Suppressing peptide of heptitis B virus polymerase lymerase protein YMDD functionalized area and its uses

A polymerase protein, hepatitis B virus technology, applied in the field of molecular virology research of hepatitis B virus, can solve the problem of inability to cure hepatitis B, inability to completely cure hepatitis B, inability to inhibit HBVcccDNA synthesis, and mutation of viral polymerase P protein YMDD functional region And other issues

Inactive Publication Date: 2009-01-21
ZHEJIANG UNIV
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  • Description
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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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  • Suppressing peptide of heptitis B virus polymerase lymerase protein YMDD functionalized area and its uses
  • Suppressing peptide of heptitis B virus polymerase lymerase protein YMDD functionalized area and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0023] (1) Synthesis of a short peptide in the YMDD functional region of the hepatitis B virus polymerase protein: According to the HBV ayw genotype sequence design (genbank access no: U95551), the short peptide was synthesized in Hangzhou Zhongpei Biochemical Co., Ltd., and 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 >0.5mg / ml.

[0024] (2) Phage peptide library screening

[0025] 1. The first day:

[0026] 1.1. With 0.1M NaHCO 3 (PH8.6) prepared target molecule solution: Hepatitis B virus polymerase protein YMDD functional region short peptide is made into a 150 μg / ml solution, first dissolved with DMSO, and then added with 0.1M NaHCO 3 (PH8.6) dissolved.

[0027] 1.2. Coating: Add 150 μl target molecule solution to each well, and v...

Embodiment 2

[0086] Example 2 Experiment of Inhibitory Peptides on Hepatitis B Virus Inhibition

[0087] (1) Cell culture

[0088] 1. The culture of 2.2.15 cells 2.2.15 cells are hepatocellular carcinoma cell lines transfected with the whole HBV gene, which contains the polyploid of the complete HBV gene, can transcribe and translate the HBV gene and produce and secrete HbsAg, HbeAg, HBV DNA and HBV particles. Cultured under pressure with RPMI1640 medium, 10% newborn calf serum, and 400mg / L G418. 2.2.15 cells by 2×10 6 / plate inoculated on 60mm cell culture plate. The experimental group was a synthetic polypeptide (Trp Try Thr Asn Asn Ser Thr (HBV YMDD functional region inhibitory peptide 1#), Gly Pro Phe Asn Asn Pro Pro (HBV YMDD functional region inhibitory peptide 2#), Gly Trp Leu Pro ProPro Asp ( HBV YMDD functional region inhibitory peptide 3#), synthesized by Hangzhou Zhongpei Biochemical Co., Ltd.), add 30 μl of inhibitory peptide solution to each plate. For the lamivudine cont...

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Abstract

This invention relates to hepatitis B virus polymerase protein YMDD function inhibitory peptide and its application, belonging to molecular biology and biochemistry field. Hepatitis B virus polymerase protein YMDD function short peptide is used as a target, and phage display technology is used to screen peptides for specific-binding with the target mentioned. The amino acid sequences of three screened inhibitory peptide of this invention were: Trp Try Thr Asn Ser Thr Asn; Gly Pro Asn Phe Asn Pro Pro; Gly Trp Leu Pro Pro Pro Asp. It is proved by experiments that the three peptide inhibition of HBV DNA is markedly inhibited, which provides a new and effective drug source for anti-HBV drugs. Relative to the existing role of blocking HBV DNA synthesis of nucleotide drugs, the inhibitory peptide fundamentally inhibits the replication of HBV, is complementary with the existing lamivudine drugs, and can be applied for anti-HBV drug development and HBV research in molecular virology.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biochemistry of biotechnology, and relates to the inhibitory peptide of the YMDD functional region of the hepatitis B virus polymerase protein screened from a phage peptide library, which can inhibit the replication of hepatitis B virus and is suitable for anti-hepatitis B Research on hepatitis virus drugs and molecular virology research on hepatitis B virus. Background technique [0002] Chronic hepatitis B is caused by hepatitis B virus (hepatitis B virus, HBV), 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 practical and effective therapeutic drugs against hepatitis B virus is a major issue to be solved urgently at present. [0003] In the HBV replication cycle, the HBV covalently closed circular DNA (cccDN...

Claims

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Application Information

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