Heat treatment induces solubility change and degradation of pml/rarα fusion protein and its mutants

A fusion protein, soluble technology, applied in the medical field to achieve the effect of speeding up the treatment

Active Publication Date: 2021-05-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Other studies have shown that As alone 2 o 3 To treat newly diagnosed APL patients, the disease recurrence rate or drug resistance rate is as high as 20-30%

Method used

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  • Heat treatment induces solubility change and degradation of pml/rarα fusion protein and its mutants
  • Heat treatment induces solubility change and degradation of pml/rarα fusion protein and its mutants
  • Heat treatment induces solubility change and degradation of pml/rarα fusion protein and its mutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: As 2 o 3 (iAs III ) in vitro on the solubility transition of mutant A216V that induces PML / RARα and arsenic resistance

[0034] 293T cells were cultured in DMEM (purchased from Gibco) medium containing 10% fetal bovine serum (FBS) (37°C, 5% CO 2 , saturated humidity), after resuscitating, take the fourth generation cells and press (5~10)×10 4 / mL concentration to inoculate 5ml in T25 culture flask. Wait for the cells to grow to the logarithmic growth phase, and use Lipofectamine2000 (purchased from Thermo Fisher Scientific) to transfect PML / RARα ( figure 1 ) and its drug-resistant mutant A216V ( figure 2 ). Add As 24h after transfection 2 o 3 (4μM) for different time and collect the samples, blot the supernatant and store the cell samples at -80°C or directly lyse. When the cells are lysed, add a certain amount of RIPA lysate to the cell mass, and immediately pipette evenly. Place on ice, pipette several times every 10 min and vortex with a vortex ...

Embodiment 2

[0035] Example 2: Effect of Heat Shock on Inducing PML / RARα, PML (1-552) and RARα's Solubility Transformation in Vitro

[0036] Overexpress PML / RARα, PML(1-552) and RARα in HeLa cells using the method of Example 1, and heat shock treatment at 43°C for different times after 24h, such as Figure 3-5 shown. Collect samples, blot dry supernatant and store cell samples at -80°C or directly lyse. When the cells are lysed, add a certain amount of RIPA lysate to the cell mass, and immediately pipette evenly. Place on ice, pipette several times every 10 min and vortex with a vortex shaker. After 30 minutes, centrifuge at 13,000 rpm and 4°C for 30 minutes, take the supernatant, which is the supernatant protein solution (S), wash the precipitate with PBS 3 times, and dissolve it with LDS lysate to obtain the precipitated protein (P). The protein was quantified to a uniform concentration by the Lowrry method, and the protein solution was stored at -80°C or directly loaded on Western bl...

Embodiment 3

[0037] Example 3: As 2 o 3 (iAs III ), heat shock and their combined application on the solubility transition of PML / RARα fusion protein

[0038] Overexpression of PML / RARα, As in HeLa cells by the method of Example 1, 24h 2 o 3 , heat shock at 43°C and combined treatment of the two for different periods of time, such as Figure 6 ,7,8. Collect samples, blot dry supernatant and store cell samples at -80°C or directly lyse. When the cells are lysed, add a certain amount of RIPA lysate to the cell mass, and immediately pipette evenly. Place on ice, pipette several times every 10 min and vortex with a vortex shaker. After 30 minutes, centrifuge at 13,000 rpm and 4°C for 30 minutes, take the supernatant, which is the supernatant protein solution (S), wash the precipitate with PBS 3 times, and dissolve it with LDS lysate to obtain the precipitated protein (P). The protein was quantified to a uniform concentration by the Lowrry method, and the protein solution was stored at ...

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Abstract

The invention provides a method for inducing solubility change and degradation of PML / RARα fusion protein and its mutants, inducing the solubility change of wild-type and arsenic-resistant PML / RARα fusion proteins through heat shock to promote their degradation. Compared with traditional chemotherapy or arsenic trioxide and all-trans retinoic acid, the method of the present invention solves the biggest problem in the field of APL treatment, and can induce As 2 o 3 The solubility transformation of the drug-resistant PML / RARα mutant protein promotes its degradation with less side effects. In addition, combined application of heat shock and As 2 o 3 Can speed up the treatment of APL patients. The method provided by the invention is used in inducing the solubility change of the wild-type and arsenic-resistant PML / RARα fusion protein so as to promote its degradation.

Description

technical field [0001] The invention belongs to the field of medicine, and specifically relates to a heat treatment-induced solubility change and degradation method of PML / RARα fusion protein and its arsenic trioxide drug-resistant mutant, which uses relatively mild heat shock to induce the solubility change and degradation of PML / RARα fusion protein, thereby This method is used in the clinical treatment of APL patients. Background technique [0002] Acute promyelocytic leukemia (APL) is a special type of acute myeloid leukemia (AML). According to the FAB standard, APL belongs to M3 of AML, accounting for about 10% of AML. APL is usually accompanied by chromosomal translocation, that is, the retinoic acid receptor alpha (RARa) gene on chromosome 17 translocates with genes on other chromosomes, and X-RARa fusion protein and RARa-X fusion protein are produced thereby. More than 98% of APL patients carry t(15;17), the X sequence is derived from the promyelocytic leukemia (PML...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C07K19/00
CPCC07K14/70567C07K14/82C07K2319/00C12N5/0693C12N5/0694C12N2500/05C12N2500/30C12N2510/00
Inventor 那仁满都拉亚森买买提依明王超郝睿陈烨嘉孙洁马丽亚杨畅金洁
Owner ZHEJIANG UNIV
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