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Membrane penetrating polypeptide for specific inhibition of hepatitis b virus assembly and replication

A technology of hepatitis B virus and membrane penetration, which is applied in the field of biomedicine, can solve the problems of low membrane penetration, impossibility of practical application, and poor dimensional stability, so as to inhibit replication and secretion, improve antiviral effect, and reduce core Effects at the protein subunit level

Inactive Publication Date: 2009-05-06
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has defects such as poor steric stability, low membrane penetration ability, easy degradation in vivo, and may induce certain immune reactions, so it cannot be used in practical applications.

Method used

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  • Membrane penetrating polypeptide for specific inhibition of hepatitis b virus assembly and replication
  • Membrane penetrating polypeptide for specific inhibition of hepatitis b virus assembly and replication
  • Membrane penetrating polypeptide for specific inhibition of hepatitis b virus assembly and replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 Preparation of recombinant protein of the present invention

[0055] According to the characteristics of HBV virus, the membrane-penetrating polypeptide drug C20-MTS, which specifically blocks the assembly and replication of hepatitis B virus, is designed with HBV core protein as the target site.

[0056] Firstly, screening of polypeptide sequences that can specifically and strongly bind to HBV core protein is used as the basis for drug development;

[0057] According to literature reports, and using the humanized peptide aptamer screening system, a polypeptide sequence consisting of 20 AAs with the strongest binding ability to HBV core protein was identified, named C20, and its amino acid sequence was:

[0058] SFYSVLFLWGTCGGFSHSWY.

[0059] In order to meet the requirements of clinical application, the sequence (C20) is molecularly modified:

[0060] 1. Select the humanized backbone protein TrxA to reduce the immunogenicity of heterologous proteins; ins...

Embodiment 2

[0077] Embodiment two in vitro pharmacodynamic studies

[0078] The HBV transgenic HepG2.215 cell line was obtained to verify the efficacy of the in vitro model, and C20 was used as a control (without adding MTS sequence) to test the membrane penetration ability of C20-MTS.

[0079] ELISA can detect the content of HBsAg in the supernatant of cell culture medium, the level of HBsAg antigen on the surface of cell culture medium and plasma, which can indicate the content of secreted virus particles.

[0080] Western blot detection of HBV core protein subunit content in cell lysate; determination of intracellular HBV core protein subunit DNA content to indicate the formation of HBV core protein subunit

[0081] The probe method was used to detect the HBV DNA content in the cell lysate and determine the intracellular HBV DNA content to indicate the replication of viral DNA.

[0082] The results are as follows: In the in vitro experiment, C20-MTS (polypeptide drug with MTS sequence...

Embodiment 3

[0085] Embodiment three in vivo pharmacodynamics research

[0086] Use the HBV transgenic Balb / c mouse model (mouse source: Guangzhou Military Region Hospital.) to detect the in vivo efficacy of the protein of the present invention, and use C20 as a control (without adding MTS sequence) to test the membrane penetration ability of C20-MTS.

[0087] The HBsAg content in peripheral blood of mice was detected by ELISA.

[0088] The content of HBV core protein subunit in mouse hepatocytes was detected by Western blot.

[0089] Detection of HBV DNA content in mouse hepatocytes by probe method.

[0090] In vivo experiments, we use the HBV transgenic mouse model for drug efficacy verification. According to the results of in vitro experiments, we chose to administer once every 2 days. The results showed that: when the dose was 2.5 mg / kg or more, the level of HBsAg secretion could be reduced; when the dose was 5 mg / kg, the level of HBsAg was detected on the 8th day Reduced by about 7...

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Abstract

The invention belongs to the biological medicine field, and in particular relates to a new membrane penetrating polypeptide capable of interdicting by specificity the assembly and reproduction of HBV and a preparation method thereof. The recombinant protein structure of the invention is as follows: the polypeptide sequence capable of combining HBV core protein is inserted to a thioredoxin sequence, and the C terminal of the thioredoxin sequence is connected with membrane translocation sequence, wherein the polypeptide sequence capable of combining the HBV core protein is inserted between the 34th amino acid and the 35th amino acid of the thioredoxin sequence. The recombinant protein can not only be combined with HBV core protein in the HBV transgene cell strain and animal model body to reduce the subunit level of the core protein, but also inhibit the reproduction and exudation of virus, thereby having good application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a membrane-penetrating polypeptide capable of specifically blocking assembly and replication of hepatitis B virus and a preparation method thereof. Background technique [0002] Hepatitis B virus (HBV) infection is a kind of disease that seriously threatens human health. Globally, more than 300 million people are infected with HBV [1], and about 1 million people die from acute and chronic hepatitis B every year [2]. The risk of liver cancer in people infected with hepatitis B is about 100 times that of normal people, and about 60-80% of patients with primary liver cancer have a history of hepatitis B [3]. At the same time, persistent HBV infection can also cause chronic liver insufficiency, liver cirrhosis and other diseases [1-4]. The latest epidemiological survey shows that the carrier rate of hepatitis B surface antigen (HBsAg) in the Chinese population is 7.18%, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61K38/16A61K48/00A61P31/20A61P1/16
Inventor 辛洪波吴思思张巍
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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