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Application of gtpbp4 protein as immunosuppressant and construction of gtpbp4 knockdown or overexpression cell line

A protein expression, β inhibitor technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve the problem of unclear natural immune relationship, and achieve the goal of promoting vaccine production, inhibiting replication, improving The effect of virus titers

Active Publication Date: 2022-04-22
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 gtpbp4 knockdown or overexpression cell line
  • Application of gtpbp4 protein as immunosuppressant and construction of gtpbp4 knockdown or overexpression cell line
  • Application of gtpbp4 protein as immunosuppressant and construction of gtpbp4 knockdown or overexpression 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, liposome reagents were used to transfect NC siRNA (75nm / well), GTPBP4 siRNA (75nm / 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, and harvested the cells after 12h of infection, The activity of IFN-β promoter was determined by luciferase detection kit.

[0073] Experimental results such as figure 1 As shown, in cells transfected with GTPBP4 siRNA, the activity of SeV-induced IFN-β promoter was significantly increased. The results showed that GTPBP4 inhibited the activity of SeV-induced IFN-β promoter, and the activity of SeV-induced IFN-β promoter was significantly increased after the expression of GTPBP...

Embodiment 2

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

[0096] 1. Preparation of HEK-293T cell samples infected with Seneca virus

[0097] Plate HEK-293T cells into individual wells of a 6-well plate. When the cells grew to 70%-80% confluence, liposome reagents were used to transfect NC siRNA and GTPBP4 siRNA respectively, transfected for 36 hours, inoculated with 1 MOI of Seneca virus, and cultured in maintenance medium after 1 hour of inoculation. Cells were harvested 12 h after infection.

[0098] 2. Detection of viral protein content

[0099] Preparation of protein samples: Discard the cell culture supernatant 12 hours after infection with Seneca virus, wash the cell samples once with PBS, scrape the cells with a cell spatula, transfer them into a 1.5mL centrifuge tube, centrifuge at 2000rpm for 5min, and discard the supernatant , keep the cell pellet, which is the harvested cell sample (...

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 be 6×10 5 HEK-293T cells were plated in a single well of a 6-well plate. When the cells grow to 70%-80% confluence, use liposome reagents to transfect 0, 1, 2 μg of FLAG-GTPBP4 plasmids respectively, transfect for 24 hours, inoculate with 1 MOI of Seneca virus, and switch to maintenance after 1 hour of inoculation liquid culture.

[0109] 2. Detection of viral protein content

[0110] The detection method is as described in 2 in Example 2.

[0111] Experimental results such as Figure 6 As shown in the middle left figure, with the increase of the amount of FLAG-GTPBP4 plasmid added, the expression of Seneca virus VP1 protein increased significantly, indicating that compared with normal HEK-293T cells, exogenous addition of GTPBP4 protein significantly promoted the expression of Seneca vi...

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to the application of GTPBP4 protein as an immunosuppressant and the construction of knockdown or overexpression GTPBP4 cell lines. First, the present invention unexpectedly discovered that GTPBP4 inhibits the activation of IFN-β promoter induced by SeV (Sendai virus), the mRNA expression of IFN-β and its downstream genes, and inhibits the expression of IFN-β protein induced by SeV; targeting IRF3 (interference Primer response factor 3) suppresses innate immunity. Secondly, overexpressing the GTPBP4 protein in the cell line significantly promoted the expression of Seneca virus VP1 protein and improved the Seneca virus titer, which can be used for the construction of Seneca virus and vaccine production cell lines; finally, in the cell line Medium knockdown of GTPBP4 protein significantly inhibits the expression of Seneca virus VP1 protein, reduces the titer of Seneca virus, and can be used for animal breeding against Seneca virus infection; and GTPBP4 protein inhibitors can be used to prepare prevention or treatment Picornaviridae virus infection drugs, pharmaceutical compositions or vaccine compositions.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of GTPBP4 protein as an immunosuppressant and the construction of knockdown or overexpression GTPBP4 cell lines. Background technique [0002] Picornaviridae is a family consisting of the smallest group of RNA viruses, mainly including enterovirus, rhinovirus, cardiovirus and aphth virus. Among them, Senecavirus A (SVA) is the only member of the newly added Senecavirus genus in the family Picornaviridae. The clinical symptoms are difficult to distinguish from foot-and-mouth disease, mainly manifested as blisters on the mouth, nose and hooves and ulcers. [0003] Before 2014, SVA only occurred sporadically in the United States and Canada, but since 2015, SVA outbreaks have occurred in Brazil, Vietnam, Colombia, Thailand, China, etc., and continue to spread. SVA was introduced into my country in 2015, and Chinese scholars first discovered cases of SVA infe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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