CN117169516B - Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof - Google Patents
Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof Download PDFInfo
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Abstract
The invention discloses an insect-resistant protein Cry1Ah colloidal gold immunochromatographic rapid test strip and a detection method thereof. The test strip comprises a bottom plate and a sample pad, a gold mark pad, a reaction membrane and a water absorption pad which are adhered on the bottom plate, wherein the sample pad, the gold mark pad, the reaction membrane and the water absorption pad are arranged from one end of the bottom plate to the other end of the bottom plate and are sequentially connected, and the test strip is characterized in that the gold mark pad is coated with an insect-resistant protein Cry1Ah monoclonal antibody-colloidal gold conjugate, and the insect-resistant protein Cry1Ah monoclonal antibody in the conjugate is secreted by a hybridoma cell strain 3C4 2E6 with the preservation number of CGMCC No.45483. The test strip has strong specificity and high sensitivity, is simple, convenient, quick and accurate to operate, and is suitable for on-site quick detection and safety evaluation of the insect-resistant protein Cry1Ah in transgenic corn and products thereof.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to an insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and a detection method thereof.
Background
Bacillus thuringiensis (Bacillus thuringiensis, bt) is a widely occurring gram-positive bacterium, and the insect-resistant crystal protein secreted by this bacterium is the main biological pesticide at present. The insect-resistant crystal proteins are classified into two classes according to amino acid sequence similarity: cry proteins and Cyt, wherein Cry proteins are toxic to larvae of a variety of insect pests (e.g., lepidopteran, dipteran, coleopteran), nematodes, protozoa, and the like. Cry proteins have been transferred into crops to render them insect-resistant, and at present, crops transformed with Cry protein genes mainly include corn, potato, rice, cotton, and the like. The Cry1Ah protein is one of the Cry proteins.
Advances and developments in transgenic technology have driven the development of biology. Although the transgenic food can meet the requirements of people on yield, insect resistance and the like, the transgenic food also brings potential threats to the life of people, for example, certain genes can cause toxicity to the food after being introduced into a host; transgenic foods generate allergen, so that people can generate drug resistance; the nutritional value of the food is changed, etc. In order to comprehensively evaluate the safety of the transgenic food while researching, developing and commercializing the transgenic food, consumers can rapidly distinguish the transgenic food from natural food, and a proper method is established for identifying and detecting the transgenic ingredients in the transgenic food, so that the safety management of agricultural transgenic organisms can be promoted, the safety of people, animals and microorganisms can be ensured, the ecological environment can be protected, and the further research of agricultural transgenic biotechnology can be promoted. In order to carry out rapid qualitative analysis on Cry1Ah proteins in transgenic crops or derivatives thereof, the development of Cry1Ah colloidal gold immunochromatography test strips has great significance.
The immune colloidal gold technology (Immune colloidal gold technique) is a novel immune labeling technology which uses colloidal gold as a tracer marker to be applied to antigen and antibody. The colloidal gold is prepared from chloroauric acid (HAuCl) 4 ) Under the action of reducing agent such as ascorbic acid, sodium citrate, etc., gold particles with a specific size are polymerized, and a stable colloid state is formed due to the electrostatic action. Colloidal gold is negatively charged in a weak alkaline environment and can form firm combination with positive charge groups of protein molecules, and the biological properties of the protein are not affected because the combination is electrostatic combination. The colloidal gold labeling technology is a core technology of immunochromatography rapid diagnosis, and is characterized in that colloidal gold is used as a tracer marker for antigen-antibody reaction, and the substance is a coating process that high molecules such as protein are adsorbed on the surfaces of colloidal gold particles. According to some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, and the immunological and biological properties of the conjugate, colloidal gold has been widely used in the fields of immunology, histology, pathology, cell biology, etc.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatography rapid test strip for detecting a transgenic corn insect-resistant protein Cry1Ah and a detection method thereof, so that the colloidal gold immunochromatography rapid test strip is suitable for on-site rapid detection and safety evaluation of the insect-resistant protein Cry1Ah in transgenic corn and products thereof.
In order to achieve the aim, the invention provides an insect-resistant protein Cry1Ah colloidal gold immunochromatographic rapid test strip, which comprises a bottom plate and a sample pad, a gold-labeled pad, a reaction membrane and a water-absorbing pad which are adhered on the bottom plate, are arranged from one end of the bottom plate to the other end of the bottom plate and are sequentially connected, and is characterized in that the gold-labeled pad is coated with an insect-resistant protein Cry1Ah monoclonal antibody-colloidal gold conjugate, and the insect-resistant protein Cry1Ah monoclonal antibody in the conjugate is secreted by a hybridoma cell strain 3C 4E 6 with the preservation number of CGMCC No.45483.
Further, the sample pad and the gold-labeled pad, the gold-labeled pad and the reaction membrane, and the reaction membrane and the water-absorbing pad overlap each other, and the width of each overlapping portion is 1-2mm.
Further, the sample pad and the gold mark pad are stacked above the gold mark pad at the adjacent ends, the gold mark pad and the reaction membrane are stacked above the reaction membrane at the adjacent ends, and the reaction membrane and the water absorption pad are stacked above the reaction membrane at the adjacent ends.
Further, the reaction membrane is a nitrocellulose membrane, the sample pad is made of a glass fiber membrane or a filter paper, and the water absorbing pad is made of a filter paper.
Further, a detection line for coating the anti-insect protein Cry1Ah monoclonal antibody and a quality control line for coating the goat anti-mouse IgG are arranged on the reaction film.
Furthermore, the coated insect-resistant protein Cry1Ah monoclonal antibody arranged on the reaction film is secreted by a hybridoma cell strain 2G11 1C7 with the preservation number of CGMCC No. 45482.
Further, the distance between the detection line and the quality control line is 0.5-1.0cm.
The hybridoma cell strain 2G11 1C7 is secreted to obtain the variable region heavy chain amino acid sequence of the antibody, which is shown in SEQ ID NO:1, and the variable region light chain amino acid sequence is shown as SEQ ID NO:2 is shown in the figure; the hybridoma cell strain 3C4 2E6 is secreted to obtain the variable region heavy chain amino acid sequence of the antibody, which is shown in SEQ ID NO:3, the variable region light chain amino acid sequence is shown as SEQ ID NO: 4. The specific sequence is as follows:
| cell strain | Heavy chain variable region amino acid sequence | Light chain variable region amino acid sequence |
| 2G11 1C7 | VQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQSPGNK LEWLGYINYSGSTDYNPSLKSRMSINRDTSKNQFFLQLNSVTTE DTATYYCAKFYYDWYFDVWGAGTTVTVSS | DIVMSQSPSSLTVSVGEKVTMSCKSSQ SLLYSYNQKNHLAWYQQKPGQSPKLLI YWASTRESGVPDRFTGSGSGTDFTLTI SSVKAEDLAVYYCQQYYSSWTFGRGTK LEIKR |
| 3C4 2E6 | QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKG LEWLGVIWSGGNTDYNAAFISRLSIRKDISKSQVFFKINSLRVN DTAIYYCATNHGGYDGAYFSYWGQGTLVTVSA | DILLTQSPAILSVSPGERVSFSCRASQ SIGTTIHWYQQRTNGSPRLLIKYAFES VSGIPSRFSGSGSGTDFTLSINSVESE DIADYYCQQSNNWPLTFGAGTKLELKR |
The invention also provides a detection method of the colloidal gold immunochromatographic rapid test strip, which comprises the steps of immersing the test strip into a sample solution to be detected, allowing the test strip to act for 5 to 10 minutes at room temperature, and then observing the result.
Further, when the sample to be detected is a liquid substance (such as plant tissue extract), 40-60 mu L of the liquid substance is directly taken as a sample solution to be detected for detection; when the sample to be detected is a non-liquid substance (such as plant leaves, stems, seeds and the like), 90-110mg of the sample to be detected is taken and ground, 0.5-1.5mL of sample treatment liquid is added for fully and uniformly mixing, and after the sample to be detected is kept stand on ice for 25-35min, 40-60 mu L of supernatant is taken as a sample solution to be detected for detection.
Further, the formula of the sample treatment fluid is as follows: 1M Tris-HCl, pH 7.5, 500. Mu.L; 1M NaCl1.5 mL; 20. Mu.L of 0.5M EDTA; 50v/v% glycerol 2 mL;10wt% SDS 1mL was added to 10mL of water, and 10mg of protease inhibitor and 50. Mu.L of 1mM phenylmethylsulfonyl fluoride were added at the time of use.
Analysis of detection results:
positive: the quality control line C and the detection line T both show strips, which indicates that Cry1Ah proteins exist in the sample to be detected;
negative: the detection line is not provided with a strip, and the quality control line is provided with a strip, which indicates that no Cry1Ah protein exists in the sample to be detected;
failure: if the detection line and the quality control line are not provided with strips or only the detection line is provided with strips, the test strip is proved to be invalid.
Compared with the prior art, the invention has the following beneficial effects: the colloidal gold immunochromatography rapid test strip for detecting the transgenic corn insect-resistant protein Cry1Ah has strong specificity and high sensitivity, is simple, convenient, quick and accurate to operate, and is suitable for on-site rapid detection and safety evaluation of the insect-resistant protein Cry1Ah in transgenic corn and products thereof.
Preservation information
The hybridoma cell strains 2G11 1C7 and 3C4 2E6 related to the invention are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms for 10 days in 2023, and are classified and named as Cry1Ah monoclonal antibody hybridoma cell strains, and the preservation addresses are as follows: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Post code: 100101 and the preservation numbers are CGMCC No.45482 and CGMCC No.45483 in sequence.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold immunochromatographic rapid test strip according to the present invention, which comprises a bottom plate 1, a sample pad 2, a gold-labeled pad 3, a Cry1Ah detection line 4, a quality control line 5, a reaction membrane 6, and a water-absorbing pad 7;
FIG. 2 is a schematic diagram showing the detection results of a colloidal gold immunochromatographic rapid test strip according to the present invention: positive, the quality control line (namely the C line) develops color, the detection line (namely the T line) is visible to naked eyes, and no matter the color depth is positive; negative, the color of the C line is developed, the color of the T line is not developed, and the judgment is negative; the color of the T line is invalid whether the color is developed or not;
FIG. 3 is a graph showing the results of testing transgenic corn positive leaf samples at room temperature using a colloidal gold immunochromatographic rapid test strip according to the present invention;
FIG. 4 is a Cry1Ah detection card sample measurement result display (test development for 5 min) of a transgenic corn leaf detected by a colloidal gold immunochromatographic rapid test strip according to the present invention;
FIG. 5 is a Cry1Ah detection card sample measurement result display (10 min of test development) of the transgenic corn leaf detected by the colloidal gold immunochromatography rapid test strip according to the present invention;
FIG. 6 is a graph showing the results of the transfection and Western blotting of Bt Cry insect-resistant proteins commonly used for the specific detection of monoclonal antibodies produced by hybridoma cell line 2G11 1C7 according to the invention;
FIG. 7 is a graph showing the results of the staining and Western analysis of a Bt Cry insect-resistant protein commonly used for the specific detection of monoclonal antibodies produced by hybridoma cell line 3C4 2E6 according to the invention.
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: colloidal gold immunochromatography test strip
The embodiment provides a colloidal gold immunochromatographic rapid test strip, which comprises a bottom plate and a sample pad, a gold-labeled pad, a reaction membrane and a water absorption pad which are sequentially connected on the bottom plate, wherein the sample pad, the gold-labeled pad, the reaction membrane and the water absorption pad are sequentially arranged from one end of the bottom plate to the other end of the bottom plate, the gold-labeled pad is coated with an insect-resistant protein Cry1Ah monoclonal antibody-colloidal gold conjugate, and the insect-resistant protein Cry1Ah monoclonal antibody in the conjugate is secreted by a hybridoma cell strain 3C 4E 6 with the preservation number of CGMCC No.45483.
The sample pad and the gold mark pad adjacent end, the gold mark pad and the reaction membrane adjacent end and the reaction membrane and the water absorption pad adjacent end are all overlapped, and the width of each overlapped part is 1-2mm. And the adjacent ends of the sample pad and the gold mark pad are the sample pad and are stacked above the gold mark pad, the adjacent ends of the gold mark pad and the reaction membrane are the gold mark pad and are stacked above the reaction membrane, and the adjacent ends of the reaction membrane and the water absorption pad are the water absorption pad and are stacked above the reaction membrane.
The reaction film is provided with a detection line for coating the anti-insect protein Cry1Ah monoclonal antibody and a quality control line for coating sheep anti-mouse IgG.
The coated insect-resistant protein Cry1Ah monoclonal antibody arranged on the reaction film is secreted by a hybridoma cell strain 2G11 1C7 with the preservation number of CGMCC No. 45482.
The interval between the detection line and the quality control line is 0.5-1.0cm.
The reaction membrane is nitrocellulose membrane, the sample pad is made of glass fiber membrane or filter paper, and the water absorbing pad is made of filter paper.
Example 2: test strip for detecting transgenic corn leaves by colloidal gold immunochromatography of example 1
100mg of transgenic corn leaves are taken, ground by liquid nitrogen, fully mixed with 1mL of sample treatment liquid, kept stand on ice for 30min, and 50 mu L of supernatant is taken for detection. During detection, the test strip is immersed into the solution of the sample to be detected, and the detection result is observed after 5min of action at room temperature. Specific results are shown in fig. 3, and as can be seen from fig. 3, the Cry1Ah protein contained in the sample solution to be detected can be specifically detected by the colloidal gold immunochromatographic rapid test strip.
Table 1 sample treatment fluid formulations
| Composition of the components | Dosage of |
| 1M Tris, pH 7.5 | 500 uL |
| 1M NaCl | 1.5 mL |
| 0.5M EDTA | 20 uL |
| 50% glycerol | 2 mL |
| 10% SDS | 1 mL |
| Double distilled water (DDH 2O) | Is prepared into 10mL |
| Protease inhibitor (Roche) | 10mg |
| 1mM PMSF (phenylmethylsulfonyl fluoride, sigma) | When in use, 50uL is added |
The specific detection method comprises the following steps:
1. taking out a sample from the refrigerator at the temperature of-80 ℃, and after thawing, detecting;
2. taking out the rapid test strip and inserting the rapid test strip into the supernatant of the sample to be detected;
3. and 5-10min after sample addition, observing the color development area, and judging the result.
4. And (3) result judgment:
positive: the color of the C line is developed, the T line is visible to naked eyes, no matter how deep the color is, the color is positive, and the color depth is in direct proportion to the concentration of the target protein.
Negative: the line C shows that the line T does not develop color, and is judged as negative.
Invalidation: the line C does not develop color, and the detection card is judged to be invalid no matter whether the line T develops color or not.
The result of 5 Cry1Ah detection card sample detection and color development for 5min is shown in FIG. 4, and the result of 10min color development is shown in FIG. 5.
6. Performance: the detection reference should meet the following criteria:
6.1 Minimum detection limit: the lowest limit of detection reference was repeatedly tested 10 times, and the results were calculated. The result meets the requirement;
the calculation method comprises the following steps:
LLD= +3×S
wherein: x-series of measurements;
-determining a mean value;
n is the number of measurements;
s is standard deviation;
LLD-minimum detection limit
6.2 Accuracy: with not less than 40 samples of different concentrations in the measurement range, each sample was measured separately by the test kit procedure and the comparison method using the manufacturer-specified analysis system as the comparison method.
The calculation method comprises the following steps: bias (%) =
Wherein: bias-relative deviation;
-determining a mean value;
x-calibrator, standard or reference substance.
6.3 Repeatability: repeated reference products CV1 and CV2 are detected, each repeated test is not less than 10 times, each horizontal variation Coefficient (CV) is calculated respectively, and the result meets the requirement:
the calculation method comprises the following steps:
wherein: x-series of measurements;
-determining a mean value;
n is the number of measurements;
s is standard deviation;
CV-intra-batch coefficient of variation.
Through the detection, the detection limit of the colloidal gold immunochromatography test strip obtained by the invention is 4 ppb (ng/mL).
Example 3: specificity verification of antibody used by colloidal gold immunochromatography speed test strip
In addition to Cry1Ah, the commonly used Cry insect-resistant proteins in transgenic crops include Bt proteins such as Cry1C, cry2Aa and Cry2 Ac. In order to detect the specificity of the monoclonal antibodies produced by the hybridoma cell lines 2G11 1C7 and 3C4 2E6, western detection of Cry1Ah (obtained by prokaryotic expression purification), cry1C, cry Aa and Cry2Ac (purchased from Shanghai Youlong Co.), and detection results are shown in FIG. 6 and FIG. 7, and the results show that the monoclonal antibodies produced by the purified (Protein A-agarose affinity chromatography column purification of ascites) 2G 11C 7 and 3C4 2E6 can specifically recognize Cry1Ah, but cannot effectively recognize other Cry insect-resistant proteins.
Claims (8)
1. The test strip comprises a bottom plate and a sample pad, a gold-labeled pad, a reaction membrane and a water-absorbing pad which are arranged from one end of the bottom plate to the other end of the bottom plate and are sequentially connected, wherein the gold-labeled pad is coated with an insect-resistant protein Cry1Ah monoclonal antibody-colloidal gold conjugate, the insect-resistant protein Cry1Ah monoclonal antibody in the conjugate is secreted by a hybridoma cell strain 3C 4E 6 with the preservation number of CGMCC No.45483,
the reaction film is provided with a detection line for coating the anti-insect protein Cry1Ah monoclonal antibody and a quality control line for coating the goat anti-mouse IgG, and the coated anti-insect protein Cry1Ah monoclonal antibody arranged on the reaction film is secreted by a hybridoma cell strain 2G 11C 7 with the preservation number of CGMCC No. 45482.
2. The test strip of claim 1, wherein the sample pad and the gold-labeled pad are each overlapped at their adjacent ends, the gold-labeled pad and the reaction membrane are each overlapped at their adjacent ends, and the width of each overlapped portion is 1-2mm.
3. The test strip of claim 2, wherein the sample pad is stacked above the gold-labeled pad at the end adjacent to the gold-labeled pad, the gold-labeled pad is stacked above the reaction membrane at the end adjacent to the reaction membrane, and the water-absorbent pad is stacked above the reaction membrane at the end adjacent to the water-absorbent pad.
4. The test strip of claim 1, wherein the reaction membrane is a nitrocellulose membrane, the sample pad is made of a glass fiber membrane or a filter paper, and the absorbent pad is made of a filter paper.
5. The test strip of claim 1, wherein the detection line and the quality control line are spaced apart by 0.5-1.0cm.
6. A detection method using the test strip according to any one of claims 1 to 5, wherein the test strip is immersed in a sample solution to be detected, allowed to act at room temperature for 5 to 10 minutes, and then observed.
7. The method according to claim 6, wherein when the sample to be detected is a liquid substance, 40-60 μl of the sample solution to be detected is directly taken as the sample solution to be detected; when the sample to be detected is a non-liquid substance, 90-110mg of the sample to be detected is taken and ground, 0.5-1.5mL of sample treatment liquid is added for fully and uniformly mixing, and after the sample to be detected is kept stand on ice for 25-35min, 40-60 mu L of supernatant is taken as a sample solution to be detected for detection.
8. The method according to claim 7, wherein the sample processing liquid has a formula of: 1M Tris-HCl, pH 7.5, 500. Mu.L; 1M NaCl1.5 mL; 20. Mu.L of 0.5M EDTA; 50v/v% glycerol 2 mL;10wt% SDS 1mL was added to 10mL of water, and 10mg of protease inhibitor and 50. Mu.L of 1mM phenylmethylsulfonyl fluoride were added at the time of use.
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