CN111363722A - Monoclonal antibody of bacillus thuringiensis Cry2A toxin and application thereof - Google Patents

Abstract

The invention discloses a preparation method and application of an antibody for detecting Bt Cry2A toxin, wherein the antibody is obtained by immunizing Balb/c mice, and the preservation number of the antibody is CCTCC NO: c2019188 hybridoma cells are secreted; the application also provides a DAS-ELISA detection method based on the antibody, the minimum detection limit of the method is 10.76ng/ml, the cross reaction rate of the Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1F toxins based on 50 mu g/ml is less than 0.1%, the cross reaction rate of the Cry1B toxins is 3.2%, the specificity is strong, the method can be used for quickly detecting the Cry2A toxins, and the blank in the field of Cry2A toxin detection is filled.

Description

Monoclonal antibody of bacillus thuringiensis Cry2A toxin and application thereof

Technical Field

The invention relates to a monoclonal antibody of a bacillus thuringiensis Cry2A toxin common in transgenic plants and application thereof, which can be used for preparing a kit for detecting the Cry2A toxin common in the transgenic plants, and belongs to the technical field of biology.

Background

Bacillus Thuringiensis (BT), a crystal-producing Bacillus containing many varieties, is a gram-positive bacterium. The BT Cry toxin is widely applied to agricultural production due to the advantages of high specificity to target insects, safety to human, livestock and beneficial organisms, no pollution to the environment and the like, but in recent years, people have increasing worry about the problems of environmental ecological safety and potential safety hazard due to the use of Bt gene-transgenic insect-resistant plants. The detection of transgenic Bt plants at present mainly aims at recombinant DNA and Cry toxin protein.

Currently, generally accepted methods for accurately and specifically detecting a transgenic Bt plant mainly include PCR, real-time PCR, High Performance Liquid Chromatography (HPLC), ELISA, colloidal gold immunochromatography test strips, and the like. However, although the PCR method has high sensitivity and specificity, the expression level of the Cry toxin cannot be determined, and it is additionally time-consuming and requires a specialized instrument to be operated by a professional. For the HPLC determination method, expensive instruments are needed, the cost is high, trained professionals are needed for operation, the pretreatment is complex, the consumed time is long, and the method cannot be used for rapid field detection. The immunological detection method, such as ELISA and colloidal gold immunochromatography test strip, has simple operation, does not need expensive instruments, and can detect the target protein quantitatively or semi-quantitatively in a short time.

Immunological detection techniques are mainly established on the basis of specific reactions of antigens and antibodies, i.e., antigen-antibody reactions, i.e., specific reactions between antigens and antibodies. Immunological assays also exist in both competitive and non-competitive formats. The non-competitive immunoassay technology is a mode superior to the competitive immunoassay technology, and has higher specificity, higher sensitivity and less antigen dosage.

DAS-ELISA (double sandwich ELISA detection) is the most widely used method in Bt Cry toxin detection, where the analyte is sandwiched between a capture antibody and a detection antibody. At present, a plurality of detection methods based on DAS-ELISA are applied to the detection of Cry toxin, such as Cry1Ab, Cry1Ac and the like. In 2015, Wuaihua and the like screen and obtain dodecapeptide capable of specifically binding Cry2A from phage display dodecapeptide, and an indirect competition ELISA method for detecting Cry2A toxin is established based on the dodecapeptide. However, due to the presence of the common structure, monoclonal antibodies detected for certain Bt toxins have some cross-reactivity to other toxins. (Roda et al development and identification of a sensitive and fantastic enzyme immunoassay for the detection of genetic modification dmaize, 2006; Zhang rt ai, Rapid isolation of single-chain antibodies from a human synthetic phase display for the detection of Bacillus thuringiensis (Bt) Cry1B toxin, 2012). At present, a Cry 1A-type detection method (Dongsa et al.production and chromatography of Monoclonal Antibody Broadly recognition Cry1 Toxins by use of Designed polypeptides as happten, 2016) exists, but a Monoclonal Antibody detection method against Cry2A toxin has not been reported.

Disclosure of Invention

Aiming at the problems of long time consumption and cross reaction of Cry2A toxin detection, the application provides a Bacillus thuringiensis Cry2A toxin monoclonal antibody, the Cry2A monoclonal antibody is used for DAS-ELISA detection, the cross reaction rate is low, on the premise of coating completion, whether Cry2A exists in a sample can be rapidly detected within 4-5 hours, the detection result shows obvious color change (blue), rapid and accurate detection of Cry2A toxin is realized, and the time is saved.

The application is realized by adopting the following technical scheme:

the application firstly provides a bacillus thuringiensis Cry2A monoclonal antibody hybridoma cell strain 1D2D11, wherein the preservation number of the cell strain 1D2D11 is CCTCC NO: c2019188, the preservation unit is China Center for Type Culture Collection (CCTCC), the address: wuhan, China, zip code: 430072, deposit name: the hybridoma cell strain 1D2D11 has a preservation time of 2019, 10 months and 30 days.

Secondly, the application also provides a new product with the preservation number of CCTCC NO: c2019188 hybridoma cell strain 1D2D11, and a Bacillus thuringiensis Cry2A monoclonal antibody.

Third, the present application provides a method of preparing a culture medium consisting of a nucleic acid sequence with a preservation number of CCTCC NO: the application of the Bacillus thuringiensis Cry2A monoclonal antibody prepared from the hybridoma cell strain 1D2D11 of C2019188, namely a DAS-ELISA non-competitive detection method aiming at Bt Cry2A established by utilizing the monoclonal antibody can be used for detecting whether Bt Cry2A genes are carried or remained in crop leaves in a laboratory or a field, the method breaks through the cross reaction phenomenon of a common structure in non-competitive detection, and can specifically detect the Bt Cry2A toxin concentration in a sample of 0.010763-4 mu g/ml range;

the detection method comprises the following specific steps:

(1) coating the capture antibody: diluting a polyclonal antibody of Cry2A toxin (the preparation method is referred to as 'preparation and application analysis of polyclonal antibody of canine procalcitonin protein', Wangman, Shandong university of agriculture '2019 Master thesis') with PBS buffer solution to 0.5 mu g/ml, adding the diluted solution into a 96-well enzyme label plate, adding the diluted solution into the 96-well enzyme label plate, and keeping the solution at 100 mu l/well overnight at 4 ℃;

(2) and (3) sealing: the next day the plates were washed 3 times with PBST. Adding 200 μ l of 5% MPBS per well, and incubating at 37 deg.C for 1 h;

(3) adding a sample: PBST washes the board three times, add to the hole after diluting the sample to be measured in CBS buffer solution 2 times, 100 mul/hole, regard CBS as the blank control at the same time, incubate 1h at 37 ℃;

(4) adding the antibody to be detected: PBST is washed for three times, and 0.5 mu g/ml of CCTCC NO: the Cry2A monoclonal antibody secreted by the hybridoma cell strain 1D2D11 of C2019188 is injected into a mouse, and the antibody (100 mu l/hole) obtained after ascites purification is placed in an incubator at 37 ℃ for incubation for 1 h;

(5) adding an enzyme-labeled antibody: washing the plate 3 times with PBST, adding 100 μ l HRP-labeled goat anti-mouse antibody (1: 5000) per well, and incubating at 37 deg.C for 1 h;

(6) color development: PBST washing plate for 5 times, adding TMB color development solution into the micropore of the ELISA plate, 100 mul/hole, and incubating in the incubator at 37 ℃ for 15 min; whether the sample contains the Cry2A toxin or not can be detected in the range of 0.010763-4 mug/ml, and the sample shows blue color change.

Fourthly, the application provides a DAS-ELISA kit for detecting Bt Cry2A, which comprises a nucleic acid sequence with a preservation number of CCTCC NO: the hybridoma cell strain 1D2D11 of C2019188 secretes the Bacillus thuringiensis Cry2A monoclonal antibody.

Aiming at the common Bt Cry2A gene in transgenic plants, protoxin is selected as an immune antigen, a Balb/c mouse is immunized, and a positive hybridoma cell strain capable of efficiently secreting monoclonal antibodies is obtained through cell fusion and subclone screening, compared with the existing detection method of Bacillus thuringiensis Cry2A, the method has the following advantages and beneficial effects:

(1) the hybridoma cell strain 1D2D11 obtained by the invention has the preservation number of CCTCC NO: the monoclonal antibody secreted by the C2019188 can specifically recognize Bt Cry2A and has the characteristic of detecting transgenic Bt Cry2A protein.

(2) The hybridoma cell strain 1D2D11 obtained by the invention has the preservation number of CCTCC NO: DAS-ELISA detection established by the monoclonal antibody secreted by the C2019188 breaks through the common cross reaction phenomenon of common structures in non-competitive detection, can specifically detect Bt Cry2A toxin, and has the lowest detection limit of 10 ng.

(3) The hybridoma cell strain 1D2D11 obtained by the invention has the preservation number of CCTCC NO: the monoclonal antibody generated by the C2019188 can be applied to detection and screening of transgenic plants such as Western blotting (Western blotting), DAS-ELISA, indirect ELISA, genetic engineering and the like, has sensitive reaction and short detection time (4-5h), and has extremely high practical application value.

Drawings

FIG. 1 is a schematic diagram showing the effect of antibody purification;

FIG. 2 is a schematic representation of the specificity of monoclonal antibodies;

FIG. 3 is a schematic diagram of the assay concentration of the optimal capture antibody;

FIG. 4 is a diagram showing a standard curve established by DAS-ELISA at an optimal antibody concentration;

FIG. 5 is a schematic representation of Cry1B cross-reaction standard curve.

Detailed Description

The present invention is further described with reference to the drawings and the detailed description below, so that those skilled in the art can more clearly understand the technical solutions of the present invention, and the present invention is not limited thereto.

Reagents, instruments, mouse sources to which the following examples refer:

freund's complete adjuvant, Freund's incomplete adjuvant, 50% by mass of PEG, HAT, all purchased from Sigma company;

the Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F proteins are all purchased from Shanghai Youlong Biotech limited;

the Cry2A protein is purchased from Envirologix, USA;

balb-c mice were purchased from the comparison center of Yangzhou university;

HiTrap Protein G HP was purchased from GE;

mouse myeloma cells sp/20 are provided by the laboratory;

RPMI-1640 medium and fetal bovine serum were purchased from Gibco;

one-component TMB color former (PR1200) was purchased from Solarbio;

HRP-labeled goat anti-mouse antibody was purchased from KPL corporation. Other reagents, unless otherwise specified, were obtained commercially.

Example 1 preparation of immunized mice and monoclonal antibodies

1. Immunization of mice

Emulsifying Cry2A protein with Freund complete adjuvant (see product instruction), injecting 6-8-year female Balb-c mice intraperitoneally at a dose of 50 μ g/mouse, then performing booster immunization once every 14 days, emulsifying Cry2A protein with Freund incomplete adjuvant at a dose of 50 μ g/mouse, cutting tail after one week of each immunization, collecting blood, measuring titer, and selecting the mouse with highest titer (6.4 × 10) after no significant increase in titer⁵) Injecting the mixture into abdominal cavity for impact immunization, and mixing the antigen with normal saline uniformly, wherein the dosage is 50 mu g per mouse.

2. Preparation of monoclonal antibodies

When the titer reaches 640000 times of serum dilution and the OD450 value is greater than 1.0, collecting ascites of a mouse, preparing the mouse monoclonal antibody according to a conventional monoclonal antibody preparation technology (the specific antibody preparation technology refers to' Liu Jing et al, preparation and characteristics of duck skeletal muscle troponin I monoclonal antibody, 2019), and finally obtaining the mouse monoclonal antibody prepared by the cell strain 1D2D 11.

The applicant reserves the hybridoma cell strain named hybridoma cell strain 1D2D111 in 2019, 10 months and 30 days in the chinese typical culture collection at address: wuhan university in Wuhan, China, zip code: 430072 with preservation number of CCTCC NO: C2019188.

3. Purification of monoclonal antibodies

Antibodies were purified from ascites fluid obtained in step 2 using HiTrap Protein G HP affinity chromatography as described. The purity of the antibody is identified by SDS-PAGE gel, the detection result is shown in figure 1, and in figure 1, a lane M is a Protein Marker; lane 1 is unmodified IgG and lane 2 is denatured IgG (denaturation method is boiling purified sample for 5min, see the literature "preparation of monoclonal and single chain antibodies to Bt Cry1 toxoid, and activity analysis thereof, 2017" for details). It can be seen from FIG. 1 that the antibody is relatively pure. The concentration measured by an ultraviolet micro-spectrophotometer is 3.65 mg/ml. Purified antibody was stored in aliquots at-80 ℃.

EXAMPLE 2 characterization of monoclonal antibodies

1. Subtype identification

The culture supernatant of the monoclonal antibody prepared in example 1 was assayed by using an immunoglobulin ELISA kit 88-50660-22 available from Thermo corporation according to the instructions.

The results show that the heavy chain subtype of the monoclonal antibody of the invention is IgG1 type, and the light chain subtype is kappa type.

2. Monoclonal antibody reaction specificity

Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1F and Cry2A proteins are selected, and the recognition specificity of the monoclonal antibody is detected by an indirect ELISA method.

The indirect ELISA method was as follows:

1. coating: seven proteins were diluted to 2. mu.g/ml with CBS buffer, coated at 100. mu.l/well and left overnight at 4 ℃.

2. And (3) sealing: the next day the plates were washed 3 times with PBST. 200. mu.l of 5% MPBS were added to each well and incubated at 37 ℃ for 1 h.

3. Sample adding: PBST washing plate three times, each hole adding PBS diluted to the final concentration of 50 u g/ml antibody, 37 degrees C were incubated for 1 h.

4. Adding an enzyme-labeled secondary antibody: PBST washing plate three times, will use 2% MPBS diluted 5000 times enzyme labeled secondary antibody (HRP-sheep anti mouse antibody) according to 100 u L/hole add to the plate hole of enzyme, 37 degrees C were incubated for 1 h.

5. Color development: PBST washing plate 5 times, adding single component TMB color developing solution into the micro-hole of the ELISA plate, 100 μ l/hole, and incubating for 15min in the incubator at 37 ℃.

6. Reading: with 2MH₂SO₄The reaction was stopped, 50. mu.l/well and absorbance was read at 450nm with a microplate reader. It can be seen that the sample reaction containing the Cry2A protein exhibited a significant blue change with a high OD value.

As shown in fig. 2, it can be seen that the 1D2D11 monoclonal antibody obtained in example 1 has a lower specificity reaction to Cry1B, but not to Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1F, indicating that the monoclonal antibody has better specificity.

EXAMPLE 3 establishment of monoclonal antibodies DAS-ELISA detection

1. The samples were analyzed by chessboard titration (Lorenzen N, Olesen N J,

p E V. Neutralizationof Egtved viruses pathology to cell cultures and fish by monoclonal antibodies to the viral G protein 1990) determination of the optimal concentration of antibodies

(1) Coating the capture antibody: the Cry2A toxin polyclonal antibody (the preparation method is referred to as 'preparation and application analysis of canine procalcitonin protein polyclonal antibody, 2019') is sequentially diluted to 0.1, 0.5, 1, 2 and 5 mu g/m by PBS buffer solution, added into a 96-well enzyme label plate, 100 mu l/well and kept at 4 ℃ overnight.

(2) And (3) sealing: the next day the plates were washed 3 times with PBST. 200. mu.l of 5% MPBS were added to each well and incubated at 37 ℃ for 1 h.

(3) Adding the antibody to be detected: PBST plates were washed three times, and 100. mu.l of Cry2A monoclonal antibody (concentration 0.1, 0.5, 1, 2, 5. mu.g/ml in this order) diluted with PBS was added to each well in a checkerboard format and incubated at 37 ℃ for 1h in an incubator.

(4) Adding an enzyme-labeled antibody: the plates were washed 3 times with PBST, 100. mu.l of HRP-labeled goat anti-mouse antibody (1: 5000) was added to each well, and incubated at 37 ℃ for 1 h.

(5) Color development: PBST washing plate for 5 times, adding TMB color development solution into the micropore of the ELISA plate, 100 μ l/hole, and incubating in the incubator at 37 ℃ for 15 min.

(6) Reading: with 2MH₂SO₄The reaction was stopped, 50. mu.l/well and absorbance was read at 450nm with a microplate reader.

In this example, the optimum concentrations of the capture antibody and the detection antibody determined from the range of momentum change for establishing the standard curve are determined as 0.5. mu.g/ml and 0.5. mu.g/ml, respectively, as shown in FIG. 3 (taking the optimum capture antibody assay as an example).

DAS-ELISA procedure

(7) Coating the capture antibody: the polyclonal antibody against Cry2A toxin was diluted to 0.5. mu.g/ml with PBS buffer, added to a 96-well microplate at 100. mu.l/well and overnight at 4 ℃.

(8) And (3) sealing: the next day the plates were washed 3 times with PBST. 200. mu.l of 5% MPBS were added to each well and incubated at 37 ℃ for 1 h.

(9) Adding a sample: the plates were washed three times with PBST, Cry2A toxin was dissolved in CBS buffer and diluted 2-fold before being added to the wells at 100. mu.l/well with CBS as a blank and incubated for 1h at 37 ℃.

(10) Adding the antibody to be detected: the PBST plates were washed three times, 0.5. mu.g/ml Cry2A monoclonal antibody (100. mu.l/well) was added to the wells, and incubated for 1h at 37 ℃ in an incubator.

(11) Adding an enzyme-labeled antibody: the plates were washed 3 times with PBST, 100. mu.l of HRP-labeled goat anti-mouse antibody (1: 5000) was added to each well, and incubated at 37 ℃ for 1 h.

(12) Color development: PBST washing plate 5 times, adding single component TMB color developing solution into the micro-hole of the ELISA plate, 100 μ l/hole, and incubating for 15min in the incubator at 37 ℃.

(13) Reading: with 2MH₂SO₄The reaction was terminated and the reaction mixture was allowed to react,50 μ l/well, absorbance was read at 450nm with a microplate reader.

The minimum Limit of detection (LOD) is defined as the blank control value. + -. 5X standard deviation, in this case the LOD value is 0.010763. The minimum Limit of quantitation (LOQ) is defined as the blank control value. + -. 10X standard deviation. The LOQ value of this case is 0.020702. In DAS-ELISA, the optimal concentrations of capture antibody and detection antibody were determined to be 0.5 and 0.5. mu.g/ml, respectively. From the lowest detection limit and the detection range visible to the naked eye, a quantitative detection range of 0.010763-4 μ g/ml can be obtained (if the detection range is exceeded, the result may be inaccurate).

After parameter fitting, a standard curve is obtained as shown in fig. 4:

standard curve equation: y ═ A-D/[ 1+ (x/C) ^ B]+ D, wherein a is 3.03818, B is-0.87429, C is 0.53720, and D is 0.19353. r is²＝0.99743。

3. DAS-ELISA specific assay

The established DAS-ELISA method is used for detecting Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F so as to verify the specificity of the established method (Cry 1B is taken as an example for carrying out cross reaction detection in the embodiment).

(1) Coating the capture antibody: the polyclonal antibody against Cry2A toxin was diluted to 0.5. mu.g/ml with PBS buffer, added to a 96-well microplate at 100. mu.l/well and overnight at 4 ℃.

(2) And (3) sealing: the next day the plates were washed 3 times with PBST. 200. mu.l of 5% MPBS were added to each well and incubated at 37 ℃ for 1 h.

(3) Adding a sample: the plates were washed three times with PBST, Cry1B toxin was dissolved in CBS buffer and diluted 2-fold before being added to the wells at 100. mu.l/well with CBS as a blank and incubated for 1h at 37 ℃.

(4) Adding the antibody to be detected: the PBST plates were washed three times, 0.5. mu.g/ml Cry2A monoclonal antibody (100. mu.l/well) was added to the wells, and incubated for 1h at 37 ℃ in an incubator.

(5) Adding an enzyme-labeled antibody: the plates were washed 3 times with PBST, 100. mu.l of HRP-labeled goat anti-mouse antibody (1: 5000) was added to each well, and incubated at 37 ℃ for 1 h.

(6) Color development: PBST washing plate for 5 times, adding TMB color development solution into the micropore of the ELISA plate, 100 μ l/hole, and incubating in the incubator at 37 ℃ for 15 min.

(7) Reading: with 2MH₂SO₄The reaction was stopped, 50. mu.l/well and absorbance was read at 450nm with a microplate reader.

The cross reaction result between the Cry1B toxin and the DAS-ELISA method established in the embodiment and Cry2A is shown in FIG. 5, and it can be seen that the cross reaction rate of the DAS-ELISA method established in the embodiment to Cry1B is 3.2%. The cross-reactivity with other proteins (Cry 1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1F at 50 μ g/ml) is less than 0.1%. The detection method is proved to have stronger specificity and can be used for single detection of Cry 2A.

4. Addition recovery experiment

The crushed rice, corn and soybean (10 g/part) are weighed into a 50ml centrifuge tube, and 10ng/g, 50ng/g and 100ng/g of standard BtCry2A toxin are respectively added into the centrifuge tube. The sample was shaken at room temperature for 30min and then placed in a refrigerator at 4 ℃ overnight. The next day, 10ml of CBS buffer containing 0.05% Tween-20 was added to the sample, and extracted with shaking at room temperature for 2 h. Subsequently, the mixture was centrifuged at 3000rpm for 10min, and the supernatant was diluted ten-fold with CBS and used directly for the double antibody sandwich ELISA assay. Blank control is a sample without added toxin. The results are shown in Table 1.

TABLE 1 additive recovery test

As can be seen from Table 1, the addition recovery rate of the detection method provided by the embodiment is between 85% and 105.4%, and the coefficient of variation is less than 8.4%, which indicates that the established method has better accuracy and can be used for quantitative detection of Cry2A toxin in agricultural products.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (5)

1. A bacillus thuringiensis Cry2A monoclonal antibody hybridoma cell strain with a preservation number of CCTCC NO: C2019188.

2. the hybridoma cell line of claim 1 secreting the obtained monoclonal antibody Bacillus thuringiensis Cry 2A.

3. The application of the monoclonal antibody of Bacillus thuringiensis Cry2A secreted by the hybridoma cell line as claimed in claim 1 in Cry2A toxin detection.

4. The application as claimed in claim 3, characterized by the specific steps of:

diluting the Cry2A toxin polyclonal antibody to 0.5 mug/ml by using PBS buffer solution, adding the diluted polyclonal antibody into an ELISA plate at 100 mug/hole, and standing overnight at 4 ℃; washing the plate with PBST the next day, adding MPBS, and incubating for 1 h; after PBST washing, diluting a sample to be detected by CBS buffer solution, adding the sample to the ELISA plate at 100 mu l/hole, and incubating for 1h by taking CBS as a blank control;

washing the plate by PBST, adding 0.5 mug/ml Cry2A monoclonal antibody into the hole, incubating for 1h at 100 mul/hole; after PBST washing, adding 100 mul of HRP-labeled goat anti-mouse antibody into each hole, and incubating for 1 h;

the Cry2A monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: c2019188 hybridoma cell strain;

PBST washing a plate, adding TMB developing solution into micropores of an ELISA plate at 100 mu l/hole, and placing the plate in an incubator at 37 ℃ for incubation for 15 min; if blue color appears, the Cry2A toxin is contained in the sample to be detected.

5. A Cry2A toxin detection kit comprising a Bacillus thuringiensis Cry2A monoclonal antibody is characterized in that the Bacillus thuringiensis Cry2A monoclonal antibody is prepared from a monoclonal antibody with a preservation number of CCTCC NO: c2019188.

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