Nano antibody Nb DX-6F capable of specifically recognizing doxycycline and application of nano antibody Nb DX-6F

Abstract

The invention discloses a nano antibody Nb DX-6F capable of specifically recognizing doxycycline and an application of the nano antibody Nb DX-6F. The amino acid sequence of the VHH of the nano antibody Nb DX-6F is as shown in SEQ ID No.1, and the nano antibody has excellent organic solvent tolerance and acid and alkali tolerance. An ELISA detection method is established based on the nano antibody, the detection range of doxycycline by the method is 0.17-14.25 ng / mL, the half inhibitory concentration (IC50) is 1.34 ng / mL, the lowest limit of detection (LOD) is 0.05 ng / mL, and the detection sensitivity is high. Therefore, when the nano antibody Nb DX-6F is used for detecting doxycycline residues in an actual sample, the detection result is good in stability, high in sensitivity and high in accuracy, and the nano antibody has extremely high application value in detection of doxycycline or preparation of doxycycline detection products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nano antibody NbDX-6F that specifically recognizes doxycycline and applications thereof. Background Art

[0002] Doxycycline, also known as doxycycline and deoxytetracycline, is a semi-synthetic tetracycline antibiotic. It boasts high antimicrobial activity, strong tissue penetration, wide distribution, high bioavailability, and a long half-life, making it widely used in veterinary clinics and aquaculture. With the increasing intensification of aquaculture in China and a significant increase in stocking density, the abuse of veterinary drugs has become increasingly serious. The misuse of doxycycline not only has mixed toxic effects on the ecological environment but can also be transmitted to humans through the food chain. Long-term consumption of livestock and poultry products containing excessive doxycycline residues can cause a variety of acute and chronic reactions in humans, including gastric ulcers, esophagitis, gastritis, and glossitis. It can also induce cell mutations leading to malformations and carcinogenesis, and even induce the development of drug-resistant strains. This poses significant health risks and seriously impacts public health. Therefore, it is necessary to develop more efficient and accurate detection methods to monitor doxycycline residues in aquatic products.

[0003] Currently, there are two methods for detecting doxycycline residues in food: instrumental analysis and immunoassay. As the mainstream detection method in food safety, instrumental analysis is the standard method for determining doxycycline residues in food and determining food safety. Large-scale precision instrument confirmation methods have the advantages of accuracy and reliability, but due to the high cost of equipment, high detection costs, and high professional quality requirements for operators, their application in large-scale on-site rapid screening and detection of samples is limited. The immunoassay method is based on the specific recognition of antigens and antibodies to achieve qualitative and quantitative detection of target substances. It has the advantages of high selectivity and sensitivity, simple operation, and low cost. It complements the instrumental analysis method to jointly maintain food safety and has become an important rapid detection method at present.

[0004] Antibodies are core reagents in rapid immunoassay technologies and products, determining the sensitivity, specificity, and stability of detection. Monoclonal antibodies, with their advantages of strong specificity, high sensitivity, ease of labeling, and mature production processes, are currently the mainstream biorecognition materials used in immunoassays. However, monoclonal antibodies based on hybridoma technology do suffer from issues such as uncertainty in cell line genetic stability, significant batch variability in antibodies, and high production costs using animal ascites. More importantly, once established, antibody performance is difficult to regulate. Changing detection requirements necessitates redesigning the hapten / antigen to produce monoclonal antibodies, which is time-consuming and costly. For example, the article "Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle, liver, and egg" discloses the use of hybridoma technology to prepare highly sensitive and specific monoclonal antibodies (McAbs) against doxycycline. Mice were immunized with a DC-PABA-BSA conjugate, and the IC values ​​of the obtained antibodies were determined by ELISA. 50 The lowest value was 2.36 ng / mL.

[0005] With the advancement of genetic engineering technology, nanobodies, with their advantages of small molecular weight, strong plasticity, large-scale production through prokaryotic expression, and high resistance to denaturants, have rapidly developed. They are expected to provide simpler, faster, and more cost-effective technologies and products for immunoassays. Enzyme-linked immunosorbent assay (ELISA), as a rapid test technique, often requires direct extraction with organic solvents as a pretreatment method. Nanobodies' excellent tolerance to organic solvents makes them promising for rapid testing. Currently, there is still no development and application of nanobodies that specifically and sensitively recognize doxycycline. Therefore, the development of a stable, accurate, and sensitive rapid detection method for doxycycline based on nanobodies has great application value. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a nanobody Nb DX-6F that specifically recognizes doxycycline.

[0007] The second object of the present invention is to provide a gene encoding the above-mentioned Nanobody Nb DX-6F.

[0008] The third object of the present invention is to provide a recombinant vector containing the above gene.

[0009] A fourth object of the present invention is to provide a recombinant cell containing the above-mentioned recombinant vector, the above-mentioned gene, or capable of expressing the above-mentioned Nanobody Nb DX-6F.

[0010] A fifth object of the present invention is to provide the use of the above-mentioned Nanobody Nb DX-6F, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant cell in detecting doxycycline for non-diagnostic purposes.

[0011] A sixth object of the present invention is to provide the use of the above-mentioned nanobody Nb DX-6F, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant cell in the preparation of a product for detecting doxycycline.

[0012] The seventh object of the present invention is to provide a kit for detecting doxycycline.

[0013] An eighth object of the present invention is to provide an immunoassay method for detecting doxycycline for non-diagnostic purposes.

[0014] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention first provides a nanobody Nb DX-6F that specifically recognizes doxycycline, and the amino acid sequence of VHH of the nanobody NbDX-6F is shown in SEQ ID No.1.

[0015] The invention prepares an artificial antigen to immunize alpacas to obtain a nano antibody gene library, and uses phage display technology and biological screening to screen out a nano antibody Nb DX-6F that specifically recognizes doxycycline from the alpaca immune antibody gene library.

[0016] Furthermore, the Nanobody Nb DX-6F includes four framework regions FR1, FR2, FR3, FR4 and three complementary determining regions CDR1, CDR2, and CDR3, and the order of the four framework regions and the three complementary determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; wherein the amino acid sequence of the FR1 is shown in SEQ ID No.2, the amino acid sequence of the FR2 is shown in SEQ ID No.3, the amino acid sequence of the FR3 is shown in SEQ ID No.4, the amino acid sequence of the FR4 is shown in SEQID No.5, the amino acid sequence of the CDR1 is shown in SEQ ID No.6, the amino acid sequence of the CDR2 is shown in SEQID No.7, and the amino acid sequence of the CDR3 is shown in SEQ ID No.8.

[0017] The present invention provides a gene encoding the nanobody Nb DX-6F, and the nucleotide sequence of VHH of the gene is shown in SEQ ID No.9.

[0018] The present invention provides a recombinant vector containing the gene.

[0019] The present invention also provides a recombinant cell comprising the above-mentioned recombinant vector, the above-mentioned gene, or being capable of expressing the above-mentioned Nanobody Nb DX-6F. Since the present invention has provided the amino acid sequence of Nanobody Nb DX-6F and the gene sequence encoding the Nanobody, those skilled in the art can obtain the Nanobody of the present invention by known recombinant DNA technology on this basis. Therefore, any recombinant vector or recombinant cell that can be used to prepare the Nanobody of the present invention should also be within the scope of protection of the present invention.

[0020] The present invention analyzes the detection performance and tolerance of the nanoantibody Nb DX-6F. The results show that the ELISA detection method based on the nanoantibody can detect doxycycline at a concentration of 0.17 to 14.25 ng / mL, and the half inhibitory concentration (IC 50 ) was 1.34 ng / mL, with a limit of detection (LOD) of 0.05 ng / mL, demonstrating high sensitivity. The antigen-binding activity of the nanobody Nb DX-6F remained above 85% at 20% methanol and ethanol concentrations, and above 60% at 40% methanol and ethanol concentrations, demonstrating excellent tolerance to organic solvents. Nb DX-6F exhibited comparable and excellent acid-base tolerance within the pH range of 2.4 to 11.4, exhibiting high antigen-binding activity (>80%) and excellent acid-base tolerance. These results demonstrate that the nanobody possesses high detection sensitivity and excellent organic and acid-base tolerance. During pretreatment of actual sample testing, it is minimally affected by organic solvents and is suitable for testing in acidic and alkaline environments.

[0021] Therefore, the present invention also provides the use of the nanobody Nb DX-6F, the gene, the recombinant vector or the recombinant cell in detecting doxycycline for non-diagnostic purposes.

[0022] The present invention also provides the use of the nanobody Nb DX-6F, the gene, the recombinant vector or the recombinant cell in the preparation of a product for detecting doxycycline.

[0023] The present invention provides a kit for detecting doxycycline, wherein the kit contains the nano antibody NbDX-6F.

[0024] The present invention provides an immunoassay method for detecting doxycycline for non-diagnostic purposes, wherein a complete antigen obtained by coupling doxycycline hapten with a carrier protein is used as a coating source, and the nano antibody Nb DX-6F or the kit is used for detection.

[0025] Furthermore, the structural formula of the doxycycline hapten is shown in formula (I): Formula (I).

[0026] Furthermore, the complete antigen is represented by formula (II): Formula (II).

[0027] Furthermore, the carrier protein (Protein) is ovalbumin (OVA).

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a nanobody Nb DX-6F that specifically recognizes doxycycline and its application. The amino acid sequence of the VHH of the nanobody is shown in SEQ ID No. 1. The nanobody has excellent organic solvent tolerance and acid-base tolerance. During the pre-treatment process of actual sample detection, it is less affected by organic solvents and can also be applied to detection needs in acid-base environments. An ELISA detection method was established based on the nanobody. The detection range of doxycycline by this method is 0.17 to 14.25 ng / mL, and the half inhibitory concentration (IC50) is 0. 50 ) was 1.34 ng / mL, and the limit of detection (LOD) was 0.05 ng / mL, demonstrating high sensitivity. Therefore, using this nanobody Nb DX-6F to detect doxycycline residues in actual samples is simple and time-consuming, with stable test results, strong sensitivity, and high accuracy. This nanobody also has great application value in detecting doxycycline or preparing doxycycline detection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the amino acid sequence and domain division of the specific nanobody Nb DX-6F.

[0030] Figure 2 This is the standard curve of the indirect competitive ELISA established based on the specific nanobody Nb DX-6F.

[0031] Figure 3 The activity curve of nanobody Nb DX-6F when different proportions of methanol, ethanol, and acetonitrile / PBS are used as diluents.

[0032] Figure 4The activity curve of nanobody Nb DX-6F when different pH values ​​are used as diluents. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] Example 1 Construction of Alpaca Immune Antibody Library 1. Experimental Methods 1. Preparation of complete antigens DX-A-LF and DX-A-OVA The structural formula of doxycycline hapten is shown in formula (I): Formula (I) The doxycycline hapten with the structural formula shown in formula (I) was coupled to lactoferrin (LF) and ovalbumin (OVA) by the active ester method to prepare the complete antigens DX-A-LF and DX-A-OVA. The complete antigen structure is shown in formula (II): Formula (II); The proteins are lactoferrin (LF) and ovalbumin (OVA).

[0036] 2. Alpaca immunization program Healthy alpacas were immunized with DX-A-LF as the immunizing antigen, administered subcutaneously in the neck. Each immunization dose was 1.0 mg. The first immunization was emulsified with 0.5 mL of complete Freund's adjuvant and the immunizing antigen. Subsequent booster immunizations were emulsified with 0.5 mL of incomplete Freund's adjuvant and the antigen. Thereafter, booster immunizations were administered every two weeks for a total of three booster immunizations.

[0037] Before immunization, 10 mL of blood was collected to separate serum for use as a negative control. Starting from the second immunization, 10 mL of blood was collected one week after immunization for serum titer and competition reaction testing. After the third and fourth immunizations, 50-100 mL of peripheral blood was collected for construction of the nanobody gene library.

[0038] 3. Isolation of alpaca lymphocytes Lymphocyte isolation should be performed as soon as possible after collecting alpaca peripheral blood. The specific procedure is as follows: dilute the alpaca peripheral blood with sterile saline in a 50 mL centrifuge tube at a ratio of 2:1. Centrifuge the diluted peripheral blood with commercial lymphocyte separation buffer. Add 15 mL of lymphocyte separation buffer to a sterile 50 mL centrifuge tube and slowly add 15 mL of diluted blood along the side of the tube using a sterile Pasteur pipette. Centrifuge at 800 g for 25 minutes. Transfer the lymphocyte layer to a new 50 mL centrifuge tube, dilute it 2-fold with saline, and centrifuge at 1500 g for 10 minutes at 4°C. Discard the supernatant. Disperse the lymphocytes with 5 mL of saline, centrifuge again at 1500 g for 10 minutes, and discard the supernatant to thoroughly wash the lymphocytes. Add lysis buffer (TRNsol) to each lymphocyte aliquot, and aliquot 1 mL into 2 mL centrifuge tubes. Store at -80°C until needed.

[0039] 4. Total RNA Extraction Total RNA was extracted from the lymphocytes stored in the TRNsol lysis solution using a commercial RNA extraction kit according to the instructions.

[0040] After total RNA extraction, a small sample was taken for nucleic acid electrophoresis and the RNA concentration was determined in a Nanodrop spectrophotometer. The ideal RNA sample should be intact and undegraded, with clear 28S and 18S bands visible on the nucleic acid electrophoresis gel and no genomic DNA contamination. The ratio of the UV absorbance at 260 nm to 280 nm (A 260 / 280 ) should be around 2.0. If genomic DNA contamination is present, remove the genomic DNA with DNase before reverse transcription. Perform another electrophoresis to verify that the genomic DNA has been removed and that the RNA has not been degraded. If the RNA has been degraded, re-extract the RNA. RNA should be reverse-transcribed into cDNA as quickly as possible or stored temporarily at -80°C.

[0041] 5. cDNA Synthesis Using RNA as a template, synthesize the first strand of cDNA according to the instructions of Takara's first strand reverse transcription kit. The specific method is as follows: A. According to the first step reaction system of cDNA synthesis shown in Table 1, mix the reagents in a nuclease-free centrifuge tube and operate under ice bath; Table 1 Reaction system for the first step of cDNA synthesis

[0042] B. Incubate the above reaction system at 65°C for 5 min and cool on ice for 2 min. C. According to the second step reaction system of cDNA synthesis shown in Table 2, add reagents to the system after the reaction in step A; Table 2 The second step reaction system of cDNA synthesis

[0043] D. Incubate at 42°C for 60 minutes and then at 70°C for 5 minutes. Store the reverse transcribed cDNA at -80°C.

[0044] 6. Amplification of Nanobody Target Gene Nested PCR was used to amplify the target gene in two steps.

[0045] The first round of PCR used cDNA as a template and primers Q1 / Q2 for the first round of PCR reaction. The nucleotide sequences of primers Q1 / Q2 are shown in Table 5 , and the reaction system and reaction system of the first round of PCR are shown in Table 3 .

[0046] Table 3 Nested PCR first step reaction system

[0047] Second-round PCR: The first-round PCR reaction product was recovered using a kit and appropriately diluted to serve as a template for the second-round PCR reaction. The second-round PCR reaction was performed using primers Q3 / Q4. The nucleotide sequences of primers Q3 / Q4 are shown in Table 5. The reaction system and reaction conditions for the second-round PCR are shown in Table 4.

[0048] Table 4 Nested PCR second step reaction system

[0049] Table 5 Primers and nucleotide sequences used for amplification of the target gene of Nanobody VHH

[0050] 7. Gene library construction (1) Enzyme digestion of VHH target gene and vector The VHH target gene and the pComb3xss vector were digested with SfiI enzyme at 50°C for 16 hours.

[0051] The pComb3xss vector digestion product was recovered by agarose gel as a band with a molecular weight of 3500 bp; the VHH gene digestion product was directly cleaned and recovered using a DNA recovery kit.

[0052] (2) Ligation of enzyme digestion products The vector pComb3xss and VHH fragment were mixed evenly (molar ratio 1:3), reacted at 16°C for 16 h, and then cleaned and recovered using a DNA recovery kit.

[0053] (3) Electric shock conversion Take 5 μL of ligation product and add 50 μL of electroporation competent medium E. coli After gently mixing in TG1, transfer to a 0.2 cm electroporation cuvette for electroporation (voltage: 1.8 kV). Immediately after electroporation, add 800 μL and 150 μL of SOC medium preheated to 37°C to the electroporation cuvette twice, collect the cells into a sterile centrifuge tube, and culture at 37°C, 250 rpm for 1 h to revive the cells.

[0054] Prepare a serial dilution series with 50 μL of the recovered bacterial solution. Spread 100 μL of each serial dilution onto a 90 mm diameter LB-Amp dish to serve as a counting plate and incubate at 37°C overnight. Spread the remaining undiluted recovered bacterial solution onto a 120 mm diameter LB-Amp dish, spreading 2-3 dishes per 1 mL of solution as amplification plates. Incubate the culture plates at 37°C overnight.

[0055] Count the number of colonies on the culture dish, calculate the total number of bacteria in the resuscitated bacterial solution, and perform multiple electric shock transformations until the total number of transformed colonies reaches 10 7 This number is the library capacity of the nanobody gene library.

[0056] Scrape the transgenic E. coli colonies from the amplification plate with a cell scraper, collect the cells by centrifugation, discard the supernatant, and resuspend in LB-Amp (add 0.5 mL per tube for electroporation). Mix thoroughly, then add sterile glycerol to a final concentration of 25% (v / v). Take 50 μL of the bacterial solution for serial dilution to determine the cell count, and aliquot the remaining solution and freeze at -80°C. This is the doxycycline nanobody gene library.

[0057] 8. Phage rescue According to the above transgenic E. coli cell number determination results, 10 times more cells than the reservoir capacity were inoculated into 150 mL LB-Amp and the OD was controlled. 600 <0.2, cultured at 37℃ and 250rpm until the logarithmic phase (OD 600 About 0.4~0.6); add 1mL to titrate 10 12 Helper phage M above cfu / mL 13 K 07After standing at 37°C for 30 minutes, incubate at 250 rpm for 1 hour. Add Kanamycin (working concentration of 50 μg / mL) and incubate overnight at 37°C and 250 rpm. Transfer the bacterial solution to a centrifuge bottle and centrifuge at 12,000 rpm for 15 minutes at 4°C. Remove the supernatant and add 1 / 4 volume of PEG / NaCl. Incubate on ice for at least 2.5 hours. Centrifuge at 12,000 rpm for 15 minutes at 4°C, discard the supernatant, resuspend the pellet in 750 μL of TBS, transfer to a 1.5 mL centrifuge tube, centrifuge at 4,000 rpm for 5 minutes at 25°C, and filter through a 0.22 μm polyethersulfone filter. Titer 10 μL of phage was titered. The remaining phage was mixed thoroughly and added to a final concentration of 50% sterile glycerol (v / v). Store at -80°C. This is the doxycycline nanobody phage library, ready for affinity panning.

[0058] Example 2 Affinity panning and identification of nanobodies 1. Experimental Methods 1. Affinity panning of nanobodies (1) Immobilization of antigen and carrier protein Affinity panning was performed using a strong adsorption microtiter plate with high adsorption capacity. One column of the plate was coated per panning round, for a total of four rounds. Wells AB were coated with the carrier protein OVA diluted to 1 mg / mL, while wells CDEF were coated with the detection antigen DX-A-OVA diluted to 10 μg / mL using CB coating buffer. 100 μL of this solution was added to each well of the strong adsorption plate and incubated at 37°C overnight. Additionally, because alpacas may have been immunized with multiple carrier proteins, a mixture of three immune carrier proteins—ConA (concanavalin), LF (lactoferrin), and KLH (keyhole limpet hemocyanin)—was diluted to a final concentration of 2 mg / mL and coated separately in one column of the immunogen carrier protein wells. The next day, the plate was washed twice with PBST (0.01 M PBS, 0.05% Tween-20). 120 μL of a 1% fish gelatin solution was added to each well and incubated at 37°C for 3 hours. Pour out the liquid in the well and pat dry on absorbent paper, dry at 37℃ for 1h, and store at 4℃ for later use.

[0059] (2) Positive phage screening Add the phage library from Example 1 to two wells containing the immunogen carrier protein, adding 150 μL to each well and incubating at 37°C with shaking for 1 hour (this step is only required for the first round; rounds 2, 3, and 4 should begin directly in wells AB). Transfer free phage to wells AB coated with the original carrier protein, adding 150 μL to each well and incubating at 37°C with shaking for 1 hour. Transfer free phage to three wells containing immobilized antigen (DX-A-OVA, 10 μg / mL), adding 100 μL to each well and incubating at 37°C with shaking for 1 hour. Discard free phage from the wells, wash the wells 10 times with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)) and then five times with PBS. Add 100 μL of 10 mg / mL trypsin-TBS solution and elute at 37°C for 30 minutes. Collect the phage, take 10µL of the eluted phage to determine the titer, and use the rest to infect 5mL of culture medium grown to the logarithmic phase. E. coli The TG1 strain was amplified. The next day, the amplified phages were precipitated with PEG / NaCl and the titer of the phages was determined.

[0060] During the second, third, and fourth rounds of panning, the coating concentration of DX-A-OVA was reduced to 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. Phages were added and incubated at 37°C for 1 hour. After washing with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)) and PBS, drug competition elution was adopted, that is, a certain concentration of drug was added, incubated at 37°C for 1 hour, and the liquid in the well was aspirated to obtain the eluted phage. The screening scheme of step (2) was repeated. The drug elution concentrations were 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. The above conditions can be adjusted according to the actual immune situation. If the serum titer is low, the concentration of the detection antigen DX-A-OVA during panning can be appropriately increased; if the inhibition rate is low, the drug concentration of the competitive reaction needs to be appropriately increased.

[0061] 2. Identification of positive clones Indirect enzyme-linked immunosorbent assay (ELISA) was used to identify positive phage clones. The specific method is as follows: (1) Antigen immobilization The detection antigen DX-A-OVA was diluted to 1 μg / mL using coating buffer, and 100 μL was added to each well. The plate was incubated at 37°C overnight. The next day, the plate was washed twice with PBST (0.01M PBS, 0.05% Tween-20). Then, 120 μL of 2% skim milk solution was added to each well and incubated at 37°C for 3 hours. The liquid in the wells was poured out and patted dry on absorbent paper. The plate was then dried at 37°C for 1 hour and stored at 4°C until ready for use.

[0062] (2) Small-scale expression of nanobodies In the third and fourth rounds of panning, 96 single colonies were randomly selected from the plate and inoculated into a 96-well deep-well plate containing 0.5 mL of LB-Amp per well. A single TG1 clone was also inoculated as a negative control. The plates were cultured overnight at 37°C and 180 rpm to serve as the bacterial solution "mother plate."

[0063] Take out 10 μL of bacterial solution from each well of the mother plate and inoculate it into another 96-well deep-well plate with 1 mL of LB-Amp in each well. The inoculated well numbers should correspond to those of the mother plate. Incubate at 37°C, 180 rpm for 4 h. Add IPTG (1:1000 ratio, v / v) to each well and incubate at 37°C, 180 rpm overnight.

[0064] (3) Enzyme-linked immunosorbent assay (ELISA) to identify positive clones On the third day, centrifuge at 4000 rpm and collect the supernatant. Add 50µL of the supernatant to the coated ELISA plate, incubate at 37°C for 40 minutes, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100µL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 40 minutes, 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 at 37°C in the dark for 10 minutes; add 50µL of stop solution (10% H2SO4, v / v) to terminate the reaction; measure the absorbance at 450nm using a microplate reader. Select the OD value. 450 Phage clones with a count three times greater than that of negative clones were considered positive clones.

[0065] Select OD 450 Phage clones with a count greater than 3 times that of negative clones were identified as positive clones for identification of positive nanoantibodies. Add titer group: 50µL supernatant of positive clones identified by indirect ELISA and 50µL PBS; inhibition group: 50µL supernatant of positive clones identified by indirect ELISA and 50µL doxycycline standard (concentration of 1µg / mL), incubate at 37°C for 40min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the well, add 100µL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 40min, wash with PBST (0.01M The wells were washed five times with PBS, 0.06% Tween-20 (v / v), and the liquid in the wells was patted dry. 100 µL of TMB substrate solution was added and color was developed at 37°C in the dark for 10 min. 50 µL of stop solution (10% H2SO4, v / v) was added to terminate the reaction. The absorbance at 450 nm was measured using a microplate reader.

[0066] Select the clone with an OD value 3 times greater than that of the negative control well in plate 1 and with obvious inhibition, record the number of the corresponding well as Nb DX-6F, and transfer the bacterial solution in the corresponding well of the mother plate to a sterile centrifuge tube, add glycerol and freeze for later use.

[0067] That is, a nanobody that can specifically recognize doxycycline was obtained and named Nb DX-6F.

[0068] Example 3 Sequencing of the gene encoding the specific nanobody Nb DX-6F and determination of its amino acid sequence 1. Experimental Methods The strain with the specific nanoantibody Nb DX-6F obtained by indirect competitive ELISA identification was sent to a sequencing company for sequencing to obtain the nucleotide sequence of the specific nanoantibody Nb DX-6F; based on the DNA sequencing results and the codon table, the amino acid sequence of the specific nanoantibody Nb DX-6F was obtained.

[0069] 2. Experimental Results The amino acid sequence of the VHH of the specific nanobody Nb DX-6F is shown in SEQ ID No. 1: EVQLVQSGGGSVQAGGSLRLSCAAPGYIYSSCMGWFRQAPGKEREGVAGISTGGGPTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAVGPGGSCPRPTRYWGQGTLVTVSS.

[0070] The amino acid sequence and domain division diagram of the specific nanobody Nb DX-6F are shown in the figure. Figure 1 As shown, it can be seen that the specific nanobody Nb DX-6F includes 4 framework regions (FR) and 3 complementarity-determining regions (CDR).

[0071] The framework regions (FR1-FR4) are as follows: SEQ ID No.2: EVQLVQSGGGSVQAGGSLLRLSCAAP, SEQ ID No.3: MGWFRQAPGKEREGVAG, SEQ ID No.4: YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC, SEQ ID No. 5: WGQGTLVTVSS.

[0072] The complementarity determining regions (CDR1-CDR3) are as follows: SEQ ID No.6: GYIYSSSC, SEQ ID No.7: ISTGGGPT, SEQ ID No. 8: AVGPGGSCPRPTRY.

[0073] The nucleotide sequence of the VHH of the specific nanobody Nb DX-6F is shown in SEQ ID No.9: GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCCTGGATACATCTACAGTAGCAGCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGGTCGCAGGTATTTCGACTGGTGGTGGTCCCACATACTATGC CGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGCGCGGTAGGTCCCGGTGGTAGCTGTCCCGACCGACGCGTTACTGGGGCCAGGGGGACCCTGGTCACCGTCTCCTCA.

[0074] Example 4 Large-scale preparation of specific nanobody Nb DX-6F 1. Experimental Methods The specific nanobody Nb DX-6F was prepared in the form of protein expression. The specific method is as follows: The sequencing-containing pComb3xss-VHH plasmid was transferred into E. coli In BL21 (DE3) culture medium, pick a single colony from the transformation plate and inoculate it into 10 mL of LB (Amp) medium. Incubate the overnight culture at a ratio of 1:100 into 750 mL of LB (Amp) medium. Incubate at 37°C, 250 rpm until the OD reaches 0. 600 When the concentration is approximately 0.4-0.6, IPTG (1:1000, v / v) is added and cultured overnight at 37°C and 250 rpm. The next day, the cell pellet is centrifuged at 12,000 rpm for 5 minutes at 4°C. The pellet is then collected and subjected to the sucrose osmotic pressure freeze-thaw method. The supernatant is then centrifuged at 12,000 rpm for 10 minutes to obtain the periplasmic soluble protein.

[0075] The histidine tag carried by Nb DX-6F and the impurity protein were used to bind Ni 2+ The antibody was isolated and purified by affinity chromatography based on differences in adsorption capacity. The specific steps were as follows: After filtering the periplasmic protein through a 0.22 μm filter, 1 mL of Ni-NTA resin was added and premixed overnight. The next day, the mixture was transferred to a gravity column. The column was washed and equilibrated with 0.01 mol / L PBS, and the mixture was added. The sample was loaded twice, and the column was then equilibrated and washed with PBS. The column was then washed with 25 mmol / L imidazole-PBS. Finally, the target protein was eluted with 200 mmol / L imidazole-PBS. The eluate was collected, which is the doxycycline nanobody Nb DX-6F. The collected eluate was transferred to a 3 kDa dialysis bag and dialyzed against PBS at 4°C for 3 days, a total of 6 times. After dialysis, the eluate was stored at -20°C until use.

[0076] The nanobody Nb DX-6F is obtained.

[0077] Example 5 Detection performance analysis of specific nanobody Nb DX-6F 1. Experimental Methods 1. Coating and sealing Dilute the DX-A-OVA coating agent to 1 μg / mL in the coating solution and coat at 37°C overnight. The next day, wash twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)). Add 120 μL of 1% fish gelatin solution to each well, block at 37°C for 3 hours, dry at 37°C for 1 hour, and store in a sealed bag at 4°C until use.

[0078] 2. Establishment of standard curve (1) Experimental methods Dilute the DX-A-OVA coating agent to 1 μg / mL in coating solution and coat overnight at 37°C. The next day, wash twice with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)). Add 120 μL / well of 2% skim milk solution and block at 37°C for 3 h. Discard the blocking solution and dry at 37°C for 1 h. Add 50 μL of nanobody and a series of 50 μL of doxycycline standards of different concentrations to each well, incubate at 37°C for 40 minutes, wash five times with PBST, pat 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 minutes, wash five times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop at 37°C in the dark for 10 minutes; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using a microplate reader. The horizontal axis is plotted with the concentration of doxycycline standard, B / B0 (OD value of the wells with doxycycline added).450 / OD of the well without doxycycline 450 ) as the vertical axis, and establish an indirect competition standard curve.

[0079] 2. Experimental Results The standard curve of the indirect competitive ELISA established based on the specific nanoantibody Nb DX-6F is shown in the figure below. Figure 2 As shown in the figure, the standard curve is S-shaped and has a good linear correlation. The detection range of doxycycline by this method is 0.17-14.25 ng / mL, and the half-inhibitory concentration (IC 50 ) was 1.34 ng / mL, and the limit of detection (LOD) was 0.05 ng / mL, indicating high detection sensitivity.

[0080] Example 6 Organic Tolerance Analysis of Nanobody Nb DX-6F 1. Experimental Methods Using different concentrations of methanol and ethanol (10%, 20%, 30%, 40%, 50%) as diluents, the nanobody NbDX-6F was diluted to the same working concentration to determine the binding ability of the antibody to the antigen. The binding ability of the antibody to the antigen without dilution with organic solvent diluent was taken as 100%, and the tolerance of the nanobody to different organic solvents and different concentrations of the same organic solvent was evaluated. The specific method is as follows: Add 50 μL of diluted nanoantibody Nb DX-6F and 50 μL of PBS to the coated ELISA plate, incubate at 37°C for 40 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 30 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance value at 450 nm using a microplate reader.

[0081] 2. Experimental Results The activity curve of nanobody Nb DX-6F when different ratios of methanol and ethanol are used as diluents is shown in the figure below. Figure 3As shown in the figure, it can be seen that the antigen binding activity of the nanobody Nb DX-6F still maintains more than 85% activity at a concentration of 20% methanol and ethanol, and still maintains more than 60% activity at a high concentration of 40% methanol and ethanol. Therefore, the nanobody NbDX-6F has excellent organic solvent tolerance. During the pretreatment process of actual sample detection, it is less affected by organic solvents and the detection results are highly accurate.

[0082] Example 7 Analysis of Acid-Base Tolerance of Nanobody Nb DX-6F 1. Experimental Methods PBS buffer solutions of different pH values ​​(pH 1.4, 2.4, 3.4, 4.4, 5.4, 6.4, 7.4, 8.4, 9.4, 10.4, 11.4, and 12.4) were prepared and used as diluents to dilute the Nbs to the working concentration. The biological activity of the antibodies in the system was measured, and the antigen-antibody binding capacity in the PBS buffer solution with a pH of 7.4 was taken as 100% to evaluate the acid-base tolerance of the nanobody. The specific method is as follows: Add 50 µL of diluted nanoantibody Nb DX-6F and 50 µL of PBS to the coated ELISA plate, incubate at 37°C for 40 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 µL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 30 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 µL of TMB substrate solution, and develop at 37°C in the dark for 10 min; add 50 µL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using a microplate reader.

[0083] 2. Experimental Results Considering the complexity of the application environment and the diversity of samples, acidic or alkaline environments are not conducive to detection. This experiment investigated the stability of doxycycline nanobody Nb DX-6F in the pH range of 1.4 to 12.4. Figure 4 NbDX-6F exhibits excellent acid-base tolerance. Within the pH range of 2.4 to 11.4, the nanobody exhibits comparable and excellent acid-base tolerance, with high antigen-binding activity (>80%). Even in acidic environments (pH 2.4 to 6.4), antigen-binding activity is enhanced, demonstrating that NbDX-6F exhibits high acid-base tolerance and is suitable for testing in both acidic and alkaline environments.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A nanobody Nb DX-6F that specifically recognizes doxycycline, characterized in that The amino acid sequence of VHH of the nanobody Nb DX-6F is shown in SEQ ID No.

1.

2. The gene encoding the nanobody Nb DX-6F according to claim 1, characterized in that The nucleotide sequence of the VHH of the gene is shown in SEQ ID No.

9.

3. A recombinant vector, characterized in that The recombinant vector contains the gene according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 3 or the gene according to claim 2 or is capable of expressing the nanobody Nb DX-6F according to claim 1.

5. Use of the nanobody Nb DX-6F according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or the recombinant cell according to claim 4 in detecting doxycycline for non-diagnostic purposes.

6. Use of the nanobody Nb DX-6F according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or the recombinant cell according to claim 4 in the preparation of a product for detecting doxycycline.

7. A kit for detecting doxycycline, characterized in that: Contains the nanobody Nb DX-6F according to claim 1.

8. An immunoassay method for detecting doxycycline for non-diagnostic purposes, characterized in that: The complete antigen obtained by coupling doxycycline hapten with carrier protein is used as coating source, and the nanobody Nb DX-6F according to claim 1 or the kit according to claim 7 is used for detection.

9. The immunoassay method according to claim 8, characterized in that: The structural formula of the doxycycline hapten is shown in formula (I): Formula (I).

10. The detection method according to claim 8, characterized in that: The carrier protein is ovalbumin.

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