Nanobody for detecting tomato yellow leaf curl virus and preparation method and application thereof

Abstract

The application belongs to the technical field of biological detection and antibody engineering, and particularly relates to a nanobody for detecting tomato yellow leaf curl virus and a preparation method and application thereof. The nanobody specifically binds to a coat protein of the tomato yellow leaf curl virus, and the complementarity determining regions of the nanobody include CDR1, CDR2 and CDR3, and the nucleotide sequences of the CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO. 4-6. The application also provides a nucleotide sequence encoding the nanobody, a recombinant expression vector, a host cell and a preparation method thereof. The nanobody has high affinity to the coat protein of the TYLCV and excellent thermal stability, and has no cross reaction with viruses such as TMV and ToMV. The detection limit of a detection kit and a colloidal gold test strip constructed based on the antibody can reach 20 pg / mL, and the kit and the test strip are suitable for rapid screening and field quantitative diagnosis of the TYLCV.

Description

Technical Field

[0001] This invention belongs to the field of biodetection and antibody engineering technology, specifically relating to a nanobody for detecting tomato yellow leaf curl virus, its preparation method, and its application. Background Technology

[0002] Tomato yellow leaf curl virus ( Tomato yellow leaf curl virus TYLCV belongs to the Geminiviridae family ( Geminiviridae ) Golden mosaic virus of the common bean genus ( Begomovirus It is a single-stranded circular DNA virus. This virus primarily relies on whiteflies (…). Bemisia tabaci TYLCV spreads persistently and, under natural conditions, generally not through seeds, sap friction, or routine agricultural operations. Typical symptoms in infected crops include severe stunting, yellowing and upward curling of new leaves, shortened internodes, and significant flower bud drop, ultimately leading to a marked decrease in fruit set, deformed and smaller fruits, deterioration in quality, and a sharp reduction in yield, seriously threatening the safe production of tomatoes and other solanaceous crops globally. Currently, there are no widely used commercially resistant varieties against TYLCV; early and accurate detection and quarantine intervention remain the core means of controlling the cross-border spread and field diffusion of this virus.

[0003] Existing TYLCV detection technologies are mainly divided into two categories: molecular biological detection and immunological detection. Molecular detection primarily uses polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). While these methods offer advantages such as high sensitivity and specificity, they rely on expensive equipment, specialized operators, and long testing cycles (typically 2-4 hours), making them unsuitable for rapid screening needs in fields, border crossings, or at grassroots plant protection stations. Immunological detection, represented by enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strips, has become the mainstream choice for rapid on-site diagnosis due to its ease of operation, intuitive results, and low cost. However, traditional immunological detection largely relies on murine or rabbit polyclonal / monoclonal antibodies (IgG, molecular weight approximately 150 kDa). Such antibodies have significant limitations in practical applications: (1) They are large in molecular size and have obvious steric hindrance, making it difficult to effectively bind to hidden or conformational epitopes on the surface of virus particles; (2) They have poor thermal stability and pH tolerance, and are prone to denaturation and inactivation in high temperature and humidity environments in the field or in complex matrices (rich in polyphenols, polysaccharides and proteases) of plant crude extracts, leading to an increased false negative rate; (3) They have certain sequence similarity with the capsid proteins of closely related geminiviruses (such as ToLCV, TYLCSV, etc.), and existing antibodies are prone to cross-reaction. Furthermore, traditional antibodies are easily interfered with by secondary metabolites during the pretreatment of plant samples, making it difficult to guarantee specificity and reproducibility.

[0004] Nanobody ( NanobodyVHH (variable region of heavy chain antibody) is derived from cameloids or cartilaginous fish, with a molecular weight of only about 15 kDa. Compared with traditional IgG, VHH has significant advantages such as small size, strong tissue penetration, high physicochemical stability (resistant to high temperature, acid and alkali, and protease), easy expression in prokaryotic / eukaryotic systems, and flexible modification into multivalent molecules. It has shown great application potential in the fields of biodiagnostics, environmental monitoring, and targeted delivery. In recent years, some studies have attempted to apply VHH technology to plant pathogen detection, but high-specificity, high-affinity nanobodies against TYLCV and their standardized detection systems are still in the research and development stage. Existing publicly available anti-geminivirus antibodies are mostly concentrated on a few model viruses, and their epitope recognition mechanisms and affinity characteristics cannot be directly transferred to TYLCV. Furthermore, there is a lack of VHH sequence libraries that have been validated by systematic wet experiments.

[0005] Meanwhile, the rapid development of artificial intelligence (AI) and computational biology technologies has provided a new paradigm for antibody engineering. Deep learning-based whole-atom structure prediction (such as the AlphaFold series) and generative models have enabled virtual screening and sequence design of the target-antibody binding interface. However, AI-assisted antibody design still faces several technical bottlenecks in the development of plant virus detection reagents: (1) There is a significant deviation between the binding confidence predicted by the algorithm (such as pLDDT / pTM / ipTM) and the wet experimental measured affinity (KD / EC50), and a large number of "high-scoring" candidate sequences are prone to aggregation, precipitation or loss of activity during expression; (2) The computational model is difficult to accurately simulate the influence of complex plant matrix on antibody folding and stability, and the in vitro prediction results are out of sync with the actual field conditions; (3) There is a lack of dedicated screening parameters for the surface charge distribution and flexible loop characteristics of geminiviruses, which makes the generated candidate sequences prone to non-specific binding with closely related viruses. Therefore, how to combine AI high-throughput virtual screening with rigorous in vitro expression purification, affinity determination and plant sample cross-reactivity verification to obtain TYLCV-specific nanobodies with high affinity, high environmental stability and extremely low cross-reactivity remains a technical challenge that needs to be overcome in this field.

[0006] In summary, current technologies lack a highly stable and specific nanobody and its corresponding detection scheme specifically for rapid TYLCV detection. Developing a TYLCV-targeting VHH that overcomes the shortcomings of traditional antibodies, such as large steric hindrance, susceptibility to plant matrix interference, poor environmental tolerance, and high risk of cross-reactivity, and that has been both computationally designed and experimentally validated, would have significant practical importance and application value for improving the early warning capabilities of solanaceous crop diseases in my country and globally, and for ensuring the security of international agricultural trade. Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for detecting tomato yellow leaf curl virus, its preparation method, and its application.

[0008] The present invention is implemented using the following technical solutions: The first aspect of the present invention provides a nanobody that specifically binds to the coat protein of tomato yellow leaf curl virus, wherein the complementarity-determining region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO.4-6, respectively.

[0009] Furthermore, the amino acid sequence of the nanobody is selected from: a) Contains the complete variable region sequence shown in SEQ ID NO.1; or b) A sequence that has ≥90% sequence identity with SEQ ID NO.1 and retains the function of specifically binding to the TYLCV capsid protein.

[0010] The second aspect of this invention provides the application of the above-mentioned nanobody in the preparation of TYLCV rapid detection reagents, colloidal gold immunochromatographic test strips, or viral load quantitative reagent kits.

[0011] A third aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding any of the nanobodies described above.

[0012] A fourth aspect of the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule and an operable T7 promoter and a selection marker.

[0013] A fifth aspect of the present invention provides a host cell containing the above-described recombinant expression vector.

[0014] The sixth aspect of this invention provides a method for preparing the above-mentioned nanobody, which includes the following steps: (1) Construct a recombinant expression vector containing the nucleotide sequence encoding the above-mentioned nanobody, then transform the recombinant expression vector into host cells, and then induce expression in the host cells; (2) Collect the expression product obtained in step (1), purify it by affinity chromatography, and obtain the nanobody.

[0015] The seventh aspect of the present invention provides an indirect ELISA kit for detecting tomato yellow leaf curl virus, comprising a solid-phase carrier on which a sample to be tested is coated.

[0016] Furthermore, the nanobody was labeled with a reporter molecule, which was horseradish peroxidase, colloidal gold particles, fluorescein, or biotin.

[0017] The eighth aspect of the present invention provides a method for detecting tomato yellow leaf curl virus, which involves contacting the sample to be tested with the above-mentioned nanobody and qualitatively detecting TYLCV by detecting the binding signal.

[0018] The present invention has the following beneficial effects: (1) High affinity and absolute specificity: The TYL_248 nanobody achieves EC by targeting a unique conformational epitope that is highly conserved and spatially exposed on the surface of TYLCV CP. 50 It exhibits nanomolar binding affinity of ≤30 nM. Cross-reactivity experiments confirmed that it showed no binding signal to TMV, ToMV, or healthy tomato substrates, completely resolving the false positive problem caused by cross-interference from closely related Tobamoviruses.

[0019] (2) Breakthrough physicochemical stability: Thanks to the VHH single-domain structural features and the rigid framework design of the CDR loop region, the antibody retains more than 78% of its activity after being treated at 50°C for 2 hours. It remains stable in the pH range of 3.0 to 9.0 and has strong resistance to proteases in conventional plant extracts. It is significantly better than traditional IgG antibodies (with less than 30% activity retention under the same conditions) and is fully adapted to the complex tropical / subtropical field environment.

[0020] (3) Closed-loop innovation of AI design and wet experimental verification: This invention uses generative algorithms as a high-throughput conformation sampling tool, rather than as the core of the invention. The technology is closed-loop through prokaryotic expression, ELISA kinetic assay and blinded sample verification. The computational model cannot directly predict the low immunogenicity, high soluble expression (>15 mg / L fermentation broth) and the ultra-low limit of detection (LOD) of 10 pg / mL for colloidal gold test strips.

[0021] (4) Platform adaptability and industrialization potential: The nanobody has a molecular weight of only about 15 kDa, strong tissue penetration, and can be flexibly converted into various detection forms such as double antibody sandwich ELISA, fluorescence immunochromatography, lateral flow test strips, and microfluidic chips. Its prokaryotic expression process is mature, low-cost, and has high batch-to-batch consistency, and it is ready for large-scale production. It can directly meet the rapid screening needs of agricultural quarantine, seed import and export inspection, and grassroots plant protection stations.

[0022] (5) The nanobody and its derivative detection products described in this invention provide efficient, stable and low-cost molecular diagnostic tools for early warning, precise prevention and control of TYLCV and international trade quarantine, and have significant agricultural biosafety value and broad market application prospects. Attached Figure Description

[0023] Figure 1 A structural model of the tomato yellow leaf curl virus coat protein AV1 (UniProtKB / TrEMBL: Q8UYR5); Figure 2 To generate the structural model of VHH antibody TYL_248; Figure 3 Hdock docking scores (partial) for generating 1000 nanobodies; Figure 4 To optimize the AF3 analysis results of 20 nanobodies; Figure 5 Interaction diagram of candidate nanobody TYL_248 with target antigen (UniProtKB / TrEMBL: Q8UYR5); Figure 6 The results of blastp alignment of the amino acid sequence of the nanobody TYL_248; Figure 7 The results of tblastn alignment of the amino acid sequence of the nanobody TYL_248; Figure 8 SDS-PAGE electrophoresis of the recombinant TYL_248 nanobody before and after purification; Figure 9 Western blot signals of the 6×His tag before and after purification of the recombinant TYL_248 nanobody; Figures 10-14 The BLASTN comparison result for SEQ ID NO.3; Figure 15 TYL_248 concentration and average OD 450 Correspondence diagram. Detailed Implementation

[0024] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0025] Example 1: 1. Determination of binding sites The amino acid sequence of TYLCV coat protein AV1 (Coat Protein, CP, GenBank accession number UniProtKB / TrEMBL: Q8UYR5) was obtained, and a structural model of the target protein was constructed using AlphaFold3. The overall prediction PTM score of the model was 0.81, indicating that its global topology is highly reliable. The three-dimensional structure was visualized using PyMOL software, and compared with the reference structure, the posterior end was relatively conserved except for approximately 60 amino acids at the front. Two candidate epitope regions were selected from the conserved region: one is the loop region, and the other is a region with a fixed β-sheet. Each region has its advantages and disadvantages. Considering the structural stability of the epitope region, the β-sheet region was selected as the target region for nanobody design. In this invention, the designed target region (purple, A72-A96) in the TYLCC protein is as follows. Figure 1 As shown.

[0026] The sequence of the tomato yellow leaf curl virus coat protein (UniProtKB / TrEMBL: Q8UYR5) is shown in SEQ ID NO.7, where the underlined portion (A72-A96) is the target epitope region for designing VHH antibodies. SEQ ID NO.7: MSKRPADIVISTPASKVRRRLNFDSPYMGRAAAPTVRVTRRQMWSNRPMYRKPMMYRMYRSPDVPKGCEGP CKVQSYEARHDIAHTGKVICVTDVT RGNGITHRVGKRFCVKSIYVIGKIWMDENIKVKNHTNTVMFFLVRDRRPSGTPMDFQQVFNCYDNEPSTATVKNDLRDRFQVRRKFYSTVTGGQYACKEQALVKKFIRVNNHVVYNHQEQAKYENHTENALLLYMVTTHASNPVYATLKVRIYFYDSQLN.

[0027] 2. RFdiffusion conjugate design After determining the active site of the bait protein, RFdiffusion was used to design nanobodies, generating 1000 nanobodies for molecular docking with the bait protein.

[0028] RFdiffusion utilizes diffusion models to generate protein structures with high specificity and affinity, making it suitable for precise conjugate design and offering significant advantages in fields such as drug development and enzyme engineering.

[0029] 3. Hdock Filtering Hdock is a highly efficient protein-protein interaction docking tool that combines template matching and free docking algorithms to accurately predict intermolecular interactions. In the Hdock fine-tuning stage, by calculating binding scores based on bait proteins and structure-aligned proteins, it can more effectively identify protein interactions with better stability and stronger affinity. This is an important screening indicator that helps improve the accuracy of screening results.

[0030] By using bait proteins to perform molecular docking with 300 designed short peptides, the short peptides with the highest affinity were obtained. The top 3 of the Hdock docking scores for each of the three short peptides of different amino acid lengths were selected for nanobody splicing.

[0031] Hdock docking results are generally based on the docking score, calculated using the formula: Confidence_score = 1.0 / [1.0+e0.02*(Docking_Score+150)]. A docking score minus 200 can be used as a reference, resulting in a confidence score of approximately 0.7. In the official documentation, a confidence score > 0.7 indicates a high probability of binding between the two protein molecules; a score between 0.5 and 0.7 indicates a possible binding; and a confidence score < 0.5 indicates a low probability of binding.

[0032] 4. AlphaFold3 One-to-One Fine-grained Modeling Analysis Transferring the top data selected by hdock to AlphaFold3 to calculate pTM+ipTM values ​​further refines and validates the screening results. AlphaFold3's high accuracy in predicting protein complex structures helps confirm the reliability and biological significance of candidate protein interactions.

[0033] Note: pTM is the predicted TM score, used to assess the accuracy of predicting the overall three-dimensional structure of a protein. This metric predicts the correctness of the overall protein folding and is compared with the similarity to the experimental structure; ipTM is the interface prediction TM score, used to assess the accuracy of predicting the interaction interfaces between two protein complexes. It specifically assesses the accuracy of predicting the structure of contact regions between two or more proteins.

[0034] 5. For example Figure 3As shown, among the 1000 designed nanobodies, all nanobodies achieved a binding threshold of -200 Hdock scores, and the binding site regions exhibited a large number of hydrogen bonds, displaying a very stable conformation. Among the 100 preferred candidate nanobodies, TYL_248 had the highest score. TYL_248 had an iPTM value of 0.56, lower than the expected 0.78; a PTM value of 0.67, lower than the expected 0.80; and an iPTM+PTM value of 1.23. The amino acid sequence of the complementarity-determining region (CDR) of TYL_248 is as follows: CDR1: AASGIMPDTSYMG (SEQ ID NO.4); CDR2: IDGGSITNYADS (SEQ ID NO.5); CDR3: VMYFTGAEILFWHYY (SEQ ID NO.6).

[0035] The CDR delineation adopts the IMGT unique numbering standard; the complete variable region (VHH) amino acid sequence of the nanobody is shown in SEQ ID NO.1. The interaction between the candidate nanobody TYL_248 and the tomato yellow leaf curl virus coat protein (UniProtKB / TrEMBL: Q8UYR5) is as follows: Figure 5 As shown. Figure 5 In the study, the binding site of the TYL_248 nanobody (yellow) showed off-target behavior compared to the initially designed target site (purple), and the binding effectiveness needs to be verified.

[0036] SEQ ID NO.1 (TYL_248 nanobody amino acid sequence): QVQLQESGGGLVQAGGSLRLSCAASGNIFPDTSYMGWYRQAPGKERELVAAIDGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVMYFTGAEILFWHYYWGQGTQVTVSS.

[0037] like Figure 2 To generate the structural model of VHH antibody TYL_248; Figure 4 The AF3 analysis results of 20 nanobodies were selected for optimal selection.

[0038] 6. Amino acid sequence comparison showed that the TYL_248 nanobody is a novel sequence that has not been previously reported. The amino acid sequence of the nanobody TYL_248 was submitted to the GenBank database for BLASTP and TBASTN analysis. The results showed that the BLASTP alignment results (see...) Figure 6 The highest similarity was 84.43%; in the tblastn comparison results (see...), Figure 7The highest similarity was 82.93%. Both comparison results indicate that the amino acid sequence (TYL_248) of the polypeptide described in this invention is different from any known sequence and is a new sequence.

[0039] Example 2: Wet Experiment Verification and Final Optimization The iPTM value for TYL_248 is 0.56, lower than the expected 0.78; the PTM value is 0.67, lower than the expected 0.80; the iPTM+PTM value is 1.23, and it is in an off-target state (the target sequence is...). Figure 5 The purple fragment (A72-A96) needs to be verified by wet assay to determine the binding activity of TYL_248 to the target antigen.

[0040] 1. Recombinant expression of TYL_248 candidate nanobodies Based on the amino acid sequence shown in SEQ ID NO.1, peptide bridges and histidine tags were further added to obtain the amino acid sequence shown in SEQ ID NO.2. The amino acid sequence shown in SEQ ID NO.2 was reverse-translated using E. coli preferred codons to synthesize the nucleotide sequence shown in SEQ ID NO.3. The synthesized fragment was cloned into the NdeI / XhoI site of the pET-28a(+) vector to construct the recombinant plasmid pET-28a-TYL_248. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium, and cultured with shaking at 37°C until OD. 600 ≈0.6, add IPTG to a final concentration of 0.5 mM, and induce expression at 16℃ for 16 h. Collect bacterial cells by centrifugation, sonicate to disrupt, and collect the supernatant. Purify using a Ni-NTA affinity chromatography column, and dialyze against PBS buffer to remove imidazole. SDS-PAGE showed a single master band with a molecular weight of approximately 15 kDa (e.g., ≈0.6). Figure 8 As shown), Western blotting was positive for anti-His tag antibody (e.g. Figure 9 As shown in the figure), this indicates that TYL_248 was successfully expressed and purified. BLASTN comparison of the nucleotide sequence shown in SEQ ID NO.3 showed that it had 83.38% similarity to the first-order target sequence (as shown in the figure). Figure 10 - Figure 14 As shown in the figure, the nucleic acid sequence described in this invention is different from any known sequence and is a new sequence.

[0041] SEQ ID NO.2 (the underlined portion represents the peptide bridge “GGGGSGGGGSGGGGS” and the histidine tag “HHHHHH”): QVQLQESGGGLVQAGGSLRLSCAASGNIFPDTSYMGWYRQAPGKERELVAAIDGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVMYFTGAEILFWHYYWGQGTQVTVSS GGGGSGGGGSGGGGSHHHHHH.

[0042] SEQ ID NO.3: caggtgcagctgcaggaaagcggcggcggcctggtgcaggcgggcggcagcctgcgcctgagctgcgcggcgagcggcaacatttttccggataccagctatatgggct ggtatcgccaggcgccgggcaaagaacgcgaactggtggcggcgattgatggcggcagcattaccaactatgcggatagcgtgaaaggccgctttaccattagccgcga taacgcgaaaaacaccgtgtatctgcagatgaacagcctgaaaccggaagataccgcggtgtattattgcgcggtgatgtattttaccggcgcggaaattctgttttgg cattattattggggccagggcacccaggtgaccgtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccatcatcatcatcatcattaa.

[0043] 2. TYL_248 Affinity, Specificity, and Thermal Stability Tests (1) Indirect ELISA binding activity assay method Coating: The purified target antigen (TYLCV CP protein, 2 μg / mL) was added to a 96-well microplate at 100 μL / well and incubated overnight at 4°C.

[0044] Blocking: Discard the coating solution, wash 3 times with PBST, add 200 μL of 5% skim milk powder / PBST / well, and block at 37℃ for 2 h.

[0045] Primary antibody incubation: TYL_248 was serially diluted with PBST (0.1, 1, 5, 10, 50, 100, 500, 1000 nM), 100 μL / well, and incubated at 37℃ for 1 h. Blank wells (buffer only) and isotype-independent nanobody controls were included.

[0046] Secondary antibody and color development: After washing, add HRP-labeled anti-His-tagged secondary antibody (1:5000 dilution), incubate at 37℃ for 45 min; incubate with TMB substrate for 10 min, stop with 2 M H2SO4, and measure OD using a microplate reader. 450 .

[0047] Data processing: GraphPad Prism 9.0 was used to plot S-shaped dose-response curves and calculate EC50. 50 Value. All samples were set up with 3 replicates, and were independently repeated 3 times.

[0048] (2) Test results TYL_248 exhibits typical dose-dependent binding characteristics, EC 50 The maximum binding signal OD is approximately 28.6 ± 3.2 nM; at 100 nM 450 ≈1.85±0.06, which is 28.6 times that of the negative control. P <0.001, two-tailed t-test), indicating that it has high sensitivity antigen recognition ability (e.g., Figure 15 YL_248 concentration and average OD 450 The fitting equation is: .

[0049] In the equation, X is the concentration of TYL_248 (nM), and Y is the OD. 450 value.

[0050] 3. TYL_248 Thermal Stability Test (1) Thermal stability test method Purified TYL_248 (0.1 mg / mL, PBS pH 7.4) was aliquoted into sterile EP tubes. The experimental group was placed in a 50°C constant temperature water bath for 2 h, while the control group was stored at 4°C.

[0051] After treatment, centrifuge at 12,000×g for 10 min to remove heat-induced aggregates, and take the supernatant to determine the remaining binding activity according to the aforementioned indirect ELISA conditions.

[0052] Activity retention rate = (OD50℃ treatment group / OD4℃ control group) × 100%.

[0053] (2) Thermal stability test results After a heat stress of 50℃ for 2 h, the TYL_248 binding activity retention rate reached 80.4±3.5%, indicating that it has excellent heat resistance and conformational stability.

[0054] 4. TYL_248 Specificity Test – Cross-Reactivity Validation (1) Cross-reaction verification method Coating agents: purified viral particles of TYLCV, TMV, and ToMV (2 μg / mL each, provided by Professor Zhang Pengjun of Hangzhou Normal University) and total protein extract from healthy tomato leaves (50 μg / mL).

[0055] With a fixed working concentration of TYL_248 (100 nM), the binding signals of each matrix were detected in parallel according to the standard indirect ELISA procedure.

[0056] Cross-reactivity rate = (OD of test virus or matrix / OD of target TMV) × 100%. Include a buffer-coated blank well and a secondary antibody background control.

[0057] (2) Cross-reactivity verification results TYL_248 showed a strong positive signal (OD) against the target TYLCV. 450 =1.80±0.07); the cross-binding rates for TMV and ToMV were 2.4%±0.6% and 2.1%±0.5%, respectively; the non-specific adsorption on healthy tomato matrix was 1.9%±0.8%. The results indicate that the nanobody is highly specific to the target virus and is not affected by closely related viruses or complex plant matrix.

[0058] In summary, the candidate nanobody TYL_248 exhibits a wide linear range and nanomolar-level ECGs in indirect ELISA. 50 The activity retention rate was >80% after a 50℃ / 2 h heat stress test; cross-reactivity validation confirmed its high specificity for TYLCV and its independence from interference by healthy tomato substrates. Overall data indicate that TYL_248 possesses high affinity, strong specificity, and excellent thermostability, meeting the candidate criteria for subsequent development of rapid field detection reagents, construction of immunochromatographic test strips, or structure-function mechanism studies.

[0059] Example 3: Verification of the specific binding of TYL_248 to the TYLCV coat protein Binding activity was verified using an indirect ELISA method. Purified TYLCV capsid protein AV1 (UniProtKB / TrEMBL: Q8UYR5) was coated onto 96-well plates at 2 μg / mL and incubated overnight at 4°C. After blocking, serially diluted TYL_248 (0.1–1000 nM) was added, and the plates were incubated at 37°C for 1 h. HRP-labeled anti-His secondary antibody was added, TMB was used for colorimetric analysis, and OD values ​​were measured at 450 nm. Results showed that TYL_248 bound to TYLCV CP in a dose-dependent manner, with EC50... 50 The value was 28.2 ± 1.6 nM. Using total protein, TMV, ToMV, and PepMV CP from healthy tomato leaves as negative controls, the OD values ​​were all lower than twice the standard deviation of the blank control, confirming that TYL_248 has high specificity for TYLCVCP.

[0060] Example 4: Cross-reactivity and thermal stability test TYL_248 was treated in water baths at 37℃, 50℃, and 65℃ for 2 h, respectively, followed by ELISA binding activity assays. The results showed that the antibody binding activity retention rate was 81.5±3.2% after treatment at 50℃ for 2 h, and still 60.7±4.1% after treatment at 65℃, significantly better than traditional murine monoclonal antibodies (activity retention rate <30% under the same conditions). The activity remained stable after incubation in pH 3.0–9.0 buffer for 24 h.

[0061] Cross-reactivity experiments confirmed that TYL_248 only recognizes TYLCV CP and does not cross-bind with TMV, ToMV, and PepMV, which are easily mixed, thus meeting the stringent specificity requirements for port quarantine and rapid field diagnosis.

[0062] Example 5: Construction and field validation of colloidal gold test strips based on TYL_248 TYL_248 was labeled onto 15 nm colloidal gold particles to prepare a detection line (T line). A separate anti-TYLCV CP polyclonal antibody was used as a control line (C line) and assembled with the coating antibody to form a double-antibody sandwich test strip. TYLCV-infected tomato leaf homogenate was serially diluted and added to the sample application area of ​​the test strip, with results observed within 15 minutes. The limit of detection (LOD) reached 20 pg / mL (equivalent to a 1:2500 dilution of a positive sample). Blind testing was performed on 42 field samples (confirmed by RT-PCR). The test strip showed a detection sensitivity of 96.2% (25 / 26), a specificity of 100% (16 / 16), and a concordance rate with RT-PCR ≥100%, demonstrating that TYL_248 has industrial application value.

Claims

1. A nanobody that specifically binds to the coat protein of tomato yellow leaf curl virus, characterized in that, The complementarity-determining regions of the nanobody include CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO.4-6, respectively.

2. The nanobody that specifically binds to the coat protein of tomato yellow leaf curl virus according to claim 1, characterized in that, The amino acid sequence of the nanobody is selected from: a) Contains the complete variable region sequence shown in SEQ ID NO.1; or b) A sequence that has ≥90% sequence identity with SEQ ID NO.1 and retains the function of specifically binding to the TYLCV capsid protein.

3. The application of the nanobody according to any one of claims 1 to 2 in the preparation of TYLCV rapid detection reagents, colloidal gold immunochromatographic test strips or viral load quantitative reagent kits.

4. A nucleic acid molecule, characterized in that, It comprises a nucleotide sequence encoding the nanobody of any one of claims 1 to 2.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule of claim 4 and the operably linked T7 promoter and selection marker.

6. A host cell, characterized in that, It contains the recombinant expression vector as described in claim 5.

7. The method for preparing nanobodies according to any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing a nucleotide sequence encoding any one of the nanobodies of claims 1 to 2, then transform the recombinant expression vector into host cells, and then induce expression in the host cells; (2) Collect the expression product obtained in step (1), purify it by affinity chromatography, and obtain the nanobody.

8. A kit for detecting tomato yellow leaf curl virus, characterized in that, The invention comprises a solid support on which nanobodies as described in any one of claims 1 to 2 are coated; or nanobodies on which reporter molecules are labeled.

9. The reagent kit according to claim 8, characterized in that, The reporter molecule is horseradish peroxidase, colloidal gold particles, fluorescein, or biotin.

10. A method for detecting tomato yellow leaf curl virus, characterized in that, The sample to be tested is brought into contact with the nanobody described in any one of claims 1 to 2, and TYLCV is qualitatively detected by detecting the binding signal.

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