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In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening inhibitors of HBV DNA polymerase

A technology of hepatitis B virus and polymerase, applied in the field of the N-terminal of the enzyme, can solve problems such as inability to implement

Inactive Publication Date: 2005-04-20
GOVERNMENT OF THE REPUBLIC OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These tedious operations are not possible due to the large number of HBV mutants

Method used

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  • In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening inhibitors of HBV DNA polymerase
  • In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening inhibitors of HBV DNA polymerase
  • In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening inhibitors of HBV DNA polymerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Polymerase Activity Test of HBV Surface Antigen Mutants

[0058]An immediate application of the facile activity assay of human HBV DNA polymerase reported in the present invention is the assessment of HBV variants with major surface antigen mutations, especially those induced by vaccination (i.e. 'S'145). active. Viral DNA was extracted from serum samples containing such HBV variants as described above. The mutants involved in the present invention include 'S'145 (glycine to arginine mutation) and 'S'146 (threonine to alanine mutation), which can evade existing immune detection systems and replicate independently. Using the viral DNA as a template, with the same oligonucleotide primers, use Taq polymerase (Promega, U.S.A.) on a DNA thermal cycler (Perkin-Elmer, Cetus) to carry out 35 cycles of PCR, each cycle including 1.5 minutes at denaturation temperature (94°C), 2 minutes at annealing temperature (53°C) and 4 minutes at extension temperature (72°C).

[0059] PCR ...

Embodiment 2

[0061] Detection of inhibitors of human HBV DNA polymerase

[0062] Screening for inhibitors of human HBV DNA polymerase will provide new therapeutic agents for HBV infection, since effective inhibition of polymerase activity will lead to inhibition of HBV viral replication. The simplified activity assay reported in the present invention enables more rapid analysis of the inhibitory effect of newly discovered molecules on the DNA polymerase of HBV variants carrying major surface antigen mutations. Briefly, candidate inhibitors were first incubated with wild-type human HBV DNA polymerase that was PCR amplified and translated in vitro as reported in the present invention. There is [α- 32 P]dTTP and the other conditions mentioned above are present. The inhibitory effect can be determined by laser densitometry followed by SDS-PAGE analysis, or by measuring the amount of radiolabeled HBV DNA polymerase bound to the nitrocellulose membrane after filtration. Obviously, analysis of...

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Abstract

The present invention provides an in vitro activity test of human hepatitis B virus (HBV) DNA polymerase, which comprises utilizing an oligonucleotide incorporated into the SP6 viral promoter as a 5' oligonucleotide to amplify HBV from a sample by PCR DNA polymerase, direct transcription and translation of the PCR product in the presence of a radiolabeled reagent, and determination of HBV DNA polymerase priming. The present invention also provides the application of such a test in the activity test of various serum samples, in the screening of HBV DNA polymerase inhibitors, and in the detection and / or screening of potential anti-HBV drugs on human HBV DNA polymerization Applications in the inhibitory capacity of DNA-priming activity of enzymes.

Description

Background of the invention [0001] Polymerase is a basic enzyme that is very important to living organisms. They are responsible for synthesizing nucleic acids and converting them into other nucleic acids necessary for the synthesis of proteins. Thus, polymerases are found in all cell types including the pathogenic DNA virus hepatitis B virus (HBV). [0002] HBV can cause hepatitis, one of the most common human infectious diseases, and the resulting hepatocellular carcinoma (HCC) is one of the most common cancers in the world. At present, the most effective measure to prevent HBV infection and pathogenesis is to resist HBV through immunization. Currently approved HBV vaccines consist of the major surface antigen (HBsAg) in native form (plasmid-derived) or recombinant form (purified from yeast cells). Vaccine-induced antibodies have been shown to effectively neutralize HBV replication by binding to most of the antigenic 'a' epitope located between residues 124 and 147 of HBs...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/50C12N9/10C12N9/12C12N15/09C12P19/34C12P21/00C12Q1/48C12Q1/68C12Q1/70G01N27/447G01N33/15G01N33/48
CPCC12N9/1252C12Q1/706C12Q2525/143C12Q1/68
Inventor 温崇仁陈维宁林玉娇梁爱玲
Owner GOVERNMENT OF THE REPUBLIC OF SINGAPORE
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