Synthetic polypeptide, synthesis method and application of synthetic polypeptide, and gene encoding the synthetic polypeptide

A technology for synthesizing polypeptides and genes, applied in the field of prevention and treatment of alcoholic liver disease, can solve problems such as toxic and side effects, and achieve the effects of strong hydrogen supply capacity, inhibition of survival rate reduction, and high ORAC activity

Active Publication Date: 2018-11-06
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment methods for alcoholic liver are gradually increasing, such as the use of colchicine, propylthiouracil, lipid-lowering drugs, anti-endotoxin agents and other therapies have certain effects, but these drugs have certain toxic and side effects

Method used

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  • Synthetic polypeptide, synthesis method and application of synthetic polypeptide, and gene encoding the synthetic polypeptide
  • Synthetic polypeptide, synthesis method and application of synthetic polypeptide, and gene encoding the synthetic polypeptide
  • Synthetic polypeptide, synthesis method and application of synthetic polypeptide, and gene encoding the synthetic polypeptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Polypeptide solid-phase synthesis

[0021] 1. Resin selection

[0022] (1) Adopt the standard fluorenylmethoxycarbonyl (Fmoc) scheme, which selects 0.0125mmol, 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin), according to the sequence characteristics of the amino acid sequence Leu-Val-Tyr-Pro, Add 0.3 mol of the first Fmoc protected amino acid, add DCC and 5% (mass fraction) DMAP into the reactor to shake the reaction, wash the resin with NMP to remove excess protected amino acid. The connection efficiency of the first amino acid to the resin, that is, the coupling rate (as shown in Table 1), was determined.

[0023] (2) Select 0.0125 mmol, 4-(hydroxymethyl) phenoxymethyl polystyrene resin (Wang resin), the method is the same as above, measure the connection efficiency of the first amino acid and the resin, that is, the coupling rate (as shown in Table 1) .

[0024] Table 1

[0025] resin type

Coupling rate (%)

2-chlor...

Embodiment 2

[0030] Embodiment 2: ORAC value determination of synthetic polypeptide

[0031] The reaction was carried out in an environment of 75mmol / L potassium phosphate buffer (pH=7.4), and the specific experimental operation was as follows: 20 μL of synthetic polypeptide, GSH solution and Trolox standard solution (6.25, 12.5, 25, 50 μM), add 200 μL of FL solution (95.68 μmol / mL) to each well, incubate at 37°C for 20 min, then quickly add 20 μL of AAPH solution (119.4 μmol / mL) to each well with a multichannel pipette, and the microwell The plate was placed in a microplate detector at 37°C with an excitation wavelength of 485nm and an emission wavelength of 538nm for continuous measurement, and the fluorescence intensity of each well was measured every 2 minutes for 90 measurements. The ORAC experiment needs to set two control groups, that is, the FL fluorescence natural attenuation control without adding free radicals (-AAPH) and the free radical action control without antioxidants (+AA...

Embodiment 3

[0034] Example 3: Liver-protecting application of synthetic polypeptide acting on ethanol-induced liver cell injury model

[0035] LO2 cells in the logarithmic growth phase were seeded in 96-well cell plates, 5×10 per well 3 cells in CO 2After culturing in the incubator for 24 hours, the experimental group was added with the culture solution with the concentration of synthetic peptides being 0.25, 0.5, 0.75, 1, 1.25 mmol / L respectively, and the blank control group was added with the same amount of culture solution. The incubator continued to cultivate for 24h. Add 20 μL of MTT solution (5 mg / mL) to each well, incubate in an incubator for 4 hours, discard the supernatant, add 150 μL DMSO to each well, and measure the absorbance value OD of each well at a wavelength of 490 nm. Calculate the survival rate of each group of cells after treatment with different ethanol concentrations, such as Figure 4 Shown is the effect of peptides on ethanol-induced LO2 cytotoxicity. One-way A...

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Abstract

The invention discloses a synthetic polypeptide, a synthesis method and an application of the synthetic polypeptide, and a gene encoding the synthetic polypeptide. The amino acid sequence of the polypeptide is Leu-Val-Tyr-Pro and is shown as in a sequence table SEQ ID No:1. The polypeptide is synthesized through solid-phase synthesis by using a polypeptide synthesizer. The oxygen radical absorbance capacity (ORAC) experiment is conducted on the polypeptide, and the result shows that the polypeptide has certain antioxidant ability. The polypeptide is applied to an ethyl alcohol induced liver injured cell model, an MTT (Methyl Thiazolyl Tetrazolium) detection method is used to verify the protection action of the polypeptide on an LO2 cell, the result shows that the polypeptide inhibits the ethyl alcohol induced LO2 cell to reduces the survival rate, and the polypeptide has a certain effect on protecting the liver and is applied to the protecting the alcoholic liver disease.

Description

technical field [0001] The invention relates to the technical field of prevention and treatment of alcoholic liver disease, in particular to a synthetic polypeptide with protective effect on ethanol-induced liver cell (LO2) damage, its application and the gene encoding the synthetic polypeptide. Background technique [0002] Alcoholic liver disease (ALD) is a chronic liver disease caused by long-term heavy drinking. Initially, it usually presents as fatty liver, which can progress to alcoholic hepatitis, alcoholic fibrosis, and alcoholic cirrhosis. The disease is more common in European and American countries, and the incidence rate in China has also increased in recent years. According to some regional epidemiological surveys, the prevalence rate of alcoholic liver disease in Chinese adults is 4%-6%, so it seriously affects the health of human beings. [0003] The pathogenesis of alcoholic liver disease is relatively complex. Studies have found that it is related to multi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/103C12N15/70C12N15/81C12N15/11A61K38/07A61P1/16A23L33/18
CPCA61P1/16A23L33/18C12N15/70C12N15/81C07K5/101A23V2002/00A61K38/00A23V2200/32A23V2250/55
Inventor 任娇艳龚聪聪尚帅明
Owner SOUTH CHINA UNIV OF TECH
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