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Gene engineering bacteria high-efficiently expressing recombined human glucagon-like peptide-1 (1-37) and construction method and application thereof

A technology of glucagon and genetically engineered bacteria, applied in the direction of microorganism-based methods, medical preparations containing active ingredients, recombinant DNA technology, etc., can solve the problems of high synthesis cost, expensive price, and difficult purification, and achieve production The effect of low cost, low cost, and simple purification steps

Inactive Publication Date: 2012-09-12
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, to study the function of recombinant human glucagon-like peptide-1 (1-37), it is necessary to synthesize recombinant human glucagon-like peptide-1 (1-37). Synthetic method, which has defects such as difficulty, high synthesis cost, and difficult purification
Products prepared by existing methods are expensive

Method used

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  • Gene engineering bacteria high-efficiently expressing recombined human glucagon-like peptide-1 (1-37) and construction method and application thereof
  • Gene engineering bacteria high-efficiently expressing recombined human glucagon-like peptide-1 (1-37) and construction method and application thereof
  • Gene engineering bacteria high-efficiently expressing recombined human glucagon-like peptide-1 (1-37) and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of a genetically engineered bacterium that highly expresses human glucagon-like peptide-1 (1-37)

[0049] Step 1 Amplification of GLP-1(1-37) DNA sequence

[0050] Entrust a professional biotechnology company to chemically synthesize the following base sequences: P1—upstream primer, introduce GLP-1 (1-6) gene sequence, P2—upstream primer, introduce Bgl II restriction site and enterokinase EK site, P3-downstream primer, introduced Hind III restriction site and stop codon TAA.

[0051] P1 (39bp):

[0052] 5'- CATGATGAATTTGAACGC CATGCCGAAGGCACCTTTACC-3'

[0053] GLP-1(1-6) gene sequence

[0054] P2 (43bp):

[0055] 5'-GG AGATCT G GACGACGACGACAAG CATGATGAATTTGAACGCC-3'

[0056] Bgl II restriction site Enterokinase EK site

[0057] P3 (32bp):

[0058] 5'-GG AAGCTT G TTA GCCTCTGCCTTTCACCAGCC-3'

[0059] Hind III restriction site stop codon

[0060] Using the plasmid pET32a(+)-GLP-1(7-37) as a template, adding primers P1 ...

Embodiment 2

[0068] Example 2 Production of human glucagon-like peptide-1 (1-37) by a genetically engineered bacterium that highly expresses human glucagon-like peptide-1 (1-37)

[0069] The affinity chromatography medium described below in this example is NTA-0 resin, purchased from Novagen Company, and the following enterokinase EK, purchased from NEB Company.

[0070] The first step liquid culture

[0071] The genetically engineered bacteria constructed in Example 1 were inoculated into 20 mL of LB medium containing 100 μg / mL Amp, cultivated overnight at 37°C and 210 r / min, and the bacterial liquid was inoculated into 200 mL of LB medium containing 100 μg / mL LB culture medium of mL Amp, cultivated until the OD600 of the bacterial liquid was 0.6-0.8, and took 1 mL of the bacterial liquid as a reserved sample before induction (see attached figure 2 2), add IPTG with a final concentration of 0.6-1.0mM to the remaining bacterial solution for induction for 4 hours, and take a 1mL sample of...

Embodiment 3

[0077] Example 3 Hypoglycemic effect of intraperitoneal administration of human glucagon-like peptide-1 (1-37)

[0078] Experimental materials and methods:

[0079] Female healthy Kunming mice (clean grade, Shanghai Slack Experimental Animal Co., Ltd.);

[0080] 40% glucose solution, 0.9% NaCl solution, human glucagon-like peptide-1 (1-37);

[0081] Blood glucose tester, blood glucose test strip (Shanghai Xinli Medical Instrument Co., Ltd.);

[0082] Disposable 1mL sterile syringe (Shanghai Kindly Enterprise Development Group Co., Ltd.);

[0083] Female healthy Kunming mice were fasted for 16 h and divided into 4 groups (n=8). 1, normal saline control group; 2-4, human glucagon-like peptide-1 (1-37) administration group, the administration doses were 6 nmol / kg, 12 nmol / kg, 24 nmol / kg respectively. The administration group was injected with 250 μL of 40% glucose solution and different doses of human glucagon-like peptide-1 (1-37), and this time was recorded as time zero. A...

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Abstract

The invention discloses gene engineering bacteria high-efficiently expressing recombined human glucagon-like peptide-1 (1-37). The gene engineering bacteria is Escherichia coli DH5alpha and BL21(DE3) carrying recombined plasmid, wherein the recombined plasmid is the plasmid pET32a (+) containing human glucagon-like peptide-1 (1-37). The invention also discloses a construction method of the gene engineering bacteria, which is characterized in that the pET32a (+) - GLP -1 (1-37) is adopted as a template, a primer is designed according to an alkaline base of the GLP-1 (1-37) gene, the gene GLP-1 (1-37) is obtained through the polymerase chain reaction (PCR) augmentation, through the enzyme digestion, the gene GLP-1 (1-37) is inserted into the plasmid pET32a(+), the recombined plasmid pET32a (+)-GLP-1(1-37) is constructed and is converted into Escherichia coli. The recombined human glucagon-like peptide-1 (1-37) prepared by the method has advantages of low cost, high expression index, simple and convenient purification step, easiness in operation and high yield.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for highly expressing recombinant human glucagon-like peptide-1 (1-37) and its construction method and application, belonging to the technical field of bioengineering. Background technique [0002] The truncated GLP-1 (7-37) is the product of proglucagon translation and processing, and is secreted by small intestinal L cells. It can not only stimulate insulin secretion depending on glucose, but also inhibit glucagon secretion to reduce meal In addition, GLP-1 (7-37) can also inhibit gastric motility and reduce appetite. These functions of GLP-1(7-37) make it an effective drug for the treatment of type 2 diabetes. Proglucagon is processed in the small intestine to produce GLP-1 (1-37), and then under the action of specific proteases, the upstream 6 amino acid residues are excised to produce GLP-1 (7-37). At present, most studies focus on the role of truncated GLP-1(7-37), and the function of ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P21/02A61K38/26A61P3/10C12R1/19
Inventor 赵丽芬吴自荣黄静李冬青劳勋
Owner EAST CHINA NORMAL UNIVERSITY
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