Porcine rotavirus specific monoclonal antibody and latex microsphere immunochromatography detection test strip containing same

Abstract

The invention belongs to the technical field of biology, and particularly relates to a porcine rotavirus (PRoV) specific monoclonal antibody and a latex microsphere immunochromatography detection test strip containing the antibody. Amino acid sequences of heavy chain variable regions of the monoclonal antibody or the antigen binding fragment I and the antigen binding fragment II are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 5; amino acid sequences of light chain variable regions of the monoclonal antibody or antigen binding fragment I and antigen binding fragment II are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 6. According to the invention, the PRoV latex microsphere detection test strip is established by utilizing a double-antibody sandwich principle. The latex microsphere immunochromatography test strip can specifically recognize the porcine rotavirus, and does not react with other porcine viruses with similar clinical symptoms, such as porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine acute diarrhea syndrome coronavirus and porcine sapelovirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a porcine rotavirus specific monoclonal antibody and a latex microsphere immunochromatographic test strip containing the antibody. BACKGROUND

[0002] Rotaviruses (RVs) belong to the family Reoviridae, genus Rotavirus, are double-stranded RNA (dsRNA) viruses with a icosahedral structure and no envelope, and are an important cause of diarrhea in children and young animals. The RV virion is about 70 nm in diameter, and the genome is composed of 11 dsRNA segments, encoding 6 structural proteins (VP1-VP4, VP6 and VP7) and 6 non-structural proteins (NSP1-NSP6). According to the antigenicity of VP6 protein, RVs can be divided into 12 serogroups (RVA-RVL), among which RVA, RVB, RVC, RVE and RVH can infect pigs, and group A is the most common subtype causing diarrhea in suckling pigs. The outer capsid proteins VP4 and VP7 have specific neutralizing epitopes, and determine G and P genotypes, respectively. Various combinations between G and P types can occur, and the cross-protection between different serotypes is low. Currently, 42 G genotypes and 58 P genotypes have been identified. In pigs, G3-G5, G9 and G11 are most common in the world, as are P[6], P[7], P

[13] , P

[19] and P

[23] . In addition, in order to better distinguish the homology and heterogeneity between strains, the Rotavirus Classification Working Group (RCWG) established a classification system based on 11 gene segments, using the symbol Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx to represent the genotypes of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 / 6 genes.

[0003] Due to the genetic reassortment of RV in nature, it brings great challenges to the prevention and control of the disease. In pig population, porcine rotaviruse (PRoV) is an important cause of epidemic diarrhea in piglets, often mixed infection with some enteroviruses such as: porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (PDCoV) and porcine sapovirus (PSV), etc., which makes the disease worsen rapidly and causes significant economic losses. In order to reduce losses and ensure the healthy development of pig industry, it is of great significance to establish an economic and efficient PRoV differential diagnosis method. At present, the detection methods for PRoV include: virus isolation culture identification, virus electron microscope morphology examination, enzyme-linked immunosorbent assay (ELISA), immunochromatography test strip detection technology, reverse transcription-polymerase chain reaction (RT-PCR), real-time fluorescent quantitative (RT-qPCR), loop-mediated isothermal nucleic acid amplification technology (LAMP), in situ hybridization technology (ISH) and nanopore sequencing, etc. Compared with other technologies, the immunochromatography test strip detection technology has the characteristics of simple operation, short time consumption, visual observation of detection results by naked eye, and no need of professional personnel and instruments for operation. The latex microspheres have high physical and chemical tolerance, deep color and high visualization degree compared with the commonly used marker colloidal gold. SUMMARY

[0004] The present application first screens a monoclonal hybridoma cell strain of porcine rotavirus, then obtains a specific monoclonal antibody, and obtains the antibody variable region gene sequence by nested PCR amplification technology (the gene engineering or protein engineering method can be used subsequently). Further, based on the specific monoclonal antibody of porcine rotavirus, the present application develops a latex microsphere immunochromatography test strip for detecting porcine rotavirus, which is convenient, fast, accurate, high in sensitivity and strong in specificity.

[0005] The present application specifically includes the following contents:

[0006] In a first aspect, the present application provides a monoclonal antibody or antigen binding fragment (the antigen binding fragment can be a Fab fragment, a F(ab)2 fragment or a single chain Fv fragment (scFv)) specifically binding to porcine rotavirus (PRoV):

[0007] The amino acid sequence of the heavy chain variable region of the monoclonal antibody or antigen binding fragment one is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen binding fragment one is shown in SEQ ID NO: 2. The heavy chain and light chain variable regions of the monoclonal antibody or antigen binding fragment one comprise CDR1, CDR2 and CDR3 as shown in Table 1:

[0008] Table 1. Amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains of monoclonal antibodies.

[0009]

[0010] The present invention also provides a nucleic acid encoding a monoclonal antibody or antigen-binding fragment one, wherein the DNA encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment one is shown in SEQ ID NO:3; and the DNA encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment one is shown in SEQ ID NO:4.

[0011] SEQ ID NO:1

[0012] PGASVSCPARLLATPSPATGCTGCSRGLDKALSGSERLILLIVILPTIKSSGARPHCLWTNPPEQPSCTSAACHLRTLRSITVQDGGLRLGILWTTGVKEPQSPSP;

[0013] SEQ ID NO:2

[0014] SLGQRATISYRASESVSTSGYSYMHWNQQKPGQPPKLLIYLVSNLQSGVPARFTGRGSGTDFTLNIHPEEEEDAATYYCQHIRELTRSEGGPSWKSN;

[0015] SEQ ID NO:3

[0016] CCTGGGGCTTCAGTGAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGcAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATACTACCTACAATCAAAAGTTCAGGGGCA AGGCCACATTGcCTGTGGACAAATCCTCCAGAACAGCCTTCATGCACCTCAGCAGCCTGCCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGGGGGATTACGACTAGGGATACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT;

[0017] SEQ ID NO:4

[0018] TCTCTGGGACAGAGGGCcACCATCTCATACAGGGCCAGTGAAAGTGTcAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAAAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGTATCCAACCTACAATCTGGGGT CCCTGCCAGGTTCACTGGCAGAGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGAGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGATCGGAGGGGGGACCAAGCTGGAAATCAAAC.

[0019] The amino acid sequence of the heavy chain variable region of monoclonal antibody or antigen-binding fragment two is shown in SEQ ID NO:5; the amino acid sequence of the light chain variable region of monoclonal antibody or antigen-binding fragment two is shown in SEQ ID NO:6. The heavy and light chain variable regions of monoclonal antibody or antigen-binding fragment two include CDR1, CDR2, and CDR3 as shown in Table 2.

[0020] Table 2. Amino acid sequences of the variable regions CDR1, CDR2, and CDR3 of the heavy and light chains of monoclonal antibodies.

[0021]

[0022] The present invention also provides a nucleic acid encoding a monoclonal antibody or antigen-binding fragment two, wherein the DNA encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment two is shown in SEQ ID NO:7; and the DNA encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment two is shown in SEQ ID NO:8.

[0023] SEQ ID NO:5

[0024] PGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEIDPSDSYTTYNQKFRGKATLTVDKSSRTAFMHLSSLTSEDSAVYYCARRGITTTRDTMDYWGQGTSVTVSS;

[0025] SEQ ID NO:6

[0026] ISYRASENVSTSGYSYMHWNQQKPGQPPKLLIYLVYNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKSN;

[0027] SEQ ID NO:7

[0028] CCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATACTACCTACAATCAAAAGTTCAGGGGCAA GGCCACATTGACTGTAGACAAATCCTCCAGAACAGCCTTCATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGGGGGATTACGACTAGGGATACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0029] SEQ ID NO:8

[0030] ATCTCTTACAGGGCCAGCGAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAAAAACCAGGACAGCCGCCCAAACTCCTCATCTATCTTGTATACAACCTAGAATCTGGGGTCCCTGCCAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAAC.

[0031] The constant region of the heavy chain of the PRoV monoclonal antibody mentioned above is all of the IgG1 type.

[0032] The light chain constant regions of the aforementioned PRoV monoclonal antibodies are all of the Kappa type.

[0033] In addition, the present invention provides a vector containing the above-mentioned nucleic acid.

[0034] Furthermore, the present invention provides a host cell containing the above-mentioned vector.

[0035] By transferring a vector containing the aforementioned nucleic acid into host cells for expression, monoclonal antibodies or antigen-binding fragments that specifically bind to PRoV can be obtained through genetic engineering.

[0036] Secondly, the present invention provides the application of the monoclonal antibody or antigen-binding fragment that specifically binds to porcine rotavirus, the nucleic acid encoding the monoclonal antibody or antigen-binding fragment, the vector, and the host cell in the preparation of a porcine rotavirus detection reagent.

[0037] Thirdly, the present invention provides a reagent for detecting porcine rotavirus, the reagent comprising a capture antibody and a detection antibody, wherein the capture antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one that specifically binds to porcine rotavirus and a monoclonal antibody or antigen-binding fragment two that specifically binds to porcine rotavirus.

[0038] Alternatively, the detection antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one that specifically binds to porcine rotavirus and the monoclonal antibody or antigen-binding fragment two that specifically binds to porcine rotavirus.

[0039] Alternatively, the capturing antibody may be a monoclonal antibody or antigen-binding fragment one that specifically binds to porcine rotavirus, and the detection antibody may be a monoclonal antibody or antigen-binding fragment two that specifically binds to porcine rotavirus.

[0040] Alternatively, the capturing antibody may be a monoclonal antibody or antigen-binding fragment two specifically binding to porcine rotavirus, and the detection antibody may be a monoclonal antibody or antigen-binding fragment one specifically binding to porcine rotavirus. The detection reagent may be a latex microsphere immunochromatographic test strip or a double-antibody sandwich ELISA kit.

[0041] Fourthly, the present invention provides a latex microsphere immunochromatographic test strip for rapid detection of porcine rotavirus, the test strip containing the aforementioned reagents for detecting porcine rotavirus. Specifically, the test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad being sequentially overlapped on the PVC base plate; the nitrocellulose membrane has a detection line T and a control line C, the detection line T being located near the conjugate pad, and the control line C being located near the absorbent pad; the conjugate pad is coated with the capture antibody labeled with latex microspheres, the detection line T is coated with the detection antibody, and the control line C is coated with goat anti-mouse IgG.

[0042] Antibody capture and antibody detection include at least the following:

[0043] The conjugate pad is coated with a latex microsphere-labeled monoclonal antibody that specifically binds to PRoV, and the detection line T is coated with a monoclonal antibody that specifically binds to PRoV, namely, the second one.

[0044] Alternatively, the conjugate pad may be coated with a second monoclonal antibody that specifically binds to PRoV labeled with latex microspheres, and the detection line T may be coated with a first monoclonal antibody that specifically binds to PRoV.

[0045] Alternatively, the conjugate pad may be coated with a latex microsphere-labeled monoclonal antibody that specifically binds to PRoV, and the detection line T may be coated with other existing monoclonal antibodies that specifically bind to PRoV.

[0046] Alternatively, the conjugate pad may be coated with a latex microsphere-labeled monoclonal antibody II that specifically binds to PRoV, and the detection line T may be coated with other existing monoclonal antibodies that specifically bind to PRoV.

[0047] Alternatively, the conjugate pad may be coated with a latex microsphere-labeled monoclonal antibody that specifically binds to PRoV, and the detection line T may be coated with the aforementioned monoclonal antibody that specifically binds to PRoV.

[0048] Alternatively, the conjugate pad may be coated with a latex microsphere-labeled monoclonal antibody that specifically binds to PRoV, and the detection line T may be coated with the aforementioned monoclonal antibody that specifically binds to PRoV.

[0049] Preferably, the method for preparing the conjunctival pad is as follows:

[0050] The conjunctival pad is sealed with a conjunctival pad sealing solution;

[0051] The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended in MES buffer, and then a capture antibody specifically binding to PRoV was added. The mixture was incubated at room temperature, and then blocked with latex microsphere labeling blocking solution. The mixture was incubated at room temperature and centrifuged. The precipitate was resuspended with latex microsphere labeling protectant to obtain the capture antibody latex microsphere labeled solution.

[0052] The prepared capture antibody latex microsphere labeling solution was uniformly sprayed onto the sealed conjugate pad;

[0053] The latex microsphere labeling blocking solution is an ultrapure aqueous solution containing 10% m / v BSA;

[0054] The latex microsphere labeling protectant is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40, and 1% v / v Tween-20.

[0055] The binding pad sealing solution is an ultrapure aqueous solution of 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, 1.5% m / v PVP-40, and 0.05% Krovin 300.

[0056] The latex microsphere labeling solution for capturing antibodies is composed of 60 μg of capturing antibody per 1 mL of latex microsphere labeling solution.

[0057] Preferably, the detection line T is coated with a detection antibody at a concentration of 1 mg / mL.

[0058] Preferably, the quality control line C is coated with 1 mg / mL of goat anti-mouse IgG.

[0059] Preferably, the sample pad is treated by soaking in a sample pad sealing solution; the sample pad sealing solution is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000, 0.5% sodium caseinate and 0.05% Krovin 300.

[0060] Fifthly, the present invention provides a method for preparing the above-mentioned latex microsphere immunoassay strip for detecting porcine rotavirus, the method comprising the following steps:

[0061] (1) Preparation of binding pads coated with latex microspheres labeled with capture antibodies;

[0062] (2) Spray the detection antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) at intervals of 5-8 mm onto the nitrocellulose membrane, which serve as the detection line T and the control line C, respectively;

[0063] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are sequentially overlapped on the PVC base plate to obtain the latex microsphere immunochromatographic test strip for detecting porcine rotavirus.

[0064] Sixthly, the method of using the test strip of the present invention comprises the following steps:

[0065] (1) Take a sample for testing.

[0066] (2) Remove the test strip and bring it to room temperature. Add the sample to be tested into the sample well and let it stand at room temperature. Then determine the result.

[0067] (3) Result determination: After the reaction is completed, the positive and negative properties are determined by visual inspection.

[0068] The beneficial effects of this invention are:

[0069] This invention screened and obtained porcine rotavirus monoclonal antibodies 2E5 and 2G2, which can be used as capture antibodies or detection antibodies to detect porcine rotavirus.

[0070] The latex microsphere immunochromatographic test strip for detecting porcine rotavirus provided by this invention can be used for the specific detection of porcine rotavirus. Furthermore, the test strip can detect the virus in cultures up to a minimum concentration of 10. 2.56 TCID 50 / mL, can differentiate porcine rotavirus from other porcine enteroviruses with similar clinical symptoms, such as SADS-CoV, TGEV, PEDV, and PSV.

[0071] The test strips prepared by this invention have the advantages of high specificity, high sensitivity, simple operation, short detection time, and intuitive result display, providing a real-time detection method for the on-site or laboratory detection of porcine rotavirus. Attached Figure Description

[0072] Figure 1 Schematic diagram of the test strip structure.

[0073] Figure 2 Diagram illustrating the determination of test strip results.

[0074] Figure 3 The results of the sensitivity test of the test strip.

[0075] Figure 4 Image of a negative anal swab test result. In the image, PC represents a positive sample and NC represents a negative sample.

[0076] Figure 5 The image shows the specificity test results of the test strip. NC in the image represents a negative sample. Detailed Implementation

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0078] The PRoV strain used in the examples was group A porcine rotavirus RVA / Pig / China / GZ / 2023 / G9P

[23] (Genebank No. PQ323310~PQ323320). Six-week-old Babl / c female mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The reverse transcription kit was PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A. Latex microspheres were purchased from Suzhou Weidu Biotechnology Co., Ltd. EDC was purchased from Sigma, catalog number: 03449. NHS was purchased from Sigma, catalog number: 8045180025.

[0079] Example 1: Preparation of PRoV monoclonal antibody and amplification of antibody sequence

[0080] 1.1 ProoV virus propagation and purification

[0081] MA104 cells were seeded into T75 cell culture flasks. After the cells formed a monolayer, they were washed twice with sterile 0.01 mol / L PBS (K2HPO4 0.26 g, Na2HPO4·12H2O 2.89 g, NaCl 8.50 g, diluted to 1 L with water, pH 7.4). Then, 0.01 MOI of PRoV was seeded. The cell culture medium was serum-free DMEM containing 0.1% 1 mg / mL trypsin. Before seeding, PRoV was treated with 1% 1 mg / mL trypsin at 37°C for 1 h. After 80% of the cells showed cytopathic effects, the samples were collected and subjected to three freeze-thaw cycles at -80°C, followed by centrifugation at 8000 rpm for 30 min to remove cell debris. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000 g for 2 h to collect the precipitate. The precipitate was resuspended in an appropriate amount of PBS, and the virus was purified using a sucrose density gradient centrifugation method. Four sucrose gradients were set up: 30%, 40%, 60%, and 70%. Sucrose was added sequentially from low to high concentration to ultracentrifuge tubes, with the virus solution added on top. The tubes were centrifuged at 100,000g for 2 hours, and samples between 40% and 60% concentration were collected. This sample was mixed with an appropriate amount of PBS and centrifuged at 100,000g for 2 hours to remove the sucrose. The precipitate was resuspended in PBS, yielding purified PRoV.

[0082] 1.2 Mouse Immunization

[0083] Purified PRoV was inactivated at 56℃ for 30 min, and Babl / c mice were immunized at a dose of 30 μL / mouse. For the initial immunization, PRoV was emulsified by mixing an equal volume of PRoV with Freund's complete adjuvant and administering the emulsion via multiple subcutaneous injections at the dorsal sites. Booster immunizations were administered every 2 weeks for a total of four immunizations. For booster immunizations, purified PRoV was emulsified by mixing an equal volume of PRoV with Freund's incomplete adjuvant and administering the emulsion via the same method as the initial immunization.

[0084] 1.3 Preparation of Monoclonal Antibodies

[0085] Spleen cells from a PRoV-immunized mouse were mixed with SP20 cells (5:1 ratio) and fused using PEG fusion agent. The fused cells were seeded in 96-well cell plates and cultured at 37°C in a CO2 incubator. Once the hybridoma cells had covered the bottom 1 / 10 of the cell plate, the supernatant was aspirated and verified using indirect immunofluorescence assay (IFA). Positive cell wells were subcloned three times using limiting dilution. Finally, anti-PRoV monoclonal hybridoma cell lines were obtained and labeled.

[0086] IFA Assay: PRoV was treated with 1% 1 mg / mL trypsin at 37°C for 1 h, and then MA104 cells in a monolayer of 96 wells were seeded at 0.01 MOI. 24 h after seeding, cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and washed three times with 0.01 mol / L PBS; then permeabilized with 0.1% Triton X-100 at room temperature for 10 min, and washed three times with PBS; subsequently, hybridoma cell supernatant and FITC-labeled goat anti-mouse IgG were incubated. The results were observed under a fluorescence microscope after the reaction was complete.

[0087] 1.4 Purification of Monoclonal Antibodies

[0088] Balb / c mice aged 10–12 weeks were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant. One week later, each mouse was injected intraperitoneally with 5 × 10⁻⁶ mL of the adjuvant. 5 After 7-10 days, significant abdominal distension occurred in the mice treated with 0.2 mL of hybridoma cells. Ascites fluid was collected and aliquoted. Purification of the ascites fluid was performed according to PIERCE NAb... TM Affinity chromatography was performed using the Protein G Spin Purification Kit.

[0089] 1.5 Subclass Identification of Monoclonal Antibodies

[0090] The obtained monoclonal antibodies were identified as antibody subclasses according to the instructions of the Southern Biotech SBAClonotyping™ System / HRP Antibody Subclass Identification Kit.

[0091] The results showed that the heavy chain constant region of the monoclonal antibodies from hybridoma cell lines 2E5 and 2G2 was of the IgG1 type, and their light chain constant region was of the Kappa type.

[0092] 1.6 PCR amplification and sequencing of the variable region gene of monoclonal antibody

[0093] RNA was extracted from hybridoma cells 2E5 and 2G2, and then reverse transcribed into cDNA using Oligo-dt or random primers.

[0094] The antibody variable region gene was amplified using nested PCR. First, using the cDNA synthesized via reverse transcription as a template, the antibody variable region gene was amplified using a first-round primer sequence of murine IgG and κ light chain antibodies. Then, using the first-round product as a template, the antibody variable region gene was amplified using a second-round primer sequence of murine IgG and κ light chain antibodies. The PCR reaction system was: 25 μL PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL cDNA, and ddH2O to a final volume of 50 μL. The reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min. Primer references for antibody variable region gene amplification (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.).

[0095] The amplified products were sequenced, and the sequencing results were compared with the antibody gene library (IMGT). The DNA sequences encoding the heavy chain variable regions of PRoV monoclonal antibodies 2E5 and 2G2 are shown in SEQ ID NO:3 and SEQ ID NO:7, respectively; the DNA sequences encoding the light chain variable regions of PRoV monoclonal antibodies 2E5 and 2G2 are shown in SEQ ID NO:4 and SEQ ID NO:8, respectively. The amino acid sequences of the heavy chain variable regions of PRoV monoclonal antibodies 2E5 and 2G2 are shown in SEQ ID NO:1 and SEQ ID NO:5, respectively; the amino acid sequences of the light chain variable regions of PRoV monoclonal antibodies 2E5 and 2G2 are shown in SEQ ID NO:2 and SEQ ID NO:6, respectively.

[0096] The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 2E5 are shown in Table 3 below.

[0097] Table 3. Amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 2E5.

[0098] Monoclonal antibody 2E5 CDR1 CDR2 CDR3 Heavy chain VH ATPSPATG LILLIVIL QDGGLRLGILWTT Light chain VL ESVSTSGYSY LVS QHIRELTR

[0099] The amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 2G2 are shown in Table 4 below.

[0100] Table 4. Amino acid sequences of the heavy and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 2G2.

[0101] Monoclonal antibody 2G2 CDR1 CDR2 CDR3 Heavy chain VH GYTFTSYW IDPSDSYT ARRGITTRDTMDY Light chain VL ENVSTSGYSY LVY QHIRELTR

[0102] Example 2: Preparation of latex microsphere immunochromatographic test strips for porcine rotavirus detection

[0103] 2.1 Preparation of PRoV monoclonal antibody latex microsphere labeling solution

[0104] Add 975 μL of 0.1M pH 5.6 MES buffer to a 2 mL centrifuge tube, then add 25 μL of latex microspheres with a solid content of 4% and mix gently. Add 10 μL of 20 mg / mL EDC and 5 μL of 20 mg / mL NHS, mix thoroughly, and activate at room temperature for 20 min. Centrifuge at 17000 rpm for 20 min. Remove the supernatant, resuspend thoroughly in 1 mL of MES buffer, and centrifuge again at 17000 rpm for 20 min. Add 60 μg of PRoV monoclonal antibody 2G2 and incubate at room temperature for 2 h. Add 100 μL of 10 w / v % BSA solution for blocking, incubate at room temperature for 1 h, centrifuge at 17000 rpm for 10 min, and discard the supernatant. Finally, use 1 mL of latex microsphere labeling protectant (3% m / v sucrose, 1% m / v PVP-40, 1% v / v...) Resuspend the Tween-20 solution in Tris-HCl; if there is a significant precipitate that cannot be mixed, sonicate at 100W for 3 seconds, pause for 3 seconds, and sonicate for a total of 1 minute. Store at 4°C for later use.

[0105] 2.2 Preparation of latex microsphere binding pads

[0106] At 37℃~38℃, the conjugate pads were completely immersed in a blocking solution (an ultrapure aqueous solution of 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, 1.5% m / v PVP-40, and 0.05% Krovin 300) for 2 hours, then removed and dried in an oven for 2~4 hours. The prepared PRoV monoclonal antibody 2G2 latex microsphere labeling solution was uniformly sprayed onto the conjugate pads using a spray film applicator, dried in an oven at 37℃ for 2~3 hours, and then sealed for storage.

[0107] 2.3 Preparation of sample pad

[0108] Immerse the sample pad in a sample pad blocking solution (2% BSA, 0.5% S17, 2% PEG200000, 0.5% sodium caseinate and 0.05% Krovin 300 in Tris-HCl solution) for 2 hours at 37℃~38℃, then remove and dry in an oven for 2~4 hours, and finally place in an aluminum foil bag to dry for later use. Figure 1 As shown, a sample application hole is provided in the middle of the sample pad.

[0109] 2.4 Preparation of nitrocellulose membranes

[0110] PRoV monoclonal antibody 2E5 and goat anti-mouse IgG antibody were diluted to 1 mg / mL and sprayed onto a nitrocellulose membrane at a distance of 5-8 mm, serving as the detection line (T) and control line (C), respectively. After the lines were drawn, they were dried at 37℃-38℃ for 2-3 hours.

[0111] 2.5 Assembly of test strips

[0112] The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially connected on a PVC base plate to obtain a test strip. The test strip is then cut into strips according to the size of the test strip holder, and the cut test strips are then inserted into the test strip holder. A schematic diagram of the test strip structure is shown below. Figure 1 As shown.

[0113] Example 3: Application of latex microsphere immunochromatographic test strips for porcine rotavirus detection

[0114] 3.1 How to use the test strip

[0115] 3.1.1 Sample preparation

[0116] (1) Sample collection swabs were used as pig anal swabs or pig feces swabs for sample collection.

[0117] (2) Insert the sampled swab into the sample tube (containing 1 mL of sample processing solution), stir thoroughly for 10–15 seconds, then remove it and shake back and forth to mix well, so that the sample dissolves and is thoroughly mixed with the sample processing solution. Then let it stand for 1 minute to allow any undissolved sample to precipitate. The sample processing solution is 0.01 mol / L pH 7.4 PBS solution.

[0118] 3.1.2 Sample Testing

[0119] Remove the sealed test strip and place it on a dry, stable surface. Use a pipette to draw an appropriate amount of the supernatant from step 3.1.1 and slowly add 6–7 drops (approximately 100–150 μL) into the sample well. Observe the results 10 minutes after sample addition.

[0120] 3.1.3 Judgment

[0121] (1) The appearance of two red bands on the test strip (T: test line, C: control line) indicates a positive result (see [link to test strip]). Figure 2 A).

[0122] (2) If only one red band appears on the test strip (C: control line), the result is negative (see...). Figure 2 B).

[0123] (3) If no red band appears at the control line of the test strip, it is considered invalid (see...). Figure 2 C).

[0124] 3.2 Sensitivity of the test strip

[0125] PRoV TCID 50 10 5.67 The virus was serially diluted 1:10 at a concentration of 1 / mL, and then serially diluted 2:10 to test the sensitivity of the established test strip.

[0126] The results showed that the test result was still positive after a 1280-fold dilution of the virus, and negative after a 2560-fold dilution. Therefore, the test strip prepared in this embodiment has a sensitivity of 1280 times, meaning that the minimum virus detection limit of the method of this invention is 10. 2.56 TCID 50 / mL, such as Figure 3 As shown.

[0127] 3.3 Specificity of the test strip

[0128] Ten negative anal swabs, numbered 1-10, were tested using test strips. The results showed a red band at the control line C, but no band was observed at the T line, indicating a negative test result. Figure 4 Furthermore, when different porcine viruses such as PEDV, TGEV, SADS-CoV, and PSV were detected using test strips, only PRoV tested positive, while the others did not react. This indicates that the method established in this invention can specifically identify PRoV. Figure 5 As shown, the test strip developed in this invention has high specificity.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment that specifically binds to porcine rotavirus, characterized in that, the heavy chain variable region of the monoclonal antibody or antigen-binding fragment one comprises CDR1 with the sequence of ATPSPATG, CDR2 with the sequence of LILLIVIL and CDR3 with the sequence of QDGGLRLGILWTT, and the light chain variable region comprises CDR1 with the sequence of ESVSTSGYSY, CDR2 with the sequence of LVS and CDR3 with the sequence of QHIRELTR; or, the heavy chain variable region of the monoclonal antibody or antigen-binding fragment two comprises CDR1 with the sequence of GYTFTSYW, CDR2 with the sequence of IDPSDSYT and CDR3 with the sequence of ARRGITTRDTMDY, and the light chain variable region comprises CDR1 with the sequence of ENVSTSGYSY, CDR2 with the sequence of LVY and CDR3 with the sequence of QHIRELTR. 2.The monoclonal antibody or antigen-binding fragment that specifically binds to porcine rotavirus according to claim 1, characterized in that, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment one is shown as SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment one is shown as SEQ ID NO: 2; or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment two is shown as SEQ ID NO: 5; and the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment two is shown as SEQ ID NO:

6.

3. A nucleic acid, characterized in that, The nucleic acid encodes the monoclonal antibody or antigen-binding fragment that specifically binds to porcine rotavirus according to claim 1 or 2. 4.The nucleic acid according to claim 3, characterized in that, the DNA encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment one is shown as SEQ ID NO: 3; and the DNA encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment one is shown as SEQ ID NO: 4; or, the DNA encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment two is shown as SEQ ID NO: 7; and the DNA encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment two is shown as SEQ ID NO:

8.

5. A vector, characterized in that, The vector contains the nucleic acid according to claim 3 or 4.

6. A host cell, characterized in that, The host cell contains the vector according to claim 5. 7.Use of the monoclonal antibody or antigen-binding fragment that specifically binds to porcine rotavirus according to claim 1 or 2, the nucleic acid according to claim 3 or 4, the vector according to claim 5 or the host cell according to claim 6 in the preparation of a reagent for detecting porcine rotavirus.

8. A reagent for detecting porcine rotavirus, characterized by, The reagent comprises a capture antibody and a detection antibody, the capture antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one and the monoclonal antibody or antigen-binding fragment two according to claim 1 or 2, or, the detection antibody is selected from at least one of the monoclonal antibody or antigen-binding fragment one and the monoclonal antibody or antigen-binding fragment two according to claim 1 or 2. or, the capture antibody is monoclonal antibody or antigen binding fragment one of claim 1 or 2, and the detection antibody is monoclonal antibody or antigen binding fragment two of claim 1 or 2; or, the capture antibody is monoclonal antibody or antigen binding fragment two of claim 1 or 2, and the detection antibody is monoclonal antibody or antigen binding fragment one of claim 1 or 2.

9. A colloidal microsphere immunochromatographic test strip for rapidly detecting porcine rotavirus, characterized in that, The test strip contains the reagent for detecting porcine rotavirus according to claim 8.

10. The colloidal microsphere immunochromatographic test strip for rapid detection of porcine rotavirus according to claim 9, characterized in that, The test strip comprises a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped on the PVC base plate; the nitrocellulose membrane is provided with a test line T and a quality control line C, the test line T is arranged at one end close to the conjugate pad, and the quality control line C is arranged at one end close to the water absorption pad; the conjugate pad is sprayed with latex microsphere-labeled capture antibodies, the test line T is coated with the detection antibodies, and the quality control line C is coated with goat anti-mouse IgG.

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