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Recombined baculovirus of insect, preparation method and application as insecticide

A virus and insecticidal crystal protein technology, applied in the field of genetic engineering, can solve problems such as weak virulence, failure to meet the requirements of agricultural production, and slow insecticidal speed

Inactive Publication Date: 2004-10-27
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN95117749 uses a series of genetic engineering operations to construct a polyhedron by inserting a P10 promoter and a partially truncated Bacillus thuringiensis σ-endotoxin cry1A (b) at the 5' end of the polyhedron gene ph containing AcNPV Recombinant viruses with complete and polyhedral outer membrane deletions, although the insecticidal spectrum of the obtained recombinant viruses has expanded, but due to the weak toxicity of the cry1A(b) gene and the slow insecticidal speed, they still cannot well meet the requirements of agricultural production

Method used

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  • Recombined baculovirus of insect, preparation method and application as insecticide
  • Recombined baculovirus of insect, preparation method and application as insecticide
  • Recombined baculovirus of insect, preparation method and application as insecticide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1 Cloning of the truncated cry1A(c) gene of Bacillus thuringiensis subspecies Kustak HD-73

[0142] Bacillus thuringiensis subsp. Kustak (BTK) HD-73 strain, plasmid vectors pXI93 (containing cry1Ab gene) and pHT3101 were provided by the China Center for Type Culture Collection, and the plasmid vector pBluescriptSK(+) was purchased from Stratagene. Dicer, random primer labeling kit was purchased from Promega Company, T4DNA ligase was purchased from GIBCO-BRL Company, α- 32 PdATP was purchased from Beijing Furui Company.

[0143] Construction of BTK HD-73 plasmid gene library: Bacillus thuringiensis (Bt) insecticidal crystal protein gene cry1A(c) was located on its largest 47Mda plasmid. In order to clone the full-length cry1A(c) gene, HindIII was used to completely digest Thuringiensis All the plasmids of Bacillus subsp. Kustak (BTK) HD-73 were cloned with pBluescriptSK(+) as vector, and the plasmid gene library of BTK HD-73 was constructed (Fig. 2).

[0144] L...

Embodiment 2

[0147] Example 2 Construction of transposable vector pFPhBc

[0148] The plasmid vector pBluePh was provided by China Type Culture Collection, and the transposable vector pFastBacl was purchased from GIBCO / BRL. pFastBacl has a mini-Tn7 transposon with a Gm inserted between its left and right arms rGene, AcNPV polyhedron promoter, multiple cloning sites and SV40 poly(A) termination structure.

[0149] Clone the polyhedrin gene (ph) to pFastBacl: From the vector pBluePh with the complete polyhedrin gene (ph) cloned, use Pst I / Sal I double enzyme digestion and recover the complete ph gene, insert it into the same Pst I / Sal I The transposition vector pFastBacl was double digested to construct the transposition vector pFPh. Positive clones were identified by Pst I / Sal I double digestion of pFPh to release a 1.3 kb ph fragment ( Figure 10 ).

[0150] The truncated cry1A(c) gene was cloned into pFPh: using the artificially synthesized DNA sequences GCGTCGACTATGGATAACAATCCGAACATC...

Embodiment 3

[0151] Example 3 Construction of recombinant Bacmid and acquisition of recombinant virus vAcPhBc:

[0152] Escherichia coli DH10Bac (containing the shuttle plasmid Bacmid and helper plasmids constructed based on the whole genome of Autographa californica nuclear polyhedrosis virus AcNPV) was purchased from GIBCO / BRL company, Sf-9 cells (Spodoptera frugiperda cells) Provided by China Center for Type Culture Collection.

[0153] Extract transposable vector pFPhBc DNA, transform Escherichia coli DH10Bac competent cells, under the action of the trans-acting factor produced by the helper plasmid, the Gm on pFPhBc r The / Pph / cry1A(c) / ph / SV40 polyA expression cassette is transposed to the mini-att site of Bacmid (Tn7 transposition contact site) by the Tn7 left and right flanking sequences on its 3' and 5' sides to obtain a recombinant Bacmid, Since the LacZ (galactosidase gene, which can decompose galactose and its analogues) of the recombinant Bacmid is insertionally inactivated, i...

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Abstract

A recombinant autographa california multiple nuclear polybedrosis virus, its preparing process, and its application as insecticide with broad spectrum and high speed are disclosed.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering. Specifically, the invention relates to a recombined Autographa californica nuclear polyhedrosis virus, a preparation method and an application as an insecticide. Background technique: [0002] Since the first wild-type baculovirus insecticide (HzNPV-cotton bollworm nuclear polyhedrosis virus) was approved and registered in the United States in 1975, there have been more than 30 commercial or experimental wild-type baculovirus insecticides was developed. Among them, Autographa californica nuclear polyhedrosis virus (AcNPV) insecticide is the most widely used. However, the disadvantage of the wild-type baculovirus is that it has a single insecticidal spectrum and a slow insecticidal speed, and it does not show insecticidal activity until 7 to 14 days after application. Therefore, in recent years, the application of genetic engineering to transform wild-type baculoviruses has become an importan...

Claims

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Application Information

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IPC IPC(8): A01N63/00C12N7/01C12N15/32C12N15/33C12N15/63C12N15/70
Inventor 齐义鹏杨复华周文科姚伦广李凌云肖化忠吕颂雅刘青珍
Owner WUHAN UNIV
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