Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for rapidly purifying calf thymus gland deoxycytidine kinase

An adenodeoxycytidine kinase and purification method technology, which is applied in the field of rapid purification of calf thymidine kinase, can solve the problems of dCK inactivation, large experimental error, complicated purification method and the like

Inactive Publication Date: 2008-02-27
DONGHUA UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to overcome the traditional calf thymus dCK purification method which is cumbersome, takes a long time, is easy to inactivate dCK and has the defects of relatively large experimental errors, and provides a fast, simple and convenient method. Purification method of calf thymus dCK with better dCK activity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for rapidly purifying calf thymus gland deoxycytidine kinase
  • Method for rapidly purifying calf thymus gland deoxycytidine kinase
  • Method for rapidly purifying calf thymus gland deoxycytidine kinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Extraction of calf thymus dCK crude enzyme:

[0027] (1) Cut the calf thymus into pieces with scissors, put it into a disrupter and homogenize it together with 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[0028] (2) After centrifuging the homogenate above (10000g / 10min), take the supernatant. A 10% streptomycin sulfate solution was added to the supernatant for precipitation.

[0029] (3) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Add 22.5% ammonium sulfate to the supernatant for precipitation.

[0030] (4) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Ammonium sulfate 13.7% was added to the supernatant to precipitate.

[0031] (5) After centrifuging (10000g / 10min) the above-mentioned sediment solution, keep the sediment. Dissolve the precipitate with a small amount of 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[0...

Embodiment 2

[0040] 1. Extraction of calf thymus dCK crude enzyme:

[0041] (1) Cut the calf thymus into pieces with scissors, put it into a disrupter and homogenize it together with 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[0042] (2) After centrifuging the homogenate above (10000g / 10min), take the supernatant. A 10% streptomycin sulfate solution was added to the supernatant for precipitation.

[0043] (3) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Add 22.5% ammonium sulfate to the supernatant for precipitation.

[0044] (4) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Ammonium sulfate 13.7% was added to the supernatant to precipitate.

[0045] (5) After centrifuging (10000g / 10min) the above-mentioned sediment solution, keep the sediment. Dissolve the precipitate with a small amount of 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[004...

Embodiment 3

[0051] 1. Extraction of calf thymus dCK crude enzyme:

[0052] (1) Cut the calf thymus into pieces with scissors, put it into a disrupter and homogenize it together with 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[0053] (2) After centrifuging the homogenate above (10000g / 10min), take the supernatant. A 10% streptomycin sulfate solution was added to the supernatant for precipitation.

[0054] (3) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Add 22.5% ammonium sulfate to the supernatant for precipitation.

[0055] (4) After centrifuging (10000g / 10min) the above precipitated solution, take the supernatant. Ammonium sulfate 13.7% was added to the supernatant to precipitate.

[0056] (5) After centrifuging (10000g / 10min) the above-mentioned sediment solution, keep the sediment. Dissolve the precipitate with a small amount of 0.01M Tris-HCl buffer solution (containing 5mM mercaptoethanol, pH 7.0).

[0...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a rapid purifying method of calf-skin thymic deoxycytidine kinase (dCK), which comprises the following steps: (1) extracting rough enzyme of calf thymus dCK; grinding the calf thymus; homogenizing; salting out; sedimenting; dissolving; dialyzing to extract rough enzyme of calf thymus dCK; (2) utilizing one or all of FPLC gel molecular sieve and anion exchange chromatograph to purify the calf thymus dCK. The invention has rapid purifying speed and simple operation, which improves the activity of the purified dCK.

Description

technical field [0001] The invention belongs to a purification method, in particular to a rapid purification method of calf thymidine kinase. Background technique [0002] Deoxycytidine kinase (dCK) is one of the four cytoplasmic kinases, which widely exists in mammals and some bacterial cells. In cells, dCK can catalyze the phosphorylation of natural nucleosides, and also catalyze the phosphorylation of some nucleoside drugs [1]~[4] . Studies have shown that antiviral nucleoside drugs are phosphorylated by dCK in the human body, and produce substrates that bind to reverse transcriptase to exert their drug effects. However, in some patients, the dCK content of cells is very low, unable to catalyze the phosphorylation of nucleoside drugs, thus leading to drug resistance of the virus [5] . Therefore, in vitro phosphorylation of nucleoside drugs is a major direction of nucleoside drug research in recent years. The traditional purification of calf thymus dCK is mainly divid...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/12
Inventor 朱利民柴艳倩
Owner DONGHUA UNIV