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CpTI enzyme-linked immunoassay reagent kit as well as preparation method and application thereof

A technology of trypsin inhibition and cowpea, which is applied in the biological field, can solve the problems of high false positives, low insect resistance efficiency, and unsatisfactory effects, and achieve the effects of maintaining inhibitory activity, reducing plant background, and improving sensitivity

Inactive Publication Date: 2012-08-08
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are only enzyme inhibitor methods and very few immunological tests for protein detection and expression, and the results reported at this level are far from the actual insect resistance efficiency in the field, and the false positives are very high, which is ultimately unsatisfactory.

Method used

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  • CpTI enzyme-linked immunoassay reagent kit as well as preparation method and application thereof
  • CpTI enzyme-linked immunoassay reagent kit as well as preparation method and application thereof
  • CpTI enzyme-linked immunoassay reagent kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of embodiment 1 cowpea trypsin inhibitor protein

[0024] 1. Extraction of cowpea trypsin inhibitor protein

[0025] Select 300g of high-quality cowpea seeds, soak them in distilled water for 3 to 4 hours in a large beaker, and peel them off. Grind with a pulverizer at 4°C, add 5 times (W / V) sodium acetate extract (0.1M NaAc+0.3M NaCl+0.01M CaCl 2, pH 4.0), placed in a 2-liter beaker and mixed with a magnetic stirrer for 16 hours in a refrigerator at 4°C. Then centrifuge at 10,000 rpm for 30 minutes at 4°C. Take the supernatant and add 60% ammonium sulfate (ie 198g / L) to precipitate in an ice bath for 30 minutes (while stirring slowly with a glass rod, or slowly stirring with a magnetic stirrer to avoid protein denaturation). Then centrifuge at 12,000 rpm at 4°C for 15 minutes to retain the pellet. Use as little as possible A eluent (50mM Tris-HCl+0.01MCaCl 2 , pH 8.0), centrifuged at 10,000 rpm for 2 minutes at 4°C, and the supernatant was passed t...

Embodiment 2

[0034] Embodiment 2, cowpea trypsin inhibitor protein antiserum acquisition and titer determination

[0035] With reference to "Veterinary Microbiology and Immunology Technology" (Cao Shuze et al., Beijing: Beijing Agricultural University Press, 1992) and "Refined Molecular Biology Experiment Guide" (F. Osper, etc., Beijing: Science Press, The cowpea trypsin inhibitor protein obtained by the method in 1998) was used as an antigen to immunize rabbits to measure serum titer, antibody specificity (indirect ELISA method), and indirect ELISA method to determine the detection accuracy of antigen.

[0036] The obtained dried protein was formulated into a protein solution with a concentration of 300 μg / ml, which was the protein antigen for injection. Refer to "Veterinary Microbiology and Immunology Techniques" to immunize rabbits, take blood to collect all serum, add NaN 3 antiseptic. The titer was determined by ELISA ( image 3 ) Cowpea trypsin inhibitor protein was diluted with C...

Embodiment 3

[0037] The assembly of embodiment 3 kits

[0038] Coated enzyme plate of cowpea trypsin inhibitor protein (prepared by the method of embodiment 1)

[0039] Cowpea trypsin inhibitor protein polyclonal antibody

[0040] Enzyme-conjugated secondary antibody (HRP-conjugated secondary antibody)

[0041] Wash solution (PBST)

[0042] Substrate solution (TMB solution)

[0043] Stop solution (2M H 2 SO 4 )

[0044] Cowpea Trypsin Inhibitor Protein Standard

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Abstract

The invention provides an enzyme-linked immunoassay reagent kit for detecting vigna trypsin inhibitor protein. With an affinity chromatography, the vigna trypsin inhibitor protein with high purity is obtained and used as an immune animal with immunogen, which is prepared into a vigna trypsin inhibitor protein antibody with high zymolyte specificity and sensitivity. The antibody is used for detecting the vigna trypsin inhibitor protein, particularly for detecting the trypsin inhibitor protein of a transgenic plant, and obtains favorable effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cowpea trypsin inhibitor (CpTI) enzyme-linked immunoassay kit, and also relates to a method for preparing the kit. Background technique [0002] Pests are the main limiting factors that endanger my country's agricultural production. The use of a large number of chemical pesticides not only pollutes the environment, but also makes the number of beneficial insects drop sharply, and the resistance of pests continues to increase. In addition, the pesticide residues after the use of chemical pesticides will cause serious harm to humans and animals. Therefore, transgenic plant insect resistance engineering has become one of the hot research fields of scientists. [0003] Cowpea proteinase Trypsin Inhibitor (CpTI) is one of the protease inhibitors with the best poisoning effect on target insects. It can usually specifically bind to a certain type of digestive enzyme, affect t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C07K1/26C07K14/81C07K16/38G01N33/68
Inventor 张永军张莹陈洋许冬吴孔明
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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