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Bt protein in-vitro insect resistance identification method

A technology of bt protein and carrier, applied in the field of biological genetic engineering, to achieve the effects of not being moldy, maintaining integrity and biological activity, and expressing efficiently

Inactive Publication Date: 2013-04-03
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method can only detect the content of Bt protein, and the actual effect of Bt protein must be bioassayed in order to accurately identify the insect resistance of Bt protein

Method used

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  • Bt protein in-vitro insect resistance identification method
  • Bt protein in-vitro insect resistance identification method
  • Bt protein in-vitro insect resistance identification method

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Embodiment 1

[0041] Embodiment 1 A method for identifying Bt protein against insects in vitro, comprising the following steps:

[0042] (1) Transform and synthesize the insect-resistant gene Bt ( figure 1) into the prokaryotic expression vector pET-28B (purchased from Shanghai Biological Engineering Co., Ltd.); the specific method is as follows:

[0043] Step 1: Digest pUC with restriction enzymes bt , the gel recovery kit recovers and purifies the Bt fragment;

[0044] Step 2: Digest pET28b with the same restriction enzyme, and recover and purify the digested pET28b fragment with the gel extraction kit;

[0045] Step 3: Ligate the two purified fragments, and construct the resulting prokaryotic expression plasmid named pET28b bt , identified by restriction endonuclease digestion, indicating that the vector was constructed correctly ( figure 2 );

[0046] (2) Transform the prokaryotic expression vector PET-28B containing the Bt gene into Escherichia coli BL21 (purchased from Shangh...

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Abstract

The invention relates to a Bt protein in-vitro insect resistance identification method which comprises the following steps: connecting a Bt gene to a protocaryon expression vector pET-28B; transferring the protocaryon expression vector containing the Bt gene into colibacillus BL21; inducing to express the Bt protein; extracting the induction-expressed Bt protein; mixing the Bt protein into an Ostrinia nubilalis larva artificial feed, and feeding Ostrinia nubilalis larvae; and after several days, making statistics on the mortality and growth conditions of the Ostrinia nubilalis larvae. The method is simple and quick to operate, is not restricted by time and seasons, has the advantage of low economic cost, and can be used for quickly carrying out Bt protein insect resistance identification.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to a method for identifying insect resistance of Bt protein in vitro. Background technique [0002] The Bt gene encodes an insecticidal crystal protein from Bacillus thuringiensis ( Bacillus thuring Hansis ). It produces delta-endotoxin insecticidal parasporal crystal proteins during sporulation, and these proteins have high insecticidal activity. The principle of action is that this anti-insect protein can be dissolved by alkaline intestinal fluid and hydrolyzed into smaller active toxin fragments—core fragments (Hofte and Whiteley, 1989). The active fragment is resistant to further hydrolysis by proteases, and the activated protein binds to the brush vesicles in the insect gut, causing perforation and affecting osmotic balance, cells swell and dissolve, target organisms stop feeding and eventually die. Studies have shown that the intestinal epithelial cells of many target ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12P21/02A01K67/033A23K1/16C12R1/21A23K10/18A23K10/30A23K20/158A23K20/163A23K20/174A23K50/90
Inventor 铁双贵岳润清卢彩霞齐建双王延召朱卫红柏松孙静
Owner HENAN ACAD OF AGRI SCI
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