A method for in vitro construction of silkworm plasmopolyhedrosis virus

A technology of plasmopolyhedron and construction method, which is applied in the field of in vitro construction of silkworm plasmopolyhedrosis virus, can solve the problem of slow progress in functional research, inability to obtain recombinant CPVs insecticides, and no in vitro construction of silkworm plasmopolyhedrosis virus And other issues

Active Publication Date: 2018-03-30
SUZHOU UNIV
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  • Summary
  • 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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  • A method for in vitro construction of silkworm plasmopolyhedrosis virus
  • A method for in vitro construction of silkworm plasmopolyhedrosis virus
  • A method for in vitro construction of silkworm plasmopolyhedrosis virus

Examples

Experimental program
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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 silkworm plasmopolyhedrosis virus. Specifically, the method of the present invention comprises the following steps: 1) Obtaining the genome of Bombyx mori plasmopolyhedrosis virus; 2) Designing and synthesizing primers; 3) Obtaining the cDNA of fragments S1 to S10 of the viral genome; 4) Performing PCR amplification on the cDNA ;5) Digest the amplified product, and then clone it into a plasmid vector to obtain a recombinant plasmid; 6) Digest and linearize the recombinant plasmid, and transcribe in vitro to obtain RNA; and 7) Mix the RNA equimolarly , use liposome to encapsulate and transfect the host, and then obtain the silkworm plasmopolyhedrosis virus constructed in vitro. The in vitro construction method of the silkworm polyhedrosis virus of the present invention not only promotes the functional research of each fragment of the silkworm polyhedrosis virus genome, but also provides technical support for the research and development of the recombinant cytoplasmic polyhedrosis virus biopesticide.

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 Patents(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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