Nanobody for detecting tomato brown rugose fruit 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 brown rugose fruit virus and a preparation method and application thereof. The application discloses a specific nanobody for detecting tomato brown rugose fruit virus and application thereof. Complementarity determining region sequences of the nanobody are shown in SEQ ID NO: 4-6; and a complete variable region amino acid sequence of the nanobody is shown in SEQ ID NO: 1. The application simultaneously provides a nucleotide sequence for encoding the nanobody, a recombinant expression vector, a host cell and a preparation method thereof. The nanobody has high affinity and excellent thermal stability to ToBRFV coat protein, and has no cross reaction with TMV, ToMV and other related viruses. A detection kit and a colloidal gold test strip constructed based on the antibody have a detection limit of 10 pg / mL, and are suitable for rapid screening and field quantitative diagnosis of ToBRFV.

Description

Technical Field

[0001] This invention belongs to the field of biodetection and antibody engineering technology, specifically relating to a specific nanobody (VHH) targeting the coat protein (CP) of Tomato brown rugose fruit virus (ToBRFV), its encoding nucleic acid sequence, recombinant expression system, and further relating to a detection kit containing the nanobody, an immunochromatographic test strip, and its application in rapid screening, field quantitative diagnosis, and port quarantine of ToBRFV. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) belongs to the Tobamovirus genus and is a single-stranded positive-sense RNA virus. This virus can be efficiently transmitted through multiple routes, including seeds, sap friction, agricultural operations, and pollinating insects. Infection causes typical brown wrinkles, deformities, necrotic spots, and sharp yield reductions in fruits, potentially leading to total crop failure. Currently, there are no commercially available resistant varieties for ToBRFV; early and accurate detection and strict quarantine are the core means to prevent its cross-border spread.

[0003] Existing ToBRFV detection technologies are mainly divided into two categories: molecular biology detection and immunological detection. Molecular detection primarily uses reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR). 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 (LFIA), 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: (1) They are large in size and have obvious steric hindrance, making it difficult to effectively bind to hidden or conformational epitopes on the surface of plant virus particles; (2) They have poor thermal stability and pH tolerance, and are prone to denaturation and inactivation in the field at high temperature and humidity or in complex matrices of plant crude extracts, leading to false negatives; (3) They are highly homologous to the coat protein (CP) sequences of Tobacco Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV), Pepper Light Mottle Virus (PepMV), etc., which belong to the same Tobamovirus family (amino acid homology is often >85%), and existing antibodies are very prone to cross-reaction, resulting in a high false positive rate.

[0004] Nanobodies (VHHs) are variable regions of heavy chain antibodies derived from camelids or cartilaginous fish, with a molecular weight of only about 15 kDa. Compared with traditional IgG, VHHs have significant advantages such as small size, strong tissue penetration, high stability (resistant to high temperatures, acids and alkalis, and proteases), ease of expression in prokaryotes / yeast, and the ability to be modified into multivalent molecules, demonstrating great potential in the fields of biodiagnostics and targeted delivery. In recent years, some studies have attempted to apply VHH technology to plant virus detection, but specific nanobodies against ToBRFV remain a gap in public research. Existing publicly available anti-Tobamovirus antibodies mostly target TMV or ToMV, and their epitope recognition mechanisms and affinity cannot directly migrate to ToBRFV, and there is a lack of systematically validated high-specificity VHH sequences.

[0005] Meanwhile, the rapid development of artificial intelligence (AI) and computational biology technologies has provided a new paradigm for antibody engineering. Deep learning-based full-atom structure prediction (such as the AlphaFold series) and generative models have enabled virtual screening and sequence generation of target binding interfaces. However, AI-assisted antibody design still faces severe challenges in industrialization: (1) there is a significant discrepancy between the binding confidence predicted by the algorithm (such as pTM / ipTM) and the wet experimental measured affinity (KD / EC50), and a large number of "high-scoring" sequences aggregate, precipitate, or become completely inactive during expression; (2) computational models have difficulty accurately simulating the impact of complex biological environments (such as the interference of proteases, polyphenols, and polysaccharides in plant crude extracts) on antibody stability; (3) there is a lack of dedicated screening parameters for the surface charge distribution and flexible loop characteristics of specific plant viruses, resulting in insufficient specificity of the generated candidate sequences. Therefore, how to combine AI high-throughput screening with rigorous in vitro / in vivo validation to obtain ToBRFV-specific nanobodies with high affinity, zero cross-reactivity, and excellent environmental stability remains a technical bottleneck that urgently needs to be solved in this field.

[0006] In summary, current technologies lack a highly stable and specific nanobody and its corresponding detection scheme specifically for rapid detection of ToBRFV. Developing a ToBRFV-targeting VHH nanobody that overcomes the shortcomings of traditional antibodies, such as large steric hindrance, easy cross-reactivity, and poor environmental tolerance, and that has been experimentally validated, is of significant practical importance for improving the disease early warning capabilities of the tomato industry in my country and globally, and for ensuring the security of international agricultural trade. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a specific nanobody that can bind to tomato brown wrinkle virus with high specificity and high affinity, as well as detection products and applications based on this nanobody pair.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a nanobody that specifically binds to the coat protein of tomato brown wrinkle fruit virus, the complementarity-determining region sequence of which is shown in SEQ ID NO:4-6: CDR1: GGSISNYTIG (SEQ ID NO.4); CDR2: ISSTGIPGVPEKTRYADS (SEQ ID NO.5); CDR3: ANSYSAYLTYTSEYDY (SEQ ID NO. 6).

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

[0010] The second aspect of this invention provides the application of the above-mentioned nanobody in the preparation of ToBRFV 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 encoding the aforementioned nanobody.

[0012] A fourth aspect of the present invention provides a recombinant expression vector comprising the above-described nucleic acid molecule and an operable promoter and 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, comprising the following steps: (1) Express the above-mentioned host cells under induction conditions; (2) Collect the expression product described in step (1), purify it by affinity chromatography, and obtain the nanobody.

[0015] The seventh aspect of the present invention provides a kit for detecting tomato brown wrinkle virus, comprising a solid-phase carrier coated with the above-mentioned nanobodies; or comprising the nanobodies labeled with a reporter molecule.

[0016] Furthermore, the reporter molecule is horseradish peroxidase, colloidal gold particles, fluorescein, or biotin.

[0017] The eighth aspect of the present invention provides a method for detecting Tobacco Brown Ruffle Virus (ToBRFV), comprising contacting the sample to be tested with the aforementioned nanobody and determining the presence or concentration of ToBRFV by detecting the binding signal.

[0018] Compared with existing technologies, the present invention has the following significant technical effects and industrialization advantages: 1. High affinity and absolute specificity: The TBF_4248 nanobody achieves EC by targeting a unique conformational epitope that is highly conserved and spatially exposed on the surface of ToBRFV 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 >78% of its activity after treatment at 50℃ for 2 hours, remains stable in the pH range of 3.0~9.0, and has strong resistance to proteases in conventional plant extracts, which is significantly better than traditional IgG antibodies (activity retention rate <30% under the same conditions), making it fully adaptable to the complex tropical / subtropical field environment.

[0020] 3. Closed-loop innovation in AI design and wet experimental validation: This invention uses generative algorithms as a high-throughput conformational sampling tool, rather than as the core of the invention. A closed-loop technology is achieved through prokaryotic expression, ELISA kinetic assays, and blinded sample validation. The invention achieves low immunogenicity, high soluble expression (>15 mg / L fermentation broth), and an ultra-low limit of detection (LOD) of 10 pg / mL for colloidal gold test strips, which cannot be directly predicted by computational models.

[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 methods such as double-antibody sandwich ELISA, fluorescence immunochromatography, lateral flow test strips (LFA), 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] The nanobody and its derived detection products described in this invention provide an efficient, stable, and low-cost molecular diagnostic tool for early warning, precise prevention and control of ToBRFV and international trade quarantine, which has significant value for agricultural biosafety and broad market application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Structural model of the capsid protein YP_009182171.1 of tomato brown wrinkled fruit virus; Figure 2 Generate an iPTM distribution of 10,000 nanobodies; Figure 3 The PAE distribution of 10,000 nanobodies was generated; Figure 4 RMSD distribution of 10,000 nanobodies generated; Figure 5 Interaction diagram of candidate nanobody TBF_4248 with target antigen (YP_009182171.1); Figure 6 BLASTP sequence alignment results of nanobody TBF-4248; Figure 7 tblastn alignment results of the amino acid sequence of nanobody TBF-4248; Figure 8 SDS-PAGE electrophoresis of recombinant TBF_4248 nanobody before and after purification; Figure 9 Western blot signals of 6× His tag before and after purification of recombinant TBF_4248 nanobody; Figures 10-13 The comparison results for BLASTN for SEQ ID NO.3. Detailed Implementation

[0025] Example 1:

[0026] The outer shell protein of the tomato brown wrinkled fruit virus (YP_009182171.1) was used for large-scale de novo nanobody design on the BoltzGen platform. 100 candidate nanobodies were selected, among which TBF_4248 was the candidate nanobody with the highest score.

[0027] 1. Construct a structural model of the target antigen protein using AlphaFold3. The amino acid sequence of the ToBRFV coat protein (CP, GenBank accession number YP_009182171.1) was obtained, and a structural model of the target protein was constructed using AlphaFold3. The overall prediction TM score of the model was as high as 0.86, indicating that its global topological structure was highly reliable. The confidence score per residue of the model is presented as PLDDT value, where the red area (usually PLDDT>80) indicates that the confidence of the structure is extremely high; while the yellow and green areas represent the increased uncertainty of local conformation (e.g., Figure 1 (As shown). Further analysis of surface electrostatic potential distribution, comparison of residue conservation, and calculation of solvent accessible surface area (SASA) were used to screen potential binding epitope regions located on the surface of viral particles that are highly conserved in sequence and spatially exposed.

[0028] The sequence of the outer coat protein (YP_009182171.1) of the tomato brown wrinkled fruit virus is shown in SEQ ID NO.7: >YP_009182171.1 (SEQ ID NO.7) MSYTIATPSQFVFLSSAWADPIELINLCTNSLGNQFQTQQARTTVQRQFSEVWKPVPQVTVRFPDSGFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRVDDATVAIRSAINNLVVELVKGTGLYNQSTFESASGLQWSPAAS.

[0029] 2. Design 10,000 candidate nanobodies from scratch. against Figure 1The structural model shown is used for nanobody design based on the BoltzGen All-atom Generative Model, a unified structure prediction and binding design platform. The potential epitopes are input into the BoltzGen antibody generation platform (or a generative antibody design system based on diffusion models / graph neural structures), using the Camelidae VHH natural backbone as a structural prior, to generate 10,000 candidate nanobody amino acid sequences in batches. The following physicochemical constraints are applied during the generation process: the CDR-H3 ring length is limited to 10–16 amino acids; the proportion of hydrophobic residues is controlled between 0.35 and 0.45; and candidates containing free cysteine ​​(Cys), N-glycosylation sites (Asn-X-Ser / Thr), and common protease-sensitive sequences are eliminated. The confidence scores of the predicted templates (pTM≥0.75) and interfaces (ipTM≥0.78) were calculated, and the molecular docking binding free energy (ΔG≤-8.0 kcal / mol) was used for initial screening. Entries with a BLASTP similarity >75% to known VHH sequences in GenBank were further excluded through sequence alignment, resulting in a preliminary screening library of ≤50 high-confidence candidate sequences. The design parameters are shown in Table 1.

[0030] Table 1. Parameter settings for the design of 1000 nanobodies .

[0031] 3. Scoring and multi-level filtering to screen high-scoring candidate nanobodies The top-ranked nanobodies were selected based on their structural quality root mean square deviation (RMSD), interface prediction TM score (IPTM score), prediction alignment error (PAE), solvent accessible surface area (SASA), and the number of interactions such as salt bridges and hydrogen bonds. The 10,000 designed nanobodies were ranked according to the rules described in Table 2. Table 3 summarizes the parameters of all 10,000 designed nanobodies, as well as the top 10 and top 100 nanobodies. Table 4 provides a detailed explanation of the parameters listed in Table 3. Figures 2-4 The distribution maps of all 10,000 generated nanobody iPTM, PAE, and RMSD are shown respectively. The corresponding thresholds in the three maps are 0.4, 5, and 2, respectively.

[0032] Table 2. Sorting rules for designing and generating nanobodies using BoltzGen. .

[0033] Table 3. Summary statistics of the parameters of the designed nanobody .

[0034] Table 4. Detailed Explanation of Parameters in the Joint Statistics Table .

[0035] 4. Obtained one high-resolution candidate nanobody, TBF_4248. After prediction, generation, scoring and ranking, the high-scoring candidate nanobody TBF_4248 was obtained. TBF_4248 has an iPTM value of 0.44, lower than the expected 0.78; a PTM value of 0.79, close to the expected value of 0.80; and an iPTM+PTM value of 1.23.

[0036] The amino acid sequences of the complementarity-determining region (CDR) of TBF_4248 are as follows: CDR1: GGSISNYTIG (SEQ ID NO.4); CDR2: ISSTGIPGVPEKTRYADS (SEQ ID NO.5); CDR3: ANSYSAYLTYTSEYDY (SEQ ID NO.6).

[0037] Preferably, the CDR delineation adopts the IMGT unique numbering standard; more preferably, the complete variable region (VHH) amino acid sequence of the nanobody is shown in SEQ ID NO.1. The interaction between the candidate nanobody TBF-4248 and the tomato brown wrinkled fruit virus coat protein (YP_009182171.1) is as follows: Figure 5 As shown.

[0038] SEQ ID NO.1 (TBF-4248 nanobody amino acid sequence): QVQLQESGGGLVQAGDSLKLSCEASGGSISNYTIGWFRQAPGKERIYLATISSTGIPGVPEKTRYADSVKGRFAVSRDNAKNTVNLQMNSLKPEDTAVYYCAAANSYSAYLTYTSEYDYWGQGTQVTVSS.

[0039] 5. Amino acid sequence comparison showed that the TBF-4248 nanobody is a novel sequence that has not been previously reported. The amino acid sequence of the nanobody TBF-4248 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 76.38%; in the tblastn comparison results (see...), Figure 7 The highest similarity was 63.08%. Both comparison results indicate that the amino acid sequence (TBF-4248) of the polypeptide described in this patent is different from any known sequence and is a new sequence.

[0040] Example 2: Wet Experiment Verification and Final Optimization The iPTM value for TBF_4248 was 0.44, lower than the expected 0.78; the PTM value was 0.79, close to the expected 0.80; and the iPTM+PTM value was 1.23. Its binding activity to the target antigen needs to be verified by wet assay.

[0041] 1. Recombinant expression of TBF_4248 candidate nanobodies Based on the amino acid sequence shown in SEQ ID NO.1, a peptide bridge and histidine tag were further added to obtain SEQ ID NO.2. SEQ ID NO.2 was reverse-translated using E. coli preferred codons to synthesize the coding sequence (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-TBF_4248. 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 with 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 TBF_4248 was successfully expressed and purified. BLASTN comparison results for SEQ ID NO.3 show that its similarity to the first-order target sequence is 79.29% (as shown in the figure). Figures 10-13 As shown in the figure, this indicates that the nucleic acid sequence described in this patent is different from any known sequence and is a new sequence.

[0042] SEQ ID NO.2 (The underlined portion represents the peptide bridge “GGGGSGGGGSGGGGS” and the histidine tag “HHHHHH”) QVQLQESGGGLVQAGDSLKLSCEASGGSISNYTIGWFRQAPGKERIYLATISSTGIPGVPEKTRYADSVKGRFAVSRDNAKNTVNLQMNSLKPEDTAVYYCAAANSYSAYLTYTSEYDYWGQGTQVTVSS GGGGSGGGGSGGGG SHHHHHH .

[0043] SEQ ID NO.3: caggtgcagctgcaggaaagcggcggcggcctggtgcaggcgggcgatagcctgaaactgagctgcgaagcgagcggcggcagcattagcaactataccattggctggtttcgc caggcgccgggcaaagaacgcatttatctggcgaccattagcagcaccggcattccgggcgtgccggaaaaaacccgctatgcggatagcgtgaaaggccgctttgcggtgagc cgcgataacgcgaaaaacaccgtgaacctgcagatgaacagcctgaaaccggaagataccgcggtgtattattgcgcggcggcgaacagctatagcgcgtatctgacctatacc agcgaatatgattattggggccagggcacccaggtgaccgtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccatcatcatcatcattaa.

[0044] 2. TBF_4248 Affinity, Specificity, and Thermal Stability Tests (1) Indirect ELISA binding activity assay method Coating: The purified target antigen (ToBRFV CP protein, 2 μg / mL) was added to a 96-well microplate at 100 μL / well and incubated overnight at 4°C.

[0045] 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.

[0046] Primary antibody incubation: TBF_4248 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.

[0047] 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 .

[0048] Data processing: GraphPad Prism 9.0 was used to plot the S-shaped dose-response curve and calculate EC50. 50Value. All samples were set up with 3 replicates, and were independently repeated 3 times.

[0049] (2) Test results TBF_4248 exhibits typical dose-dependent binding characteristics, EC 50 The maximum binding signal OD is approximately 28.6 ± 3.2 nM; at 100 nM 450 The value was approximately 1.85 ± 0.06, which was 28.6 times that of the negative control (P < 0.001, two-tailed t-test), indicating that it has a high sensitivity to antigen recognition.

[0050] 3. Thermal stability test of TBF_4248 (1) Thermal stability test method Purified TBF_4248 (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 = (OD) 50 (℃ treatment group / OD4℃ control group) × 100%.

[0053] (2) Thermal stability test results After a heat stress of 50℃ for 2 h, the TBF_4248 binding activity retention rate reached 78.5±2.7%, indicating that it has excellent heat resistance and conformational stability.

[0054] 4. TBF_4248 Specificity Test - Cross-reactivity Validation (1) Cross-reaction verification method Coating agents: purified viral particles of ToBRFV, 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 TBF_4248 (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 TBF_4248 showed a strong positive signal (OD) for the target ToBRFV.450 =1.72±0.08); the cross-binding rates for TMV and ToMV were 3.1%±0.4% and 2.4%±0.3%, respectively; the non-specific adsorption on healthy tomato matrix was only 1.8%±0.2%. 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] 5. Surface Plasmon Resonance (SPR) Kinetic Analysis (1) Antigen / antibody binding kinetic analysis assay Instrument: Biacore T200 system. Chip: CM5. Run buffer: HBS-EP+ (PBS containing 0.05% Tween 20).

[0059] Immobilization: The target antigen is coupled to the chip surface via EDC / NHS amino groups, with a response value of approximately 8500 RU.

[0060] Kinetic injection: TBF_4248 was injected in a single-cycle kinetic (SCM) mode with dual concentration gradients (31.25, 62.5, 125, 250, 500 nM), with a binding phase of 120 s, a dissociation phase of 600 s, and a flow rate of 30 μL / min.

[0061] Regeneration: 10 mM Glycine-HCl (pH 2.0) for 30 s. Data were fitted using a 1:1 Langmuir model to calculate ka, kd, and KD.

[0062] (2) Results of kinetic analysis TBF_4248 exhibits high affinity kinetics for the target antigen: The binding rate constant ka = (2.34±0.18)×10 5 M -1 ·s -1 Dissociation rate constant kd = (1.87±0.15)×10 -3 s -1 The equilibrium dissociation constant KD = 8.0 ± 0.9 nM The half-life of the complex, t1 / 2, is approximately 6.2 min. The dissociation phase curve was flat, with a fitting residual of <5%, which was highly consistent with the ELISA quantitative results, confirming the excellent stability of the complex.

[0063] In summary, the candidate nanobody TBF_4248 exhibits a wide linear range and nanomolar-level ECGs in indirect ELISA. 50The activity retention rate was >78% after a 50℃ / 2 h heat stress test. Cross-reactivity validation confirmed its high specificity for ToBRFV, unaffected by TMV, ToMV, and healthy tomato substrate. SPR kinetics further quantified its KD≈8 nM and slow dissociation characteristics (t1 / 2≈6.2 min). Overall data indicate that TBF_4248 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.

[0064] Example 3: Verification of the specific binding of TBF_4248 to the ToBRFV coat protein Binding activity was verified using an indirect ELISA method. Purified ToBRFV capsid protein (YP_009182171.1) was coated onto 96-well plates at 2 μg / mL and incubated overnight at 4°C. After blocking, serially diluted TBF_4248 (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 color development, and OD values ​​were measured at 450 nm. The results showed that TBF_4248 bound to ToBRFV CP in a dose-dependent manner, with EC50... 50 The value was 28.6 ± 3.2 nM. Using total protein, TMV, ToMV, and PepMVCP from healthy tomato leaves as negative controls, the OD values ​​were all lower than twice the standard deviation of the blank control, confirming that TBF_4248 has high specificity for ToBRFV CP.

[0065] Example 4: Cross-reactivity and thermal stability test TBF_4248 was treated in water baths at 37℃, 50℃, and 65℃ for 2 h, respectively, followed by ELISA binding activity assays. Results showed that the antibody binding activity retention rate was 79.3±4.1% after 2 h treatment at 50℃ and 62.5±5.8% after treatment at 65℃, significantly superior to 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. Cross-reactivity experiments confirmed that TBF_4248 only recognizes ToBRFV CP and shows no cross-binding with TMV, ToMV, and PepMV, all belonging to the Tobamovirus family, meeting the stringent specificity requirements for port quarantine and rapid field diagnosis.

[0066] Example 5: Construction and field validation of colloidal gold test strips based on TBF_4248 TBF_4248 was labeled onto 15 nm colloidal gold particles to prepare a detection line (T line). A separate anti-ToBRFV 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. A serially diluted solution of ToBRFV-infected tomato leaves was added to the sample application area of ​​the test strip, and the results were observed within 15 minutes. The limit of detection (LOD) reached 10 pg / mL (equivalent to a 1:5000 dilution of a positive sample). Blind testing was conducted on 42 field samples (confirmed by RT-PCR) collected from Guangdong and Shandong provinces. 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 ≥97.6%, demonstrating that TBF_4248 has industrial application value.

Claims

1. A nanobody that specifically binds to the coat protein of tomato brown wrinkle fruit virus, characterized in that, Its complementary determinant region sequence is shown in SEQ ID NO: 4-6.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is selected from: (a) The complete variable region sequence as shown in SEQ ID NO: 1; (b) A sequence that has ≥90% sequence identity with SEQ ID NO: 1 and retains the function of specifically binding to the ToBRFV capsid protein.

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

4. A nucleic acid molecule, characterized in that, The nanobody as described in any one of claims 1 to 2 is encoded.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 3 and an operable promoter and screening 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) Expressing the host cell described in claim 6 under induction conditions; (2) Collect the expression product described in step (1), purify it by affinity chromatography, and obtain the nanobody.

8. A kit for detecting tomato brown wrinkle virus, characterized in that, It comprises a solid support on which the nanobody as described in any one of claims 1 to 2 is coated; or comprises the nanobody labeled with a reporter molecule.

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 brown wrinkle virus, characterized in that, This includes contacting the sample to be tested with the nanobody described in any one of claims 1 to 2, and determining the presence or concentration of ToBRFV by detecting the binding signal.

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