Method for preparing antibody aiming at beet armyworm caspase-1

A technology of beet armyworm and large subunit, applied in the field of molecular biology, can solve problems such as unmet need, intolerance of rabbits, undetectable large subunit of caspase-1, etc.

Active Publication Date: 2017-02-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

The inventor once tried to select the full-length caspase-1 as the immunogen to make antibodies, but found that the rabbits could not tolerate it during the process of immunizing rabbits, and the inventors repeatedly selected some peptide segments as antigens as antibodies, Antibody not needed
For example, see Figure 4 , the inventor selected part of the N-terminal prodomain and the entire large subunit sequence (29-195aa) as an immunogen to immunize rabbits, and the resulting serum could only detect the caspase-1 holoenzyme, but the caspase-1 large subunit could not be detected

Method used

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  • Method for preparing antibody aiming at beet armyworm caspase-1
  • Method for preparing antibody aiming at beet armyworm caspase-1
  • Method for preparing antibody aiming at beet armyworm caspase-1

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Embodiment Construction

[0027] The principles and features of the present invention are described below in conjunction with the examples and accompanying drawings, and the given examples are only used to explain the present invention, and are not intended to limit the scope of the present invention.

[0028] 1. Cloning and expression of Caspase-1 gene fragment of beet armyworm

[0029] 1.1 Total RNA extraction

[0030] RNA extraction kit from QIAGEN ( MiniKit) and reverse transcription kit ( RT Kit), operate according to the instruction manual.

[0031] (1) Soak various pipette tips and centrifuge tubes in prepared 0.1% DEPC water, and treat them overnight at 37°C. The next day, autoclave the processed utensils at 121°C for 30 minutes, and store them at room temperature for RNA extraction. ready;

[0032] (2) Lyse cells to release RNA: put 4.5mL of IOZCAS-Spex-II cell culture medium after 3 days in a 2mL small tube, if the number of cells is less than 5×10 6 Take 350μL buffer RLT to lyse, if...

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Abstract

The invention relates to a method for preparing antibody aiming at beet armyworm caspase-1. The method comprises the following steps: immunizing an animal through polypeptide as shown in SEQ ID NO: 1 of an amino acid sequence, collecting serum of the animal, and purifying antibody from the serum. The invention further discloses the antibody obtained through the method. The invention further discloses a method for detecting beet armyworm caspase-1 large-subunit through the antibody. Through the adoption of the method and the antibody, the beet armyworm caspase-1 large-subunit in a sample can be specifically detected, and moreover, excitation of caspase-1 protein during insect cell apoptosis is initially detected, that is, breakage of large and small-subunit is detected. The method has important significance for the research on the toxicological meaning of the insect caspase gene family and the development of a new pesticide taking caspase as a target.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for preparing antibodies against the large subunit of caspase-1 of beet armyworm. Background technique [0002] Caspases are a class of conserved aspartate-specific cysteine ​​proteases present in multicellular organisms. As the central effector of apoptosis, caspase plays a key role in the initiation and process of apoptosis. Members of the caspase family complete the cascade reaction among caspase members through the recruitment, transmission and execution of apoptotic signals to promote the process of cell apoptosis. The caspase family is divided into two subfamilies of inflammatory and apoptotic caspases according to their physiological functions. Apoptotic caspases can be divided into initiator caspases and effector caspases. Upon initiation of apoptosis, activation of initiating caspases is a critical point at which cells choose between apoptosis or survival, ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C07K16/06C07K1/22G01N33/573
CPCC07K16/40
Inventor 张兰王丽萍杨菁菁张燕宁毛连纲蒋红云
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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