An avrcap1b nanobody and uses thereof

Abstract

The present application relates to an AVRcap1b nanobody and application thereof, and belongs to the field of biological medicine, wherein the present application develops an anti-AVRcap1b nanobody through multiple steps such as detection and screening by using a yeast two-hybrid screening system, the screening cost is greatly reduced compared with traditional antibodies, the research and development cycle is shorter, the requirements for experimental environment and equipment are simpler, and the applicability is wide. Compared with traditional AVRcap1b antibodies, the developed AVRcap1b nanobody has smaller molecular weight, better tissue permeability, is more stable, and has low immunogenicity. In summary, the AVRcap1b nanobody has outstanding advantages in terms of antibody affinity and targeting specificity, small molecule size, stability and transformability, production cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an AVRcap1b nanobody obtained by yeast two-hybrid screening and its application. Background Technology

[0002] AVRcap1b is an effector protein secreted by oomycetes, targeting the membrane transport-related protein NtTOL9a within plant cells. By targeting NtTOL9a, AVRcap1b inhibits the cell death response mediated by NLR proteins NRC2 and NRC3, thereby facilitating the successful infection and colonization of plants by pathogens. Simultaneously, by suppressing the plant's immune defense response, it makes the pathogens more susceptible to spread and cause disease within the plant, severely impacting plant growth and development, potentially leading to reduced crop yields and significant agricultural losses. The structural characteristics of AVRcap1b are as follows: ① It possesses a specific amino acid sequence: Like many proteins, AVRcap1b is composed of specific amino acids arranged in a particular order. The types, quantities, and sequence of these amino acids determine its unique physicochemical properties and biological functions. ② It contains a signal peptide domain: Signal peptides are generally located at the N-terminus of proteins, guiding their directional transport and localization within cells. For AVRcap1b, the signal peptide domain guides its secretion from the oomycete cell to the extracellular space, where it then enters the plant cell to exert its effects. ③ The presence of effector domains: The effector domains are the key sites where AVRcap1b exerts its regulatory role on the physiological functions of plant cells. By interacting with target proteins in plant cells, it interferes with physiological processes such as the plant's immune response, thereby helping pathogens infect plants.

[0003] Research on AVRcap1b is of great significance. First, it reveals the interaction mechanism between plants and pathogens: studying the interaction between AVRcap1b and proteins within plant cells helps to understand how the plant immune system recognizes and resists pathogen invasion, and how pathogens evolve corresponding effector proteins to evade plant immune responses. This is crucial for understanding the co-evolutionary relationship between plants and pathogens. Second, it provides targets for plant disease resistance breeding: clarifying the key role of AVRcap1b in pathogenic processes allows for the design of corresponding disease resistance strategies targeting this effector protein. For example, gene editing technology can be used to breed plant varieties that can specifically recognize and resist AVRcap1b, improving plant resistance to related pathogens, thereby reducing the use of chemical pesticides and achieving sustainable agricultural development. Third, it provides structural complementarity: the effector domain of AVRcap1b has a specific spatial conformation, with its surface amino acid residues forming a specific shape and charge distribution. The target protein NtTOL9a within plant cells also has a complementary binding site. Like a "lock and key," the two can precisely match and bind specifically.

[0004] Since Hamers et al. discovered naturally occurring heavy chain antibodies lacking light chains in camel blood in 1993, single-domain antibodies (sdAbs) have gradually replaced other small antibodies and become a hot topic in the development of novel antibody drugs. Single-domain antibodies, also known as nanobodies, are typically only about 15 kDa, approximately one-tenth the size of traditional antibodies. They contain disulfide bonds internally and have numerous hydrophilic residues on their surface, exhibiting strong resistance to heat and pH. The absence of Fc fragments and light chains in sdAbs allows them to recognize cryptic or small epitopes that traditional antibodies cannot, while avoiding complement reactions. Furthermore, single-domain antibodies possess numerous advantages, including high stability, low toxicity, high solubility, ease of target screening, ease of direct expression in prokaryotic microorganisms, and good cost-effectiveness. Sequence homology analysis showed that the VHH germline gene sequence of camel sdAb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than those of humans. CDR3 protrudes outward in the tertiary structure, thus suggesting that it has higher antigen binding specificity and affinity.

[0005] Developing nanobodies targeting AVRcap1b is of great significance due to their excellent properties. At the basic research level, it can reveal the immune mechanisms related to the AVRcap1b target: AVRcap1b is an effector protein from *Phytophthora indicum*, the causal agent of potato late blight. Research on nanobodies targeting AVRcap1b helps to deepen our understanding of the interaction mechanisms between plants and pathogens, particularly how pathogens interfere with plant immune responses through effector proteins, and how the plant immune system recognizes and defends against pathogen invasion. Nanobodies have small molecular weights and simple structures. By studying the binding mode between AVRcap1b nanobodies and the AVRcap1b protein, we can more clearly elucidate the structure-function relationship of the AVRcap1b protein, clarifying its key sites and mechanisms of action in the pathogenic process. At the disease diagnostic level, nanobodies possess high specificity and affinity. AVRcap1b nanobodies can be used to develop highly sensitive and specific detection methods to rapidly and accurately detect the presence of *Phytophthora indicum* or its related antigens, facilitating early diagnosis of plant diseases and timely implementation of control measures to reduce losses in agricultural production. AVRcap1b nanobodies can be combined with various detection technologies, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunochromatography, providing a powerful tool for rapid on-site detection and precise laboratory diagnosis of plant diseases, improving detection efficiency and accuracy. In terms of disease treatment, the excellent cell penetration and tissue permeability of nanobodies enable them to effectively reach lesion sites. AVRcap1b nanobodies can serve as targeted carriers, specifically delivering therapeutic drugs or other active substances to the infection site, achieving precise targeting of pathogens, reducing damage to normal tissues, and improving treatment efficacy. Simultaneously, nanobodies can also regulate the body's immune response through interaction with the immune system. AVRcap1b nanobodies may have the ability to activate or enhance the plant's own immune defense mechanisms, thereby helping plants better resist infection by late blight pathogens and providing insights for developing novel plant immunotherapy methods. In terms of drug development, they can contribute to the design of novel drugs and the establishment of drug screening models. Research on AVRcap1b nanobodies lays the foundation for developing novel antifungal drugs based on nanobodies. Modifying and transforming nanobodies, such as conjugating them with drug molecules like antibiotics and antifungal peptides, or designing nanobodies with enzymatic activity or cytotoxicity, holds promise for developing more efficient and less toxic drugs against Potato Late Blight. Utilizing the specific interaction between AVRcap1b nanobodies and pathogens, high-throughput drug screening models can be established to rapidly screen small molecule compounds or biological agents that can inhibit AVRcap1b protein activity or block its interaction with host cells, accelerating the development of novel antifungal drugs.

[0006] The development of existing traditional AVRcap1b antibodies faces numerous limitations. Firstly, production costs are high: the production process of traditional antibodies is complex, requiring techniques such as animal cell culture, demanding high standards for the production environment and equipment, and involving long cell culture cycles and limited yields, leading to significantly increased production costs and hindering large-scale production. The long production cycle, from immunizing animals and screening for positive hybridoma cells to obtaining stable antibody-secreting cell lines and then large-scale antibody production, is time-consuming, typically taking several months or even longer, making it difficult to quickly meet market demand. Secondly, antibody performance is limited in terms of stability: traditional antibodies are prone to denaturation and inactivation under high temperature, acidity, and alkalinity conditions, making it difficult to meet the stability requirements of some special applications, such as complex physiological environments in vivo or long-term storage. Poor tissue penetration: the large molecular weight of traditional antibodies makes it difficult to effectively penetrate physiological barriers such as biological membranes and the blood-brain barrier, limiting the therapeutic effect for pathogens in some deep tissues or special sites. Insufficient affinity and specificity: despite screening and optimization, the affinity and specificity of traditional antibodies still have room for improvement, potentially leading to cross-reactions with other unrelated antigens, affecting the accuracy of detection and treatment. Thirdly, in terms of immunogenicity, they are prone to triggering immune responses: Traditional antibodies are mostly derived from animals and are exogenous proteins for the human body. They have strong immunogenicity and are prone to triggering immune responses and producing anti-antibodies. This can not only reduce the effectiveness of treatment but also cause adverse reactions such as allergies, and even lead to treatment failure. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an anti-AVRcap1b nanobody developed based on yeast two-hybrid technology and its application. It has a short development cycle, high antibody quality, and outstanding advantages in antibody affinity and targeting specificity, small molecule size, stability and modifiability, and production cost.

[0008] The specific solution adopted in this invention is as follows:

[0009] In a first aspect, the present invention provides an AVRcap1b nanobody, wherein the AVRcap1b nanobody is AVRcap1b nanobody I, AVRcap1b nanobody II, AVRcap1b nanobody III or AVRcap1b nanobody IV;

[0010] The complementary binding region of the AVRcap1b nanobody I contains amino acid sequences such as CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4;

[0011] The complementary binding region of the AVRcap1b nanobody II contains amino acid sequences such as CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9;

[0012] The complementary binding region of the AVRcap1b nanobody III contains amino acid sequences such as CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14;

[0013] The complementary binding region of the AVRcap1b nanobody IV contains amino acid sequences such as CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 19.

[0014] As a further optimization of the above-mentioned AVRcap1b nanobody, the amino acid sequence of AVRcap1b nanobody I is shown in SEQ ID NO: 1; the amino acid sequence of AVRcap1b nanobody II is shown in SEQ ID NO: 6; the amino acid sequence of AVRcap1b nanobody III is shown in SEQ ID NO: 11; and the amino acid sequence of AVRcap1b nanobody IV is shown in SEQ ID NO: 16.

[0015] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned AVRcap1b nanobody. Furthermore, the nucleic acid molecule sequence encoding AVRcap1b nanobody I is shown in SEQ ID NO: 5; the nucleic acid molecule sequence encoding AVRcap1b nanobody II is shown in SEQ ID NO: 10; the nucleic acid molecule sequence encoding AVRcap1b nanobody III is shown in SEQ ID NO: 15; and the nucleic acid molecule sequence encoding AVRcap1b nanobody IV is shown in SEQ ID NO: 20.

[0016] Thirdly, the present invention provides a nucleic acid construct comprising the aforementioned nucleic acid molecules. Furthermore, the nucleic acid construct is a vector or a host cell.

[0017] Fourthly, this invention provides the application of the above-mentioned AVRcap1b nanobody, nucleic acid molecule, or nucleic acid construct in the study of revealing the immune mechanism related to the AVRcap1b target.

[0018] Fifthly, the present invention provides the application of the above-mentioned AVRcap1b nanobody, nucleic acid molecule or nucleic acid construct in the development of early diagnostic products for plant diseases.

[0019] Sixthly, the present invention provides the application of the above-mentioned AVRcap1b nanobody, nucleic acid molecule or nucleic acid construct in the development of antibacterial infection drugs.

[0020] Beneficial Effects: This invention employs a yeast two-hybrid screening system, which significantly reduces screening costs compared to traditional antibodies, shortens the screening cycle, simplifies experimental environment and equipment requirements, and has wide applicability. This system studies protein-protein interactions within living cells, reflecting the true interactions of proteins under physiological conditions, more closely resembling the natural in vivo environment, and making the research results more convincing. The use of expression vectors with high copy numbers and strong promoters, combined with sensitive detection methods such as yeast phenotype, X-gal, and His3 protein expression, enables the detection of even weak protein-protein interactions. Multiple screening steps, such as reporter gene validation and dual selection of yeast strains, effectively reduce false positive results caused by non-specific interactions, improving the accuracy of screening results. Compared to traditional AVRcap1b antibodies, AVRcap1b nanobodies have smaller molecular weights, better tissue permeability, are more stable, and have lower immunogenicity. Attached Figure Description

[0021] Figure 1 This is a screenshot of the self-activation detection results.

[0022] Figure 2 This is a graph showing the results of the screening.

[0023] Figure 3 This is a graph of the results of the rotational verification; in the graph, (+) represents the positive control pGBKT7-p53+pGADT7-largeT, and (-) represents the negative control pGBKT7-laminC+pGADT7-largeT. Detailed Implementation

[0024] This invention uses the AVRcap1b gene constructed on the pGBKT7 vector as bait to screen yeast two-hybrid nanobody libraries. Through multiple reporter gene detection, DNA sequencing and BLAST comparison analysis of positive clones, nanobodies that interact with AVRcap1b are identified.

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Unless otherwise specified, all reagents or materials used below are commercially available, and all methods used are conventional technical means.

[0026] I. Transformation of bait plasmid into recipient bacteria AH109 and detection of its self-activation

[0027] 1.1 Yeast Conversion

[0028] The following plasmids were transformed into AH109:

[0029]

[0030] 1.2 Preparation and Transformation Methods of Yeast Competent Cells

[0031] 1. Pick a single colony of AH109 from a YPDA plate and inoculate it into 4 ml of YPDA liquid medium. Incubate at 30°C with shaking at 225 rpm for 18-20 hours (overnight), until OD reaches zero. 600 >1.5.

[0032] 2. Transfer to YPDA liquid culture medium, with a culture volume of 50 ml, to allow initial OD... 600 =0.2, 30℃, 225rpm, shake and incubate for 4-5 hours until OD 600 =0.6.

[0033] 3. Centrifuge to collect bacteria at room temperature, 4000 rpm, for 5 minutes.

[0034] 4. Resuspend the bacterial cells in 20 ml of sterile water, mix well, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0035] 5. Resuspend the bacterial cells in 5 ml of 0.1 M LiAc, mix well, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0036] 6. Resuspend the bacterial cells in 500ul of 0.1M LiAc, mix well, and dispense 50ul into 1.5ml centrifuge tubes (per transformation) for later use.

[0037] 7. Add the following reagents to each 1.5ml centrifuge tube in sequence, mix by pipetting or vigorous shaking for about 1 minute until completely mixed.

[0038]

[0039] 8. Incubate in a water bath at 30℃ for 30 minutes.

[0040] Heat shock in a water bath at 9.42℃ for 25 minutes.

[0041] Resuscitate in a 10.30℃ water bath for 30 minutes.

[0042] 11. Centrifuge to collect bacteria at room temperature, 4000 rpm for 5 min, and discard the supernatant.

[0043] 12. For each transformation, suspend the bacterial cells in 200 μL of sterile water, mix gently as much as possible, and spread on the corresponding defect screening plate.

[0044] Incubate at 13.30℃ for 4 days.

[0045] 1.3 Self-activation detection

[0046] Eight single colonies were randomly selected from each of the yeast transformants grown from pGBKT7-AVRcap1b+pGADT7 co-transformed with AH109 for PCR detection and preservation. Three spots were randomly selected from each of the PCR-detected single colonies and plated on SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal plates, and incubated at 30℃ for 3-5 days.

[0047] The experimental results show that the positive control can grow on SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal plates, and can develop color on SD-TLHA+X-α-gal plates. The negative control grows on SD-TL plates but not on SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal plates. The consistent growth between the experimental and negative control groups indicates that pGBKT7-AVRcap1b+pGADT7 does not exhibit self-activation. See attached results. Figure 1 .

[0048] II. Document Filtering

[0049] Competent cells were prepared using the AH109 yeast strain containing the correct pGBKT7-AVRcap1b bait plasmid as the recipient, and the library plasmid pGADT7-VHH was transferred into them and plated on SD-TLH screening plates.

[0050] 2.1 Library DNA Transformation Methods:

[0051] 1. Pick a single bacterial strain from an SD-T plate and inoculate it into 50 ml of liquid SD-T medium. Incubate at 30°C and 225 rpm for 24 h with shaking.

[0052] 2. Transfer to 500ml of YPDA liquid to allow initial OD. 600 =0.2, 30℃, 225rpm, shake and incubate for 4-5 hours until OD 600 =0.6.

[0053] 3. Centrifuge to collect bacteria at room temperature, 4000 rpm, for 5 minutes.

[0054] 4. Resuspend the bacterial cells in 30 ml of sterile water, mix well, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0055] 5. Resuspend the bacterial cells in 20 ml of 0.1 M LiAc, mix well, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0056] 6. Resuspend the bacterial cells in 10 ml of 0.1 M LiAc, mix well, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0057] 7. Add the following reagents to the centrifuge tube in sequence, mix by pipetting or vigorous shaking for about 1 minute until completely mixed.

[0058]

[0059] 8. Incubate in a water bath at 30℃ for 30 minutes.

[0060] Heat shock in a water bath at 9.42℃ for 25 minutes.

[0061] Resuscitate in a 10.30℃ water bath for 1 hour.

[0062] 11. Centrifuge to collect the bacteria at room temperature, 4000 rpm for 5 min. Discard the supernatant, resuspend the bacteria in 6 ml of sterile water, and mix gently. Take 20 μL of the culture, dilute it, and plate it on SD-TL plates to detect library transformation efficiency. Plate the rest on SD-TLH plates, for a total of 20 plates.

[0063] Incubate at a constant temperature of 12.30℃ for 3-7 days and observe colony growth.

[0064] 13. Pick out the newly grown single colonies and transfer them to SD-TLHA selection plates for further culture for 3-5 days.

[0065] 2.2 Screening results

[0066] Positive yeast clones were obtained by screening on SD-TLHA selection plates. Colonies grew on the SD-TLHA plates containing the screening library, so a total of 96 growing clones were picked from the plates for PCR verification. Results are attached. Figure 2 .

[0067] III. Identification and Sequencing Alignment of Yeast Positive Clones

[0068] To identify the genes of the positive clones screened in the SD-TLHA plate, these positive clones need to be amplified from yeast cells for DNA sequencing and then compared with sequences in the GenBank database using BLAST.

[0069] Sequencing and alignment results: Positive yeast clones were amplified by PCR and sequenced. After Seqman and BLAST alignment, five gene sequences were finally obtained.

[0070] IV. Verification of positive yeast clones by inversion

[0071] Positive clones grown on SD-TLHA-deficient plates were diluted with sterile water and spotted onto SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal-deficient plates, respectively, and incubated at 30°C for 3-4 days. Results are attached. Figure 3 Rotational validation results showed that the positive control could grow on SD-TL, SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal deficient plates, and showed a blue color on the SD-TLHA+X-α-gal deficient plate; the negative control could only grow on SD-TL plates and could not grow on other plates. All five selected yeast positive clones grew normally on SD-TL deficient plates, and all four selected yeast positive clones grew normally on SD-TLH, SD-TLHA, and SD-TLHA+X-α-gal deficient plates and showed a blue color on the SD-TLHA+X-α-gal plate.

[0072] The present invention was verified by a rotational experiment, which proved that all four positive yeast clones obtained by screening were indeed positive.

[0073] This invention successfully screened four AVRcap1b nanobodies using a yeast two-hybrid screening system. By combining yeast two-hybrid technology, this invention obtains AVRcap1b nanobody genes in a short time, effectively reducing the development and production costs of AVRcap1b antibodies, indicating that the AVRcap1b nanobodies obtained by this invention have further development value.

[0074] The four AVRcap1b nanobodies are AVRcap1b nanobodies I, AVRcap1b nanobodies II, AVRcap1b nanobodies III, and AVRcap1b nanobodies IV.

[0075] The amino acid sequence of AVRcap1b nanobody I is shown in SEQ ID NO: 1, and its CDR1 is shown in SEQ ID NO: 2, CDR2 in SEQ ID NO: 3, and CDR3 in SEQ ID NO: 4. The nucleotide sequence is shown in SEQ ID NO: 5.

[0076] The amino acid sequence of AVRcap1b nanobody II is shown in SEQ ID NO: 6, and its CDR1 is shown in SEQ ID NO: 7, CDR2 in SEQ ID NO: 8, and CDR3 in SEQ ID NO: 9. The nucleotide sequence is shown in SEQ ID NO: 10.

[0077] The amino acid sequence of AVRcap1b nanobody III is shown in SEQ ID NO: 11, and its CDR1 is shown in SEQ ID NO: 12, CDR2 in SEQ ID NO: 13, and CDR3 in SEQ ID NO: 14. The nucleotide sequence is shown in SEQ ID NO: 15.

[0078] The amino acid sequence of AVRcap1b nanobody IV is shown in SEQ ID NO: 16, and its CDR1 is shown in SEQ ID NO: 17, CDR2 in SEQ ID NO: 18, and CDR3 in SEQ ID NO: 19. The nucleotide sequence is shown in SEQ ID NO: 20.

[0079] The full amino acid sequence information and partitioned sequence information of the four AVRcap1b nanobodies of this invention are as follows:

[0080] The amino acid sequence of AVRcap1b nanobody I (SEQ ID NO: 1):

[0081] QVQLQESGGGLVQAGGSLRLSCAASGSIFCSCFMGWYRQAPGKEREFVAGINFGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVTLRATTTHDYWGQGTQVTDSS

[0082] [FR1:QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTQVTDSS; CDR1: GSIFCSCFM; CDR2: EFVAGINFGGSTYY; CDR3: AVTLRATTTHDY]

[0083] The amino acid sequence of AVRcap1b nanobody II (SEQ ID NO: 6):

[0084] QVQLQESGGGLVQAGGSLRLSCAASGTIFHHYNMGWYRQAPGKERELVASINDGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAACRSQITQLPYWGQHPGHSQ

[0085] [FR1: QVQLQESGGGLVQAGGSLRLSCAAS; FR2: MGWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQHPGHSQ; CDR1: GTIFHHYN; CDR2: ELVASINDGGNTYY; CDR3: AACRSQITQLPY]

[0086] Amino acid sequence of Nanobody III of AVRcap1b (SEQ ID NO: 11):

[0087] QVQLQESGGGLVQAGGSLRLSCAASGSIFGPLLMGWYRQAPGKEREFVASISSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVTSYWHLLIARIHQYWGQGTRSQS

[0088] [FR1: QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTRSQS; CDR1: GSIFGPLLM; CDR2: EFVASISSGGITYY; CDR3: AVTSYWHLLIARIHQY]

[0089] Amino acid sequence of Nanobody IV of AVRcap1b (SEQ ID NO: 16):

[0090] QVQLQESGGGLVQAGGSLRLSCAASGYISSPNLMGWYRQAPGKERELVAGISRGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPGTYCKFHWYWGQGTRVTVSS

[0091] [FR1: QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTRVTVSS;

[0092] CDR1: GYISSPNLM; CDR2: ELVAGISRGSSTYY; CDR3: AAPGTYCKFHWY]

[0093] The DNA sequence information of the four AVRcap1b nanobodies of the present invention is as follows:

[0094] DNA sequence of AVRcap1b nanobody I (SEQ ID NO: 5):

[0095] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCTCTATATTTTGTTCTTGTTTTATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAATTTGTGGCAGGTATCAATTTTGGAGGTAGTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGTTACGTTGAGGGCGACGACTACTCATGATTATTGGGGGCAAGGCACCCAGGTCACAGACAGTAGT

[0096] DNA sequence of AVRcap1b nanobody II (SEQ ID NO: 10):

[0097] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCACTATATTTCATCATTATAATATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAACTTGTGGCAAGTATCAATGATGGAGGTAATACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGCTTGTAGGTCGCAGATTACTCAGCTTCCGTATTGGGGGCAGCACCCAGGTCACAGTCAG

[0098] DNA sequence of AVRcap1b nanobody III (SEQ ID NO: 15):

[0099] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTCTTGTGCTGCATCCGGCTCTATATTTGGTCCTCTTCTGATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAATTTGTGGCAAGTATCAGTTCTGGAGGTATTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAAT TTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGTACGTCGTATTGGCATTTGCTGATTGCGCGTATTCATCAGTATTGGGGGCAAGGCACCAGGTCACAGTCA

[0100] DNA sequence of AVRcap1b nanobody IV (SEQ ID NO: 20):

[0101] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTCTTGTGCTGCATCCGGCTATATATCTTCGCCTAATCTGATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAACTTGTGGCAGGTATCAGTCGGGGAAGTAGTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCAC AATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGCTCCGGGGACGTATTGTAAGTTTCATTGGTATTGGGGGCAAGGCACCCGGGTCACAGTCAGTAGT

[0102] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. An AVRcap1b nanobody, characterized in that: The AVRcap1b nanobody is AVRcap1b nanobody IV; The complementary binding region of the AVRcap1b nanobody IV contains amino acid sequences such as CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO:

19.

2. The AVRcap1b nanobody according to claim 1, characterized in that: The amino acid sequence of the AVRcap1b nanobody IV is shown in SEQ ID NO:

16.

3. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the AVRcap1b nanobody as described in claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecular sequence encoding the AVRcap1b nanobody IV is shown in SEQ ID NO:

20.

5. A nucleic acid construct, characterized in that: Includes the nucleic acid molecule as described in claim 4.

6. The nucleic acid construct according to claim 5, characterized in that: The nucleic acid construct is a vector or a host cell.

7. The application of the AVRcap1b nanobody according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, or the nucleic acid construct according to any one of claims 5-6 in the development of early diagnostic products for plant diseases, wherein the plant disease is potato late blight.

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