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DPV (duck plague virus) gene gE markless deletion strain DPV CHv-delta gE and construction method thereof

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: 2019-04-09
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
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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 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

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  • DPV (duck plague virus) gene gE markless deletion strain DPV CHv-delta gE and construction method thereof
  • DPV (duck plague virus) gene gE markless deletion strain DPV CHv-delta gE and construction method thereof
  • DPV (duck plague virus) gene gE markless deletion strain DPV CHv-delta gE and construction method thereof

Examples

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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 DPV (duck plague virus) gene gE markless deletion strain DPV CHv-delta gE and a construction method thereof. With utilization of a GS1783 escherichia coli strain and pEPkan-Splasmid, homologous recombination for deletion of a DPV gene gE is performed twice on a BAC (bacterial artificial chromosome) recombination DPV rescue system platform, deletion of MiniF (minimal fertility factor replicon) elements is performed with an intracellular spontaneous homologous recombination method, and construction of the DPV markless deletion strain without residues of exogenous basesand MiniF elements is finished for the first time. With the adoption of the technical scheme, the problem of base residues at a deletion site during deletion of the DPV gene is solved, the deletion ofthe MiniF elements is realized, and sufficient technical support is provided for accurate exploration of DPV gene functions and construction of attenuated live vaccines.

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 Applications(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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