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Superoxide dismutase and preparation method thereof

A technology of superoxide and dismutase, applied to recombinant plasmids and strains containing this sequence, its coding sequence, expression and fermentation production in yeast cells, the field of superoxide dismutase, can solve the problem of increased risk and difficult It has been widely used, and the yield is small, and it has achieved the effects of good pH stability, good resistance to protease hydrolysis, and good thermal stability.

Inactive Publication Date: 2010-01-27
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to limited raw materials, difficult purification and other reasons, the purity of SOD is low and the yield is low, especially with the frequent reports of malignant infectious diseases such as mad cow disease, avian influenza, foot-and-mouth disease and SARS transmitted by animals around the world, the risk of producing animal-derived blood products In addition, the increase in product purity requirements also increases production costs
SOD from natural microorganisms and plants is also difficult to be widely used because of its few types, low expression, large molecular weight of enzymes, and low homology with SOD in animals and humans.

Method used

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  • Superoxide dismutase and preparation method thereof
  • Superoxide dismutase and preparation method thereof
  • Superoxide dismutase and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] The acquisition of embodiment 1Cu / Zn-SOD gene

[0048] (1) Separation and extraction of total RNA:

[0049] Take small pieces of sika deer liver, freeze them with liquid nitrogen, and crush them in a mortar. Take about 100 mg of powder in a 1.5 ml centrifuge tube, add 1 mL of TRIzol solution ( Reagent, Invitrogen TM Cat No.15596-026), and extract total RNA according to the instructions.

[0050] Take 1uL to dilute 100 times and perform quantitative detection, take the necessary amount for reverse transcription (RT), and add the remaining amount to three times the volume of ethanol to mix and store at -80°C.

[0051] (2) Synthesis of the first strand of cDNA:

[0052] RT-PCR is to reverse transcribe mRNA into cDNA first, and then amplify it by PCR. cDNA refers to the complementary DNA (complementary DNA, referred to as cDNA) formed under the action of reverse transcriptase using mRNA as a template. There are two key factors in the synthesis of the first strand of ...

Embodiment 2

[0061] Expression and amplification of embodiment 2Cu / Zn-SOD coding gene in escherichia coli

[0062] The target gene obtained in the embodiment 1-(4) is connected with the pET21a (+) plasmid through EcoR I and NotI double digestion to obtain the recombinant plasmid pET21a (+)-SOD (such as image 3 shown).

[0063] Take 10 ul of the constructed plasmid DNA and add it to 100 ul of prepared competent cells (Escherichia coli Rosetta2 (DE3) and JM109), shake well and place on ice, ice bath for 30 min; place in a 42°C water bath for 90 s heat shock; Quickly move the centrifuge tube to the ice-water mixture for 2 minutes; add 400ul SOC medium (2% peptone, 0.5% yeast powder, 10mM NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4, 20mM glucose, pH7.0 to each tube) ~7.2), lightly sucked with a pipette, then revived on a shaker at 37°C for 1h (80rpm~200rpm); centrifuged, 4000rpm×5min, removed 400ul supernatant, and mixed the rest; spread on a plate (LB-agar plate, containing 100ug / ml Amp), plac...

Embodiment 3

[0066] Example 3 Construction of Yeast Recombinant Expression Vector

[0067] The target gene obtained in embodiment 2 is connected with the pPIC9k plasmid through EcoR I and NotI double digestion, to obtain recombinant plasmid pPIC9k-SOD (such as image 3 shown).

[0068] Using the obtained pPIC9k recombinant plasmid as a template, PCR was performed with the primer pair composed of primer P1 and primer P2, and at the same time, PCR was performed with the primer pair composed of the pET21a (+) recombinant plasmid prepared in Example 2 and primer P1 and primer P2, from Verify at the DNA level whether the foreign gene insertion is correct. The sequence length of the two products obtained by PCR is 456bp, which is consistent with the sequence and other characteristics of the original SOD gene obtained from the sika deer liver cells in Example 1, so it can be known that the insertion site, direction and sequence of the target gene are correct .

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Abstract

The invention provides a SOD gene from animal and a method for cloning and expressing the SOD gene in colibacillus and yeast cells, wherein a nucleotide sequence of superoxide dismutase is shown as SEQ NO.1; an amino acid sequence of the superoxide dismutase is shown as SEQ NO.2; carriers of nucleotide molecules are colibacillus plasmids or yeast plasmids; cells of the nucleotide molecules are formed by carrier conversion; and cells of the nucleotide molecules of the superoxide dismutase contain colibacillus containing the nucleotide molecules or converted by the carriers or pichia yeast containing the nucleotide molecules or converted by the carriers. The invention can prepare recombinant production strains which can efficiently express and secrete Cu / Zn-SOD, realizes the production industrialization of the Cu / Zn-SOD, and achieves good pH stability, favorable thermal stability and Cu / Zn-SOD products with anti-protease hydrolyzation capacity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a superoxide dismutase, its coding sequence, recombinant plasmids and bacterial strains containing the sequence, and the expression and expression of superoxide dismutase encoded by the sequence in Escherichia coli Fermentative production in yeast cells. technical background [0002] SOD (superoxide dismutase) is a class of metalloenzymes widely present in organisms. So far, people have isolated many enzymes from various organisms such as bacteria, fungi, algae, fish, insects, plants and mammals. According to the different types of metal ions combined, SOD can be divided into three types, namely Cu / Zn-SOD, Mn-SOD and Fe-SOD. The first type contains Cu and Zn, which is blue-green and mainly exists in the cytoplasm of eukaryotic cells. The second type contains Mn, which is pink and has been isolated from the mitochondria of eukaryotic cells and the cytoplasm of prokar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/81C12N1/21C12N1/19C12P19/34C12R1/19C12R1/84
Inventor 叶秀云李仁宽靳伟刚张洋罗鋆琳
Owner FUZHOU UNIV
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