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Duck plague virus ge and gi double gene traceless deletion strain dpv CHv-ΔgE+ΔgI and its construction method

A duck plague virus and construction method technology, applied in the field of genetic engineering, can solve problems such as residual bases and residual MiniF elements

Active Publication Date: 2022-06-17
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of the above-mentioned problems existing in the prior art, the present invention provides a duck plague virus gE and gI double-gene traceless deletion strain DPV CHv-ΔgE+ΔgI and its construction method, which can effectively solve the problem of duck plague virus gene deletion. The problem of leaving bases at the deletion site and leaving MiniF elements at the same time

Method used

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  • Duck plague virus ge and gi double gene traceless deletion strain dpv CHv-ΔgE+ΔgI and its construction method
  • Duck plague virus ge and gi double gene traceless deletion strain dpv CHv-ΔgE+ΔgI and its construction method
  • Duck plague virus ge and gi double gene traceless deletion strain dpv CHv-ΔgE+ΔgI and its construction method

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Embodiment 1

[0060] Example 1 Preparation of duck plague virus gE and gI double gene scarless deletion strain DPV CHv-ΔgE+ΔgI

[0061] DPV CHv-ΔgE+ΔgI double gene scarless deletion strain DPV CHv-ΔgE+ΔgI of duck plague virus gE and gI, and its construction method includes the following steps:

[0062] 1. Preparation of GS1783 electrotransformation competence, and electrotransformation of pBAC-DPV plasmid

[0063] (1) Resuscitate E. coli with pBAC-DPV plasmid in LB solid medium containing chloramphenicol, and culture at 37°C overnight; single colonies are inoculated into LB liquid medium containing chloramphenicol, and culture at 37°C overnight;

[0064] (2) Extract the pBAC-DPV plasmid according to the QIAGEN Plasmid Midi Kit operating instructions;

[0065] (3) Resuscitate GS1783 cryopreserved bacteria in LB solid medium, and cultivate overnight at 30°C;

[0066] (4) Pick a single colony of GS1783 and inoculate it in 5 mL of LB liquid medium, and culture at 30°C overnight to obtain seed...

Embodiment 2

[0202] Example 2 Detection of virus quantity after inoculation of 7-day-old ducks with traceless deletion strain DPV CHv-ΔgE+ΔgI

[0203] The gE, gI gene and MiniF element traceless deletion strain DPV CHv-ΔgE+ΔgI was inoculated into DEF cells at 0.01 MOI, and the cells were collected 120 hours after inoculation, and the cells were frozen and thawed twice, and the TCID was determined. 50 Afterwards, 2MOI DPV CHv-ΔgE+ΔgI deletion virus was used to inoculate 7-day-old ducks intramuscularly, and the blood, liver and spleen of the ducks were collected at 24h, 48h, 72h, and 96h respectively, and extracted according to the instructions of TaKaRaMiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 tissue DNA. The identification primers gE-F and gI-R were used to detect whether duck liver virus was a double deletion virus of gE and gI genes at 48h after challenge (see Figure 5 ), primers and Taq probes were identified by UL30 QPCR, and the amount of virus in blood, liver and spleen at diff...

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Abstract

The invention provides a duck plague virus gE and gI double gene traceless deletion strain DPV CHv-ΔgE+ΔgI and a construction method thereof. The present invention utilizes GS1783 Escherichia coli strain and pEPkan-S plasmid to delete duck plague virus gE gene and gI gene through two homologous recombination on the bacterial artificial chromosome recombination duck plague virus rescue system platform, and then through intracellular spontaneous homologous recombination method The MiniF element was deleted, and the construction of a double-gene seamless deletion strain of duck plague virus without exogenous bases and MiniF element residues was completed for the first time. The technical scheme of the present invention solves the problem of residual bases at the deletion site when the duck plague virus gene is deleted and the MiniF element is deleted, and provides sufficient technical support for accurately exploring the function of the duck plague virus gene and the construction of an attenuated live vaccine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a duck plague virus gE and gI double gene traceless deletion strain DPV CHv-ΔgE+ΔgI and a construction method thereof. Background technique [0002] Bacterial artificial chromosome (BAC) is a newly developed DNA vector system. It has the advantages of large capacity, stable genetic characteristics and easy operation. It has a wide range of applications in gene library construction and gene function analysis. The complete viral genome DNA molecule is inserted into the BAC vector, and the molecular cloned virus is obtained by using the minimal fertility factor replicon (Mini-F) encoded by the vector, and combined with the mature gene location modification technology in Escherichia coli, thereby Realize the deletion of viral genes and the insertion of foreign genes in prokaryotic systems. At present, the commonly used E. coli gene location modification techn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/63C12R1/93
CPCC12N7/00C12N15/63C07K14/005C12N2710/16321C12N2710/16322C12N2800/30C12N2800/204
Inventor 程安春刘田汪铭书
Owner SICHUAN AGRI UNIV
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