Insect-resistant protein Cry1Da colloidal gold immunochromatographic rapid test strip and detection method thereof

Abstract

The application belongs to the technical field of bioengineering, and discloses a kind of anti-insect protein Cry1Da colloidal gold immunochromatography rapid test strip and its detection method.The test strip includes a base plate and is adhered to the base plate, and is provided from one end of the base plate to the other end of the base plate and is sequentially connected with a sample pad, a gold label pad, a reaction membrane and a water absorption pad, the gold label pad is coated with anti-insect protein Cry1Da monoclonal antibody-colloidal gold conjugate, and the anti-insect protein Cry1Da monoclonal antibody in the conjugate is obtained by hybridoma cell strain 7F1 with the preservation number of CGMCC No.46852.The test strip of the application has high specificity and sensitivity, and is simple, fast and accurate to operate, and is suitable for on-site rapid detection and safety evaluation of anti-insect protein Cry1Da in transgenic corn and its products.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and specifically relates to a colloidal gold immunochromatographic rapid test strip for the insect-resistant protein Cry1Da and its detection method. Background Technology

[0002] Bacillus thuringiensis (Bt) is a widespread Gram-positive bacterium whose insect-resistant crystal proteins are currently the main biological pesticides. These insect-resistant crystal proteins are divided into two categories based on amino acid sequence similarity: Cry proteins and Cyt proteins. Cry proteins are toxic to the larvae of various harmful insects (such as Lepidoptera, Diptera, and Coleoptera), nematodes, and protozoa. Cry proteins have been transferred into crops to give them insect resistance; currently, crops with Cry protein genes mainly include corn, potatoes, rice, and cotton. Cry1Da protein is one type of Cry protein.

[0003] The development and advancement of genetically modified (GM) technology have propelled the progress of biology. While GM foods can meet people's demands for yield and insect resistance, they also pose potential threats to human life. For example, certain genes introduced into the host can cause food toxicity; GM foods can produce allergens, leading to antibiotic resistance; and the nutritional value of food can be altered. Simultaneously with the research, development, and commercialization of GM foods, it is crucial to conduct comprehensive safety assessments, enable consumers to quickly distinguish between GM and natural foods, and establish appropriate methods for identifying and detecting GM components in GM foods. This will promote the safe management of agricultural GM organisms, protect the safety of humans, animals, and microorganisms, protect the ecological environment, and further advance research in agricultural GM biotechnology. The development of Cry1Da colloidal gold immunochromatographic rapid test strips is of great significance for the rapid qualitative analysis of Cry1Da protein in GM crops or their derivatives.

[0004] Immunochromatographic gold labeling is a novel immunolabeling technique that uses colloidal gold as a tracer marker for antigen-antibody reactions. Colloidal gold is produced by polymerizing chloroauric acid (HAuCl4) into gold particles of a specific size under the action of reducing agents such as ascorbic acid and sodium citrate, forming a stable colloidal state due to electrostatic interactions. Colloidal gold carries a negative charge in a weakly alkaline environment, allowing it to form a strong bond with the positively charged groups of protein molecules. Because this bond is electrostatic, it does not affect the biological properties of the protein. Colloidal gold labeling is a core technology in rapid immunochromatographic diagnosis. It uses colloidal gold as a tracer marker in antigen-antibody reactions; its essence is the coating process where macromolecules such as proteins are adsorbed onto the surface of colloidal gold particles. Based on the physical properties of colloidal gold, such as high electron density, particle size, shape, and color reactivity, combined with the immunological and biological properties of the conjugates, colloidal gold has been widely used in immunology, histology, pathology, and cell biology. Summary of the Invention

[0005] The purpose of this invention is to provide a colloidal gold immunochromatographic rapid test strip and its detection method for detecting the insect-resistant protein Cry1Da in genetically modified maize, thereby making it suitable for rapid on-site detection and safety evaluation of the insect-resistant protein Cry1Da in genetically modified maize and its products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a colloidal gold immunochromatographic rapid test strip for the insecticidal protein Cry1Da. The test strip includes a base plate and a sample pad, a gold-labeled pad, a reaction membrane, and an absorbent pad, which are attached to the base plate and sequentially connected from one end of the base plate to the other. The gold-labeled pad is coated with a Cry1Da monoclonal antibody-colloidal gold conjugate. The Cry1Da monoclonal antibody in the conjugate is secreted by hybridoma cell line 7F1 with accession number CGMCC No. 46852. The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by hybridoma cell line 7F1 is shown in SEQ ID NO:3 (EVQLVESGGDLVKPGGSLKVSCAASGFTFSSYAMSWVRQTPEKRLEWVATITSGGTYTHYPDSVKGRFTISRDNAKDTLYLQMSSLRSEDTAMYFCVRGVRGVYAIDYWGQGTSVTVSS), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3. NO:4 (DIVMTQSHKLMSTSVGDRVSITCKASKDVSNAVAWYQQKPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCQQHYNTPYTFGGGTKMEIK);

[0008] The reaction membrane is equipped with a detection line coated with a monoclonal antibody against the insecticidal protein Cry1Da and a control line coated with goat anti-mouse IgG. The monoclonal antibody against the insecticidal protein Cry1Da on the reaction membrane is secreted by hybridoma cell line 7B1 with accession number CGMCC No. 46851. The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by hybridoma cell line 7B1 is shown in SEQ ID NO:1 (EVQLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVATITSGGSHTHYPDSVKGRFTISRDNAKNTLDLKMSSLRSEDTAMYYCVRGVRGLYAMDYWGQGTSVTVSS), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:1. NO:2 (DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLTVYSCQQHYSTPYTFGGGTKLEIR) shown;

[0009] Wherein, the amino acid sequence of the insect-resistant protein Cry1Da is as SEQ ID NO:5(SQRIEFARNQAISRLEGLSNLYKVYVRAFSDWEKDPTNPALREEMRIQFNDMNSALITAIPLFRVQNYEVALLSVYVQAANLHLSILRDVSVFGERWGYDTATINNRYSDLTSLIHVYTNHCVDTYNQGLRRLEGRFLSDWIVYNRFRRQLTISVLDIVAFFPNYDIRTYPIQTATQ LTREVYLDLPFINENLSPAASYPTFSAAESAIIRSPHLVDFLNSFTIYTDSLARYAYWGGHLVNSFRTGTTNLIRSPLYGREGNTERPVTITASPSVPIFRTLSYITGLDNSNPVAGIEGVEFQNTISRSIYRKSGPIDSFSELPPQDASVSPAIGYSHRLCHATFLERISGPRIAGT).

[0010] Furthermore, the sample pad and the gold label pad adjacent to each other, the gold label pad and the reaction membrane adjacent to each other, and the reaction membrane and the absorbent pad adjacent to each other all overlap, with each overlapping portion having a width of 1-2 mm.

[0011] Furthermore, the sample pad is stacked on top of the gold label pad at the end adjacent to the gold label pad, the gold label pad is stacked on top of the reaction membrane at the end adjacent to the reaction membrane, and the absorbent pad is stacked on top of the reaction membrane at the end adjacent to the absorbent pad.

[0012] Furthermore, the reaction membrane is a nitrocellulose membrane, the sample pad is made of glass fiber membrane or filter paper, and the absorbent pad is made of filter paper.

[0013] Furthermore, the detection line and the quality control line are spaced 0.5-1.0 cm apart.

[0014] Secondly, the present invention provides a detection method using the above-mentioned test strip, wherein the test strip is immersed in the sample solution to be tested, and the solution is allowed to stand at room temperature for 5-10 minutes, and then the results are observed.

[0015] Furthermore, when the sample to be tested is a liquid substance, 40-60 μL is directly taken as the sample solution for detection; when the sample to be tested is a non-liquid substance, 90-110 mg of the sample to be tested is ground, 0.5-1.5 mL of sample processing solution is added and mixed thoroughly, and after standing on ice for 25-35 min, 40-60 μL of the supernatant is taken as the sample solution for detection.

[0016] Furthermore, the sample processing solution is formulated as follows: 500 μL of 1M Tris-HCl, pH 7.5; 1.5 mL of 1M NaCl; 20 μL of 0.5M EDTA; 2 mL of 50 v / v% glycerol; 1 mL of 10 wt% SDS, with water added to 10 mL. When using, 10 mg of protease inhibitor and 50 μL of 1 mM benzyl sulfonyl fluoride are added.

[0017] Analysis of test results:

[0018] (1) Positive, both the control line (C) and the test line (T) show bands, indicating that Cry1Da protein is present in the sample to be tested;

[0019] (2) Negative, no band appears on the test line, but a band appears on the control line, indicating that there is no Cry1Da protein in the sample to be tested;

[0020] (3) Failure: If no bands appear on the test line or the control line, or only the test line appears, the test strip is invalid.

[0021] The hybridoma cell line 7B1 provided by this invention was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46851.

[0022] The hybridoma cell line 7F1 provided by this invention was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as a monoclonal antibody hybridoma cell line. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46852.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The colloidal gold immunochromatographic rapid test strip provided by the present invention for the detection of insect-resistant protein Cry1Da in genetically modified maize has high specificity and high sensitivity, and is simple, fast and accurate to operate. It is suitable for rapid on-site detection and safety evaluation of insect-resistant protein Cry1Da in genetically modified maize and its products. Attached Figure Description

[0024] Figure 1 The diagram shows the structure of the colloidal gold immunochromatographic rapid test strip provided by the present invention, which includes a base plate 1, a sample pad 2, a gold label pad 3, a Cry1Da detection line 4, a quality control line 5, a reaction membrane 6, and an absorbent pad 7.

[0025] Figure 2This is a schematic diagram of the detection results of the colloidal gold immunochromatographic rapid test strip provided by the present invention; positive, the control line (i.e., C line) is colored and the test line (i.e., T line) is visible to the naked eye, and it is judged as positive regardless of the color intensity; negative, the C line is colored and the T line is not colored, and it is judged as negative; invalid, the C line is not colored and the T line is invalid regardless of whether it is colored.

[0026] Figure 3 The results of the colloidal gold immunochromatographic rapid test strip provided by the present invention were obtained by detecting positive leaf samples of transgenic maize at room temperature.

[0027] Figure 4 The monoclonal antibody produced by hybridoma cell line 7B1 provided by this invention is a capture antibody, and the monoclonal antibody produced by hybridoma cell line 7F1 is a detection antibody. The result of sandwich ELISA detection of Bt Cry anti-insect protein, which is commonly used for specific detection, is shown in the figure. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Colloidal gold immunochromatographic rapid test strip

[0032] This embodiment provides a colloidal gold immunochromatographic rapid test strip, including a base plate and a sample pad, a gold-labeled pad, a reaction membrane, and an absorbent pad that are sequentially connected to the base plate. The sample pad, gold-labeled pad, reaction membrane, and absorbent pad are arranged sequentially from one end of the base plate to the other end. The gold-labeled pad is coated with an insect-resistant protein Cry1Da monoclonal antibody-colloidal gold conjugate. The insect-resistant protein Cry1Da monoclonal antibody in the conjugate is secreted by hybridoma cell line 7F1 with accession number CGMCC No. 46852.

[0033] The sample pad and the gold-labeled pad, the gold-labeled pad and the reaction membrane, and the reaction membrane and the absorbent pad all overlap, with each overlap being 1-2 mm wide. Specifically, at the end adjacent to the sample pad and the gold-labeled pad, the sample pad is placed on top of the gold-labeled pad; at the end adjacent to the gold-labeled pad and the reaction membrane, the gold-labeled pad is placed on top of the reaction membrane; and at the end adjacent to the reaction membrane and the absorbent pad, the absorbent pad is placed on top of the reaction membrane.

[0034] The reaction membrane has a detection line coated with Cry1Da monoclonal antibody and a control line coated with goat anti-mouse IgG.

[0035] The monoclonal antibody coated with the insect-resistant protein Cry1Da on the reaction membrane was secreted by hybridoma cell line 7B1 with accession number CGMCC No.46851.

[0036] The test line and the quality control line should be spaced 0.5-1.0 cm apart.

[0037] The reaction membrane is a nitrocellulose membrane, the sample pad is made of glass fiber membrane or filter paper, and the absorbent pad is made of filter paper.

[0038] Example 2: Detection of Cry1Da protein in transgenic maize leaves

[0039] Take 100 mg of genetically modified maize leaves, grind them with liquid nitrogen, mix them thoroughly with 1 mL of sample processing solution (formula shown in Table 1), let them stand on ice for 30 min, and then take 50 μL of the supernatant for detection.

[0040] Table 1 Sample processing solution formulation

[0041]

[0042] During testing, the test strip described in Example 1 is immersed in the sample solution to be tested, ensuring that the sample solution level does not exceed the gold label pad area. The results are observed after 5 minutes at room temperature.

[0043] The specific testing method and process are as follows:

[0044] 1. Remove the sample from the -80°C freezer, thaw it, and then test it;

[0045] 2. Remove the rapid test strip and insert it into the supernatant of the sample to be tested;

[0046] 3. After adding the sample, observe the color development area 5-10 minutes later and determine the result.

[0047] (1) Positive: C line is visible and T line is visible to the naked eye. Regardless of the color intensity, it can be judged as positive. The color intensity is directly proportional to the concentration of the target protein.

[0048] (2) Negative: The C line is visible and the T line is not visible, which is considered negative.

[0049] (3) Invalid: If the C line does not show color, the test card is invalid regardless of whether the T line shows color.

[0050] For detailed results, please see [link / details]. Figure 3 .pass Figure 3 It is known that the Cry1Da protein contained in the sample solution can be specifically detected by the colloidal gold immunochromatographic rapid test strip of the present invention.

[0051] 4. To verify the detection performance of the test strip of the present invention, the limit of detection was determined.

[0052] Repeat the test for the minimum detection limit reference sample 10 times and calculate the results.

[0053] Calculation method: , In the formula, X represents a series of measured values. The mean value is given by n, the number of measurements is given by S, the standard deviation is given by S, and the limit of detection is given by LLD.

[0054] Based on the above tests, the detection limit of the colloidal gold immunochromatographic rapid test strip provided by the present invention is 100 ng / mL.

[0055] Example 3: Antibody Specificity Verification

[0056] Besides Cry1Da, other commonly used insect-resistant Cry proteins in transgenic crops include Cry1Fa and Cry1Ah Bt proteins. To detect the specificity of the monoclonal antibodies produced by the hybridoma cell lines 7B1 and 7F1 of this invention, sandwich ELISA was performed using monoclonal antibody (7B1) as the capture antibody and another monoclonal antibody (7F1) as the detection antibody to detect insect-resistant maize materials transgenic with the Cry1Ah (HGK60) and Cry1Ab+Cry1Fa (BFL4-2) genes.

[0057] The results showed that ( Figure 4 The monoclonal antibodies produced by the purified 7B1 and 7F1 can specifically recognize Cry1Da, but cannot effectively recognize other Cry anti-insect proteins.

Claims

1. A colloidal gold immunochromatographic rapid test strip for the insect-resistant protein Cry1Da, the test strip comprising a base plate and a sample pad, a gold label pad, a reaction membrane, and an absorbent pad, which are attached to the base plate and arranged sequentially from one end of the base plate to the other end, characterized in that, The gold-coated pad is coated with a colloidal gold conjugate containing an insect-resistant protein Cry1Da monoclonal antibody. The Cry1Da monoclonal antibody in the conjugate is secreted by hybridoma cell line 7F1 with accession number CGMCC No. 46852. The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by hybridoma cell line 7F1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4. The reaction membrane is equipped with a detection line coated with an anti-insect protein Cry1Da monoclonal antibody and a control line coated with goat anti-mouse IgG. The anti-insect protein Cry1Da monoclonal antibody on the reaction membrane is secreted by hybridoma cell line 7B1 with accession number CGMCC No. 46851. The amino acid sequence of the heavy chain variable region of the monoclonal antibody secreted by hybridoma cell line 7B1 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2. The amino acid sequence of the insect-resistant protein Cry1Da is shown in SEQ ID NO:

5.

2. The test strip according to claim 1, characterized in that, The sample pad and the gold label pad adjacent to each other, the gold label pad and the reaction membrane adjacent to each other, and the reaction membrane and the absorbent pad adjacent to each other all overlap, with each overlapping portion having a width of 1-2 mm.

3. The test strip according to claim 2, characterized in that, The sample pad is stacked on top of the gold label pad at the end adjacent to the gold label pad, the gold label pad is stacked on top of the reaction membrane at the end adjacent to the reaction membrane, and the absorbent pad is stacked on top of the reaction membrane at the end adjacent to the absorbent pad.

4. The test strip according to claim 1, characterized in that, The reaction membrane is a nitrocellulose membrane, the sample pad is made of glass fiber membrane or filter paper, and the absorbent pad is made of filter paper.

5. The test strip according to claim 1, characterized in that, The detection line and the quality control line are spaced 0.5-1.0 cm apart.

6. A detection method using the test strip according to any one of claims 1-5, characterized in that, Immerse the test strip in the sample solution to be tested and allow it to stand at room temperature for 5-10 minutes, then observe the results.

7. The detection method according to claim 6, characterized in that, When the sample to be tested is a liquid substance, take 40-60 μL directly as the sample solution for detection; when the sample to be tested is a non-liquid substance, take 90-110 mg of the sample to be tested, grind it, add 0.5-1.5 mL of sample processing solution, mix thoroughly, let it stand on ice for 25-35 min, and then take 40-60 μL of the supernatant as the sample solution for detection.

8. The detection method according to claim 7, characterized in that, The sample processing solution was formulated as follows: 500 μL of 1M Tris-HCl, pH 7.5; 1.5 mL of 1M NaCl; 20 μL of 0.5M EDTA; 2 mL of 50 v / v% glycerol; 1 mL of 10 wt% SDS, and water to a final volume of 10 mL. Before use, 10 mg of protease inhibitor and 50 μL of 1 mM benzyl sulfonyl fluoride were added.

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