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HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line and preparation method thereof

A technology of stable expression and cell line, applied in the field of high expression wild-type HBV virus liver cancer cell line and its preparation, can solve the problems of low expression level, more influence, unfavorable virus protein research, etc., and achieve the effect of stable expression level

Inactive Publication Date: 2012-12-12
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, HepG2.2.15 still has the following defects in HBV research: (1) The expression level of HepG2.2.15 HBV proteins such as HBsAg and HBeAg is relatively low, which is not conducive to the research of related viral proteins, and like HBV-DNA, its expression The level is unstable, and is more affected by factors such as the culture environment; (2) The integrated HBV genotypes are not B and C genotypes, and there are considerable limitations in the research on the pathogenesis or drug resistance mechanism of HBV in the Chinese population

Method used

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  • HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line and preparation method thereof
  • HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line and preparation method thereof
  • HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Establishment of HepG2.HSWT cell line

[0043] After the HBV eukaryotic expression plasmid pREP-HBV-WT 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 expressing HBV wild strain, and obtained the high-expression wild-type HBV virus liver cancer cell line HepG2.HSWT, which was preserved on June 1, 2011. (CGMCC), address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No.4909, classification name: human liver cancer cell line HepG2.HSWT.

[0044] The described cell line HepG2.HSWT is prepared by the following methods and steps:

[0045] (1) Plasmid transfection into HepG2 cells

[0046] ① Cell culture: HepG2 cells were cultured in DMEM med...

Embodiment 2

[0067] Embodiment 2 tests HepG2.HSWT cell line

[0068] Such as figure 1 As shown, the expression levels of HBsAg and HBeAg in the supernatant of the pREP-HBV (WT) HBV cell line, the HBsAg level is maintained between 20-40IU / ml, which is about 2-4 times that of Hep2.2.15 cells; the HBeAg level is 40-70PeIU / ml Between, about 4-8 times as Hep2.2.15 cells, HBV-DNA maintained at 10 4 -10 5 , slightly lower than that of Hep2.2.15 cells, and there was no significant decrease with the increase of passage times.

[0069] Such as figure 2 As shown, the results of pREP-HBV (WT) HBV cell line drug resistance phenotype analysis showed that the HBV-DNA level in the cell supernatant increased from 10 to 100 with the concentration gradient of LAM and ADV. 4 Dropped below the lower limit of detection, indicating that it is sensitive to both LAM and ADV.

[0070] Such as image 3 As shown, pREP-HBV (WT) HBV cell supernatant HBV-DNA gene sequencing results show that the wild type is the ...

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Abstract

The invention belongs to the field of microorganism animal cell lines, and relates to a HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line, in particular to a high-expression wild HBV virus liver cancer cell line HepG2.HSWT and a preparation method thereof. The high-expression wild HBV virus liver cancer cell line HepG2.HSWT is obtained by detecting HBV reproduction in cells, specific antigen expression and generation of virus particles through hygromycin screening expression after HepG2 cell transfection of HBV eukaryotic expression plasmid pREP-HBV-WT. The cell line is evidently higher than HepG2.HSWT in expression level of HBsAg and HBeAg and is stable in expression level and less susceptible to factors of culturing environments and the like. The integrated HBV genotype is B or C genotype, cells are sensitive to LAM (lamivudine) and ADV (adenovirus), and HBV intra-cell reproduction level is gradually decreased along with increasing of LAM and ADV concentration. The HepG2-based stable-expression HBV wild strain cell line can be used for researches on Chinese people HBV morbidity or drug resistance mechanisms and screening anti-HBV drugs.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, in particular to a HepG2-based cell line stably expressing HBV wild strains, in particular to a high-expression wild-type HBV virus liver cancer cell line (HepG2.HSWT) and a preparation method thereof. Background technique [0002] It is well known in the art that the HBV cell model is an essential tool for screening drug resistance, studying its biological characteristics and its interaction with cells. Especially, it plays an important role in the study of the pathogenesis, immune mechanism, and screening of antiviral drugs related to liver diseases; among them, Hep2.2.15 cells are a milestone in the development and research of HBV cell models. The pDolt-HBV-1 recombinant vector of the tail dimer was transfected into HepG2 cells, and a cell clone producing high levels of HBsAg and HBeAg was obtained after selection by G418, and HBV-DNA and infectious whole virus could be detected both...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/79C12Q1/02C12R1/91
Inventor 张继明张轶俊
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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