Method for fixing scorpion toxin protein in silkworm BmNPV polyhedrosis crystal

A technology of scorpion toxin protein and polyhedron, which is applied in the field of DNA recombination technology and protein analysis, and can solve the problems of protein instability and loss of activity.

Inactive Publication Date: 2013-02-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Scorpion toxin protein is a kind of protein produced by cells that is toxic to insects and can be used to k

Method used

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  • Method for fixing scorpion toxin protein in silkworm BmNPV polyhedrosis crystal
  • Method for fixing scorpion toxin protein in silkworm BmNPV polyhedrosis crystal
  • Method for fixing scorpion toxin protein in silkworm BmNPV polyhedrosis crystal

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Embodiment Construction

[0020] Relevant materials involved in the following examples and their sources are as follows:

[0021] (1) Bombyx mori wild-type virus BmNPV: provided by the Silkworm and Bee Research Institute, School of Animal Science, Zhejiang University.

[0022] (2) RNA extraction kit: purchased from Qiagene.

[0023] (3) DNA processing and PCR amplification kit: purchased from Takara Biomedicals (Kyoto, Japan).

[0024] (4) TA cloning kit, baculovirus transfer vector pBlue BacHisa and lipofectin: all in the United States Invitrogen company's product.

[0025] (5) DNA sequencing kit: purchased from PE Applied Biosystems .

[0026] (6) Fetal calf serum FCS, silkworm culture medium TC-100: both Gibco BRL The product.

[0027]

[0028] The method for fixing the scorpion toxin protein of the silkworm BmNPV polyhedron crystal of the present invention is described in detail below with specific examples, and it comprises the following parts:

[0029] (1) Scorpion toxin protein...

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Abstract

The invention discloses a method for fixing the scorpion toxin protein in the silkworm BmNPV polyhedrosis crystal, comprising the following steps: (1) cloning the scorpion toxin protein BmK gene; (2) digesting the pCR2.1-BmK gene by the restriction endonucleases BamHI and HindIII to obtain a BmK gene fragment, cloning the BmK gene fragment into the baculovirus transfer vector pBlueBacHisa to obtain the recombinant transfer vector, cotransferctign the recombinant transfer vector and the wild nuclear nuclear polyhedrosis virus BmNPVDNA of the silkworm into the silkworm culture cells, and generating the recombinant baculovirus containing the scorpion toxin protein genes; and (3) inoculating the recombinant baculovirus containing the scorpion toxin protein gene and the wild nuclear nuclear polyhedrosis virus BmNPVDNA agaist the silkworm culture cells, infecting for 3 days at 27 DEG C, and collecting the silkworm culture cells by centrifuging. The method overcomes the defect that the scorpion toxin protein is easy to lose and creates a condition for developing the scorpion toxin protein used as the biological insecticide.

Description

technical field [0001] The invention relates to DNA recombination technology and protein analysis technology, in particular to a method for fixing scorpion toxin protein in silkworm BmNPV polyhedron crystal. Background technique [0002] Scorpion toxin protein is a kind of protein produced by cells that is toxic to insects and can be used to kill agricultural pests. However, the protein is unstable and easily decomposed in the environment and loses its activity. [0003] The nuclear polyhedrosis virus in the Bombyx mori baculovirus can produce a large amount of polyhedrosis protein in the late stage after infecting the host, and aggregate to form a unique super macromolecular protein crystal - polyhedron. Current research shows that the function of polyhedron is mainly to protect the virus particles embedded in polyhedron, so that it can preserve the activity for a long time. [0004] The present invention utilizes this phenomenon, utilizes wild-type virus to express polyhe...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N5/10
Inventor 王国宝沈运旺吴小锋
Owner ZHEJIANG UNIV
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