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Functional gene segment for reducing blood uric acid level, recombinant strain and application

A uric acid level and functional gene technology, applied in gene therapy, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problem that recombinant bacteria can not reduce blood uric acid level, etc., to broaden the application value and enhance the activity of urate oxidase Effect

Pending Publication Date: 2022-05-13
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of modified intestinal probiotics to reduce uric acid levels. A patent (CN108103080) has constructed a nucleic acid sequence and expression vector for recombinant urate oxidase protein, which mainly realizes high-level expression and purification in Escherichia coli Uric acid oxidase, but can not be directly applied to reduce blood uric acid level of recombinant bacteria

Method used

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  • Functional gene segment for reducing blood uric acid level, recombinant strain and application
  • Functional gene segment for reducing blood uric acid level, recombinant strain and application
  • Functional gene segment for reducing blood uric acid level, recombinant strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Activity Test of Uric Acid Oxidase Expressed by Plasmids in Different Locations

[0032] In this example, we cloned and optimized uric acid oxidase from three different sources, and provided their sequence characteristics, UA (SEQ.ID.NO:002) from Candida, and UaZ (SEQ.ID.NO:002) from Aspergillus niger. .NO:003), Uox from Arthrobacter globosa (SEQ.ID.NO:004).

[0033] First, a plasmid expressing Candida-derived UA was introduced into the intestinal probiotic Escherichia coli EcN for in vitro uric acid degradation test, and it was found that the intracellular expression of urate oxidase in the strain did not show strong enzymatic activity ( figure 1 ), this phenomenon may be limited by the amount of uric acid entering the cell, so we tried to give UA different secretion signal peptides, trying to secrete and express urate oxidase to improve its enzyme activity.

[0034] In this example, the UA expression plasmid was constructed using pKT100 (Hu, Y., et al. (20...

Embodiment 2

[0046] Example 2: Different Urate Oxidase Activity Tests Expressed by Plasmids under FtsP Signal Peptide Positioning

[0047] In this example, in order to test whether the signal peptide FtsP is universal in enhancing the function of uric acid oxidase, we introduced UaZ from Aspergillus niger and Uox sequence from Arthrobacter globosa into the signal peptide, and cloned it into The transformed pKT100 plasmid was screened for positive plasmids and transformed into EcN to obtain recombinant strains. Different recombinant strains were cultured overnight at 37°C in LB kana-resistant liquid (50 μg / ml), transferred to 3ml of fresh LB-resistant medium at a ratio of 1:100, cultured at 37°C until OD600~0.6, and added 0.3mM IPTG , induced expression for 3 hours. Centrifuge at room temperature for 3 minutes at 5000rpm to collect the bacteria, resuspend the bacteria with 6ml MU medium, take samples every 1 hour, and monitor the change of uric acid level in the solution by measuring the c...

Embodiment 3

[0061] Example 3: Enzyme activity test of EcN C6 degrading uric acid in vitro

[0062] In this example, the recombinant strain EcN C6 (deposit number: CCTCC NO. M20211517) was obtained by integrating the UA-encoding gene derived from Candida into the HIS-1 site of the Escherichia coli EcN genome.

[0063] The specific operation process is as follows:

[0064]1. Construct the EcN strain carrying the temperature-sensitive pKD46 plasmid (Amp). The pKD46 plasmid (Datsenko, K.A. and B.L.Wanner (2000). "One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products." ProcNatlAcadSci US A 97 (12): 6640-6645.) was electroporated by electrotransfer Cultivate in EcN at 30°C to obtain an EcN strain carrying Amp resistance.

[0065] 2. Construction of pDM4-UAm. With pDM4 suicide plasmid (Milton, D.L., R.O'Toole, P.Horstedt and H.Wolf-Watz (1996). "Flagellin A is essential for the virulence of Vibrio anguillarum." J Bacteriol 178(5):1310-1319. ) starting vector t...

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Abstract

The invention discloses a functional gene segment for reducing the blood uric acid level, a recombinant strain and application. The gene segment is composed of an FtsP signal peptide sequence and a urate oxidase coding gene located behind the FtsP signal peptide sequence. The activity of urate oxidase can be effectively enhanced, so that the urate oxidase has an efficient degradation function. On the basis of the functional gene, the functional gene is introduced into an escherichia coli EcN genome for expression, an engineering strain EcN C6 capable of stably expressing urate oxidase is constructed and obtained, in-vivo and in-vitro models verify that the engineering strain EcN C6 has the capability of efficiently reducing uric acid, a new thought is provided for treating hyperuricemia from the perspective of intestinal microecology, and the engineering strain EcN C6 has a wide application prospect. And the application value of the probiotic escherichia coli EcN in metabolic diseases is widened.

Description

technical field [0001] The invention relates to the field of microbial synthetic biology, in particular to a functional gene segment for reducing blood uric acid level, recombinant bacterial strain and application. Background technique [0002] Uric acid is a key product in the purine metabolic pathway, and the pathway from purine degradation to uric acid production is highly conserved in the biological world. Most mammals express urate oxidase in the liver, which can further hydrolyze uric acid into allantoin, which is more soluble and easier to excrete. However, the gene encoding uric acid oxidase in higher primates has been inactivated during the long-term evolution process, so the concentration of uric acid in higher primates is much higher than that of other mammals. [0003] Hyperuricemia is a disorder of purine metabolism in the body, excessive production of uric acid or renal excretion dysfunction, leading to metabolic diseases in which uric acid accumulates in the ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N9/06C12N1/21C12N15/70A61K48/00A61K38/44A61P19/06C12R1/19
CPCC12N9/0048C12Y107/03003C07K14/00C12N15/70C12N1/20A61K48/005A61K38/44A61P19/06C07K2319/02Y02A50/30
Inventor 胡杨波何丽娜陈士云唐薇
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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