A new delhi tomato leaf curl virus coat protein polypeptide, antigen, antibody and application thereof

Abstract

The application discloses a New Delhi tomato leaf curl virus coat protein polypeptide, antigen, antibody and application thereof, and belongs to the technical field of organism immunity detection. The application is based on ToLCNDV CP to design a polypeptide sequence, and provides a New Delhi tomato leaf curl virus coat protein polypeptide. The antigen prepared by coupling the polypeptide with keyhole limpet hemocyanin (KLH) can obtain a New Delhi tomato leaf curl virus antibody with high sensitivity and specificity, and can be used for screening the virus, and realizes instant detection and early warning of the ToLCNDV virus. Meanwhile, the application provides a theoretical basis and technical support for establishment of a virus serological diagnosis method and related detection product research and development, and is helpful to development of more accurate, efficient and portable detection products, so as to better serve the virus monitoring, quarantine and prevention and control work.

Description

Technical Field

[0001] This invention belongs to the field of biological immune detection technology, specifically relating to a polypeptide, antigen, antibody, and application of the outer shell protein of New Delhi tomato leaf curl virus. Background Technology

[0002] Tomato leaf curl New Delhi virus (ToLCNDV) is a double-stranded DNA virus belonging to the Geminiviridae family. Geminiviridae ), Common Bean Golden Mosaic Virus ( Begomovirus ToLCNDV primarily infects various economic crops, including tomatoes, peppers, and cucurbits. This virus can cause severe stunting of plants, wrinkled, uneven, smaller, yellowed, and curled leaves, as well as deformed fruits, severely impacting crop yield and quality. ToLCNDV is mainly transmitted through whiteflies in a cyclical and persistent manner. Besides whiteflies, it can also be transmitted through mechanical friction and even through seeds on some crops. Therefore, the trade and transportation of infected seeds and seedlings can lead to long-distance transmission of the virus. In recent years, this virus has spread globally, becoming a newly emerging virus of great concern both domestically and internationally.

[0003] Currently, the main detection methods for ToLCNDV include molecular biological detection methods and immunological detection methods. Molecular biological detection methods primarily include PCR and quantitative real-time PCR (qPCR). PCR detection: By designing specific primers, the DNA-A and DNA-B components of the viral genome are amplified. If a specific band is detected, it indicates that the sample is infected with ToLCNDV. This method has high sensitivity and can detect trace amounts of viral nucleic acid, making it a commonly used laboratory method. Quantitative real-time PCR (qPCR): Based on PCR, it combines fluorescent labeling technology to quantitatively analyze viral load and more accurately assess the degree of infection, but its high cost limits its application in field monitoring and forecasting. Immunological detection methods mainly include enzyme-linked immunosorbent assay (ELISA): This method utilizes the binding of specific antibodies to viral antigens, and the colorimetric result from the enzyme reaction determines whether the sample contains the virus. It is simple to operate and suitable for large-scale screening, but its sensitivity is relatively low.

[0004] It is evident that existing detection methods generally suffer from limitations such as relatively slow detection processes, insufficient sensitivity, and unsuitability for on-site testing. To achieve the prevention and control of ToLCNDV virus transmission and ensure agricultural production safety, there is an urgent need to propose a ToLCNDV virus testing scheme that is more accurate, convenient, and faster. Summary of the Invention

[0005] This invention designs a polypeptide sequence based on ToLCNDV CP to provide a New Delhi tomato leaf curl virus coat protein polypeptide. The antigen immunoassay prepared by conjugating the polypeptide with keyhole hemocyanin (KLH) can produce New Delhi tomato leaf curl virus antibodies with high sensitivity and specificity. This is expected to build a detection scheme for rapid detection of New Delhi tomato leaf curl virus, thereby solving the limitations of general detection methods such as relatively slow detection process, insufficient sensitivity, and unsuitability for on-site detection.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a New Delhi tomato leaf curl virus capsid protein polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0008] SEQ ID NO.1: CWTNRPMNRKPRMYR.

[0009] Secondly, the present invention also provides the application of the New Delhi tomato leaf curl virus coat protein polypeptide described in the first aspect in the preparation of New Delhi tomato leaf curl virus immunoantigen.

[0010] Thirdly, the present invention also provides a New Delhi tomato leaf curl virus immunogenic antigen, which is prepared by coupling the New Delhi tomato leaf curl virus capsid protein polypeptide described in the first aspect with keyhole hemocyanin.

[0011] Fourthly, the present invention also provides the use of the antigen described in the third aspect in the preparation of New Delhi tomato leaf curl virus immune antibodies.

[0012] Fifthly, the present invention also provides a New Delhi tomato leaf curl virus immune antibody, which is prepared by immunizing animals with the New Delhi tomato leaf curl virus immune antigen described in the third aspect.

[0013] Furthermore, the animal in question is a New Zealand white rabbit.

[0014] In a sixth aspect, the present invention also provides the use of the New Delhi tomato leaf curl virus immunoantigen as described in the third aspect and the New Delhi tomato leaf curl virus immunoantibody as described in the fifth aspect in the New Delhi tomato leaf curl virus detection method.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] This invention designs a polypeptide sequence based on the ToLCNDV CP, providing a ToLCNDV coat protein polypeptide. Conjugating this polypeptide with keyhole hemocyanin (KLH) yields an antigen immunoassay for ToLCNDV with high sensitivity and specificity, enabling screening for the virus and achieving immediate detection and early warning of ToLCNDV. Simultaneously, it provides theoretical basis and technical support for establishing viral serological diagnostic methods and developing related detection products, contributing to the development of more accurate, efficient, and portable detection products, thereby better serving virus monitoring, quarantine, and control efforts. Attached Figure Description

[0017] Figure 1 The figure shows the results of determining the titer of rabbit polyclonal antibody serum after four immunizations using the indirect ELISA method.

[0018] Figure 2 The graph shows the results of determining the antibody titer of the finished product using the indirect ELISA method.

[0019] Figure 3 Comparison of tobacco and melons infected with and uninfected with ToLCNDV.

[0020] Figure 4 The image shows Western blotting results of ToLCNDV CP antibody diluted multiple times. In the image, M represents the protein molecular weight standard; 1-7 represent ToLCNDV CP antibody diluted 2000, 4000, 8000, 16000, 32000, 64000, and 128000 times, respectively.

[0021] Figure 5 This is a graph showing the specificity of ToLCNDV CP antibody detection. In the graph, M represents the protein molecular weight standard control. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the invention.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. The materials and reagents, experimental conditions, and methods involved in the following examples are as follows:

[0025] Materials: New Zealand White Rabbits are sourced from Hangzhou Huaan Biotechnology Co., Ltd.

[0026] Reagents: BCA Protein Assay Kit and KLH were purchased from Thermo Fisher Scientific. TRIS, SDS, ammonium persulfate, glycine, Tween 20, and BSA were purchased from Sangon Biotech (Shanghai) Co., Ltd. Washing buffer, TMB chromogenic solution, goat anti-rabbit-HRP, goat anti-rat-HRP, and goat anti-guinea pig-HRP were purchased from Hangzhou Huaan Biotechnology Co., Ltd. NC membrane, color pre-stained protein maker, and ECL luminescence solution were purchased from Bio-Tech (Beijing) Technology Co., Ltd.

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] Example 1: This example describes the design of a polypeptide sequence based on ToLCNDV CP, providing a New Delhi tomato leaf curl virus coat protein polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1. The synthesizer is Hangzhou Huaan Biotechnology Co., Ltd. For the subsequent application of this New Delhi tomato leaf curl virus coat protein polypeptide, we designed the following experiments:

[0029] (a) Antigen preparation

[0030] 1. Peptide conjugation with KLH

[0031] 1.1 Dissolve 20 mg KLH in 2 mL of 5 mM EDTA aqueous solution.

[0032] 1.2 Weigh 8 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well.

[0033] 1.3 Add the Sulfo-SMCC solution dropwise to the KLH solution while gently shaking (vigorous shaking will produce a precipitate), and let it stand at room temperature for 1 h.

[0034] 1.4 Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze for 1 h in 2 L of 1×PBS at 4°C with magnetic stirring.

[0035] 1.5 Replace with fresh 1×PBS and dialyze for 2 h, repeating once. Place the activated and dialyzed KLH into a 15 mL imported centrifuge tube, label the tube with the reagent name, time, and concentration, and store at 4°C.

[0036] 1.6 Weigh 4 mg of peptide, dissolve it in 50 μL LDMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add KLH at a ratio of peptide:KLH = 1 mg: 680 μg. Incubate overnight at 4°C or react at room temperature for 2 h.

[0037] 1.7 Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clamp, and dialyze overnight in 4 L of 1×PBS at 4°C with magnetic stirring.

[0038] 1.8 Transfer the dialyzed KLH-peptide into a clean 1.5 mL centrifuge tube, aliquot according to the immunization dose, and store at -20°C.

[0039] (II) Immunological and Serological Evaluation

[0040] 1. Animal Selection: Select healthy animals with glossy fur and free movement. After selecting the animals, pre-raise them for about two weeks. The purpose is to weed out any unsuitable animals to ensure the smooth progress of later experiments.

[0041] 2. Preparation before the experiment: Mark the animals.

[0042] 3. Antigen preparation:

[0043] 3.1 Remove the antigen from the -20°C freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0044] 3.2 Extract the antigen (the antigen must be completely mixed). The initial immunization dose is 0.1-1.0 mg (depending on the species and weight of the immunized animal), 0.5 ml / animal. The antigen dose is halved for the second to fourth immunizations.

[0045] 3.3 Draw the adjuvant, with a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. Ensure the adjuvant is thoroughly mixed before drawing it into the syringe.

[0046] 3.4 After connecting the two syringes with the syringe connecting tube, complete emulsification is performed. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37 ℃ water.

[0047] 4. Immunization: Animals were given multiple subcutaneous injections, with 0.2 ml at each point.

[0048] Immunization schedule: The second immunization is administered 14 days after the first immunization, with a 7-day interval between the second and third immunizations. A small serum sample is collected from the middle ear artery 7 days after the third immunization. If the sample passes the test, a booster immunization is administered 7 days later, and whole blood can be collected 7 days after the booster immunization.

[0049] 4.1 Procedure for collecting small serum samples

[0050] The experimental rabbit was restrained in a frame, and its ears were gently tapped to dilate the central auricular artery. The area was disinfected with 75% alcohol. The rabbit's ear was held still with the left hand, and the syringe was held in the right hand. The needle was inserted into the central auricular artery at a point one-third of the way down from the distal end, parallel to the artery and directed towards the heart. 8 ml of blood was collected at a time, and pressure was applied with a cotton ball to stop the bleeding.

[0051] 4.2 Whole blood collection procedure

[0052] 4.2.1 Capture the animals to be immunized, check the ear tag number, weigh them, and anesthetize them by intravenous injection of 1 ml of 3% sodium pentobarbital per kilogram.

[0053] 4.2.2 After anesthesia, the immunized animal was placed with its abdomen facing upwards, and its limbs were restrained on a stainless steel mesh frame. Blood was drawn from the heart using the cardiac blood sampling method.

[0054] 4.2.3 After blood collection, place the centrifuge tube containing the blood in a 37℃ water bath for 15-30 minutes, then remove and cool it before placing it in a 4℃ refrigerator. Wait for the blood to separate automatically, then transfer the supernatant to a clean 50ml centrifuge tube.

[0055] 4.2.4 Centrifuge at 12000 rpm for 2 min, transfer the supernatant to a clean centrifuge tube, add 100 μL of 10% sodium thimerosal solution (final concentration 0.02%) to 50 mL of supernatant, mix well, and store at -20 °C.

[0056] 5. ELISA detection (indirect method)

[0057] 5.1 Plate coating: Dilute the known antigen to 1 μg / ml with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μl to each reaction well of the polystyrene plate, incubate overnight at 4°C, and the next day, discard the solution in the wells and wash once with 180 μl of 1xPBST washing buffer per well.

[0058] 5.2 Blocking: Add 150 μl of 1% BSA (prepared with PBST) to each well for blocking, and incubate at 37°C for 1 hour. Then discard the blocking solution.

[0059] 5.3 Sample Addition: Add 50 μl of the diluted sample to be tested (dilute the sample according to a certain ratio) to the sealed reaction wells. Also, set up negative control wells (1% BSA). Incubate at 37°C for 30 min, then wash three times with 150 μl of 1xPBST washing buffer per well.

[0060] 5.4 Add enzyme-labeled antibody: Add 50 μl of freshly diluted secondary antibody-HRP (diluted with 1% BSA) to each well of the ELISA plate, incubate at 37°C for 45 min, and wash three times with 150 μl of 1xPBST buffer per well.

[0061] 5.5 Adding substrate solution for color development: Add 50 μl of the temporarily prepared TMB substrate solution to each reaction well and incubate at 37 °C for 5 min.

[0062] 5.6 Termination of reaction: Add 50 μl of 1M sulfuric acid to each reaction well.

[0063] 5.7 Plate Reading: Place the ELISA plate in a preheated ELISA reader (450nm) for reading, save the data, and perform analysis. Serum titer results from two randomly selected New Zealand white rabbits were statistically analyzed. The results are as follows: Figure 2 As shown, the serum titers of both New Zealand white rabbits reached a high level of 1:1,024,000, with rabbit 2 exhibiting a higher titer than rabbit 1. Therefore, subsequent research selected the purification of rabbit 2's serum for preparing the final antibody.

[0064] (III) Antibody purification

[0065] 1. Wash the affinity chromatography column thoroughly with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0066] 2. Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0067] 3. Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0068] 4. Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument's transmittance (T setting) to 100.

[0069] 5. Adjust the absorbance of the protein detector (1A setting) to 0. At this time, turn on the HD-A computer acquisition device on the computer desktop and set the full-screen range to 5. Elute the antibody with glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0070] 6. During antibody collection, adjust the pH of the antibody to around 7 with 1M Tris-HCl in a timely manner, and record the highest peak value of the elution peak.

[0071] 7. After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tubing connected to the collector with purified water.

[0072] 8. Wash the affinity chromatography column sequentially with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store at 4°C.

[0073] 9. The purified antibodies should be sent for testing according to different requirements.

[0074] 10. Once the titer of the purified antibody semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator to achieve a certain concentration and volume.

[0075] 11. Transfer the antibody to a clean centrifuge tube. Filter the antibody through a 0.22 μm disposable low-adsorption filter in a clean bench. Send a small sample for testing. Determine the titer of the finished antibody using an indirect ELISA method, setting a 4-fold serial dilution gradient (1:250 to 1:1024000). Results are as follows: Figure 2 As shown, at the highest dilution of 1:1,024,000, the antibody's P / N value was still as high as 6.2. Based on the positive criterion of P / N ≥ 2, its titer is higher than 1:1,024,000. The concentration of the finished antibody was determined to be 2.47 mg / mL using the A280 protein quantification module of a DeNovix DS-11 micro-spectrophotometer.

[0076] (iv) Analysis of antibody sensitivity and specificity

[0077] like Figure 3The image shows a comparison of tobacco and melon samples infected and uninfected with ToLCNDV. Total protein was extracted from the ToLCNDV-infected samples and subjected to SDS-PAGE electrophoresis. After electrophoresis, the viral proteins were transferred to an NC membrane and detected by Western blotting. Different dilutions of ToLCNDV polyclonal antibody were used as the primary antibody (1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000), and a 1:5000 dilution of HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. The sensitivity of the antibody was evaluated by analyzing the band signal intensity at different antibody dilutions. Results are shown below. Figure 4 As shown, antibody sensitivity indicates that the protein band signal gradually weakens as the ToLCNDV CP antibody dilution increases. It is still effectively detectable at a dilution of 1:32,000, but the band basically disappears at a dilution of 1:128,000, suggesting that the highest effective detection dilution of this antibody in Western blotting is 1:32,000.

[0078] Total protein extracted from samples infected with ToLCNDV and TYLCV was transferred to an NC membrane by SDS-PAGE electrophoresis. After blocking with 5% skim milk powder, the membrane was incubated sequentially with a 1:5000 dilution of ToLCNDV polyclonal antibody as the primary antibody and a 1:5000 dilution of HRP-labeled goat anti-rabbit IgG as the secondary antibody. The membrane was then developed using ECL chemiluminescence. A specific band of ToLCNDV CP protein (approximately 29.5 kDa) appeared on the NC membrane, indicating a positive result; otherwise, a negative result was observed. The same treatment was applied to ToLCNDV and TYLCV to evaluate the specificity of this method. Results are as follows: Figure 5 As shown in Figure 3, Western blotting detected a specific protein band of approximately 29.5 kDa in samples infected with ToLCNDV, and there was no cross-reactivity with TYLCV samples, indicating that the antibody has good detection specificity.

[0079] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A polypeptide containing the outer coat protein of New Delhi tomato leaf curl virus, characterized in that, The amino acid sequence of the New Delhi tomato leaf curl virus coat protein polypeptide is shown in SEQ ID NO.

1.

2. The application of the New Delhi tomato leaf curl virus coat protein polypeptide as described in claim 1 in the preparation of New Delhi tomato leaf curl virus immunoantigen.

3. A New Delhi tomato leaf curl virus immunogen, characterized in that, The immunoantigen is prepared by coupling the New Delhi tomato leaf curl virus coat protein polypeptide as described in claim 1 with keyhole hemocyanin.

4. The use of the New Delhi tomato leaf curl virus immunoantigen as described in claim 3 in the preparation of New Delhi tomato leaf curl virus immunoantibodies.

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