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High-expression lamivudine-resistant hepatitis B virus (HBV) liver cancer cell strain

A technology of liver cancer cells and high expression, which can be applied to cells modified by the introduction of foreign genetic material, the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve unseen problems.

Inactive Publication Date: 2014-02-12
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no report about a suitable HepG2-based stable high-expression HBV YVDD variant cell line

Method used

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  • High-expression lamivudine-resistant hepatitis B virus (HBV) liver cancer cell strain
  • High-expression lamivudine-resistant hepatitis B virus (HBV) liver cancer cell strain
  • High-expression lamivudine-resistant hepatitis B virus (HBV) liver cancer cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Establishment of HepG2.HS204V cell line

[0050] After the HBV eukaryotic expression plasmid pREP-HBV-YVDD was transfected into HepG2 cells, it was screened by hygromycin, and then the HBV replication, specific antigen expression and virus particle production in the cells were detected, and compared with the Hep2.2.15 cell line , and finally established a cell line based on HepG2 stably and highly expressing HBV YVDD mutant strain, and obtained a high-expressing lamivudine-resistant HBV virus liver cancer cell line HepG2.HS204V, which was preserved on May 13, 2011. Deposit unit: China Microorganism Culture Collection General Microbiology Center of the Management Committee (CGMCC), Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Collection Number: CGMCC No.4859, Classification Name: Highly Expressed Lamivudine-resistant HBV Virus Liver cancer cell lines.

[0051] The preparation met...

Embodiment 2

[0075] Embodiment 2 tests HepG2.HS204V cell line

[0076] Such as figure 1 As shown, the expression levels of HBsAg, HBeAg and HBV-DNA in the supernatant of the pREP-HBV-YVDD HBV drug-resistant variant cell line, the HBsAg level was maintained between 150-300IU / ml, about 15-30 times that of Hep2.2.15 cells; HBeAg The level is between 150-250PeIU / ml, which is about 10-20 times that of Hep2.2.15 cells, and HBV-DNA is maintained at 10 5 -10 6 between, slightly higher than that of Hep2.2.15 cells, and did not decrease significantly with the increase of passage times.

[0077] Such as figure 2 As shown, the results of drug-resistant phenotype analysis of pREP-HBV-YVDD HBV drug-resistant mutant cell lines showed that the level of HBV-DNA in the cell supernatant did not decrease significantly with the increase of the LAM concentration gradient, and increased from 10 to 10 with the increase of the ADV concentration gradient. 5 down to 10 3 Below, it shows insensitivity to LAM bu...

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Abstract

The invention belongs to the field of microbial animal cell lines, relates to human hepatocellular liver carcinoma cell line (HepG2)-based high-expression stable hepatitis B virus valine (HBV YVDD) variant strain cell line, and relates to a HepG2-based high-expression stable lamivudine-resistant HBV liver cancer cell strain, in particular to a high-expression lamivudine-resistant HBV liver cancer cell strain HepG2.HS204V and a preparation method thereof. The cell strain can express hepatitis B surface antigen (HBsAg) and hepatitis Be Antigen (HBeAg) continuously, the expression of the cell strain on the HBsAg and the HBeAg is higher than that of Hep2.2.15, cells have HBV replication, HBV granules are generated in the cells, and the level of the HBV granules of cell supernatant is the same as that of the Hep2.2.15 basically; the cells are insensitive to lamivudine (LAM) but are sensitive to adefovir; and the intracellular HBV replication level is not reduced obviously with the ascending of LAM concentration and is reduced gradually with the ascending of ADV concentration. The cell strain can be used for an HBV cell model for screening anti-HBV medicines and studying HBV biological characteristics and having a mutual effect between the HBV cell model and the cells.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, and relates to a HepG2-based stable and high-expression HBV YVDD variant cell line, in particular to a HepG2-based stable and high-expression lamivudine-resistant HBV virus liver cancer cell line, especially to a high-expression lamivudine-resistant HBV Viral liver cancer cell line HepG2.HS204V and its preparation method. The cell strain of the invention can be used for screening anti-HBV drugs, studying the HBV biological characteristics and the HBV cell model of interaction with cells. Background technique [0002] The prior art discloses that the HBV cell model is a necessary tool for screening anti-drugs, studying its biological characteristics and its interaction with cells, especially in the study of pathogenesis of liver diseases, immune mechanism, and screening of antiviral drugs. Among them, Hep2.2.15 cells are a milestone in the development and research of HBV cell models. St...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02C12R1/91
Inventor 张继明张轶俊
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV