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High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase

A high-density fermentation, superoxide technology, applied in fermentation, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of difficulty in ensuring SOD yield, SOD yield limitation, low recombinant expression, etc., and achieve high yield , High heat resistance, simple purification process

Inactive Publication Date: 2012-04-04
YANGTZE DELTA REGION INST OF TSINGHUA UNIV ZHEJIANG +1
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Problems solved by technology

[0003] At present, SOD is mainly obtained by extracting from animal blood or plant tissue, so the output of SOD is largely limited by raw materials
Many researchers have also carried out a lot of explorations to produce SOD by genetic engineering methods, but the existing methods still have the following problems: 1) most of the recombinantly expressed SOD exists in the form of inactive inclusion bodies, which must be added in the purification process. Therefore, it is difficult to guarantee the yield of SOD; 2) The SOD produced has problems such as low activity, poor heat tolerance, and easy inactivation, and needs to be chemically modified before it can be used; 3) The amount of recombinant expression Relatively low, generally at the level of 20-40%

Method used

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  • High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase
  • High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase
  • High-density fermentation and purification process for recombination high temperature-resistant hyperoxide dismutase

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, construction and induced expression of SOD engineering bacteria

[0045] (1) The heat-resistant SOD gene was amplified by PCR, and then ligated into the plasmid vector pET28a after the same digestion treatment to construct a recombinant plasmid, which was named pSOD. Then, the plasmid pSOD was transformed into competent Escherichia coli BL21(DE3) by chemical transformation method, and a strain with high SOD production was obtained after screening to complete the construction of SOD engineering bacteria.

[0046] (2) Add kanamycin to the LB medium to a final concentration of 30 mg / L, inoculate a single colony of SOD engineered bacteria, culture with shaking at 37°C for 2 hours, add 0.5mM IPTG, and continue to cultivate at 30°C for 8 hours;

[0047] (3) The cells were collected by centrifugation and resuspended in 500 ml of lysis buffer; lysis buffer: 10 mM Tris-HCl, pH 8.0.

[0048](4) Cell disruption was performed with an ultrasonic cell disruptor, the po...

Embodiment 2

[0050] Embodiment 2, the high-density fermentation of SOD engineering bacteria

[0051] (1) The composition of the basic culture medium of the fermenter is as shown in Table 2, and the composition of the feed is as shown in Table 3;

[0052] (2) Primary seed culture: inoculate 5ul of glycerol bacteria in 20ml LB liquid medium, the final concentration of kana is 50mg / L, 37°C, 220rpm, and cultivate for 10h;

[0053] (3) Secondary seed culture: transfer the cultured bacteria liquid in the previous step to 200ml LB liquid medium, the final concentration of kana is 50mg / L, 37°C, 220rpm, and cultivate for 4h;

[0054] (4) Upper tank fermentation: Inoculate the 200ml secondary seed bacterial liquid cultivated in the previous step into a 6.6L fermenter. The fermentation conditions are: dissolved oxygen 30%, temperature 30°C, pH 7.0, speed between 200rpm and 800rpm automatically adjusted according to dissolved oxygen;

[0055] (5) The fermentation is carried out for 5-6 hours, and wh...

Embodiment 3

[0056] Embodiment 3: the electrophoresis detection of SOD engineering bacteria high-density fermentation product

[0057] After the cells were sonicated under 300W ultrasonic waves, they were centrifuged at 8000g for 20 minutes, and the supernatant and precipitate after the cells were crushed were subjected to 12.5% ​​SDS-PAGE electrophoresis, and the results were as follows: figure 2 . The label descriptions in the figure are respectively 1-split supernatant before induction; 2-split supernatant after induction 5 hours; 3-split supernatant after induction 10 hours; 4-split supernatant after induction 14 hours; 5-before induction lysis precipitate; 6-induction 5 hours lysis precipitation; 7-induction 10 hours lysis precipitation; 8-induction 14 hours lysis precipitation; 9-fermentation supernatant; 10-Marker(14400-97000).

[0058] The results showed that no SOD target protein was produced before induction, and all SOD protein appeared in the form of soluble protein after ind...

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Abstract

The present invention provides a high density fermentation and a purification process of a recombination high temperature resistance superoxide dismutase, the construction method of the invention includes: using gene coded for SOD in a thermophilic bacteria as a template, designing specific primer amplification target gene having restriction enzyme sites, after double digestion, connecting to plasmid vector pET28a after the same double digestion, constructing a recombinant plasmid, named for pSOD, transforming plasmid pSOD to competence escherichia coli BL21(DE3) by chemical transformation method, obtaining strain having high SOD yield after screening, completing the construction of SOD engineering bacteria; the fermentation process includes four steps of first order seed culture, secondary order feed culture, batch fermentation and induced expression, fermentation product SOD is finally obtained; the fermentation process realizes high level expression of SOD, the expression of the target protein is more than 60% of the bacterial protein total; SOD has excellent thermal stability and heat resistance, the expression product accounts for more than 60% of the whole proteins, and fully soluble protein, avoiding any trouble in the course of inclusion body renaturation; the purification process is simple, having high yield, lower cost, the final product SOD has high purification, high activity and strength stability.

Description

technical field [0001] The invention relates to a method for constructing engineering bacteria of recombinant high-temperature-resistant superoxide dismutase and a high-density fermentation and purification process, belonging to the technical field of genetic engineering pharmaceuticals. Background technique [0002] Superoxide Dismutase (Superoxide Dismutase, hereinafter referred to as SOD) is a metalloenzyme widely present in organisms; it is named Cu / Zn-SOD, Mn-SOD, Fe-SOD according to the different metal ions it binds. and Ni-SOD. SOD is an efficient scavenger of oxygen free radicals, and has anti-tumor, anti-fatigue, anti-disease and anti-aging effects; this enzyme has important applications in the fields of food, cosmetics and medicine. [0003] At present, SOD is mainly obtained by extracting from animal blood or plant tissue, so the output of SOD is largely limited by raw materials. Many researchers have also carried out a lot of explorations to produce SOD by gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/70C12N9/08C12N1/21C12R1/19
Inventor 孟凡国夏勇王天文胡卫江周海梦
Owner YANGTZE DELTA REGION INST OF TSINGHUA UNIV ZHEJIANG
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