Inhibition peptide in domain of polymerase protein of hepatitis b virus, and application

A technology of hepatitis B virus and polymerase protein, which is applied in the fields of molecular biology and biochemistry of biotechnology, and can solve problems such as the variation of the YMDD functional region of the virus polymerase P protein, virus drug resistance, and inability to cure hepatitis B

Inactive Publication Date: 2009-04-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fundamental reason why hepatitis B cannot be completely cured is that the currently used antiviral drugs cannot inhibit the synthesis of HBV cccDNA in the nucleus of liver cells
HBV reverse transcriptase inhibitors block most new HBV DNA synthesis but have little effect on the cccDNA pool in the nucleus of up to 50 or more copies
This is also the reason why nucleoside analogues such as lamivudine (Lamivudine, 3TC) currently used clinically cannot cure hepatitis B.
Moreover, it has been found that some patients have mutations in the functional region of the viral polymerase P protein YMDD after treatment with lamivudine, resulting in virus resistance to lamivudine

Method used

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  • Inhibition peptide in domain of polymerase protein of hepatitis b virus, and application
  • Inhibition peptide in domain of polymerase protein of hepatitis b virus, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Screening of inhibitory peptides for the YMDD functional region of hepatitis B virus polymerase protein

[0023] (1) Synthesis of short peptides in the YMDD functional region of hepatitis B virus polymerase protein: Designed according to the HBV ayw genotype sequence (genbank access no: U95551), synthesized short peptides in Hangzhou Zhongpi Biochemical Co., Ltd. The sequence is: Arg Arg Ala PhePro His Cys Leu Ala Phe Ser Try Met Asp Asp Val Val Leu Gly Ala. The purity of the short peptide is> 85%, and the solubility is> 0.5 mg / ml.

[0024] (2) Screening of phage peptide library

[0025] 1. The first day:

[0026] 1.1. With 0.1M NaHCO 3 (PH8.6) Prepared target molecule solution: prepared by short peptide of YMDD functional region of hepatitis B virus polymerase protein DMSO solution first, then add 0.1M NaHCO 3 (PH8.6) dissolved.

[0027] 1.2. Coating: add in each hole The target molecule solution, vortex repeatedly until the surface is completely wetted. In a ...

Embodiment 2

[0085] Example 2 Experiment of inhibitory peptide on the inhibitory effect of hepatitis B virus

[0086] (1) Cell culture

[0087] 1. Cultivation of 2.2.15 cells 2.2.15 cells are hepatocellular carcinoma cell lines transfected with full HBV genes, containing polyploidy of complete HBV genes, capable of transcribing and translating HBV genes and producing and secreting HbsAg, HbeAg, HBV DNA and HBV Particles. It was cultured under pressure with RPMI1640 medium, 10% newborn calf serum, 400mg / L G418. 2.2.15 cells press 2×10 6 / Plate inoculated on a 60mm cell culture plate. The experimental group consists of synthetic peptides (Trp Try Thr Asn Asn Ser Thr (HBV YMDD functional area inhibitory peptide 1#), Gly Pro Phe Asn Asn Pro Pro (HBV YMDD functional area inhibitory peptide 2#), Gly Trp Leu Pro ProPro Asp( HBV YMDD functional domain inhibitory peptide 3#), synthesized by Hangzhou Zhongpi Biochemical Co., Ltd.), added per plate Inhibitory peptide solution. Lamivudine control group p...

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Abstract

This invention discloses inhibitory peptides of HBV polymerase protein YMDD functional domain, and their application. This invention utilizes short peptides of HBV polymerase protein YMDD functional domain as the target, and screens peptides that can specifically bind the target by phage surface display technique. The screened three kinds of inhibitory peptides are Trp Try Thr Asn Asn Ser Thr, Gly Pro Phe Asn Asn Pro Pro, and Gly Trp Leu Pro Pro Pro Asp. Experiments show that the three kinds of inhibitory peptides have obvious inhibitive effect on DNA of HBV, thus provide new raw materials for anti-HBV drugs. Compared with the present nucleotide drugs that act on HBV DNA synthesis, the inhibitory peptides in this invention can inhibit the duplication of HBV. The inhibitory peptides are complementary with present lamivudine drugs, and are suitable for development of anti-HBV drugs and research of HBV molecular virology.

Description

Technical field [0001] The present invention belongs to the fields of molecular biology and biochemistry of biotechnology, and relates to inhibitory peptides of hepatitis B virus polymerase protein YMDD functional region screened from a phage peptide library, which can inhibit hepatitis B virus replication and is suitable for anti-B Research on hepatitis virus drugs and molecular virology of hepatitis B virus. Background technique [0002] Chronic hepatitis B is caused by hepatitis B virus (HBV). It is a common disease that seriously endangers human health. The prevention and treatment of chronic hepatitis B is a global public health problem, which has attracted the attention of countries all over the world. . The development of effective anti-hepatitis B virus therapeutic drugs is a major issue that needs to be solved urgently. [0003] In the HBV replication cycle, the HBV covalently closed circular DNA (cccDNA) molecule in the hepatocyte nucleus is the synthesis template of HB...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K38/08A61P1/16A61P31/12
Inventor 朱海红周林福贾红宇陈智
Owner ZHEJIANG UNIV
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