotechnology Co., Ltd. and housed in an SPF-grade mouse filtration system. Throughout the animal experiments, the operators strictly adhered to the regulations established by the Experimental Animal Management and Ethics Committee of Hebei Agricultural University, and the experimental procedures were conducted entirely in accordance with the National Guidelines for Laboratory Animal Welfare.

[0033] 1.3 Primer Design and Synthesis Primers for PCR amplification of the VP8 nucleotide sequence were designed based on the PoRV reference sequence (MH137266.1) published in GenBank. Nucleotide restriction endonucleases were inserted at the 5′ ends of the upstream and downstream primers, respectively. Bam HI and Sal I site (Table 1). Primers were synthesized by Sangon Biotech Co., Ltd. In addition, universal primers (M13-F and M13-R) for recombinant plasmid identification and PoRVA detection primers (F-PoRVA-VP6 and R-PoRVA-VP6) were synthesized (Table 1).

[0034] Table 1 PCR primer sequences

[0035] 1.4 Expression and identification of recombinant VP8 protein 1.4.1 Preparation of VP8 recombinant baculovirus PoRVA RNA was extracted using a viral RNA extraction kit (Beijing Tiangen Biotech Co., Ltd., China). After reverse transcription into cDNA, the VP8 gene sequence was obtained by PCR amplification using primers eF-VP8 and eR-VP8.

[0036] Then, the gene fragment was cloned into the pFastBac HTB vector plasmid, transfected into DH10Bac competent cells, and recombinant rod granules were obtained.

[0037] Subsequently, the recombinant rod-like particles were identified by PCR using VP8 gene sequence-specific primers (eF-VP8 and eR-VP8) and M13 primers (M13-F and M13-R). The correctly identified recombinant rod-like particles were transfected into Sf9 cells using ExpiFectamine SF (Thermo Fisher Scientific, USA). After 96 h, the culture supernatant was harvested by centrifugation at 1500 rpm / min for 10 min, representing the first generation (P1) virus. The cells were then continuously cultured at 27 °C for three passages.

[0038] 1.4.2 Identification of recombinant proteins 1.4.2.1 SDS-PAGE Analysis Sf9 cells were infected with P3 generation virus at a dose of 8 multiples of infection (MOI) and cultured at 27 °C for 96 h. Cell culture supernatant and cell pellet were harvested separately. The cell pellet was resuspended in 0.01 mol / L phosphate-buffered saline (PBS, pH 7.2-7.4), sonicated, and centrifuged at 10,000 rpm for 10 min. The supernatant was the lysed cell solution. Protein expression was analyzed by SDS-PAGE of both the cell culture supernatant and the lysed cell solution.

[0039] 1.4.2.2 Western blot analysis The supernatant of recombinant baculovirus culture and the supernatant of lysed cells were subjected to SDS-PAGE, and the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane for antigen-antibody reaction. For Western blotting analysis, PVDF membranes were blocked at room temperature for 2 h with Tris buffered saline (TBST) containing 5% skim milk powder and 0.05% Tween-20 (Beijing Solarbio Biotechnology Co., Ltd., China). The primary antibody, porcine PoRV antiserum (prepared and stored in our laboratory), was diluted 1:50 and incubated at room temperature for 2 h. After washing with TBST for 10 min each time, for a total of 3 times, HRP-labeled goat anti-porcine IgG (Beijing Bio-Long Immunotherapy Co., Ltd., China) was added at a 1:5000 dilution and incubated at room temperature for 1.5 h. After washing 3 times, chemiluminescence ultrasensitive colorimetric reagent kit (Yisheng Biotechnology Co., Ltd., Shanghai, China) was used for color development, and the specific reaction bands were observed using a chemiluminescence imaging system (Gene Company Limited, USA) to identify the expression of the target recombinant protein (26 kDa).

[0040] 1.4.2.3 Indirect Immunofluorescence Assay (IFA) Add 100 μL of Sf9 cells to the corresponding wells of a 96-well cell culture plate, 5 × 10⁶ 4 Cells / well were simultaneously infected with P3 generation virus at a dose of 0.1 MOI and cultured at 27 ℃ for 48 h; 50 μL of pre-chilled anhydrous methanol was added to each well and the cells were fixed at -20 ℃ for 10 min; the cells were washed 3 times with PBS and blocked with PBS containing 2% bovine serum albumin (BSA, Guangzhou Saiguo Biotechnology Co., Ltd., China) at 37 ℃ for 1 h; the membrane was immersed in a solution of specific VP8 mouse antiserum (prepared in our laboratory) diluted 100 times with 1% BSA-PBS and incubated at 37 ℃ for 1 h; the membrane was washed 3 times with PBS, 3 min each time; the membrane was immersed in a solution of YF594-labeled goat anti-mouse fluorescent antibody (Suzhou Youyilandi Biotechnology Co., Ltd., China) diluted 1:500 and incubated at 37 ℃ for 45 min; after washing, 1:2000 diluted 4',6-diamidinyl-2-phenylindole (10 μg / mL) was added. DAPI (50 μL per well) was stained at room temperature in the dark for 10 min, washed twice with PBS, and the specific fluorescence signal was observed under a fluorescence microscope (Carl Zeiss, Germany).

[0041] The amino acid sequence of the recombinant VP8 protein is shown in SEQ ID NO.7.

[0042] SEQ ID NO.7: MASLIYRQLLSNSYTVDLSDEIQTIGSEKTQNVTINPGPFAQTGYAPVNWGPGETSDSTVEPVLDGPYQPTTFNPPVSYWILLSPSNAGVVVEGTNNSDRWLATILIEPNVT SQNRTYTLFGQQEQITVENVSTTKWKFVDLAKTDVNGTFTQHGLLLSDTKLYGVMKFSGRLYTYNGETPNATTGYYTTTNYDAVNMTSHCDFYIIPRSEENACTNYINNGLPPIQNTR.

[0043] 1.5 Preparation of Monoclonal Antibodies Four 6-8 week old Balb / c mice were injected subcutaneously at multiple sites on their backs with recombinant VP8 protein emulsified with Freund's adjuvant (Sigma, USA), with each mouse receiving 50 μg of protein. The injections were repeated three times, 14 days apart.

[0044] Three days prior to cell fusion, 80 μg of VP8 protein was injected intraperitoneally. Spleens were harvested from immunized mice, and spleen cell suspensions were prepared. These spleen cell suspensions were then fused with SP2 / 0 cells using PEG4000 (Solomon Syringe Technology Co., Ltd., China).

[0045] The fused cells were added to 96-well cell culture plates and cultured in a 37°C incubator under 5% CO2 conditions. For the first two weeks, the fused cells were cultured in DMEM medium containing 1% HAT (Gibco, USA) and 10% FBS, followed by DMEM medium containing 1% HT (Gibco, USA) and 10% FBS. The supernatant from the wells showing fused hybridoma cell growth was observed and collected. ELISA was used to detect specific antibodies, and antibody-secreting positive hybridoma cells were screened.

[0046] The specific steps of ELISA are as follows: Coat each well of the microplate with 1 μg / mL VP8 recombinant protein, and block with PBS (PBST) containing 5% skim milk powder and 0.05% Tween-20 at 37 ℃ for 1 h; discard the liquid in the wells, add hybridoma cell supernatant and SP2 / 0 cell supernatant (1:2 dilution), and incubate at 37 ℃ for 1 h; wash the plate 3 times with PBST, 3 min each time; add HRP-labeled goat anti-mouse IgG (1:10000 dilution) (Beijing Bio-Long Immunotherapy Co., Ltd., China), and incubate at 37 ℃ for 45 min; wash the plate 3 times, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, Beijing Solarbio Science & Technology Co., Ltd., China) to each well, and develop color at 37 ℃ in the dark for 15 min; add 50 μL of 2mol / L... Sulfuric acid was used to terminate the color development. The absorbance at 450 nm was read using a full-wavelength microplate reader (Gene Company Limited, USA) to screen for positive hybridoma cells that secrete specific antibodies.

[0047] Then, the positive hybridoma cells selected by ELISA were subcloned five times using the limiting dilution method to obtain monoclonal hybridoma cell lines that could stably secrete VP8-specific antibodies.

[0048] 1.6 Identification of Monoclonal Antibodies 1.6.1 Western blotting Western blotting was performed on the culture supernatant of the selected hybridoma cells (D3) and the recombinant VP8 protein or PoRVA protein transferred onto the PVDF membrane after SDS-PAGE.

[0049] The main steps of Western blotting are as follows: PVDF membranes transferred with target proteins are blocked at room temperature for 2 h with TBST containing 5% skim milk powder; D3 hybridoma cell supernatant (1:100 dilution) is added and incubated at room temperature for 2 h; the membrane is washed 3 times, HRP-labeled goat anti-mouse IgG (1:10000 dilution) is added and incubated at room temperature for 1.5 h; the membrane is washed, and chromogenic working solution is added according to the instructions of the chemiluminescence ultrasensitive colorimetric reagent kit. After incubation at room temperature, the reaction bands are observed on a chemiluminescence imaging system.

[0050] 1.6.2 IFA Qualification MA-104 cells with good growth in 96-well cell culture plates were infected with PoRVA at 0.2 MOI and cultured in a 5% CO2 incubator at 37°C for 24 h. A blank control (NC) without virus infection was also established. After fixation with pre-cooled anhydrous methanol, cells were subjected to in vitro immunoassay (IFA). During IFA, the supernatant of the hybridoma cells to be tested (D3 monoclonal antibody) was diluted 1:50 and incubated at 37°C for 1 h. YF594-labeled goat anti-mouse fluorescent antibody was diluted 1:500 and incubated at 37°C for 45 min. Then, DAPI diluted 1:2000 was added to stain the cell nuclei, and the fluorescence signal was observed.

[0051] 1.6.3 Antibody type identification The antibody types secreted by the positive hybridoma cell line D3 were detected using the ELISA method according to the instructions of the mouse monoclonal antibody Ig class / subclass / identification ELISA kit (Beijing Bio-Long Immunotherapy Co., Ltd., China).

[0052] 1.6.4 Identification of the specificity of antibody-antigen reactions PoRVA VP6 protein, the S1 region (S1) and nucleocapsid (N) protein of porcine epidemic diarrhea virus (PEDV), P72 and CD2V proteins of African swine fever virus (ASFV), N protein of transmissible gastroenteritis virus (TGEV), and capsid protein of porcine circovirus type 2 (PCV2) were coated onto each well of an ELISA plate (0.1 μg / well). A VP8 protein control was also included. The cross-reactivity of the obtained VP8 monoclonal antibody with these proteins was detected by indirect ELISA to analyze the specificity of the monoclonal antibody-antigen binding.

[0053] PoRVA VP6 protein sequence (GenBank ID: 581873, SEQ ID NO.10): MEVLYSLSKTLKDARDKIVEGTLYSNVSDLIQQFNQMIVTMNGNDFQTGGIGNLPIRNWTFDFGLLGTTLLNLDANYVENARTTIEYFIDFIDNVCMDEIARESQRNGIAPQSEALRKLSGIKFKRINFDNSSDYIENWNLQNRRQRTGFVFHKPNILPYSASFTLNRSQPAHDNLMGTMWINAGSEIQVAGFDYSCAFNAPANIQQFEHVVPLRRALTTATITLLPDAERFSFPRVINSADGTTTWYFNPVILRPSNVEVEFLLNGQIINTYQARFGTIIARNFDTIRLSFQLVRPPNMTPAVANLFPQAPPFIFHATVGLTLRIESAVCESVLADASETLLANVTAVRQEYAIPVGPVFPPGMNWTELITNYSPSREDNLQRVFTVASIRSMLIK。

[0054] PEDV S1 protein sequence (GenBank ID: JX163294, SEQ ID NO.11): MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGRHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYSANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDTSVILAEGSIVLHTALGTNFSFVCSNSSDPHLATFAIPLGAIQVPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIHRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGRSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFRGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNIGIPTNFS。

[0055] PEDV N protein sequence (JN601056, SEQ ID NO.12): MASVSFQDRGRERVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNLGVRKASEKPIIPKFSQQLPSVVEIVEPNTPPASRTNSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNKSRNQSKNRNQSNDRGGMTSRDDLVAAVKDALKSLGIGENPDRHKQQQKPKQEKSDNSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAIPQRKKEKKNKRETTQQQNEEAIYDDVGVPSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN。

[0056] ASFV P72 protein sequence (MK333180, SEQ ID NO.13): MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST。

[0057] ASFV CD2V protein sequence (MK333180, SEQ ID NO.14): MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETTLINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPLPKPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRII。

[0058] TGEV N protein sequence (GenBank ID: KX499468, SEQ ID NO.15): MANQGQRVSWGDESTKTRGRSNSRGRKNNNIPLSFFNPITLQQGSKFWNLCPRDFVPKGIGNRDQQIGYWNRQTRYRMVKGQRKELPERWFFYYLGTGPHADAKFKDKLDGVVWVAKDGAMNKPTTLGSRGANNESKALKFDGKVPGEFQLEVNQSRDNSRSRSQSRSRSRNRSQSRGRQQFNNKKDDSVEQAVLAALKKLGVDTEKQQQRSRSKSKERSNSKTRDTTPKNENKHTWKRTAGKGDVTRFYGARSSSANFGDTDLVANGSSAKHYPQLAECVPSVSSILFGSYWTSKEDGDQIEVTFTHKYHLPKDDPKTGQFLQQINAYARPSEVAKEQRKRKSRSKSAERSEQDVVPDALIENYTDVFDDTQVEIIDEVTN。

[0059] PCV2 Cap protein sequence (MK585076, SEQ ID NO.16): MTYPRRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPPLTVPFEYYRIRKVKVEFWPCSPITQGDRG VGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK.

[0060] 1.7 Determination of antibody affinity D3 ascites was prepared using the in vivo mouse ascites induction method. The harvested ascites was purified using the ammonium octanoate-sulfate method, and the affinity of the monoclonal antibody was determined by indirect ELISA. Specifically, ELISA plates were coated with 0.1 and 0.2 μg / mL VP8 protein, respectively, and blocked with PBST containing 5% skim milk powder at 37 °C for 1 h. Serially diluted ascites monoclonal antibody (10 μg / mL ~ 0.078 μg / mL) was added and incubated at 37 °C for 1 h. After washing three times with PBST, HRP-labeled goat anti-mouse IgG (1:10000 dilution) was added and incubated at 37 °C for 45 min. After three washes, TMB chromogenic buffer was added, and the plates were incubated at 37 °C in the dark for 15 min. Finally, 50 μL of 2mol / L sulfuric acid was added to each well to stop the color development. The absorbance at 450 nm was read, the dissociation constant Kd was calculated, and the affinity of the D3-mAb was analyzed.

[0061] 1.8 Detection of antibody neutralizing activity D3-mAb was diluted 1:2 to 1:8192 times and mixed with an equal volume of 200 TCID50. 50After mixing with PoRVA, the mixture was incubated at 37°C for 1 h. A negative control (empty cell control, NC) and a positive control (PoRVA control, PC) were also established. The mAb-virus mixture was inoculated onto well-grown MA-104 cells and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times with PBS. Virus culture maintenance medium was added, and the cells were cultured for another 24 h. After fixing and blocking the cells, 1:100 diluted specific VP8 mouse antiserum was added, and the cells were incubated at 37°C for 1 h. After washing with PBS, YF594-labeled goat anti-mouse fluorescent antibody (1:500 dilution) was added, and the cells were incubated at 37°C for 45 min. DAPI was added, and the cell nuclei were stained. The fluorescence signal was observed under a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ (National Institutes of Health, USA) software.

[0062] D3-mAb at different dilutions (1:2, 1:8, and 1:32) was mixed with 200 TCID2 50 After incubation with PoRVA, the virus was inoculated into MA-104 cells, and the viral fluid was harvested. Viral nucleic acid concentration was determined by qRT-PCR. The qRT-PCR used PoRVA VP6 detection primers (F-PoRVA-VP6 and R-PoRVA-VP6). The amplification program was: pre-denaturation at 95 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 5 s, and 72 °C for 25 s. Additionally, the TCID of the virus was determined using the Reed-Muench two-factor assay. 50 The inhibitory effect of mAb on PoRVA was analyzed.

[0063] Furthermore, the neutralizing titer of D3-mAb against PoRVA was determined using the fixed-virus dilution antibody method, which involves serially diluting D3-mAb at concentrations of 1:8 to 1:2048 and reacting it with 200 TCID50 solutions. 50 PoRVA was mixed, and six replicates were performed for each antibody dilution. The antibody-virus mixture was incubated at 37°C for 1 h, then seeded into a monolayer of MA-104 cells in 96-well plates and cultured at 37°C with 5% CO2 for 72 h. The reduction in cytopathic effects was counted, and the neutralization efficiency of D3-mAb against PoRVA at each dilution was calculated using the Reed-Muench two-factor method to determine the antibody's neutralizing titer.

[0064] 1.9 Identification and Conservation Analysis of Antibody-Recognized Antigenic Epitopes Linear epitopes of the VP8 protein were predicted using the website http: / / imed.med.ucm.es / Tools / antigenic.html, and five peptides were designed and synthesized (Table 2). Keyhole hemocyanin (KLH) was conjugated to the C-terminus of each peptide. Epitopes were identified by indirect ELISA and dot blot hybridization. For ELISA, the peptides and VP8 protein were coated onto the microplates, 0.1 μg / well; D3-mAb (1:1000 dilution) and HRP-labeled goat anti-mouse IgG (1:10000 dilution) were used as primary and secondary antibodies, respectively. For Dot-blot assays, 10 μL of each synthesized epitope peptide (10 μg) and 10 μL of VP8 protein (2 μg) were added to an activated NC membrane, which was then allowed to air dry at room temperature. After blocking with 5% skim milk powder for 2 h at room temperature, the membrane was reacted sequentially with D3-mAb (1:500 dilution) and HRP-labeled goat anti-mouse IgG (1:10000 dilution) for 1.5–2 h at room temperature. A negative control of SP2 / 0 culture supernatant was also included. Finally, the formation of specific spots was observed using chemiluminescent solution.

[0065] Table 2 VP8 fragmented peptide sequences

[0066] To analyze the conservation of the antigenic epitope bound by D3-mAb, the VP4 amino acid sequence of the PoRVA isolate was obtained from the NCBI database and compared using MAGE7 (7.0.26) software to analyze the conservation of the antigenic epitope bound by D3-mAb among different strains.

[0067] 1.10 Prediction of Monoclonal Antibody-Antigen Epitope Interaction Sites The prepared D3-mAb was sent to Sangon Biotech (Shanghai) Co., Ltd., where high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine and analyze the amino acid sequence of the antibody (both light and heavy chains, excluding the signal peptide). The sequencing results were consistent with the predicted molecular weight. The sequencing results showed that the amino acid sequence of the monoclonal antibody D3-mAb contained the light chain as shown in SEQ ID NO. 8 and the heavy chain as shown in SEQ ID NO. 9.

[0068] SEQ ID NO.8: DVLMTQTPLSLPVSLGDQASISCRSSQSMVHSDGNTYLEWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGT KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID NO.9: QVQLQQSGPELARPWASVKISCQAFYTFSRRVYFAIGDTNYWMQWVKQRPGQGLEWIGAISPGNGDTTYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTLYGS SQYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG PTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0069] Then, based on the amino acid sequences of D3-mAb and VP8 protein, the location of the epitope recognized by D3-mAb on the VP8 protein was analyzed using PyMOL molecular graphics software (v2.6.0), and the D3-mAb (VH and VL) was compared with... 16 AQTGYAPV 23 Epitope sequences were submitted to the AlphaFold3 server to analyze key interaction sites between epitope amino acids and variable regions of the antibody light and heavy chains.

[0070] 1.11 Statistical Analysis GraphPad Prism 9.0 software was used to perform statistical analysis on the research data. The t-test was used to analyze statistical significance. p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference. p <0.001 indicates a highly significant difference.

[0071] 2 Results 2.1 Expression and identification of VP8 protein The constructed VP8 recombinant expression plasmid pFastBac HTB-VP8, after PCR amplification and enzyme digestion verification, can release a fragment consistent with the size of the target gene (626 bp). Figure 1 (A) indicates that the recombinant expression plasmid pFastBac HTB-VP8 was successfully constructed. The recombinant plasmid was transformed into DH10Bac competent cells, and single colonies were picked. PCR amplification using VP8 gene-specific primers and universal primer M13 successfully amplified the expected size nucleic acid fragment (…). Figure 1 (B) indicates that the identified bacteria are recombinant rod-like particles carrying the VP8 protein gene.

[0072] SDS-PAGE revealed that the target protein of approximately 26 kDa was present in both the culture supernatant and cell lysate of Sf9 insect cells transfected with VP8 recombinant rod cells, while no protein band of the corresponding size was found in the culture supernatant and cell lysate of the Sf9 cell control. Figure 1 (C). Western blot analysis showed that after reacting VP8 virus culture supernatant and lysed cell slurry with porcine PoRV antiserum, a reaction band of approximately 26 kDa was observed, consistent with the expected target protein size. However, no corresponding reaction band was observed in the Sf9 cell control lane. Figure 1 (D). The above results indicate that a recombinant baculovirus expressing the VP8 protein was obtained (named VP8 recombinant baculovirus).

[0073] After infecting Sf9 cells with VP8 recombinant baculovirus for 48 h, IFA identification showed that Sf9 cells inoculated with recombinant baculovirus emitted red fluorescence after reacting with VP8 mouse antiserum, but did not emit red fluorescence after reacting with empty cells. Figure 1 (E). This result indicates that the VP8 protein is expressed in Sf9 cells.

[0074] 2.2 Preparation and Identification of Monoclonal Antibodies The culture supernatant of the obtained D3 hybridoma cell line, after being reacted with recombinant VP8 protein and PoRVA, showed a clear reaction band of 26 kDa on the PVDF membrane, while no reaction band of the same size appeared after being reacted with SP2 / 0 cell supernatant. Figure 2 (A). Additionally, IFA results showed that after the D3 hybridoma cell culture supernatant was reacted with PoRVA-infected MA-104 cells, a red fluorescent signal appeared in the cytoplasm, while no fluorescent signal was observed in the cytoplasm of the uninfected blank cell control. Figure 2 (B). The above experimental results indicate that the monoclonal antibody (D3-mAb) secreted by D3 hybridoma cells can specifically recognize recombinant VP8 protein and PoRVA-infected cells.

[0075] ELISA analysis showed that D3-mAb reacted with anti-IgG2a antibody and anti-κ light chain antibody, belonging to the IgG2a class κ chain (IgG2aκ). The titer of D3-mAb in the ascites fluid was 1:6.4×10⁻⁶. 6 ( Figure 2 (C and D), and specifically binds only to the VP8 protein, showing no cross-reactivity with eight other different proteins including PoRVA VP6 and PEDV. Figure 2 (E). Antibody-antigen binding affinity assays showed that the dissociation constant of D3-mAb was 6.46 nmol / L (E). Figure 2 (Middle F).

[0076] 2.3 Virus neutralizing activity of D3-mAb After serially diluted D3-mAb was incubated with the virus, it was inoculated into MA-104 cells. IFA detection was performed 24 h later. The results showed that antibodies diluted 1:2 to 1:4 exhibited strong inhibitory effects on the virus, with almost no fluorescent signal. As the antibody dilution factor increased, the fluorescent signal gradually increased, and the fluorescence intensity also increased. Figure 3 (A and B) indicates a gradual increase in viral load. After incubation with D3-mAb diluted 1:2 to 1:32, the viral nucleic acid load and TCID50 were significantly lower than those in the positive control group. Figure 3 (C and D). The neutralizing titer of D3-mAb was determined to be 1:10² using the fixed virus dilution antibody method. Figure 3 (E). The above results demonstrate that the prepared D3-mAb has a significant inhibitory effect on PoRVA.

[0077] 2.4 Recognition of monoclonal antibody and antigen-binding region Five synthetic peptide fragments and VP8 protein were coated onto an ELISA plate. ELISA detection showed that the monoclonal antibody D3-mAb only reacted with the VP8-1 peptide (…). 16 AQTGYAPV23 ) and VP8 protein (positive control) bind ( Figure 4 (A). Meanwhile, Dot-blot assays also demonstrated that D3-mA reacts only with the VP8-1 peptide and VP8 protein, appearing as black spots at the corresponding sites on the NC membrane. It does not react with the other four peptides, and the VP8-1 peptide does not react with the SP2 / 0 cell supernatant. Figure 4 (B). Amino acid sequence alignment analysis of the VP8 protein revealed... 16 AQTGYAPV 23 The amino acid sequence of the epitope is highly conserved among common PoRVA genotypes. Figure 4 (C)

[0078] 2.5 Analysis of antibody epitope interaction sites The spatial locations of five synthesized linear epitopes in the VP8 protein, predicted using PyMOL software, are shown. 16 AQTGYAPV 23 The other four epitopes are all interspersed within the VP8 protein, exposed on its surface. Figure 5 A). Amino acid sequencing analysis revealed that the molecular weights of the light (L) and heavy (H) chains of D3-mAb were 24.151 kDa and 50.063 kDa, respectively. Molecular docking showed that the variable regions of the L and H chains of D3-mAb corresponded to epitopes. 16 AQTGYAPV 23 There are 6 interacting hydrogen bonds between them, namely VH. 109 G and epitope 16 A, 110 S and epitope 19 G, 36 I, 38 D and 108 Y and epitope 23 Hydrogen bonds form between N atoms, VL 61 S and epitope 20 Y forms hydrogen bonds ( Figure 5 B).

[0079] To ensure the high immunogenicity of the VP8 protein, this invention first selects an insect baculovirus expression system for protein expression. Proteins expressed in eukaryotic systems are generally considered to approximate their native structure and activity. Furthermore, the VP8 protein expressed in this invention exists in the cell culture supernatant, retaining the innate structure of VP8. The prepared VP8 protein exhibits good reactivity with porcine PoRV antiserum.

[0080] mAbs targeting the VP8 fragment of rotavirus from different host sources all exhibit significant neutralizing activity and in vivo protective potential. Human VP8-specific mAbs can efficiently neutralize the virus in human intestinal epithelial cells and provide protection in suckling mice. The porcine rotavirus VP8 monoclonal antibody prepared in this invention, as detected by ELISA, has a dissociation constant Kd = 6.46 nM (6.46 × 10⁻⁶). -9 The monoclonal antibody (M) meets the criteria for high-affinity antibody and exhibits highly efficient neutralizing activity against porcine rotavirus in vitro, suggesting its potential to neutralize the virus in vivo.

[0081] Studies on antigenic epitopes on the VP8 protein of rotavirus have focused on circulating human strains, with few studies identifying PoRVA epitopes. Five consecutive epitopes were found on the human rotavirus VP8 protein: M1-L10, I35-R44, I55-D66, V115-G123, and L223-P234. Further analysis revealed that three epitopes are involved in virus neutralization: M1-L10, I55-D66, and L223-P234, with 41-A, 42-Q, and 44-R considered key amino acids. This invention first demonstrated that D3-mAb prepared in MA-104 cells can neutralize PoRVA. Subsequently, potential epitopes on the VP8 protein were predicted using a website, and five peptides were designed. These peptides were then identified using ELISA and Dot-blot results. 16 AQTGYAPV 23 The peptide can be recognized by D3-mAb. This epitope sequence is similar to that identified by human rotavirus. 35 INPSPFAQTR 44 They have the same amino acid sequence. The VP8 sequence selected in this invention consists of amino acids 26-231, with amino acid residue 26 ranked as position 1. 16 AQTGYAPV 23 Compared with those reported in the literature 35 INPSPFAQTR 44 Although the amino acids are similar, their amino acid sequences are different. 35 INPSPFAQTR 44 Although it is an epitope, it did not have a neutralizing effect. Compared with this invention, it is speculated that it may be related to the GYAPVN amino acid sequence. PyMOL software was used to analyze... 16 AQTGYAPV 23Visualization of the VP8 protein revealed that its epitope is located on the outer side of the VP8 protein. Molecular docking of the peptide with the antibody showed that there are a total of 6 hydrogen bonds on the peptide that interact with the antibody. Epitope 16-A forms hydrogen bonds with VH 109-G, epitope 19-G forms hydrogen bonds with VH 110-S, epitope 23-N forms hydrogen bonds with VH 36-I, 38-D, and 108-Y respectively, and epitope 20-Y forms hydrogen bonds with VL 61-S.

[0082] In summary, the PoRV VP8 protein was successfully expressed using an insect baculovirus expression system. The expressed VP8 protein was released into Sf9 cell culture supernatant while retaining its original structure. A monoclonal antibody D3 with neutralizing activity against the VP8 protein was screened, and subsequently, the neutralizing epitope was identified. 16 AQTGYAPV 23 Recognized by D3-mAb, sequence comparison analysis of currently prevalent PoRVA strains revealed that this antigenic epitope is absolutely conserved. Furthermore... 16 AQTGYAPV 23 The epitope is located on the surface of the VP8 protein and has six hydrogen bonds that interact with the variable region of the antibody. This invention provides valuable insights for the design of novel epitope vaccines and antiviral strategies.

[0083] 2.6 Application of Monoclonal Antibodies Using D3-mAb as a diagnostic reagent, it can be applied in Western blot, immunohistochemistry, and immunofluorescence assays to accurately detect rotavirus-specific antigens and isolated rotavirus. Figure 6 and Figure 7 The working concentration range of D3-mAb is between 1:500 and 1:5000, and it has no non-specific cross-reactivity with PEDV S, TGEV N, and PCV2 Cap protein antigens.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody D3-mAb, characterized in that, The monoclonal antibody D3-mAb specifically binds to the antigenic epitope of porcine rotavirus VP8 protein. 16 AQTGYAPV 23 It has neutralizing activity and contains a light chain as shown in SEQ ID NO.8 and a heavy chain as shown in SEQ ID NO.

9.

2. The monoclonal antibody D3-mAb according to claim 1, characterized in that, The monoclonal antibody D3-mAb is of the IgG2b subtype, and the light chain is the κ chain.

3. The use of the monoclonal antibody D3-mAb according to claim 1 or 2 in the preparation of vaccines or drugs, characterized in that, The vaccine or drug is used to prevent and / or treat porcine rotavirus infection.

4. A detection reagent or kit, characterized in that, It contains the monoclonal antibody D3-mAb as described in claim 1 or 2.

5. The use of the monoclonal antibody D3-mAb according to claim 1 or 2 in the preparation of reagents or kits for detecting porcine rotavirus.

6. The use of the monoclonal antibody D3-mAb of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of porcine rotavirus infection.

7. A drug for the prevention and / or treatment of porcine rotavirus infection, characterized in that, It comprises the monoclonal antibody D3-mAb as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

Images