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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 inability to cure hepatitis B, virus drug resistance, and variation of the YMDD functional region of the virus polymerase P protein

Inactive Publication Date: 2007-07-25
ZHEJIANG UNIV
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  • Abstract
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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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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 solution of 150g / ml, first dissolved with DMSO, then added with 0.1M NaHCO 3 (PH8.6) dissolved.

[0027] 1.2. Coating: add 1501 target molecule solution to each well, and repe...

Embodiment 2

[0085] The experiment of embodiment two inhibitory peptides to hepatitis B virus inhibitory effect

[0086] (1) Cell culture

[0087] 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 domain inhibitory peptide 3#), synthesized by Hangzhou Zhongpei Biochemical Co., Ltd.), add 30l inhibitory peptide solution to each plate. For the lam...

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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 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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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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