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Non-receptor binding protein composition of insecticidal crystal protein as well as extracting method and application of non-receptor binding protein composition

A technique of combining insecticidal crystal proteins and receptors, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of no relevant reports and achieve the effect of overcoming the problem of resistance

Inactive Publication Date: 2016-06-15
HUAQIAO UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, does the pest body, especially the midgut, which is the main battlefield between pests and Cry, contain Cry-binding proteins (non-receptor-binding protein compositions) other than receptors? There are no relevant reports at home and abroad in this regard

Method used

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  • Non-receptor binding protein composition of insecticidal crystal protein as well as extracting method and application of non-receptor binding protein composition
  • Non-receptor binding protein composition of insecticidal crystal protein as well as extracting method and application of non-receptor binding protein composition
  • Non-receptor binding protein composition of insecticidal crystal protein as well as extracting method and application of non-receptor binding protein composition

Examples

Experimental program
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Effect test

Embodiment 1

[0032] (1) Take 100 healthy fourth-instar diamondback moth larvae cultured artificially and put them on ice for 5 minutes; dissect them under a stereomicroscope, and peel off the complete midgut (operate carefully, pay attention to retain the midgut lumen fluid) , placed in 20mM Tris-HCl buffer; homogenate on ice for 5min, 4°C, 12000rpm, centrifuge for 5min, take the supernatant, and store at -80°C.

[0033] (2) Separation and purification of Cry1Ab1 active region protein ( figure 1 ); covalently cross-link it on the chromatographic medium Sepharose4B; take 0.2g of activated Sepharose4B (1g of Sepharose4B is fully swollen to 5mL after activation), add it to the Cry1Ab1 protein solution, and incubate on a shaker at 25°C for 6h; Add 10mg of glycine, react for 6h to block the remaining groups; put the covalently cross-linked Cry1Ab1 chromatography medium Sepharose4B into the chromatography column, wash 10 column volumes with 1M NaCl solution, then wash 10 column volumes with deio...

Embodiment 2

[0038] Get 50 of the healthy fifth instar beet armyworm larvae of artificial culture, all the other operations are the same as embodiment 1, it can be seen that the non-receptor binding protein composition of Cry1Ab1 in the midgut of different pests has something in common and also has differences ( image 3 ).

Embodiment 3

[0040] Using short peptides derived from insect crystal protein sequences, combined with the corresponding non-receptor binding protein composition in the pest midgut (such as Figure 4Shown in is the docking interface between Cry1Ab and a binding protein, short peptides can be designed according to the 184-197 amino acids on Cry1Ab, and added to the product, the short peptide can block the binding of the binding protein to Cry1Ab), or use any binding protein that can bind The chemical molecules of the corresponding non-receptor binding protein composition in the midgut of the pest (for example, the insecticidal crystal protein in the midgut is a lectin, and the lectin can be bound by a specific monosaccharide, so this monosaccharide can be added In the product, blocking the binding of lectin to insecticidal crystal protein) can improve the insecticidal activity of Cry (insecticidal crystal protein) in the form of additive without changing the existing products of the factory. ...

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Abstract

The invention discloses a non-receptor binding protein composition of an insecticidal crystal protein as well as an extracting method and application of the non-receptor binding protein composition. The non-receptor binding protein composition comprises but not limited to tubulin, glutathione synthetase, ATP (Adenosine Triphosphate) synthetase, arginine kinase, lectin, trypsin, translation elongation factor and cytochrome C oxidase; in addition, the non-receptor binding protein composition is extracted from water-soluble matters in pest midgut containing midgut cavity liquid by affinity chromatography; affinity molecules for the affinity chromatography are insecticidal crystal proteins Cry. By applying the non-receptor binding protein composition, on the basis of not changing existing products in a factory, the insecticidal activity of the Cry can be improved, and the problems that pests generate resistance to the existing products, and the existing products have low activity for important pests are solved.

Description

technical field [0001] The invention belongs to the technical field of biopesticides, and in particular relates to a non-receptor binding protein composition of insecticidal crystal protein and its extraction method and application. Background technique [0002] Bacillus thuringiensis (Bt) preparation is the most widely used microbial insecticide in the world, and its main active ingredient is insecticidal crystal protein (Cry protein). [0003] The insecticidal mechanism of Cry protein is a complicated process. Currently, there are two hypothetical models: the pore-forming model and the signal transduction model. Both models believe that Cry is first activated by insect midgut proteases, and then interacts with receptor proteins such as aminopeptidase N (APN) and alkaline phosphatase on the surface of midgut BBMV (brushbordermembranevesicle, brush border membrane vesicle) cells. (ALP) and cadherin binding. In the pore-forming model, after Cry binds to the receptor, it for...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N9/00C12N9/14C12N9/12C12N9/76C12N9/02C07K1/22A01N47/44A01N61/00A01P7/04
CPCC07K14/00A01N47/44A01N61/00C12N9/0053C12N9/1223C12N9/14C12N9/6408C12N9/93C12Y109/03001C12Y207/03003C12Y304/21004C12Y306/01C12Y603/02003
Inventor 林毅刘肖萍古宇清余小强
Owner HUAQIAO UNIVERSITY
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