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
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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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