Nanobodies against bacillus cereus and uses thereof

Abstract

The application relates to an anti-Bacillus cereus nanobody and application thereof, the nanobody comprising a framework region FR and a complementarity determining region CDR, wherein the complementarity determining region CDR is CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, CDR2 with an amino acid sequence as shown in SEQ ID NO. 7 and CDR3 with an amino acid sequence as shown in SEQ ID NO. 8. The nanobody can recognize and combine with Bacillus cereus, and has the advantages of easy expression and high expression efficiency, so that the nanobody can be applied to an enzyme-linked immunoassay method for detecting Bacillus cereus.

Description

Technical Field

[0001] This application relates to the field of immunological detection technology for foodborne pathogens, and in particular to a nanobody against Bacillus cereus and its application. Background Technology

[0002] Bacillus cereus is a common Gram-positive conditional foodborne pathogen. Food poisoning caused by it can be mainly categorized into vomiting and diarrhea. However, in addition to gastrointestinal infections, it can also cause eye infections, bacterial sepsis, pneumonia, and central nervous system infections. It has been reported that Bacillus cereus contaminates rice-based foods relatively frequently, posing a significant risk of food poisoning. Therefore, testing for Bacillus cereus is crucial to ensuring food safety.

[0003] The traditional method for detecting Bacillus cereus is plate culture, which identifies the bacteria through colony morphology and physiological and biochemical methods. However, this method requires multiple centrifugations and dilutions, is time-consuming and labor-intensive, and is not suitable for the rapid screening needs of food safety. Molecular biology methods include traditional PCR, real-time quantitative PCR (RT-PCR), and loop-mediated isothermal amplification (LAMP), but these often require sophisticated instruments and highly trained technicians. Antibody-based immunological detection methods are also used in microbial detection. Antibodies are the core material of immunoassay methods, playing a crucial role in antigen recognition and method sensitivity. Currently, immunoassay methods for Bacillus cereus mainly rely on polyclonal and monoclonal antibodies. Although polyclonal antibody preparation is relatively simple, the variability and randomness of animal immune responses lead to poor uniformity and specificity. Monoclonal antibodies often have disadvantages such as long and complex initial screening cycles, high costs, and difficulty in processing and modification, which to some extent limits their further application in immunoassay methods. In addition, the later production process of traditional antibodies does not align with current animal protection trends, prompting increasing attention to genetically engineered antibodies.

[0004] Nanobodies are a novel type of genetically engineered antibody, derived from variable region fragments of natural heavy chain antibodies found in animals such as camels and sharks using molecular biology methods. They offer advantages such as lower production costs, higher expression efficiency, better water solubility and stability, and ease of genetic modification, leading to greater economic benefits and application prospects. Phage display nanobody technology involves inserting DNA fragments encoding exogenous proteins or antibodies into the gene fragments of phage coat proteins through genetic engineering, fusing the target protein onto the phage surface. This method is simple, rapid, and allows for mass production. However, nanobodies targeting Bacillus cereus have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nanobody against Bacillus cereus and its application.

[0006] The first objective of this invention is to provide a nanobody against Bacillus cereus.

[0007] A second objective of this invention is to provide a biomaterial.

[0008] A third objective of this invention is to provide the application of the nanobody or the biomaterial in the preparation of an immunological detection kit for Bacillus cereus.

[0009] A fourth objective of this invention is to provide the application of the nanobody or the biomaterial in establishing an immunological detection method for Bacillus cereus.

[0010] The fifth objective of this invention is to provide a kit for the immunological detection of Bacillus cereus for non-diagnostic purposes.

[0011] The sixth object of the present invention is to provide a method for detecting Bacillus cereus for non-diagnostic purposes.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for a nanobody against Bacillus cereus, the nanobody comprising a framework region (FR) and a complementarity-determining region (CDR), wherein the complementarity-determining region (CDR) is: CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, CDR2 with an amino acid sequence as shown in SEQ ID NO. 7, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 8.

[0013] The amino acid composition of the complementarity-determining region CDR1 of the nanobody is shown in SEQ ID NO.6: GYAINSNSMA; The amino acid composition of the complementarity-determining region CDR2 of the nanobody is shown in SEQ ID NO.7: RIYPASGGTN; The amino acid composition of the complementarity-determining region CDR3 of the nanobody is shown in SEQ ID NO.8: AAGLGGALESGRYNYW.

[0014] Preferably, the frame region FR is: FR1 with the amino acid sequence shown in SEQ ID NO.2, FR2 with the amino acid sequence shown in SEQ ID NO.3, FR3 with the amino acid sequence shown in SEQ ID NO.4, and FR4 with the amino acid sequence shown in SEQ ID NO.5.

[0015] The amino acid composition of the framework region FR1 of the nanobody is shown in SEQ ID NO.2: EVQLVESGGGSVQAGGSLRLSCVAS; The amino acid composition of the framework region FR2 of the nanobody is shown in SEQ ID NO.3: WFRQAPGKEREGVA; The amino acid composition of the framework region FR3 of the nanobody is shown in SEQ ID NO.4: YADSVKGRFTISQDSAQRKVYLQMNSLKPEDTAMYYC; The amino acid composition of the framework region FR4 of the nanobody is shown in SEQ ID NO. 5: GQGTQVTVSS.

[0016] More preferably, the amino acid sequence of the nanobody is shown in SEQ ID NO.1.

[0017] The amino acid sequence of the nanobody is shown in SEQ ID NO.1: EVQLVESGGGSVQAGGSLRLSCVASGYAINSNSMAWFRQAPGKEREGVARIYPASGGTNYADSVKGRFTISQDSAQRKVYLQMNSLKPEDTAMYYCAAGLGGALESGRYNYWGQGTQVTVSS.

[0018] This invention also claims protection for a biological material, which is one or more of the following: (1) The nucleic acid molecule encoding the nanobody described above; (2) An expression cassette containing the nucleic acid molecules described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) Recombinant microorganisms containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0019] As a specific embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.9.

[0020] This invention also claims protection for the following applications: The application of the nanobody or the biomaterial in the preparation of an immunological detection kit for Bacillus cereus.

[0021] Application of the nanobody or the biomaterial in establishing an immunological detection method for Bacillus cereus.

[0022] The present invention also claims a kit for the immunological detection of Bacillus cereus for non-diagnostic purposes, containing the nanobody.

[0023] Preferably, the kit is a double-antibody sandwich kit containing a detection antibody and a capture antibody, wherein both the detection antibody and the capture antibody are the nanobodies.

[0024] Preferably, the detection antibody is the nanobody displayed on the surface of the bacteriophage; the capture antibody is coated on a solid-phase carrier.

[0025] As a specific implementation, the kit also contains enzyme-labeled secondary antibody, chromogenic solution, and stop solution.

[0026] Specifically, the color developing solution is color developing solution A and color developing solution B.

[0027] More preferably, the stop solution is 10% H2SO4 (v / v).

[0028] More preferably, the enzyme-labeled secondary antibody is Anti-phage M13 antibody (HRP).

[0029] The present invention also claims a method for detecting Bacillus cereus for non-diagnostic purposes, utilizing the nanobody.

[0030] Preferably, the method is a sandwich ELISA method based on dual nanobodies, using the antibody displayed on the surface of the bacteriophage as the detection antibody and the antibody coated on the solid-phase carrier as the capture antibody.

[0031] The detection antibody is the nanobody; the capture antibody.

[0032] As a specific implementation method, the sample to be tested is added to the ELISA plate coated with the nanobody, and after the reaction is complete, the liquid is discarded and the plate is washed; the detection antibody is added, and after the reaction is complete, the liquid is discarded and the plate is washed; the enzyme-labeled secondary antibody is added, and after the reaction is complete, the liquid is discarded and the plate is washed; a colorimetric reaction is performed, and the reaction is terminated; the 450nm OD value is read.

[0033] Specifically, the TMB colorimetric solution, which is a mixture of colorimetric solution A and colorimetric solution B, is used for the colorimetric reaction, and the reaction is terminated with 10% H2SO4 (v / v).

[0034] Specifically, the enzyme-labeled secondary antibody is Anti-phage M13 antibody (HRP).

[0035] Compared with the prior art, the present invention has the following beneficial effects: This invention yields a nanobody that recognizes Bacillus cereus, possessing a unique variable region sequence. It can recognize and bind to Bacillus cereus and exhibits advantages such as ease of expression and high expression efficiency, making it suitable for application in enzyme-linked immunosorbent assay (ELISA) methods for detecting Bacillus cereus. The anti-Bacillus cereus nanobody provided by this invention has advantages such as short preparation cycle, low preparation cost, high expression yield, strong specificity, and good stability. It addresses the problems of poor specificity of current polyclonal antibodies against Bacillus cereus, and the high cost and time required for monoclonal antibodies, as well as the inconsistency in titers between different batches of antibodies. This fills a gap in the immunological field of Bacillus cereus nanobodies and facilitates the expansion of their application in practical immunoassay scenarios. The Bacillus cereus sandwich ELISA method based on dual nanobodies established in this invention fully utilizes the advantages of nanobodies, minimizing the use of traditional antibodies, reducing animal casualties, and aligning with animal welfare trends. Attached Figure Description

[0036] Figure 1 This is an electrophoresis image of the VHH gene amplified in the first round of PCR in the example. Figure 2 This is an electrophoresis image of the VHH gene amplified in the second round of PCR in the example. Figure 3 The results of ELISA identification of positive clones selected in the examples; Figure 4 This is an SDS-PAGE electrophoresis image of the anti-Bacillus cereus nanobody Nb-Bc01 in the examples; Figure 5 For the specificity analysis of the anti-Bacillus cereus nanobody Nb-Bc01 in the examples; Figure 6 The standard curve for detecting Bacillus cereus using the anti-Bacillus cereus nanobody Nb-Bc01 in the examples is shown. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0038] Example 1: Construction of a Bacillus cereus nanobody library 1. Bactrian camel immunization program Immunize healthy Bactrian camels with inactivated Bacillus cereus (10)8 The immunogen (CFU / mL) was administered subcutaneously to the back and neck of Bactrian camels via injection, with each immunization dose being 1 mL. For the initial immunization, 1 mL of complete Freund's adjuvant was mixed with the immunogen and emulsified before administration. Subsequent booster immunizations were administered using 1 mL of incomplete Freund's adjuvant mixed with the antigen, with each immunization spaced 2 weeks apart, for a total of 4 booster immunizations. Starting with the second immunization, 10 mL of Bactrian camel blood was collected one week after each immunization to separate serum for assessing the immune response. One week after the third, fourth, and fifth immunizations, 50 mL of peripheral blood from Bactrian camels was collected to separate lymphocytes for later use.

[0039] 2. Isolation of Bactrian camel lymphocytes Peripheral blood from Bactrian camels was diluted with an equal volume of sterile saline in a clean, RNase-free container. The diluted peripheral blood was centrifuged using commercially available lymphocyte separation medium. 20 mL of this solution was added to a sterile 50 mL centrifuge tube, and another 20 mL of diluted blood was slowly added along the tube wall using a sterile Pasteur pipette. The mixture was centrifuged at 500 g for 30 min at room temperature (25°C). Blood cells of different densities were distributed at different depths in the lymphocyte separation medium after centrifugation, with lymphocytes forming a white cell layer approximately one-third of the way below the surface. The lymphocyte layer was transferred to a new 50 mL centrifuge tube, diluted twice with saline, and centrifuged at 2000 g for 10 min at 4°C. The supernatant was discarded. The lymphocytes were dispersed by blowing with 5 mL of saline and centrifuged again at 2000 g for 10 min. The supernatant was discarded to thoroughly wash the lymphocytes. Lysis buffer (TRNsol) was added to each lymphocyte fraction, and the fractions were aliquoted into 2 mL centrifuge tubes at 1 mL each and stored at -80°C for later use.

[0040] 3. Extraction of total RNA Total RNA was extracted from lymphocytes preserved in TRNsol lysis buffer using a commercially available RNA extraction kit, following the instructions of the kit. After extraction, a small sample was subjected to nucleic acid electrophoresis, and the RNA concentration was determined using a Nanodrop microspectrophotometer.

[0041] 4. cDNA Synthesis Using extracted total RNA as a template, cDNA first-strand synthesis was performed according to the instructions of a commercial reverse transcription kit. After reverse transcription, the products were mixed thoroughly and then aliquoted into different sterile centrifuge tubes and stored at -80°C.

[0042] 5. Amplification of the target gene VHH of nanobody The target gene VHH of the nanobody was amplified by nested PCR in two steps. The primer sequences used are shown in Table 1.

[0043] Table 1. Primer sequences for Bactrian camel heavy chain antibody genes:

[0044] The first round of PCR used cDNA as its PCR template for nested PCR. The specific reaction parameters are shown in Table 2.

[0045] Table 2 Reaction system and reaction conditions for the first round of PCR

[0046] The first round of PCR products showed two bands after nucleic acid electrophoresis: 1000bp and 750bp. The 750bp band was excised and recovered, and its concentration was determined.

[0047] See the specific electrophoretic identification images. Figure 1 , Figure 1 In the diagram, M represents DL 2000+ DNA marker, and 1 represents the product of the VHH gene amplified in the first round of PCR.

[0048] The second round of PCR used the recovered products from the first round of PCR as templates for nested PCR to obtain the VHH target gene. The specific reaction parameters are shown in Table 3.

[0049] Table 3. Second round PCR reaction system and reaction conditions

[0050] See the electrophoresis identification image of the second round of PCR products. Figure 2 , Figure 2 In the diagram, M represents the DL 2000 DNA marker, and 1 represents the product of the VHH gene amplified by the second round of PCR.

[0051] 6. Gene library construction (1) Enzyme digestion of the target gene VHH and the vector pComb3xss: The VHH target gene and the pComb3xss vector were digested with SfiI enzyme. Digestion conditions: reaction at 50℃ for 16 h. The pComb3xss vector digestion product was recovered by agarose gel electrophoresis, and the 3500 bp band was recovered directly by a DNA recovery kit.

[0052] (2) Ligation of enzyme digestion products: Mix the vector pComb3xss and VHH fragment (molar ratio 1:3), react at 16℃ for 16h, and then clean and recover using a DNA recovery kit.

[0053] (3) Electroporation: Take 5 μL of ligation product and add it to 50 μL of electrocompetent state. E.coilIn TG1, after gentle mixing, transfer to a 0.1 cm electroporation cuvette for electroporation (1.8 kV). Immediately after electroporation, add 950 μL of preheated SOC medium to 37°C and incubate at 37°C and 250 rpm for 1 hour to revive the cells. Take 100 μL of the revived bacterial solution and perform serial dilutions, then plate the solution onto LB-Amp plates and incubate inverted at 37°C overnight.

[0054] Count the number of colonies on the culture dish and calculate the total number of bacteria in the revived bacterial solution. Perform multiple electroporation transformations until the total number of transformed colonies reaches 10. 7 The number of CFU or higher represents the library capacity of the nanobody gene library. Transgenic E. coli colonies are scraped from the amplification plate using a cell scraper, mixed thoroughly, and then glycerol (v / v) is added to a final concentration of 25%. After aliquoting, the mixture is frozen at -80°C to obtain the Bacillus cereus nanobody gene library.

[0055] 7. Construction of a phage-display nanobody library against Bacillus cereus 1 mL of the anti-Bacillus cereus nanobody library was inoculated into 150 mL of LB (Amp) culture and cultured at 37°C and 250 rpm until the OD600 value reached approximately 0.4–0.6. Helper phage M13KO7 (multiple of infection ratio of 20:1) was added, and the mixture was incubated at 37°C for 30 min, followed by incubation at 37°C and 250 rpm for 1 h. Kanamycin was then added to a final concentration of 70 μg / mL, and the mixture was incubated overnight at 37°C and 250 rpm. The next day, the mixture was centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was collected. 1 / 5 volume of PEG / NaCl (100 g PEG-8000 and 73.05 g sodium chloride diluted with water to a final volume of 500 mL) was added, and the mixture was incubated on ice for 2–3 h. Centrifuge at 12000 rpm for 15 min at 4℃, discard the supernatant, resuspend the precipitate in 1 mL TBS, transfer to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 5 min at 4℃, and filter through a 0.22 μm polyethersulfone membrane to obtain the phage display nanobody library against Bacillus cereus. Store at -80℃ for later use.

[0056] Example 2: Panning and Identification of Bacillus cereus Nanobodies I. Experimental Methods 1. Selection of anti-Bacillus cereus nanobodies Inactivated Bacillus cereus was used as the coating agent, with a coating amount of 100 μL / well and a concentration of 10. 9 CFU / mL (10 in round 2) 8 CFU / mL, 10 in round 3. 7CFU / mL), incubate overnight at 4℃, and wash twice the next day with washing buffer PBST (0.01M PBS containing 0.05% Tween-20). Add 300 μL of 3% skim milk powder-PBS (w / v) to each well and block at 37℃ for 3 h. Pour out the liquid from the well, pat dry on absorbent paper, and invert to dry at 37℃ for 30 min before storing at 4℃. This well is the original coating well. Separately, add 300 μL of 3% skim milk powder to new microwells, incubate at 37℃ for 3 h, pour out the liquid from the well, pat dry on absorbent paper, invert to dry at 37℃ for 30 min, and store at 4℃. This well is the background well.

[0057] The Bacillus cereus phage-displaying nanobody library obtained in Example 1 was added to the background wells at a rate of 100 μL / well and incubated at 37°C for 1 h. Then, 100 μL of phage from the background wells was transferred to the original coated wells for the first round of panning, and incubated at 37°C for 1 h. The wells were washed 10 times with PBST, then 5 times with PBS. Elution was performed with 100 μL of glycine-HCl solution (pH=2.2), and after incubation at 37°C for 10 min, an appropriate amount of Tris-HCl solution (pH 9.1) was immediately added to neutralize the eluted phage. 10 μL of the eluted phage was used to determine the titer, and the remainder was used to infect 4 mL of bacteria grown to the logarithmic growth phase. E.coil The TG1 strain was amplified. The amplified phage was precipitated with 1 / 5 volume of PEG / NaCl solution (100g PEG-8000 and 73.05g sodium chloride diluted with water to 500mL), and the phage titer was determined. The amplified phage was immediately used for subsequent panning.

[0058] The above screening process was performed in three rounds. After the third round of selection, 10 μL of phage was taken to determine the titer. The next day, 48 clones were randomly selected from the plate and inoculated into 96-well deep-well plates containing 1 mL of LB (containing Amp) medium per well. The plates were sealed and incubated overnight at 37°C and 250 rpm. This plate served as the "mother plate." The next day, 10 μL of bacterial culture was taken from each well of the mother plate and inoculated into another deep-well plate containing 1 mL / well of LB (containing Amp) medium. The plates were incubated at 37°C and 250 rpm for 4 hours until the logarithmic growth phase. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to each well to a final concentration of 1 mM, and the plates were incubated overnight with shaking. The next day, the deep-well plates were centrifuged at 4000 rpm for 20 minutes at 4°C, and the supernatant was collected for positive clone identification.

[0059] Positive phage clones were identified using enzyme-linked immunosorbent assay (ELISA). The specific method was as follows: (1) Antigen immobilization: The concentration of 10 8CFU / mL inactivated Bacillus cereus was added at 100 μL / well and incubated overnight at 4°C. The next day, the cells were washed twice with PBST (0.01 M PBS, 0.05% Tween-20). Then, 300 μL of freshly prepared 3% skim milk powder (w / v) was added to each well and the cells were blocked at 37°C for 2 hours. The blocking solution was discarded, and the cells were dried at 37°C for 30 minutes and stored at 4°C for later use.

[0060] (2) Indirect ELISA identification of positive clones: Take an enzyme-labeled plate with immobilized antigen, add 100 μL of the supernatant of the 96-well plate after centrifugation to each well (as a sample well), add 100 μL of PBS buffer to each well (as a blank well), incubate at 37°C for 1 h, wash 5 times with washing buffer PBST (0.01M PBS, 0.05% Tween-20), and pat dry the liquid in the well; dilute Anti-VHH-HRP secondary antibody 5000 times with PBST, add 100 μL to each well, and incubate at 37°C for 40 min. Wash 5 times with washing buffer PBST (0.01M PBS, 0.05% Tween-20), and blot dry the liquid in the wells; add 100μL of TMB chromogenic solution to each well, which has been pre-mixed with an equal volume of chromogenic solution A and chromogenic solution B (Solarbio, PR1210), and develop at 37℃ for 10 min; add 50μL of stop solution 10% H2SO4 (v / v) to terminate the reaction; measure the absorbance at 450nm using a microplate reader.

[0061] Clones with OD values ​​three times greater than those of the negative control wells were selected, and their corresponding well numbers were recorded. The bacterial culture from the corresponding wells in the mother plate was transferred to sterile centrifuge tubes, and glycerol was added for cryopreservation. The phage clones displaying nanobodies, identified by ELISA, were sent to a sequencing company for gene sequencing.

[0062] II. Experimental Results The OD value results in the plate are as follows Figure 3 As shown. Phage clones with an OD value greater than 1 in the sample wells and an OD value / blank OD value (P / N value) greater than 2.1 were selected as positive clones. The positive clones were sequenced and expressed, resulting in two nanobodies with different amino acid sequences.

[0063] Based on DNA sequencing results and codon table alignment analysis, one of the nanobodies, Nb-Bc01, possesses the amino acid sequence shown in SEQ ID NO. 1. This nanobodies comprise four FR framework regions and three CDR complementarity-determining regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid sequences of the framework regions FR1 to FR4 are shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; the amino acid sequences of the complementarity-determining regions CDR1 to CDR3 are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively. The corresponding positive clone is designated as strain Nb-Bc01.

[0064] The amino acid sequence of VHH in the nanobody Nb-Bc01 is shown in SEQ ID NO.1: EVQLVESGGGSVQAGGSLRLSCVASGYAINSNSMAWFRQAPGKEREGVARIYPASGGTNYADSVKGRFTISQDSAQRKVYLQMNSLKPEDTAMYYCAAGLGGALESGRYNYWGQGTQVTVSS The amino acid sequence of the framework region FR1 of the nanobody Nb-Bc01 is shown in SEQ ID NO.2: EVQLVESGGGSVQAGGSLRLSCVAS; The amino acid sequence of the framework region FR2 of the nanobody Nb-Bc01 is shown in SEQ ID NO.3: WFRQAPGKEREGVA; The amino acid sequence of the framework region FR3 of the nanobody Nb-Bc01 is shown in SEQ ID NO.4: YADSVKGRFTISQDSAQRKVYLQMNSLKPEDTAMYYC; The amino acid sequence of the framework region FR4 of the nanobody Nb-Bc01 is shown in SEQ ID NO.5: GQGTQVTVSS; The amino acid sequence of the complementarity-determining region CDR1 of the nanobody Nb-Bc01 is shown in SEQ ID NO. 6: GYAINSNSMA; The amino acid sequence of the complementarity-determining region CDR2 of the nanobody Nb-Bc01 is shown in SEQ ID NO.7: RIYPASGGTN; The amino acid sequence of the complementarity-determining region CDR3 of the nanobody Nb-Bc01 is shown in SEQ ID NO. 8: AAGLGGALESGRYNYW; Simultaneously, the nucleotide sequence encoding the nanobody Nb-Bc01 was obtained as shown in SEQ ID NO.9: TGAGGAGACGGTGACCTGGGTCCCCTGGCCCCAGTAGTTATAGCGGCCCGACTCCAAGGCACCACCCAAACCGGCCGCACAGTAGTACATGGCAGTGTCCTCAGGTTTCAGGCTGTTCATTTGCAGATACACCTTTCTCTGGGCGCTGTCTTGGGAAATGGTGAATCGGCCCTTCACGGAGTC GGCATAGTTGGTGCCCCACTAGCAGGATAAATACGCGCGACCCCCTCGCGCTCCTTCCCTGGAGCCTGGCGGAACCAGGCCATGGAGTTGCTATTGATGGCGTATCCAGAGGCTACACAGGAGAGTCTCAGAGACCCTCCAGCCTGCACCGAGCCTCCCCCAGACTCCACCAGCTGCACCTC.

[0065] Example 3: Large-scale preparation of phage-displayed nanobodies phage-Bc01 and Nb-Bc01 I. Experimental Methods 1. Preparation of phage-displayed nanobody phage-Bc01 by phage amplification The Nb-Bc01 strain obtained in Example 2 was inoculated into 150 mL of LB (Amp) culture medium and cultured at 37°C with shaking at 250 rpm until OD. 600 The value was 0.6. 1 mL of helper phage M13KO7 (multiple of infection of 20:1) was added, and the mixture was incubated at 37°C for 30 min, then incubated at 37°C and 250 rpm for 1 h. 150 μL of kanamycin stock solution (70 mg / mL) was added, and the mixture was incubated overnight at 37°C and 250 rpm with shaking. The next day, the supernatant was collected by centrifugation, and 1 / 5 volume of PEG / NaCl solution was added to the supernatant. After mixing well, the phage was precipitated. The precipitate was collected by centrifugation to obtain the phage-displayed nanobody phage-Bc01. 10 μL was used for titer determination.

[0066] 2. Preparation of soluble nanobody Nb-Bc01 via protein expression The plasmid of strain Nb-Bc01 obtained in Example 2 was extracted using a kit and chemically transformed into E. coil BL21. A single colony was picked from the transformation plate and inoculated into 10 mL of LB (Amp) medium, and cultured overnight at 37°C and 250 rpm. The overnight culture was inoculated into 750 mL of LB (Amp) medium at a ratio of 1:100 (v / v), and cultured at 37°C and 250 rpm until the OD600 value was approximately 0.4–0.6. IPTG was then added to a working concentration of 1 mM, and the culture was incubated overnight at 37°C and 250 rpm. The next day, the bacterial pellet was collected by centrifugation at 12,000 rpm for 5 min at 4°C. The pellet was then centrifuged at 12,000 rpm for 10 min using the sucrose osmotic pressure freeze-thaw method. The supernatant was collected and purified by affinity chromatography to obtain the nanobody Nb-Bc01 (amino acid sequence as shown in SEQ ID NO.1). The nanobody was identified by SDS-PAGE.

[0067] II. Experimental Results The protein electrophoresis results of the nanobody Nb-Bc01 are as follows: Figure 4 As shown, Figure 4 In the diagram, M represents Protein marker, and 1 represents Bacillus cereus nanobody Nb-Bc01. The nanobody concentration was measured using Nanodrop, and the yield of the nanobody was calculated to be 17.05 mg / L of culture medium.

[0068] Example 4: Specificity of anti-Bacillus cereus nanobody I. Experimental Methods The specificity of the Bacillus cereus nanobody Nb-Bc01, with an amino acid sequence as shown in SEQ ID NO.1, was detected using indirect ELISA. Ten Bacillus cereus species and seven other microorganisms were used as analytes, including Bacillus cereus F4810 and Bacillus cereus ATCC. 14579, Bacillus cereus GW-1-1520-1920-02, Bacillus cereus GSJ / 2014-Bc-45, Bacillus cereus 892-1, Bacillus cereus C04, Bacillus cereus C10, Bacillus cereus BD1-1, Bacillus cereus BD1-2, Bacillus cereus YNB1-2, as well as Bacillus thuringiensis, Bacillus megaterium, Bacillus licheniformis, Bacillus mycosis fungoides, Salmonella enteritidis, Salmonella pullorum, and Escherichia coli O157:H7 were used to evaluate the binding ability of the nanobody Nb-Bc01 to other foodborne pathogens.

[0069] The specific testing methods are as follows: With a concentration of 10 8Various inactivated bacterial solutions with CFU / mL were coated onto ELISA plates at 100 μL / well and incubated overnight at 4°C. The next day, the plates were washed twice with PBST (0.01 M PBS, 0.05% Tween-20). Then, 300 μL of freshly prepared 3% skim milk powder (w / v) was added to each well, and the plates were blocked at 37°C for 2 hours. The blocking solution was discarded, and the plates were dried at 37°C for 30 minutes. If necessary, the plates could be stored at 4°C. 100 μL of the anti-Bacillus cereus nanobody Nb-Bc01 (amino acid sequence as shown in SEQ ID NO.1) was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were then washed twice with PBST (0.01 M PBS, 0.05% Tween-20). Wash 5 times with PBS (0.05% Tween-20) and blot dry the liquid in the wells; dilute Anti-VHH-HRP secondary antibody 5000 times with PBST, add 100 μL to each well, incubate at 37℃ for 40 min, wash 5 times with washing buffer PBST (0.01M PBS, 0.05% Tween-20), and blot dry the liquid in the wells; add 100 μL of TMB chromogenic solution pre-mixed with equal volumes of chromogenic solution A and chromogenic solution B (Solarbio, PR1210) to each well, and incubate at 37℃ for 10 min; add 50 μL of stop solution 10% H2SO4 (v / v) to terminate the reaction; measure the absorbance at 450 nm using a microplate reader.

[0070] II. Experimental Results The results are as follows Figure 5 As shown, the nanobody Nb-Bc01 can effectively recognize multiple strains of Bacillus cereus, and its specificity is good.

[0071] Example 5: Enzyme-linked immunosorbent assay (ELISA) with dual nanobody sandwich for the detection of anti-Bacillus cereus I. Experimental Methods Using the anti-Bacillus cereus nanobody Nb-Bc01 with the amino acid sequence as shown in SEQ ID NO.1 as the capture antibody, and the phage-displayed nanobody phage-Bc01 prepared in Example 3 as the detection antibody, Bacillus cereus was detected by a double-antibody sandwich enzyme-linked immunosorbent assay.

[0072] The specific method is as follows: Using 10 μg / mL of anti-Bacillus cereus nanobody Nb-Bc01 as the capture antibody, 100 μL / well was coated onto an ELISA plate and incubated overnight at 4°C. The plate was washed twice with washing buffer PBST (0.01M PBS, 0.05% Tween-20). Blocking buffer (Sangon Biotech, C520041) was added to 300 μL / well, and the plate was blocked at 37°C for 3 h. The blocking buffer was discarded, and the plate was dried at 37°C for 30 min. If necessary, the plate could be stored at 4°C for later use. Add 100 μL / well to the serially diluted 10 μL solution. 4 ~108 Inactivated Bacillus cereus at CFU / mL was incubated at 37°C for 1 h, followed by washing three times with PBST (0.01M PBS, 0.05% Tween-20); 5×10⁻⁶ CFU / mL of the solution was added to each well. 10 Phage-displaying nanobody phage-Bc01 (PFU / mL) was incubated at 37°C for 1 h, and washed 10 times with PBST (0.01M PBS, 0.05% Tween-20). Anti-phage M13 antibody (HRP) diluted 5000 times with PBST was added at 100 μL / well, and the mixture was incubated at 37°C for 40 min, followed by washing 5 times with PBST (0.01M PBS, 0.05% Tween-20). TMB chromogenic solution (pre-mixed with equal volumes of chromogenic solution A and chromogenic solution B (Solarbio, PR1210)) was added at 100 μL / well, and the mixture was incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 10% H2SO4 (v / v). The absorbance at 450 nm was measured using a microplate reader. A standard curve was obtained by fitting the bacterial concentration as the x-axis and the absorbance as the y-axis using a logistic regression equation.

[0073] II. Experimental Results Standard curve such as Figure 6 As shown, y = 4.22934 - 4.04974 / (1 + (x / 14855000)^0.93252), the result shows that the detection limit of the method is 4.54 × 10⁻⁶. 5 CFU / mL.

[0074] Example 6 An immunological detection kit for Bacillus cereus I. Composition Removable ELISA plates pre-coated with anti-Bacillus cereus nanobody Nb-Bc01: Add 10 μg / mL of the anti-Bacillus cereus nanobody Nb-Bc01 (amino acid sequence as shown in SEQ ID NO.1) to each well of the ELISA plate using PBS, 100 μL per well, and incubate overnight at 4°C. The next day, wash the plate twice with washing buffer PBST (0.01M PBS, 0.05% Tween-20), add 300 μL of blocking buffer (Sangon Biotech, C520041) to each well, incubate at 37°C for 3 h, discard the blocking buffer, dry at 37°C for 30 min, and store at 4°C for later use. Detection antibody: Phage-Bc01 nanobody prepared according to Example 3; Enzyme-labeled secondary antibody: Anti-phage M13 antibody (HRP), which needs to be diluted 5000 times before use; Antibody and sample dilution buffer: 0.01M PBS; Enzyme-labeled antibody dilution solution: 0.01M PBST (containing 0.05% Tween-20); Developing solutions: Developing solution A and developing solution B; Stop solution: 10% H2SO4 (v / v); Washing solution: 0.01M PBST (containing 0.05% Tween-20).

[0075] II. Instructions for Use Add 100 μL of the sample to be tested to each well, incubate at 37°C for 1 h, and wash the plate three times with PBST (0.01 M PBS, 0.05% Tween-20). Add 100 μL of a 5 × 10⁻⁶ m² solution to each well. 10 Phage-Bc01 nanobody (PFU / mL) was incubated at 37°C for 1 h, and washed 10 times with PBST (0.01M PBS, 0.05% Tween-20). Anti-phage M13 antibody (HRP) diluted 5000 times with PBST was added at 100 μL / well, and the plate was incubated at 37°C for 40 min, followed by washing 5 times with PBST (0.01M PBS, 0.05% Tween-20). TMB chromogenic solution (pre-mixed with equal volumes of chromogenic solution A and chromogenic solution B (Solarbio, PR1210)) was added at 100 μL / well, and the plate was incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 10% H2SO4 (v / v). The absorbance at 450 nm was measured using a microplate reader, and the detection concentration was calculated based on the standard curve and absorbance value.

Claims

1. A nanobody against Bacillus cereus, characterized in that, The nanobody includes a framework region (FR) and a complementarity-determining region (CDR), wherein the complementarity-determining region (CDR) is: CDR1 with an amino acid sequence as shown in SEQ ID NO.6, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

8.

2. The nanobody according to claim 1, characterized in that, The frame region FR is: FR1 with the amino acid sequence shown in SEQ ID NO.2, FR2 with the amino acid sequence shown in SEQ ID NO.3, FR3 with the amino acid sequence shown in SEQ ID NO.4, and FR4 with the amino acid sequence shown in SEQ ID NO.

5.

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

1.

4. A biomaterial, characterized in that, One or more of the following: (1) A nucleic acid molecule encoding the nanobody of claim 1; (2) An expression cassette containing the nucleic acid molecules described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) Recombinant microorganisms containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

5. The use of the nanobody of claim 1 or the biomaterial of claim 4 in the preparation of an immunological detection kit for Bacillus cereus.

6. The use of the nanobody of claim 1 or the biomaterial of claim 4 in establishing an immunological detection method for Bacillus cereus.

7. A kit for the immunological detection of non-diagnostic Bacillus cereus, characterized in that, Contains the nanobody as described in claim 1.

8. The reagent kit according to claim 7, characterized in that, The kit is a double antibody sandwich kit containing a detection antibody and a capture antibody, both of which are nanobodies as described in claim 1.

9. The reagent kit according to claim 8, characterized in that, The detection antibody is the nanobody described in claim 1 displayed on the surface of the phage; the capture antibody is coated on a solid-phase carrier.

10. A method for detecting Bacillus cereus for non-diagnostic purposes, characterized in that, The nanobody described in claim 1 is used.

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