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In vitro construction method of cytoplasmic polyhedrosis virus of Bombyx mori

The technology of a plastid polyhedrosis and a construction method is applied to the field of in vitro construction of the silkworm plastid polyhedrosis virus, which can solve the problem that the silkworm plastid polyhedrosis virus has not been constructed in vitro, the functional research progress is slow, and the recombinant CPVs insecticide cannot be obtained. And other issues

Active Publication Date: 2015-07-22
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Usually, the method of obtaining silkworm plasmopolyhedrosis virus is to purify from the midgut of silkworms suffering from plasmopolyhedrosis, which occurs naturally or artificially, but this method must use silkworm as a host
In addition, the genome of the silkworm plasmopolyhedrosis virus is a segmented double-stranded RNA virus, and it is difficult to modify the genome of the silkworm plasmopolyhedrosis virus using conventional genetic manipulation techniques to obtain recombinant viruses, resulting in the fragmentation of each segment of the CPVs genome. Functional studies are slow and recombinant CPVs insecticides are not available
[0005] So far, there is no report on the construction of silkworm plasmopolyhedrosis virus in vitro

Method used

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  • In vitro construction method of cytoplasmic polyhedrosis virus of Bombyx mori
  • In vitro construction method of cytoplasmic polyhedrosis virus of Bombyx mori
  • In vitro construction method of cytoplasmic polyhedrosis virus of Bombyx mori

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: In vitro construction and performance testing of silkworm plasmopolyhedrosis virus.

[0066] 1. In vitro construction of virus:

[0067] (1) Extract the silkworm plasmopolyhedrosis virus genome:

[0068] Collect the midgut tissue of silkworm diseased polyhedrosis silkworm, add double distilled water according to the ratio of 1g midgut tissue: 10mL double distilled water (self-made, Chengdu Ultrapure Technology Co., Ltd., UPT-Ⅲ-5T ultrapure water machine) , after homogenization, filter with gauze, and the filtrate is centrifuged at a differential speed by a CF15D2 centrifuge (KuBoTa Company, Japan) to obtain pure silkworm plastid polyhedron; add water to suspend the pure silkworm plastid polyhedron, and adjust the concentration to per mL of water Contains at least 10 8 Polyhedron; Take 0.5mL of polyhedron suspension, add an equal volume of Tris-balanced phenol (Beijing Suo Laibao Technology Co., Ltd.), shake the mixture on a QL-901 oscillator (Qilin Beier In...

Embodiment 2

[0126] Example 2: In vitro construction of silkworm plasmopolyhedrosis virus.

[0127] 1. Extracting the silkworm plasmopolyhedrosis virus genome: the same as step (1) in Example 1.

[0128] 2. Design and synthesis of primers: same as step (2) in Example 1.

[0129] 3. Reverse transcription to obtain cDNA of fragments S1 to S10: same as step (3) in Example 1.

[0130] 4. PCR amplification of the full-length cDNA of S1 to S10 fragments: same as step (4) in Example 1.

[0131] 5. In vitro transcription: take the full-length cDNA of the PCR-amplified S1 to S10 fragments in step 4 as templates, use mMESSAGE mMACHINE T7 Ultra Kit (Ambion) to perform in vitro transcription according to the product instructions, and use RNase-free DNase for in vitro transcription products Digested to remove the DNA template, the in vitro transcribed RNA was ethanol-precipitated, and the RNA concentration was determined using a UV spectrophotometer.

[0132] 6. Obtaining the Bombyx mori plasmopolyhed...

Embodiment 3

[0133] Example 3: Construction of Bombyx mori plasmopolyhedrosis virus in vitro.

[0134] (1) Extraction of the silkworm plasmopolyhedrosis virus genome: the same as step (1) in Example 1.

[0135] (2) Design and synthesis of primers: same as step (2) in Example 1.

[0136] (3) Reverse transcription to obtain cDNA of S1 to S10 fragments: same as step (3) in Example 1.

[0137] (4) PCR amplification of the full-length cDNA of S1 to S10 fragments: same as step (4) in Example 1.

[0138] (5) Construction of recombinant plasmid: same as step (5) in Example 1.

[0139] (6) In vitro transcription: use the recombinant plasmids pT-S1 to pT-S10 obtained in step (5) as templates, and use the 10 pairs of primers synthesized in step (2) for PCR amplification, that is, the recombinant plasmid pT-S1 The primer pair PS1F / PS1R amplifies, and so on. Each amplified product was used as an in vitro transcription template, and mMESSAGE mMACHINE T7 Ultra Kit (Ambion Company) was used for in vit...

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Abstract

The invention discloses an in vitro construction method of a cytoplasmic polyhedrosis virus of Bombyx mori. Particularly, the in vitro construction method comprises the following steps: 1) obtaining a cytoplasmic polyhedrosis virus genome of the Bombyx mori; 2) designing and combining primers; 3) obtaining cDNA of S1-S10 fragments of the cytoplasmic polyhedrosis virus genome; 4) carrying out PCR amplification on the cDNA; 5) carrying out endonuclease digestion on an amplified product, and cloning into a plasmid vector to obtain a recombinant plasmid; 6) after carrying out endonuclease digestion and linearization on the recombinant plasmid, obtaining RNA by virtue of in vitro transcription; and 7) equal mol mixing RNA, wrapping by using a liposome, transfecting a host, and thus obtaining the cytoplasmic polyhedrosis virus of Bombyx mori constructed in vitro. The in vitro construction method of the cytoplasmic polyhedrosis virus of the Bombyx mori has the action of promoting function research of each fragment of the cytoplasmic polyhedrosis virus genome and can also provide technical support to develop a recombinant cytoplasmic polyhedrosis virus biological insecticide.

Description

technical field [0001] The invention belongs to the field of virus genetic engineering, and in particular relates to an in vitro construction method of silkworm cytoplasmic polyhedrosis virus. Background technique [0002] Reoviridae viruses are a class of double-stranded RNA (dsRNA) viruses that can infect animals, plants, and fungi. Cytoplasmic polyhedrosis virus (CPVs for short) is a member of the genus Cypovirus in the Reoviridae family, and its genome consists of 9 to 11 double-stranded RNA segments. CPVs are important pathogens of agricultural and forestry pests and can infect Lepidoptera ( Lepidoptera ), Hymenoptera ( Hymenoptera ) and Coleoptera ( Coleoptera ) insects, which play an important role in the natural population control of pests, are a biopesticide with great development potential. [0003] According to the migration pattern of CPVs genomic dsRNA in polyacrylamide gel electrophoresis, CPVs can be divided into 20 different types. Bombyx mori plasmopo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/88C12N15/46C12N7/00C12N15/11C12R1/93
Inventor 贡成良薛仁宇曹广力胡小龙郭睿
Owner SUZHOU UNIV
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