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Preparation method for Margaritana copper-zinc superoxide dismutase

A technology of superoxide and dismutase, applied in botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of low enzyme specific activity and tedious refolding process, and achieve high activity and preparation The effect of simple technological process and easy organization of production

Inactive Publication Date: 2010-12-22
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The above-mentioned invention obtains the SOD enzyme of the mature protein containing 199 amino acid residues from Anabaena, and does not add a certain concentration of CuSO during the expression process 4 and ZnCl 2 , so the specific activity of the enzyme is not too high, and the expression product mainly exists in the form of inclusion bodies, so a relatively tedious refolding process is required to obtain an active enzyme product

Method used

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  • Preparation method for Margaritana copper-zinc superoxide dismutase

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Embodiment 1

[0026]Select a healthy crested clam, and draw 10 mL of blood from the mussel adductor muscle sinus; centrifuge at 4°C, 3600 rpm for 5 min; discard the supernatant, and obtain cell pellets for RNA extraction. The total RNA in the blood cells was extracted with Trizol reagent from Invitrogen Company, the cell pellet obtained above was taken, and 1 mL of Trizol was added, and the blood cells were fully lysed by Trizol by pipetting repeatedly. Let stand at room temperature for 10 minutes, centrifuge at 4°C and 12,000×g for 10 minutes; take the supernatant, add 0.2 mL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C and 12,000×g for 15 minutes; take the supernatant and transfer to a clean Add 0.5 mL of isopropanol to the centrifuge tube, let stand at room temperature for 10 min, and centrifuge at 4°C, 10000×g for 10 min. After the precipitate was washed with 75% ethanol, it was stored in 100% ethanol at -35°C for lat...

Embodiment 2

[0028] Select a healthy crested clam, and draw 10 mL of blood from the mussel adductor muscle sinus; centrifuge at 4°C, 3600 rpm for 5 min; discard the supernatant, and obtain cell pellets for RNA extraction. The total RNA in the blood cells was extracted with Trizol reagent from Invitrogen Company, the cell pellet obtained above was taken, and 1 mL of Trizol was added, and the blood cells were fully lysed by Trizol by pipetting repeatedly. Let stand at room temperature for 10 minutes, centrifuge at 4°C and 12,000×g for 10 minutes; take the supernatant, add 0.2 mL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C and 12,000×g for 15 minutes; take the supernatant and transfer to a clean Add 0.5 mL of isopropanol to the centrifuge tube, let stand at room temperature for 10 min, and centrifuge at 4°C, 10000×g for 10 min. After the precipitate was washed with 75% ethanol, it was stored in 100% ethanol at -35°C for la...

Embodiment 3

[0030] Select a healthy crested clam, and draw 10 mL of blood from the mussel adductor muscle sinus; centrifuge at 4°C, 3600 rpm for 5 min; discard the supernatant, and obtain cell pellets for RNA extraction. The total RNA in the blood cells was extracted with Trizol reagent from Invitrogen Company, the cell pellet obtained above was taken, and 1 mL of Trizol was added, and the blood cells were fully lysed by Trizol by pipetting repeatedly. Let stand at room temperature for 10 minutes, centrifuge at 4°C and 12,000×g for 10 minutes; take the supernatant, add 0.2 mL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C and 12,000×g for 15 minutes; take the supernatant and transfer to a clean Add 0.5 mL of isopropanol to the centrifuge tube, let stand at room temperature for 10 min, and centrifuge at 4°C, 10000×g for 10 min. After the precipitate was washed with 75% ethanol, it was stored in 100% ethanol at -35°C for la...

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Abstract

The invention discloses a preparation method for Margaritana copper-zinc superoxide dismutase, which extracts a total RNA of pearl-breading cristaria plicata, obtains full-length cDNA of a cristaria plicata copper-zinc superoxide dismutase gene by reverse transcription and a PCR technology, clones the cDNA on a carrier of pET30, and converts escherichia coli BL21 (DE3) to obtain recombinant engineering bacteria. The preparation method comprises the following steps: carrying out high-density culturing on the engineering bacteria in an LB culturing medium; inducing by IPTG; adding Cu<2+> and Zn<2+> at a certain concentration in the culturing medium; expressing recombination genes in a large amount; and passing a product through an affinity chromatography column to obtain the purified recombination superoxide dismutase. The invention can accurately obtain a maturation protein sequence containing 155 amino acid residues; and the superoxide dismutase has the effects of antioxidation and anti-aging. The invention has high thallus output, expression level and activity, is stable to acid, alkali, temperature and denaturant, has obvious protection effect on LO-2 hepatic cells damaged by ethanol, is easy to purify, and has wide industrial application prospect.

Description

technical field [0001] The invention relates to the construction of a copper-zinc superoxide dismutase genetic engineering bacterium of the pearl mussel pleated crown mussel and the production and purification process of an enzyme product, belonging to the technical field of biological enzyme preparation. Background technique [0002] With the development of modern biological science, scientists have finally uncovered the mystery that affects human health and longevity. Free radicals are the original culprits that cause various human diseases. At the same time, the enzyme superoxide dismutase SOD, which scavenges free radicals in the body, was also discovered. SOD is an active enzyme recognized by the international medical community that can scavenge free radicals in the body. [0003] Superoxide dismutase (SOD) has the ability to specifically remove superoxide ions in organisms, balance oxygen free radicals in the body, and protect human cells from environmental factors. M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N9/08
Inventor 文春根胡宝庆范小勇徐欢欢
Owner NANCHANG UNIV
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