Antigens and polyclonal antibody for discriminating Tilletia controversa Kuehn and Tilletia foetida

A technology of dwarf black powder fungus and polyclonal antibody is applied in the field of plant disease prevention and control, and can solve the problems of indistinguishable, destructive damage to wheat production, complicated procedures and the like, and achieves the effect of easy operation.

Active Publication Date: 2016-02-24
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

The disease is very harmful and difficult to control. Once it is introduced, it will cause devastating damage to wheat production. Usually, the incidence rate is approximately equal to the loss rate of wheat yield.
[0004] The field infection symptoms of T. tritici and T. dwarf as well as the spore morphology and color are very similar, and it is difficult to distinguish them in field observation
At present, the commonly used identification methods are PCR reaction or microscope detection, but these two methods require the use of instruments and can only be carried out in the laboratory, and the PCR reaction takes a long time and the process is complicated

Method used

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  • Antigens and polyclonal antibody for discriminating Tilletia controversa Kuehn and Tilletia foetida
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  • Antigens and polyclonal antibody for discriminating Tilletia controversa Kuehn and Tilletia foetida

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Preparation of polyclonal antibodies for identifying TCK and TFL

[0038] Reagents and solutions:

[0039] Freund's complete adjuvant, Freund's incomplete adjuvant (Beijing Qinbang Biotechnology Co., Ltd.), phosphate buffer (PBS, 0.02M, pH7.2), coating solution (phosphate buffer, 0.05M, pH7 .4), reaction diluent (PBS1, 0.03M, pH7.4+0.05% Tween 20), washing working solution (dilute the 20× concentrated washing solution with deionized water at a volume ratio of 1:19); blocking solution (Add 0.05% BSA in 0.02mol / LPBS1), substrate chromogenic solution: composed of A solution and B solution, A solution is hydrogen peroxide, and B solution is tetramethylbenzidine; stop solution (2mol / LH 2 SO 4 ), enzyme-labeled secondary antibody (Beijing Qinbang Biotechnology Co., Ltd.).

[0040] Tilletia triticum suspension 1*10 5 Spore / ml

[0041] Main equipment:

[0042]Vacuum freeze dryer, electric thermostat incubator, ProteinA / G column, electrophoresis tank, electropho...

Embodiment 2

[0088] Embodiment 2. Adopt antibody of the present invention to distinguish Tilletia tritici and Tilletia tritici

[0089] The spores of T. tritici and T. tritici preserved in the laboratory were used as antigens to react with the purified antibody obtained in Example 1 respectively, and the antibody blocking solution was used as a negative control. The steps were as follows:

[0090] a, wrapping plate: the polypeptide coating in embodiment 1 is diluted mass volume ratio 1:1000 times with antigen diluent, dilutes Tilletia tritici with antigen diluent 200 times, adds enzyme-labeled plate respectively, every hole 100ul, incubate at 37°C for 2 hours, wash the plate once, and pat dry;

[0091] b. Blocking: Add blocking solution for blocking, 150ul per well, incubate at 37°C for 2 hours, discard the solution, pat dry, and set aside;

[0092] c, add antibody: dilute the polyclonal antibody prepared in Example 1 to a certain concentration with antibody diluent (polypeptide coating o...

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Abstract

The invention relates to antigens and a polyclonal antibody used for discriminating Tilletia controversa Kuehn and Tilletia foetida. A semiantigen used for preparing the polyclonal antibody for discriminating Tilletia controversa Kuehn and Tilletia foetida has an amino acid sequence as shown in SEQ ID No. 1. An immunogen used for preparing the polyclonal antibody for discriminating Tilletia controversa Kuehn is characterized in that the semiantigen mentioned above is reconstructed and coupled so as to obtain the immunogen, the immunogen is used to immunize domestic rabbits, domestic rabbits generating high-titer whole blood are screened and whole blood is acquired from a selected domestic rabbit and undergoes separating and purifying so as to obtain the polyclonal antibody. The antibody can specifically identify Tilletia controversa Kuehn without undergoing an antigen-antibody reaction with Tilletia foetida, so the polyclonal antibody can be used for discriminating Tilletia controversa Kuehn and Tilletia foetida difficult to distinguish in a field.

Description

technical field [0001] The invention relates to an antigen and a polyclonal antibody used for differentiating Tilletia tritici and Tilletia tritici, belonging to the field of plant disease prevention and control. Background technique [0002] Tilletia foetida (Wallr.) Liro, TFL, caused by Tilletiafoetida (Wallr.) Liro, TFL, is one of the most destructive wheat diseases in the world. Supplementary agricultural phytosanitary pests. If the spore content of this pathogen exceeds 0.6%, severe poisoning can be caused, and severe poisoning symptoms such as nausea, vomiting and even coma can be caused by humans and animals after ingestion (Goats, 1999; Hoffman, 1982), which seriously affects the quality and commercial value of wheat. [0003] Tilletia controversa Kühn (TCK) is an important international quarantine disease caused by Tilletia controversa Kühn (TCK), and it is also a kind of dangerous plant disease in foreign quarantine in my country. The disease is very harmful and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/34C07K16/18C07K16/06G01N33/569
Inventor 高利沈慧敏李超陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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