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System for inducing formation of HBV cccDNA and construction method thereof

A construction method and fragment technology, which are applied in the field of inducing the formation of HBV cccDNA, can solve the problems of harsh infection conditions, low sensitivity, high cost, etc., and achieve the effect of simple biological background.

Pending Publication Date: 2020-01-10
GUANGZHOU EIGHTH PEOPLES HOSPITAL
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Problems solved by technology

[0007] All in all, there are few direct research models of HBV cccDNA, the main reasons are as follows: 1. cccDNA and RC-DNA are highly similar in DNA sequence, and the copy number of HBV cccDNA in infected cells is far lower than that of RC-DNA, and PCR detection cannot Effectively distinguish HBV cccDNA, the Southern Blot detection method takes a long time and has low sensitivity; 2. There is a lack of cell models and animal models supporting HBV infection, and the efficiency of HBV cell infection models is low, and the infection conditions are harsh, so it is not suitable for high-throughput screening; Derived mouse model is the only animal model supporting HBV infection, but it is expensive and only suitable for preclinical evaluation of drugs

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  • System for inducing formation of HBV cccDNA and construction method thereof
  • System for inducing formation of HBV cccDNA and construction method thereof
  • System for inducing formation of HBV cccDNA and construction method thereof

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Embodiment Construction

[0042] In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0043] 1. Obtain genomic DNA in patient samples

[0044] Use nucleic acid extraction or purification kit (Sun Yat-sen University Daan Gene) to extract hepatitis B virus RNA from patient samples, the operation is as follows:

[0045] 1) Take 200ul sample into the centrifuge tube, add 50ul proteinase K;

[0046] 2) Add 200ul lysis buffer, vortex for 15sec, centrifuge for 10sec, 72℃ for 10min;

[0047] 3) Add 250ul of absolute ethanol and vortex for 15sec;

[0048] 4) Suction the mixed solution to a spin column, centrifuge at 12,000g for 1 min at room temperature, and transfer the spin column to a new collection tube;

[0049] 5) Add 500ul inhibitor removal solution to the spin column, centrifuge at 12,000g for 1 min at room temperature, and transfer the spin column to a...

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Abstract

The invention discloses a system for inducing formation of HBV cccDNA and a construction method thereof. According to the invention, a stable cell line is constructed by integrating a Phic31 recombinase gene; the 5'end and the 3'end of the linear HBV are provided with complementary luciferase gene segments and specific sites recognized by the Phic31 recombinase, under the action of the Phic31 recombinase, cccDNA is formed through cyclization, and meanwhile, the complementary luciferase is also expressed; the luciferase provides a quick way for quickly detecting the active HBV cccDNA, and is suitable for high-throughput screening.

Description

Technical field [0001] The present invention relates to a system for inducing the formation of HBV cccDNA and a construction method. Specifically, a linear genome can produce HBV cccDNA containing luciferase in liver cancer cells expressing PhiC31 recombinase, and the luciferase provides rapid detection of active HBV cccDNA The shortcut is suitable for high-throughput screening. Background technique [0002] Hepatitis B virus (hepatitis B virus, HBV) is a DNA virus belonging to the Hepadnaviridae family. The HBV genome is about 3.2 kb in length and encodes 7 functional proteins: HBV core antigen (HBcAg), e antigen (HBeAg), surface antigen (L, M, S HBsAg), polymerase (pol) and X protein (HBx). About 240 million people worldwide are infected with HBV, of which about 25 million are chronically infected (Global Hepatitis Report, 2017). Chronic infection will greatly increase the risk of liver cirrhosis and liver cancer, and more than 700,000 people die from HBV-related diseases ever...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/52C12N15/36C12N15/867C12N15/65C12Q1/02C12Q1/66C12R1/91
CPCC07K14/005C12N9/00C12N15/86C12N15/65C12Q1/66G01N33/5067C12N2730/10122C12N2740/15043G01N2500/10
Inventor 李锋冯成千
Owner GUANGZHOU EIGHTH PEOPLES HOSPITAL
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