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TPR structural motifs and cellular localization of swine ISG60 gene associated with antiviral immunity

An immune-related, cell-localized technology, applied in the field of livestock genetic engineering, can solve the problem that swine fever cannot be completely eradicated

Inactive Publication Date: 2012-07-11
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although the currently used swine fever vaccine has a good protective effect on pigs, swine fever cannot be completely eradicated, and new clinical features have appeared in the infection of swine fever virus, except for the acute clinical symptoms commonly seen. In addition to symptoms, there are also some mild or subclinical swine fever that do not show obvious clinical symptoms, but both antibody testing and genetic testing are positive, which is the so-called persistent infection state, carrying the virus for a long time without showing clinical symptoms pigs become an important source of infection

Method used

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  • TPR structural motifs and cellular localization of swine ISG60 gene associated with antiviral immunity
  • TPR structural motifs and cellular localization of swine ISG60 gene associated with antiviral immunity
  • TPR structural motifs and cellular localization of swine ISG60 gene associated with antiviral immunity

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Embodiment 1

[0090] The location of the chromosome of the EST sequence of embodiment 1, pig ISG60 gene

[0091] The porcine whole genome chip was used to analyze the gene expression profile of porcine peripheral blood lymphocytes, and it was found that the expression level of ISG60 gene increased after infection with classical swine fever virus, and the expression level of the gene was the highest 6 days after infection. Through the nucleotide blast program analysis of the EST sequence of the ISG60 gene and the NCBI website http: / / blast.ncbi.nlm.nih.gov / database, it was found that the ISG60 gene was located on the pig chromosome 14.

Embodiment 2

[0092] Embodiment 2, the cloning of porcine ISG60 gene

[0093] Firstly, the recombinant human type I interferon IFN-α2b was used to stimulate the pig kidney epithelial cell line PK-15 to mediated the production of ISG60 gene. Interferon is an antiviral protein produced by mammalian cells. It is mainly divided into type I and type II interferon, among which α and β interferon belong to type I interferon, and gamma interferon belongs to type II interferon. The ingredient is type I interferon. After the interferon stimulates the pig kidney cell PK-15, the mRNA of the ISG60 gene is transcribed in large quantities. Since the cells contain molecules such as RNA, DNA and protein, the total cellular RNA needs to be extracted from it, and the mRNA of the ISG60 gene is contained in the cell in total RNA. After interferon binds to the interferon receptor on the cell, it activates the JAK-STAT signaling pathway, making STAT1 and STAT2 heterodimers, and the interferon regulatory factor ...

Embodiment 3

[0103] Example 3, identifying the location of the ISG60 gene in PK-15 cells

[0104] The nucleotide encoding 6×his was marked on the ISG60 gene by PCR (see image 3 ), the marked ISG60 gene was subcloned to the mammalian eukaryotic expression vector pCDNA3.0 under the effect of T4 ligase (see Figure 4 ), transfected PK-15 cells with liposome-mediated method, and identified the cellular localization of ISG60 gene by indirect immunofluorescence (see Figure 5 ). Indirect immunofluorescence method: suck off the PK-15 cell culture medium 36 hours after transfection, wash the cells three times with PBS, fix the volume of formaldehyde and methanol at 1:1, wash the cells three times with PBS; use 6×his monoclonal antibody at 37°C Incubate for 60 min, wash cells with PBS three times; incubate with FITC-labeled goat anti-mouse IgG at 37°C for 60 min, wash cells with PBS three times, observe cells with an inverted fluorescence microscope.

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Abstract

The invention provides TPR (tetratricopeptide repeat) structural motifs of swine ISG60 gene associated with antiviral immunity, which contains viral infection-related regulatory element sequences that has no significant homology with existing reported TPR structure of a swine gene. The invention also specifically relates to the strategy of swine ISG60 gene, and identifies cellular location of ISG60 gene. The TPR structural motifs of swine ISG60 gene can be used for studying on the infection mechanism of classical swine fever virus. A medicine for treating swine fever can be developed through revealing the anti-swine fever virus mechanism of the gene.

Description

technical field [0001] The invention belongs to the field of livestock genetic engineering, and in particular relates to a gene structural motif related to pig antiviral immunity. Background technique [0002] Pigs are one of the most important livestock products in the world. Swine fever is a severe infectious disease of great economic significance in pigs, and the economic loss caused by swine fever is huge every year all over the world. Therefore, it is of great significance to study the immunity of pigs against CSFV. Classical Swine Fever Virus (Classical Swine Fever Virus) is the causative agent of swine fever. The incubation period is 24-72 hours. Its virus particles are round and belong to the Flaviviridae Pestivirus genus. The size is 38-44nm. The nucleocapsid is stereosymmetric. Surface body, floating density in cesium chloride 1.15 ~ 1.17g / ml, enveloped, replicated in the cytoplasm. Swine fever virus harms pigs and wild boars, commonly known as "rotten intestina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/79C07K14/47G01N33/53
Inventor 罗廷荣蔡新斌孙石开李晓宁苏丽娟尹珊李晓泉李延生
Owner GUANGXI UNIV
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