Supercharge Your Innovation With Domain-Expert AI Agents!

Duck plague virus gI gene traceless deletion strain DPV CHv-[delta]gI and construction method thereof

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

Active Publication Date: 2019-04-12
SICHUAN AGRI UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the above-mentioned problems existing in the prior art, the present invention provides a duck plague virus gI gene traceless deletion strain DPV CHv-ΔgI 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Duck plague virus gI gene traceless deletion strain DPV CHv-[delta]gI and construction method thereof
  • Duck plague virus gI gene traceless deletion strain DPV CHv-[delta]gI and construction method thereof
  • Duck plague virus gI gene traceless deletion strain DPV CHv-[delta]gI and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0052] Duck plague virus gI gene traceless deletion strain DPV CHv-ΔgI, 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] The determination of the growth curve of DPV CHv-ΔgI of embodiment 2 traceless deletion strain

[0161] 1. Determination of one-step growth curve

[0162] The parental virus DPV CHv and the gI gene traceless deletion strain DPV CHv-ΔgI were inoculated into DEF cells at 2MOI 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 gI gene would not significantly affect the replication of the DPV CHv virus.

[0163] 2. Determination of multi-step growth curve

[0164] The parental virus DPV CHv and gI gene traceless deletion strain DPV CHv-ΔgI 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 thr...

Embodiment 3

[0165] Example 3 Plaque formation experiment of DPV CHv-ΔgI strain without trace deletion

[0166] The parental virus DPV CHv and gI gene traceless deletion strain DPV CHv-ΔgI were inoculated at a MOI of 0.01 on a 6-well plate covered with DEF cells at 37°C, 5% CO 2 After absorbing for 2 hours, remove the supernatant, add 2 mL of 1% methylcellulose, 37 ° C, 5% CO 2 After culturing for 48 h, remove 1% methylcellulose, wash 3 times with PBS, fix overnight at 4°C with 4% paraformaldehyde, wash 3 times with PBS, add H 2 o 2 The mixture mixed with methanol at a volume ratio of 1:50 was incubated at room temperature for 30 minutes, washed 3 times with distilled water, added 5% BSA blocking solution, incubated at room temperature for 30 minutes, added rabbit anti-DPV, incubated overnight at 4°C, washed 3 times with PBS, added Biotinylated goat anti-rabbit IgG, incubated at 37°C for 30 minutes, washed 3 times with PBS, added dropwise SABC substrate, incubated at 37°C for 30 minutes,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a duck plague virus gI gene traceless deletion strain DPV CHv-[delta]gI and a construction method thereof. According to the construction method, a GS1783 Escherichia coli strainand a pEPkan-S plasmid are used to delete a duck plague virus gI gene by twice homologous recombination on a bacterial artificial chromosome recombinant duck plague virus rescue system platform, thenan MiniF element is deleted by an intracellular spontaneous homologous recombination method, so that construction of the duck plague virus traceless deletion strain without an exogenous base and theMiniF element is completed for the first time. The technical scheme of the invention solves the problem that when the duck plague virus gene is deleted, bases remain at the deleted site and the MiniFelement is deleted, thus providing a sufficient technical support for accurately exploring the function of the duck plague virus gene and construction of live attenuated vaccines.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a duck plague virus gI gene traceless deletion strain DPVCHv-ΔgI 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/85C12N15/38C12N7/00C12R1/93
CPCC07K14/005C12N7/00C12N15/85C12N2710/16321C12N2710/16322C12N2710/16352C12N2800/106C12N2800/204
Inventor 程安春刘田汪铭书
Owner SICHUAN AGRI UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com