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Preparation and application of human superoxide dismutase hSOD1 mutant

A superoxide and mutant technology, applied in the field of bioengineering, can solve the problems of difficult to maintain activity, unstable expression level, poor stability, etc.

Active Publication Date: 2022-05-13
SOUTHWEST JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, Invention Patent No. 201010169323.5 describes the recombinant expression of yak SOD1, but it is still the SOD extracted from traditional animals, which has defects such as poor stability, unstable expression level, and difficulty in maintaining activity.

Method used

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  • Preparation and application of human superoxide dismutase hSOD1 mutant
  • Preparation and application of human superoxide dismutase hSOD1 mutant
  • Preparation and application of human superoxide dismutase hSOD1 mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The construction method of recombinant human superoxide dismutase hSOD1 and its mutant Mt1SOD1, the steps are as follows:

[0034] 1. Construction of recombinant human SOD1 and its mutants

[0035]Sequences were obtained from NCBI, codon optimization was performed according to the codon usage preference of Escherichia coli, 6×His-tag tags were added to the 5’ end, and hSOD1 and Mt1SOD1 genes with 6×His-tag coding sequences at the 5’ end were chemically synthesized; sequenced confirmed. Add BamHI and XhoI restriction sites to both ends of the sequence, and clone into the pET-28a(+) vector. This step is done by Qingke Company. Transform Escherichia coli BL21(DE3) by heat shock method, pick a single colony and culture it, and use the pET-28a(+) vector universal primers T7 and T7-ter to carry out colony PCR identification, and temporarily store the positive clones identified correctly in Southwest Jiaotong University School of Life Science and Engineering -80°C refrigerat...

Embodiment 2

[0053] 1. Enzymatic properties of recombinant human superoxide dismutase hSOD1 and its mutant Mt1SOD1

[0054] (1) Introduction of measuring principle and method:

[0055] The catalytic activity of recombinant hSOD1 and Mt1SOD1 was detected by the total superoxide dismutase (T-SOD) assay kit by hydroxylamine method. The superoxide anion free radical is generated through the xanthine and xanthine oxidase reaction system, and the latter oxidizes hydroxylamine to form nitrite, which is purple-red under the action of the chromogen. According to the requirements of the manufacturer, the reagent IV application solution was prepared in advance. Preliminary experiments have determined the optimal amount of hSOD1 added, so that the inhibition rate of SOD is between 15% and 55%. Mix a certain amount of SOD sample and each reagent and incubate at 37°C for 40min, leave it at room temperature for 30min and measure the absorbance at 450nm. The activities of hSOD1 and Mt1SOD1 were calcula...

Embodiment 3

[0059] Electrophoresis detection of recombinant hSOD1 and Mt1SOD1

[0060] Collect the bacterial solution and eluate in the above expression and purification process respectively, add 5× protein loading buffer, and place in a metal bath at 95°C for 8 minutes. Centrifuge at 10,000 rpm for 10 min, and draw 8 μl of the supernatant for routine SDS-PAGE detection (5% stacking gel, 12% separating gel). The working mode of the electrophoresis instrument (Beijing Liuyi Instrument Factory) was 80V for 30min and then adjusted to 120V for 1h30min. After electrophoresis, stain with Coomassie Brilliant Blue staining solution for 2 h, and then replace the destaining solution until the bands are clear. figure 1 It is the hSOD1 expression and purification process, where M is the protein pre-stained Maker, 1 is the soluble fragment of the uninduced bacterial solution, 2 is the whole bacterial cell lysate induced by adding IPTG, 3 is the supernatant of ultrasonic induction after induction, and...

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Abstract

The invention provides a preparation method and application of a human superoxide dismutase hSOD1 mutant, and relates to the technical field of bioengineering. The invention provides a coding protein of a human-derived superoxide dismutase hSOD1 mutant, wherein the coding protein has an amino acid sequence shown as SEQ ID NO.Mt1SOD1aa; the invention provides an Mt1SOD1 gene for coding the protein, and the Mt1SOD1 gene has a nucleotide sequence as shown in SEQ ID NO.Mt1SOD1. According to the invention, human-derived hSOD1 is taken as a template, an hSOD1 mutant Mt1SOD1 (E25G, P29T, E101V and C112S) gene is obtained through gene mutation, and human-derived superoxide dismutase hSOD1 mutant Mt1SOD1 protein is recombined; and the heterologous problem is solved in application. And the polypeptide has high activity and high stability, and provides a new way for the development of novel functional anti-aging cosmetics and series products and the development of other anti-aging products such as functional foods and anti-aging drugs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the preparation and application of a mutant of human superoxide dismutase hSOD1. Background technique [0002] Superoxide dismutase (Superoxide Dismutase, SOD) is an active substance ubiquitous in various organisms. It is the core free radical scavenging enzyme in the organism, which can specifically remove superoxide anion in the process of cell metabolism in the body. , known as the first line of defense of the organism's antioxidant system. SOD can reduce the oxygen free radicals produced by the excessive oxygen consumption of melanin under light, prevent the formation of excessive melanin, and also continuously remove the oxygen free radicals produced by lipofuscin and ceroid to avoid excessive lipid Peroxidation occurs, reducing the formation of lipofuscin and ceroid. In addition, the increase in the activity of SOD in the body can reduce the peroxidativ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N9/02C12N1/21A61K8/66A61K38/44A61P17/16A61Q19/08A23L33/00C12R1/19
CPCC12N9/0089C12N15/70C12Y115/01001A61K8/66A61K38/446A61Q19/08A61P17/16A23L33/00A23V2002/00A23V2200/318Y02A50/30
Inventor 黄新河杨方瑶简甜甜段嘉欣刘汶东
Owner SOUTHWEST JIAOTONG UNIV
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