A nanobody binding both bacitracin a and bacitracin b and uses thereof

Abstract

This invention provides a nanobody that simultaneously binds bacitracin A and bacitracin B and its applications, belonging to the field of food and feed analysis and detection technology. The nanobody specifically binds to bacitracin A and bacitracin B. The VHH chain of the nanobody includes a complementarity-determining region (CDR) and a frame region, with the CDR separated by the frame region. The CDR includes CDR1, CDR2, and CDR3; the frame region includes FR1, FR2, FR3, and FR4. This nanobody can simultaneously target two bacitracins and exhibits excellent resistance to organic solvents and acids / alkalis. During sample pretreatment for actual detection, it is less affected by organic solvents and can also be used for detection in acidic and alkaline environments, providing a new solution for the detection of bacitracin-related substances.

Description

Technical Field

[0001] This invention belongs to the field of food and feed analysis and testing technology, specifically a nanobody that simultaneously combines bacitracin A and bacitracin B and its application. Background Technology

[0002] Bacitracin is a polypeptide antibiotic synthesized by Bacillus licheniformis or Bacillus subtilis. It possesses a relatively broad spectrum and good antibacterial efficacy, inhibiting most Gram-positive bacteria and a few Gram-negative bacteria. Therefore, bacitracin was once widely used as a growth promoter in livestock and poultry diets to improve growth and slaughter performance, enhance the immune barrier and intestinal environment; it was also used to treat bacterial diseases in livestock and poultry. However, with the long-term and extensive use of bacitracin, the problem of bacterial resistance has become increasingly prominent. Studies have shown that some Gram-positive bacteria can acquire resistance by producing resistance genes (such as the bacA gene) that encode transport proteins, pumping bacitracin out of the cell, thus leading to decreased clinical treatment efficacy. Furthermore, bacitracin residues in food and feed may enter the human body through the food chain, triggering allergic reactions or disrupting the balance of intestinal flora, posing a potential threat to human health.

[0003] To ensure the safety of animal-derived foods and public health, a series of maximum residue limits (MRLs) for bacitracin have been established both domestically and internationally for monitoring. For example, my country's national standard GB31650-2019, "National Food Safety Standard: Maximum Residue Limits for Veterinary Drugs in Food," stipulates a maximum residue limit of 500 μg / kg for bacitracin in animal muscle, liver, kidney, fat, milk, and eggs. Bacitracin is a mixture of several similar polypeptides. It has many components with similar and complex structures. The effective active ingredients are bacitracin A and bacitracin B, accounting for approximately 96%. Both have the same cyclic peptide chain structure; the main difference lies in the amino acid structure at the linear end of the chain. Current literature, patents, and national standards mostly target the determination of bacitracin A, bacitracin methylene salicylate synthesized from bacitracin A, or bacitracin zinc. The vast majority of these methods rely on expensive, sophisticated instruments. Some literature has screened mouse monoclonal antibodies against bacitracin zinc, and some patents (publication number: CN120795139A) have screened nanobodies against bacitracin methylene salicylate. However, the antigenic epitopes in these methods are complex, typically related to conjugated zinc ions or methylene salicylate, and cannot simultaneously recognize bacitracin B. Therefore, there is currently no rapid immunoassay-based method for the simultaneous detection of bacitracin A and bacitracin B.

[0004] Nanobodies (VHH) are single-domain antibodies derived from heavy chain antibodies of camelids, with a molecular weight of only about 15 kDa, exhibiting high affinity and good stability. Currently, there are no research studies on rapid immunoassay methods targeting both bacitracin A and B simultaneously. Furthermore, due to the high structural similarity of bacitracin A and B, and their frequent presence in mixtures in practical applications, simultaneous recognition and regulation of both is difficult, limiting their application in complex scenarios. Summary of the Invention

[0005] In view of the fact that there are currently no nanobodies that simultaneously target two bacitracin A and B, this invention provides a nanobodies that simultaneously bind bacitracin A and bacitracin B and their applications.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A nanobody that simultaneously binds bacitracin A and bacitracin B, wherein the nanobody specifically binds bacitracin A and bacitracin B, and the VHH chain of the nanobody includes a complementarity-determining region and a frame region, wherein the complementarity-determining region is separated by the frame region. The complementary determination regions include CDR1, CDR2, and CDR3: The amino acid sequence of CDR1 is shown in SEQ ID No. 2, the amino acid sequence of CDR2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR3 is shown in SEQ ID No. 4. The frame region includes FR1, FR2, FR3, and FR4: The amino acid sequence of FR1 is shown in SEQ ID No. 5, the amino acid sequence of FR2 is shown in SEQ ID No. 6, the amino acid sequence of FR3 is shown in SEQ ID No. 7, and the amino acid sequence of FR4 is shown in SEQ ID No. 8.

[0007] The above-mentioned three complementary determinant regions (CDRs) CDR1, CDR2, CDR3 and four frame regions (FRs) FR1, FR2, FR3, FR4 are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0008] The CDR region is a key area within the VHH domain that directly binds to the antigen, and its amino acid sequence determines the antigen binding affinity. Precisely defining the VHH CDR sequence ensures that the nanobody exhibits high binding capacity for both bacitracin A and bacitracin B.

[0009] The nanobody that specifically binds to bacitracin A and bacitracin B in this invention is named nanobody BAC-A138.

[0010] A nanobody that simultaneously binds bacitracin A and bacitracin B, wherein the amino acid sequence of the VHH of the nanobody is shown in SEQ ID No. 1.

[0011] This invention uses cyclic polypeptides (CP) with identical structures of bacitracin A and bacitracin B as haptens. After conjugating proteins, immunizing alpacas yields a nanobody gene library. Using phage display technology and bio-panning, a nanobody that simultaneously recognizes bacitracin A and bacitracin B is screened from the alpaca immune antibody gene library.

[0012] As an example, the VHH of the nanobody also includes an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 1, or an amino acid sequence having 1-6 amino acid additions, deletions and / or substitutions (preferably conservative substitutions) compared with SEQ ID No. 1, and must retain the ability to bind bacitracin A and bacitracin B simultaneously.

[0013] A polynucleotide molecule that encodes the aforementioned nanobody.

[0014] As an example, the sequence of the polynucleotide molecule is shown in SEQ ID No. 9.

[0015] The polynucleotide sequence can be optimized based on the amino acid sequence of the nanobody and the codon preference of the host cell. For example, the base sequence can be adjusted to suit the codon preference of CHO cells or HEK293 cells to increase the expression level of the antibody in the host cell. The polynucleotide can be in the form of DNA or RNA and can be obtained through conventional molecular biology methods such as gene synthesis and PCR amplification.

[0016] A recombinant vector comprising the above-mentioned polynucleotide molecule, wherein the recombinant vector is preferably an expression vector.

[0017] The expression vector can be constructed based on commercially available vectors such as pComb 3X, cDNA3.4, pCHO1.0, and pLVX. It must include elements such as a promoter (e.g., CMV promoter, EF1α promoter), a terminator (e.g., BGH terminator), a multiple cloning site, selection markers (e.g., ampicillin resistance gene (Amp), neomycin resistance gene), and a replication origin. Simultaneously, a histidine tag, a flag tag, or an Fc tag is inserted. Inserting the polynucleotide sequence encoding the nanobody into the multiple cloning site of the expression vector yields a recombinant expression vector suitable for host cell transformation.

[0018] A host cell containing the aforementioned polynucleotides or recombinant vectors. The host cells are preferably mammalian cells, such as HEK293 cells (HEK293T, HEK293F), CHO cells (CHO-K1, CHO-S), or NSO cells, which possess good antibody expression capabilities and the ability to perform correct post-translational protein modifications (such as glycosylation). Alternatively, yeast cells (such as Pichia pastoris), insect cells (such as Sf9 cells), or prokaryotic cells (such as Escherichia coli) can also be selected as host cells for nanobody expression. Since the amino acid sequence of the nanobody BAC-A138 and the gene sequence encoding it have been provided in this invention, those skilled in the art can obtain the nanobody described in this invention using known recombinant DNA techniques. Therefore, any recombinant vectors or recombinant cells that can be used to prepare the nanobody described in this invention should also be within the scope of protection of this invention.

[0019] A method for preparing nanobodies, the method comprising the following steps: Host cell culture and transfection: The recombinant vector described above is introduced into host cells (such as E. coli, HEK293F cells or CHO-S cells) via electrotransfection, liposome transfection (such as Lipofectamine 3000) or viral infection, and then cultured in a suitable culture medium (such as LB medium, ExpiCHO™ Expression Medium, CD-CHOMedium) and induced to express. Antibody expression and collection: After culturing to a suitable cell density, collect bacterial cells (bacterial cells need to be lysed) or cell culture supernatant, and centrifuge to collect the supernatant; Antibody purification: The nanobodies in the supernatant were purified by nickel column ion affinity chromatography, anti-Flag affinity chromatography column or Protein A affinity chromatography column to finally obtain high-purity nanobodies; Antibody identification: Antibody purity was determined by SDS-PAGE (reducing / non-reducing conditions).

[0020] A detection composition comprising the above-described nanobody.

[0021] The above-mentioned nanobodies are used in the preparation of reagents for detecting the presence or level of bacitracin A and bacitracin B in a sample.

[0022] This reagent can be used as a core component in the preparation of diagnostic kits for the detection of bacitracin residues in food (such as meat, dairy products, and eggs), feed, or environmental samples (such as soil and water). Detection methods include ELISA, immunochromatographic strips, Western blotting, or immunofluorescence assays; detection materials include immunoaffinity columns, biosensor chips, or detection probes.

[0023] The present invention has the following beneficial effects: 1. This invention provides a nanobody that simultaneously binds bacitracin A and bacitracin B and its application. Using bacitracin cyclic peptide chains as antigens, the VHH domains that simultaneously bind bacitracin A and bacitracin B are obtained through immunological library screening. A nanobody that can simultaneously target two bacitracins is then designed and constructed, providing a new solution for the detection applications related to bacitracin-like substances.

[0024] 2. The present invention provides a nanobody that simultaneously combines bacitracin A and bacitracin B and its application. The final screened nanobody BAC-A138 has excellent organic solvent resistance and acid-base resistance. In the pretreatment process of actual sample detection, it is less affected by organic solvents and can also be used for detection needs in acidic and alkaline environments.

[0025] 3. This invention provides a nanobody that simultaneously binds bacitracin A and bacitracin B and its application. A colloidal gold immunochromatographic detection method is established based on the nanobody BAC-A138. This method has a detection limit of 0.01 μg / mL for bacitracin A and bacitracin B and has high detection sensitivity. Therefore, when using the nanobody BAC-A138 to detect bacitracin A and bacitracin B in actual samples, the operation is simple, the time consumption is short, and the detection results have good stability, high sensitivity, and high accuracy. Attached Figure Description

[0026] Figure 1 Schematic diagram of a cyclic polypeptide hapten; Figure 2 A schematic diagram of the SDS-PAGE electrophoresis results for the complete antigen; Figure 3 A schematic diagram of the amino acid sequence and domain division of the nanobody BAC-A138; Figure 4 This is a schematic diagram of the SDS-PAGE electrophoresis results after nanobody purification; Figure 5 Activity curves of the nanobody BAC-A138 when methanol and ethanol / PBS were used as diluents at different ratios; Figure 6 The activity curves of the nanobody BAC-A138 when different pH values ​​were used as diluents; Figure 7 This is a schematic diagram of the time-resolved fluorescence immunochromatographic strip used in Example 7 to detect bacitracin A and bacitracin B. Figure 8 Time-resolved fluorescence standard curves for detecting bacitracin A and bacitracin B.

[0027] Figure label: 1. Conjugate release pad; 2. Nitrocellulose membrane detection pad; 3. Absorbent pad; 4. Base plate. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer. The reagents, enzymes, culture media, and other chemical materials used in the following embodiments of the present invention are all commercially available products. Some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are also commercially available products. Some synthetic biological materials, such as cyclic polypeptides, primers, and sequences, which require artificial synthesis, are all outsourced to synthetic companies. For example, cyclic polypeptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0029] The reagents represented by the abbreviations in the specific embodiments are as follows: CP: Cyclic polypeptide; BSA: Bovine serum albumin; OVA: Ovalbumin; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; VHH chain: Variable domain of heavy chain; Nanobody: A naturally occurring antibody lacking a light chain, found in the peripheral blood of camels, i.e., a "heavy chain single-domain antibody." This type of antibody contains only one variable domain of heavy chain (VHH), and the light chain is naturally absent compared to other antibodies; DEPC water: Ultrapure water treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure, free of impurities such as RNA, DNA, and protein; PBST: Phosphate buffer (PBS), containing 0.05% Tween 20, free of Ca2+. 2+ Mg 2+ The pH value is 7.4, and it is mostly used as an ELISA washing solution; CBS buffer solution: carbonate buffer solution with a concentration of 0.1 mol / L and a pH of 9.6; Sulfo-NHS-Biotin: sulfosuccinimide biotin.

[0030] Example 1: Construction of a nanobody phage library 1.1 Preparation of cyclic polypeptide hapten conjugated with BSA complete antigen (cyclic polypeptide-BSA), bacitracin A complete antigen and bacitracin B complete antigen Dissolve 10 mg of BSA protein in 10 mL of MES buffer solution (0.1 mol / L pH 5.5), then add 5 mg of EDC and 3 mg of NHS, and mix vertically for 15 min to complete the preparation of the activation solution.

[0031] 10 mg of cyclic polypeptide (synthesized by Sangon Biotech (Shanghai) Co., Ltd., structure shown in [reference needed]) was synthesized. Figure 1 The cyclic polypeptide solution was prepared by dissolving it in 1 mL of 0.05 mol / L carbonate buffer solution (CBS buffer solution, concentration 0.1 mol / L, pH 9.6).

[0032] Add 60 µL of the cyclic polypeptide solution to 1 mL of the above activation solution and mix vertically overnight at 4°C to obtain the mixture. Dialyze the mixture in phosphate buffer (0.01 mol / L, pH 7.4, volume 5-10 L) for 3 days to obtain the complete BSA protein-conjugated antigen. Confirm the concentration of the complete antigen is greater than 80% by SDS-PAGE. The SDS-PAGE electrophoresis results of the complete antigen are shown below. Figure 2 As shown in the figure, M represents the protein marker, BSA represents the electrophoresis band of bovine serum albumin sample, and BSA-CP represents the sample band of BSA protein-conjugated complete antigen. The complete antigen is stored in a -20°C freezer.

[0033] The synthesis steps for cyclic polypeptide hapten conjugated with OVA complete antigen (cyclic polypeptide-OVA) are the same as those for the preparation of cyclic polypeptide hapten conjugated with BSA complete antigen, except that bovine serum albumin is replaced with chicken ovalbumin.

[0034] Bacitracin A and bacitracin B were chemically conjugated to OVA to prepare bacitracin A-OVA and bacitracin B-OVA conjugated antigens for coating the detection line on test strips. 10 mg of OVA protein was dissolved in 1 mL of MES buffer (0.1 mol / L, pH 5.5), followed by the addition of 5 mg of EDC and 3 mg of NHS. The mixture was vertically mixed for 15 min to prepare the activation solution. 10 mg of bacitracin A and 10 mg of bacitracin B were dissolved in 1 mL of 0.05 mol / L carbonate buffer (CBS buffer, 0.1 mol / L, pH 9.6) to prepare 10 mg / mL bacitracin A hapten solutions and 10 mg / mL bacitracin B hapten solutions, respectively. 1 mL of each bacitracin A hapten solution and 1 mL of each bacitracin B hapten solution were then added to 1 mL of the above activation solutions and vertically mixed overnight at 4°C to obtain the final mixture. The two mixtures were dialyzed separately in phosphate buffer (0.01 mol / L, pH 7.4, 5-10 L) for 3 days to obtain the complete OVA protein-conjugated antigen. The complete antigen was stored at -20°C.

[0035] 1.2 Immunity The above-mentioned conjugated cyclic polypeptide was conjugated with BSA complete antigen and adjuvant in a 1:1 mixture, emulsified, and then used to immunize alpacas. The first immunization used complete Freund's adjuvant, and from the second immunization onwards, incomplete Freund's adjuvant was used. Immunization was carried out every two weeks for a total of 4 immunizations. Blood titers were measured starting from the second immunization. The collected alpaca blood was placed at 4°C for 2 hours, then centrifuged at 8000 rpm for 5 minutes. The supernatant was collected, and the antiserum titer was determined by enzyme-linked immunosorbent assay.

[0036] 1.3 Isolation of peripheral blood lymphocytes (PBMCs) After ensuring the serum titer was not lower than 1:64000, blood was collected from the neck of alpacas to isolate lymphocytes for subsequent experiments. The specific procedure was as follows: Alpaca peripheral blood was diluted 2:1 with sterile physiological saline in a 50mL centrifuge tube without RNase. The diluted peripheral blood was centrifuged with commercially available lymphocyte separation medium. 15mL of the lymphocyte separation medium was added to a sterile 50mL centrifuge tube, and 15mL of diluted blood was slowly added along the tube wall using a sterile Pasteur pipette. Centrifugation was performed at 800g for 25min. The lymphocyte layer was transferred to a new 50mL centrifuge tube, diluted twice with physiological saline, and centrifuged at 1500g for 10min at 4℃. The supernatant was discarded. The lymphocytes were dispersed by blowing with 5mL of physiological saline and centrifuged again at 1500g for 10min. The supernatant was discarded to thoroughly wash the lymphocytes. Lysis buffer (TRNsol) was added to each lymphocyte fraction, and 1mL was aliquoted into 2mL centrifuge tubes and stored at -80℃ for later use.

[0037] 1.4 Extraction of total RNA The PBMCs isolated in section 1.3 were used for total RNA extraction from cells. The total RNA extraction method was the TRZOL manual extraction method, specifically as follows: 1 mL of Trizol buffer was added to the PBMCs. After complete lysis, 0.2 mL of chloroform was added, and the mixture was thoroughly mixed. The mixture was incubated at room temperature for 20 min, then centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 min, then centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was discarded, and the precipitate was washed twice with 75% ethanol. The precipitate was centrifuged at 12000 rpm for 5 min at 4°C. The aqueous solution was discarded, and the precipitate was retained. After air-drying at room temperature, the precipitate was resuspended in DEPC water to obtain RNA.

[0038] 1.5 cDNA Synthesis The RNA obtained in step 1.4 was reverse transcribed into cDNA. Using the reverse-transcribed cDNA as a template, PCR primers were designed using Primer Premier software based on the upstream and downstream sequences of the VHH gene. Nested PCR was used to amplify the VHH gene. The first-round primers were CALL001 (forward primer): GTCCTGGCTGCTCTTCTACAAGG, and CALL002 (reverse primer): GGTACGTGCTGTTGAACTGTTCC. Using the recovered product from the first-round PCR as a template, the VHH gene was amplified using the second-round primers: VHH-F (forward primer): TTAAGCCATGGAGGTGCGC, and VHH-R (reverse primer): ATAGTGCGGCCGCTGAGGA. A band with a molecular weight of approximately 400 bp was finally obtained. The band was excised and recovered from the gel, which is the VHH fragment.

[0039] 1.6 Gene Library Construction The VHH target gene and pComb 3X vector were digested using NcoI and NotI enzymes. Digestion conditions: 37℃ for 2 h. The pComb 3X vector and VHH fragment were mixed (molar ratio 1:3), reacted at 22℃ for 0.5 h, and then cleaned and recovered using a DNA recovery kit. 5 μL of the ligation product was added to 50 μL of electroporation-competent E. coli TG1 cells, gently mixed, and transferred to a 0.2 cm electroporation cuvette for electroporation (1.8 kV). Immediately after electroporation, 800 μL and 150 μL of preheated 2YT medium (37℃) were added to the cuvette in two separate portions. The cells were collected in sterile centrifuge tubes and cultured at 37℃, 250 rpm for 1 h to revive the cells. 50 μL of the revived bacterial culture was serially diluted, and 100 μL of each serially diluted culture was spread onto 90 mm diameter LB-Amp culture dishes as counting chambers and incubated overnight at 37℃. The remaining undiluted resuscitated bacterial solution was spread onto 120 mm diameter LB-Amp culture dishes, with 1 mL of bacterial solution spread onto 2-3 dishes to form an amplification plate. Amplification was carried out overnight at 37°C. The number of colonies on the culture dishes was counted, and the total number of bacteria in the resuscitated bacterial solution was calculated. Multiple electroporation transformations were performed until the total number of transformed colonies reached at least 10⁷ CFU. This number is the library capacity of the nanobody gene library. Transgenic E. coli colonies from the amplification plate were scraped off with a cell scraper, centrifuged to collect the cells, and the supernatant was discarded. The cells were resuspended in LB-Amp (0.5 mL per electroporation tube), mixed thoroughly, and then sterile glycerol (v / v) was added to a final concentration of 25%. 50 μL of the bacterial solution was serially diluted to determine the cell count. The remaining bacterial solution was aliquoted and stored at -80°C to form the nanobody phage antibody library.

[0040] Example 2: Screening of phage libraries 2.1 Affinity Screening of Nanobodies The cyclic peptide-OVA was coated onto 96-well plates and incubated overnight at 4°C. The next day, 100 µL of 2% BSA was added to each well for 1 hour to block the plate. The VHH phage antibody library prepared in Example 1 was then added and incubated for 2 hours. After washing 4-10 times, positive clones specifically binding to the cyclic peptide-OVA were eluted with elution buffer (pH 2.2). Phages were collected, and 10 µL of the eluted phage was used to determine the titer. The remainder was used to infect 5 mL of E. coli TG1 strain grown to the logarithmic growth phase for amplification. The next day, the amplified phages were precipitated with PEG / NaCl, and the phage titer was determined, completing the first round of panning. Subsequent rounds of panning were performed using the same methods as the first round.

[0041] 2.2 Identification of positive clones The positive phages obtained from screening in 2.1 were plated, and single clones were directly induced to express by IPTG. The expression products were extracted and detected as follows: cyclic peptides-OVA, bacitracin A, and bacitracin B at a concentration of 2 μg / mL were coated with 0.1 mol / L pH 9.6 CBS buffer, 100 μl / well, and incubated overnight at 4℃; blocked with 5% BSA at 37℃ for 1 h; 100 μl of each positive clone diluted with PBST was added to each well and incubated at 37℃ for 1 h; then Goat pAb to Llama HRP secondary antibody was added, and incubated at 37℃ for 1 h. After 10 min of color development, the OD450 value was read on the microplate reader.

[0042] Ultimately, three positive nanobodies that simultaneously bind to three antigens—cyclic polypeptide-OVA, bacitracin A, and bacitracin B—were screened.

[0043] Example 3: Sequencing of the nanobody encoding gene and determination of its amino acid sequence 3.1 Sequencing of the nanobody encoding gene The positive nanobodies obtained in Example 2 were identified by PCR using VHH-F and VHH-R primers (primer sequences are shown in Example 1). The target fragment size was approximately 500 bp. The positive clones were sequenced to determine the diversity of nanobodies.

[0044] The positive nanobody sequence that simultaneously binds to three antigens—cyclic polypeptide-OVA, bacitracin A, and bacitracin B—was translated into amino acid sequences and compared. After sequence analysis, one correctly expressed nanobody was obtained and named BAC-A138.

[0045] 3.2 Sequence Determination The amino acid sequence of VHH in the nanobody BAC-A138 is shown in SEQ ID No. 1: EVRRQESGQGVVEAQGGLSRLLACVASRVSHTFGLGWAKDPPGFRQEFGAGIWRGKVSGTFYAGRFDSVKTIYLQSRDNAKKPEDNTNSLTVMALPAPRGQTGVTVGRRIPTRTPETLVTCVS.

[0046] A schematic diagram of the amino acid sequence and domain division of the nanobody BAC-A138 is shown below. Figure 3 As shown, the specific nanobody BAC-A138 includes four framework regions (FRs) and three complementarity-determining regions (CDRs).

[0047] The amino acid sequences of the complementarity-determining regions (CDR1-CDR3) are as follows: SEQ ID No. 2: VASRVSHTFGLG SEQ ID No. 3: GIWRGKVSGTF As shown in SEQ ID No. 4: RGQTGVTVGRRIPT.

[0048] The amino acid sequences of the framework regions (FR1-FR4) are as follows: SEQ ID No.5: EVRRQESGQGVVEAQGGLSRLLAC, SEQ ID No.6: WAKDPPGFRQEFGA, SEQ ID No.7: YAGRFDSVKTIYLQSRDNAKKPEDNTNSLTVMALPAP, As shown in SEQ ID No. 8: RTPETLVTCVS.

[0049] The nucleotide sequence of VHH in the specific nanobody BAC-A138 is shown in SEQ ID No. 9: GAGGTGCGCCGCCAGGAAAGCGGCCAGGGCGTGGTGGAAGCGCAGGGCGGCCTGAGCCGCCTGCTGGCGTGCGTGGCGAGCCGCGTGAGCCATACCTTTGGCCTGGGCTGGGCGAAAGATCCGCCGGGCTTTCGCCAGGAATTTGGCGCGGGCATTTGGCGCGGCAAAGTGAGCGGCACCTTTTA TGCGGGCCGCTTTGATAGCGTGAAAACCATTTATCTGCAGAGCCGCGATAACGCGAAAAAACCGGAAGATAACACCAACAGCCTGACCGTGATGGCGCTGCCGGCGCCGCGCGGCCAGACCGGCGTGACCGTGGGCCGCCGCATTCCGACCCGCACCCCGGAAACCCTGGTGACCTGCGTGTCA.

[0050] Example 4: Large-scale preparation of nanospecific nanobody BAC-A138 The pComb 3X-VHH plasmid was chemically transformed into BL21 competent cells (TAKARA, catalog number 9126). Single clones were selected and cultured, and expression was induced by IPTG (0.5 mM, 2 μL). The cells were cultured overnight at room temperature and 250 rpm. The next day, the bacterial culture was centrifuged at 4°C and 4000 rpm for 20 min, and the supernatant was discarded. The bacterial pellet was lysed with B-PER cell lysis buffer (Thermo Fisher Scientific, 89821), and the supernatant was further purified using HisPur Ni-NTA resin (Thermo Fisher Scientific, 88222). Nanobodies with a purity of over 90% were prepared by nickel column ion affinity chromatography. The antibody concentration was determined by SDS-PAGE, yielding the nanobodies. The SDS-PAGE electrophoresis image of the nanobodies is shown below. Figure 4 In the figure, BAC-A138 represents the nanobody BAC-A138, and M is the protein marker.

[0051] Example 5: Organic Tolerance Analysis of Nanobody BAC-A138 The nanobody BAC-A138 was diluted to the same working concentration using methanol and ethanol at different concentrations (10%, 20%, 30%, 40%, 50%) as diluents to determine the antibody-antigen binding ability. The binding ability of the antibody to the antigen without dilution with organic solvents was taken as 100%, evaluating the nanobody's tolerance to different organic solvents and different concentrations of the same organic solvent. The specific method is as follows: Add 50 µL of diluted BAC-A138 nanobody and 50 µL of PBS to the pre-packaged ELISA plate, incubate at 37 °C for 40 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 µL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37 °C for 30 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 µL of TMB substrate solution, and develop color at 37 °C in the dark for 10 min; add 50 µL of stop solution (10% H2SO4, v / v) to stop the reaction; read the absorbance at 450 nm using an ELISA reader.

[0052] like Figure 5 As shown in the diagram, the activity curves of the BAC-A138 nanobody with different proportions of methanol and ethanol as diluents demonstrate that the antigen-binding activity of the BAC-A138 nanobody remains above 75% at a methanol and ethanol concentration of 30%, and above 60% at a high concentration of 50%. Therefore, the BAC-A138 nanobody exhibits excellent resistance to organic solvents, showing minimal impact from organic solvents during sample pretreatment for actual detection, resulting in high accuracy of the detection results.

[0053] Example 6: Acid-base tolerance analysis of nanobody BAC-A138 Different pH PBS buffer solutions (pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.5, 8.0, 9.0, 10.0, 11.0, 12.0) were prepared and used as diluents to dilute the nanobody BAC-A138 to the working concentration. The bioactivity of the antibody in the system was measured. The antigen-antibody binding capacity in the PBS buffer solution at pH 7.5 was taken as 100% to evaluate the acid-base tolerance of the nanobody. The specific method is as follows: Add 50µL of diluted nanobody BAC-A138 and 50µL of PBS to the pre-packaged ELISA plate, incubate at 37℃ for 40 min, then add 100µL of HRP-labeled anti-VHH secondary antibody diluted 1:5000 with PBST, incubate at 37℃ for 30 min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100µL of TMB substrate solution, and develop color at 37℃ in the dark for 10 min; add 50µL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450nm using an ELISA reader.

[0054] Considering the complexity of the application environment and the diversity of samples, neither acidic nor alkaline environments are conducive to detection. This experiment investigated the stability of the nanobody BAC-A138 within the pH range of 2.0–12.0. Figure 6 It can be seen that BAC-A138 has excellent acid and alkali tolerance. In the pH range of 3.0 to 10.0, the acid and alkali tolerance of the nanobody is relatively good and it has high antigen binding activity (>80%).

[0055] Example 7: Time-resolved fluorescent immunochromatographic test strips for bacitracin A and bacitracin B like Figure 7 As shown, this application provides a time-resolved fluorescence immunochromatographic test strip for the simultaneous detection of bacitracin A and bacitracin B. The test strip structure includes a base plate 4, on which a conjugate release pad 1, a nitrocellulose membrane detection pad 2, and an absorbent pad 3 are sequentially disposed. The conjugate release pad 1 and the absorbent pad 3 are respectively stacked on both ends of the nitrocellulose membrane detection pad 2. The conjugate release pad 1 is coated with time-resolved fluorescent microsphere-labeled anti-bacitracin A and bacitracin B nanobodies (BAC-A138) prepared in Example 4. The nitrocellulose membrane... The test pad 2 has a T1 line, a T2 line, and a control line C. The T2 line, T1 line, and control line C are arranged sequentially and parallel to each other along the length of the nitrocellulose membrane test pad 2. The control line C is closest to the absorbent pad 3. The T1 and T2 lines are coated with the complete antigen of the measured index, and the control line C is coated with goat anti-camel IgG. The nitrocellulose membrane test pad 2 is a nitrocellulose membrane with a pore size of 5~12μm. The absorbent pad 3 is made of absorbent filter paper. The conjugate release pad 1 is made of glass cellulose membrane.

[0056] The time-resolved fluorescent microspheres are modified polystyrene microspheres with carboxyl groups as the functional group on the surface. The particle size of the polystyrene microspheres is approximately 200 nm. The interior of the time-resolved fluorescent microspheres is filled with 1% (w / w) lanthanum chelate. The labeling method of the time-resolved fluorescent microspheres is as follows: 1 mg of time-resolved fluorescent microspheres are washed twice with 200 μL of 100 mM MES buffer (pH=6) and resuspended. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to a concentration of 0.08% and 0.15%, respectively. The microspheres are activated by shaking at 30 °C for 60 min. Add 20 μL of 10% ethanol, mix well, centrifuge and wash to remove the activator, then add 200 μL of 60 mM borate buffer (pH=7.5) to resuspend the microspheres, mix well, and then add 80 μg of antibacitracin A and antibacitracin B nanobodies BAC-A138 respectively. Incubate at 37℃ with shaking for 2 h, centrifuge, resuspend with reconstitution buffer, add 50 μL of 10% BSA (mass percentage) and block for 30 min to obtain time-resolved fluorescent microsphere-labeled antibacitracin A and antibacitracin B nanobodies complexes.

[0057] The reconstitution buffer is 0.03M Tris-HCl (pH 7.5-8.5), containing 0.08% BSA, 20.4% trehalose and 0.05% NaN3, the percentages being mass percentages.

[0058] The above test strips can be prepared by the following methods: Treatment of conjugate release pad 1: Conjugate release pad 1: A glass cellulose membrane was pre-blocked by soaking it in a buffer solution containing surfactant (formulation: 100mM PB, pH 7.4, containing 2% NaCl, 2% BSA, 0.5% casein, 0.1% Tween-20 and 5% sucrose, the aforementioned percentages are mass percentages) and then dried at 37°C for 3 hours; using a Biodot instrument, antibodies labeled with time-resolved fluorescent microspheres were ultrasonically sprayed onto the glass cellulose membrane at a rate of 5uL / cm, and dried at 37°C for 3 hours to prepare conjugate release pad 1.

[0059] Treatment of nitrocellulose membrane (NC membrane) test pad 2: Using a Biodot streaking device, the diluted antigen and diluted antibody were uniformly streaked onto the NC membrane at a streaking volume of 2.0 μL / cm to prepare the T1 and T2 lines (e.g., ...). Figure 7 As shown, the coating material for line T1 is bacitracin A complete antigen, and the coating material for line T2 is bacitracin B complete antigen (specifically prepared as described in 1.1 of Example 1 above). Line C and the treated NC membrane were placed in a drying oven at 37°C and dried for 2 hours.

[0060] Assembly: The prepared conjugate release pad 1 is fixedly stacked on one end of the prepared nitrocellulose membrane detection pad 2, and the absorbent pad 3 is fixedly stacked on the other end of the nitrocellulose membrane detection pad 2. The stacking length is 1~4mm. The strips are cut to a width of 3~5mm each using a membrane cutter and fixed on the base plate 4 to obtain the time-resolved fluorescent immunochromatographic test strip that can detect bacitracin A and bacitracin B simultaneously.

[0061] Creating a standard curve: Using the test strips prepared above, the fluorescence values ​​of eight different concentrations of bacitracin A and bacitracin B standards (0.8 µg / mL, 0.4 µg / mL, 0.2 µg / mL, 0.1 µg / mL, 0.05 µg / mL, 0.02 µg / mL, 0.01 µg / mL, and 0.005 µg / mL) were measured. An S-shaped curve of the antigen-antibody reaction was plotted with the logarithm of the standard concentration on the x-axis and the T-line signal ratio on the y-axis. The curve is shown in [Figure number missing]. Figure 8 This demonstrates that the nanobody BAC-A138 prepared in this invention can simultaneously detect bacitracin A and bacitracin B, that is, it can simultaneously target and bind bacitracin A and bacitracin B.

[0062] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nanobody that simultaneously binds bacitracin A and bacitracin B, characterized in that, The nanobody can specifically bind bacitracin A and bacitracin B. The VHH chain of the nanobody includes a complementarity-determining region and a frame region, with the complementarity-determining region separated by the frame region. The complementary determination regions include CDR1, CDR2, and CDR3: The amino acid sequence of CDR1 is shown in SEQ ID No. 2, the amino acid sequence of CDR2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR3 is shown in SEQ ID No.

4. The frame region includes FR1, FR2, FR3, and FR4: The amino acid sequence of FR1 is shown in SEQ ID No. 5, the amino acid sequence of FR2 is shown in SEQ ID No. 6, the amino acid sequence of FR3 is shown in SEQ ID No. 7, and the amino acid sequence of FR4 is shown in SEQ ID No.

8.

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

1.

3. The nanobody according to claim 2, characterized in that, While retaining the ability to simultaneously bind bacitracin A and bacitracin B, the amino acid sequence of the VHH of the nanobody further includes at least one of the following: 1) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No. 1; 2) Or an amino acid sequence having 1-6 added, deleted and / or substituted amino acids compared to SEQ ID No.

1.

4. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the nanobody according to any one of claims 1-3.

5. The polynucleotide molecule according to claim 4, characterized in that, The sequence of the polynucleotide molecule is shown in SEQ ID No.

9.

6. A recombinant vector, characterized in that, It comprises the polynucleotide molecule according to any one of claims 4-5.

7. A host cell, characterized in that, It comprises the polynucleotide molecule described in any one of claims 4-5 or the recombinant vector described in claim 6.

8. A method for preparing nanobodies, characterized in that, The preparation method includes the following steps: Host cell culture and transfection: The recombinant vector containing the polynucleotide sequence of the nanobody encoding bacitracin A and / or bacitracin B is introduced into the host cell via liposome transfection, electroporation or viral infection, and then cultured in a suitable culture medium. Antibody expression and collection: After culture, the cell culture supernatant is collected, and cell debris is removed by centrifugation and filtration; Antibody purification: The nanobodies in the supernatant were initially purified using nickel column ion affinity chromatography, anti-Flag affinity chromatography column, or Protein A affinity chromatography column to finally obtain high-purity nanobodies; Antibody identification: Antibody purity was detected by SDS-PAGE.

9. A detection composition, characterized in that, The detection composition comprises a nanobody according to any one of claims 1-3, a polynucleotide molecule according to any one of claims 4-5, a recombinant vector according to claim 6, or a host cell according to claim 7.

10. Use of a nanobody according to any one of claims 1-3, a polynucleotide molecule according to any one of claims 4-5, a recombinant vector according to claim 6, or a host cell according to claim 7, characterized in that, Application in the preparation of reagents for detecting the presence or level of bacitracin A and bacitracin B in a sample.

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