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Application of GTPBP4 protein as immunosuppressant and construction of knockdown or overexpression GTPBP4 cell line

An immunosuppressant and protein expression technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve problems such as unclear relationship between natural immunity, so as to promote vaccine production and increase virus titer Effect

Active Publication Date: 2021-02-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relationship of GTPBP4 to innate immunity remains unclear

Method used

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  • Application of GTPBP4 protein as immunosuppressant and construction of knockdown or overexpression GTPBP4 cell line
  • Application of GTPBP4 protein as immunosuppressant and construction of knockdown or overexpression GTPBP4 cell line
  • Application of GTPBP4 protein as immunosuppressant and construction of knockdown or overexpression GTPBP4 cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Immunosuppressive effect of GTPBP4

[0071] 1. GTPBP4 inhibits SeV-induced activation of the IFN-β promoter

[0072] HEK-293T cells were plated in a single well of a 24-well plate, and when the cells reached 70%-80% confluence, NC siRNA (75nm / well) and GTPBP4 siRNA (75nm) were transfected with liposome reagents. / well), IFN-β promoter luciferase plasmid (IFN-β-luc, 100ng / well) and internal reference TK plasmid (10ng / well), transfected for 36h, inoculated with an appropriate amount of SeV, 12h after infection, harvested cells, The activity of the IFN-β promoter was measured using a luciferase assay kit.

[0073] The experimental results are as figure 1 shown that SeV-induced activity of the IFN-β promoter was significantly increased in cells transfected with GTPBP4 siRNA. The results showed that GTPBP4 inhibited the activity of SeV-induced IFN-β promoter, while the activity of SeV-induced IFN-β promoter was significantly increased after GTPBP4 siRNA interfer...

Embodiment 2

[0095] Example 2 Evaluation of the effect of down-regulating expression of GTPBP4 protein to inhibit Seneca virus replication

[0096] 1. Preparation of HEK-293T Cell Samples Infected with Seneca Virus

[0097] HEK-293T cells were plated in a single well of a 6-well plate. When the cells reached 70%-80% confluence, NC siRNA and GTPBP4 siRNA were transfected with liposome reagents for 36 h, inoculated with 1 MOI of Seneca virus, and then inoculated with maintenance medium for 1 h. Cells were harvested 12 h after infection.

[0098] 2. Detection of viral protein content

[0099] Preparation of protein samples: Discard the cell culture supernatant after 12h infection with Seneca virus, wash the cell samples with PBS once, scrape the cells with a cell spatula, transfer them into a 1.5mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant , retain the cell pellet, which is the harvested cell sample (all operated on ice); add an appropriate amount of cell lys...

Embodiment 3

[0106] Example 3 Evaluation of the effect of GTPBP4 protein on promoting Seneca virus replication

[0107] 1. Preparation of HEK-293T Cell Samples Infected with Seneca Virus

[0108] Will 6x10 5 HEK-293T cells were plated in a single well of a 6-well plate. When the cells grow to 70%-80% confluence, 0, 1, and 2 μg of FLAG-GTPBP4 plasmids were transfected with liposome reagents, transfected for 24 hours, inoculated with 1MOI of Seneca virus, and then switched to maintenance after inoculation for 1 hour. liquid culture.

[0109] 2. Detection of viral protein content

[0110] The detection method is the same as that described in 2 of Example 2.

[0111] The experimental results are as Image 6 As shown in the middle and left figures, with the increase of the amount of FLAG-GTPBP4 plasmid added, the expression of Seneca virus VP1 protein was significantly increased, indicating that compared with normal HEK-293T cells, exogenous addition of GTPBP4 protein significantly promote...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an application of GTPBP4 protein as an immunosuppressant and construction of a knockdown or overexpression GTPBP4 cell line. Firstly, it is accidentally found that GTPBP4 inhibits activation of an IFN-beta promoter induced by SeV (Sendai virus) and mRNA expression of IFN beta and downstream genes of IFN beta, andinhibits IFN-beta protein expression induced by SeV; and targeted IRF3 (interferon reaction factor 3) inhibits innate immunity; secondly, overexpression of the GTPBP4 protein in the cell line significantly promotes expression of the Senecavirus VP1 protein, improves the Senecavirus titer, and can be used for construction of Senecavirus and vaccine production cell lines; and finally, the GTPBP4 protein is knocked down in the cell line, expression of the Senecavirus VP1 protein is obviously inhibited, the titer of the Senecavirus is reduced, and the cell line can be used for breeding animals resisting Senecavirus infection. The GTPBP4 protein inhibitor can be used for preparing medicines, pharmaceutical compositions or vaccine compositions for preventing or treating virus infection of the small ribonucleic acid viruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the application of GTPBP4 protein as an immunosuppressant and the construction of a GTPBP4 knockdown or overexpression cell line. Background technique [0002] Picornaviridae is a family composed of the smallest groups of RNA viruses, mainly including Enterovirus, Rhinovirus, Cardiovirus and Aphthovirus. Among them, Senecavirus A (SVA) is the only member of the new Senecavirus genus in the Picornaviridae family. and ulcers. [0003] Before 2014, SVA only occurred sporadically in the United States and Canada, but since 2015, SVA outbreaks have appeared in Brazil, Vietnam, Colombia, Thailand, and China, and have continued to spread. SVA was introduced to my country in 2015, and Chinese scholars first discovered cases of pigs infected with SVA in Guangdong Province. Subsequently, epidemics occurred in Hubei, Heilongjiang, Fujian, Henan, Guangxi, Hebei, Liaoning, Shandong, Zhejiang, A...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/713A61P37/06A61P31/14C12N7/01C12N15/867C12N5/10C12R1/91
CPCA61K45/00A61K31/713A61P37/06A61P31/14C12N7/00C12N15/86C12N5/0686C12N9/14C12N2740/15021C12N2740/15043C12N2510/02C12N2800/107
Inventor 郑海学刘会胜薛巧唐闻达朱紫祥薛钊宁曹伟军杨帆刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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