A method for in vitro construction of silkworm plasmopolyhedrosis virus based on dna vector

A technology of plastid polyhedrosis and construction method, which is applied to the field of in vitro construction of silkworm plastid polyhedrosis virus, can solve problems such as difficulty in obtaining sequence comparison, affecting virus efficiency, and easy degradation of RNA molecules.

Active Publication Date: 2020-12-04
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing technical solutions all need to use in vitro transcription technology. Due to technical bottlenecks, in vitro transcription is sometimes difficult to obtain complete RNA molecules with long sequences, and RNA molecules are easy to degrade, which affects the efficiency of virus acquisition.
For some viruses, the viral RNA obtained by in vitro transcription through cell transfection cannot rescue the virus in vitro.

Method used

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  • A method for in vitro construction of silkworm plasmopolyhedrosis virus based on dna vector
  • A method for in vitro construction of silkworm plasmopolyhedrosis virus based on dna vector
  • A method for in vitro construction of silkworm plasmopolyhedrosis virus based on dna vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: In vitro construction and characteristic detection 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 Beie...

Embodiment 2

[0130] Example 2: In vitro construction of silkworm plasmopolyhedrosis virus.

[0131] 1. According to the RNA sequence of S1 to S10 fragments, the cDNA of S1 to S10 is fully chemically synthesized;

[0132] 2. Construction of recombinant plasmids:

[0133] Take the cDNAs from S1 to S10 fully chemically synthesized in step (1), and clone them into the pIZT-V5 / His vector respectively, wherein:

[0134] The full-length cDNA of the S1 fragment was cloned into the SacI and SacII sites of pIZT-V5 / His;

[0135] The full-length cDNA of the S2 fragment was cloned into the SacI and SacII sites of pIZT-V5 / His;

[0136] The full-length cDNA of the S3 fragment was cloned into the SacI and SacII sites of pIZT-V5 / His;

[0137] The full-length cDNA of the S4 fragment was cloned into the kpnI and SacII sites of pIZT-V5 / His;

[0138] The full-length cDNA of the S5 fragment was cloned into the kpnI and SacII sites of pIZT-V5 / His;

[0139] The full-length cDNA of the S6 fragment was cloned ...

Embodiment 3

[0149] Embodiment three: In vitro construction of silkworm plasmopolyhedrosis virus

[0150] (1) Same as the step (1) in the in vitro construction of the virus in Example 1;

[0151] (2) The genome of silkworm plasmopolyhedrosis virus was separated by 0.75% agarose gel electrophoresis, and the results were as follows: Figure 5 As shown, from bottom to top, each nucleic acid band is the S1 to S10 fragments, respectively. Use the nucleic acid gel recovery kit to recover the RNA of each fragment, and use it in RNase-free double distilled water to obtain the RNA from S1 to S10 respectively;

[0152] (3) Take 20 μL of RNA from S1 to S10 in step (2), boil at 100°C for 10 minutes, then ice-bath for 2 minutes, take 1 μL as a template, and use the RNA in step (2) in the in vitro construction of the virus in Example 1 Primers PS1R, PS2R, PS3R, PS4R, PS5R, PS6R, PS7R, PS8R, PS9R, and PS10R were reverse-transcribed using a reverse transcription kit (Transcriptor Reverse Transcriptase, ...

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Abstract

The invention discloses a DNA carrier based in vitro construction method of a nombyx mori cytoplasmic polyhedrosis virus. Specifically, the method comprises the following steps: 1) acquiring a genomeof a nombyx mori cytoplasmic polyhedrosis virus; 2) designing and synthesizing a primer; 3) acquiring cDNA of fragments S1 to S10 of the genome of the virus; 4) conducting PCR (Polymerase Chain Reaction) amplification on the cDNA; 5) implementing enzyme digestion on an amplification product, then cloning to a plasmid vector, acquiring a recombinant plasmid; 6) implementing enzyme digestion and linearization on the recombinant plasmid; 7) mixing the linearized plasmid at equal molar ratio, wrapping by using a lipidosome, transfecting and expressing a host cell of a T7-RNA polymerase, and further acquiring the nombyx mori cytoplasmic polyhedrosis virus constructed in vitro. The DNA carrier based in vitro construction method of the nombyx mori cytoplasmic polyhedrosis virus not only has an effect on promoting the functional study of various fragments of the genome of the nombyx mori cytoplasmic polyhedrosis virus, but also provides a novel method for acquiring the nombyx mori cytoplasmicpolyhedrosis virus in vitro, and provides theoretical direction and technical support for the researches and development of the construction of a 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 a silkworm plasmopolyhedrosis virus based on a DNA carrier. Background technique [0002] Reoviridae viruses are a class of double-stranded RNA (dsRNA) viruses that can infect a variety of animal, plant, and fungal hosts. Cytoplasmic polyhedrosis virus (CPVs for short) belongs to the Cytoplasmic polyhedrosis virus genus (Cypovirus) in the family Reoviridae in classification, and its genome usually consists of 9 to 11 dsRNA fragments with different molecular weights. CPVs can infect species belonging to the order Lepidoptera ( Lepidoptera ), Hymenoptera ( Hymenoptera ) and Coleoptera ( Coleoptera ) and other agricultural and forestry pests, it is a kind of biopesticide with great development potential. However, due to technical bottlenecks, there is no recombinant cytoplasmic polyhedrosis insecticide obtained by gene rec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N7/00C12R1/93
CPCC12N7/00C12N15/85C12N2720/12052C12N2800/105
Inventor 贡成良曹广力薛仁宇胡小龙余蕾冯永杰
Owner SUZHOU UNIV
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