Application of African swine fever virus E120R protein as immunosuppressant and construction of immunosuppression site knockout strain

An African swine fever virus, E120R technology, applied in the direction of antiviral agents, viruses, virus antigen components, etc., to achieve the effect of inducing high-level antibody production, improving biological safety, and weakening the ability of natural immunity

Active Publication Date: 2020-12-11
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

ASFV is not capable of inducing neutralizing antibodies and therefore has not been serotyped

Method used

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  • Application of African swine fever virus E120R protein as immunosuppressant and construction of immunosuppression site knockout strain
  • Application of African swine fever virus E120R protein as immunosuppressant and construction of immunosuppression site knockout strain
  • Application of African swine fever virus E120R protein as immunosuppressant and construction of immunosuppression site knockout strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 E120R protein inhibits the activation of IFN-β induced by cGAS / STING and poly(dA:dT)

[0059] 1. The effect of E120R protein on the expression of IFN-β induced by cGAS / STING

[0060] Plate HEK-293T cells into individual wells of a 24-well plate. When the cells grow to 70%-80% confluence, use liposome reagents to transfect IFN-β promoter plasmid (100ng / well) and TK plasmid (10ng / well), HA-cGAS / HA-STING plasmid respectively (100ng / well), and FLAG-E120R plasmid (0, 50, 100, 200ng), transfected for 24h, and using a luciferase kit to detect the activity of IFN-β.

[0061] The result is as figure 1 As shown, where Vec is not transfected with HA-cGAS / HA-STING plasmid and FLAG-E120R plasmid. The results showed that only after transfection of HA-cGAS / HA-STING plasmid (100ng / well), the expression level of IFN-β was higher; while co-transfection of HA-cGAS / HA-STING plasmid (100ng / well) and After FLAG-E120R plasmid (50, 100, 200ng), the expression of IFN-β decreased s...

Embodiment 2

[0066] Example 2 E120R protein inhibits the expression of IFN-β and its downstream factor mRNA induced by cGAS / STING and poly(dA:dT)

[0067] 1. Preparation of cell samples co-transfected with E120R protein, cGAS / STING and poly(dA:dT)

[0068] Plate HEK-293T cells into individual wells of a 24-well plate. When the cells grow to 70%-80% confluence, use liposome reagents to transfect, HA-cGAS / HA-STING plasmid (100ng / well), FLAG-E120R plasmid (200ng / well), transfection 24h Receive samples later.

[0069] Plate HEK-293T cells into individual wells of a 24-well plate. When the cells grew to 70%-80% confluence, liposome reagent was used to transfect FLAG-E120R plasmid (200ng / well). After 24h of transfection, liposome was used to transfect poly(dA:dT)( 1000ng / well), the samples were collected 12 hours after transfection.

[0070] 2. qPCR detection of IFN-β and its downstream factors

[0071] Samples of cells co-transfected with FLAG-E120R plasmid, HA-cGAS / HA-STING plasmid and po...

Embodiment 3

[0081] Example 3 E120R protein inhibits cGAS / STING and poly(dA:dT)-induced IRF3 phosphorylation

[0082] 1. Preparation of cell samples co-transfected with E120R protein, cGAS / STING and poly(dA:dT)

[0083] As described in (1) in Example 2.

[0084] 2. Western blot detection of protein expression level

[0085] Preparation of protein samples: Discard the supernatant of cell samples co-transfected with FLAG-E120R, HA-cGAS / HA-STING plasmid or poly(dA:dT) plasmid, wash the cell samples once with PBS, and scrape off with a cell spatula. Transfer the cells into a 1.5mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant, and keep the cell pellet, which is the harvested cell sample (all operated on ice); add an appropriate amount of cell lysate according to the amount of collected cell pellet, and quickly Repeatedly blow and beat the resuspended cell sample, lyse on ice for 5 minutes, and ultrasonically break it instantaneously (operate on ice, sonicate 2-3 ti...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to application of African swine fever virus E120R protein as an immunosuppressant and construction of an immunosuppression site knockout strain. The invention firstly discovers that the African swine fever virus E120R protein can inhibit activation of IFN-beta induced by cGAS / STING and poly (dA: dT), inhibit mRNA expression of IFN-beta induced by cGAS / STING and poly (dA: dT) and downstream factors ISG56 and ISG15 thereof, inhibit phosphorylation of IRF3 induced by cGAS / STING and poly (dA: dT), has a relatively strong immunosuppression effect, and can be used as an immunosuppressant; and secondly, the invention discovers an immunosuppression site of the African swine fever virus E120R protein, and constructs an African swine fever recombinant virus with the knockout of the immunosuppression site of the E120R protein through a genetic engineering means, the natural immunity of the African swine fever recombinant virus is significantly enhanced, and the African swine fever recombinant virus can be used as a recombinant vaccine strain, can promote early immune response, induce the production of high-levelantibodies, improve the biological safety and immune protection efficacy, and has good application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of an African swine fever virus E120R protein as an immunosuppressant and the construction of an immunosuppressive site knockout virus strain. Background technique [0002] African swine fever (African swine fever, ASF) is caused by the infection of African swine fever virus (ASFV), and is characterized by fever and hemorrhage in pigs. The fatality rate for domestic pigs is as high as 100%. . The disease first broke out in Kenya in 1921 and subsequently became widespread among domestic and wild pigs throughout Africa. It was introduced into Europe in the 1950s, and it took 40 years to eliminate the disease throughout Europe. However, the disease was introduced to Georgia from East Africa again in 2007, and then spread widely in Eastern Europe, and in 2017, it was introduced to Irkutsk in the Russian Far East. In early August 2019, researcher Hu Ronglian...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/20C12N7/01C12N15/85C12R1/93
CPCA61K39/12A61K2039/552A61P31/20C12N7/00C12N15/85C12N2710/12021C12N2710/12034C12N2710/12052C12N2800/107
Inventor 郑海学刘会胜薛巧朱紫祥李攀冯涛李豪马昭党文杨帆曹伟军刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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