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Cyanea capillata thioredoxin and coding gene and application thereof

A technology of jellyfish jellyfish and thioredoxin, applied in the fields of application, genetic engineering, oxidoreductase, etc., can solve problems such as no research reports on jellyfish thioredoxin, achieve significant antioxidant activity, and simple preparation method , low cost effect

Active Publication Date: 2013-08-07
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no relevant research reports on jellyfish thioredoxin at home and abroad

Method used

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  • Cyanea capillata thioredoxin and coding gene and application thereof
  • Cyanea capillata thioredoxin and coding gene and application thereof
  • Cyanea capillata thioredoxin and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Screening and preparation of thioredoxin from Aequorea habilis.

[0053] 1. Construction and analysis of cDNA library of jellyfish tentacles

[0054] 1) The extraction of total RNA from the tentacle tissue of Jellyfish jellyfish was carried out according to the instructions of Invitrogen's Trizol reagent, and the protein was removed with chloroform to obtain about 7 μg of total RNA;

[0055] 2) The isolation of mRNA was carried out according to the instructions of Oligotex mRNA Spin-column Kit of QIANGEN Company, and the synthesis of cDNA was carried out according to the instructions of SMART cDNA Library Construction Kit of Clontech Company;

[0056] 3) Insert the cDNA into the pUC19 plasmid vector (purchased from Takara Company), and then transform it into Escherichia coli DH5α (purchased from Beijing Bomaide Company), spread it on a 15CM petri dish for blue-white screening, and then construct the jellyfish jellyfish Tentacle tissue cDNA library.

[0057]...

Embodiment 2

[0078] Example 2: Detection of Antioxidative Ability of Recombinant Aequorea hacivus thioredoxin (ABTS + free radical scavenging method)

[0079] After the relatively single target protein was obtained by nickel ion affinity chromatography, the antioxidant capacity of the purified thioredoxin was determined according to the method described in the antioxidant capacity test kit produced by Beyontian Company. First add 200 μl of ABTS to all detection wells of the 96-well plate + solution, then add 10 μl of eluent to the blank control group, add 10 μl of vitamin C equivalent Trolox standard solution of different concentrations to the standard curve group, add 10 μl of purified Aequorea thioredoxin of different concentrations to the sample group, and mix well. A750 was measured after incubation at room temperature for 2-6 minutes. Obtain a standard curve as Figure 5 shown.

[0080] According to the standard curve, the antioxidant capacity of thioredoxin from jellyfish was 6.5...

Embodiment 3

[0081] Example 3: Detection of Disulfide Bond Reducing Ability of Recombinant Aequorea habilis thioredoxin

[0082]This experiment was carried out according to the method introduced by Holmgren (J. Boil. Chem, 1979, 254:9627-9632.). 1ml reaction buffer contains bovine insulin with a concentration of 1.25mg / ml, 0.1M PBS, 2mMEDTA, add 100μl eluate to the blank control group, and add 100μl recombinant hairdressing agents with concentrations of 4μM, 8μM, and 12μM to the sample detection group Aequorea jellyfish thioredoxin, and then add DDT at a final concentration of 2 mmol / l to each reaction solution and mix well to start the reaction. Immediately start to measure A655 every 1min, and make a response curve according to the change of light absorption value with time (such as Figure 6 ).

[0083] The results show that the A655 curve of the control group is very flat and its value is close to zero, while the detection curves of each sample have a rising trend with time, and the ...

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Abstract

The invention belongs to the technical field of biomedicine and provides a cyanea capillata thioredoxin. Currently, researches and reports related to jellyfish thioredoxin are not seen yet. The cyanea capillata thioredoxin provided by the invention has protein formed by an amino acid sequence shown in SEQ ID NO: 2. The invention further provides a coding gene of the cyanea capillata thioredoxin, wherein the coding gene has a nucleotide sequence shown in SEQ ID NO: 1. Meanwhile, the invention further provides application of the cyanea capillata thioredoxin and the coding gene of the cyanea capillata thioredoxin in preparation of anti-oxidation drugs, anti-radiation drugs, skin protectants, food preservatives, anti-aging drugs and the like. The cyanea capillata thioredoxin and the coding gene of the cyanea capillata thioredoxin have greater clinical application values.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a jellyfish thioredoxin and its coding gene and application. Background technique [0002] Jellyfish belong to the phylum Cnidaria, which is a kind of marine plankton with a wide distribution and a very large biomass. A large number of studies have shown that jellyfish are rich in various biologically active substances, including jellyfish toxin protein and some new functional proteins with strong activity and good development prospects. Jellyfish float in the surface layer of seawater at a depth of 4-6 meters, and live in a strong light radiation environment for a long time. Oxidative damage is one of the important mechanisms of body damage caused by ultraviolet radiation. Studies have shown that in order to avoid or reduce the damage caused by ultraviolet radiation, plankton have undergone long-term adaptive selection to produce a wide variety of structurally s...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70A61K38/44A61K8/66A61P39/06A61Q19/08A23L3/3571
Inventor 阮增良柳国艳张黎明赵杰温小娟尹慢慢陆佳刘丹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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