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Duck plague virus us1 gene seamless deletion virus strain and its construction method

A duck plague virus and deletion virus technology, applied in the field of genetic engineering

Active Publication Date: 2021-02-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the above-mentioned problems existing in the prior art, the present invention provides a duck plague virus US1 gene traceless deletion virus strain and its construction method. The present invention utilizes GS1783 Escherichia coli strain and pEPkan-S plasmid to recombine duck plague virus in bacterial artificial chromosome On the virus rescue system platform, the single and double copy genes of duck plague virus US1 were deleted through two homologous recombination, and the construction of the virus strain without trace deletion of this gene was completed for the first time

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  • Duck plague virus us1 gene seamless deletion virus strain and its construction method
  • Duck plague virus us1 gene seamless deletion virus strain and its construction method
  • Duck plague virus us1 gene seamless deletion virus strain and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067]Example 1 Construction of the US1 gene traceless deletion strain GS1783-pBAC-DPV-ΔUS1

[0068]The US1 single-copy gene traceless deletion strain GS1783-pBAC-DPV-ΔUS1 was constructed on E. coli GS1783 by Red / ET modification technology. The construction method includes the following steps:

[0069]1. Prepare GS1783 electrocompetent and electrotransform pBAC-DPV plasmid

[0070](1) Resuscitate Escherichia coli with pBAC-DPV plasmid in LB solid medium containing chloramphenicol and culture overnight at 37°C; pick a single colony and inoculate it in LB liquid medium containing chloramphenicol and cultivate overnight at 37°C;

[0071](2) Extract the pBAC-DPV plasmid according to the QIAGEN Plasmid Midi Kit operating instructions;

[0072](3) Resuscitate cryopreserved bacteria of GS1783 in LB solid medium and cultivate overnight at 30°C;

[0073](4) Pick a single colony of GS1783 and inoculate it in 5mL LB liquid medium, and cultivate it overnight at 30°C to obtain seed liquid;

[0074](5) Add 5mL seed s...

Embodiment 2

[0114]Example 2 Construction of the US1 gene traceless deletion strain GS1783-pBAC-DPV-2ΔUS1 strain

[0115]The US1 double-copy gene traceless deletion strain CHv-BAC-G-2ΔUS1 was constructed on E. coli GS1783 by Red / ET modification technology. The construction method includes the following steps:

[0116]1. Amplify I_sceI-Kana-US1 targeting fragment

[0117](1) Resuscitate Escherichia coli with pEPkan-S plasmid in LB solid medium containing kanamycin and culture overnight at 37°C; pick a single colony and inoculate it in LB liquid medium containing kanamycin, 37°C Cultivate overnight, and extract pEPkan-S plasmid with a plasmid small extraction kit (see the pEPkan-S plasmid mapfigure 1 );

[0118](2) Using the pEPkan-S plasmid as the template, 1808F and 1808R as primers, PCR was used to amplify the base fragments containing the I-SceI restriction site and Kana elements, as well as the 40bp homology arms in the upstream and downstream of the US1 gene. Target fragment 1808, use 1% ordinary agaros...

Embodiment 3

[0147]Example 3 Rescue CHv-BAC-G-ΔUS1, CHv-BAC-G-2ΔUS1 virus

[0148]1. Resuscitate GS1783-pBAC-DPV-ΔUS1 and GS1783-pBAC-DPV-2ΔUS1 cryopreserved bacteria in LB solid medium containing chloramphenicol and cultivate overnight at 30°C.

[0149]2. Extract pBAC-DPV-ΔUS1 and pBAC-DPV-2ΔUS1 according to the instructions of QIAGEN Plasmid Midi Kit:

[0150](1) Pick a single colony of GS1783-pBAC-DPV-ΔUS1, GS1783-pBAC-DPV-2ΔUS1 and inoculate it in 100 mL of LB liquid medium containing chloramphenicol, and cultivate overnight at 30°C;

[0151](2) Harvest cells: centrifuge at 4°C for 15 minutes, with a centrifugal force of 6000×g.

[0152](3) Resuspend the bacteria in 4mL Buffer P1 (add RNase A to Buffer P1 before use).

[0153](4) Add 4mL buffer P2, quickly flip the tube 4-6 times, and let it stand at room temperature (15-25°C) for 5 minutes.

[0154](5) Add 4 mL of pre-cooled buffer P3, mix it immediately and turn it over 4-6 times vigorously, and incubate on ice for 15 minutes.

[0155](6) Centrifuge for 30 minute...

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Abstract

The invention provides a duck plague virus US1 gene traceless deletion virus strain and a construction method thereof. The present invention utilizes GS1783 Escherichia coli strain and pEPkan‑S plasmid to delete the single and double copy gene of duck plague virus US1 respectively through two homologous recombination on the bacterial artificial chromosome recombination duck plague virus rescue system platform, and completes the geneless detection of the gene for the first time. A virus strain with scar deletion was constructed, and the effect of this gene on virus replication was explored. The technical scheme of the invention lays the foundation for accurately exploring the function of the US1 gene of duck plague virus, so as to provide a reference for molecular biology research of duck plague virus.

Description

Technical field[0001]The invention belongs to the field of genetic engineering, and specifically relates to a duck plague virus US1 gene single- and double-copy traceless deletion virus strain CHv-BAC-G-ΔUS1, CHv-BAC-G-2ΔUS1 and a construction method thereof.Background technique[0002]Bacterial artificial chromosome (BAC) is a plasmid vector constructed based on E. coli F factor. F factor, also known as F plasmid, is a "sex plasmid" that can transfer host chromosomal genes to another host cell. Natural F factor is a supercoiled closed-loop DNA molecule with a size of about 100 kb and encoding about 100 proteins. BAC can accommodate foreign inserts with a size of 100 to 300 kb, and can be inherited stably. The genome cloned into the BAC vector will not appear in the yeast artificial chromosome (YAC) vector during the passage and is prone to deletion, recombination and mosaicism, and Manual genetic manipulation can be performed in E. coli, which is convenient and safe.[0003]Duck plague...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N7/01C12R1/93
CPCC07K14/005C12N7/00C12N15/63C12N2710/16321C12N2710/16322C12N2800/204C12N2800/30
Inventor 吴英李阳光程安春汪铭书
Owner SICHUAN AGRI UNIV