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Application and method of E199L protein in promoting apoptosis

A cell and apoptosis technology, applied in the field of cell biology, can solve problems such as decreased activity

Pending Publication Date: 2022-04-26
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cleavage of these proteins results in a decrease in their activity

Method used

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  • Application and method of E199L protein in promoting apoptosis
  • Application and method of E199L protein in promoting apoptosis
  • Application and method of E199L protein in promoting apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Materials and methods

[0054] 1.1 Construction of recombinant plasmids

[0055] 94 expression ASFV encoded proteins (pCAGGS-Flag-X) and BCL-2 family members (pCAGGS-HA / Flag-Y, BAK, BAX, BCL-XL, MCL-1, BCL-W, BCL-2, BCL-2A1) plasmids. E199L and its mutant plasmids (pEGFP-E199L, pEGFP-E199L-dMTD, pEGFP-E199L-dBH3, pEGFP-E199L-dBH1, pCAGGS-HA-E199L) were constructed into pEGFP-C1 or pCAGGS- HA carrier. All constructed plasmids were verified by DNA sequencing.

[0056] 1.2 cells

[0057] HEK293T and HeLa cells were stored in our laboratory at 37°C, 5% CO 2 cultured in an incubator with 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin in the medium. HeLa-ΔBAK and its parental cells were purchased from EDIGENE (catalogue number: LS0032850802A).

[0058] 1.3 Antibody

[0059] The primary antibodies used in this study were specific TOM20 (612278, BD Biosciences), PDI (MA3-19, Thermo), HA (3724S, Cell Signaling Technologies), FLAG (14793...

Embodiment 2

[0078] Example 2: ASFV E199L induces cell death in vitro

[0079] according to figure 1 Based on the results, we proposed that the protein encoded by ASFV may be involved in the process of ASFV-induced cell death. In order to verify this hypothesis, 94 plasmids expressing ASFV-encoded proteins were transfected into HEK293T cells, and then used CellTiter-Glo TM Fluorescent cell viability assay. Screening results showed that the overexpression of 4 genes (B117L, CP123L, E183L, E199L) had a significant effect on cell viability ( figure 1 A). In particular, E199L-induced cell death was as high as 60%. Therefore, this study chose E199L for further study. Our results showed that in PAM cells infected with the HLJ / 18 isolate, the mRNA level of E199L was significantly increased starting from 12 hours post-infection (HPI) ( figure 1 B), which indicates that E199L is a late gene expressed by the virus. To further verify the effect of E199L on inducing cell death, the E199L exp...

Embodiment 3

[0080] Example 3: Cell death induced by ASFV E199L presents apoptosis characteristics

[0081] To investigate whether E199L induces cell death by inducing apoptosis in vitro, time-lapse confocal microscopy, TUNEL labeling and Annexin V-FITC / PI staining were used to detect the morphological changes of apoptosis, DNA fragments, phosphatidylserine (PS ) eversion. The characteristic morphological changes of apoptosis were observed by real-time confocal microscopy. After E199L aggregated in HeLa cells, the cell morphology began to shrink, bleb, and eventually form apoptotic bodies ( figure 2 A). In addition, apoptosis-induced DNA fragmentation was marked by TUNEL assay. HEK293T cells overexpressing E199L produced TUNEL positive signal ( figure 2 B), the proportion of TUNEL positive cells is as high as 60%, which is significantly higher than that of HEK293T cells expressing GFP ( figure 2 C). In addition, we also found that the apoptosis level of cells transfected with E199...

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Abstract

The invention discloses application and a method of E199L protein in promoting cell apoptosis. The invention provides an application of E199L in promoting cell apoptosis, the E199L competitively destroys the interaction between anti-apoptosis proteins and BAK and / or BAX through the wide interaction between the E199L and the anti-apoptosis proteins such as BCL-XL, MCL-1, BCL-W and BCL-2A1, and finally induces cell apoptosis.

Description

technical field [0001] The present invention relates to cell biology, and more particularly relates to a kind of use and method for the substance of inducing cell apoptosis Background technique [0002] Programmed cell death (PCD) refers to an active extinction process that occurs in order to maintain a stable internal environment after cells receive certain signals or are stimulated by certain factors. Apoptosis, autophagy, programmed necrosis, and pyroptosis are all manifestations of programmed death. [0003] Apoptosis, also known as "pyknotic necrosis," "programmed cell death," or "cellular suicide," is a series of gene-mediated changes by which a cell actively causes its own destruction. The apoptotic process that normally eliminates senescent cells or lymphocytes not involved in the immune response can play a role in tumorigenesis if pathologically disturbed. [0004] Apoptosis can be physiological or induced by chemotherapy drugs and radiation, and it is a self-dest...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P35/00A61P37/02A61P17/06A61P17/00A61P35/02A61P31/00
CPCA61K38/1761A61P35/00A61P37/02A61P17/06A61P17/00A61P35/02A61P31/00
Inventor 翁长江郑君
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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