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Preparation method of human-derived copper-zinc superoxide dismutase

A superoxide and dismutase technology, applied in biochemical equipment and methods, oxidoreductase, enzymes, etc., can solve the problems of low target protein expression, complicated purification process, affecting target protein expression, etc., to avoid Safety risks, improved expression, and beneficial effects on purification

Inactive Publication Date: 2018-03-09
苏州东泉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a precedent for the production of human copper-zinc superoxide dismutase (hSOD1) by genetic engineering technology, but there are still many problems, mainly including: (1) The expression level of the target protein is not high, because the heterologous expression system affects the target protein (2) The purification process is complicated. Those without purification tags need to be purified by ion exchange, molecular exclusion and / or a series of chromatographic means, while those with purification tags exist in the form of inclusion bodies and need to be purified Active forms are obtained through tedious processes such as denaturation and renaturation

Method used

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  • Preparation method of human-derived copper-zinc superoxide dismutase
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  • Preparation method of human-derived copper-zinc superoxide dismutase

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

[0044] The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

[0045] The nucleotide fragment comprising hSOD1 gene coding sequence used in the present invention can be obtained by PCR amplification, artificial synthesis and other methods. For artificially synthesizing the target gene, those skilled in the art know techniques such as analyzing codon bias by software such as Emboss and designing the target gene sequence. Different organisms have a certain preference for codons encoding the same amino acid. Through the analysis of codon usage preference, the design of these optimal codons can improve the expression of the target gene. In the present invention, the nucleotide fragment comprising the coding sequence of the hSOD1 gene is preferably artificially syn...

Embodiment 2

[0063] Embodiment 2: Construction of recombinant escherichia coli

[0064] 1) Preparation of Escherichia coli Competent Cells

[0065] with CaCl 2 Preparation of Escherichia coli competent cells by chemical method, the steps include: taking 1ml of activated Escherichia coli bacteria liquid and inoculating it into 100ml LB medium; shaking culture at 37°C until OD 600 = 0.35-0.5 hours; collect the bacteria by centrifugation at 4000g at 4°C; add 40ml of pre-cooled 0.1MCaCl 2 Resuspend the cells and keep on ice for 10 minutes; collect the cells by centrifugation at 4000g at 4°C; add 2ml of pre-cooled 0.1M CaCl 2 Resuspend the cells; add glycerol with a final concentration of 15% to 20%; aliquot into 50 μL per tube; store at 4°C for 24 hours and transfer to a -80°C refrigerator for freezing.

[0066] 2) Recombinant vector pET28a-N-His-TEV-hSOD1 transforms Escherichia coli competent cells

[0067] Mix 2 μL of the recombinant vector pET28a-N-His-TEV-hSOD1 (the content does not ex...

Embodiment 3

[0071] Embodiment 3: recombinant bacterial strain produces hSOD1

[0072] 1) The recombinant strain was induced by IPTG to produce hSOD1

[0073] Take 20 μL of the bacterial solution in the glycerol preservation tube in Example 2 and inoculate it into 200 ml of LB liquid medium containing antibiotics, cultivate it at 37° C., 140 rpm to OD 600 Up to 0.4 ~ 0.6. Transfer to 1000ml LB liquid culture medium containing antibiotics according to 2.5% inoculum amount. When cultured with shaking at 37°C to OD 600 When the concentration reaches 0.4-1.0, add IPTG to a final concentration of 0.3 mM, and induce at 30° C. for 4 hours. Bacteria were collected by centrifugation. SDS-PAGE detection was carried out to analyze the expression and enzyme activity of hSOD1.

[0074] image 3Middle (a) is the production of hSOD1 induced by IPTG. As shown in the figure, M is the protein molecular weight standard; Lane 1 is the whole cell; Lane 2 is the broken whole cell; Lane 3 is the broken su...

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Abstract

The invention discloses a preparation method of human-derived copper-zinc superoxide dismutase. The preparation method includes: inserting a sequence as shown in SEQ ID No. 2 into an original carrierto build a recombinant carrier, performing expanding culture, then extracting an expression product, performing N-His-TEV-hSOD1 enzymolysis, and purifying to obtain hSOD1. The preparation method has the advantages that the target gene sequence is designed according to codon preference, a His-TEV label is added to the N tail end, and expression heterology is avoided while the safety risks of animalor human sources are avoided; the His purification label and the TEV recognition site of the label is removed during sequence design, purification is benefited, and the His label can be removed conveniently to meet the requirements of pharmaceutical protein; the expression of the hSOD1 expressed by an expression strain is soluble expression, expression quantity is increased evidently when Cu<2+>and Zn<2+> are added for induction, and the requirements of large-scale production are satisfied.

Description

technical field [0001] The invention belongs to the technical field of superoxide dismutase preparation, in particular to a preparation method of human copper-zinc superoxide dismutase. Background technique [0002] Superoxide dismutase (SOD) is a metalloenzyme widely present in organisms, which can catalyze the dismutation reaction of superoxide anion free radicals and balance oxygen free radicals in the body. SOD shows unique functions in radiation protection, anti-aging, anti-inflammation, tumor and cancer suppression, autoimmune therapy, etc., and has been used more and more in fields such as medicine, food, and cosmetics. [0003] So far, SOD has been isolated from organisms such as bacteria, protozoa, algae, molds, plants, insects, birds, fish and mammals. According to the different metal ions bound by its active center, SOD is mainly divided into three categories: copper-zinc superoxide dismutase (Cu / Zn-SOD), iron superoxide dismutase (Fe-SOD) and manganese superoxid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N9/02
CPCC12N9/0089C07K2319/21C12N15/70C12N2800/22C12Y115/01001
Inventor 朱斌辉朱艳娟许仁杰韩蓉
Owner 苏州东泉生物科技有限公司
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