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Bioactivity polypeptide and application thereof to preparation of alpha-glucosidases inhibitor

A bioactive peptide, glucosidase technology, applied in the fields of peptides, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as side effects and metabolic disorders in patients

Active Publication Date: 2019-05-07
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that although acarbose and miglitol can be used to prevent abnormal glucose tolerance from developing into type II diabetes, they also have certain side effects, such as causing metabolic disorders in patients, etc.

Method used

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  • Bioactivity polypeptide and application thereof to preparation of alpha-glucosidases inhibitor
  • Bioactivity polypeptide and application thereof to preparation of alpha-glucosidases inhibitor
  • Bioactivity polypeptide and application thereof to preparation of alpha-glucosidases inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1 Yeast two-hybrid method to obtain positive clones and self-activation verification

[0013] Yeast two-hybrid experiments were performed using the MATCHMAKER GAL Two-Hybrid System 3 system from Clontech. The specific steps are: 1) Transform the bait plasmid (BD) containing the glucoamylase domain in human intestinal α-glucosidase into the yeast strain AH109, ​​and prepare it into a competent state by using the PEG / LiAc method; 2) Take 10 μg of the plasmid DNA of the random eicosopeptide library was transformed into the competence of the previous step; 3) Finally, the competent cells were spread on the growth-deficient medium and cultured at 30°C until the positive clones grew; 4) The positive clones Carry out small-scale expansion culture, extract the plasmid, transfer it into AH109, ​​and spread it on a suitable defective medium for repeated experiments and self-activation verification; 5) Send the positive plasmid without self-activation to the sequencing com...

Embodiment 2

[0015] Example 2 Chemical Peptide Synthesis of Candidate Peptide Sequences

[0016] Peptides were synthesized on a CEM Liberty microwave-assisted automatic peptide synthesizer using the Fmoc protection strategy, with a microwave power of 35 W and a maximum reaction temperature of 75 °C. Rink Amide NovaPEG resin was used, 20% piperidine / DMF solution was used as deprotecting agent, peptide bond was formed by HBTU coupling, and reactants were fed according to 4 times the molar number of resin loading. After the construction of the polypeptide chain is completed, 2 g of peptide resin is obtained. The peptide resin was cleaved, TFA was removed under reduced pressure, and the crude peptide was precipitated. The crude peptide was then purified by RP-HPLC ( figure 1 RP-HPLC purification profile, acetonitrile concentration is eluted from 5%-30%), MALDI-TOF-MS analysis of the target product (see figure 2 ), freeze-drying and other steps, and finally obtain high-purity polypeptide fr...

Embodiment 3

[0017] Example 3 In vitro enzyme activity test to detect the α-glucosidase inhibitory activity of the polypeptide

[0018] Add 112 μL of 0.1 mol / L PBS (pH=6.8), 20 μL of 0.2 U / mL α-glucosidase and 8 μL of peptide solution in sequence to the wells of the 96-well ELISA plate (prepare different concentrations with PBS) 2.5 mmol / L 4-nitrophenyl-α-D-glucopyranoside (PNPG), mixed and kept at 37°C for 15 min. After reacting for 15 min, add 80 μL of 0.2 mol / L Na 2 CO 3 The solution terminated the reaction, and its absorbance value at a wavelength of 405 nm was detected by a microplate reader. The experiment was repeated 3 times, and the average value was obtained.

[0019] The formula for enzyme activity inhibition rate: I%=[1-(A1-A2) / (A3-A4)]×100%; where: A1 is the absorbance value of the sample solution group, and A2 is measured by replacing the enzyme solution with PBS A3 is the absorbance value of the blank control group measured by replacing the sample solution with PBS soluti...

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Abstract

The invention discloses bioactivity polypeptide and an application thereof to preparation of an alpha-glucosidases inhibitor. The amino acid sequence of the bioactivity polypeptide is as shown in SEQID NO.1, the name of the bioactivity polypeptide is AGI-1, the AGI-1 consists of 10 amino acid residues, wherein the 10 amino acid residues contain 2 pieces of cysteine which form a pair of intramolecular disulfide bonds, and through MALDI-TOF mass spectrometric detection, the molecular weight is 1102.3 Da. The physical and chemical properties of purified freeze-dried powder of the polypeptide AGI-1 are white or off-white loose bodies which are odorless and dissolve in water easily, and an aqueous solution is nearly colorless and transparent. The AGI-1 can restrain activity of alpha-glucosidase, and the IC50 value is 1.54 mM.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a biologically active polypeptide and its application in the preparation of alpha-glucosidase inhibitors. Background technique [0002] Diabetes is a metabolic disease caused by high blood sugar and long-term high blood sugar. Diabetes can cause damage to the patient's heart, liver, kidneys, nerves and other organs and tissues, and may also cause endocrine disorders in the body. Currently, more than 300 million people in the world suffer from diabetes, and this number is expected to increase to more than 500 million by 2030. In China, about 114 million people suffer from diabetes, and China has become the largest country with diabetes in the world. [0003] α-glucosidase is a hydrolase present in the brush border of the small intestine, which can hydrolyze α-1, 4-glucosidic bonds from the non-reducing end of starch to produce glucose, and can also slowly hydrolyze α-1, ...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11
Inventor 宋延民龙莉莉
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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