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Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof

A technology of Hansenula and expression vector is applied in the field of recombinant polytype Hansenula and its special recombinant expression vector to achieve the effect of high-efficiency expression

Inactive Publication Date: 2008-09-03
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the expression of urate oxidase in Hansenula at home and abroad

Method used

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  • Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof
  • Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof
  • Recombinant polymorphism hansenula as well as special recombinant expression vectors and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, construct the recombinant multitype Hansenula polymorpha (Hansenula polymorpha) that produces uric acid oxidase

[0041] 1. Construction of recombinant expression vector pMOXZ-alpha-UOXu containing urate oxidase coding sequence

[0042] 1. PCR amplification of the MOX promoter sequence of methanol oxidase gene

[0043] According to the nucleotide sequence of the MOX1 promoter reported in the literature (GenBank Access No.A11156, the sequence shown by the deoxyribonucleotides at positions 1-1510 from the 5' end), design primers:

[0044] MOXp-L: 5'-TCA AGATCT TCGACGCGGAGAACGATCT-3'

[0045] MOXp-R: 5'-CCG AAGCTT TGTTTTTGTACTTTTAGATTGATG-3'

[0046]The underlined parts are BglII and HindIII recognition sites, respectively. The PCR reaction uses MOXp-L and MOXp-R as primers, and the genomic DNA of Hansenula polymorpha CGMCC 2.2498 as a template, 25 μl Pfu Mix in 50 μl PCR reaction mixture, 2 μL each of 10 μmol / l primers, and 0.3 μg / μl template DNA 10μ...

Embodiment 2

[0070] Example 2. Optimization of conditions for producing urate oxidase by recombinant Hansenula polymorpha HPMU6 CGMCC 2351

[0071] In shake flasks, single-factor fermentation experiments and multi-factor orthogonal experiments were used to optimize the conditions for the production of urate oxidase by recombinant Hansenula polymorpha HPMU6CGMCC 2351. Determining the optimum condition is: with YPG (2g / 100ml peptone, 1g / 100ml yeast powder, 1ml / 100ml glycerin) as medium, medium pH is 7.0; Cell wet weight is 6 grams of wet cells / litre when adding methanol (final The concentration is 1ml / 100ml) to induce the production of uricase, and methanol is added every 12 hours to a final concentration of 1ml / 100ml; 37°C, 200rpm induction culture for 72 hours. Under this optimal condition, the experiment was repeated 3 times, and 3 shake flasks were inoculated each time. Each shake flask (200 ml) was equipped with 20 ml of medium, and the fermentation culture was carried out for 72 hours,...

Embodiment 3

[0076] Example 3, High-density fermentation of recombinant Hansenula polymorpha HPMU6 CGMCC 2351 producing urate oxidase

[0077] Recombinant Hansenula polymorpha HPMU6 CGMCC 2351 was cultured in YPG (2g / 100ml peptone, 1g / 100ml yeast powder, 1ml / 100ml glycerol) medium at 37°C for 18 hours, and transferred to 400ml YPG at 10% inoculum size, 37 Cultivate on shaker to OD 600 is 10-12, the bacterium solution is the seed solution. The seed solution was placed in a fermenter equipped with 2L YPG with a glycerol concentration of 4% (v / v), cultured at 37°C, pH was controlled at 6.5, and dissolved oxygen was 20%. When glycerol will be consumed (after culturing for 24 hours), add glycerol at a rate of 20ml / l / h for 3 hours to make the cell dry weight reach 50g / L fermentation broth. Then feed methanol to keep the concentration of methanol in the fermentation broth at about 0.5%, and take samples every 12 hours to measure the cell biomass (cell dry weight) and urate oxidase activity. Th...

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Abstract

The invention discloses a recombinant multi shape hansenula yeast, specific recombinant carrier and application thereof. The disclosed recombinant multi shape hansenula yeast is recombinant strain capable of secreting expressive uricoxidase obtained by introducing uricoxidase gene of candida producing prion into multi shape hansenula yeast CGMCC2.2498; encoding gene of the uricoxidase is inserted between MOX promoter and AOX1 terminator in pMOXZ-alpha-A to obtain recombinant expression vector, then uricoxidase expression cassette is integrated onto chromosome of multi shape hansenula yeast by transformation. The recombinant multi shape hansenula yeast can produce uricoxidase on high level by high density fermentation, wherein yield of uricoxidase is 8.5 times of reported yield. Uricoxidase produced by constructed recombinant multi shape hansenula in the invention is high in yield, low in immunogenicity, highly benefit for wide application of uricoxidase in the medical and pharmaceutical field.

Description

technical field [0001] The invention relates to a recombinant multitype Hansenula and its special recombinant expression vector and application. Background technique [0002] Urate oxidase (Urateoxidase, Uricase, EC.1.7.3.3) is an enzyme in the purine degradation metabolic pathway in organisms, which catalyzes the oxidation of uric acid to allantoin and hydrogen peroxide. Excreta of purine metabolism in other mammals. However, there is no urate oxidase in higher mammals (apes and humans), and uric acid is used as the end product of purine metabolism. The solubility of uric acid and its salts in the blood is very low. If the purine metabolism in the body is disordered, excessive uric acid is produced, or the excretion of uric acid is blocked, the concentration of uric acid in the blood increases, forming hyperuricemia, and long-term excessive uric acid will cause gout. Uric acid oxidase is an important enzyme used in medicine. Clinically, it is widely used in the detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/53C12N9/04C12N1/19C12R1/78
Inventor 何秀萍陈志禹王肇悦郭雪娜张博润
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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