A kind of methylene salicylic acid bacillus peptide-resistant nanobody and its preparation method and application

Abstract

The application belongs to the technical field of food and feed analysis and detection, and specifically provides a nanobody for detecting methylenesalicylic acid bacillus peptide, which comprises a nanobody BMD-BA19 and a nanobody BMD-CA45; the amino acid sequence of the VHH chain of the nanobody BMD-BA19 is shown as SEQ ID NO:1, and the amino acid sequence of the VHH chain of the nanobody BMD-CA45 is shown as SEQ ID NO:2. The application takes methylenesalicylic acid bacillus peptide as a research object, immunizes a llama after coupling proteins, and uses phage display technology to screen two strains of nanobodies against methylenesalicylic acid bacillus peptide, thereby providing materials for establishing a detection method of methylenesalicylic acid bacillus peptide.

Description

Technical Field

[0001] This invention belongs to the field of food and feed analysis and testing technology, specifically a nanobody against methylene salicylic acid bacitracin, its preparation method and application. Background Technology

[0002] Cameloids possess a naturally occurring antibody in their blood that lacks the light chain and contains only the heavy chain. This antibody fragment, approximately 12-15 kDa in size and only one-tenth the size of a traditional IgG antibody, is called a nanobody. Nanobodies offer advantages such as simple structure, high antigen specificity, stable physicochemical properties, easy availability, and the ability to be cultured and expanded using microorganisms.

[0003] Bacitracin is a product obtained through fermentation from a strain of Bacillus licheniformis, an antibacterial bacterium isolated by Johnson et al. at Columbia University during the treatment of tibial trauma cases. Bacitracin has an antibacterial spectrum similar to penicillin, exhibiting strong antibacterial activity against Gram-positive bacteria and also effective against some Gram-negative bacteria, actinomycetes, and spirochetes. Bacitracin promotes animal growth, improves feed conversion ratio, and prevents and treats various intestinal diseases in animals, and is widely used in livestock and poultry farming and feed production. Bacitracin is unstable and requires complexation with metal ions such as zinc ions or methylene disalicylic acid to increase its stability. Bacitracin zinc can effectively prevent animal mortality and liver lesions caused by high doses of Clostridium perfringens in feed. Methylene salicylic acid bacitracin, due to its solubility in the slightly acidic environment of the small intestine, can better exert its antibacterial and growth-promoting effects than bacitracin zinc.

[0004] Bacitracin, due to its sensitizing, nephrotoxic, and neurotoxic properties, can leave residues that enter the human body through milk or animal tissues, posing a health risk. However, the greatest threat is antibiotic resistance caused by the emergence and development of drug-resistant strains. If the trend of increasing antibiotic resistance continues, it can lead to simple infections that are no longer treatable. According to GB 31650-2019, the maximum residue limit for bacitracin in edible animal tissues, milk, and eggs is 500 μg / kg.

[0005] To better regulate the use of bacitracin preparations as feed additives, the detection of bacitracin zinc and bacitracin methylenedisalicylate in feed has become a hot topic. A review of domestic and international literature currently only reveals methods for detecting bacitracin zinc in milk and feed. These methods include precision instrument methods and immunoassays. Immunoassays primarily use traditional mouse monoclonal and polyclonal antibodies as recognition elements, resulting in complex, time-consuming, and labor-intensive preparation processes, significant batch-to-batch variations, and stringent storage conditions. There are no reports on bacitracin methylenedisalicylate (BMD), and given the significant differences in physicochemical properties between bacitracin zinc and BMD, the detection of bacitracin zinc has limited reference value. Summary of the Invention

[0006] In view of the fact that there is currently no accurate detection method for methylene salicylic acid bacitracin, this invention provides a nanobody against methylene salicylic acid bacitracin, its preparation method and application.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] A nanobody against methylene salicylic acid bacitracin, comprising nanobody BMD-BA19 and nanobody BMD-CA45.

[0009] The anti-methylene salicylic acid bacitracin nanobody provided by this invention is a nanobody targeting different antigenic epitopes of methylene salicylic acid bacitracin, and is a typical VHH chain containing only the heavy chain and lacking the light chain.

[0010] The present invention provides the VHH chain of the anti-methylene salicylic acid bacitracin nanobody. The amino acid sequence of the VHH chain corresponding to nanobody BMD-BA19 is shown in SEQ ID NO:1, and the amino acid sequence of the VHH chain corresponding to nanobody BMD-CA45 is shown in SEQ ID NO:2.

[0011] Furthermore, the nanobody BMD-BA19 includes at least one of monomeric nanobody and multivalent nanobody; the nanobody BMD-CA45 includes at least one of monomeric nanobody and multivalent nanobody.

[0012] The multivalent nanobody is a bivalent nanobody, and the structure of the bivalent nanobody is composed of two monovalent nanobodies linked by a linker to an anti-rabbit serum albumin nanobody. The amino acid sequence of the anti-rabbit serum albumin nanobody is shown in SEQ ID NO.5.

[0013] The linker in the divalent nanobody is an amino acid sequence, preferably AAA, GGGGSGGGS or GGGGSGGGGSGGGS, and more preferably GGGGSGGGS.

[0014] Further, the amino acid sequences of SEQ ID NO:1 and / or SEQ ID NO:2 are replaced by amino acid sequences selected from the following a) to d):

[0015] a) The amino acid sequence is at least about 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2;

[0016] b) The amino acid sequence encoded by the nucleotide sequence hybridizes with the nucleotide sequence encoding the amino acid shown in SEQ ID NO:1 and / or SEQ ID NO:2;

[0017] The above nucleotide sequence hybridization refers to DNA or RNA sequences that can bind to the parental strand, not just one of them.

[0018] c) The amino acid sequence differs from the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2 by no more than 5, 4, 3, 2 or no more than 1 amino acid;

[0019] d) The amino acid sequence has the amino acid sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2, including substitutions, deletions and / or insertions of 1 to 5 amino acid residues.

[0020] A nucleic acid molecule, wherein the nucleic acid molecule encodes the VHH chain of the above-mentioned nanobody BMD-BA19 and the VHH chain of the above-mentioned nanobody BMD-CA45, or the nucleic acid molecule encodes the above-mentioned nanobody BMD-BA19 and the above-mentioned nanobody BMD-CA45;

[0021] Preferably, the nucleotide sequence of the nucleic acid molecule comprises at least one of the following 1) to 3):

[0022] 1) The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO:3 and SEQ ID NO:4;

[0023] 2) A nucleotide sequence having at least about 95%, 96%, 97%, 98%, or at least 99% identity with the nucleotides shown in SEQ ID NO:3 and SEQ ID NO:4;

[0024] 3) The nucleotide sequences encoding the amino acid sequences a) to d) above are specifically as follows:

[0025] Nucleotide sequences that hybridize with the nucleotides shown in SEQ ID NO:3 and SEQ ID NO:4, where the nucleotide sequence hybridizing refers to a DNA or RNA sequence that can bind to the parental strand, and not one of them;

[0026] Its amino acid sequence is at least about 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2;

[0027] The nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence differs from the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 by no more than 5, 4, 3, 2 or 1 amino acid residues.

[0028] A nucleotide sequence encoding an amino acid sequence, the differences of which from the sequences of SEQ ID NO:1 and SEQ ID NO:2 include substitutions, deletions and / or insertions of 1 to 5 amino acid residues.

[0029] A biomaterial comprising any one of A) to C):

[0030] A) A recombinant vector containing two nucleic acid molecules: the VHH chain of nanobody BMD-BA19 and / or the VHH chain of nanobody BMD-CA45.

[0031] B) Recombinant microorganisms containing the recombinant vector in A);

[0032] C) Transgenic animal cell lines containing the recombinant vector in A).

[0033] A method for preparing anti-methylene salicylic acid bacitracin nanobody, the method comprising the following steps:

[0034] Step 1: Insert the two VHH chains or the bivalent nanobody encoding the above-mentioned anti-methylene salicylic acid bacitracin nanobody (nanobody BMD-BA19, nanobody BMD-CA45) into the cloning vector to construct the recombinant vector.

[0035] The vector includes, but is not limited to: viral vectors, such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors; and non-viral vectors, such as plasmids and transposon vectors. Preferably, the vector is an expression vector. The vector is preferably a plasmid vector, and more preferably the pPICZαA vector.

[0036] Step 2: The recombinant vector constructed in Step 1 is introduced into recipient cells to obtain transgenic cells. The transgenic cells contain nucleic acids encoding any of the aforementioned nanobodies or bivalent nanobodies and a vector. Preferably, the cells are host cells containing the aforementioned expression vector or nucleic acid.

[0037] The host cells include, but are not limited to, mammalian cells, insect cells, plant cells, fungal cells, and prokaryotic cells. Examples of host cells include: *Escherichia coli*, *Streptomyces*, bacterial cells of *Salmonella typhimurium*, fungal cells such as yeast, insect cells of *Drosophila S2* or *Sf9*, and animal cells of CHO, COS7, and 293. Preferably, the host cell provided by this invention for expressing the VHH chain of the antibody binding to methylene salicylate bacitracin is *Pichia pastoris* cells.

[0038] Step 3: Cultivate the transgenic cells to obtain the VHH chain of the anti-methylene salicylic acid bacitracin nanobody or the anti-methylene salicylic acid bacitracin nanobody.

[0039] Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, amino acid sequence expression is induced using suitable methods (such as temperature adjustment or chemical induction).

[0040] A method for screening VHH chains or anti-methylene salicylic acid bacitracin nanobodies, the method comprising the following steps:

[0041] The purified bacitracin methylenesalicylate complete antigen was emulsified with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant, and then administered via subcutaneous injection at multiple sites in the neck to immunize alpacas 6-8 times, once a week. After the 8th immunization, peripheral blood of alpacas was collected, and peripheral blood lymphocytes were isolated to construct a VHH phage antibody library against bacitracin methylenesalicylate. The time point for constructing the VHH phage antibody library against bacitracin methylenesalicylate was when the antibody titer in the alpaca serum after immunization was detected by indirect ELISA, and the antibody titer against bacitracin methylenesalicylate in the alpaca serum was not less than 1:64000.

[0042] The VHH phage antibody library was screened for binding and competitive activity, and sequence difference analysis and characterization of candidate antibodies were performed to obtain two nanobodies targeting different epitopes of methylene salicylic acid bacitracin.

[0043] A detection composition comprising the VHH chain of the aforementioned anti-methylene salicylic acid bacitracin nanobody or the aforementioned anti-methylene salicylic acid bacitracin nanobody. Specifically, an ELISA or test strip detection composition comprising nanobody BMD-BA19 and / or nanobody BMD-CA45.

[0044] A recombinant protein comprising the sequence of the VHH chain of the aforementioned anti-methylene salicylic acid bacitracin nanobody or the sequence of the aforementioned anti-methylene salicylic acid bacitracin nanobody; and a tag sequence to assist in expression and / or purification.

[0045] Furthermore, the tag sequence includes a 6His tag and / or a HA tag.

[0046] The use of the above-mentioned anti-methylene salicylic acid bacitracin nanobody VHH chain, or the above-mentioned anti-methylene salicylic acid bacitracin nanobody, for the preparation of reagents, detection plates, test strips or kits for detecting methylene salicylic acid bacitracin molecules.

[0047] Furthermore, the test strip is a competitive time-resolved fluorescence test strip with a detection limit of 0.1 μg / mL;

[0048] The kit is a competitive time-resolved fluorescence detection kit with a detection limit of 0.1 μg / mL. The kit is a combination of test strip, pretreatment solution, and dropper.

[0049] The present invention has the following beneficial effects:

[0050] This invention provides a nanobody against methylene salicylic acid bacitracin, its preparation method, and its application. This invention uses methylene salicylic acid bacitracin as the research object, conjugates it with protein, and then immunizes alpacas. Two nanobodies against methylene salicylic acid bacitracin are obtained by screening using phage display technology.

[0051] This invention provides materials for establishing a detection method for methylene salicylic acid bacitracin through the preparation of the anti-methylene salicylic acid bacitracin nanobody. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the SDS-PAGE electrophoresis results of the methylene salicylic acid bacitracin-conjugated BSA complete antigen prepared in Example 1 of the present invention, where: M is the marker, BSA is the protein band of the bovine serum albumin sample, and BMD-BSA is the protein band of the methylene salicylic acid bacitracin-conjugated BSA complete antigen sample.

[0053] Figure 2This is a schematic diagram of the SDS-PAGE electrophoresis results of the methylene salicylic acid bacitracin-conjugated OVA complete antigen prepared in Example 1 of the present invention, where: M is the marker, OVA is the protein band of the chicken egg white protein sample, and BMD-OVA is the protein band of the methylene salicylic acid bacitracin-conjugated OVA complete antigen sample.

[0054] Figure 3 The liquid chromatography result is the detection result of the purity of the methylene salicylic acid bacitracin conjugated with BSA complete antigen prepared in Example 1 of the present invention.

[0055] Figure 4 This is a schematic diagram showing the results of detecting the binding activity of four candidate nanobodies using the ELISA method in Example 4 of the present invention.

[0056] Figure 5 This is a schematic diagram showing the results of the ELISA method for detecting the competitive activity of four candidate nanobodies in Example 4 of the present invention;

[0057] Figure 6 This is a schematic diagram of the structure of the bivalent nanobody in Embodiment 5 of the present invention;

[0058] Figure 7 This is a schematic diagram of the SDS-PAGE electrophoresis results of the purified bivalent nanobody BMD-BA19D and bivalent nanobody BMD-CA45D in Example 5 of the present invention, where M represents the marker.

[0059] Figure 8 This is a schematic diagram of the structure of the time-resolved fluorescence immunochromatographic test strip of Embodiment 6 of the present invention, wherein: 1 is a conjugate release pad; 2 is a nitrocellulose membrane detection pad; 3 is an absorbent pad; 4 is a base plate; 5 is a sample absorption pad; T is a detection line; and C is a control line.

[0060] Figure 9 for Figure 8 A top-down view;

[0061] Figure 10 This is a time-resolved fluorescence standard curve for detecting methylene salicylic acid bacitracin in Example 6 of the present invention. Detailed Implementation

[0062] 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. Furthermore, the reagents used in the present invention can be obtained by purchasing commercially available raw materials or by conventional preparation methods. Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as those skilled in the art.

[0063] The abbreviations used in the specific embodiments are explained below:

[0064] BSA: Bovine serum albumin; OVA: Chicken oocyte albumin; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; VHH chain: Heavy chain variable region; Nanobody: A naturally occurring antibody lacking a light chain, found in the peripheral blood of camels; Monovalent nanobody: A single nanobody obtained through screening; Bivalent nanobody: Composed of two monovalent nanobodies linked to an anti-mouse / rabbit serum protein nanobody via a linker; Multivalent nanobody: Composed of at least one monovalent nanobody linked to an anti-mouse / rabbit serum protein nanobody via a linker; DEPC water: Ultrapure water treated with DEPC (diethyl pyrocarbonate) and autoclaved, free of impurities such as RNA, DNA, and protein; PBST: Phosphate-buffered saline (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.

[0065] Example 1: Generation of anti-methylene salicylic acid bacitracin nanobody

[0066] Preparation of methylene salicylic acid bacitracin conjugated with BSA complete antigen

[0067] 10 mg of BSA protein was dissolved in 10 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 methylene salicylate (Greencon Biochemical Co., Ltd., CAS: 8027-21-2) was dissolved in 1 mL of 0.05 mol / L carbonate buffer (CBS buffer, concentration 0.1 mol / L, pH 9.6). 60 µL of the bacitracin methylene salicylate solution was added to 1 mL of the above activation solution, and the mixture was vertically mixed overnight at 4°C. The mixture was dialyzed against phosphate buffer (0.01 mol / L, pH 7.4, volume 5-10 L) for 3 days to obtain the bacitracin methylene salicylate-conjugated BSA complete antigen.

[0068] The purity of the methylene salicylic acid bacitracin conjugated with BSA complete antigen was confirmed to be greater than 80% by liquid chromatography. The chromatographic column used for liquid chromatography was a Zenix-C SEC-300 7.8×300mm, the mobile phase was 0.15M PB + NaCl pH 6.0, and the flow rate was 0.8 mL / min. The detection chromatogram results are shown below. Figure 3 As shown, the specific retention time of the methylene salicylic acid bacitracin-conjugated BSA complete antigen is 8.8 min. Furthermore, the SDS-PAGE electrophoresis results of the methylene salicylic acid bacitracin-conjugated BSA complete antigen are as follows: Figure 1 As shown. The prepared methylene salicylic acid bacitracin-conjugated BSA complete antigen was stored in a -20°C freezer.

[0069] Preparation of methylene salicylic acid bacitracin conjugated with OVA complete antigen

[0070] 10 mg of OVA protein was dissolved in 0.1 mol / L MES buffer, followed by the addition of 5 mg EDC and 3 mg NHS. The mixture was vertically mixed for 15 min to prepare the activation solution. 10 mg of bacitracin methylenesalicylate was dissolved in 1 mL of 0.05 mol / L carbonate solution, and the activation solution was added. The mixture was vertically mixed overnight at 4°C. The mixture was dialyzed against 0.01 mol / L phosphate buffer for 3 days to obtain the complete OVA antigen conjugated with bacitracin methylenesalicylate. The concentration of the complete antigen was confirmed to be greater than 80% by SDS-PAGE. The purity was determined using the same liquid chromatography method as for the bacitracin methylenesalicylate conjugated with BSA complete antigen. The antigen was stored at -20°C. The specific SDS-PAGE electrophoresis results of the complete OVA antigen conjugated with bacitracin methylenesalicylate are shown below. Figure 2 As shown.

[0071] immunity

[0072] The BSA complete antigen, conjugated with the aforementioned methylene salicylic acid bacitracin, was mixed with adjuvant at a 1:1 volume ratio, emulsified, and then used to immunize alpacas. The first immunization used complete Freund's adjuvant, while subsequent immunizations used incomplete Freund's adjuvant, once a week for a total of 8 immunizations. Blood titers were measured starting from the second immunization. Alpaca blood was incubated at 4°C for 2 hours, then centrifuged at 8000 rpm for 5 minutes. The supernatant was collected, and the antiserum titer was determined using an enzyme-linked immunosorbent assay (ELISA). Once the serum titer was not lower than 1:64000, blood was collected from the alpaca's neck, and lymphocytes were isolated for subsequent experiments.

[0073] Establishment of VHH phage antibody library

[0074] Alpaca peripheral blood lymphocytes (PBMCs) were isolated using peripheral blood lymphocyte separation medium. 5 mL of the medium was added to a 15 mL centrifuge tube. Blood from the neck obtained after immunization was carefully aspirated using a pipette and added to the surface of the separation medium. The tube was centrifuged at 450-650g for 20-30 minutes. After centrifugation, the second layer of ring-shaped milky-white lymphocytes was carefully transferred to another centrifuge tube using a pipette to obtain alpaca peripheral blood lymphocytes. The isolated PBMCs were used for total RNA extraction.

[0075] The method for extracting total RNA was the TRZOL manual extraction method, and the specific operation was as follows: Trizol solution was added to PBMCs, and after complete lysis, chloroform was added and mixed thoroughly. After incubation at room temperature for 20 min, centrifugation was performed at 4°C and 12,000 rpm for 20 min. The supernatant was collected, and an equal volume of isopropanol was added. After incubation at room temperature for 20 min, centrifugation was performed at 4°C and 12,000 rpm for 20 min. The supernatant was discarded, and the sample was washed twice with 75% ethanol. After centrifugation at 4°C and 12,000 rpm for 5 min, the sample was discarded, and the precipitate was retained. After air drying at room temperature, the precipitate was resuspended in DEPC water to obtain total RNA.

[0076] The obtained total RNA was reverse transcribed into cDNA. Using the reverse-transcribed cDNA as a template, PCR primers were designed based on the upstream and downstream sequences of the VHH gene (the VHH gene contains conserved regions whose sequences are known). Nested PCR was used to amplify the VHH gene. The first round of primers were:

[0077] CALL001 (forward primer): GTCCTGGCTGCTCTTCTACAAGG

[0078] CALL002 (reverse primer): GGTACGTGCTGTTGAACTGTTCC.

[0079] Using the recovered product from the first round of PCR (the band around 750 bp) as a template, the VHH gene was amplified using the primers for the second round of amplification. The primers for the second round of amplification were:

[0080] VHH-F (forward primer): CATGCCATGACTGTGGCCCAGGCGGCCCAGGTGCAGCTCGTGGAGTC,

[0081] VHH-R (reverse primer): CATGCCATGACTCGCGGCCGGCCTGGCCGGAGACGGTGACC.

[0082] The final result is a specific band with a molecular weight of approximately 400 bp. The specific band is then recovered by gel cutting, which is the VHH fragment.

[0083] The obtained VHH fragment and pComb 3X linearized phage vector were ligated using homologous recombination, following the instructions of a non-ligase-dependent one-step multi-fragment cloning kit (Norvoza, catalog number C113-01). The VHH phage antibody library was constructed according to the method of Els Pardon et al. (Nature Protocols, VOL 9 NO.3, 2014), with a phage library size of 3.5*10⁻⁶ cells / mL constructed using alpacas. 8 The VHH phage antibody library was established.

[0084] Example 2: Screening of VHH phage antibody library

[0085] Positive clones that specifically bind to the complete antigen of the VHH phage antibody library constructed in Example 1 and the methylene salicylic acid bacitracin-conjugated OVA were screened using the following two methods.

[0086] 1. Plate screening: 96-well plates were coated with 0.2 μg / well of the methylene salicylic acid bacitracin-conjugated OVA complete antigen and incubated overnight at 4°C. The next day, 100 µL of 2% BSA was added to each well to block the plates for 1 h. The VHH phage antibody library prepared in Example 1 was added and incubated for 2 h. After washing 4-10 times, positive clones that specifically bound to the methylene salicylic acid bacitracin-conjugated OVA complete antigen were eluted with elution buffer (pH 2.2).

[0087] 2. Magnetic bead screening: Sulfo-NHS-Biotin was added to the methylene salicylic acid bacitracin-conjugated OVA complete antigen for biotinylation and reacted at room temperature for 30 minutes. The conjugate was separated from the reactants by gel filtration. The biotinylated methylene salicylic acid bacitracin-conjugated OVA complete antigen was then bound to Thermo's magnetic beads and incubated with the VHH phage antibody library prepared in Example 1 to obtain positive clones that specifically bind to the methylene salicylic acid bacitracin-conjugated OVA complete antigen.

[0088] By using the two screening methods described above, magnetic bead screening can overcome the problem of antigen epitope masking in plate screening, thereby obtaining positive clones that completely and specifically bind to the OVA conjugated with methylene salicylic acid bacitracin.

[0089] Example 3: Identification of sequencing clones by detecting binding and competitive activity using ELISA.

[0090] The ELISA detection method is as follows:

[0091] The positive phages obtained from Example 2 were plated, and single clones were picked and directly induced to express by IPTG. The expression products were extracted and detected as follows: 0.2 μg / mL methylene salicylic acid bacitracin conjugated with OVA complete antigen was coated with 0.1 mol / L pH 9.6 CBS buffer at 100 μl / well overnight at 4°C; the wells were blocked with 3% skim milk powder at 37°C for 1 h; 100 μl of each positive clone prepared in Example 2 diluted with PBST was added to each well and incubated at 37°C for 1 h; then, GoatpAb to Llama HRP secondary antibody was added and incubated at 37°C for 1 h. After 10 min of color development, the OD450 value was read on the microplate reader.

[0092] The following is a method for detecting competitive activity using ELISA:

[0093] 0.1 mol / L pH 9.6 CBS buffer was used to coat 0.5 μg / mL methylene salicylic acid bacitracin-conjugated OVA complete antigen, 100 μL / well, overnight at 4°C; blocked with 3% skim milk powder at room temperature for 1 h; simultaneously, 100 μL each of methylene salicylic acid bacitracin (0.2 μg / mL) and PBST diluted with PBST prepared in Example 2 were incubated at 37°C for 1 h, then added to the blocked ELISA plate and incubated at 37°C for another 1 h; then streptomycin / HRP was added and incubated at 37°C for 1 h; after 10 min of color development, the OD450 value was read on the microplate reader.

[0094] Ultimately, 90 positive phages with binding activity and 25 positive phages with competitive activity were screened, and the sequences of the 25 positive phages were determined.

[0095] Sequence determination and expression of candidate nanobodies

[0096] PCR identification was performed using VHH-F and VHH-R primers (primer sequences are shown in Example 1). The target fragment size was approximately 400 bp. The 25 positive phages were sequenced to determine the diversity of nanobodies.

[0097] The amino acid sequences of 25 positive phages with competitive activity were compared. After sequence analysis, four phage clone plasmids with significant sequence differences were obtained (CA represents plate screening, BA represents magnetic bead screening). The specific amino acid sequence information of the four clones with blocking activity is shown in Table 1 below.

[0098] Table 1. Cloned amino acid sequences with blocking activity

[0099]

[0100] The four phage clones were transformed into BL21 competent cells (TAKARA, catalog number 9126). Single clones were selected for culture and induced to express the cells with 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 affinity chromatography (named BMD-CA17, BMD-CA45, BMD-BA12, and BMD-BA19, respectively; the prefix BMD stands for bacitracin methylene salicylate). The antibody concentration was determined by SDS-PAGE.

[0101] Example 4 Characterization of candidate nanobodies

[0102] ELISA detection of the binding activity of candidate nanobodies to the methylene salicylic acid bacitracin complete antigen

[0103] The binding activity of the four candidate nanobodies purified in Example 3 was tested to determine the binding sensitivity of the candidate nanobodies to the methylene salicylic acid bacitracin complete antigen.

[0104] The ELISA method for detecting the binding activity of the candidate antibody to the methylene salicylic acid bacitracin complete antigen is the same as the ELISA method described in Example 1. The detection results are as follows: Figure 4 The four nanobodies, BMD-CA17, BMD-CA45, BMD-BA12, and BMD-BA19, all exhibited good binding activity.

[0105] ELISA detection of the competitive activity of candidate nanobodies bound to methylene salicylic acid bacitracin against complete antigens

[0106] The purified candidate nanobodies were subjected to competitive activity assays to determine their competitive sensitivity in binding to the complete antigen. The competitive activity assay was performed using the ELISA method described in Example 1, and the results are as follows: Figure 5 BMD-CA45 and BMD-BA19 exhibit relatively good competitive activity.

[0107] Example 5 Expression of nanobodies in Pichia pastoris

[0108] This embodiment demonstrates the expression of a bivalent nanobody. The structure of the bivalent nanobody is shown in the attached figure. Figure 6It consists of two monovalent nanobodies linked by a linker to an anti-rabbit serum albumin nanobodily antibody (anti-RSA sdAb). The amino acid sequence of the anti-RSA sdAb is shown in SEQ ID NO.5, and the specific sequence information of SEQ ID NO.5 is as follows:

[0109] EVQLVESGGGLVQYADSVKGRFTISRDNAKTTLYLQMNSLRPEDPGNSLRLSCAASGSSFGMSWFTFVRDTLTAVYYCTIGGSLSRQAPGKGLEWVSQGTLVSISGSGSSSTVSS

[0110] The connector sequence is GGGGSGGGS

[0111] Bivalent nanobodies were constructed using BMD-BA19 and BMD-CA45 sequences, respectively, and are referred to as BMD-BA19D (structure: BMD-BA19-linker-anti-RSA sdAb-linker-BMD-BA19) and BMD-CA45D (structure: BMD-CA45-linker-anti-RSA sdAb-linker-BMD-CA45).

[0112] After full gene synthesis, the plasmids were constructed into the pPICZαA vector. The plasmids were linearized with SalI restriction endonuclease and then electroporated into Pichia pastoris cells. Cells were cultured in YPD medium for 22 hours, followed by methanol induction for 3–5 days. The expression supernatant was purified by affinity chromatography using a nickel column (6His tag purification) or an anti-HA purification system (HA tag purification), and the purity was assessed by SDS-PAGE protein electrophoresis.

[0113] The SDS-PAGE protein electrophoresis detection method is as follows: Pipette 1 mL of fermentation broth into a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 2 min; take 20 μL of the supernatant and add 20 μL of 10x non-reducing loading buffer, heat at 80℃ for 5 min, and then perform electrophoresis on the sample using an 8%–16% polyacrylamide gel. The electrophoresis results are as follows: Figure 7 The results showed that the bivalent nanobody BMD-BA19D and bivalent nanobody BMD-CA45D antibodies had high purity in yeast supernatant, which facilitates subsequent large-scale purification.

[0114] Example 6: Time-Resolved Fluorescent Immunochromatographic Test Strip for Methylene Salicylic Acid Bacitracin

[0115] like Figure 8As shown, this application provides a time-resolved fluorescence immunochromatographic test strip for the specific detection of methylene salicylic acid bacitracin. The test strip structure includes a base plate 4, on which a sample absorption pad, a conjugate release pad 1, a nitrocellulose membrane detection pad 2, and an absorbent pad 3 are sequentially disposed. The sample absorption pad, the conjugate release pad 1, and the absorbent pad 3 are respectively stacked on both ends of the nitrocellulose membrane detection pad 3. The sample absorption pad and the conjugate release pad 1 are coated with the anti-methylene salicylic acid bacitracin nanobody (BMD-BA19 or BMD) prepared in Example 3, which is labeled with time-resolved fluorescent microspheres. -CA45) or the multivalent nanobody prepared in Example 5; the nitrocellulose membrane detection pad 2 is provided with a detection line T line and a control line C line, the T line and the control line C line are arranged sequentially along the length direction of the nitrocellulose membrane detection pad and are parallel to each other, the control line C line is closest to the absorbent pad 3, the detection line T line is coated with the complete antigen of the measured index, the control line C line is coated with goat anti-camel IgG, the nitrocellulose membrane detection pad is a nitrocellulose membrane and is a porous membrane with a pore size of 5~12μm; the absorbent pad is made of absorbent filter paper; the sample absorption pad and the conjugate release pad are made of glass cellulose membrane.

[0116] The time-resolved fluorescent microspheres are modified polystyrene microspheres with carboxyl groups as the functional groups on the surface of the polystyrene microspheres and a particle size of about 200 nm. The time-resolved fluorescent microspheres are filled with 1% (w / w) lanthanum chelates.

[0117] The method for labeling 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 make their concentrations 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 anti-methylene salicylic acid bacitracin nanobody or multivalent nanobody (in this example, the nanobody BMD-BA19 prepared in Example 3), label at 37°C with shaking for 2 h, centrifuge, resuspend with reconstitution buffer, add 50 μL of 10% BSA (mass percentage) to block for 30 min, and obtain time-resolved fluorescent microsphere-labeled anti-methylene salicylic acid bacitracin nanobody or multivalent nanobody complex.

[0118] 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.

[0119] The above test strips can be prepared by the following methods:

[0120] Treatment of conjugate release pads and sample absorption pads:

[0121] Conjugate release pad: 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 the conjugate release pad.

[0122] Sample absorption pad: Soak the sample absorption pad (glass cellulose membrane) in 0.1 mol / L phosphate buffer containing 0.5% bovine serum albumin (percentage is by mass fraction), pH 7.2 for 2 hours, and then dry it at 37°C for 2 hours.

[0123] Nitrocellulose membrane (NC membrane) treatment:

[0124] The NC membrane was attached to the designated position on the substrate. The methylene salicylic acid bacitracin-conjugated OVA complete antigen was diluted to 200 μg / mL with 0.01 mol / L PBS buffer (pH 7.4) to prepare the T line. The goat anti-camel IgG antibody was diluted to 500 μg / mL with 0.01 mol / L PBS buffer (pH 7.4) to prepare the C line. The diluted complete antigen and diluted antibody were uniformly streaked onto the NC membrane using a Biodot streaking device at a streaking volume of 2.0 μL / cm to prepare the T line and C line. The streaked NC membrane was placed in a 37°C drying oven and dried for 2 hours.

[0125] Assembly:

[0126] The prepared sample absorption pad and conjugate release pad are fixedly stacked on one end of the prepared nitrocellulose membrane detection pad, and the absorbent pad is fixedly stacked on the other end of the nitrocellulose membrane detection pad. The stacking length is 1-4 mm. The strips are then cut to a width of 3-5 mm using a membrane cutter to obtain a time-resolved fluorescent immunochromatographic test strip that specifically detects methylene salicylic acid bacitracin. The specific structure of the time-resolved fluorescent immunochromatographic test strip is as follows: Figure 8 As shown, where Figure 8 The markings are as follows: 1 for conjugate release pad; 2 for nitrocellulose membrane detection pad; 3 for absorbent pad; 4 for base plate; 5 for sample absorption pad; T for test line; C for control line; and the top view of the corresponding test strip is shown below. Figure 9 As shown.

[0127] Meanwhile, by combining the test strip of this embodiment with the pretreatment solution (extraction solution before sample detection) and the dropper (dropper used to add the sample for detection), a kit can be prepared.

[0128] Creating a standard curve:

[0129] The fluorescence values ​​of six different concentrations of methylene salicylic acid bacitracin standards were determined according to the operating procedures (a: 0 µg / mL, b: 0.1 µg / mL, c: 0.5 µg / mL, d: 2 µg / mL, e: 10 µg / mL, f: 50 µg / mL, g: 100 µg / mL). The logarithm of the standard concentration was plotted on the x-axis, and the T / C fluorescence intensity was plotted on the y-axis. The results were processed using a double logarithmic mathematical model, Log~Log function, and the linear regression equation was y = -0.2615x + 0.522, R0. 2 = 0.9923, see standard curve. Figure 10 .

Claims

1. A nanobody against methylene salicylic acid bacitracin, characterized in that, Including nanobody BMD-BA19 and / or nanobody BMD-CA45; The amino acid sequence of the VHH chain of the nanobody BMD-BA19 is shown in SEQ ID NO:1, and the amino acid sequence of the VHH chain of the nanobody BMD-CA45 is shown in SEQ ID NO:

2.

2. The nanobody against methylene salicylic acid bacitracin according to claim 1, characterized in that, The nanobody BMD-BA19 includes at least one of monovalent nanobody and multivalent nanobody; the nanobody BMD-CA45 includes at least one of monovalent nanobody and multivalent nanobody. The multivalent nanobody is a bivalent nanobody; The structure of the bivalent nanobody consists of two monovalent nanobodies linked by a linker to an anti-rabbit serum albumin nanobody anti-RSA sdAb, wherein the bivalent nanobody is BMD-BA19D or BMD-CA45D. The structure of the bivalent nanobody BMD-BA19D is BMD-BA19-linker-anti-RSA sdAb-linker-BMD-BA19, and the structure of the bivalent nanobody BMD-CA45D is BMD-CA45-linker-anti-RSA sdAb-linker-BMD-CA45. The amino acid sequence of the anti-rabbit serum albumin nanobody anti-RSA sdAb is shown in SEQ ID NO.5; The amino acid sequence of the linker is GGGGSGGGS.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the VHH chain of the nanobody of claim 1; The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO:3 and SEQ ID NO:

4.

4. A biomaterial, characterized in that, The biomaterial comprises at least one of the following A) to B): A) A recombinant vector containing two nucleic acid molecules: the VHH chain of nanobody BMD-BA19 and / or the VHH chain of nanobody BMD-CA45. B) Recombinant microorganisms containing the recombinant vector in A).

5. A method for preparing an anti-methylene salicylic acid bacitracin nanobody, characterized in that, The preparation method includes the following steps: Step 1: Insert the two VHH chains encoding the nanobody BMD-BA19 or BMD-CA45 of claim 1, or the bivalent nanobody of claim 2, into the cloning vector to construct a recombinant vector; Step 2: The recombinant vector constructed in Step 1 is introduced into recipient cells to obtain transgenic cells, which contain nucleic acids encoding the nanobody of claim 1 or the bivalent nanobody of claim 2 and a vector; Step 3: Culture the transgenic cells to obtain the VHH chain of the anti-methylene salicylic acid bacitracin nanobody or the anti-methylene salicylic acid bacitracin nanobody; The cloning vector mentioned in step one is the pPICZαA vector, and the recipient cell mentioned in step two is Pichia pastoris cell.

6. A detection composition, characterized in that, The detection composition comprises the anti-methylene salicylic acid bacitracin nanobody as described in any one of claims 1-2.

7. A recombinant protein, characterized in that, The recombinant protein comprises the anti-methylene salicylic acid bacitracin nanobody as described in any one of claims 1-2, and a tag sequence to assist in expression and / or purification.

8. The recombinant protein according to claim 7, characterized in that, The tag sequence includes a 6His tag and / or a HA tag.

9. The use of the anti-methylene salicylic acid bacitracin nanobody as described in any one of claims 1-2, characterized in that, This is used to prepare test strips for detecting methylene salicylic acid bacitracin molecules.

10. The use of the anti-methylene salicylic acid bacitracin nanobody according to claim 9, characterized in that, The test strip is a competitive time-resolved fluorescence test strip with a detection limit of 0.1 μg / mL.

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