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Method for precisely and quantitatively detecting function effect of anti-prion medicament at protein level

A technology for quantitative detection of prion, which is applied in the field of detection of the effect of anti-prion drugs, can solve problems such as the inability to quantitatively measure the change in the molecular weight of prion protein aggregates, and achieve the effect of overcoming incomplete transfer and saving time

Active Publication Date: 2010-10-13
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this is a proven method, it is not possible to quantify changes in the molecular weight of prion protein aggregates using this method

Method used

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  • Method for precisely and quantitatively detecting function effect of anti-prion medicament at protein level
  • Method for precisely and quantitatively detecting function effect of anti-prion medicament at protein level
  • Method for precisely and quantitatively detecting function effect of anti-prion medicament at protein level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 quantitative detection [PSI + ], [psi - ] Aggregation levels of Sup35p in cells

[0022] 1) Activation of Saccharomyces cerevisiae cells:

[0023] Take the [PSI + ], [psi - ] The single colony of the strain was inoculated into 10ml YPD liquid medium respectively, and cultured with shaking in a water bath at 30°C for 18h. Then use YPD to debug the initial OD value of the yeast, and adjust the final concentration OD of the bacterial suspension 600 = 0.5;

[0024] Inoculate the adjusted [PSI+] and [psi-] into 50ml YPD liquid medium with 1% inoculum amount, culture at 30°C, shake at 180 rpm for about 12 hours, until OD 600 =1.5 or so, the culture was stopped.

[0025] 2) Using SDD-AGE / Western blot method, quantitative detection [PSI + ], [psi - ] Aggregation levels of Sup35p in cells:

[0026] Preparation of Saccharomyces cerevisiae cell lysate: [PSI + ], [psi - ] The yeast culture solution was centrifuged respectively, 5000rpm, 5min, the supernatan...

Embodiment 2

[0031] Example 2 Quantitative detection of the effect of guanidine hydrochloride on prions

[0032] 1) Activation of yeast cells:

[0033] Take the [PSI + ], [psi - ] The single colony of the strain was inoculated into 10ml YPD liquid medium respectively, and cultured with shaking in a water bath at 30°C for 18h. Then use YPD to debug the initial OD value of the yeast, and adjust the final concentration OD of the bacterial suspension 600 = 0.5;

[0034] 2) Primary screening of anti-prion drugs:

[0035] Take 2ml of sterile cryopreservation tube, add guanidine hydrochloride, 0.9ml YPD liquid medium and 0.1ml adjusted yeast cell suspension, so that the final concentration of guanidine hydrochloride is 5mM. Under the condition of 24°C, shake culture on a water bath shaker, take an appropriate amount of yeast cells from the cryopreservation tube of the previous day at regular intervals every day, put them into a new sterile cryopreservation tube containing YPD culture medium ...

Embodiment 3

[0044] Embodiment 3 Quantitative detection phenanthridine is to the action effect of prions

[0045] 1) Activation of Saccharomyces cerevisiae cells: same as Example 2;

[0046] 2) Primary screening of anti-prion drugs:

[0047] Get 2ml sterile cryopreservation tubes, add guanidine hydrochloride, phenanthridine, 0.9ml YPD culture solution and 0.1ml adjusted yeast cell suspension respectively, so that the final concentration of guanidine hydrochloride is 0.5mM (at this concentration, guanidine hydrochloride Prion has no curative effect, but acts simultaneously with other drugs to act as a synergist), so that the final concentration of phenanthridine is 0.2mM. Under the condition of 24°C, shake culture in a water bath shaker, regularly take an appropriate amount of yeast cells from the cryopreservation tube the day before, put them into a new sterile cryopreservation tube containing YPD culture medium and the drug to be tested, and shake the culture 1 to 5 days;

[0048] Duri...

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Abstract

The invention relates to a method for precisely and quantitatively detecting function effect of an anti-prion medicament at a protein level. The invention adopts the technical scheme that the method comprises the following steps of: 1) activating saccharomyces cerevisiae cells; 2) primarily screening the anti-prion medicament, namely culturing the medicament to be detected, YPD liquid culture medium and prepared saccharomyces cerevisiae cell bacteria suspension for 1 to 5 days with shaking, and primarily judging the treatment effect of the medicament to be detected according to the change of the colony color; 3) quantitatively detecting the function effect of the anti-prion medicament by using an SDD-AGE / Western blot method; and 4) scanning and analyzing images by using a full-automatic image analysis system. The method can precisely evaluate the function effect of the medicament by detecting the soluble change of prion protein aggregates in the cells at the protein level, and judge the effect magnitude of the medicament on the prion at the protein level by quantitatively detecting the change of molecular weight of the prion protein aggregates.

Description

technical field [0001] The invention relates to a method for detecting the effect of anti-prion drugs in yeast cells, in particular to a method for accurately quantitatively detecting the effect of anti-prion drugs at the protein level. Background technique [0002] Prions are a class of pathogens that can cause transmissible encephalopathy (including mad cow disease, scrapie, kuru, Creutzfeldt-Jakob disease, etc.) in animals and humans. great harm. Research on the molecular pathogenic mechanism of prions and the screening and development of therapeutic drugs are the frontiers and hot spots in the fields of biology and medicine in the world. At present, SDS-PAGE / Western Blot is generally used in the world to evaluate the effect of anti-prion drugs by detecting the soluble changes of intracellular prion protein aggregates at the protein level. The monomeric prion protein in the state is separated from the aggregate prion protein in the insoluble state, and then SDS-PAGE / Wes...

Claims

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Application Information

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IPC IPC(8): G01N33/561C12Q1/02
Inventor 宋有涛兰万军宋尧李辉
Owner LIAONING UNIVERSITY
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