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New application of drug screening cell model targeting HBV core promoter, Tricirine and structural analogue

A core promoter and cell model technology, applied in the fields of molecular biology and biomedicine, can solve problems such as drugs that cannot effectively inhibit the transcriptional activity of core promoters

Active Publication Date: 2021-06-25
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is no drug that can effectively inhibit the transcriptional activity of the core promoter on the market

Method used

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  • New application of drug screening cell model targeting HBV core promoter, Tricirine and structural analogue
  • New application of drug screening cell model targeting HBV core promoter, Tricirine and structural analogue
  • New application of drug screening cell model targeting HBV core promoter, Tricirine and structural analogue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of a drug screening cell model targeting the HBV core promoter

[0052] 1. Design idea and working principle

[0053] The HBV core promoter functions on cccDNA, and cccDNA exists in cells in the form of minichromosomes independent of cell chromosomes. At this point, all cell models based on transgenes (integrated in the chromosomes of cells) are far from reality. To better model cccDNA transcription events, it is preferable to place the core promoter outside the chromosome.

[0054] The cccDNA simulation system based on plasmid vector (or plasmid-like) transfection, such as the mcHBV-Gluc cccDNA model, and the integration-deficient lentiviral vector of Fluc based on Fluc, etc., are indeed located outside the chromosome, However, its significant defect is that the cccDNA mimic molecule cannot replicate, but is gradually lost with cell division and cannot exist stably. This results in the need for a transfection or infection step for each experi...

Embodiment 2

[0091] Example 2. Screening of compounds targeting HBV transcription

[0092] 1. Preliminary screening of compounds

[0093] After the stable passage cell pCore-AML12 was obtained, it was used for compound screening. The screening procedure is as follows: pCore-AML12 cells were revived and cultured in 10 cm culture dish with DMEM medium containing 10% fetal bovine serum (cultivated by conventional methods). After the cells have been passaged for 3 times, when the growth state is better, the cells are digested and seeded into 96-well plates. On the second day after cell inoculation, the compounds to be tested were respectively added to the 96-well plate with a final concentration of 5 μM, and two replicate wells were made for each compound. About 48 hours after adding the drug, aspirate the medium, add cell lysate to each well, incubate with shaking at room temperature for 20 minutes, then take the lysate and use HiBiT detection reagent for detection (the process is as follow...

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Abstract

The invention discloses a plasmid pcore-HU-SMAR, a framework vector of the plasmid pcore-HU-SMAR is plasmid PCMV-Gluc, a HiBiT-unaG fusion gene is inserted into the framework vector, the HiBiT-unaG fusion gene is connected with a puromycin resistance gene through a T2A sequence, an SMAR sequence is inserted between the puromycin resistance gene and a polyA sequence, and the HiBiT-unaG fusion gene is a fusion gene of a luciferase tag HiBiT and a fluorescent protein reporter gene UnaG. The invention further discloses a drug screening cell model of the targeted HBV core promoter, wherein the drug screening cell model is obtained by transfecting the AML12 with the plasmid pcore-HU-SMAR. The invention further discloses application of Tricirine and a structural analogue thereof to preparation of a medicine for inhibiting HBV core promoter transcription and / or HBV replication or application of the Tricirine and the structural analogue thereof to preparation of a medicine for treating hepatitis B.

Description

Technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, and specifically relates to a drug screening cell model targeting the HBV core promoter, a new application of Triciribine and its structural analogs. Background technique [0002] Currently, there are approximately 350 million hepatitis B virus carriers worldwide, and 6-7% of the population in my country (approximately 80 million people) are virus carriers. Virus carriers can develop chronic hepatitis B. Compared with the general population, patients with chronic hepatitis B have a higher risk of developing cirrhosis and liver cancer. About 1 million people worldwide die from liver diseases related to HBV infection every year. Therefore, treating chronic HBV infection and preventing its fatal consequences are important issues related to public health. [0003] Currently, drugs used for the treatment of hepatitis B include alpha interferon (IFN-α, PEG-IFNα) and six nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N5/10C12Q1/02C07H19/167C07D487/14C07H19/23C07D473/16A61P31/20
CPCC12N15/85C12N15/65C12N5/067G01N33/5067C07H19/167C07D487/14C07H19/23C07D473/16A61P31/20C12N2510/00C12N2503/02G01N2500/10
Inventor 胡接力黄爱龙甘春杨魏霞飞黄露义
Owner CHONGQING MEDICAL UNIVERSITY
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