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Cell model capable of supporting hepatitis B virus replication and infection and establishment method

A cell model, hepatitis B virus technology, applied in genetically modified cells, chemical instruments and methods, botanical equipment and methods, etc., can solve problems that have not yet been applied, and achieve the effect of simple culture conditions

Pending Publication Date: 2021-11-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] BEL-7404 is a liver cancer cell line derived from a 69-year-old Chinese male. It is often used in liver cancer research, but its application in HBV replication and infection research has not been seen yet

Method used

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  • Cell model capable of supporting hepatitis B virus replication and infection and establishment method
  • Cell model capable of supporting hepatitis B virus replication and infection and establishment method
  • Cell model capable of supporting hepatitis B virus replication and infection and establishment method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Observe the transfection efficiency after BEL-7404 transfected pCMV-EGFP plasmid by fluorescence microscope and cell flow experiments:

[0064] 1. Inoculate BEL-7404 cells in a 6-well plate (the BEL-7404 cells are provided by the Chinese Academy of Sciences Cell Bank / Stem Cell Bank, catalog number is TCHu64) suspension (8 × 10 5 cells / well), the culture plate was pre-cultured in the incubator for 12 hours, and the cells adhered to the wall.

[0065] 2. Use 4 μl of Turbofect transfection reagent (Thermo Fisher Scientific, Waltham, USA) transfection reagent and 2 μg pCMV-EGFP plasmid in each well for transfection experiments, culture at 37°C for 48 hours, and observe the fluorescent expression effect under a fluorescent microscope. The experimental results are as follows: figure 1 As shown in A. The pCMV-EGFP plasmid in this step was constructed by the applicant himself, and the specific method was as follows: the EGFP cDNA sequence was cloned into the pCDN3.1 (V79520, I...

Embodiment 2

[0071] Southern blot experiments detected that overexpression of the HNF4α gene in BEL-7404 can support the replication of complete HBV. Specific experiments include:

[0072] 1. Inoculate BEL-7404 cell suspension (1×10 6 cells / dish), the culture dish was pre-cultured in the incubator for 12 hours, and the cells adhered to the wall.

[0073] 4 μg pHNF4α and 4 μg p1.3×HBV replicon plasmid were co-transfected into BEL-7404 cells using 16 μl Turbofect transfection reagent, and the control group was transfected with 4 μg empty plasmid and 4 μg p1.3×HBV replicon plasmid. After 96 hours, intracellular HBV DNA was extracted for Southern blot experiment. Wherein, the construction method of pHNF4α is to clone the HNF4α cDNA sequence into pCDN3.1 (V79520, Invitrogen) vector by using PCR combined with restriction enzyme digestion and ligation.

[0074] The p1.3×HBV replicon plasmid was constructed by using the HBV strain sequence as a template and cloning 1.3 copies of the HBV genome i...

Embodiment 3

[0097] The 7404NT-HNF4α cell line was constructed, and the expression levels of hNTCP and HNF4α in the cells were detected by real-time quantitative PCR (qPCR) and Western blot

[0098] 1. Construction of 7404NT-HNF4α cell line: The construction of a stable cell line was carried out step by step by lentivirus infection.

[0099] Schematic diagrams for the construction of hNTCP and HNF4α recombinant lentiviral plasmids are as follows Figure 5 and Figure 6 shown. in, Figure 5 It is the HNF4α plasmid map constructed based on the Tet-on expression regulation system. The expression of this system requires the expression of two genes, Tet-on and TRE-HNF4α. The principle is as follows: TRE is the responsive promoter of Tet-on. In the presence of tetracycline (DOX), Tet-on expression can be activated, thereby activating the TRE promoter to start the transcription of HNF4α. Therefore, two steps were carried out in the process of cell line construction lentiviral infection proce...

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Abstract

The invention provides a cell model capable of supporting hepatitis B virus replication and infection and an establishment method. The inventor finds that BEL-7404 cells have higher transfection efficiency in experiments, and can better support replication and infection of hepatitis B virus after overexpression of human sodium taurocholate cotransporter polypeptide (hNTCP) and hepatocyte nuclear factor (HNF4alpha) genes, so that a 7404NT-HNF4alpha cell model capable of stably expressing hNTCP and HNF4alpha is constructed. According to the cell model capable of supporting hepatitis B virus replication and infection, the expression of the HNF4alpha can be regulated and controlled by adding doxycycline (DOX) into the culture medium, so that the replication and infection of the HBV are regulated and controlled, and a new cell model is provided for the research of the HBV.

Description

technical field [0001] The invention relates to the technical field of cell transformation, in particular to the application of a 7404NT-HNF4α cell model constructed on BEL-7404 cells with high expression of hNTCP and HNF4α genes in the study of replication and infection of hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) is a hepatotropic enveloped virus containing incomplete double-stranded DNA. It is reported that there are at least 257 million people with chronic HBV infection worldwide. Chronic HBV infection can cause clinical diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, which greatly endanger human life and health. Existing antiviral therapy (interferon and nucleoside (acid) analogues) can only inhibit the replication of the virus to a certain extent, but cannot effectively eliminate the virus, and cannot yet achieve the purpose of curing chronic hepatitis B. [0003] After HBV enters liver cells by binding...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867C12Q1/02C12R1/01
CPCC07K14/4702C07K14/705C12N5/067C12N5/0693C12N15/86G01N33/5067G01N33/5044C12N2510/00C12N2740/15043C12N2830/003C12N2503/02G01N2500/10
Inventor 谢幼华宋迎迎刘晶
Owner FUDAN UNIV
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