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A kind of preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome

A cyclobutane pyrimidine dimer, photorepair enzyme technology, applied in the directions of liposome delivery, botanical equipment and method, biochemical equipment and method, etc., can solve the problem of unsuitable large-scale preparation and purification of chromatography column matrix Expensive and other problems, to achieve the effect of improving the repair effect, the effect is good, and the price is cheap

Inactive Publication Date: 2016-01-06
曹毅 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome, which overcomes the expensive price of the purification column matrix in the existing method and is not suitable for large-scale preparation of cyclobutane pyrimidine di The defects of polymer photorepair enzymes, and improve the ability of cyclobutane pyrimidine dimer photorepair enzymes to penetrate the cell membrane or skin barrier, greatly improving the effect of DNA damage repair

Method used

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  • A kind of preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome
  • A kind of preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome
  • A kind of preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome

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Embodiment 1

[0020] a. Expression of cyclobutane pyrimidine dimer photorepair enzyme

[0021] The cyclobutane pyrimidine dimer photorepair enzyme gene (International Publication No., GenBank: EF090912.1) was cloned from Dunaliella salina, and the gene was constructed into the prokaryotic expression vector pGEX4T-1, and then transformed into the expression strain of Escherichia coli E. coliBL21 competent cells, to obtain bacterial liquid; add 0.03ml of the above bacterial liquid to 3ml of LB medium containing ampicillin, and culture at 37°C until OD 600Then add IPTG to a final concentration of 0.1mol / L, induce expression at 23°C for 3 hours, centrifuge and precipitate the bacteria under the condition of 10,000 gravitational accelerations, collect the bacteria, and ultrasonically disrupt the bacteria with an ultrasonic cell disruptor. The crushing conditions are: under the power of 200-400W, the working time is 1-5s, the intermittent time is 2-5s, and the crushing time is 20-40min; then the ...

Embodiment 2

[0028] a. Expression of cyclobutane pyrimidine dimer photorepair enzyme

[0029] The cyclobutane pyrimidine dimer photorepair enzyme gene (International Publication No., GenBank: EF090912.1) was cloned from Dunaliella salina, and the gene was constructed into the prokaryotic expression vector pGEX4T-1, and then transformed into the expression strain of Escherichia coli E. coliBL21 competent cells, to obtain the bacterial liquid; add 0.5ml of the above bacterial liquid to 50ml of LB medium containing ampicillin, culture at 37°C until OD 600 Then add IPTG to a final concentration of 0.8mol / L, induce expression at 28°C for 4 hours, centrifuge and precipitate the bacteria under the condition of 12,000 gravitational accelerations, collect the bacteria, and ultrasonically break the bacteria with an ultrasonic cell disruptor. The crushing conditions are: under the power of 200-400W, the working time is 1-5s, the intermittent time is 2-5s, and the crushing time is 20-40min; then the c...

Embodiment 3

[0036] a. Expression of cyclobutane pyrimidine dimer photorepair enzyme

[0037] The cyclobutane pyrimidine dimer photorepair enzyme gene (International Publication No., GenBank: EF090912.1) was cloned from Dunaliella salina, and the gene was constructed into the prokaryotic expression vector pGEX4T-1, and then transformed into the expression strain of Escherichia coli E. coliBL21 competent cells, to obtain bacterial liquid; add 0.3ml of the above bacterial liquid to 10ml of LB medium containing ampicillin, and culture at 37°C until OD 600 Then add IPTG to a final concentration of 0.9mol / L, induce expression at 25°C for 3.5 hours, centrifuge and precipitate the bacteria under the condition of 12,000 accelerations of gravity, collect the bacteria, and ultrasonically disrupt the bacteria with an ultrasonic cell disruptor. The conditions for ultrasonic crushing are: under the power of 200-400W, the working time is 1-5s, the intermittent time is 2-5s, and the crushing time is 20-4...

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Abstract

The invention discloses a preparation method of cyclobutane pyrimidine dimmer photolyase liposome, which comprises the following steps: an expression of cyclobutane pyrimidine dimmer photolyase, a purification of cyclobutane pyrimidine dimmer photolyase, and a preparation of cyclobutane pyrimidine dimmer photolyase liposome; the method employs a Q column for separating the cyclobutane pyrimidine dimmer photolyase, and the medium price is low, moreover, the effects for separating and removing glutathione-S-transferase are better, and the method is suitable for industrialization production of cyclobutane pyrimidine dimmer photolyase. The cyclobutane pyrimidine dimmer photolyase is prepared into a liposome according to the invention, thereby the cyclobutane pyrimidine dimmer photolyase can penetrate the barrier of cell membrane or skin and enter into cells, thereby substantially improving restoration effect of DNA damage.

Description

technical field [0001] The invention belongs to the field of DNA photorepair enzyme preparation, in particular to a preparation method of cyclobutane pyrimidine dimer photorepair enzyme liposome. Background technique [0002] Cyclobutane pyrimidine dimer photorepair enzyme (cuclobutanepyrimidinedimmerphotolyase, CPDase) is the main tool for organisms to use light to repair DNA damage. CPDase is widely distributed in various species and is a monomeric protein with a molecular weight between 55-65kD. CPDase has not been found in the human body. At present, on a global scale, with the environmental damage and the increase in the dose and intensity of ultraviolet rays reaching the ground, the incidence of skin diseases, especially skin cancer, is increasing year by year. The reason is that ultraviolet rays in sunlight can cause damage to macromolecules in organisms. Make DNA produce photoproducts such as cuclobutanepyrimidinedimmer (CPD). CPD damage is difficult to be repaired...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/51A61K9/127C12N15/60C12N15/70C12N9/88A61P17/00
Inventor 曹毅乔代蓉徐辉
Owner 曹毅
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