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Production method for recombinant humanized superoxide dismutase

A superoxide and production method technology, applied in the field of bioengineering, can solve problems such as limited sources of human SOD, production of bacterial endotoxin, cumbersome SOD purification methods, etc.

Inactive Publication Date: 2014-03-12
SHANDONG BOAOKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the SOD circulating in the market is extracted from animal blood, which is easily contaminated, produces bacterial endotoxin, and causes pyrogenic problems. The research and development of recombinant human superoxide dismutase can not only solve the problem of limited sources of human SOD and pollution caused by blood products The safety problems brought about, and can avoid the immune allergic reaction that may occur in the clinical application of heterologous SOD
The traditional SOD purification method is cumbersome, time-consuming and labor-intensive, the SOD yield is low, and the production cost is expensive, which is not conducive to large-scale factory production

Method used

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  • Production method for recombinant humanized superoxide dismutase
  • Production method for recombinant humanized superoxide dismutase
  • Production method for recombinant humanized superoxide dismutase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Centrifuge the 60L fermentation broth with a tube centrifuge at a speed of 16000rpm and a flow rate of 0.6-0.8mL / min. Collect the fermented bacteria, suspend them according to the ratio of the wet weight of the bacteria to the volume of pure water 1:10, and use a high-pressure homogenizer Broken, crushing pressure is 880bar, broken once. Add 2%, 3%, 4%, 5%, 6% volume of 10mmol / L CuCl to the mixed solution after crushing 2 Then pass it into the spiral tube heater, 65°C, 20min. After centrifugation, discard the pellet. The supernatant is treated with a membrane, firstly through a 5μm pretreatment membrane to remove impurities and cell debris; then through a 0.22μm filter membrane to remove bacteria; finally with a 6kDa ultrafiltration membrane to remove salt and small molecular impurities, and concentrate to 15L. The concentrated solution is subjected to DEAE-Sephadex A50 column chromatography, and gradient elution is performed with a potassium phosphate buffer solutio...

Embodiment 2

[0019] Centrifuge 60L fermentation broth with a tubular centrifuge, speed: 16000rpm, flow rate: 0.6-0.8mL / min, collect the fermented bacteria, suspend according to the ratio of the wet weight of the bacteria to the volume of pure water 1:10, and use a high-pressure homogenizer Broken, crushing pressure is 880bar, broken once. Add 5% volume of 10mmol / L CuCl to the mixed solution after crushing 2 Then pass it into the spiral tube heater, set the temperature at 65°C, 70°C, 75°C, 80°C, control the speed of the peristaltic pump so that the time from entry to exit is controlled as 10min, 15min, 20min, 25min, 30min respectively, centrifuge Afterwards, the precipitate was discarded. The supernatant is treated with a membrane, firstly through a 5μm pretreatment membrane to remove impurities and cell debris; then through a 0.22μm filter membrane to remove bacteria; finally with a 6kDa ultrafiltration membrane to remove salt and small molecular impurities, and concentrate to 15L. The ...

Embodiment 3

[0021] Centrifuge 60L fermentation broth with a tubular centrifuge, speed: 16000rpm, flow rate 0.6-0.8mL / min: collect the fermented bacteria, suspend them according to the ratio of wet weight of bacteria to volume of pure water 1:10, and crush them with a high-pressure homogenizer , crushing pressure is 800bar, crushing once. Add 5% volume of 10mmol / L CuCl to the mixed solution after crushing 2 Then pass it into the spiral tube heater, set the temperature at 70°C, control the speed of the peristaltic pump so that the time from entering to exiting is 15 minutes, centrifuge, and discard the precipitate. The supernatant is treated with a membrane, firstly through a 5μm pretreatment membrane to remove impurities and cell debris; then through a 0.22μm filter membrane to remove bacteria; finally with a 6kDa ultrafiltration membrane to remove salt and small molecular impurities, and concentrate to 15L. The concentrate was subjected to column chromatography on DEAE-32 and DEAE-Sepha...

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Abstract

The invention relates to a production method for a recombinant humanized superoxide dismutase and belongs to the bioengineering technology field. In the method, most of impure proteins are removed by utilization of copper salt heat denaturation; after centrifugation, bacteria and impurities are removed by microfiltration; concentration is carried out by utilization of ultrafiltration, salts and micromolecule impure proteins are removed; after anion exchange chromatographic purification, freeze drying is carried out. The purity of the superoxide dismutase can reach 92%, and the activity is 3170U / mg. During the separation process, no organic solvents are used, and protein denaturation caused by organic solvents is avoided. The method has simple operation and low cost, and is suitable for industrial production.

Description

(1) Technical field [0001] The invention relates to a production method of recombinant human superoxide dismutase, which belongs to the technical field of bioengineering. (2) Background technology [0002] Superoxide dismutase (SOD) can effectively remove superoxide free radicals in the body in a timely manner, so that the organism can avoid oxidative damage. It is the first line of defense for the organism to resist the poisoning effect of active oxygen. Anti-inflammatory, anti-tumor and cancer functions. At present, SOD enzyme has been widely used in medicine, food, beverage, cosmetics and other industries as a health care product, and has a very broad market. [0003] Most of the SOD circulating in the market is extracted from animal blood, which is easily contaminated, produces bacterial endotoxin, and causes pyrogenic problems. The research and development of recombinant human superoxide dismutase can not only solve the problem of limited sources of human SOD and pollu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12R1/19
CPCC12N9/0089C12Y115/01001
Inventor 刘钦松王绍文刘勇尹萌萌王德金李小刚张丛丛刘孟刚尚艳艳李华利
Owner SHANDONG BOAOKE BIOTECH
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