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Duck plague virus ge gene seamless deletion strain dpv CHv-ΔgE 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: 2021-02-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 gene traceless deletion strain DPV CHv-ΔgE and its construction method, which can effectively solve the problem of remaining in the deletion site when the duck plague virus gene is deleted. Base, while remaining MiniF element problem

Method used

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  • Duck plague virus ge gene seamless deletion strain dpv CHv-ΔgE and its construction method
  • Duck plague virus ge gene seamless deletion strain dpv CHv-ΔgE and its construction method
  • Duck plague virus ge gene seamless deletion strain dpv CHv-ΔgE and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of duck plague virus gE gene traceless deletion strain DPV CHv-ΔgE

[0052] Duck plague virus gE gene traceless deletion strain DPV CHv-ΔgE, its construction method comprises the following steps:

[0053] 1. Prepare GS1783 electroporation competent, electrotransform pBAC-DPV plasmid

[0054] (1) Escherichia coli with pBAC-DPV plasmid was revived in LB solid medium containing chloramphenicol, cultured overnight at 37°C; single colonies were inoculated in LB liquid medium containing chloramphenicol, and cultivated overnight at 37°C;

[0055] (2) Extract the pBAC-DPV plasmid according to the operating instructions of the QIAGEN Plasmid Midi Kit;

[0056] (3) Resuscitate GS1783 frozen-preserved bacteria in LB solid medium, culture overnight at 30°C;

[0057] (4) Pick a single colony of GS1783 and inoculate it in 5 mL of LB liquid medium, and culture it overnight at 30°C to obtain a seed solution;

[0058] (5) Add 5mL seed liquid to 100mL LB liquid m...

Embodiment 2

[0160] Example 2 Detection of virus quantity after inoculation of 7-day-old ducks with traceless deletion strain DPV CHv-ΔgE

[0161] The gE gene and MiniF element traceless deletion strain DPV CHv-ΔgE was inoculated into DEF cells at 0.01 MOI, and the cells were collected 120 hours after inoculation, frozen and thawed twice, and TCID was determined 50 Afterwards, 7-day-old ducks were inoculated with 2MOI of DPV CHv-ΔgE deletion virus, and the blood, liver, and spleen of the ducks were collected at 24h, 48h, 72h, and 96h respectively, and the tissues were extracted according to the instructions of TaKaRa MiniBEST ViralRNA / DNA Extraction Kit Ver.5.0 DNA. Use identification primers gE-F and gE-R to carry out PCR amplification to detect whether the duck liver virus is a gE gene deletion virus 48 hours after challenge, use UL30QPCR to identify primers and Taq probes, and QPCR to detect blood, liver and spleen at different time points virus count. The results showed that DPV CHv-Δg...

Embodiment 3

[0162] The determination of the growth curve of DPV CHv-ΔgE of embodiment 3 traceless deletion strain

[0163] 1. Determination of one-step growth curve

[0164] The parental virus DPV CHv and the gE gene traceless deletion strain DPV CHv-ΔgE were inoculated into DEF cells at 2 MOI respectively, and the supernatant and cells were collected 6h, 12h, 18h, and 24h after inoculation, and each time point was repeated three times. After the collection was complete, the freeze-thaw was repeated twice, and the virus titer was detected in a 96-well plate. The results showed that the deletion of the gE gene would not significantly affect the replication of the DPV CHv virus.

[0165] 2. Determination of multi-step growth curve

[0166] The parental virus DPV CHv and gE gene traceless deletion strain DPV CHv-ΔgE were inoculated into DEF cells at 0.01 MOI respectively, and the supernatant and cells were collected 12h, 24h, 48h, and 72h after inoculation, and each time point was repeated ...

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Abstract

The invention provides a duck plague virus gE gene traceless deletion strain DPV CHv‑ΔgE and a construction method thereof. The present invention utilizes GS1783 Escherichia coli strain and pEPkan‑S plasmid to delete the gE gene of duck plague virus through two homologous recombination on the bacterial artificial chromosome recombination duck plague virus rescue system platform, and then delete the MiniF element through intracellular spontaneous homologous recombination method , for the first time completed the construction of a duck plague virus seamless deletion strain without exogenous bases and MiniF elements. 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 gene traceless deletion strain DPVCHv-ΔgE and a construction method thereof. Background technique [0002] Bacterial artificial chromosome (BAC) is a newly developed DNA carrier system. It has the advantages of large capacity, stable genetic characteristics, and easy operation. It is widely used in gene library construction and gene function analysis. The complete viral genome DNA molecule is inserted into the BAC vector, and the minimal fertility factor replica (Minimal fertility factor replica, Mini-F) encoded by the vector is used to obtain molecularly cloned virus, and combined with the mature gene positioning modification technology in Escherichia coli, thereby Realize the deletion of viral genes and the insertion of foreign genes in the prokaryotic system. At present, the commonly used E. coli gene positioning modification tec...

Claims

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Application Information

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