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Cell system for simply and efficiently generating hepatitis B virus (HBV) recombinant cccDNA

A cell line and cell technology, which can be applied to cells modified by introducing foreign genetic material, biochemical equipment and methods, applications, etc., and can solve the problems of difficult signal detection, high price, complicated and time-consuming cultivation operations, etc.

Pending Publication Date: 2019-06-18
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the in vitro cell models currently used to study HBV, primary human hepatocytes are difficult to obtain and expensive; non-infected models such as HepG2.2.15, HepAD38, and HepDES19 have little or no cccDNA formation. In these models, plasmids replace cccDNA Become the main template for virus replication; while the cccDNA formed in HepaRG, HepG2 / Huh7-NTCP and other infection systems is still very small, it is difficult to detect the signal by Southern blot hybridization (Southern Blot), and the culture operation of these infected cell models is relatively complicated Time consuming, limiting their use in high-throughput screening

Method used

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  • Cell system for simply and efficiently generating hepatitis B virus (HBV) recombinant cccDNA
  • Cell system for simply and efficiently generating hepatitis B virus (HBV) recombinant cccDNA
  • Cell system for simply and efficiently generating hepatitis B virus (HBV) recombinant cccDNA

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The establishment of embodiment 1 HepG2-HBV / loxP cell line

[0035] ①Plasmid construction: The pSBbi-rcccDNA plasmid was constructed by inserting the pSBbi-GP vector plasmid (Addgene#60511) into the HBV single-copy genome with loxP sites at both ends; pCMV-SB100 is a Cre recombinase expression plasmid (Addgene#34879) .

[0036] ② Cell transfection: HepG2 cells were cultured in high-glucose DMEM medium (BI Company, containing 10% fetal bovine serum, 2 mM glutamine). The cells in logarithmic phase of growth were trypsinized and counted. Press 1×10 6 The concentration of cells / well was seeded into 6-well culture plates. Cultivate overnight, and use for transfection when the cells are about 80-90% confluent into sheets. According to the transfection amount of 2 μg per well, 1.8 μg pSBbi-rcccDNA and 0.2 μg pCMV-SB100 were diluted in 100 μl pure DMEM medium, and 100 μl pure DMEM medium was mixed with 12 μl PEI transfection reagent (Polysciences). Mix the diluted PEI with...

Embodiment 2

[0039] Example 2 pAd-Cre transduces HepG2-HBV / loxP cells to induce rcccDNA generation

[0040] ⑤Construction of pAd-Cre recombinant adenovirus: insert Cre recombinase expression sequence into adenovirus vector pAdPLDest to construct pAd-Cre plasmid, pAd-Cre plasmid was digested with PacI endonuclease and then transfected into HEK293 cells to produce pAd-Cre recombinant adenovirus Virus.

[0041] ⑥ pAd-Cre transduction: HepG2-HBV / loxP cells with 5×10 6 The concentration of cells / dish was inoculated on a 10cm culture dish, and 1×10 7 pAd-Cre recombinant adenovirus (MOI=2) was used for transduction. The cells were cultured in high-glucose DMEM medium containing 2% fetal bovine serum, and the medium was changed every 2 days.

[0042] ⑦ Southern Blot detection of rcccDNA: Hirt extraction method was used to extract rcccDNA, and Roche company's digoxin probe labeling and detection system was used for Southern Blot detection (DIG High Prime DNA Labeling and Detection Starter Kit II...

Embodiment 3

[0044] Example 3 Detection of related characteristics of rcccDNA (taking HepG2-HBV / loxP No. 6 clone as an example)

[0045] ①The generation of rcccDNA is fast and stable: the cells were collected at the corresponding time point after pAd-Cre transduction, the genomic DNA and rcccDNA were extracted, and the HBV sequence and rcccDNA integrated in the genome were detected by quantitative PCR; Primers for specific intronic sequences were used for detection. The results showed that the integrated HBV genome copy number in HepG2-HBV / loxP No. 6 clone was ~60 copies; 3 days after pAd-Cre transduction, the integrated HBV sequence in the cell genome was basically undetectable, while the generated rcccDNA copy number Reached ~60 copies / cell, indicating that after 3 days of pAd-Cre transduction, Cre cleavage and rcccDNA generation were basically completed ( image 3 A). The generated rcccDNA can exist stably in the cell for at least 12 days ( image 3 B).

[0046] ② The structural cha...

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Abstract

The invention belongs to the field of microorganism animal cell lines, and relates to a cell system for generating hepatitis B virus (HBV) recombinant cccDNA. Through a transposon system, HBV single-copy genomes with two ends provided with loxP sites are integrated into HepG2 cells, a clone HepG2-HBV / loxP cell line integrated with the different-copy-number HBV genomes is obtained, the cell line does not express HBsAg, after cyclization recombination enzyme (Cre) is introduced through adenovirus transduction, the rcccDNA is generated and the HBsAg is expressed, and expression of the HBsAg is closely relevant to the generation and level of the rcccDNA; the rcccDNA is rapidly and stably generated, the structure feature of the rcccDNA is similar to that of real HBV cccDNA, and transcription, copy and protein expression of viruses can be supported; the rcccDNA can be quantitatively detected through quantitative PCR by designing a specific primer, and Southern Blot detection can be carried out by utilizing a digoxin marking system. The cell system can be used for establishing a platform for HBV cccDNA relevant biological research and screening and evaluation of anti-HBV medicines.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, and relates to a cell system for producing recombinant cccDNA of hepatitis B virus, in particular to a cell system capable of simple and efficient production of hepatitis B virus (r)cccDNA and its establishment method and application. Background technique [0002] The prior art discloses that hepatitis B caused by hepatitis B virus (Hepatitis B virus, HBV) infection is a major infectious disease that seriously endangers human health. According to statistics, about 2 billion people in the world are infected or have been infected with HBV, and 240 million of them are patients with chronic hepatitis B (CHB) infection. According to the survey, the risk of chronic hepatitis B patients suffering from cirrhosis, liver failure and liver cancer is significantly increased. Although the treatment of chronic hepatitis B has made some progress in recent years, a series of problems such as the cours...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/51
Inventor 袁正宏邬敏张小楠陈捷亮
Owner FUDAN UNIV
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