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Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate

A technology of trypsin and protoxin, applied in the biological field, can solve problems such as lack of pertinence, low sensitivity, and inability to directly elucidate the effect, and achieve the effects of clear specificity and pertinence, improved sensitivity, and great practical value

Inactive Publication Date: 2014-07-16
NANJING AGRICULTURAL UNIVERSITY
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In traditional assay methods, the information obtained is about the ability of protease, trypsin or chymotrypsin to hydrolyze the model substrate, which is not specific and cannot directly elucidate the effect of midgut protease on Bt toxin
Moreover, in the determination of protease activity, color reactions are used for the above three model substrates, and the sensitivity is relatively low.

Method used

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  • Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate
  • Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate
  • Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate

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Embodiment 1

[0027] The first step: extract the enzyme liquid of the midgut of the larvae of Lepidoptera insects.

[0028] Taking the Lepidoptera insect cotton bollworm as an example, take the 5th instar larva of the cotton bollworm on the second day, put it on ice for 30 minutes, cut off the head and tail with a scalpel, and pick up the midgut (including the contents) with tweezers The midguts of each 3-5 test worms were combined as a sample, and 1ml of pre-cooled 0.15M sodium chloride solution was added, homogenized in an ice bath, and then the homogenate was transferred to a centrifuge tube and centrifuged for 15 minutes (12,000g, 4°C) , take the supernatant as the enzyme source, and store it at -20°C for future use.

[0029] The second step: extracting the BBMV from the midgut of the larvae of Lepidoptera insects.

[0030] Taking the Lepidoptera insect cotton bollworm as an example, take the 5th instar larva on the second day of the cotton bollworm, put it on ice for 30 minutes, remov...

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Abstract

The invention discloses a fluorescent substrate for detecting the activity of trypsin acting on Cry1A protoxin and an application of the fluorescent substrate. The fluorescent substrate consists of fluorescence quenching group-Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu-fluorescence group. The invention also discloses the application of the fluorescent polypeptide substrate R28 in detecting the activity of insect midgut trypsin which directly acts on activated bacillus thuringiensis Cry1A protoxin. Compared with a conventional protease detection method, the fluorescent substrate disclosed by the invention has definite specificity and pertinence for detecting the activity of the insect midgut trypsin which directly acts on the activated bacillus thuringiensis Cry1A protoxin and detecting the activity of the trypsin which only acts on the corresponding fluorescent substrate R28, and intense fluorescence signals are generated in an enzymatic hydrolysis reaction, so that the sensitivity of the detection is greatly improved.

Description

technical field [0001] The invention relates to biotechnology, and relates to a fluorescent substrate for detecting the activity of trypsin acting on Cry1A protoxin and its application, in particular to a fluorescent substrate for detecting Cry1A protoxin acting on Bacillus thuringiensis (Bacillus thuringiensis) Fluorescent substrates for trypsin activity and their applications. technical background [0002] Bacillus thuringiensis is a Gram-positive, rod-shaped, spore-forming soil bacterium. Accompanied by the formation of spores during its growth, the cells can synthesize protein crystals. Because these crystal proteins have a poisonous effect on insects, they are also called insecticidal crystal proteins (ICPs). Among them, δ-endotoxin Is the most important class of insecticidal protein. δ-endotoxins are divided into Cry toxins and Cyt toxins according to their structural characteristics and mechanism of action. And Whiteley in 1989, mainly based on the difference in i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12Q1/37C12R1/07
Inventor 杨亦桦武淑文吴益东
Owner NANJING AGRICULTURAL UNIVERSITY
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