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Hepatitis C virus NS5B RNA polymerase inhibitory polypeptide sequence and application thereof

A technology of hepatitis C virus and polymerase, which is applied in the direction of antiviral agents, peptides, peptide/protein components, etc., to achieve the effect of increasing the probability of successful screening, low cost, and reducing manual screening

Active Publication Date: 2015-04-22
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the current research has not found that enzymes with similar functions to NS5B RNA polymerase are expressed in mammalian cells, which makes the inhibitors against NS5B RNA polymerase have high specificity, which makes the inhibitors against NS5B RNA polymerase Research on anti-HCV drug development has become a hot spot

Method used

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  • Hepatitis C virus NS5B RNA polymerase inhibitory polypeptide sequence and application thereof
  • Hepatitis C virus NS5B RNA polymerase inhibitory polypeptide sequence and application thereof
  • Hepatitis C virus NS5B RNA polymerase inhibitory polypeptide sequence and application thereof

Examples

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Effect test

example 1

[0032] Example 1: Virtual docking analysis of polypeptides and HCV NS5B RNA polymerase

[0033] The interaction forces between polypeptides and proteins mainly include van der Waals forces, hydrophobic interactions, hydrogen bonding forces and electrostatic interactions. Therefore, in order to understand the results obtained through virtual docking and evaluate the interaction between peptides and proteins, we selected the peptide with the best docking effect obtained by virtual screening and the target protein as a reference, and the interaction analysis was as follows:

[0034] Compared with the chemical bonds between molecules, van der Waals force is relatively weak, it is only the attraction between molecules, and it has a great relationship with the distance between molecules. The greater the van der Waals force, the higher the melting point and boiling point of the substance. For substances with similar composition and structure, as the relative molecular mass increas...

example 2

[0040] Example 2: ELISA Binding of Polypeptides and Fusion Expressed Proteins

[0041] The polypeptide sequence of the present invention is Ile-Asn-Gln-Arg-Lys-Val-Trp. That is INQRKVW in the table.

[0042] In this experiment, the polypeptide was artificially synthesized, coupled with horseradish peroxidase, and then reacted with the fusion expression protein-coated microtiter plate to determine the binding ability of the synthetic polypeptide and the fusion expression protein. Through this mode, the polypeptide of the present invention can also be used to detect the content of HCV NS5B protein in samples such as blood, and then implement the detection application of the polypeptide. During the determination process, the polypeptide-HRP was diluted 500 times, and the expressed protein was coated at 10 μg / ml. Specific steps are as follows:

[0043] (1) Dilute the expressed protein with CBS to 10ug / mL, 5ug / mL and 1ug / mL respectively, add to the microtiter plate, 50μl per w...

example 3

[0050] Example 3: The present invention establishes an evaluation system for HCV NS5B RNA polymerase activity, and then screens the designed polypeptides in vitro with the help of this system. The process is as follows:

[0051] The detection steps of polypeptide inhibition of HCV NS5B enzyme activity are as follows:

[0052] (1) Take 5 μl of Poly(A) magnetic beads (100ug / ml), add 0.5uL of RNasin, and at the same time add 5μl of 10×RNA Buffer, Mg2+ (25mM) 4ul, add water to make up to 50μl. Water bath at 37°C for 30min.

[0053] (2) Dissolve all synthesized peptides to be screened in DMSO at a specific concentration; take 15 μl of synthetic peptides (10 mM) and 15 μl of NS5B corresponding expression protein to react at 37°C for 30 min, and then take 10 μl for the next reaction.

[0054] (3) Add 0.5 μl Bio-11-UTP (1 mM), 5 μl rUTP (10 mM), and place in a water bath at 37°C for 30 minutes. Only 5 μl of rUTP (10 mM) was added to the negative control group. At the same time, 1...

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Abstract

The invention designs a series of polypeptide combinational libraries with different lengths. Polypeptides with functions of inhibiting the activity of HCV NS5B RNA polymerase can be screened out by performing molecular docking on the polypeptides and specific inhibitory sites on HCV NS5B RNA polymerase by using molecular docking software and performing comprehensive analysis on results, and can be used for laying a foundation for further researching and developing anti-HCV medicines. Combined experimental results of modified polypeptide and NS5B expression protein show that the designed polypeptides can be combined with expression protein to express that fusion protein measurement results have correlations with virtual docking results. The polypeptides designed by the invention can be used for detecting HCV NS5B protein, and results obtained by performing polypeptide inhibition screening by using an HCV NS5B RNA polymerase in-vitro activity identification system show that the polypeptides designed by the invention have significant inhibition effects on the polymerase activity of HCV NS5B protein, IC50 of the polypeptides can reach 9.85 microns, and the polypeptides can be used as potential medicines for inhibiting HCV.

Description

technical field [0001] The invention relates to the field of virus-suppressing drug development, and is mainly used for detection of HCV NS5B polymerase, inhibition of biological activity, treatment of HCV and the like. Background technique [0002] At present, whether domestic or foreign, the infection rate of hepatitis C virus (HCV) continues to increase; according to statistics, more than 3% of the people in the world are infected with HCV, and the proportion in my country has reached 3.2%, which is much higher than world average. Most HCV-infected patients carry the virus for life, and more than half of them will eventually develop chronic hepatitis, and even further develop into liver cirrhosis; among them, about 2% of patients with liver cirrhosis will develop into primary liver cancer. Therefore, HCV has become a major disease threatening public safety. [0003] Since there are no vaccines and specific drugs against HCV at present, the combination of interferon and r...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K38/08A61P31/14G01N33/68G01N33/569
Inventor 王春峰张连峰王方雨吕军姚建宁程鹏高冰
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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