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Cloning of irg6 gene with potential anti-swine fever virus effect and construction of its stable expression cell line

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

Active Publication Date: 2015-09-23
GUANGXI UNIV
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  • 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

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  • Cloning of irg6 gene with potential anti-swine fever virus effect and construction of its stable expression cell line
  • Cloning of irg6 gene with potential anti-swine fever virus effect and construction of its stable expression cell line
  • Cloning of irg6 gene with potential anti-swine fever virus effect and construction of its stable expression cell line

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The acquisition of the gene fragment of embodiment 1:

[0083] (1) Lymphocyte separation

[0084] 10ml of peripheral blood of Guangxi Bama Xiang pigs infected with swine fever virus was collected intravenously and diluted with an equal amount of PBS. Take 4 centrifuge tubes, add 5ml of lymphocyte separation solution respectively, divide 20ml of diluted blood into four equal parts, and carefully inject them along the tube wall into the centrifuge tubes that have been added with lymphocyte separation solution, making sure to separate them. 2000rpm, 20min. Carefully aspirate the second layer of white lymphocytes from the separation layer in the centrifuge tube, dilute with an equal volume of PBS, and centrifuge at 2000 rpm for 5 min. Discard the supernatant, add 5ml of erythrocyte lysate, mix well, and act at room temperature for 5-10min, 2000rpm, 5min. Discard the supernatant, add 5ml PBS, blow off the cells, centrifuge at 2000rpm for 5min. Discard the supernatant, ad...

Embodiment 2

[0100] The construction of embodiment 2 carrier:

[0101] pcDNA3.1+ (stored in our laboratory) was digested with Xho I and Xba I double enzymes, and the digested product (5.4kb) was recovered. The GFP-E plasmid was also double-enzyme digested with Xho I and Xba I, and a fragment of about 700 bp was recovered. Two-stage recovery product with T 4 DNA ligase ligase. Transform E. coli DH5α. This vector was named pC-GFP. The pC-GFP plasmid was double digested with Hind III I and Xho I, and the digested product (6 kb) was recovered. The IRG6 E plasmid was also double digested with IIind III and Xho I, and a fragment of about 1102 bp was recovered. Two-stage recovery product with T 4 DNA ligase ligase to transform E. coli DH5α. This vector was named pC-IRG6-GFP (Figure 4).

Embodiment 3

[0102] Example 3 Screening of cell lines stably expressing IRG6 protein:

[0103] The vector 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 the cells adhered. Add G418 with a final concentration of 1000-1600 μg / mL for screening, and continue to culture for about 7 days. During this period, the medium is changed every three days, and an appropriate amount of G418 is supplemented. The cells in the control wells (cells without plasmids) all fall off. After that, the screening concentration was adjusted down to 400-800 μg / mL, and the culture was continued for about 7 days. Observation and screening of the target cell line by fluorescence microscope, labeling the cell clusters expressing green fluorescence, adding a small amount of trypsin to digest for 10 seconds, carefully covering it with a small filter paper, taking b...

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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, in particular to the construction of a gene cloning against swine fever virus and a stable expression cell line thereof. Background technique [0002] Swine fever virus (CSFV) is a small-envelope, single-stranded positive-stranded RNA virus belonging to the Flaviviridae family, the Pestivirus genus. The genome of CSFV is about 12.5kb, with only one large open reading frame (ORF), and all structural and non-structural proteins are encoded by this ORF, which encodes a variety of proteins that are processed and modified by cellular and viral proteases. 12 mature viral proteins, about 4000 amino acid residues, with a molecular weight of about 438ku (Moormann R.J.M et al., 1990). Swine fever virus (CSFV) is the causative agent of swine fever, a highly contagious and lethal viral disease of pigs. CSFV infection can cause severe leukopenia, immunosuppression, extensive thrombosis and endothel...

Claims

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Application Information

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Patent Type & Authority Patents(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
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