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Oxalate oxidase with vitality under physiological PH condition and application thereof

A technology of oxalate oxidase and oxalic acid, applied in the field of genetic engineering, can solve the problem of no oxalate oxidase activity

Active Publication Date: 2015-06-03
WUHAN KANGFUDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventor of this patent has extracted the oxalate oxidase in barley of the same species and found that it has no oxalate oxidase activity at pH7.4

Method used

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  • Oxalate oxidase with vitality under physiological PH condition and application thereof
  • Oxalate oxidase with vitality under physiological PH condition and application thereof
  • Oxalate oxidase with vitality under physiological PH condition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] [Example 1] Screening and Vitality Test of Plants Producing Oxalate Oxidase

[0041] The inventor of this patent collected more than a thousand plant samples from Guangxi Medicinal Botanical Garden, Kunming Botanical Garden, Wuhan Botanical Garden and Hainan Subtropical Botanical Garden, and carried out the screening experiment of oxalate oxidase.

[0042] When oxalate oxidase-producing plants are screened, the samples are processed first, and the processing process is as follows: cut a certain amount of plant samples (leaves, roots, stems or flowers) with scissors, put them in a quantitative beaker filled with pure water, and use The high-speed homogenizer is homogenized into a suspension (particles <100 μm), and the homogenate is divided into a soluble part (supernatant) and an insoluble part (precipitation) after centrifugation, and the insoluble part is resuspended with pure water. They were used to measure the activity of degrading oxalic acid.

[0043] There are ...

Embodiment 2

[0048] [Example 2] Cloning of Banana and Beet Oxalate Oxidase Gene

[0049] (1) Extract crude oxalate oxidase from sugar beet stem and banana peel samples

[0050] Weigh a certain amount of sugar beet stem and banana peel samples, chop them and put them into a beaker filled with pure water, homogenize them into particles less than 100 μm through a high-speed homogenizer, and centrifuge to remove the supernatant (no activity or very low Oxalate oxidase activity), the pellet was resuspended in a buffer solution (2mM PBS, pH7.0) containing surfactant (1% sodium sulfodeoxycholate) and sucrose (50% w / v), and extracted at 4°C After 2 to 5 days, the crude extract of oxalate oxidase is purified by Q-sepharose chromatographic column, and the eluted sample peak is collected, and then the surfactant and salt components in the sample are removed by dialysis. The oxalate oxidase sample still has oxalate oxidase activity after separation by Native-PAGE, and the elution peak and oxalate oxi...

Embodiment 3

[0061] [Example 3] Escherichia coli system expresses recombinant oxalate oxidase

[0062] (1) Construction of plasmid

[0063] The oxalate oxidase (OxOx) B5100, B5102, B5601, M30640 gene clones were constructed into the pAT vector (the vector was derived from the pET-32a vector), and then the recombinant plasmid was transferred into E.coli Origami B competent cells.

[0064] (2) Shake flask small amount expression

[0065] Spread the glycerol strains of B5100, B5102, B5601, and M30640 on LB agarose plates (containing 100 μg / ml ampicillin antibiotic), and culture them upside down at 37°C overnight until monoclonal plaques appear. Pick a single colony and inoculate in 200ml shake flask medium (containing 50ml LB medium, 100μg / ml ampicillin antibiotic), 220rpm, 37°C culture, OD 600 to 0.6~0.8, add MnCl 2 The final concentration was 1mM, and the final concentration of IPTG was 0.6mM to induce expression. After induction for 5 hours, the bacteria were collected by centrifugation...

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Abstract

The invention discloses an oxalate oxidase with vitality under physiological PH condition with the gene sequence shown in SEQ ID NO.1-4 and the amino acid sequence shown in SEQ ID NO.5-8, and an application thereof, and particularly discloses an application in preparation of medicine for reducing the oxalic acid concentration of the oxalosis patients by using the oxalate oxidase, and belongs to the technical field of gene engineering. Because the oxalate oxidase has vitality when the physiological PH is 7.4, the medicine containing the oxalate oxidase can be prepared into injection or embedded by an enteric-coating material. The vitality of the oxalate oxidase with the PH of 7.4 is not lower than 20% of the maximum specific activity, and the oxalate oxidase with vitality under the PH of 7.4(PH7.0-8.0) is so far uniquely disclosed and verified. The found of the oxalate oxidase provides possibility for researching the recombinant oxalate oxidase medicine to treat the oxalosis. The medicine for preventing the oxalosis can be prepared by using the oxalate oxidase.

Description

technical field [0001] The present invention relates to a group of active oxalate oxidase genes and their coded proteins and applications under physiological pH conditions, in particular to the use of the oxalate oxidase in the preparation of drugs for reducing the concentration of oxalic acid in the blood or urine of patients with hyperoxalate disease The application in medicine belongs to the technical field of genetic engineering. Background technique [0002] Urinary stone disease is a major health disease worldwide, and most urinary stones are composed of calcium oxalate or calcium oxalate and calcium phosphate. Many diseases are related to the excessive amount of oxalic acid in the human body, such as primary hyperoxaluria, second hyperoxaluria, autism, vulvar pain, advanced kidney stone disease caused by excessive oxalic acid, cardiac conduction disorder, Rohn's disease, inflammatory bowel disease, colitis, urinary stones, asthma, chronic obstructive pulmonary diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53A61K38/44A61P3/00A61P13/02
CPCA61K38/00C12N9/0008C12N15/8257C12Y102/03004
Inventor 王怡汪小锋刘海峰汪卫
Owner WUHAN KANGFUDE BIOTECH CO LTD
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