A nanobody nb dx-6f specifically recognizing doxycycline and application thereof

Abstract

The application discloses a nanobody Nb DX-6F specifically recognizing doxycycline and an application thereof. An amino acid sequence of a VHH of the nanobody Nb DX-6F is shown as SEQ ID No. 1, and the nanobody has excellent organic solvent resistance and acid and alkali resistance. An ELISA detection method is established based on the nanobody, the method has a doxycycline detection range of 0.17-14.25 ng / mL, a half-inhibitory concentration (IC 50 ) of 1.34 ng / mL, and a minimum detection limit (LOD) of 0.05 ng / mL, and the detection sensitivity is high. Therefore, when the nanobody Nb DX-6F is used for detecting doxycycline residues in actual samples, the detection result has good stability, high sensitivity and high accuracy, and the nanobody has extremely high application value in detection of doxycycline or preparation of a doxycycline detection product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to a nanobody NbDX-6F specifically recognizing doxycycline and application thereof. BACKGROUND

[0002] Doxycycline, also known as strong doxycycline and deoxytetracycline, is a semi-synthetic tetracycline antibiotic. It has high antibacterial activity, strong tissue penetration ability, wide distribution in vivo, high bioavailability and long half-life, and is widely used in veterinary clinics and aquaculture. With the development of domestic intensive breeding, the breeding density has been significantly improved, and the phenomenon of veterinary drug abuse has become increasingly serious. The abuse of doxycycline not only has mixed toxicity to the ecological environment, but also can be transmitted to humans through the enrichment of the food chain. Long-term consumption of livestock and poultry food with high doxycycline residues by humans can cause various acute and chronic reactions such as stomach ulcers, esophagitis, gastritis, and glossitis, and can induce cell mutations to cause deformities and carcinogenesis, and even induce the production of drug-resistant strains. This brings great hidden dangers to the body and seriously affects public health. Therefore, it is necessary to establish a more efficient and accurate detection method to monitor doxycycline residues in aquatic products.

[0003] At present, the detection methods for doxycycline residues in food include instrument analysis method and immune analysis method. Instrument analysis method is the standard method for determining doxycycline residues in food and ensuring food safety. Large precision instruments have the advantages of accuracy and reliability, but due to the problems of expensive equipment, high detection cost and high requirements for the professional quality of operators, its application in large-scale sample field rapid screening detection is limited. Immune analysis method is based on the specific recognition of antigen-antibody, which realizes the qualitative and quantitative detection of target substances, has high selectivity and sensitivity, simple operation and low cost, and is complementary to instrument analysis method to maintain food safety. It has become an important rapid detection method at present.

[0004] Antibodies are the core reagents of immunoassay technology and products, which determine the sensitivity, specificity and stability of the detection. Monoclonal antibodies have the advantages of strong specificity, high sensitivity, easy to label, mature production process, etc., and are the mainstream biological recognition materials in the current immunoassay method. However, monoclonal antibodies based on hybridoma technology indeed have the problems of uncertain cell line stable heredity, significant batch difference of antibodies, high production cost by using animal ascites method, etc. More importantly, once the cell line is established, it is difficult to regulate the performance of the antibody, and if the detection requirements change, a new monoclonal antibody needs to be designed and prepared by using a hapten / antigen, which is time-consuming and costly. In 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", a highly sensitive and specific monoclonal antibody (McAb) against doxycycline was prepared by using hybridoma technology. The IC 50 value of the antibody obtained by ELISA detection of the DC-PABA-BSA conjugate mouse immunization was as low as 2.36 ng / mL.

[0005] With the development of genetic engineering technology, nanobodies with the advantages of small molecular weight, strong plasticity, large-scale production by prokaryotic expression, high resistance to denaturant, etc. have been rapidly developed, and are expected to provide simpler, faster and lower-cost technology and products for immunoassay methods. Enzyme-linked immunosorbent assay (ELISA) as a rapid detection technology, the pretreatment method is mostly direct extraction with organic solvent, and the superior organic solvent tolerance of nanobodies makes them have good application prospect in rapid detection. At present, there is still no development and application of specific, high-sensitivity nanobodies for recognizing doxycycline, so it has good application value to develop a method for rapid detection of doxycycline with good stability, high accuracy and strong sensitivity based on nanobodies. SUMMARY

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

[0007] The second purpose of the present application is to provide a gene encoding the above-mentioned nanobody Nb DX-6F.

[0008] The third purpose of the present application is to provide a recombinant vector containing the above-mentioned gene.

[0009] A fourth object of the present application 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 application 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 for detecting doxycycline for non-diagnostic purposes.

[0011] A sixth object of the present application 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 for preparing a product for detecting doxycycline.

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

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

[0014] The above-mentioned objects of the present application are achieved by the following technical solutions:

[0015] The present application first provides a nanobody Nb DX-6F specifically recognizing doxycycline, the amino acid sequence of the VHH of the nanobody Nb DX-6F is shown in SEQ ID No. 1.

[0016] The present application obtains a nanobody gene library by preparing an artificial antigen to immunize a llama, and obtains a nanobody Nb DX-6F specifically recognizing doxycycline from the llama immunized antibody gene library through bio-panning by using phage display technology.

[0017] Further, the nanobody Nb DX-6F comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR3, and the arrangement order of the 4 framework regions and 3 complementarity determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, 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 SEQ ID 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 SEQ ID No. 7, and the amino acid sequence of the CDR3 is shown in SEQ ID No. 8.

[0018] The present application provides a gene encoding the nanobody Nb DX-6F, and the nucleotide sequence of the VHH of the gene is shown in SEQ ID No. 9.

[0019] The present application provides a recombinant vector containing the above-mentioned gene.

[0020] The present application also provides a recombinant cell containing the above-mentioned recombinant vector, the above-mentioned gene, or capable of expressing the above-mentioned nanobody Nb DX-6F. Since the present application has provided the amino acid sequence of the nanobody Nb DX-6F and the gene sequence encoding the nanobody, those skilled in the art can obtain the nanobody of the present application on this basis by known recombinant DNA technology. Therefore, any recombinant vector or recombinant cell that can be used to prepare the nanobody of the present application should also be within the protection scope of the present application.

[0021] The present application analyzes the detection performance and tolerance of the nanobody Nb DX-6F, and the results show that the ELISA detection method based on the nanobody has a detection range of 0.17-14.25 ng / mL for doxycycline, a half-inhibitory concentration (IC 50 ) of 1.34 ng / mL, and a minimum detection limit (LOD) of 0.05 ng / mL, with high detection sensitivity. The antigen binding activity of the nanobody Nb DX-6F remains above 85% under a methanol and ethanol concentration of 20%, and remains above 60% under a methanol and ethanol concentration of 40%, with excellent organic solvent tolerance; the nanobody has quite good acid-base tolerance and shows high antigen binding activity (>80%) in the range of pH=2.4-11.4, with excellent acid-base tolerance. The above results show that the nanobody has high detection sensitivity and excellent organic tolerance and acid-base tolerance, and is less affected by organic solvents in the pretreatment process of actual sample detection, and can also be applied to detection requirements in acid-base environments.

[0022] Therefore, the present application also provides the application of the nanobody Nb DX-6F, the gene, the recombinant vector, or the recombinant cell in the detection of doxycycline for non-diagnostic purposes.

[0023] The present application also provides the application of the nanobody Nb DX-6F, the gene, the recombinant vector, or the recombinant cell in the preparation of products for detecting doxycycline.

[0024] The present application provides a kit for detecting doxycycline, which contains the nanobody Nb DX-6F.

[0025] The application provides an immunoassay method for detecting doxycycline for non-diagnostic purposes, wherein a complete antigen obtained by coupling a doxycycline hapten with a carrier protein is used as a coating agent, and the nanobody Nb DX-6F or the kit is used for detection.

[0026] Further, the structural formula of the doxycycline hapten is shown as formula (I): Formula (I).

[0027] Further, the complete antigen is shown as formula (II):

[0028] Formula (II).

[0029] Further, the carrier protein (Protein) is ovalbumin (OVA).

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] The application provides a nanobody Nb DX-6F specifically recognizing doxycycline and an application thereof. The amino acid sequence of the VHH of the nanobody is shown as SEQ ID No. 1, the nanobody has excellent organic solvent resistance and acid-base resistance, is less affected by an organic solvent in a pretreatment process of actual sample detection, and can also be applied to detection requirements in an acid-base environment. An ELISA detection method is established based on the nanobody, the detection range of the method for doxycycline is 0.17-14.25 ng / mL, the half-inhibitory concentration (IC 50 ) is 1.34 ng / mL, and the minimum detection limit (LOD) is 0.05 ng / mL, and the detection sensitivity is high. Therefore, when the nanobody Nb DX-6F is used to detect doxycycline residues in an actual sample, the operation is simple, the time consumed is short, the detection result is stable, the sensitivity is strong, the accuracy is high, the nanobody has extremely high application value in detection of doxycycline or preparation of a doxycycline detection product. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is an amino acid sequence and domain division schematic diagram of the specific nanobody Nb DX-6F.

[0033] Figure 2 It is an indirect competitive ELISA standard curve diagram established based on the specific nanobody Nb DX-6F.

[0034] Figure 3 It is an activity curve diagram of the nanobody Nb DX-6F when methanol, ethanol, acetonitrile / PBS in different proportions are used as diluents.

[0035] Figure 4 Activity curve of nanobody Nb DX-6F at different pH as diluent. DETAILED DESCRIPTION

[0036] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The examples do not impose any form of limitation on the application. Unless otherwise specified, the reagents, methods and devices employed in the present application are conventional reagents, methods and devices in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Example 1 Construction of a llama immunized antibody library

[0039] I. Experimental Methods

[0040] 1. Preparation of complete antigens DX-A-LF and DX-A-OVA

[0041] The structural formula of the doxycycline hapten is shown as formula (I):

[0042] Formula (I)

[0043] The doxycycline hapten with the structural formula shown as formula (I) is coupled with lactoferrin (LF) and ovalbumin (OVA) by the active ester method to prepare complete antigens DX-A-LF and DX-A-OVA. The structural formula of the complete antigens is shown as formula (II):

[0044] Formula (II)

[0045] Wherein Protein is lactoferrin (LF) and ovalbumin (OVA).

[0046] 2. Llama immunization scheme

[0047] Healthy llamas are used for animal immunization, and DX-A-LF is used as the immunogen for subcutaneous injection on the back of the neck of the llama, with an immunization dose of 1.0 mg of immunogen per time. The first immunization is performed using 0.5 mL of complete Freund's adjuvant mixed and emulsified with the immunogen, and the subsequent booster immunizations are performed using 0.5 mL of incomplete Freund's adjuvant mixed and emulsified with the antigen, followed by a booster immunization every 2 weeks, for a total of 3 booster immunizations.

[0048] Before immunization, 10 mL of blood is taken to separate the serum as a negative control. Starting from the second immunization, 10 mL of blood is taken after each immunization to detect the serum titer and competitive reaction. After the third and fourth immunizations, 50-100 mL of peripheral blood is collected for the construction of a nanobody gene library.

[0049] 3. Isolation of Lymphocytes from Alpacas

[0050] Lymphocyte isolation from alpaca peripheral blood should be performed as soon as possible. The procedure is as follows: Mix alpaca peripheral blood with sterile saline in a 50 mL centrifuge tube without RNase at a ratio of 2:1. Centrifuge the diluted peripheral blood using a commercial lymphocyte isolation solution. Add 15 mL of the lymphocyte isolation solution to a sterile 50 mL centrifuge tube and slowly add 15 mL of the diluted blood along the tube wall using a sterile Pasteur pipette. Centrifuge at 800 g for 25 min. Take the lymphocyte layer to a new 50 mL centrifuge tube, dilute with saline at a ratio of 2:1, and centrifuge at 1500 g for 10 min at 4°C. Discard the supernatant. Blow the lymphocytes apart with 5 mL of saline, centrifuge again at 1500 g for 10 min, and discard the supernatant to thoroughly wash the lymphocytes. Add lysis solution (TRNsol) to each portion of lymphocytes, 1 mL per portion, and store in a 2 mL centrifuge tube at -80°C for later use.

[0051] 4. Extraction of Total RNA

[0052] Extraction of total RNA from the lymphocytes stored in the TRNsol lysis solution described above can be performed using a commercial RNA extraction kit according to the manufacturer's instructions.

[0053] After extraction of the total RNA, take a small sample for nucleic acid electrophoresis and determine the RNA concentration in a Nanodrop spectrophotometer. An ideal RNA sample should be intact and not degraded, with clear 28S and 18S bands visible on the nucleic acid electrophoresis gel, and no genomic DNA contamination, with an ultraviolet absorbance ratio (A 260 / 280 ) at 260 nm and 280 nm of about 2.0. If genomic DNA contamination occurs, remove the genomic DNA using DNase before reverse transcription and verify again by electrophoresis that the genomic DNA has been removed and that the RNA has not been degraded in the process; if the RNA has been degraded, extract the RNA again. The RNA should be reverse transcribed into cDNA as soon as possible or stored briefly at -80°C.

[0054] 5. Synthesis of cDNA

[0055] Synthesize the first strand of cDNA using RNA as a template according to the instructions for the Takara first-strand reverse transcription kit. The procedure is as follows:

[0056] A. Mix the reagents in a nuclease-free centrifuge tube according to the first-step reaction system for cDNA synthesis shown in Table 1, and perform the operation on ice.

[0057] Table 1. First-step reaction system for cDNA synthesis

[0058]

[0059] B. The reaction system above was incubated at 65°C for 5 min, and cooled in ice bath for 2 min;

[0060] C. The reagents were added to the reaction system after step A according to the second step reaction system for cDNA synthesis shown in Table 2;

[0061] Table 2 Second step reaction system for cDNA synthesis

[0062]

[0063] D. Incubated at 42°C for 60 min, and at 70°C for 5 min. The reverse transcription product cDNA was stored at -80°C.

[0064] 6. Amplification of the target gene of nanobody

[0065] The target gene was amplified by two-step method using nested PCR.

[0066] The first round of PCR used cDNA as template, and primer Q1 / Q2 was used 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 conditions of the first round of PCR are shown in Table 3.

[0067] Table 3 First step reaction system of nested PCR

[0068]

[0069] The second round of PCR used the product of the first round of PCR reaction recovered by the kit as template after appropriate dilution, and primer Q3 / Q4 was used for the second round of PCR reaction. The nucleotide sequences of primers Q3 / Q4 are shown in Table 5, and the reaction system and reaction conditions of the second round of PCR are shown in Table 4.

[0070] Table 4 Second step reaction system of nested PCR

[0071]

[0072] Table 5 Primers used for amplification of nanobody VHH target gene and their nucleotide sequences

[0073]

[0074] 7. Construction of gene library

[0075] (1) Enzymatic digestion of VHH target gene and vector

[0076] The VHH target gene and pComb3xss vector were digested using the SfiI enzyme. Digestion conditions: incubation at 50℃ for 16 hours.

[0077] The pComb3xss vector digestion product was recovered by agarose gel electrophoresis, and the 3500bp band was recovered directly by a DNA recovery kit.

[0078] (2) Ligation of enzyme digestion products

[0079] The vector pComb3xss and the VHH fragment were mixed (molar ratio 1:3), reacted at 16°C for 16 h, and then cleaned and recovered using a DNA recovery kit.

[0080] (3) Electroshock conversion

[0081] Take 5 μL of the ligation product and add it to 50 μL of electrocompetent state. E. coli In TG1, after gentle mixing, 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 in two portions. Collect the contents into sterile centrifuge tubes and incubate at 37°C and 250 rpm for 1 h to revive the cells.

[0082] Take 50 μL of resuscitated bacterial culture and perform serial dilutions. Spread 100 μL of each serially diluted bacterial culture onto 90 mm diameter LB-Amp culture dishes as counting plates and incubate overnight at 37°C. Spread the remaining undiluted resuscitated bacterial culture onto 120 mm diameter LB-Amp culture dishes, with each 1 mL of bacterial culture spread onto 2–3 culture dishes as amplification plates, and incubate overnight at 37°C for amplification.

[0083] Count the number of colonies on the culture dish, calculate the total number of bacteria in the revived bacterial solution, and perform multiple electroporation transformations until the total number of transformed colonies reaches 10. 7 The number of CFU or higher represents the library capacity of the nanobody gene library.

[0084] The 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 then 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 suspension was serially diluted to determine the cell count. The remaining bacterial suspension was aliquoted and stored at -80°C. This constitutes the doxycycline nanobody gene library.

[0085] 8. Phage rescue

[0086] Based on the above results of transgenic E. coli cell count determination, cells at a volume greater than 10 times the library size were inoculated into 150 mL of LB-Amp to control OD. 600<0.2, 250 rpm culture at 37°C to logarithmic phase (OD 600 about 0.4-0.6); 1 mL of helper phage M13 at a titer of 10 12 cfu / mL or more was added 13 K 07 After standing at 37°C for 30 min, 250 rpm culture was carried out for 1 h, Kana (kanamycin, working concentration 50 μg / mL) was added, and 250 rpm culture was carried out at 37°C overnight. The bacterial solution was transferred to a centrifuge bottle, centrifuged at 12,000 rpm at 4°C for 15 min, the supernatant was taken, 1 / 4 volume of PEG / NaCl was added, and ice bath was carried out for 2.5 h or more. Centrifugation was carried out at 12,000 rpm at 4°C for 15 min, the supernatant was discarded, the precipitate was resuspended with 750 μL of TBS, transferred to a 1.5 mL centrifuge tube, centrifuged at 4,000 rpm at 25°C for 5 min, and filtered through a 0.22 μm polyether sulfone filter membrane. 10 μL of phage was taken to determine the titer, the rest was mixed evenly, 50% sterile glycerol (v / v) was added at a final concentration, and it was stored at -80°C. Thus, the doxycycline nanobody phage library was obtained, which can be directly used for affinity panning.

[0087] Example 2 Affinity panning of nanobodies and identification thereof

[0088] I. Experimental methods

[0089] 1. Affinity panning of nanobodies

[0090] (1) Antigen and carrier protein immobilization

[0091] Affinity panning uses a strong adsorption enzyme-labeled plate with strong adsorption. One column is coated for each round of panning, and four columns are coated for four rounds. AB wells were coated with the original carrier protein OVA diluted to 1 mg / mL, and CDEF wells were coated with CB coating solution to dilute the detection antigen DX-A-OVA to 10 μg / mL, added to the micro-wells of the strong adsorption plate, 100 μL per well, and incubated at 37°C overnight. In addition, since the llama may be immune to multiple carrier proteins, three immune carrier proteins, ConA (concanavalin A), LF (lactoferrin), and KLH (keyhole limpet hemocyanin), were mixed and diluted to a final concentration of 2 mg / mL, and one column of immune antigen carrier protein was coated separately. The next day, after washing the plate twice with PBST (0.01M PBS, 0.05% Tween-20), 120 μL of 1% fish gelatin solution was added to each well and incubated at 37°C for 3 h. The liquid in the wells was poured out and dried on absorbent paper, and the plate was dried at 37°C for 1 h and stored at 4°C for later use.

[0092] (2) Positive phage screening

[0093] The phage library in Example 1 was added to 2 immunogen carrier protein wells, 150 μL per well, and incubated at 37°C for 1 h (only the first round needs this step, the second, third, and fourth rounds directly start from the AB well). The free phage was transferred to the coated original carrier protein AB well, 150 μL per well, and incubated at 37°C for 1 h. The free phage was transferred to 3 micro-wells with immobilized antigen (DX-A-OVA, 10 μg / mL), 100 μL per well, and incubated at 37°C for 1 h. The free phage in the wells was discarded, the micro-wells were washed with PBST (0.01M PBS, 0.05% Tween-20 (v / v)) for 10 times, and then washed with PBS for 5 times. 100 μL of 10 mg / mL trypsin-TBS solution was added for elution at 37°C for 30 min. The phage was collected, 10 μL of the eluted phage was taken to determine the titer, and the rest was used to infect 5 mL of TG1 strain growing to the logarithmic phase. E. coli The next day, the amplified phage was precipitated by PEG / NaCl, and the titer of the phage was determined.

[0094] In the second, third, and fourth rounds of the screening process, the plate coating concentration of DX-A-OVA was reduced to 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. After incubation with the phage at 37°C for 1 h, the micro-wells were washed with PBST (0.01M PBS, 0.05% Tween-20 (v / v)) and PBS, and then the drug competition elution method was used, that is, a certain concentration of drug was added, incubated at 37°C for 1 h, and the liquid in the wells was aspirated, which was the eluted phage. The screening scheme of step (2) was repeated. The drug elution concentration was 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. The above conditions can be adjusted according to the actual immunization situation. If the serum titer is low, the detection antigen DX-A-OVA concentration during the screening process can be appropriately increased; if the inhibition rate is low, the drug concentration of the competition reaction can be appropriately increased.

[0095] 2. Identification of positive clones

[0096] An indirect enzyme-linked immunosorbent assay was used to identify positive phage clones. The specific method is as follows:

[0097] (1) Antigen immobilization

[0098] The detection antigen DX-A-OVA was diluted with coating solution to 1 μg / mL, 100 μL per well, and incubated at 37°C overnight. The next day, the plate was washed twice with PBST (0.01M PBS, 0.05% Tween-20), 120 μL of 2% skim milk powder solution was added to each well, and incubated at 37°C for 3 h. The liquid in the wells was poured out and dried on absorbent paper, the plate was dried at 37°C for 1 h, and stored at 4°C for standby.

[0099] (2) Nanobody small expression

[0100] Output titer determination of the 3rd, 4th round of panning Randomly picked 96 single colonies from the plate of each round, inoculated into 96-well deep well plates containing 0.5 mL LB-Amp per well, inoculated a TG1 monoclonal as a negative control at the same time, 37°C, 180 rpm overnight culture, as a bacterial liquid "mother plate".

[0101] Take 10 μL of bacterial liquid from each well of the mother plate and inoculate it into another 96-well deep well plate containing 1 mL of LB-Amp per well. The well number should correspond to the mother plate. Incubate at 37°C, 180 rpm for 4 hours. Add IPTG (1:1000 ratio, v / v) per well. Incubate at 37°C, 180 rpm overnight.

[0102] (3) ELISA identification of positive clones

[0103] Centrifuge at 4000 rpm on the third day to collect the supernatant. Add 50 μL of supernatant to the already coated ELISA plate, incubate at 37°C for 40 min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), 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 40 min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the wells, add 100 μL of TMB substrate solution, develop color at 37°C for 10 min in the dark; add 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; measure the absorbance value at 450 nm with an ELISA reader. Select OD 450 Phage clones with OD450nm value greater than negative control by 3 times are positive clones.

[0104] Select OD 450Phage clones more than 3 times of negative were positive clones for positive nanobody identification. Add titer group: 50 μL supernatant of positive clones identified by indirect ELISA and 50 μL PBS; inhibition group: 50 μL supernatant of positive clones identified by indirect ELISA and 50 μL doxycycline standard (concentration 1 μg / mL), 37°C incubation for 40 min, washing five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), tapping dry the liquid in the well, adding 100 μL HRP-labeled anti-VHH secondary antibody diluted 1:5000, 37°C incubation for 40 min, washing five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), tapping dry the liquid in the well, adding 100 μL TMB substrate solution, 37°C color development for 10 min in the dark; adding 50 μL stop solution (10% H2SO4, v / v) to terminate the reaction; measuring the absorbance value at 450 nm with a microplate reader.

[0105] Selecting the clones with OD value more than 3 times of negative control hole and obvious inhibition in plate 1, recording the corresponding hole number of the clones as Nb DX-6F, and transferring the bacterial liquid in the corresponding hole of the mother plate to a sterile centrifuge tube, and adding glycerol for storage.

[0106] Thus, a nanobody capable of specifically recognizing doxycycline was obtained, which was named Nb DX-6F.

[0107] Example 3 Sequencing of the specific nanobody Nb DX-6F coding gene and determination of the amino acid sequence thereof

[0108] I. Experimental method

[0109] The strain of the specific nanobody 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 nanobody Nb DX-6F; and the amino acid sequence of the specific nanobody Nb DX-6F was obtained according to the DNA sequencing result and the codon table.

[0110] II. Experimental results

[0111] The amino acid sequence of the VHH of the specific nanobody Nb DX-6F is shown in SEQ ID No. 1:

[0112] EVQLVQSGGGSVQAGGSLRLSCAAPGYIYSSSCMGWFRQAPGKEREGVAGISTGGGPTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAVGPGGSCPRPTRYWGQGTLVTVSS.

[0113] The amino acid sequence and domain division of the specific nanobody Nb DX-6F are shown in the following table: Figure 1 As can be seen from the table, the specific nanobody Nb DX-6F comprises 4 framework regions (FR) and 3 complementarity-determining regions (CDR).

[0114] The framework regions (FR1-FR4) are respectively shown in the following table:

[0115] SEQ ID No. 2: EVQLVQSGGGSVQAGGSLRLSCAAP,

[0116] SEQ ID No. 3: MGWFRQAPGKEREGVAG,

[0117] SEQ ID No. 4: YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC,

[0118] SEQ ID No. 5: WGQGTLVTVSS.

[0119] The complementarity-determining regions (CDR1-CDR3) are respectively shown in the following table:

[0120] SEQ ID No. 6: GYIYSSSC,

[0121] SEQ ID No. 7: ISTGGGPT,

[0122] SEQ ID No. 8: AVGPGGSCPRPTRY.

[0123] The nucleotide sequence of the VHH of the specific nanobody Nb DX-6F is shown in SEQ ID No. 9:

[0124] GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCCTGGATACATCTACAGTAGCAGCTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGGTCGCAGGTATTTCGACTGGTGGTGGTCCCACATACTATGC CGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGCGCGGTAGGTCCCGGTGGTAGCTGTCCCGACCGACGCGTTACTGGGGCCAGGGGGACCCTGGTCACCGTCTCCTCA.

[0125] Example 4: Large-scale preparation of specific nanobody Nb DX-6F

[0126] I. Experimental Methods

[0127] The specific nanobody Nb DX-6F was prepared by protein expression, and the specific method is as follows:

[0128] The pComb3xss-VHH plasmid containing the sequencing data was transformed into [a specific gene / enter] using chemical transformation. E. coli In BL21(DE3). Pick a single colony from the transformation plate and inoculate it into 10 mL of LB (Amp) medium, incubating overnight at 37°C and 250 rpm. Dilute the overnight culture 1:100 into 750 mL of LB (Amp) medium and incubate at 37°C and 250 rpm until OD21 (distillation time is reached). 600 When the protein level is approximately 0.4–0.6, add IPTG (1:1000 ratio, v / v) and incubate overnight at 37°C and 250 rpm. The next day, centrifuge at 4°C and 12,000 rpm for 5 min, collect the bacterial pellet, and centrifuge at 12,000 rpm for 10 min using the sucrose osmotic pressure freeze-thaw method. Collect the supernatant to obtain the periplasmic cavity soluble protein.

[0129] Utilizing the histidine tag carried by Nb DX-6F to interact with contaminating proteins on Ni 2+The difference in the adsorption capacity of the affinity chromatography column realizes the separation and purification of the antibody. The specific operation steps are as follows: after the periplasmic cavity protein is filtered through a 0.22 μm filter membrane, 1 mL of Ni-NTA resin filler is added and is allowed to stand overnight; the next day, it is transferred to a gravity column; the gravity column is washed and balanced using 0.01 mol / L PBS, the above-mentioned mixed solution is added, and the loading is repeated twice, then PBS is added to balance and wash the gravity column, then 25 mmol / L imidazole-PBS is added for washing of impurities, finally, 200 mmol / L imidazole-PBS is used for elution of the target protein, and the eluate is collected, which is the doxycycline nanobody Nb DX-6F. The collected eluate is transferred to a 3 kDa dialysis bag and dialyzed in PBS at 4°C for 3 days, for a total of 6 times. After dialysis, it is stored at -20°C for standby.

[0130] The nanobody Nb DX-6F is obtained.

[0131] Example 5 Analysis of the detection performance of the specific nanobody Nb DX-6F

[0132] I. Experimental method

[0133] 1. Coating and blocking

[0134] The DX-A-OVA is diluted to 1 μg / mL with the coating solution, and is coated at 37°C overnight. The next day, after washing twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), 1% fish gelatin solution is added at 120 μL per well, and is blocked at 37°C for 3 h, is dried at 37°C for 1 h, and is stored at 4°C in a sealed bag.

[0135] 2. Establishment of a standard curve

[0136] (1) Experimental method

[0137] The DX-A-OVA coating agent was diluted to 1 μg / mL with coating buffer and coated overnight at 37°C. The next day, the sample was washed twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), then 120 μL / well of 2% skim milk powder solution was added, and the sample was blocked at 37°C for 3 h. The blocking solution was then discarded, and the sample was dried at 37°C for 1 h. Add 50 μL of nanobody and a series of 50 μL of doxycycline standards at different concentrations to each well, incubate at 37°C for 40 min, wash five times with PBST, 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, 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 a microplate reader. Plot the concentration of doxycycline standards against the x-axis, B / B0 (OD of the wells containing doxycycline). 450 OD of pores without doxycycline 450 Using y as the ordinate, establish an indirect competition standard curve.

[0138] II. Experimental Results

[0139] The indirect competitive ELISA standard curve based on the specific nanobody Nb DX-6F is shown in the figure. Figure 2 As shown, the standard curve exhibits an S-shape, indicating good linear correlation. This method detects doxycycline in the range of 0.17–14.25 ng / mL, with a half-inhibitory concentration (IC50) of [missing value]. 50 The detection limit is 1.34 ng / mL, the detection limit (LOD) is 0.05 ng / mL, and the detection sensitivity is high.

[0140] Example 6: Organic Tolerance Analysis of Nanobody Nb DX-6F

[0141] I. Experimental Methods

[0142] The nanobody NbDX-6F was diluted to the same working concentration using methanol and ethanol at different concentrations (10%, 20%, 30%, 40%, 50%) to determine its binding affinity to the antigen. The binding affinity of the antibody to the antigen without dilution with organic solvents was taken as 100%, and the nanobody's tolerance to different organic solvents and different concentrations of the same organic solvent was evaluated. The specific method is as follows:

[0143] Add 50 µL of diluted Nb DX-6F 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.

[0144] II. Experimental Results

[0145] The activity curves of the nanobody Nb DX-6F with different ratios of methanol and ethanol as diluents are shown in the figure. Figure 3 As shown, the antigen-binding activity of the nanobody NbDX-6F remains above 85% at 20% methanol and ethanol concentrations, and above 60% at high concentrations of 40% methanol and ethanol. Therefore, the nanobody NbDX-6F exhibits excellent resistance to organic solvents, and is less affected by organic solvents during the pretreatment process for actual sample detection, resulting in high accuracy of the detection results.

[0146] Example 7: Acid-base tolerance analysis of nanobody Nb DX-6F

[0147] I. Experimental Methods

[0148] Different pH PBS buffer solutions were prepared (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). These solutions were used as diluents to dilute Nbs to the working concentration. The bioactivity of the antibody in these systems was measured, with the antigen-antibody binding capacity in PBS buffer solution at pH 7.4 considered as 100%. The acid-base tolerance of the nanobody was evaluated. The specific method is as follows:

[0149] Add 50 μL of diluted nanobody Nb DX-6F and 50 μL of PBS into the packaged enzyme-labeled plate, incubate at 37℃ for 40 min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the hole, add 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, 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 hole, add 100 μL of TMB substrate solution, develop color at 37℃ for 10 min in the dark; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance value at 450 nm with an enzyme-labeled instrument.

[0150] II. Experimental results

[0151] Considering the complexity of the application environment and the diversity of samples, neither acidic nor alkaline environment is conducive to detection. In this experiment, the stability of doxycycline nanobody Nb DX-6F in the pH range of 1.4-12.4 was investigated, and the results are shown in Table 1. Figure 4 It can be seen that Nb DX-6F has excellent acid and alkali resistance, and the acid and alkali resistance of the nanobody is quite good and performs better in the pH range of 2.4-11.4, with very high antigen binding activity (>80%). Even in an acidic environment (pH=2.4-6.4), the antigen binding activity will increase, which indicates that Nb DX-6F has high acid and alkali resistance and can be used for detection in acid and alkali environments.

[0152] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

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

1.

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

9.

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

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

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

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

7. A kit for detecting doxycycline, characterized by, containing the nanobody Nb DX-6F of claim 1.

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

9. The immunoassay method of claim 8, wherein, The structural formula of the doxycycline hapten is shown as formula (I): Formula (I).

10. The method of claim 8, wherein the step of detecting is characterized by, The carrier protein is ovalbumin. The structural formula of the doxycycline hapten is shown as formula (I):

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