Anti-callose nano antibody and application thereof

Abstract

The invention discloses a nano antibody specifically bound with callose or an antigen binding fragment thereof, the callose nano antibody provided by the invention can be specifically bound with callose and has a wide application prospect in identification of plant immunity, the antibody purification process is relatively simple, the production cost is lower, and the method is suitable for industrial production. Wide application potential and technical advantages can be shown in biological scientific research.

Description

Technical Field

[0001] This invention relates to biological and immunological technologies, specifically to a specific callosity nanobody and its application. The specific callosity nanobody is mainly prepared by phage panning and genetic engineering techniques. Background Technology

[0002] Plant callose is a glucan linked by β-(1,3)-D bonds, playing a crucial regulatory role in plant sieve tube metabolism, immune defense, and gametophyte development. Its synthesis and degradation mechanisms are closely linked to plant growth and metabolic balance. When plants face adverse environmental conditions or stimuli, such as mechanical damage or high temperatures, callose reacts immediately, promoting rapid synthesis within the sieve tubes and deposition on the sieve plates or pores, forming a barrier to maintain the overall material flow of the sieve tube system. Simultaneously, when plants encounter pathogens, they recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), triggering a series of early defense mechanisms. At this time, callose accumulates in the infected area, rapidly sealing the infection site and effectively curbing pathogen spread.

[0003] Currently, aniline blue staining is an important method for detecting plant immunity, used to observe callose deposition. However, aniline blue staining is slow, making high-throughput experiments difficult, and the final results are hard to quantify, leading to significant errors. Therefore, finding new methods for quantifying callose is of great importance.

[0004] Nanobodies (Nb), also known as single-domain antibodies, have seen groundbreaking advancements in recent years in areas such as anti-inflammation, anti-infection, anti-tumor activity, and drug delivery. Nb is an antibody isolated and prepared from the serum of sharks or camels using genetic engineering techniques. Although naturally lacking the light and heavy chain constant regions, it retains its antibody properties and can target and bind to antigens. Compared to monoclonal antibodies, nanobodies are simpler and less expensive to prepare. More importantly, nanobodies have a smaller molecular weight (16kDa), resulting in stronger tissue penetration, high solubility and stability, high affinity, and excellent tissue penetration. There are currently no reports in this field of nanobodies containing anti-callosine. Summary of the Invention

[0005] Purpose of the invention: In order to solve the above problems, the present invention provides an anti-callosin nanobody or its antigen-binding fragment, its preparation method, and its use in identifying plant immunity.

[0006] Technical Solution: The present invention comprises a nanobody or antigen-binding fragment of a nanobody that specifically binds to callosity, wherein the amino acid sequence of the heavy chain variable region CDR1 is as shown in any one of SEQ ID NO:19-27 or has at least 85% identity with any amino acid sequence of SEQ ID NO:19-27, the amino acid sequence of the heavy chain variable region CDR2 is as shown in any one of SEQ ID NO:37-45 or has at least 85% identity with any amino acid sequence of SEQ ID NO:37-45, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in any one of SEQ ID NO:55-63 or has at least 85% identity with any amino acid sequence of SEQ ID NO:55-63.

[0007] The nanobody or its antigen-binding fragment that specifically binds to callosity has the following amino acid sequences: heavy chain variable region CDR1 as shown in any one of SEQ ID NO:19-27; heavy chain variable region CDR2 as shown in any one of SEQ ID NO:37-45; and heavy chain variable region CDR3 as shown in any one of SEQ ID NO:55-63.

[0008] Nanobodies or antigen-binding fragments thereof that specifically bind to callosity, wherein the amino acid sequence of the nanobodies or antigen-binding fragments thereof is as shown in any one of SEQ ID NO:1-9 or has at least 85% sequence identity with any one of SEQ ID NO:1-9.

[0009] The nanobody or its antigen-binding fragment that specifically binds to callosity has the following amino acid sequences: FR1 as shown in any one of SEQ ID NO: 10-18; FR2 as shown in any one of SEQ ID NO: 28-36; FR3 as shown in any one of SEQ ID NO: 46-54; and FR4 as shown in any one of SEQ ID NO: 64-72.

[0010] Recombinant protein comprising the aforementioned callosine nanobody.

[0011] The method for detecting callosity utilizes the aforementioned nanobodies or antigen-binding fragments that specifically bind to callosity.

[0012] The method for detecting callosity utilizes the specific callosity-binding nanobody or its antigen-binding fragment to bind with callosity for detection.

[0013] A callosity detection kit comprising the aforementioned nanobody or its antigen-binding fragment.

[0014] A β-D-glucan assay kit containing the aforementioned nanobody or its antigen-binding fragment.

[0015] The application of the nanobody that specifically binds to callose or its antigen-binding fragment or the recombinant protein in the identification of plant immunity.

[0016] Beneficial effects: The callosine nanobody provided by this invention can specifically bind to callosine, and has broad application prospects in identifying plant immune functions. In addition, the antibody purification process is relatively simple and the production cost is lower. It can show broad application potential and technical advantages in bioscience research. Attached Figure Description

[0017] Figure 1 Electrophoresis diagrams for constructing phage vectors; where A is a gel electrophoresis diagram of total RNA extracted from peripheral blood lymphocytes after alpaca 5th and 6th immunizations; B is a first-round electrophoresis diagram of nested PCR; C is a second-round electrophoresis diagram of nested PCR.

[0018] Figure 2 The results are for determining the diversity and capacity of the library after electroporation; A is the plate for determining the number of transformants in the library; B is the bacterial culture PCR electrophoresis image for determining the insertion rate of the target gene in the library; C is the result of VHH amino acid sequence alignment of single-clone bacterial culture.

[0019] Figure 3 ELISA colorimetric results for phage clones;

[0020] Figure 4 This is a Coomassie Brilliant Blue detection result image of the nanobody prokaryotic expression in Example 4 of the present invention (SEQ ID NO:8);

[0021] Figure 5 For potency testing;

[0022] Figure 6 This study investigated the use of nanobodies to identify callose content in plant immunity. Detailed Implementation

[0023] Example 1: Construction of an antibody library specific to anti-callosin antigen

[0024] Because β-D-glucan is a small molecule compound and lacks immunogenicity, we prepared a β-D-glucan hapten to immunize alpacas. 20 mg of β-D-glucan and 5 mg of succinic anhydride were dissolved in 1 ml of pyridine and reacted at 100°C for 5 h. The carboxyl product was separated by silica gel column chromatography. 5 mg of the carboxyl derivative and 20 mg of BSA / OVA were dissolved in 1 ml of MES (0.1 M pH 4.7). 15 mg of carbodiimide hydrochloride (EDC) was added to the reaction solution, and the mixture was stirred at room temperature for 24 h. After dialyzing with PBS (0.05 M pH 7.4) buffer to remove uncoupled small molecules, the β-D-glucan hapten product was obtained. After emulsification, two BALB / c mice were immunized. After three immunizations, the antiserum titer was measured by ELISA. It was found that the antiserum of mice immunized with callosity-BSA had a good titer against callosity-BSA and a certain titer against callosity-OVA. Therefore, callosity-BSA was used for immunization and callosity-OVA was used for detection.

[0025] Single-domain antibodies were prepared by immunizing alpacas with β-D-glucan-BSA. Serum was collected as a negative control before the first immunization. One alpaca was immunized subcutaneously with an initial dose of 1.0 mg / alpaca and a booster dose of 500 μg / alpaca. Immunizations were repeated every 3 weeks for a total of 6 immunizations. Peripheral blood was collected 7 days after each immunization for serum titer testing. After 6 immunizations, a good immune response was achieved, with the Alpaca IgG(H+L) HRP titer against β-D-glucan-BSA reaching 364.5 K at dilution, meeting the experimental criteria for downstream phage display library construction.

[0026] Anticoagulated alpaca peripheral blood was added in equal volume to diluent and gently mixed by inverting. 5 mL of separation buffer was added to a 15 mL PBMC separation tube and injected through the center well of the separation surface. Diluted blood was slowly added; the tube was centrifuged at 2000 rpm for 30 min; the lymphocyte layer was aspirated, removing all cells from the lymphocyte layer down to the separation surface, and placed in a centrifuge tube. More than one volume of washing buffer was added, and the tube was gently mixed by inverting. The tube was then centrifuged at 700 g for 10 min. The supernatant was discarded, and all cell pellets were resuspended in washing buffer. Samples were taken, and RNA was extracted. mRNA was reverse transcribed into cDNA, and nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene of alpaca heavy chain antibody. The target band was approximately 500-750 bp in size, and there was also a non-target band (traditional antibody fragment) at 900 bp. The 500-750 bp band was recovered by gel extraction and purified. Using the gel product as a template, a second round of PCR amplification of the VHH library fragment (approximately 450 bp target band) was performed. After purification, the VHH library DNA fragment was digested with SacI / SpeI, purified, quantified, aliquoted, and stored for subsequent experiments. Figure 1 ).

[0027] The enzyme-digested vector was ligated with the Scfv fragment. The ligation product was purified and electroporated into E. coli TG1 to obtain 10 mL of transformation product. Take 10 μL (i.e., 10...) -2 Perform a series of 10-fold serial dilutions (ml), and take 10 ml of the solution. -4 10 -5 and 10 -6 Three gradients were used for Amp resistance plate counting, and the assessed library size was 2.0 × 10⁻⁶. 9 The remaining transformation products were plated onto Amp antibody plates and incubated overnight. The next day, the plate was scraped off, mixed thoroughly, and then 20% glycerol was added to a final concentration. The plates were aliquoted and stored at -80°C. Thirty single clones were randomly selected from the serially diluted library plates for colony PCR detection. The 30 randomly selected single clones were sequenced using the forward sequencing primer pComb3XTT-F, yielding 30 valid sequencing clones. The overall accuracy of the library reached 100% for the antibody fragments containing the correct reading frames. Figure 2 ).

[0028] Example 2: Screening of phage libraries

[0029] The phage library was screened using a solid-phase screening method, and the specific screening method is as follows:

[0030] 100 μL of target molecules per well were coated onto the surface of a 96-well plate at 4°C overnight. Unadsorbed target molecules were washed four times with PBST (PBS + 0.05% Tween-20), followed by blocking with 300 μL of 5% skim milk powder at 37°C for 1 hour. Then, 100 μL of a phage antibody library (background subtraction) was added to the wells for binding. The plate was washed 10 times with PBST to remove non-specifically bound phages, and then eluted with 0.2 M glycine-hydrochloric acid to obtain affinity phages. High-affinity phages were obtained by decreasing the concentration of coating target molecules and increasing the washing intensity in each round. Each round of panning and amplification enriched the phage library with nanobodies capable of binding to the target molecules. Three rounds of in vitro targeted screening were performed against the OVA-β-glucan immunogen. Results showed that after three rounds of screening, the enrichment level reached 1000-fold, allowing for phage-ELISA single-clone validation.

[0031] From the third batch of plates, pick a single colony and transfer it to 1 mL of 2×YT-AI-hp medium. Incubate overnight at 37°C and 200 rpm. Centrifuge at 5000 rpm for 10 min. Carefully aliquot 600 μL of the supernatant into 1.5 mL centrifuge tubes, plate the tubes, and pick single colonies.

[0032] Example 3: Screening of Nanobody Molecules

[0033] 960 clones were picked from the plate and incubated overnight at 37°C. Positive clones were identified by ELISA. OVA-β-glucan was diluted with CBS and coated overnight at 4°C, followed by washing three times with PBS. Blocking was performed with 0.5% casein at 37°C for 1 hour, and excess blocking solution was removed, followed by washing three times with PBS. Primary antibody: 100 μL of sample / well was incubated at 37°C for 1 hour. Secondary antibody: anti-M13 antibody (HRP) was diluted 1:20000 with 1% M-PBS, 100 μL / well, and incubated at 37°C for 1 hour. Colorimetric development: 4.5 mL each of 0.2 M Na₂HPO₄ and 0.1 M citric acid were added to a small amount of OPD, mixed with 60 μL H₂O, and 50 μL / well was added. 2 M sulfuric acid was added to stop the reaction, 50 μL / well. The OD450 value was then measured. Figure 3 ).

[0034] Positive clones are amplified by PCR and sequenced, and the nucleotide sequence of the nanobody can be obtained based on the sequencing results.

[0035] The obtained sequence was cloned to construct an E. coli expression system, and IPTG was used to induce expression at 37°C. After centrifugation, the bacterial cells were collected, and then the bacterial cells were broken up and purified to obtain the desired anti-callosin nanobody.

[0036] The CDR region sequences of the nine nanobody molecules obtained in this invention are shown in Table 1.

[0037] Table 1. Amino acid sequences of the variable region and backbone region of the heavy chain of 9 nanobody samples.

[0038]

[0039]

[0040] The amino acid sequence of the nanobody molecule obtained by this invention is as follows:

[0041] NB1-1:RCAAGVGGGLVQAGGSLRLSCTASGRTFSSYTMAWFRQAPGKEREFVSSISWIGGSTYYAASVKG RFTISGDNAKNAQYLQMNSLKPEDTAVYYCAACYRVITDTTPSSCDYWGQGTQVTVSS(SEQ ID NO:1)

[0042] NB1-3:RCAAQESGGGLVQAGGSLRLSCADSGSTFSINTMGWYRQAQGKQRELVATISSGGGSTQYADSV KGRFTISRDNGKNTMYLQMHSLKPEDAAVYYCNLDRVATPAGVTEYWGQGTQVTVSS(SEQ ID NO:2)

[0043] NB1-5:RCAAQESGGGLVQPGGSLRLSCAVSGSILRIAEMGWYRQAPGKQREPVAGITSTGTTNYADSVKG RFTISRDNARRVYLQMASLKPEDTAVYYCNVRGNFFSGETFDSWGQGTQVTVSS(SEQ ID NO:3)

[0044] NB2-1:DVQLQESGGGLVQPGGSLRLSCAASGFTFSDYAVSWYRQAPGKERELVAAITTAGSYTNYADSVT GRFTISSRDNAKNTVYLQMNSLKLEDTAVYYCNARLGAGRIVAGTTGAYNYWGQGTQVTVSS(SEQ ID NO:4)

[0045] NB2-2: DVQLQESGGGLVQPGGSLRLSCAASGFTFSDYAMSWYRQAPGKERELVAAITTAGSYTNYADSVM GRFTISRDNAKNTVYLQMNSLKLEDTAVYYCNARLGAGRIVAGTTGAYNYWGQGTQVTVSS(SEQ ID NO:5)

[0046] NB2-3: DVQLQESGGGLVQPGGSLRLSCAASGFTFSDYAMSWYRQAPGKERELVAAITTAGSYTNYADSVT GRFTISRDNAKNTVYLQMNSLKLEDTAVYYCNARLGAGRIVAGTTGAYNYWGQGTQVTVSS(SEQ ID NO:6)

[0047] NB2-4: DVQLQESGGGLVQPGGSLRLSCAFSGLTLNYYYIGWFRQAPGKEREGVSCISSGGGRTNYADSVKG RFTISRDNAAKMVYLTMNSLKPEDTGVYYCAAYRTAVEAMCTRSVAWYGVWGQGTQVTVSS(SEQ ID NO:7)

[0048] NB2-8: DVQLQESGGGLVQPGGSLRLSCVAAGFTFSDYAMSWYRQAPGKERELVAAITTAGSYTNYADSVT GRFTISRDNAKNTVYLQMNSLKLEDTAVYYCNARLGAGRIVAGTTGAYNYWGQGTQVTVSS(SEQ ID NO:8)

[0049] NB2-9: DVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSDDGSTYYADAVK GRFTISRDNAKNTMYLQMNSLKAEDTAVYYCAAEPVIHGRYTDCLNAHEYDYWGQGTQVTVSS(SEQ IDNO:9)

[0050] Example 4 Expression and Purification of Anti-Callose Nanobody

[0051] The above nine nanobody sequences were constructed into the pET-22b-nanobody expression plasmid and transformed into the BL21 prokaryotic expression strain of *E. coli*. 10 μL of correctly sequenced BL21 culture stored at -80℃ was added to 30 mL of LB broth containing 50 μg / mL AMP resistance and cultured at 37℃ and 175 rpm for 12 hours with shaking. 3 mL of the culture was added to 200 mL of LB broth containing 50 μg / mL AMP resistance and cultured at 37℃ and 175 rpm for 3-4 hours with shaking, until the bacterial concentration reached approximately OD600 = 0.4-0.7. The culture was then cooled to 4℃ for 10 minutes. IPTG was added to a final concentration of 0.2 mM, and the culture was incubated at 16℃ and 175 rpm for 12 hours to induce expression.

[0052] Before and after induction, aspirate 1 mL of bacterial culture, centrifuge at 12000 rpm for 5 minutes at 4°C, discard the supernatant, add 40 μL ddH2O, and store at 4°C. After incubation, transfer the bacterial culture to a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 minutes at 4°C, and collect the bacterial pellet. Resuspend the pellet in 20 mL of protein buffer, place the resuspending in an ice-water mixture, and sonicate using an ultrasonic homogenizer with a 3-second interval between sonic breaks, an amplitude of 25%, and a duration of 20-30 minutes. The instrument can be temporarily stopped during this process for observation until the bacterial culture becomes clear and transparent. Centrifuge the homogenized bacterial culture at 15000 rpm for 30 minutes at 4°C, aspirate 40 μL of the supernatant, dissolve the pellet in 100 μL ddH2O, and store at 4°C.

[0053] Add the appropriate protein loading buffer to the samples before and after induction, as well as the supernatant and precipitate after disruption. Denature the protein in boiling water at 100°C for 10 minutes and store at -20°C. Take 5 μL of the denatured protein sample and perform SDS-PAGE protein electrophoresis. Then, stain with Coomassie Brilliant Blue and destain thoroughly to analyze the expression pattern of the recombinant protein.

[0054] The purification process of the antibody protein was performed at 4℃. An affinity chromatography column sieve was added to the column, followed by the slow addition of 1 mL of Ni-NTA agarose resin. 5 mL of ddH2O was slowly added to the Ni-NTA agarose resin for washing, followed by the slow addition of 5 mL of protein buffer to equilibrate the resin. The supernatant collected after sonication was slowly added to the Ni-NTA agarose resin, and the flow-through was collected simultaneously. 5 mL of imidazole elution buffer at different concentrations (5, 20, 50, 100, 250 mM) was added sequentially to elute the Ni-NTA agarose resin, and the protein eluent was collected simultaneously. 5 μL of the denatured protein sample was subjected to SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining and thorough destaining to analyze the purification efficiency of the antibody protein. Figure 4 ).

[0055] Antibody titers were determined using a gradient method. β-D-glucan (10 μg / mL) was used as the antigen, with 100 μL per well, dried overnight, and blocked with 2% skim milk powder (200 μL / well) at 37°C for 1 h. Prokaryotic expression antibody (100 μL / well) was added, with concentrations serially diluted twofold (200, 100, 50, 25, 12.5 μg / mL), and incubated at 37°C for 1 h. The prokaryotic expression antibody was detected using mouse anti-HIS tag monoclonal antibody-HRP (100 μL / well) at 37°C for 1 h. After the reaction, a colorimetric reaction was performed, and the OD450 value was measured. Figure 5 ).

[0056] Example 5: Application of anti-callosin nanobodies in identifying plant immunity

[0057] The three-leaf stage rice plants were treated with 0.5 ml of the scientifically recognized plant immune activator BTH. One day after the treatment, the plants were treated with spores of the standard strain of rice blast fungus Guy11. The inoculated rice plants were then placed in a dark environment with 80% humidity and 28℃ for one day. On the second day, the rice plants were transferred from the dark environment to a 12-hour light and 12-hour dark environment.

[0058] Rice leaf tissue was sampled 48 hours after being sprayed with rice blast spores. The rice leaf tissue was ground with liquid nitrogen, and 1 ml of PBS was added to every 0.1 g of tissue. The mixture was then centrifuged at 1000 x g for 20 minutes. The supernatant was the plant extract.

[0059] Plant extract was used as the antigen, 100 μL per well, coated overnight at 4°C, blocked with 200 μL of 2% skim milk powder per well, and incubated at 37°C for 1 h. 10 μM nanobody was used as the primary antibody for incubation, with 100 μL of PBS as a negative control, and incubated at 37°C for 1 h. Mouse anti-HIS tag monoclonal antibody-HRP was used as the secondary antibody, 100 μL per well, and incubated at 37°C for 1 h. The TMB method was used for color development, with 50 μL each of TMB and H2O2 added to each well, and incubated at 37°C in the dark for 15 min (a plate washing step was performed between each step: discard the liquid, blot dry on absorbent paper, fill each well with washing buffer (0.1% Tween water), let stand for 1 min, discard the washing buffer, blot dry on absorbent paper, and repeat this washing process 5 times). 50 μL of stop solution was added to each well, and the OD value of each well was measured at 450 nm within 15 min. The results showed that BTH treatment significantly increased callosity content, which was accurately measured by nanobodies. Figure 6 ).

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody or antigen-binding fragment thereof that specifically binds to callosity, characterized in that, The amino acid sequence of the heavy chain variable region CDR1 is as shown in any one of SEQ ID NO:19-27 or has at least 85% identity with any amino acid sequence of SEQ ID NO:19-27; the amino acid sequence of the heavy chain variable region CDR2 is as shown in any one of SEQ ID NO:37-45 or has at least 85% identity with any amino acid sequence of SEQ ID NO:37-45; and the amino acid sequence of the heavy chain variable region CDR3 is as shown in any one of SEQ ID NO:55-63 or has at least 85% identity with any amino acid sequence of SEQ ID NO:55-63.

2. The nanobody or antigen-binding fragment thereof that specifically binds to callosity according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region CDR1 is shown in any one of SEQ ID NO:19-27, the amino acid sequence of the heavy chain variable region CDR2 is shown in any one of SEQ ID NO:37-45, and the amino acid sequence of the heavy chain variable region CDR3 is shown in any one of SEQ ID NO:55-63.

3. A nanobody or antigen-binding fragment thereof that specifically binds to callosity, characterized in that, The amino acid sequence of the nanobody or its antigen-binding fragment is as shown in any one of SEQ ID NO:1-9 or has at least 85% sequence identity with any one of SEQ ID NO:1-9.

4. The nanobody or antigen-binding fragment thereof that specifically binds to callosity according to claim 3, characterized in that, The amino acid sequence of the backbone region FR1 is shown in any one of SEQ ID NO:10-18, the amino acid sequence of the backbone region FR2 is shown in any one of SEQ ID NO:28-36, the amino acid sequence of the backbone region FR3 is shown in any one of SEQ ID NO:46-54, and the amino acid sequence of the backbone region FR4 is shown in any one of SEQ ID NO:64-72.

5. A recombinant protein, characterized in that, Nanobodies or antigen-binding fragments thereof that specifically bind to callosity as described in any one of claims 1-4.

6. A method for detecting callosity, characterized in that, The detection was performed using the nanobody that specifically binds to callosity or its antigen-binding fragment as described in any one of claims 1-4.

7. A method for detecting callosity, characterized in that, The nanobody that specifically binds to callosity, or its antigen-binding fragment, as described in any one of claims 1-4, is used for detection.

8. A callosity detection kit, characterized in that, It includes the nanobody or its antigen-binding fragment as described in any one of claims 1-4.

9. A β-D-glucan detection kit, characterized in that, It includes the nanobody or its antigen-binding fragment as described in any one of claims 1-4.

10. The use of the nanobody that specifically binds to callosity as described in any one of claims 1-4 or its antigen-binding fragment, or the recombinant protein as described in claim 5, in the identification of plant immunity.

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