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Construction method of novel gene engineering recombinant virus for expressing gp64 gene

A recombinant virus and genetic engineering technology, applied in the field of genetic improvement of biological insecticides, can solve the problems of narrow insecticidal spectrum, slow insecticidal speed of baculovirus, hindering large-scale application, etc. The effect of improving the activity of insects

Inactive Publication Date: 2012-03-21
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the slow insecticidal speed and narrow insecticidal spectrum of baculovirus seriously hinder its large-scale application

Method used

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  • Construction method of novel gene engineering recombinant virus for expressing gp64 gene
  • Construction method of novel gene engineering recombinant virus for expressing gp64 gene
  • Construction method of novel gene engineering recombinant virus for expressing gp64 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of a genetically engineered recombinant virus co-expressing two envelope glycoproteins

[0037] 1. Reorganize the construction of bacmid:

[0038] pFB-Op166- gp64 - Polh The plasmid was electrotransformed into Escherichia coli DH10B (HaBacHz8 + helper) competent cells, spread onto LB solid medium plates, and cultured upside down at 37 °C for 24-48 hours. Pick a single white colony, insert it into 5ml LB medium, and culture overnight at 37°C. The culture was collected by centrifugation at 12,000 rpm for 1 min, and the genomic DNA of the recombinant bacmid was extracted according to the Bac-to-Bac manual (Invitrogen). Take 100 ng bacmid DNA as a template, carry out PCR identification with M13F, M13R on the carrier and gp64-For primers on the fragment respectively, and identify the correct recombinant bacmid named HaBac- gp64 - Polh ( figure 1 ).

[0039] 2. Transfection-infection experiment:

[0040] will be 5 x 10 5 HzAM1 cells were se...

Embodiment 2

[0043] Example 2. The application of the recombinant virus co-expressing two envelope glycoproteins of GP64 and F protein in reducing the dose applied to cotton bollworm larvae

[0044] Determination of the median lethal concentration LC of the recombinant virus by droplet feeding 50 . Select cotton bollworm larvae of uniform size at the end of the second instar, place them in a sterile 96-well plate and starve them overnight at 28 °C to obtain larvae of uniform age at the beginning of the third instar. The recombinant virus vHaBac- gp64 - Polh and control virus vHaBac- egfp - Polh The polyhedron was diluted to 3 x 10 6 PIBs / ml, 1 × 10 6 PIBs / ml, 3 × 10 5 PIBs / ml, 1 × 10 5 PIBs / ml, 3 × 10 4 PIBs / ml, 1 × 10 4 PIBs / ml, 3 × 10 3 PIBs / ml, 1 × 10 3 A series of concentration gradient suspensions of PIBs / ml. The diluted polyhedron suspension was fed to starved cotton bollworm larvae, and the larvae whose midgut turned blue were transferred to a 24-well plate c...

Embodiment 3

[0045] Example 3. The application of the recombinant virus co-expressing two envelope glycoproteins of GP64 and F protein in reducing the killing time of cotton bollworm larvae

[0046] According to LC in Example 2 50 The measured value of , select 3 × 10 6 PIBs / ml, 3 × 10 5 Two concentrations of PIBs / ml virus polyhedron suspension were used to infect third instar early cotton bollworm larvae by droplet feeding method, and the half survival time ST was carried out respectively. 50 determination. During this period, the death of infected larvae was observed every 4 hours until all the tested larvae died or pupated. Experiments were repeated twice. Plot time-mortality curves based on infection time and larval mortality. When using 3 x 10 6 When PIBs / ml infects cotton bollworm larvae, the recombinant virus vHaBac- gp64 - Polh reached 100% mortality faster than the control virus ( Figure 6 ), and when using a lower concentration of 3 × 10 5 When PIBs / ml infects cot...

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Abstract

The invention relates to a method for implementing gene engineering genetic improvement on a gp64-free baculovirus of a genome by using a Bac-to-Bac system. The method comprises construction of a recombinant virus, amplification of a recombinant virus polyhedrosis and biological assay of the recombinant virus; and according to the construction strategy, a gp64 gene controlled by an autographa californica multiplenucleopolyhedrovirus (AcMNPV) polyhedrosis virus gene promoter and a cyst membrane glycoprotein gene promoter of an orgyia pseudotsugata multiplenucleopolyhedrovirus (OpMNPV) polyhedrosis virus together and a helicoverpa armigera single nucleopolyhedrovirus (HearNPV) polyhedrin gene controlled by an AcMNPV polyhedrin gene promoter and a HearNPV polyhedrin gene promoter together are inserted into a polyhedrin gene site of HearNPV bacmid to construct a recombinant HearNPV bacmid for co-expressing two cyst membrane glycoproteins, namely GP64 and F proteins. The recombinant virus has better insecticidal activity and higher biological safety than a wild virus.

Description

technical field [0001] The invention belongs to the field of genetic improvement of biopesticides, in particular to an envelope glycoprotein gene expressing group I nuclear polyhedrosis virus (group I NPV) in baculovirus group II nuclear polyhedrosis virus (group II NPV) gp64 The construction method. The expression group I nuclear polyhedrosis virus (group I NPV) strain is preserved by the General Microbiology Center of China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101) . The date of deposit is June 24, 2011, and the deposit number is: CGMCC No: 4976. Background technique [0002] Most of the natural hosts of baculoviruses are agricultural and forestry pests. In 1973, baculovirus was recommended by the Food and Agriculture Organization of the United Nations and the World Health Organization as an ideal substitute for chemical pestic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866C12R1/93
Inventor 王华林王曼丽干盈盈邓菲胡志红
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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