Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod

A technology of Thermoascomycetes and Pichia pastoris, applied in the field of bioengineering

Inactive Publication Date: 2009-02-18
SHANDONG AGRICULTURAL UNIVERSITY
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  • Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod
  • Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod
  • Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod

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Embodiment Construction

[0023] 1. Isolation and identification of Thermoascus aurantiacus var.levisporus

[0024] (1) Specimen collection: collected from compost in Beijing.

[0025] (2) Separation and cultivation: 0.5 g of the collected specimens were placed on a PDA plate and cultured at 50° C. for 3 days before separation and purification. The operating steps refer to Cooney and Emerson (1964) literature.

[0026] (3) Identification: refer to Cooney and Emerson (1964) literature.

[0027] 2. Cloning of Mn-SOD enzyme gene Mn-sod from Thermoascus aurantiacus var.levisporus

[0028] (1) Extraction of total RNA from Thermoascomyces var. photospora: refer to the instructions of the Trizol kit.

[0029] (2) Synthesis of the first strand of cDNA: the first strand of cDNA was synthesized using Oligo(dT)20 as a primer according to the instructions of the Reverse Transcription Reaction kit from Promega. The reaction conditions are: 42°C for 1 hour; 85°C for 10 minutes.

[0030] (3) PCR reaction: upstre...

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Abstract

The invention relates to a pichia yeast engineering strain Pichia pastoris MS18 expressing thermostable manganese superoxide dismutase (Mn-SOD) gene Mn-sod obtained from Thermoascus aurantiacus var. levisporus. The manganese superoxide dismutase gene Mn-sod, which is obtainded from Thermoascus aurantiacus var. levisporus through the RT-PCR method, is cloned and inserted into pichia yeast integration expression vectorp PIC9K, and then the resulting manganese superoxide dismutase gene Mn-sod expression vector pPIC9K/Mn-sod is induced into pichia yeast GS115. After that, a yeast engineering bacteria MS18 expressing manganese superoxide dismutase is seleceted. The engineering bacteria is induced and cultured for six days, so the enzyme expression quantity is 0.92mg/mL, the enzyme activity is 2324 U/mL, and the molecular weight is 86.0 kDa. The engineering strain has stable activity at 50 DEG C and 60 DEG C, the enzyme activity remains 50% after heat preservation at 80 DEG C for 40 min and remains 20% after heat preservation at 90 DEG C for 60 min. The engineering strain of the invention has thermal stability, which can be used for production strain of thermostable manganese superoxide dismutase and has important economic value and social value.

Description

(1) Technical field: [0001] The invention relates to bioengineering, in particular to a Pichia pastoris engineering strain expressing the thermostable manganese superoxide dismutase (Mn-SOD) gene Mn-sod of Thermoascus aurantiacus var. levisporus Pichia pastoris MS18. (two) background technology: [0002] Superoxide dismutase (Superoxide Dismutase, referred to as SOD) (EC.1.15.1.1) is a kind of metalloenzyme, which widely exists in organisms. It can catalyze the disproportionation reaction of superoxide anions, thereby scavenging superoxide anions. It plays an important role in the process of organism defense against oxidative damage. At present, SOD is mainly used in the treatment of inflammation, tumor irradiation patients, autoimmune system diseases, and certain cardiovascular diseases. It is reported that SOD also plays an important role in anti-aging, anti-cell damage and prevention of carcinogenesis. In the treatment of renal cell carcinoma, Mn-SOD can effectively r...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N9/08C12R1/84
Inventor 李多川宋宁宁李安娜李华赵春青
Owner SHANDONG AGRICULTURAL UNIVERSITY
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