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Escherichia coli Nissle 1917 genetically engineered bacterium as well as preparation method and application thereof

A technology of genetically engineered bacteria and Escherichia coli, applied in the field of genetic engineering, can solve problems such as limiting physiological performance, and achieve the effects of increasing anti-inflammatory factors, reducing brain inflammation, and reducing inflammatory factors

Active Publication Date: 2021-09-21
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a site recognized by dipeptidyl peptidase IV (DPP-IV) in the amino acid sequence of GLP-1, which is easily degraded by DPP-IV in vivo, and its half-life is only a few minutes, which greatly limits its physiological efficacy; , the pain caused by repeated injections of GLP-1 deters many patients

Method used

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  • Escherichia coli Nissle 1917 genetically engineered bacterium as well as preparation method and application thereof
  • Escherichia coli Nissle 1917 genetically engineered bacterium as well as preparation method and application thereof
  • Escherichia coli Nissle 1917 genetically engineered bacterium as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: engineering bacteria E. coli Nissle 1917 acquisition and identification

[0020] 1, Nissle 1917 acquisition and identification of

[0021] Healthy human stool using 50% Glycerol, in sterile phosphate buffered saline (PBS) to make dilutions, 3-5 to select the appropriate gradients draw performed 0.1mL streaked on LB medium, each dilution gradient do two repetitions. After the petri dish was placed 37 ℃ incubator for 48 hours aerobically selection smooth, convex, regular edge, white colonies semipermeable microscopy. Suspected colonies were isolated and purified in LB medium again the above steps, until the colonies were completely purified. Purified colonies were serially passaged 3 times and then with 50% glycerol and bacterial suspension 1: 1 mixture stored at -80 ℃.

[0022] 2, building engineering bacteria E. coli Nissle 1917

[0023] Synthesis of GLP-1 target gene, the nucleotide sequence as shown in SEQ ID NO.2 by chemical synthesis, which is then integrated...

Embodiment 2

[0049] Example 2: Experiment acid bacterium Escherichia coli Nissle 1917 Engineering

[0050] Inoculated with 1 / 100 volume of E. coli Nissle 1917 and engineering bacteria, 18h subcultured in LB liquid medium. 8000rpm centrifuge 5min, discarded the supernatant retained precipitate was washed with sterile PBS buffer twice, respectively, each of which was dispensed into five 1.5mL centrifuge tube, centrifuged at 8000rpm 5min, wild type E. coli to give Niss le 1917 and Engineering 5 parts of each of the bacterial cell. Were then added to the pre-configured PBS buffer pH 7.0 and 2.0,3.0,4.0,5.0,6.0, stationary culture incubator at 37 ℃ 2h, 2h and cultured bacterial suspension taken 0h times over (after 10 fold) dilution, viable cell count was determined by plate count method, and calculates a wild type E. coli strain Nissle1917 survival and engineering.

[0051] Such as figure 2 As shown, in the acid-resistant experiment, in the PBS environment of pH = 4, the number of wild-type E. coli...

Embodiment 3

[0052] Example 3 Asphalt Stri Salt Experiment of Erobacter Nissle 1917

[0053] E. coli Nissle 1917 and the engineering bacteria were seeded at 1 / 100, and the biopsy was incubated in LB liquid medium for 18 h. 8000 rpm was centrifuged for 5 minutes, retaining the deposit, and then washed twice with a sterile PBS buffer, and each of them were dispensed into 5 1.5 ml of centrifuge tube, 8000 rpm centrifuge 5 min, obtained from wild-type E. coli Nissle 1917 and Engineering Five parts of the bacteria. Then, a pre-configured preferred pBS buffer is added to the PBS buffer of 0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, and placed in a 37 ° C incubator to stand for 2 h, and take 0 h after culture. 2H bacteria suspension ratio (10 times) dilution, the number of live bacteria was determined by a flat plate count, and the ventronium Nissle 1917 and the survival rate of Engineering bacterium strains were calculated.

[0054] Such as image 3 As shown, in the aspiral salt experiment, the engineering ...

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Abstract

The invention discloses an Escherichia coli Nissle 1917 genetically engineered bacterium as well as a preparation method and application thereof. The Escherichia coli Nissle 1917 genetically engineered bacterium with a function of improving sport injury is constructed by inserting a glucagon-like peptide gene sequence secreted by L cells from human intestinal tracts into a nucleophilic chromosome genome of the Escherichia coli Nissle 1917. The engineering probiotics can efficiently express GLP-1 in vitro, the defects that a plasmid overexpression system is unstable, drug resistance is prone to occurring and the like are overcome, meanwhile, the engineering probiotics have good acid resistance, cholate resistance and oxidation resistance, the motor injury of Parkinson mice can be obviously relieved, and neuroinflammation in the brain can be reduced. The Escherichia coli Nissle 1917 genetically engineered bacterium can be used for preparing food or drugs capable of improving sport injury, and has important practical significance and economic value in the aspect of Parkinson's disease treatment.

Description

Technical field [0001] The present invention belongs to the technical field of genetic engineering, an E. coli Nissle 1917 primarily relates to genetically engineered bacteria and its preparation method and application. Background technique [0002] Neurological disease and injury is a leading cause of disability worldwide, of which Parkinson's disease (PD) of the fastest growing in prevalence, disability and mortality in patients to family and society a heavy burden . Unfortunately, it has not yet found a reliable treatment can stop or reverse the progression of PD medication. Therefore, to find a safe, it can slow, stop or even reverse the neurodegenerative drugs protect neurons from damage, thereby treating such diseases is of great significance. [0003] As the host probiotics beneficial microbial activity, intestinal flora balance can be adjusted so as to enhance the integrity of the intestinal epithelium, intestinal barrier protection, adjust the mucosal immune system, inhi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A23L33/135A61K38/26A61K48/00A61P25/00A61P25/16C12R1/19
CPCC07K14/605A61K48/0008A61K48/005A61K38/26A61P25/16A61P25/00A23L33/135A23V2002/00A23V2200/30A23V2200/322Y02A50/30
Inventor 陈廷涛魏静吴恒
Owner NANCHANG UNIV
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