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A kind of glp-1 mutant and its preparation method and use

A GLP-1 and mutant technology, applied in the field of genetic engineering, can solve the problems of patients' physical and psychological pain, poor medication compliance, inconvenient use and carrying, etc., and achieve good clinical application prospects, reduce degradation, and avoid pain Effect

Active Publication Date: 2020-03-10
福州市台江区希吉亚投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, GLP-1-like peptide peptide drugs, including liraglutide, all need to be administered by injection, and direct oral administration is ineffective, and there are disadvantages such as inconvenience in medication
However, the treatment of diabetes usually requires long-term, continuous medication
The way of injection is inconvenient to use and carry, the drug compliance is poor, and there may be risks such as irritation and allergic reactions, which bring huge physical and psychological pain to patients

Method used

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  • A kind of glp-1 mutant and its preparation method and use
  • A kind of glp-1 mutant and its preparation method and use
  • A kind of glp-1 mutant and its preparation method and use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 Screening and activity detection of mutant sequences of the present invention

[0030] Starting from the original sequence of GLP-1, 4 mutant polypeptide sequences were modified and designed, and through chemical synthesis (Zhongpeptide Biochemical), a polypeptide product with a purity of up to 85% was obtained. The peptide sequence is as follows:

[0031] GLP-1 Fragment (7-37aa): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0032] GM (SEQ ID NO: 1):

[0033] HHEGTFTSDVSSYLEGQAAKKFIAWLVRGGKKKKKYGRKKRRQRRREF

[0034] C33: HACGFTTSDVSSYLEGQAAKEFIAWLCKGRG

[0035] K34: HAEGTFTSDVSSYLEGQAAKEFIAWLVKKKK

[0036] R34: HAEGTFTSDVSSYLEGQAAKEFIAWLVRRRR

[0037] GM is based on the original sequence of GLP-1, mutating the 8th alanine of GLP-1 to histidine, the 27th glutamic acid to lysine, and the 34th lysine to arginine acid, arginine at position 36 is mutated to glycine, glycine at position 37 is mutated to lysine, and a sequence is added at the end.

[0038] C33 is base...

Embodiment 2

[0050] Embodiment 2 Recombinant Escherichia coli expression and activity verification of mutants of the present invention

[0051] 1. Escherichia coli expression of the GLP-1 mutant of the present invention

[0052](1) Add a signal peptide sequence before the sequence of GLP-1 mutant GM, add HIV membrane-penetrating peptide sequence and 6 His-tag sequences at the 3'-end, and construct a protein that can be secreted and expressed and has the ability to pass through the cell membrane The nucleotide sequence of the GLP-1 mutant is shown in SEQ ID NO:2.

[0053] SEQ ID NO: 2:

[0054] atgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatcccaccacgagggcaccttcacctccgacgtgtcctcctacctggagggccaggccgccaagaagttcatcgcctggctggtgcgcggcggcaagaagaagaagaagtacggccgcaagaagcgccgccagcgccgccgcctcgaggacgacgacgacaagcaccatcaccatcaccattaa

[0055] (2) Cloning the nucleotide sequence shown in SEQ ID NO: 2 into the BamHI and SalI sites of the Escherichia coli expression vector pGEX t...

Embodiment 3

[0060] Embodiment 3 Recombinant lactic acid bacteria expression and activity verification of mutants of the present invention

[0061] 1. Lactic acid bacteria expression of the GLP-1 mutant of the present invention

[0062] (1) Cloning GLP-1GM, namely the nucleotide sequence shown in SEQ ID NO: 2, into the SalI and HindIII sites of the lactic acid bacteria expression vector pMG36e plasmid, and picking a single colony of the recombinant lactobacillus after electrotransformation of the lactic acid bacteria After culturing, the plasmid is extracted, and the GLP-1GM gene is detected in the recipient bacterium by PCR inspection and sequencing.

[0063] (2) Inoculate the above-mentioned recombinant lactic acid bacteria on the MRS medium supplemented with erythromycin at a final concentration of 20 μg / ml, and culture statically at 30°C until OD 600 When the value reaches 0.8-1.0, centrifuge, discard the supernatant, take the bacteria, and set aside.

[0064] 2. Activity verificatio...

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Abstract

The invention discloses a GLP-1 mutant. The invention also discloses a nucleotide sequence encoding the GLP-1 mutant, and a recombinant vector and recombinant bacteria containing the nucleotide sequence. The invention further discloses a preparation method and use of the GLP-1 mutant. The GLP-1 mutant can effectively reduce blood sugar concentration, can be used for treating type II diabetes and has good clinical application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a GLP-1 mutant and its preparation method and application. Background technique [0002] Diabetes is a worldwide epidemic. According to the World Health Organization, there will be 366 million people with diabetes worldwide in 2030. And China has become the world's largest diabetes country, and with the increase of the aging population in our country, the incidence of diabetes will also show a trend of increasing year by year. As a chronic multiple disease, diabetes has become a major public health problem of global concern. [0003] The current treatment method for diabetes is to control blood sugar to a standard level by applying a variety of oral drugs or insulin on the basis of diet and exercise control. However, traditional treatment methods have limitations such as the risk of hypoglycemia and weight gain. In recent years, the trend in the treatme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/605C12N15/16C12N15/70C12N15/74C12N1/21A61K38/13A61P3/10A61P3/04
CPCA61K38/00A61P3/04A61P3/10C07K14/605C12N15/70C12N15/74
Inventor 马永平顾建文马婕
Owner 福州市台江区希吉亚投资有限公司