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Recombinant superoxide dismutase as well as construction method and application thereof

A superoxide and construction method technology, applied in the field of bioengineering, can solve the problems of poor SOD stability, difficult separation and purification, and large loss of enzyme activity, and achieve the effects of simplified purification method, high purification multiple, and efficient mass production.

Active Publication Date: 2021-08-31
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, SOD has poor stability, prone to bacterial contamination, difficult separation and purification, high production cost, and large loss of enzyme activity.

Method used

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  • Recombinant superoxide dismutase as well as construction method and application thereof
  • Recombinant superoxide dismutase as well as construction method and application thereof
  • Recombinant superoxide dismutase as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of recombinant expression plasmid pET28a(+)SOD-Linker-Rec

[0024] Suzhou Synbio Biotechnology Co., Ltd. whole gene synthesis SOD ( Homo sapiens (human), accession number CR541742.1) nucleotide sequence, the nucleotide sequence is shown in SEQ ID NO: 4, and the encoded amino acid sequence is shown in SEQ ID NO: 3. The 5' and 3' of the above SOD sequence are respectively equipped with Xba I and Nhe I restriction sites to obtain the SOD nucleotide sequence with Xba I and Nhe I restriction sites, and then the Xba I and Nhe The SOD nucleotide sequence of the I restriction site was constructed on the pET28a(+) plasmid, which was denoted as pET28a(+)-SOD.

[0025] Suzhou Synbio Biotechnology Co., Ltd. synthesized the Linker-Rec nucleotide sequence from the whole gene, with a Nhe I restriction site at the 5' of the linker, and a Hind I restriction site at the 3' of the Rec, between Linker and ELP There is a Sac I restriction site, and then the above ...

Embodiment 2

[0050] Embodiment 2: expand the escherichia coli expressing SOD-Linker-Rec

[0051] (1) Transfer freshly transformed recombinant Escherichia coli to solid LB medium with 50 μg / mL kanamycin (agar powder 15g / L, tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH7.4), and cultured at 37 ℃, and then transfer the single colony after overnight culture to 5ml liquid LB medium containing 50 μg / mL kanamycin (tryptone 10g / L, yeast Extract 5g / L, sodium chloride 10g / L, pH 7.4), and cultivate overnight at 37 ℃, 200 rpm orbital shaker, then inoculate 3 mL of overnight cultured bacteria in 300 mL containing 50 μg / Grow liquid LB medium with mL kanamycin at 37°C, 200 rpm for 3 hours or until the OD600 of the bacteria reaches between 0.4-0.6.

[0052] (2) Place the liquid medium finally obtained in step (1) on ice for 20 minutes, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to it to a final concentration of 0.4 mM, Then shake culture at 16°C, 25°C and 37°C at 180rpm for 16-20h...

Embodiment 3

[0055] Example 3: Purification of recombinant superoxide dismutase SOD-Linker-Rec by cooling agglutination

[0056] In this example, cooling coagulation is used to purify SOD, so as to verify the self-purification performance and purification efficiency of the SOD prepared in Example 2. An appropriate concentration of 0.3-2.0 M (NH 4 ) 2 SO 4 Add to 500 μL soluble protein containing supernatant, mix well and place on ice for 30-60 min, then centrifuge at 12000 rpm at 4 °C for 15 min, separate supernatant and precipitate, and resuspend the precipitate in Tris-HCl (50 mM , pH 8.0) buffer. Place the resuspended Tris-HCl buffer at 0 and 4 °C for 1, 2, 12, and 24 hours, observe the appearance of coacervate, and absorb the coacervate, which is the target protein. Transfer the coacervate to a new EP tube. Save a portion of the purified sample for SDS-PAGE analysis.

[0057] figure 2 This is the SDS-PAGE image of resuspended Tris-HCl buffer placed at different temperatures for...

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Abstract

The invention provides recombinant superoxide dismutase as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, the recombinant superoxide dismutase SOD-Linker-Rec is constructed by linking SOD with Rec, and the recombinant superoxide dismutase can be self-purified efficiently, and is good in stability and high in antioxidant enzyme efficiency.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant superoxide dismutase and its construction method and application. Background technique [0002] Low concentrations of superoxide anion radicals (O 2- ) is necessary to maintain life activities. When its concentration is too high, it can cause oxidative damage to tissue cells in the body, leading to disease and even death in the body. Superoxide dismutase (superoxide dismutase; SOD) is an antioxidant metalloenzyme that exists in organisms. It can catalyze the dismutation of superoxide anion radicals to generate oxygen and hydrogen peroxide, and plays an important role in the balance of oxidation and antioxidation in the body. important role. Moreover, superoxide dismutase is inseparable from the occurrence and development of many diseases. [0003] Free radicals have unpaired electrons, atoms or ions, which are chemically active and have extremel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/02C12N15/62C12N15/70C12N1/21A61P39/06C12R1/19
CPCC12N9/0089C07K14/78C12N15/70A61P39/06C12Y115/01001C07K2319/00
Inventor 周阳张新爽刘岳霖张轩榕高力陈东锋施海峰
Owner JIANGSU UNIV
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