Engineering probiotic capable of degrading uric acid in intestinal tract, and preparation method and application of engineering probiotic

A kind of probiotics and engineering technology, applied in the field of probiotics preparation, can solve the problems of no practical application value, no expression efficiency of uric acid permeation enzyme gene, no discovery of natural probiotics to efficiently absorb exogenous uric acid, etc.

Active Publication Date: 2020-02-04
宁波倍益嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The uric acid permeating enzymes from different microbial sources have different molecular weights, but the transmembrane structure and functional regions have a high degree of similarity. The research results show that if the function of uric acid permeating enzymes is missing, the intake of uric acid will be significantly reduced (Diallinas and Scazzocchio, 1989)
In the genome sequencing of many probiotics, there are genes predicted to be uric acid permeating enzymes, but the inventors of this patent did not find the ability of natural probiotics to efficiently absorb exogenous uric acid during the uric acid reduction test. Although a few probiotics Can reduce exogenous uric acid, but the effect is very weak and has no practical application value (internal data, unpublished)
The possible reasons why natural probiotics do not have the ability to efficiently reduce uric acid are: (1) the uric acid permeation enzyme gene is not expressed or the expression efficiency is low in natural probiotics; (2) the predicted uric acid permeation enzyme has no uric acid permeation function
So far, no studies have applied uric acid permeating enzymes to the field of engineered probiotics for the treatment of hyperuricemia and gout

Method used

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  • Engineering probiotic capable of degrading uric acid in intestinal tract, and preparation method and application of engineering probiotic
  • Engineering probiotic capable of degrading uric acid in intestinal tract, and preparation method and application of engineering probiotic
  • Engineering probiotic capable of degrading uric acid in intestinal tract, and preparation method and application of engineering probiotic

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Obtaining of urate oxidase coding sequence

[0064] Bacillus subtilis urate oxidase (GenBank: WP_003242600.1), Candida utilis urate oxidase (GenBank: P78609.1), Aspergillus niger urate oxidase (GenBank: XP_001399739.1), Trichoderma reesei urate oxidase (GenBank: XP_006963697.1), Cordyceps militaris urate oxidase (GenBank: XP_006674629.1), and Kluyveromyces marxense urate oxidase (GenBank: XP_022674397.1) coding sequences were extracted from the genomic DNA of the corresponding species. The primers in Table 1 amplified the full-length sequence of urate oxidase, and after sequence comparison and analysis, the sequence was spliced ​​after manually removing introns from eukaryotic genes.

[0065] Aspergillus flavus urate oxidase (GenBank: XP_001826198.1), Arthrobacter globosa urate oxidase (GenBank: D0VWQ1.1), Microbacterium urate oxidase (GenBank: AEY68606.1), porcine urate oxidase (GenBank: NP_999435.1 ) sequence was synthesized by Wuhan Tianyi Huiyuan Biotech...

Embodiment 2

[0069] The construction of the Lactobacillus casei engineering bacterium of embodiment 2 recombinant expression uric acid permeation enzyme

[0070] 2.1pEBErm194-GFP-TFR vector construction

[0071] Using Lactobacillus casei ATCC393 genomic DNA as a template, using the primers TF-up-F / TF-up-R, TF-down-F / TF-down-R in Table 2, PCR amplification of the upstream homology arm TF-up and the downstream homology arm TF-down, and then use TF-up-F / TF-down-R as primers to amplify TF-up+down by overlapping PCR, and recover the PCR fragment with a DNA agarose gel recovery kit; The pEBErm194-GFP plasmid was used as a template, and the primers pEBErm194-F / pEBErm194-R in Table 2 were used to amplify a vector backbone fragment of about 5300 bp by PCR, digested with DpnI for 2 hours, and recovered the vector fragment with a DNA agarose gel recovery kit; The digested and recovered vector backbone and up+down overlapping fragments were assembled according to the method of the seamless cloning ki...

Embodiment 3

[0084] The construction of the Lactobacillus casei engineering bacterium of embodiment 3 recombinant expressing uric acid oxidase

[0085] 3.1 Construction of urate oxidase integration vector

[0086] Using the HCE promoter synthesized from the whole gene of the database sequence (GenBank: DJ022754.1) as a template, use the primers HCE-F / HCE-R in Table 4 to amplify the strong constitutive promoter HCE promoter (SEQ No.20) by PCR; Using the CU-UOX gene sequence as a template, use the primers CUUOX-F / CUUOX-R in Table 4 to amplify the CU-UOX gene sequence by PCR; use the rrnBT1T2 synthesized by the whole gene as a template, and use the primers rrnBT1T2-F / rrnBT1T2-R, PCR amplification terminator rrnBT1T2 (SEQNo.21). Then use these three fragments as templates, use primers HCE-F / rrnBT1T2-R, overlap PCR to amplify HCE promoter+CUUOX+rrnBT1T2, and use DNA agarose gel recovery kit to recover PCR fragments. Using the plasmid pEBErm194-GFP-TFR as a template, using the primers P-down-...

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Abstract

The present invention discloses an engineering probiotic capable of degrading uric acid in intestinal tract, a preparation method and an application of the engineering probiotic, and belongs to the technical field of biology. Uric acid permease and uric acid oxidase are respectively constructed into different intestinal probiotic strains, abilities of the intestinal probiotic strains to reduce uric acid are compared, and compared with original probiotics, the probiotic recombining and integrating the uric acid permease can significantly increase intake of exogenous uric acid and thus reduces extracellular uric acid concentration. The uric acid-lowering probiotic has significantly higher uric acid-lowering ability than the previously reported uric acid-lowering probiotics, besides, animal uric acid-lowering effect tests show that the engineering probiotic has the significant uric acid-lowering effect. As a new method to reduce blood uric acid and treat gout, the engineering probiotic has broad application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of probiotics recombinantly expressing enzymes related to uric acid degradation through genetic engineering. Background technique [0002] Hyperuricemia is a chronic metabolic disease, clinically manifested by blood uric acid levels higher than the normal range (male > 420 μmol / L, female > 360 μmol / L). In addition to gout caused by uric acid crystals, patients with hyperuricemia may also develop kidney disease, urinary tract stones, cardiovascular disorders, and cerebrovascular disorders. Under normal physiological conditions, the total amount of uric acid in the human body is about 1200 mg. There are two main ways to excrete uric acid, about 2 / 3 is excreted through the kidneys in the form of urine, and 1 / 3 is excreted in the form of intestines. More and more clinical research data show that the metabolic disorder of intestinal pathway of u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A23L33/135A61K35/741A61P19/06C12R1/245C12R1/125C12R1/01
CPCC12N9/0048C12N9/00C12Y107/03003A23L33/135A61K35/741A61P19/06A23V2002/00A23V2400/231A23V2400/125A23V2200/3204
Inventor 刘艳红刘海峰黄荷李青山
Owner 宁波倍益嘉生物科技有限公司
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