Monoclonal antibodies for detecting potato virus Y and their applications

Abstract

The present application discloses a monoclonal antibody for detecting potato virus Y and its application. The heavy chain variable region comprises: the heavy chain CDR1 shown in SEQ ID No. 1, the heavy chain CDR2 shown in SEQ ID No. 2, and the heavy chain CDR3 shown in SEQ ID No. 3; the light chain variable region comprises: the light chain CDR1 shown in SEQ ID No. 4, the light chain CDR2 shown in SEQ ID No. 5, and the light chain CDR3 shown in SEQ ID No. 6. The monoclonal antibody can be directly made into a detection device, such as a colloidal gold test strip, for rapid detection of large quantities of samples. The colloidal gold test strip made with the antibody has a detection limit of up to 300 ng / mL for PVY-CP protein; and has good specificity, with negative detection results for viruses such as PVS, PLRV, and PVM.

Description

Technical Field

[0001] The present application relates to the technical field of tobacco virus detection, and in particular to a monoclonal antibody for detecting potato virus Y and its application. Background Art

[0002] Potato virus Y (PVY) is a representative species of the Potyvirus genus, the largest plant RNA virus. It infects a wide range of Solanaceae crops, including potatoes, tobacco, and peppers, as well as a variety of cash crops, including Chenopodiaceae and legumes. The symptoms caused by PVY vary depending on the host species and virus strain. Typical symptoms include severe mosaic, vein necrosis, and leaf streak necrosis.

[0003] Currently, commonly used methods for detecting potato virus Y (PVY) include biological detection, electron microscopy, molecular biology, and serological methods. Both biological and electron microscopy methods require culturing or preparing suitable subjects for observation, which is time-consuming and subjective, making them unsuitable for rapid detection. RT-PCR, known for its high sensitivity, is a molecular biology method commonly used for PVY detection in recent years. While it has high sensitivity and specificity, its practical application requires high RNA extraction and cDNA reverse transcription, making it unsuitable for high-throughput sample testing. For example, CN201110047406.1 discloses a kit and method for rapid detection of potato virus Y.

[0004] Serological testing technology offers advantages such as ease of use, rapidity, sensitivity, and specificity, and holds great promise for application in virus-free crop production and field surveys. Therefore, developing highly specific, sensitive, and specialized antibodies and establishing a sensitive and rapid serological test for PVY is crucial for monitoring and preventing the virus.

[0005] CN201610096251.3, "Hybridoma Cell Line Secreting Anti-Potatovirus Y Monoclonal Antibody and Application of the Monoclonal Antibody Thereof," discloses that BALB / c mice were immunized with PVY virus particles purified by differential centrifugation. Through cell fusion, screening, and cloning, a stably propagated hybridoma cell line, 3B2, was obtained that secretes an anti-PVY monoclonal antibody. Its deposit number is CGMCC No. 12001. This cell line secretes a monoclonal antibody with an ascites indirect ELISA titer of 10e-7. The antibody type and subclass are IgG1, kappa light chain, and the monoclonal antibody specifically reacts with the 30kDa PVY coat protein.

[0006] However, this method uses hybridoma cell technology to produce antibodies, which carries the risk of decreased antibody production and loss. Furthermore, the method begins with cell culture, so any contamination or loss of stored cells during the culture process can halt antibody production. Existing test strips used for PVY virus detection also suffer from a high incidence of false positives. Summary of the Invention

[0007] In response to the above-mentioned technical problems, the present application provides an anti-potato virus Y monoclonal antibody with strong specificity, high sensitivity and suitability for detecting tobacco plant PVY virus, and its application. The coding sequence is obtained by sequencing, and after obtaining the antibody coding sequence, the antibody is stably obtained in the form of a recombinant protein, so that the antibody does not need to be retained in the form of a hybridoma cell as a carrier for research and development or production, but can be preserved in the form of a base sequence, which can effectively ensure the stability of the detection results of the obtained antibody. In addition, accurate antibody sequence information is also the basis for subsequent antibody affinity maturation and other antibody modifications, effectively reducing false positive test results.

[0008] The present application provides a monoclonal antibody for detecting potato virus Y, wherein the heavy chain variable region comprises: a heavy chain CDR1 shown in SEQ ID No. 1, a heavy chain CDR2 shown in SEQ ID No. 2, and a heavy chain CDR3 shown in SEQ ID No. 3;

[0009] The light chain variable region includes: a light chain CDR1 shown in SEQ ID No. 4, a light chain CDR2 shown in SEQ ID No. 5, and a light chain CDR3 shown in SEQ ID No. 6.

[0010] The heavy chain variable region of the monoclonal antibody includes a heavy chain CDR1 as shown in SEQ ID No. 1: Gly Phe ThrPhe Ser Ser Tyr Ala;

[0011] Heavy chain CDR2 shown in SEQ ID No. 2: Ile Ser Ser Gly Gly Ser Thr;

[0012] Heavy chain CDR3 shown in SEQ ID No. 3: Ala Arg Tyr Gly Asn Leu Tyr Ala Met AspTyr.

[0013] The light chain variable region of the monoclonal antibody includes a light chain CDR1 as shown in SEQ ID No. 4: Gln Ser LeuVal His Arg Asn Gly Asp Thr Tyr;

[0014] Light chain CDR2 shown in SEQ ID No. 5: Arg Val Ser;

[0015] The light chain CDR3 shown in SEQ ID No. 6: Ser Gln Ser Thr His Leu Pro Phe Thr.

[0016] Monoclonal antibodies with the amino acid sequence of the complementary determining region (CDR) described above can effectively detect potato virus Y. The CDR sequence binds to the antigenic determinant on the antigen, specifically binding to that site. Detection is achieved through the binding of the antigen to the antibody against that sequence.

[0017] Preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7; and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 8.

[0018] SEQ ID No.7:

[0019]

[0020] SEQ ID No.8:

[0021]

[0022] Preferably, the DNA sequence of the gene encoding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 9 in the sequence listing; and the DNA sequence of the gene encoding the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 10 in the sequence listing.

[0023] SEQ ID No.9:

[0024]

[0025] SEQ ID No.10:

[0026]

[0027]

[0028] When the monoclonal antibody was used to determine the titer of ascites by ELISA, the titer reached 1:(2.187×10 -7 ). The anti-potato virus Y monoclonal antibody is IgG 1(κ) .

[0029] Another aspect of the present application provides a detection device for detecting potato virus Y, comprising: the above-mentioned monoclonal antibody; the detection device is a reagent kit or a colloidal gold test strip.

[0030] The monoclonal antibodies provided in this application can be directly used in the kit structure to achieve large-scale, rapid and accurate detection.

[0031] Preferably, it is used for the detection of potato virus Y in tobacco plants.

[0032] Preferably, 5 to 50 μg of the monoclonal antibody is added to the colloidal gold label in the colloidal gold test strip per milliliter of colloidal gold solution.

[0033] Preferably, the method for preparing the colloidal gold test strip comprises the following steps: mixing the monoclonal antibody with the colloidal gold solution, adding 10% BSA to a final concentration of 1% BSA in the solution, allowing the mixture to stand and centrifuging to obtain a precipitate, and washing and resuspending the precipitate with a reconstitution buffer;

[0034] After preparing the conjugate release pad, the detection sample absorption pad and the reaction membrane, the colloidal gold detection test strip is assembled.

[0035] Preferably, the reconstitution buffer used is a 0.02 mol / L phosphate buffer containing 0.1% to 0.5% by mass of BSA, 2% to 4% by mass of sucrose, and a pH of 7.2.

[0036] Preferably, the method for using the colloidal gold test strip comprises the following steps: dripping the pretreated tobacco leaf supernatant into the sample addition well, timing for 10 minutes, and reading the result.

[0037] The detection limit of the above test paper for PVY-CP protein is 300 ng / mL, and the results of the detection of PVS, PLRV, and PVM were negative, indicating that the detection method has good specificity.

[0038] Preferably, the colloidal gold test strips are stored at 2-30° C. for 12 months.

[0039] The beneficial effects of this application include:

[0040] 1) The anti-potato virus Y monoclonal antibody provided in this application can be directly made into a detection device, such as a colloidal gold test strip, for rapid detection of large quantities of samples. The colloidal gold test strip made with this antibody has a detection limit of up to 300 ng / mL for PVY-CP protein; the test strip has good specificity and tests negative for viruses such as PVS, PLRV, and PVM. When the antibody is made into a test strip, the false positive rate of the test result can be effectively reduced, and the accuracy of the test result can be effectively improved.

[0041] 2) The anti-potato virus Y monoclonal antibody provided in this application can specifically detect potato virus Y, and its antibody titer reaches 1:(2.187×10 -7), has good specificity for potato virus Y that infects tobacco plants, and can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay, laying the foundation for the subsequent research, development and promotion of potato virus Y immunoassay methods.

[0042] 3) The anti-potato virus Y monoclonal antibody coding sequence provided in this application enables the antibody to be stably obtained in the form of a recombinant protein in the later stage, without having to retain the antibody in a hybridoma cell vector, which can effectively ensure the stability of the obtained antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the electrophoresis diagram of PVY-CP protein expression detection obtained in Example 1 of this application; the sample processed by M band is: Marker (Page Ruler TM Prestained protein molecular weight standard, 10-180 kDa, Thermo Scientific); Band 1: bacteria before PVY-CP induction; Bands 2 and 3: bacteria after PVY-CP induction; Bands 4 and 5: precipitate after PVY-CP induction; Bands 6 and 7: supernatant after PVY-CP induction;

[0044] Figure 2 This is the result of PVY-CP protein purification obtained in Example 1 of this application. The sample processed with band M is the marker; the samples processed with bands 1-5 are the purified protein; and the BSA band is bovine serum albumin.

[0045] Figure 3 This is a homology alignment diagram of the heavy chain gene sequences of the anti-potato virus Y monoclonal antibody in Example 2 of the present application.

[0046] Figure 4 This is a homology alignment diagram of the anti-potato virus Y monoclonal antibody light chain gene sequences in Example 2 of the present application.

[0047] Figure 5 This is a homology alignment of the heavy chain amino acid sequences of the anti-potato virus Y monoclonal antibodies in Example 2 of the present application.

[0048] Figure 6 This is a homology alignment of the light chain amino acid sequences of the anti-potato virus Y monoclonal antibodies in Example 2 of the present application.

[0049] Figure 7 This is a schematic diagram of the main and cross-sectional structure of the colloidal gold test strip for potato virus Y in Example 3 of the present application.

[0050] Figure 8 This is a schematic diagram of the result determination of the colloidal gold test strip for potato virus Y in Example 4 of the present application. DETAILED DESCRIPTION

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0052] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0053] The technical means that are not described in detail in this application and are not used to solve the technical problems of this application are all set according to the common knowledge in this field, and can be implemented in a variety of common knowledge settings.

[0054] Example

[0055] Unless otherwise specified, the reagents and plants used in the following examples were obtained from commercial channels. Operations not described in detail in the following examples were performed according to conventional methods.

[0056] Example 1 Preparation of anti-potato virus Y monoclonal antibodies

[0057] 1. Expression and purification of PVY-CP protein

[0058] (1) Gene synthesis design

[0059] The optimized DNA was constructed into the pET-28a(+) vector using BamHI and HindIII as restriction sites.

[0060] The optimized gene sequence is shown in SEQ ID No.11:

[0061]

[0062]

[0063] (2) Protein expression identification

[0064] ① Add 2 μL of plasmid to 50 μL of BL21 competent bacteria and incubate on ice for 30 minutes.

[0065] ②Heat shock at 42℃ for 90s, quickly place on ice for 5min, and add 500μL LB culture medium.

[0066] ③Shake at 37℃ and 220r / min for 1h, spread on LB plate containing kanamycin resistance, and culture upside down at 37℃ overnight.

[0067] ④ Pick a single clone from the plate and inoculate it into a test tube containing 5 mL LB culture medium containing kanamycin resistance.

[0068] ⑤Shake at 37℃, 200r / min until the culture medium OD 600 is 0.7.

[0069] ⑥Add IPTG to a final concentration of 0.1 mmol / L, and set a control without IPTG. Shake at 16°C and 200 rpm for 16 h to induce the expression of the fusion protein.

[0070] ⑦ Take out 1 mL of culture, centrifuge at 12000×g for 10 min at room temperature, discard the supernatant, resuspend the bacterial pellet with 50 μL of 1× PBS buffer and add 25 μL of 3× Loading Buffer, and ultrasonically disrupt the remaining culture.

[0071] ⑧Analyze by 12% SDS-PAGE electrophoresis. The results are shown in the attached Figure 1 As shown, the fusion protein was clearly expressed, mainly in the precipitate.

[0072] (3) Large-scale protein expression and purification

[0073] ① Take the activated bacteria and inoculate them into 2L of LB medium containing kanamycin resistance, shake at 37℃ and 200r / min until the OD 600 The mixture was stirred at 16 °C and 200 r / min for 16 h after adding IPTG to a final concentration of 0.1 mmol / L.

[0074] ② Centrifuge at 10000r / min for 10min, discard the supernatant and collect the bacteria.

[0075] ③ Resuspend the cells in Buffer A (20 mmol / L Tris, 20 mmol / L Imidazole, 500 mmol / L NaCl, pH 8.0), disrupt with a high-pressure homogenizer (900 bar, three times), and centrifuge at 14,000 rpm for 20 min.

[0076] ④ Collect the supernatant and filter to remove impurities.

[0077] ⑤ Equilibrate the Ni column with Buffer A for 10 column volumes, then load the sample.

[0078] ⑥Wash away impurities with Buffer A.

[0079] ⑦ Elute with Buffer B (20 mmol / L Tris, 250 mmol / L Imidazole, 500 mmol / L NaCl, pH 8.0) and collect the protein peak.

[0080] ⑧SDS-PAGE electrophoresis detection, as attached Figure 2 As shown, the target protein was purified.

[0081] 2. Preparation of monoclonal antibodies against potato virus Y

[0082] (1) Immunized animals

[0083] The expressed PVY-CP protein was diluted to 1 mg / mL in sterile PBS (pH 7.0) and then fully emulsified at a 1:1 ratio of recombinant protein to Freund's adjuvant (FCA) to prepare an oil-emulsion vaccine. The first immunization was performed with Freund's complete adjuvant vaccine, administered subcutaneously at multiple points in the nape of the neck at a dose of 200 μg per mouse.

[0084] Booster immunizations were then administered subcutaneously at multiple sites on the back of the neck every two weeks, using Freund's incomplete adjuvant (FICA) emulsified in the immunization. The final immunization was performed without adjuvant, using the recombinant protein directly via intraperitoneal injection at the same dose as the previous immunizations. Specific immunization procedures are shown in Table 1.

[0085] Table 1 Mouse immunization program

[0086] Number of immunizations Time / d Immunization dose / (μg / mouse) Immunization methods adjuvant Initial immunization 0 200 Multiple subcutaneous injections at the back of the neck Freund's complete adjuvant Second exemption 15 200 Same as above Freund's incomplete adjuvant Three exemptions 30 200 Same as above Same as above Four free 44 200 Same as above Same as above strengthen 58 (three days before fusion) 200 intraperitoneal injection Without adjuvant

[0087] (2) Mouse polyclonal antibody detection

[0088] ①Panel

[0089] PVY-CP protein was diluted to 1 μg / mL with 0.05 mol / L, pH 9.6 CB buffer, and 100 μL was added to each well of the ELISA plate and incubated at 4°C overnight. After coating, the plate was washed once with PBST solution, patted dry, and 150 μL of blocking solution (pH 9.0 carbonate buffer) was added to each well. The plate was blocked at 37°C for 2 h, patted dry, and stored at 4°C until use.

[0090] ②Positive detection of mouse polyclonal antibody serum

[0091] The mouse serum was diluted 1000 times with antibody diluent, and then diluted threefold six times. Seven serum samples of different concentrations were added to the PVY-CP protein-coated enzyme-labeled plate at 100 μL per well from top to bottom, and the plate was sealed with a cover film. The reaction was carried out at 37°C for 30 minutes, the plate was washed three times, and the plate was patted dry. 100 μL of goat anti-mouse IgG enzyme-labeled secondary antibody was added to each well, the reaction was carried out at 37°C for 30 minutes, the plate was washed four times, and the plate was patted dry. 100 μL of substrate solution was added to each well, the reaction was carried out at room temperature for 15 minutes, and 50 μL / well of stop solution was added. The OD value of each well was detected by a microplate reader at dual wavelengths of 450 nm / 630 nm, and fusion mice were selected according to the OD value.

[0092] The criteria for selecting fusion mice are as follows: the dilution factor corresponding to the wells with an OD value of around 1.0 (≥1.0) is the titer of the antibody, and the OD value of the negative serum is less than 0.4.

[0093] (3) Cell fusion

[0094] ①Preparation

[0095] The fusion agent was aliquoted into 1 mL portions and placed in a 37°C CO2 incubator before fusion.

[0096] HAT medium: Prepare before fusion (500 mL HAT medium contains 5 mg HAT, 20% FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin) and place in a 37°C incubator to warm up.

[0097] HT medium: Prepare before the first cloning (500 mL DMEM medium containing 5 mg HT, 20% FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin) and place in a 37°C incubator to warm up.

[0098] Cloning medium: Use for all clones except the first clone (500 mL DMEM medium containing 20% FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin).

[0099] Cell expansion medium: used for expansion culture after cell establishment (500 mL DMEM medium containing 15% FBS, penicillin 100 U / mL, streptomycin 0.1 mg / mL).

[0100] ②Resuscitation and cultivation of SP2 / 0

[0101] Thaw SP2 / 0 cells 2 weeks before fusion. Remove cryogenically stored SP2 / 0 cells and quickly heat them in a 37°C water bath for 2 minutes. Once thawed, add them to a centrifuge tube containing 8 mL of DMEM. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the pellet in expanded culture medium, and transfer to a cell flask for culture. Observe the cells and perform culture medium changes as needed. Handle the SP2 / 0 cells as needed one day before fusion. Replace with fresh culture medium approximately 8 hours before fusion.

[0102] ③Preparation of feeder cells

[0103] Peritoneal macrophages were used as feeder cells. BALB / c mice were sacrificed by cervical dislocation and then immersed in 75% alcohol for 5 minutes for disinfection. The mice were removed and placed upright for half a minute until no alcohol dripped from them. The mice were then transferred to a clean bench with the abdomen facing upwards. The abdominal skin was cut open to expose the peritoneal endothelium. An appropriate amount of DMEM was drawn up with a syringe and injected into the mouse's peritoneal cavity, making sure not to puncture the internal organs. The mice were shaken several times, then the DMEM was aspirated and transferred to a 50 mL centrifuge tube. This was repeated three times.

[0104] Place the centrifuge tube containing the feeder cells into a centrifuge at 1000 r / min for 5 minutes, discard the supernatant, add an appropriate amount of HAT culture medium, and blow the feeder cells in the tube evenly for use.

[0105] ④ Preparation of SP2 / 0 cells

[0106] Take SP2 / 0 cells in good growth condition, pour out the culture medium, add appropriate amount of DMEM culture medium, blow off the cells with a curved pipe to make SP2 / 0 suspension for later use.

[0107] ⑤ Preparation of spleen cells

[0108] Immunized BALB / c mice (fused mice) were sacrificed by cervical dislocation and then disinfected by immersion in 75% alcohol for 5 minutes. The spleen was removed, excess tissue removed, and the spleen was rinsed with DMEM before being placed in a sterile culture dish. DMEM culture medium was drawn up with a syringe and injected into the spleen to dissipate splenocytes. This was repeated five times to dissipate as many splenocytes as possible. The spleen cell suspension was then transferred to a centrifuge tube.

[0109] ⑥Cell fusion

[0110] Add the SP2 / 0 suspension to the spleen cell suspension, mix thoroughly, and centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant, keeping the tube facing downward. Use filter paper to remove any remaining liquid from the tube walls. Tap the cells at the bottom of the tube to disperse them evenly in a monolayer. Add 1 mL of fusion agent uniformly along the tube walls over 1 minute, rotating the tube as you add. After addition, allow the tube to rest flat for 1 minute to allow fusion to proceed. Terminate fusion with 15 mL of DMEM medium, adding 1 mL uniformly over the first minute, 2 mL over the second minute, and so on.

[0111] After addition, centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant and mix the pellet with an appropriate amount of HAT culture medium. Add the appropriate HAT working solution as needed, plate the cells at 120 μL / well on a 96-well plate. Then, plate the prepared feeder cells at 150 μL / well on the 96-well plate containing the fused cells. Culture in a 5% CO2, 37°C incubator for 9 days. Observe cell morphology and determine the assay time based on cell number and size.

[0112] (4) Screening and cloning of hybridoma cells

[0113] ① Fusion plate detection

[0114] Observe the status of the fused cells 4 to 5 days after fusion and prepare the antigen coating plate, following the same steps as (2)①.

[0115] After 7 days of fusion, the liquid was collected for detection. 50 μL of sample was taken from each well and added to the ELISA plate. The plate was sealed with a cover film. The subsequent steps were the same as (2)②.

[0116] ② Cloning and detection

[0117] Based on the test results, cells were selected for cloning and cultured in HT medium.

[0118] When cloning, prepare the culture medium in advance, prepare a new cell plate, blow the selected cell wells to blow the cells evenly, aspirate part of the cell suspension to dilute the cells, count them on a counting plate, and dilute the cells to 4 per microliter based on the count results. Calculate the amount of suspension required for 1 to 2 cells per well based on the amount of plating, and aspirate it into HT culture medium, mix well, plate into a new 96-well cell plate, and plate feeder cells.

[0119] After 7 days of cloning, the cells can be tested. The plates should be wrapped 1-2 days in advance. The wrapping steps are the same as (2) ①. The testing steps are the same as (2) ②. If the test results are qualified and there are single cells in the cell plate wells, the cells are transferred to 24-well cell plates for "③ expansion culture". After the titer and cross-culture are tested, the cells are determined and expanded. If there are no single cells, the second and third cloning are carried out until single cells appear.

[0120] ③ Expand training

[0121] Culture the cells in a 24-well plate and observe them. When the cells have spread to more than 80% of the wells, perform the test. Take the supernatant and dilute it 30-fold. Then dilute it 3-fold seven times. The subsequent steps are the same as the polyclonal antibody inhibition test (2) ②. After the test, transfer the cells to a cell flask containing expansion medium for expansion culture.

[0122] (5) Cryopreservation

[0123] Before freezing, ensure that the cells are plump and in good condition (round, translucent, and abundant), occupying 80% of the total volume. Use a curved pipette to blow off the cells, transfer them to a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add freezing solution to mix the cells, and pipette them into a cryovial. Label the tube and place it in a cryovial. Store in a -80°C freezer overnight. Short-term storage is possible at -80°C, but long-term storage requires transfer to liquid nitrogen.

[0124] (6) Preparation and detection of monoclonal antibody ascites

[0125] Measures to induce ascites in animals were adopted. A certain number of BALB / c mice were prepared. Before injecting cells, 0.5 mL of sterile paraffin oil was injected into the mouse's abdominal cavity. Cell injection could be performed at least 4 days later. When the cells were full and in good condition, cells in the bottle were blown out with a curved tube, transferred to a centrifuge tube and centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded, physiological saline was added and mixed, and the mixed cell suspension was drawn up with a syringe and injected into the mouse's abdominal cavity. 0.5 mL of liquid could be injected into the abdominal cavity of each mouse. Depending on the number of cells, 1×10 6 Around cells.

[0126] Ascites will develop 8 days after cell inoculation. The health and signs of ascites in the mice should be closely monitored. When the abdomen swells, the ascites is withdrawn with a syringe. This process can be repeated several times until the mouse dies. The ascites is centrifuged at 8000 rpm for 10 minutes in a low-temperature centrifuge, aliquoted, and frozen at -20°C until use. The titer of the ascites was determined by ELISA, reaching 1:(2.187×10 -7 ).

[0127] (7) Antibody purification

[0128] Ammonium sulfate precipitation plus affinity chromatography was performed. Mouse ascites was centrifuged to remove impurities, transferred to a fresh centrifuge tube, and mixed thoroughly with an equal volume of PB buffer. A saturated ammonium sulfate solution equal to the volume of the buffered solution was added dropwise until thoroughly mixed, resulting in a final ammonium sulfate concentration of 50%. Precipitation was allowed to proceed overnight at 4°C. After precipitation, the solution was centrifuged and the supernatant discarded. The precipitate was reconstituted with PB buffer (60% of the volume of the ascites fluid) and transferred to a dialysis bag for dialysis. After dialysis, the precipitate was removed by centrifugation and purified using a Protein G affinity column. The resulting protein solution was then assayed, preservatives added, and aliquots were stored frozen for later use.

[0129] (8) Subclass identification of monoclonal antibodies against potato virus Y

[0130] Subclass identification: The subclass of anti-potato virus Y monoclonal antibody was detected using IgG subclass detection kit (Sigma, USA). The results showed that the anti-potato virus Y monoclonal antibody was IgG. 1(κ) .

[0131] Example 2 Genetic Verification of Anti-Potatovirus Y Monoclonal Antibodies

[0132] 1. Light chain and heavy chain variable region gene cloning

[0133] (1) Hybridoma cell culture and total RNA extraction

[0134] 1×10 hybridoma cells were cultured in DMEM complete medium at 37°C and 5% carbon dioxide. 6 , cultured hybridoma cells were collected by centrifugation, cells were lysed with TRIZOL, and total RNA of cultured cells was extracted with phenol-chloroform.

[0135] (2) Synthesis of the first strand of cDNA

[0136] cDNA was synthesized using Thermo reverse transcription kit.

[0137] (3) Gene amplification

[0138] Design the upstream and downstream primers of Kappa chain and Heavy chain.

[0139] Primer: HF: CTCCACGTCCACCACCTC, as shown in SEQ ID No. 12:

[0140] HR: GTCAGTGGCAGAGGAGCC, as shown in SEQ ID No. 13:

[0141] κF: CTATAGTACGACTGGGTT, as shown in SEQ ID No. 14:

[0142] κR: GTTTCAACCTTTATTTTG, as shown in SEQ ID No. 15:

[0143] PCR was performed using the first-strand cDNA as a template in a 50 μL reaction system.

[0144] PCR reaction system: 2×I5 Master Mix 25.0 μL, upstream primer 2.0 μL, downstream primer 2.0 μL, cDNA template 2.0 μL, deionized water 16.0 μL.

[0145] The PCR reaction conditions were as follows: 98°C for 30 s; 98°C for 10 s, 60°C / 58°C for 30 s, 72°C for 30 s; 35 cycles; 72°C for 7 min.

[0146] (4) Cloning and screening of PCR amplification products

[0147] The PCR products were subjected to 1.0% agarose gel electrophoresis, and the antibody kappa chain and heavy chain fragments were recovered using a PCR product recovery kit (Omega). After double digestion with EcoRV and NheI, they were ligated with the linearized vector pFUSEss-mG1 and ligated with NEB's T4 DNA ligase. The resulting fragments were transformed into DH5α competent cells (Zeocin), and recombinant positive clones were screened and sequenced.

[0148] The PCR product was subjected to 1.5% agarose gel electrophoresis, and the results showed that the nucleotide sequence of the heavy chain was shown as SEQ ID No. 9, and the nucleotide sequence of the light chain was shown as SEQ ID No. 10.

[0149] (5) Variable region amino acid sequence and homology analysis

[0150] Comparison analysis was performed in the NCBI database. The results showed that the monoclonal antibody heavy chain variable region gene sequence had the highest homology with the mouse immunoglobulin heavy chain variable region mRNA (Sequence ID: Z11163.1), with a homology of 335 / 350 and a homology percentage of 96%. Figure 3 .

[0151] The monoclonal antibody light chain variable region gene sequence has the highest homology with the mouse immunoglobulin light chain variable region mRNA (SequenceID: EF672224.1), with a homology of 321 / 330 and a homology percentage of 97%. Figure 4 .

[0152] The amino acid sequence of the variable region of the monoclonal antibody heavy chain (SEQ ID No. 7) has the highest homology with the amino acid sequence of the variable region of the mouse immunoglobulin heavy chain (Sequence ID: AAD47039.1), with a homology of 106 / 117 and a homology percentage of 91%. Figure 5 .

[0153] The amino acid sequence of the light chain variable region of the monoclonal antibody (SEQ ID No. 8) has the highest homology with the amino acid sequence of the light chain variable region of the mouse immunoglobulin Kappa chain (Sequence ID: pir|B30577|), with a homology of 100 / 112 and a homology percentage of 89%. Figure 6 .

[0154] Homology analysis of the gene sequences and amino acid sequences encoding the heavy and light chain variable regions of the monoclonal antibody showed that no sequences identical to those of the present invention were found. This result is consistent with the identification of the anti-potato virus Y monoclonal antibody as IgG1(κ) using an IgG subclass detection kit (Sigma, USA).

[0155] The heavy chain variable region and light chain variable region sequences were obtained from the online website IMGT

[0156] (https: / / www.imgt.org / IMGT_vquest / analysis) analysis, the CDR region was obtained, and its amino acid sequence is shown in SEQ ID No. 1 to SEQ ID NO. 6.

[0157] Example 3 Preparation of Potato Virus Y Colloidal Gold Test Strips

[0158] 1. Preparation of anti-potato virus Y monoclonal antibody-colloidal gold label

[0159] (1) Preparation of colloidal gold

[0160] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double-distilled deionized water, place 100 mL in a conical flask, and heat to boiling using a thermostatic electromagnetic stirrer. Add 1.5 mL of 1% trisodium citrate while continuing to stir at high temperature until the solution turns a translucent red. Cool to room temperature, then return to the original volume with deionized water and store at 4°C. The prepared colloidal gold should be pure, translucent, free of precipitates and floating matter, and appear wine red when observed in sunlight.

[0161] (2) Preparation of anti-potato virus Y monoclonal antibody-colloidal gold label

[0162] Under magnetic stirring, adjust the pH of the colloidal gold to 7.2 with 0.2 mol / L potassium carbonate solution. Add the aforementioned anti-potato virus Y monoclonal antibody to the colloidal gold solution at a standard concentration of 45 μg of antibody per ml of colloidal gold solution, and continue stirring for 30 minutes. After 10 minutes of simmering, add 10% BSA to a final concentration of 1% in the colloidal gold solution, and let it stand for 10 minutes. Centrifuge at 12,000 rpm and 4°C for 40 minutes, discard the supernatant, and wash the pellet twice with reconstitution buffer. Resuspend the pellet in reconstitution buffer at 1 / 10 the initial colloidal gold volume and store at 4°C until ready for use.

[0163] Reconstitution buffer: 0.02 mol / L phosphate buffer containing 0.2% BSA, 3% sucrose, pH 7.2.

[0164] 2. Preparation of conjugate release pad

[0165] The conjugate release pad was soaked in 0.02 mol / L phosphate buffer containing 0.5% BSA, 5% sucrose, and pH 7.4 for 2 hours, then dried at 37°C for later use. The prepared anti-potato virus Y monoclonal antibody-colloidal gold marker was evenly sprayed onto the conjugate release pad using a Bio dot streaker, with 0.01 mL of anti-potato virus Y monoclonal antibody-colloidal gold marker applied per 1 cm of the conjugate release pad. The pad was then placed in a 37°C environment (humidity <20%) for 2 hours, removed, and stored in a dry environment (humidity <20%) until further use.

[0166] 3. Preparation of sample absorption pad

[0167] The sample absorption pad was soaked in 0.02 mol / L phosphate buffer solution containing 1% BSA and pH 7.2 for 2 h, and dried at 37° C. for 2 h before use.

[0168] 4. Preparation of reaction membrane

[0169] The anti-potato Y monoclonal antibody was coated on the reaction membrane to form the detection line, and the goat anti-mouse antibody was coated on the reaction membrane to form the quality control line.

[0170] Coating process: Dilute anti-potato virus Y monoclonal antibody to 1 mg / mL in 0.01 mol / L, pH 7.2 phosphate buffer and coat the antibody on the test line (T line) of nitrocellulose membrane using a Bio dot stripper at a coating volume of 1.0 μL / cm. Dilute goat anti-mouse antibody to 200 μg / mL in 0.01 mol / L, pH 7.2 phosphate buffer and coat the antibody on the control line (C line) of nitrocellulose membrane using a Bio dot stripper at a coating volume of 1.0 μL / cm. Dry the coated membrane at 37°C for 16 hours before use.

[0171] 5. Assembly of test strips

[0172] Assemble according to the existing commonly used test strip structure, see Figure 7 The cross-sectional structure of the test strip shown in FIG. 1 shows a sample absorption pad 1, a conjugate release pad 2, a reaction membrane 3, and a water absorbent pad 4 adhered in sequence to a PVC base plate 7. The conjugate release pad has 1 / 3 of its area covered by the sample absorption pad from its starting end. The end of the conjugate release pad is connected to the starting end of the reaction membrane, which is then connected to the starting end of the water absorbent pad. The starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the end of the water absorbent pad is aligned with the ending end of the PVC base plate. A test line 5 and a quality control line 6 are provided on the reaction membrane.

[0173] The test line (T line) and the control line (C line) are both strips perpendicular to the length of the test strip; the test line is located on the side close to the end of the conjugate release pad; the control line is located on the side away from the end of the conjugate release pad; the test strip is cut into small strips 3.95 mm wide by a machine, placed in a special plastic card case, sealed in an aluminum foil bag, and stored at 4°C until used.

[0174] Example 4 Detection of Potato Virus Y in Tobacco Leaf Samples

[0175] 1. Sample pretreatment

[0176] Place 0.15g of tobacco leaf in a centrifuge tube and rotate and squeeze it several times with a grinding rod to ensure that the leaf tissue is fully ground. Add 2mL of 0.02mol / L PB buffer and repeat the grinding to thoroughly mix the sample and buffer. Remove the grinding rod and let it stand for at least 1 minute. The supernatant is the sample to be tested (the sample to be tested is an unknown sample).

[0177] 2. Use test strips to test

[0178] Use a micropipette to draw 100 μL of the sample solution to be tested and drop it vertically into the sample well; start timing when the liquid flows, react for 10 minutes, and then determine the result.

[0179] 3. Analyze the test results. The test strip display results are consistent with the existing commonly used display results. For details, see Figure 8 :

[0180] Positive (+): C line shows red or purple stripes, T line also shows red or purple stripes, regardless of the color depth, it means that the sample is infected with potato virus Y. Figure 8 a, 8b, 8c.

[0181] Negative (-): The C line shows a red or purple band, and the T line does not show color, indicating that the sample is not infected by potato virus Y or the concentration is too low. Figure 8 d.

[0182] Invalid: No C line appears, indicating incorrect operation process or the test strip has deteriorated and become invalid, such as Figure 8 e, 8f.

[0183] 4. Detection sensitivity analysis

[0184] PVY-CP protein was diluted from 10000 ng / mL to 100 ng / mL using 0.01 mol / L PB buffer as diluent and detected using test strips. Each concentration was measured three times.

[0185] The results showed that at low PVY-CP protein concentrations, the T line on the test strip did not develop color, indicating a negative result. However, at concentrations of 300 ng / mL, a T line appeared on the test strip, and the color of the T line became increasingly darker as the concentration increased. Therefore, the detection limit of the test strip for PVY-CP protein in this study was 300 ng / mL.

[0186] 5. Specificity analysis

[0187] The test strips were used to test for potato virus S (PVS), potato virus M (PVM), and potato leaf-roll virus (PLRV), with three replicates per sample. The results showed that the test strips tested negative for PVS, PLRV, and PVM, demonstrating the good specificity of the detection method.

[0188] Example 5

[0189] The difference from Example 3 is that the reconstitution buffer used is a 0.02 mol / L phosphate buffer containing 0.1% BSA by mass, 4% sucrose by mass, and pH 7.2.

[0190] 5 μg of the monoclonal antibody was added per ml of colloidal gold solution to the colloidal gold label in the colloidal gold test strip.

[0191] Example 6

[0192] The difference from Example 3 is that the reconstitution buffer used is a 0.02 mol / L phosphate buffer containing 0.5% BSA by mass, 2% sucrose by mass, and pH 7.2. 50 μg of the monoclonal antibody is added per ml of colloidal gold solution to the colloidal gold marker in the colloidal gold test strip.

[0193] Example 7

[0194] 160 tobacco leaf samples were prepared according to the method in Example 4, and each sample was tested using the colloidal gold test strips and PCR detection method obtained in the examples of this application.

[0195] The PCR detection method refers to the standard GB / T 36816-2018 "Potato virus Y quarantine and identification method", and the results are shown in the table below:

[0196]

[0197] From the above, it can be seen that the test results of the test strips provided in this application are basically consistent with those of the PCR method. The test strips made of this antibody are conducive to improving the accuracy of the test results and effectively solving the problem of false positives.

[0198] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody for detecting potato virus Y, characterized in that The heavy chain variable region includes: heavy chain CDR1 shown in SEQ ID No. 1, heavy chain CDR2 shown in SEQ ID No. 2, and heavy chain CDR3 shown in SEQ ID No. 3; The light chain variable region includes: a light chain CDR1 shown in SEQ ID No. 4, a light chain CDR2 shown in SEQ ID No. 5, and a light chain CDR3 shown in SEQ ID No.

6.

2. The monoclonal antibody for detecting potato virus Y according to claim 1, wherein The amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 7; The amino acid sequence of the light chain variable region is shown in SEQ ID No.

8.

3. A detection device for detecting potato virus Y, characterized in that: include: The monoclonal antibody according to claim 1 or 2; the detection device is a kit or a colloidal gold test strip.

4. The detection device for detecting potato virus Y according to claim 3, characterized in that 5 to 50 μg of the monoclonal antibody is added to the colloidal gold marker in the colloidal gold detection test strip per milliliter of colloidal gold solution.

5. The detection device for detecting potato virus Y according to claim 3, characterized in that The preparation method of the colloidal gold test strip comprises the following steps: mixing the monoclonal antibody with the colloidal gold solution, adding 10% BSA until the final concentration of BSA in the solution is 1%, allowing the mixture to stand and centrifuging to obtain a precipitate, and washing and resuspending the precipitate with a resolubilization buffer; After preparing the conjugate release pad, the detection sample absorption pad and the reaction membrane, the colloidal gold detection test strip is assembled.

6. The detection device for detecting potato virus Y according to claim 5, characterized in that The reconstitution buffer used is a 0.02 mol / L phosphate buffer solution containing 0.1% to 0.5% BSA by mass, 2% to 4% sucrose by mass, and pH 7.

2.

7. A method for using the device for detecting potato virus Y according to any one of claims 3 to 6, characterized in that: When the detection device for detecting potato virus Y is a colloidal gold test strip, the method of use includes the following steps: dripping the pretreated tobacco leaf supernatant into the sample addition hole, timing for 10 minutes and reading the result.

8. A storage method for the detection device for potato virus Y according to any one of claims 3 to 6, characterized in that: When the detection device for potato virus Y detection is a colloidal gold test strip, it can be stored in an environment of 2 to 30°C for 12 months.

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