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Method of establishing hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus

A primary hepatocyte, recombinant lentivirus technology, applied in cell dissociation methods, artificial cell constructs, reverse transcription RNA viruses, etc., can solve problems such as establishing a hepatitis B virus infection cell model, and achieve small differences between cell batches Effect

Active Publication Date: 2018-05-04
立沃生物科技(深圳)有限公司
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  • Abstract
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Problems solved by technology

However, at present, there is no method to establish a HBV-infected cell model using pig primary hepatocytes and hNTCP recombinant lentivirus

Method used

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  • Method of establishing hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus
  • Method of establishing hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus
  • Method of establishing hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus

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Embodiment

[0078] The method for establishing a hepatitis B virus-infected cell model using pig primary liver cells and hNTCP recombinant lentivirus in this embodiment comprises the following steps:

[0079] Step 1: Prepare hNTCP recombinant lentivirus concentrate

[0080] Using the calcium phosphate transfection method, the lentiviral packaging plasmid pCMV dr8.91, the lentiviral packaging plasmid pMD.2G and the hNTCP overexpression plasmid pWPI-hNTCP-Puro were simultaneously transfected into 293T cells at a mass ratio of 2:1:3 48 hours after transfection, the culture supernatant containing lentivirus was collected and concentrated to obtain hNTCP recombinant lentivirus concentrate.

[0081] (1) hNTCP expression plasmid construction

[0082] The lentiviral packaging plasmid pCMV dr8.91, the lentiviral packaging plasmid pMD.2G and the hNTCP overexpression plasmid pWPI-Puro were all obtained from the global plasmid sharing Addgene website (http: / / www.addgene.org / ). Obtain human Na from ...

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Abstract

The invention discloses a method of establishing a hepatitis B virus infection cell model by using porcine primary hepatocyte and hNTCP recombinant lentivirus and belongs to the technical field of cell modification. The method comprises the following steps: S1, preparing an hNTCP recombinant lentivirus concentrated liquid; S2, preparing liver tissues which are fully digested; S3, preparing the porcine primary hepatocyte; S4, preparing a porcine primary hepatocyte single cell suspension; S5, preparing a cell suspension for paving a plate; S6, paving cell plate and culturing; S7, carrying out hNTCP recombinant lentivirus infection on the porcine primary hepatocyte; and S8, establishing the hepatitis B virus infection cell model. According to the invention, the recombinant lentivirus containing an hNTCP gene is constructed in vitro, the hNTCP gene is integrated into the primary hepatocyte genome through the lentivirus with high efficiency of infection to achieve stable overexpression of hNTCP, so that the primary hepatocyte supports hepatitis B virus infection.

Description

technical field [0001] The invention relates to a method for establishing a hepatitis B virus-infected cell model by using pig primary liver cells and hNTCP recombinant lentivirus, and belongs to the technical field of cell transformation. Background technique [0002] Hepatitis B virus (abbreviated as HBV) infection is China's "national disease". There are about 90 million hepatitis B carriers in China, and about one million people die from hepatitis B infection-related diseases every year. Due to the lack of a suitable HBV-infected cell model, the development of anti-HBV drugs is relatively lagging behind. Although hepatitis B vaccine can protect healthy people from hepatitis B, there is currently no curative drug for the large number of hepatitis B carriers. HBV-infected cell models and animal models are crucial for the development of anti-HBV drugs. [0003] For a long time, the cell models for screening and evaluating anti-HBV drugs were limited to liver cancer cell l...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/071
CPCC12N5/067C12N15/86C12N2509/00C12N2740/15043
Inventor 周明
Owner 立沃生物科技(深圳)有限公司
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