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Superoxide dismutase from pig blood cells and preparation method thereof

A technology of superoxide and blood cells, applied in the biological field, can solve problems such as difficult to be widely used, increased risk, and low yield, and achieve good protease hydrolysis resistance, good pH stability, and good thermal stability. Effect

Inactive Publication Date: 2013-09-18
LIAONING PANCO TECH DEV
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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 from pig blood cells and preparation method thereof
  • Superoxide dismutase from pig blood cells and preparation method thereof
  • Superoxide dismutase from pig blood cells and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1C

[0041] Embodiment 1Cu, the acquisition of Zn-SOD gene

[0042] (1) Separation and extraction of mRNA

[0043] Take 5ml of normal pig fresh blood, add 0.4ml of EDTA solution with a concentration of 15g / L to prevent blood coagulation, draw 0.25ml with a micropipette, dilute with DEPC-treated water at a ratio of 1:1, take 0.25ml after dilution of fresh blood in a 1.5ml centrifuge tube, add 1ml TRIzol solution ( Reagent, Invitrogen TM Cat NO.15596-026), and extract total RNA according to the instructions.

[0044] Take 1ul to dilute 100 times and carry out 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.

[0045] (2) Synthesis of the first strand of cDNA

[0046] 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 rev...

Embodiment 2

[0054] Embodiment 2 Pichia pastoris fermentation produces recombinant SOD

[0055] The pPIC9k-SOD obtained in Example 1 was digested with Sac I to obtain the linearized plasmid pPIC9k-SOD1.

[0056] Take 50ug of the constructed linear recombinant plasmid DNA and directly add it to the competent cells (Pichia pastoris GS115) which are still below zero; add 1.0ml solution II containing 5ug / ml salmon sperm DNA (40% (w / v) Polyethylene glycol 1000, 0.2M N, N-dihydroxyethylglycine, pH8.35); keep warm in a water bath at 30°C for more than 1 hour, and mix gently every 15 minutes; keep warm at 42°C for 10 minutes; centrifuge at room temperature for 5 minutes at 3000×g , discard the supernatant, resuspend the cells with 1.0ml solution III (0.15M NaCl, 10mM N,N-bishydroxyethylglycine, pH8.35); centrifuge at 3000×g for 5min at room temperature, remove 800ul supernatant, Use the remaining 200ul supernatant to resuspend the bacteria; apply 200ul of the bacteria to the YPD plate (YP and 20%...

Embodiment 3

[0061] The purification of embodiment 3 recombinant SOD

[0062] The fermentation culture liquid prepared in Example 2 was centrifuged at 10000rpm for 10min to remove the thallus, and the supernatant was taken as the crude enzyme liquid, and ultrafiltration was carried out with an external pressure type hollow fiber ultrafiltration membrane with a molecular weight cut-off of 8000Da to remove small Molecular impurities and concentrate them 3-5 times.

[0063] Place the concentrate of the crude enzyme solution obtained above in an ice bath, slowly add ammonium sulfate to 85% while stirring, centrifuge at 13000rpm for 15min, take the precipitate, redissolve it with buffer, and place it in a dialysis bag with a molecular weight cut-off of 8000Da. Use pH 8.0, 10mM Tris-HCl as the external fluid for dialysis, the volume ratio of the external fluid to the internal fluid is greater than 50, dialyze at 4°C for 12-16 hours, change the external fluid every 4 hours in the middle, and take...

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Abstract

The invention provides superoxide dismutase from pig blood cells and a preparation method thereof. The nucleotide sequence of the superoxide dismutase is shown in a figure 1; the amino acid sequence of the superoxide dismutase is shown in a figure 2 in the specification; the vector of the nucleotide molecules is yeast plasmid; the cells of the nucleotide molecules are converted from the vector; and the nucleotide molecular cells of the superoxide dismutase contain nucleic acid molecules or pichia pastoris converted from the vector. The method can be used for preparing a recombinant production strain which can effectively express and secret Cu, Zn-SOD to realize production industrialization of Cu, Zn-SOD (superoxide dismutase) to obtain a Cu, Zn-SOD product with good pH stability, excellent thermal stability and protease hydrolysis capability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a superoxide dismutase derived from pig blood cells and a preparation method thereof. Background technique [0002] In 1938, Mann and Keilin first isolated a blue copper protein (Hemocuprein) from bovine erythrocytes. In 1969, MeCord and Fridovich found that this copper-containing protein purified from red blood cells has the ability to scavenge superoxide anion free radicals (O 2 -. ) function, named it superoxide dismutase (Superoxide Dismutase, EC1.15.1.1, referred to as SOD). [0003] SOD is a class of metalloenzymes widely present in organisms, and can be divided into Cu, Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD according to different metal prosthetic groups. It catalyzes the following reaction Disproportionate superoxide anion free radicals that are redundant and extremely destructive to cells in the organism to produce hydrogen peroxide and oxygen, and hydrogen perox...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/81C12N1/19C12N15/66C12R1/84
Inventor 李东旭苏珊李晓军荣嘉鑫
Owner LIAONING PANCO TECH DEV
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