Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Clone of IRG6 gene having potential anti-classical swine fever virus effect, and construction of stable expression cell line of IRG6 gene

A stable expression, swine fever virus technology, applied in genetic engineering, plant gene improvement, application, etc., can solve the problem of uninhibited replication

Active Publication Date: 2013-01-30
GUANGXI UNIV
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although vesicular oropharyngeal virus can induce the expression of Viperin, its replication is not inhibited (Pierre Boudinot et al., 2000), and vesicular oropharyngeal virus is not released through the lipid raft pathway

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
  • Clone of IRG6 gene having potential anti-classical swine fever virus effect, and construction of stable expression cell line of IRG6 gene
  • Clone of IRG6 gene having potential anti-classical swine fever virus effect, and construction of stable expression cell line of IRG6 gene
  • Clone of IRG6 gene having potential anti-classical swine fever virus effect, and construction of stable expression cell line of IRG6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Obtaining of gene fragments:

[0083] (1) Lymphocyte separation

[0084] 10 ml of peripheral blood from Guangxi Bamaxiang pigs infected with CSFV was collected intravenously and diluted with an equal amount of PBS. Take 4 centrifuge tubes, add 5ml of lymphocyte separation solution to each, divide 20ml of diluted blood into four parts, carefully pour them into the centrifuge tubes that have been added with lymphocyte separation solution along the tube wall, and make sure to make them stratify. 2000rpm, 20min. Carefully draw the second layer of white lymphocytes in the separation layer in the centrifuge tube, dilute with an equal volume of PBS, and centrifuge at 2000rpm for 5min. Discard the supernatant, add 5ml of erythrocyte lysate, mix thoroughly, and act at room temperature for 5-10min, then 2000rpm for 5min. Discard the supernatant, add 5ml PBS, blow off the cells, centrifuge at 2000rpm for 5min. Discard the supernatant, add an appropriate amount of PBS...

Embodiment 2

[0099] Example 2 Construction of the carrier:

[0100] pcDNA3.1+ (preserved in our laboratory) was digested with Xho I and Xba I double enzymes, and the digested product (5.4kb) was recovered. GFP-E plasma was also digested with Xho I and Xba I double enzymes, and a fragment of about 700bp was recovered. T 4 DNA ligase ligation. Escherichia coli DH5α was transformed. This vector was named pC-GFP. pC-GFP plasma was digested with Hind III I and Xho I double enzymes, and the digested product (6kb) was recovered. IRG6-E plasmad was also digested with Hind III and Xho I double enzymes, and a fragment of about 1102bp was recovered. T 4 DNA ligase ligation, transformed into Escherichia coli DH5α. This vector was named pC-IRG6-GFP (Fig. 4).

Embodiment 3

[0101] Example 3 Screening of cell lines stably expressing IRG6 protein:

[0102] The carrier p-IRG6-GFP was introduced into PK-15 cells by lipofection, and 24 hours after transfection, the transfected cells were digested with trypsin, passaged at a ratio of 1:4, and added to the cell supernatant after cell attachment. Add G418 with a final concentration of 1000-1600 μg / mL for screening, continue to culture for about 7 days, during which time the medium is changed every three days, and an appropriate amount of G418 is added, and the cells in the control well (cells without plasmid transfection) all fall off Afterwards, the screening concentration was adjusted down to 400-800 μg / mL, and the culture was continued for about 7 days. Observation and screening of target cell lines by fluorescence microscope, mark the cell cluster expressing green fluorescence, add a small amount of trypsin to digest for 10 seconds, carefully cover it with a small filter paper, take back the small fi...

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 present invention relates to clone of IRG6 gene having a potential anti-classical swine fever virus effect, and construction of a stable expression cell line of the IRG6 gene. The construction is characterized by comprising the following steps: (1) obtaining a gene fragment; (2) constructing a vector; (3) screening a stable IRG6 protein expression cell line; and (4) detecting reporter gene expression. The cell line can be used for classical swine fever virus infection mechanism research, wherein drugs for treating classical swine fever virus can be developed by revealing the anti-classical swine fever virus mechanism of the protein gene.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering, and in particular relates to the gene cloning of anti-swine fever virus and the construction of stable expression cell lines. Background technique [0002] Classical swine fever virus (CSFV) is a small enveloped, single-stranded positive-sense RNA virus belonging to the family Flaviviridae and the genus Pestivirus. The genome of CSFV is about 12.5kb, and there is only one large open reading frame (ORF). All structural and non-structural proteins are encoded by this ORF, and various proteins are processed and modified by proteases in cells and viruses. There are 12 mature viral proteins with about 4000 amino acid residues and a molecular weight of about 438ku (Moormann R.J.M et al., 1990). Classical swine fever virus (CSFV) is the causative agent of swine fever, a highly contagious and fatal viral disease. Infection with CSFV can lead to severe leukopenia, immunosuppression, extensive t...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/10C12N15/85C12N9/10C12R1/91C12R1/93
Inventor 罗廷荣孙石开蔡新斌李晓宁苏丽娟尹珊李晓泉李延生
Owner GUANGXI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com