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In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof

A technology for measuring hepatitis C virus and enzyme activity, which is applied in the field of genetic engineering technology, can solve the problems of complex and impractical activity measuring methods, and achieves the effects of simple method, enrichment and amplification of detection signals.

Inactive Publication Date: 2008-04-30
李越希
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Problems solved by technology

However, the established activity assay method is complicated and impractical, mainly using isotope labeling technology

Method used

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  • In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof
  • In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof
  • In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof

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Embodiment Construction

[0049] Detailed description of the embodiment of the present invention:

[0050] Cloning, expression and identification of hepatitis C virus C-terminal truncated NS5B protein with 21 amino acids

[0051] PCR technology was used to amplify HCV NS5B-C21, the gene fragment of NS5B protein truncated by 21 amino acids at the C-terminus, the gene fragment was cloned into the BamHI / Xho I site in plasmid pET28a(+), and the recombinant plasmid was transformed into Escherichia coli BL21( DE3), screened and obtained engineering bacteria expressing NS5B highly, and the expressed NS5B protein accounted for about 20% of the total bacterial protein. IPTG induces expression, and the target protein expression product is purified by affinity chromatography and identified by SDS-PAGE electrophoresis.

[0052] Materials and methods

[0053] 1. Plasmids and bacterial strains: Escherichia coli BL21 (DE3), HCV full-length cDNA plasmids, and prokaryotic expression vector pET-28a (+) are all preserv...

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Abstract

The invention provides a detecting method and an application of the extraneous activity of the RNA polymerase relied on by the hepatitis C virus RNA, which relates to an secure and practical establishing method for the detection of the activity extraneous detection of the RNA polymerase relied on by the hepatitis C virus RNA, and the application relates to the genetic engineering technology and the nanometer segregation enrichment and RNA related technology field. The invention expresses the soluble NS5B protein through the utilization of the genetic engineering technology, and prepares the nanometer magnetic particle. One end of a RNA template is fixed on the nanometer magnetic particle, and added in the 96 hole reaction plate micromesh, and added with NS5B protein to compose a RNA duplication system, and a duplicated RNA double chain on the nanometer magnetic particle is mixed with marked biological elements. After the RNA is duplicated, the nanometer magnetic particle is attached at the micromesh bottom through an external magnetic field magnet, after repeatedly cleaning and attaching, the nanometer magnetic particle is combined with the marked combining factor of the horse radish peroxidase, after attaching and cleaning, the substrate constant of the horse radish peroxidase is added to develop color. New NS5B protease activity inhibitor can be screened from a traditional Chinese storeroom and a compound storeroom by the established method.

Description

technical field [0001] The hepatitis C virus RNA-dependent RNA polymerase in vitro activity assay method and application of the present invention relate to a kind of utilization of genetic engineering technology to prepare soluble hepatitis C virus RNA-dependent RNA polymerase (RDRP enzyme or NS5B) protein, and Preparation of magnetic nanoparticles. Fix one end of the RNA template on the magnetic nanoparticles, add it into the microwell of the 96-well reaction plate, and then add RNA-dependent RNA polymerase protein to form the RNA replication system. biotin. After RNA replication, the magnetic nanoparticles are adsorbed on the bottom of the microwell with a magnet of an external magnetic field, and after repeated washing and adsorption, they bind to avidin labeled with horseradish peroxidase (HRP). After adsorption and washing, horseradish peroxide is added The substrate of the enzyme develops color. Using the established method, new inhibitors of RNA-dependent RNA polymer...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/25G01N33/563C40B30/00
Inventor 李越希杨华凤潘明洁吕敏戚菁王正茂
Owner 李越希
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