Construction method of stably expressing clinical lamivudine-resistant hepatitis B virus cell line

A hepatitis B virus and lamivudine technology, applied in the field of stably expressing clinical lamivudine-resistant hepatitis B virus cell lines, can solve the problem of inability to truly reflect biological characteristics and differences in drug sensitivity in vitro And other issues

Active Publication Date: 2016-07-06
河南省医药科学研究院
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Problems solved by technology

[0006] However, using wild-type HBV as the backbone and using artificial site-directed mutagenesis to generate mutant HBV cannot truly reflect the biological characteristics of HBV that has spontaneously mutated or mutated under the pressure of drug screening in clinical practice. There are also some differences

Method used

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  • Construction method of stably expressing clinical lamivudine-resistant hepatitis B virus cell line
  • Construction method of stably expressing clinical lamivudine-resistant hepatitis B virus cell line
  • Construction method of stably expressing clinical lamivudine-resistant hepatitis B virus cell line

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Experimental program
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Embodiment 1

[0047] The construction of embodiment 1 HBV whole gene eukaryotic expression vector pcDNA3.1-1.3HBV

[0048] 1. Design and synthesis of primers: According to the complete HBV gene sequence in GenBank, the primers required for PCR were designed (Table 1). P1 and P2 are the upstream and downstream primers for amplifying the whole HBV gene. A1 and A2 are the upstream and downstream primers for amplifying fragment A, B1 and B2 are the upstream and downstream primers for amplifying fragment B, C1 and C2 are the upstream and downstream primers for amplifying fragment C, and D1 and D2 are the upstream and downstream primers for amplifying fragment D primers. The primer sequences are shown in Table 1 (the underlined part is the corresponding enzyme cutting site).

[0049] Table 1 PCR amplification primers for different gene fragments of HBV

[0050]

[0051] 2. PCR amplification of fragments A, B, C, D and A+B, C+D Using T-HBV containing the rtL180M+rtM204V double mutant HBV gen...

Embodiment 2

[0054] The screening of embodiment 2 stable cell lines

[0055] 1. Determination of the optimal screening concentration of G418: adjust the density of HepG2 cells to 1000 cells / ml, inoculate 1 ml per well into a 24-well culture plate. After 24 hours, add DMEM selection medium containing 100 μg / ml to 1000 μg / ml G418, change the medium every 3 days, and screen for a total of 14 days. The concentration of G418 that can just kill all cells is the optimal screening concentration of the drug. Finally, 600μg / ml was determined as the optimal screening concentration.

[0056] 2. Cell transfection and resistance screening Apply lipofectamion-2000 transfection reagent from Invitrogen Company, and transfect human with eukaryotic expression plasmid pcDNA3.1-1.3HBV containing rtL180M+rtM204V double variant without endotoxin in strict accordance with the operating instructions Liver cancer cell line HepG2 cells. 24 h after transfection, the transfected cells were passaged to a new culture ...

Embodiment 4

[0062] The drug resistance identification of embodiment 4 stable cell lines

[0063] 1. Identification of phenotypic drug resistance Taking the currently recognized HepG2.2.15 cell line stably expressing wild-type HBV as a control, evaluate the stable cell line HepG2.RL1 established by the present invention against lamivudine and adefovir dipivoxil in vitro sensitivity. Adjust the cell concentration of HepG2.2.15 and HepG2.RL1 to 2×10 4 cells / ml, 200 μl per well was inoculated into a 96-well cell culture plate. At the same time adjust the cell concentration to 2×10 5 cells / ml, 2ml per well was inoculated into a 96-well cell culture plate. After culturing for 24 hours, culture solutions containing different concentrations of lamivudine or adefovir dipivoxil (0, 0.01, 0.1, 1, 10, 100 μM) were added, and three replicate wells were set for each concentration in a 96-well plate. Two replicate wells were set up for each concentration in the 6-well plate. Cell culture medium was...

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Abstract

The invention discloses a construction method of a hepatitis B virus cell strain stably expressing tolerance of clinical lamivudine. The method comprises the following steps of: integrating HBV (Hepatitis B Virus) whole gene to HepG2 cell genome of a human hepatoma cell line through a molecular biological technique of cells to generate autonomous replication of virus and integral life cycle. The cell strain constructed by the invention can support high-level replication of DNA (Deoxyribonucleic Acid) and stable expression of antigens and is highly tolerant to lamivudine, so that the cell strain is suitable for in vitro screening research of HBV biological characteristics, tolerant mechanism and novel antiviral medicines.

Description

technical field [0001] The invention relates to a cell strain stably expressing nucleoside analogue lamivudine drug-resistant hepatitis B virus (hepatitisBvirus, HBV), and the virus contained in the cell strain is derived from chronic lamivudine-resistant chronic In the serum of patients with hepatitis B, the exact nucleoside drug lamivudine resistance mutation site exists in the DNA polymerase / reverse transcriptase region of the virus. Background technique [0002] HBV infection has a high degree of species and tissue specificity, and it only infects the liver cells of primates such as humans and chimpanzees, and it is difficult to directly culture it in vitro. At present, introducing HBV gene fragments into host cells through DNA transfection technology and establishing cell models that can support HBV replication and expression are the main ways to study the pathogenic mechanism of HBV and screen antiviral drugs in vitro. [0003] The HBV genome has a compact structure, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12R1/93
Inventor 王庆端周玉冰王亚峰江金花郑立运杨小昂张晶敏孙长宇
Owner 河南省医药科学研究院
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