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Producing method of gene recombination nucleopolyhedrosis virus pesticide and application thereof

A nuclear polyhedrosis virus and gene recombination technology, applied in the field of bioengineering, can solve problems such as a lot of work, environmental safety has not been affirmed, etc., achieve good insecticidal effect, reduce half of the survival time, and reduce the effect of growth rate

Inactive Publication Date: 2008-08-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have improved the insecticidal activity of the wild-type AcNPV virus to a certain extent, the genes used in these methods are the neurotoxin gene of insectivorous mite and the toxin gene of Bacillus thuringiensis, and the safety of the environment has not been obtained. Certainly, people need to find more environmentally safe genes to transform wild-type baculovirus insecticides. For example, Harrison and Bonning recombined the cathepsin L gene into wild-type AcMNPV, improved the insecticidal activity of AcMNPV, and achieved relatively good results. Good effect insecticide (Harrison RL, BonningBC.2001.Use of proteases to improve the insecticidal activity of baculoviruses.Biol Control.20: 199-209), however, it is still necessary to develop more and better gene recombinant virus insecticides a lot of work

Method used

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  • Producing method of gene recombination nucleopolyhedrosis virus pesticide and application thereof
  • Producing method of gene recombination nucleopolyhedrosis virus pesticide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] For the three insertion genes of green fluorescent protein gene, insect functional gene (such as HHR3 gene, HCB gene or other genes) and polyhedrin gene, design and synthesize primers with enzyme cutting sites according to the gene sequence, and amplify them respectively by PCR method. Green fluorescent protein gene, cotton bollworm hormone acceptor 3 gene and cotton bollworm nuclear polyhedrosis virus polyhedrin gene; amplify and extract pFASTBAC plasmid in Escherichia coli; use restriction enzymes SmaI and KpnI to cut the green fluorescent protein respectively Protein gene PCR product and pFASTBAC plasmid, connect the green fluorescent protein gene to the plasmid to form a pFASTBAC recombinant plasmid with the green fluorescent protein gene; on this basis, cut the cotton bollworm hormone with EcoRI and NotI in the same way 3 gene and the pFASTBAC plasmid with the green fluorescent protein gene, and connect the two; in the same way, use NotI to cut the nuclear polyhedri...

Embodiment 2

[0044] Replace the cotton bollworm hormone acceptor 3 gene in Example 1 with other functional genes such as the cotton bollworm chitinase gene (http: / / www.ncbi.nlm.nih, acceptance number: AF515667), for different insertion genes , respectively design primers with restriction sites, amplify green fluorescent protein gene, other genes and polyhedrosis protein gene of cotton bollworm nuclear polyhedrosis virus respectively by PCR method; amplify and extract pFASTBAC plasmid in Escherichia coli; The endonucleases SmaI and KpnI cut the PCR product of the green fluorescent protein gene and the pFASTBAC plasmid respectively, and connected the green fluorescent protein gene to the plasmid to form a pFASTBAC recombinant plasmid with the green fluorescent protein gene; on this basis, the same method was used to , use EcoRI and NotI to cut other functional genes and the pFASTBAC plasmid with the green fluorescent protein gene, and connect the two; in the same way, use NotI to cut the nucl...

Embodiment 3

[0048] The nuclear polyhedrosis protein gene of cotton bollworm nuclear polyhedrosis virus in embodiment 1 and 2 is changed into the nuclear polyhedrosis protein gene of other polyhedrosis virus, as AcPolh (Genbank, http: / / www.ncbi.nlm.nih, acceptance number : K02700) or BmPolh, for the inserted gene, respectively design primers with restriction sites, respectively amplify the green fluorescent protein gene, other functional genes and other nuclear polyhedrosis virus polyhedrin genes with the PCR method; amplify in Escherichia coli Increase and extract the pFASTBAC plasmid; use restriction endonuclease SmaI and KpnI to cut the PCR product of the green fluorescent protein gene and the pFASTBAC plasmid respectively, connect the green fluorescent protein gene to the plasmid, and form the pFASTBAC recombinant plasmid with the green fluorescent protein gene; On this basis, use the same method to cut other genes and the pFASTBAC plasmid with the green fluorescent protein gene with Ec...

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Abstract

The invention discloses a pesticides of gene recombination of nuclear polyhedrosis virus and its preparation method, including building a green fluorescent protein donator plasmid, inserting foreign genes into donator plasmid, reducing polyhedrin gene, transporting green fluorescent protein and exogenous gene and polyhedrin gene into the nuclear polyhedrosis virus, transfecting insect cell, amplificating virus in the insect body, collecting and storing viruses and other steps.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, relates to DNA recombination technology and its application in the technical field of bioengineering, in particular to a method for creating a gene recombinant nuclear polyhedrosis virus insecticide and its application. Background technique [0002] China is a large agricultural country. In order to ensure high agricultural production, a large amount of chemical pesticides are used to control pests every year. Most of the chemical pesticides pollute the environment and seriously threaten human health. After entering the WTO, my country's agricultural production is facing increasingly fierce international competition, and a large number of toxic chemical pesticides are used, resulting in excessive pesticide residues, which seriously restricts the export of agricultural products. Therefore, it is imperative to reduce the use of toxic chemical pesticides. Biological pesticides are one of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/09A01N63/00A01P7/04
Inventor 赵小凡王金星
Owner SHANDONG UNIV
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