Inhibition peptide of hepatatitis B virus polyzymoprotein YMDD functional region and its application

A technology of hepatitis B virus and polymerase protein, applied in the field of molecular biology and biochemistry of biotechnology, can solve the problem that hepatitis B cannot be cured, hepatitis B cannot be completely cured and cannot inhibit the synthesis of HBVcccDNA, and the function of virus polymerase P protein YMDD area variation etc.

Inactive Publication Date: 2007-05-02
ZHEJIANG UNIV
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  • Summary
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  • 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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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 (PH 8.6) 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, then added with 0.1M NaHCO 3 (PH 8.6) dissolved.

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

Embodiment 2

[0085] Example 2 Experiment of Inhibitory Peptides on Hepatitis B Virus Inhibition

[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 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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PUM

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Abstract

The inhibiting peptide of hepatitis B virus polymerase protein YMDD function zone and application belong to the field of molecular biology and biological chemistry of biological technology. Said invention uses short peptide of hepatitis B virus polymerase protein YMDD function zone as target position, and utilizes bacteriophage surface spreading technique to screen the peptide which can be combined with specificity of the described target position. The amino acid sequences of three inhibiting peptides screened by said invention respectively are Trp Try Thr Asn Asn Ser Thr; Gly Pro Phe Asn Asn Pro Pro; and Gly Trp Leu Pro Pro Pro Asp. The tests show that said three inhibiting peptides have obvious inhibiting action fro DNA of HBV.

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): C07K7/06A61K38/08A61P31/12
Inventor 朱海红周林福贾红宇陈智
Owner ZHEJIANG UNIV
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