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Hybrid cell lines suitable for human hepatitis-B viral natural infective duplication and capable of subculturing

A hybrid cell line, subculture technology, applied in tissue culture, application, biochemical equipment and methods, etc., can solve the problems of HBV insensitivity, long-term subculture, etc.

Inactive Publication Date: 2008-04-09
SHANDONG UNIV QILU HOSPITAL
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Problems solved by technology

Human primary hepatocytes are sensitive to HBV, but cannot be subcultured in vitro for a long time; human liver tumor cells can be cultured in vitro for a long time but are not sensitive to HBV; transfection and expression technology of some HBV genes under the control of strong exogenous promoters It cannot be used to study the process of HBV infection in the natural state; in view of the above reasons, a cell line that is sensitive to HBV and can be subcultured in vitro for a long time is needed to satisfy the research on human HBV and its natural state infected host cells needs

Method used

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  • Hybrid cell lines suitable for human hepatitis-B viral natural infective duplication and capable of subculturing

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Embodiment Construction

[0048] In order to screen out the required hybrid cells, according to the principle that HGPRT-negative HepG2 cells cannot survive in HAT selection medium, and human primary hepatocytes cannot be subcultured: first select HGPRT from HepG2 cells - HepG2, then HGPRT - HepG2 is fused with primary human hepatocytes, and the fused hybrid cells can contain HGPRT from the latter and thus can survive in HAT selection medium. Unfused or fused homokaryotic HGPRT - HepG2 cells cannot survive in the HAT selection medium. Unfused or fused homokaryotic primary human hepatocytes can survive in the HAT selection medium but cannot be passaged, so the hybrid cells that can survive are the hybrid cells we selected .

[0049] 1. Induced mutation of HepG2 cells and screening of HGPRT-negative HepG2

[0050] HepG2 cells were induced by 0.3g / L ethyl methanesulfonic acid (EMS) for 16 hours, EMS was removed, DMEM medium was cultured for 7-10 days, and 6-mercaptopurine (6-MP) was added to make the f...

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Abstract

The invention relates to a hybrid cell line which can adapt to human HVB virus natural infection and replication and can be subcultured and a preparation method thereof. The hybrid cell line of HepChLine are obtained by fusion and hybridization of parent cells of human primary hepatocyte and negative HepG2 filtrated from HGPRT, which is new matter. The HepCHLine succeeds sensitivity of parental human hepatocyte by HBV infection and is characterized in that HepG2 can vaccination animal and subculture, which is not only suitable for natural infection but can be long - term subcultured. The invention, for the first time, prepares the hybrid cell lines of HepChLine, which are not only suitable for natural infection of human HVB virus, but can be subcultured. The invention meets requirements for research of host cells infected at the condition of HVB or nature.

Description

technical field [0001] The invention relates to a hybrid cell line Hepatocyte hybrid line (HepCHLine for short) adapted to natural infection and replication of human hepatitis B virus (HBV) and capable of subculture and a preparation method thereof, belonging to the technical field of HBV cell culture in life science and medicine. Background technique [0002] Hepatitis B is one of the important infectious diseases in my country. So far, the early infection of HBV and the generation and regulation mechanism of covalently closed circular DNA (cccDNA) in human hepatitis B virus (HBV) etiology are still unclear. Most of the current understanding of HBV comes from its molecular cloning, or the transfection and expression of some genes under the control of an exogenous strong promoter; or the study of primary duck liver cells infected by duck hepatitis B virus (DHBV). These technical means and methods cannot meet the requirements of the research on human HBV and its infected hos...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/22C12N15/07
Inventor 马立宪王刚邵丽华江渊王爱华
Owner SHANDONG UNIV QILU HOSPITAL
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