Preparation of novel thiacloprid and ochratoxin A recognition element bispecific antibody and application of novel thiacloprid and ochratoxin A recognition element bispecific antibody in enzyme-linked immunosorbent assay synchronous detection

Abstract

The invention discloses preparation of a novel thiacloprid and ochratoxin A recognition element bispecific antibody and application of the novel thiacloprid and ochratoxin A recognition element bispecific antibody to enzyme-linked immunosorbent assay synchronous detection, and belongs to the technical field of molecular biology. The invention provides a bispecific antibody of thiacloprid and ochratoxin A. The bispecific antibody of thiacloprid and ochratoxin A is expressed in Expi293F cells and is used for preparing an ELISA (enzyme-linked immuno sorbent assay) detection reagent, so that bispecific detection of thiacloprid and ochratoxin A can be effectively realized. The linear ranges of thiacloprid and ochratoxin A detected by the bispecific antibody provided by the invention are respectively 0.05 to 1000ng / mL and 0.05 to 500ng / mL, and the IC50 (half maximal inhibitory concentration) is respectively 17.10 ng / mL and 16.69 ng / mL.

Description

Technical Field

[0001] The invention relates to the preparation of a novel recognition element bispecific antibody of thiacloprid and ochratoxin A and its application in enzyme-linked immunosorbent assay synchronous detection, belonging to the technical field of molecular biology. Background Art

[0002] Pesticide residues and mycotoxin contamination are important hidden dangers in the production, storage, processing and sales of agricultural products. Thiacloprid (TCL) is widely used in agricultural product production due to its broad-spectrum and high-efficiency properties, but its residue is high. Ochratoxin A (OTA), as a typical mycotoxin, is highly harmful and has a wide range of contamination, with residues in cereals and fruit and vegetable agricultural products. However, the immunoassay method for the simultaneous detection of these two harmful substances usually uses a combination of multiple monospecific antibodies, which has the disadvantage of high detection costs. Therefore, it is very important to establish a simultaneous detection method for these two pollutants based on bispecific antibodies.

[0003] Due to the complex relationship between the environmental system and the food supply chain system, thiacloprid in the environment will inevitably enter the human food chain, so the detection of thiacloprid residues in food is of great significance. At present, the detection of TCL in food mainly includes large-scale instrumental methods such as chromatography and mass spectrometry and immunoassays such as enzyme-linked immunosorbent assay and fluorescent immunoassay. Ochratoxin A has many transmission pathways in the food chain, so there are many types of food contaminated by ochratoxin A. In order to reduce the risk of human exposure to OTA and reduce the spread of OTA in the food chain, more and more researchers are committed to the development of OTA detection technology, including instrumental analysis and immunoassay. However, the more common rapid test products are mostly single-component detection based on traditional immune antibodies, which cannot achieve simultaneous detection of pesticides and toxins, and the antibody preparation is complex and the cycle is long. The bispecific antibody recognition element prepared based on genetic engineering recombinant technology has solved the above problems well, and can be quickly expressed in vitro to achieve simultaneous detection of cross-class harmful substances. Summary of the invention

[0004] The first object of the present invention is to provide a thiacloprid and ochratoxin A detection antibody, the amino acid sequence of which is shown in SEQ ID NO.6.

[0005] In one embodiment, the thiacloprid detection antibody comprises a single-chain antibody and a nanobody variable region, the amino acid sequence of the single chain is shown in SEQ ID NO.4; the nanobody variable region sequence is shown in SEQ ID NO.5.

[0006] The present invention also provides genes encoding the thiacloprid and ochratoxin A detection antibodies.

[0007] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.3.

[0008] The present invention also provides a recombinant vector containing the above gene.

[0009] In one embodiment, pcDNA3.1 is used as the expression vector.

[0010] The present invention also provides cells containing the above gene or the above recombinant vector.

[0011] In one embodiment, the cell is a mammalian cell line.

[0012] In one embodiment, the cell is an Expi293F cell.

[0013] The present invention also provides a method for preparing a combined antibody of thiacloprid and ochratoxin A, wherein the method comprises expressing the nucleotide sequence shown in SEQ ID NO.3 in Expi293F cells.

[0014] In one embodiment, the method is to use pcDNA3.1 as an expression vector to express the gene encoding the combined antibody of thiacloprid and ochratoxin A.

[0015] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.3.

[0016] The present invention also provides the use of the thiacloprid and ochratoxin A combined antibody in detecting thiacloprid and ochratoxin A.

[0017] The present invention also provides a kit for jointly detecting thiacloprid and ochratoxin A. The kit contains the thiacloprid and ochratoxin A combined antibody, or the recombinant vector, or the cell.

[0018] In one embodiment, the kit further comprises any one of the following (a) to (c):

[0019] (a) a conjugate of a hapten of thiacloprid and ochratoxin A and a carrier protein, and an enzyme marker of the thiacloprid and ochratoxin A detection antibody;

[0020] (b) a conjugate of a hapten of thiacloprid and ochratoxin A and a carrier protein, and an enzyme-labeled antibody for detecting the thiacloprid and ochratoxin A antibody;

[0021] (c) enzyme markers for the thiacloprid and ochratoxin A detection antibodies.

[0022] In one embodiment, the kit further contains thiacloprid and ochratoxin A standards.

[0023] The present invention also provides the use of the gene, or the recombinant vector, or the cell, or the kit in detecting thiacloprid and ochratoxin A, and the use is not for the purpose of diagnosing and / or treating a disease.

[0024] Beneficial effects:

[0025] The present invention provides a bispecific antibody for thiacloprid and ochratoxin A, wherein the bispecific antibody is composed of a single-chain antibody and a nano antibody variable region, wherein the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.4, and the nucleotide sequence of the nano antibody variable region is shown in SEQ ID NO.5. The linear ranges of the bispecific antibody provided by the present invention for detecting thiacloprid and ochratoxin A are 0.05-1000 ng / mL and 0.05-500 ng / mL, respectively, the LODs are 0.337 ng / mL and 0.788 ng / mL, respectively, and the IC50s are 17.10 ng / mL and 16.69 ng / mL, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a target gene design scheme for thiacloprid-ochratoxin A;

[0027] Figure 2 The figure is the result of SDS-PAGE of the bispecific antibody of thiacloprid and ochratoxin A obtained by the eukaryotic expression system of the present invention;

[0028] Figure 3 The comparison chart of the thiacloprid and ochratoxin A antibodies expressed in the present invention and antibodies expressed in other expression systems is shown in Figure 2. At 5 μg / mL OTA-BSA and 2.5 μg / mL TCL-OVA coated antigen concentrations, the OD value of the antibody after color development after 15 minutes of recognition of blank and antigen 450 Numeric value;

[0029] Figure 4 This is the enzyme-linked immunosorbent assay effect established based on the expressed thiacloprid and ochratoxin A bispecific antibody of the present invention. DETAILED DESCRIPTION

[0030] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0031] Unless otherwise specified, the cells, culture media, transformation reagents, etc. used in the present invention are all purchased through normal channels.

[0032] Expi293 in the present invention TMThe expression medium was purchased from Gibco, Opti-MEM I was purchased from thermofisher official website, Transfection-reagent was purchased, enhancer was purchased from thermofisher official website, and DON-BSA antigen was purchased from thermofisher official website.

[0033] Example 1: Construction of antibody expression plasmid

[0034] The heavy chain of the nucleotide sequence shown in SEQ ID NO.3 was constructed between the EcoRI and BamHI restriction sites of the pcDNA3.1 vector to obtain a plasmid expressing the thiacloprid and ochratoxin A bispecific antibody.

[0035] Example 2: Construction of a eukaryotic expression system

[0036] The purchased Expi293F cells (1×10 7 ), thawed at room temperature (37°C), and then centrifuged rapidly at 350 g, the excess supernatant removed, and transferred to fresh Expi293 TM (Thermo Fisher Scientific) expression medium at 37°C, 5-8% CO2. The cells were passaged every 3-4 days after thawing. Slower growth rate and lower cell viability may be observed in the first two passages of cells after thawing. After 3-4 passages, a certain amount of cells were harvested and transferred to fresh Expi293 TM Expression medium awaits transfection.

[0037] 24 h before transfection, cells were counted and analyzed using Expi293 TM The cell density was adjusted to 2.5 × 10 6 In this example, 30 mL of culture medium was transfected and 75×10 6 cells were added to a new 125 mL flask.

[0038] On the day of transfection, count the cells and determine viability. 6 cells were added to a new 125 mL flask and used Expi293 TM Expression medium: Adjust the volume to 25.5 mL, according to the volume of transfection medium.

[0039] Example: According to the transfection volume of 30mL, in order to make the light chain and heavy chain assemble better, the ratio between the two is set to 3:2, take 30μg of thiacloprid and ochratoxin A plasmid, dissolve in 1.5mL of Opti-MEM I, add 81μL of Transfection-reagent to 1.5mL of Opti-MEM I, let it stand for 10min, mix and let it stand for 10min, then slowly add it to the culture medium, culture at 125rpm for 16-22h, add 80μL of enhancer I and 1.5mL of enhancer II and continue to culture.

[0040] Example 3: Protein purification

[0041] When designing the target gene, a 6×His tag was designed at the C-terminus of the heavy chain gene to facilitate purification. Figure 1 , the nucleotide sequence is shown in SEQ ID NO.3.

[0042] The successfully transfected Expi293F cells were cultured for 7 days, and the entire culture system was collected. The culture system was centrifuged at 3200r / min for 20min, and the supernatant was taken and filtered with a 0.22μm filter membrane. The supernatant after filtration was passed through a nickel column 3-5 times to allow the bispecific antibody with a 6×His tag to fully bind to the nickel column. After fully washing with a washing solution containing 25mM imidazole to remove non-specifically bound impurities, the target protein bound to the nickel column was eluted with an elution solution containing 250mM imidazole, and the protein concentration was 50μg / mL.

[0043] The eluted target protein was run on SDS-PAGE, and obvious bispecific antibody protein bands were observed on the protein gel ( Figure 2 ).

[0044] Example 4: ELISA detection of antibody activity

[0045] 1. Determine the linear range of the test

[0046] (1) Coating: 100 μL / well of TCL-BSA and OTA-BSA antigens dissolved in phosphate buffer were used for coating. The concentrations of TCL-BSA and OTA-BSA were adjusted to 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL and 7.5 μg / mL, respectively. The reaction was incubated at 37°C for 2 h.

[0047] (2) Washing: Pour off the solution in the plate and wash 4 to 6 times with washing solution PBST, then pat dry.

[0048] (3) Blocking: After washing and patting dry, add 250 μL / well of 5% (V / V) skim milk powder blocking solution to the 96-well plate and react at 37°C for 2 h. After washing, dry and set aside.

[0049] (4) Antibody incubation: Add the expression antibody purified in Example 3 to the coated wells, add 100 μL of the expression antibody to each well, react at 37° C. for 1 h, then wash 4 to 6 times and pat dry.

[0050] (5) Secondary antibody incubation: Add 100 μL of goat anti-IgG-HRP enzyme-labeled secondary antibody (HRPConjugated Anti His-Tag Mouse Monoclonal Antibody) of appropriate dilution concentration to each well. Incubate the ELISA plate at 7°C for 30 min, wash the plate 4 to 6 times with PBST and pat dry.

[0051] (6) Color development: Add 50 μL of TMB color development solution A and solution B to each well and react at 37°C in the dark for 15 min.

[0052] (7) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then use an ELISA reader to measure the OD450nm value of each well ( Figure 3 ).

[0053] IC-ELISA was used to determine the IC50 of the bispecific antibody expressing thiacloprid and ochratoxin A. The IC50 values ​​of thiacloprid and ochratoxin A were 17.10 ng / mL and 16.69 ng / mL, respectively, and the linear ranges were 0.05-1000 ng / mL and 0.05-500 ng / mL, respectively, indicating that the antibody has a wide linear range for thiacloprid and ochratoxin A and can be used for immunoassay detection of thiacloprid and ochratoxin A ( Figure 4 ).

[0054] 2. Spike recovery experiment

[0055] In order to evaluate the accuracy of the developed simultaneous quantitative OTA and TCL ELISA kit, 0.1, 0.5, and 1 mg / kg of OTA and TCL standard solutions were added, respectively. The ELISA kit developed in this study was used for detection and verified by instrumental analysis. The results are shown in Table 1.

[0056] Table 1 Detection results of bispecific antibodies on samples

[0057]

[0058] Comparative Example:

[0059] In view of the difference in protein modification between Escherichia coli and animal cells in expression ability, single-chain antibodies and nanobodies are small molecule antibodies that are easy to express, so antibodies against thiacloprid and ochratoxin A were expressed in Escherichia coli.

[0060] E. coli expression: The expression vector of PET-28a(+) was used, and E. coli BL21 was used as the expression strain to express the antibodies shown in the nucleotide sequences of SEQ ID NO.7 and SEQ ID NO.8. The constructed recombinant E. coli cells were cultured in LB medium to an OD of 0.6, induced with a final concentration of 0.1 mM IPTG at 16°C for 18 h, the fermentation broth was collected, and the protein was purified using a purification kit.

[0061] The verification steps for antibody recognition ability refer to Example 4.

[0062] The results showed that the affinity constant of the antibody was 2.3×10 5 L / mol, indicating that the antibody recognition ability is extremely weak and the binding ability is poor, and it cannot be applied to actual detection.

[0063] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An antibody for the combined detection of thiacloprid and ochratoxin A, characterized in that: The amino acid sequence is shown in SEQ ID NO.

6.

2. A gene encoding the antibody according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. A recombinant vector carrying the gene according to claim 2 or 3.

5. A cell expressing the antibody of claim 1.

6. A method for preparing a combined antibody of thiacloprid and ochratoxin A, characterized in that: The nucleotide sequence shown in SEQ ID NO.3 was expressed in Expi293F cells using pcDNA3.1 as an expression vector.

7. The method according to claim 6, characterized in that Antibodies produced by the cells were also purified.

8. Use of the antibody according to claim 1 in detecting thiacloprid and ochratoxin A.

9. A kit for the combined detection of thiacloprid and ochratoxin A, characterized in that: Contains any of the following (a) to (c): (a) a conjugate of a hapten of thiacloprid and ochratoxin A with a carrier protein, and an enzyme marker of the antibody according to claim 1; (b) a conjugate of a hapten of thiacloprid and ochratoxin A with a carrier protein, or an enzyme-labeled antibody of the antibody of claim 1; (c) an enzyme-labeled substance of the antibody according to claim 1.

10. Use of the antibody according to claim 1, or the gene according to any one of claims 2 to 3, or the recombinant vector according to claim 4, or the cell according to claim 5, or the kit according to claim 9 in the detection of thiacloprid and ochratoxin A, wherein the use is not for the purpose of diagnosis and / or treatment of a disease.

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