Escherichia coli Nissel 1917 genetically engineered bacterium with hypoglycemic effect, and preparation method and application of escherichia coli Nissel 1917 genetically engineered bacterium

A technology of genetically engineered bacteria and Escherichia coli, applied in the field of genetic engineering, can solve problems such as limiting physiological efficacy

Pending Publication Date: 2021-09-14
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 Nissel 1917 genetically engineered bacterium with hypoglycemic effect, and preparation method and application of escherichia coli Nissel 1917 genetically engineered bacterium
  • Escherichia coli Nissel 1917 genetically engineered bacterium with hypoglycemic effect, and preparation method and application of escherichia coli Nissel 1917 genetically engineered bacterium
  • Escherichia coli Nissel 1917 genetically engineered bacterium with hypoglycemic effect, and preparation method and application of escherichia coli Nissel 1917 genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Acquisition and identification of Escherichia coli Nissle 1917 engineering bacteria

[0021] 1. Acquisition and identification of Nissle 1917

[0022] The feces of healthy human beings are stored in 50% glycerol, and diluted with sterile phosphate buffered saline (PBS). Do two repetitions. Put the petri dish into a 37°C incubator for aerobic culture for 48 hours, and then select smooth, convex, neat edges, and semipermeable white colonies for microscopic examination. The suspected colonies were isolated and purified again in LB medium according to the above steps until the colonies were completely purified. The purified colony was subcultured for 3 times, and then stored at -80°C with a 1:1 mixture of 50% glycerol and bacterial solution.

[0023] 2. Construction of Escherichia coli Nissle 1917 engineering bacteria

[0024] The GLP-1 target gene was synthesized by chemical synthesis, and the nucleotide sequence was shown in SEQ ID NO.2, and then integra...

Embodiment 2

[0051] Embodiment 2: the acid resistance test of Escherichia coli Nissle 1917 engineering bacteria

[0052]Escherichia coli Nissle 1917 and engineering bacteria were inoculated at a volume of 1 / 100, and subcultured in LB liquid medium for 18 hours. Centrifuge at 8000rpm for 5min, retain the precipitate and discard the supernatant, wash twice with sterile PBS buffer, divide each into five 1.5mL centrifuge tubes, and centrifuge at 8000rpm for 5min to obtain wild-type Escherichia coli Nissle 1917 and engineering 5 copies of each bacterial cell. Then add pre-configured PBS buffer solutions with pHs of 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 respectively, place them in a 37°C incubator and cultivate them for 2 hours. times) dilution, the number of viable bacteria was determined by plate counting method, and the survival rate of wild-type E. coli Nissle 1917 and engineering bacterial strains was calculated.

[0053] The result is as figure 2 As shown, in the acid resistance experiment, ...

Embodiment 3

[0054] Example 3 Escherichia coli Nissle 1917 Engineering Bacteria Bile Salt Resistance Experiment

[0055] Escherichia coli Nissle 1917 and engineering bacteria were inoculated at a volume of 1 / 100, and subcultured in LB liquid medium for 18 hours. Centrifuge at 8000rpm for 5min, save the precipitate and discard the supernatant, wash twice with sterile PBS buffer, divide each into five 1.5mL centrifuge tubes, centrifuge at 8000rpm for 5min, and obtain wild-type E. coli Nissle 1917 and engineered bacteria 5 copies of bacteria. Then add pre-configured PBS buffer solutions with bile salt concentrations of 0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, respectively, place them in a 37°C incubator for 2h, and take 0h and After 2h, the bacterial suspension was diluted in multiple ratios (10 times), and the number of viable bacteria was determined by plate counting, and the survival rate of wild-type E. coli Nissle 1917 and engineered strains was calculated.

[0056] The result is as imag...

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Abstract

The invention provides an escherichia coli Nissel 1917 genetically engineered bacterium with a hypoglycemic effect, and a preparation method and application of the escherichia coli Nissel 1917 genetically engineered bacterium. According to the invention, a glucagon-like peptide gene sequence from a human body is inserted into a genome of Escherichia coli Nissle 1917 to construct an Escherichia coli Nissle 1917 genetically engineered bacterium with a hypoglycemic function. 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; pancreatitis can be obviously reduced; and blood sugar and sugar resistance can be improved. The Escherichia coli Nissle 1917 genetically engineered bacterium disclosed by the invention can be used for preparing food or medicines with a hypoglycemic function, and has important practical significance and economic value in the aspect of human body type 2 diabetes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and mainly relates to an Escherichia coli Nissle 1917 genetic engineering bacterium with hypoglycemic effect, a preparation method and application thereof. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. In recent years, the global prevalence of diabetes has been gradually increasing. In 2019, the number of patients in China has reached 116.4 million, of which about 90% of patients are type 2 diabetes (T2DM), becoming the country with the largest number of diabetes patients in the world. At present, although the therapeutic drugs for T2DM such as biguanides, glinides, sulfonylureas, α-glucosidase inhibitors and thiazolidinediones have certain therapeutic effects, they are prone to cause hypoglycemia and gastrointestinal discomfort and other side effects. Glucagon-like peptide-1 (GLP-1) analogues, as a new class of T2DM treatmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70A23L33/135A61K35/741A61K38/26A61P3/10A61P1/18A61P29/00C12R1/19
CPCC07K14/605C12N15/70A23L33/135A61K35/741A61K38/26A61P3/10A61P1/18A61P29/00A23V2002/00A23V2200/328
Inventor 陈廷涛魏静胡鸿
Owner NANCHANG UNIV
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