Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof

A technology of toxoids and antibodies, applied in the field of biochemistry

Active Publication Date: 2015-12-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to literature reports, about 90% of monoclonal antibodies have certain cross-reactions against antigens with common structures, and monoclonal or polyclonal antibodies or single-chain antibodies detected against a certain Bt toxin are not effective for other toxins with high homology. There is also a certain cross-reactivity, which is based on the principle that although different Cry toxins have large differences in amino acid sequences, their three-dimensional structures are very similar. At present, there are no related reports on the use of broad-spectrum antibodies for the detection of BtCry1 toxoids.

Method used

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  • Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof
  • Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof
  • Antibody for broad-spectrum detection of Bt Cry1 toxoids as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0022] The preparation method that relates to solution in the embodiment:

[0023] (1) Saturated ammonium sulfate solution:

[0024] Add 500g of ammonium sulfate to 500mL of distilled water, heat until completely dissolved; overnight at room temperature, precipitate crystals and leave them in the bottle, take the required amount before use, and adjust the pH to 7.8 with NaOH;

[0025] (2) Acetate buffer (0.06mol / LpH4.8):

[0026] Dissolve 0.29g of NaAc and 0.141mL of HAc in deionized water, dilute to 100mL, and adjust the pH value to 4.8.

[0027] (3) Phosphate buffered saline (PBS, pH7.4):

[0028] NaCl8.0g, KCl0.2g, NaCl 2 HPO4·12H 2 O2.9g, KH 2 PO 4 0.2g, add distilled water to make up to 1L;

[0029] (4) Washing solution (PBST): PBS containing 0.05% Tween;

[0030] (5) Blocking solution (MPBS): PBS containing 3% skimmed milk powder;

[0031] (6) Citrate buffer (CPBS, substrate buffer, pH5.5):

[0032] C 6 h 7 o 8 (citric acid) 21g, Na 2 HPO 4 12H 2 O71.6g, ...

Embodiment 1

[0046] Embodiment 1 prepares monoclonal antibody

[0047] (1) The amino acid sequences of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F were retrieved in the GenBank database;

[0048] (2) Use DNAMAN software to compare the amino acid sequences of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F to find out the most similar peptide sequences. The amino acid sequence comparison results of the selected peptides are as follows: figure 1 shown;

[0049] (3) Use DNAStar software to find out the parts with strong antigenicity and hydrophilicity in Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F. The antigenicity and hydrophilicity analysis results of the peptides are as follows: figure 2 shown;

[0050] (4) Finally, use the SWISS-MODEL website to construct the three-dimensional structure models of the six toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F, and use the Swiss-PdbViewer4.1.0 software to analyze the three-dimensional structure models to find out the three-dimensional...

Embodiment 2

[0065] Example 2 Antibody Sensitivity Detection

[0066] (1) Inoculate the hybridoma cell line obtained in Example 1 into mice, and prepare ascites by in vivo induction method. The specific steps are as follows:

[0067] The well-grown hybridoma cells were gently blown off, collected by centrifugation, resuspended in RPMI-1640 basal culture medium and counted, and the number of cells was controlled at 1-2×10 6 Within the range of cells / mL, intraperitoneally inject mice that have been pretreated with paraffin oil in advance, and inject 2 mice per cell line, 0.5 mL per mouse. Closely observe the health status of the mice and signs of ascites. About 10 days later, the abdomen of the mice began to expand.

[0068] Centrifuge the collected ascites at 5000 rpm for 10 min at 4°C to remove fat and blood cells, add an equal amount of glycerol to the centrifuged ascites, and store it in a -20°C refrigerator.

[0069] (2) Purify the ascites obtained in step (1) by octanoic acid-ammoniu...

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Abstract

The invention discloses an antibody for broad-spectrum detection of Bt Cry1 toxoids as well as a preparation method and application thereof. The antibody is generated by secretion of hybridoma cells with the collection number of CCTCC NO:C2015145, obtained after coupling antigen polypeptide SEQ ID NO.2 and carrier protein KLH and then immunizing Balb/c mice. The ratio of the antibody to OD450 of six kinds of Bt Cry1 toxoids to negative holes (PBS solution) is far larger than 2.1, and the antibody has relatively high titer, has a broad-spectrum detection effect on the Bt Cry1 toxoids and makes up the blank of the broad-spectrum detection field of the Bt Cry1 toxoids.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to an antibody used for broad-spectrum detection of BtCry1 toxoids and its preparation method and application. Background technique [0002] Since 1987, Hilder et al first reported the successful development of Bacillus thuringiensis ( Bacillus thuringiensis , Bt) toxin gene insect-resistant plants, more than 400 Bt toxin genes have been cloned and sequenced, and more than a dozen types of BtCry family gene crops (including Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B, Cry34, Cry35, etc.), Bt toxin gene-transformed insect-resistant crops continue to appear and have been widely promoted, resulting in extremely significant economic, social and ecological benefits , but with the large-scale promotion of transgenic Bt insect-resistant plants, their ecological safety risks and potential safety hazards to humans and oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N5/20C12N15/06G01N33/68G01N33/577
Inventor 刘贤金董飒张霄仲建锋徐重新刘贝贝
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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