Construction method of a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein

A technology of red fluorescent protein and plasmopolyhedron, which is applied in the field of virus genetic engineering, can solve the problems of constructing recombination and expressing foreign genes that have not been seen in vitro.

Active Publication Date: 2018-06-19
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Since the silkworm cytoplasmic polyhedrosis virus genome consists of 10 double-stranded RNA fragments from S1 to S10, it is difficult to construct recombinant silkworm cytoplasmic polyhedrosis virus to express foreign genes through genetic manipulation.
So far, there is no report on the construction of recombinant silkworm cytoplasmic polyhedrosis virus in vitro to express foreign genes, and there is no report on the construction of other recombinant cytoplasmic polyhedrosis viruses to express foreign genes

Method used

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  • Construction method of a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein
  • Construction method of a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein
  • Construction method of a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Construction and performance testing of recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein.

[0078] 1. Virus construction:

[0079] (1) Extract the silkworm plasmopolyhedrosis virus genome:

[0080] 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, it was filtered with gauze, and the filtrate was centrifuged at a differential speed by a CF15D2 centrifuge (KuBoTa Company, Japan) to obtain pure silkworm polyhedron; the pure silkworm polyhedron was added with water (self-made, Chengdu Chaopure Technology Co., Ltd. company, UPT-Ⅲ-5T ultrapure water machine) suspension, adjust the concentration to contain at least 10 per mL of water 8 For each polyhedron, take 0.5 mL of the polyhedron suspension, add an equ...

Embodiment 2

[0145] Example 2: Construction of recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein.

[0146] (1) Extracting the silkworm plasmopolyhedrosis virus genome: the same as step (1) in Example 1.

[0147] (2) Design and synthesis of primers: same as step (2) in Example 1.

[0148] (3) Reverse transcription to obtain cDNA of S1 to S10 fragments: same as step (3) in Example 1.

[0149] (4) PCR amplification of the full-length cDNA of S1 to S10 fragments: same as step (4) in Example 1.

[0150] (5) Construction of recombinant plasmid: same as step (5) in Example 1.

[0151] (6) Preparation of RNA from S1 to S9 fragments: Take the PCR-amplified full-length cDNA from S1 to S9 fragments in step 4 as templates, use mMESSAGE mMACHINE T7 Ultra Kit (Ambion Company) to transcribe in vitro according to the product instructions, and in vitro transcribe products The DNA template was removed by RNase-free DNase digestion, the in vitro transcribed RNA was precipita...

Embodiment 3

[0154] Example 3: Construction of recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein.

[0155] (1) Extracting the silkworm plasmopolyhedrosis virus genome: the same as step (1) in Example 1.

[0156] (2) Design and synthesis of primers: same as step (2) in Example 1.

[0157] (3) Reverse transcription to obtain cDNA of S1 to S10 fragments: same as step (3) in Example 1.

[0158] (4) PCR amplification of the full-length cDNA of S1 to S10 fragments: same as step (4) in Example 1.

[0159] (5) Construction of recombinant plasmid: same as step (5) in Example 1.

[0160] (6) Preparation of RNA fragments from S1 to S9: use the recombinant plasmids pT-S1 to pT-S9 obtained in step (5) as templates, and use the 9 pairs of primers synthesized in step (2) for PCR amplification, that is, recombinant Plasmid pT-S1 uses primers to amplify PS1F / PS1R, recombinant plasmid pT-S2 uses primers to amplify PS2F / PS2R, and so on. Then use each amplified product as an...

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Abstract

The invention discloses a method for constructing a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein. Specifically, the method of the present invention includes the following steps: 1) obtaining the viral genome; 2) designing and synthesizing primers; 3) obtaining the cDNA of the viral genome; 4) performing PCR amplification on the cDNA; 6) Digest the recombinant plasmids pT‑S1 to pT‑S9, and transcribe in vitro to obtain the corresponding RNA; 7) Construct the recombinant plasmid pT‑S10‑DsRed with the red fluorescent protein gene and obtain S10‑ DsRed RNA; and 8) equimolar mixing of the above RNAs, encapsulating and transfecting the host to obtain the virus. The method established by the invention can express the red fluorescent protein in the silkworm cell, and then it is possible to obtain the recombinant cytoplasmic polyhedrosis virus biopesticide required by people.

Description

technical field [0001] The invention belongs to the field of virus genetic engineering, and in particular relates to a method for constructing a recombinant silkworm plasmopolyhedrosis virus expressing red fluorescent protein. 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 diff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/88C12N15/46C12N7/01C12N15/11C12R1/93
Inventor 贡成良曹广力薛仁宇胡小龙郭睿
Owner SUZHOU UNIV
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