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C genotype adefovir dipivoxil drug-resistant HBV (Hepatitis B Virus) stable replication and expression cell line

An adefovir dipivoxil and cell line technology, which is applied in the field of stable virus replication and expression cell lines, can solve the problem that it is difficult to fully reflect the phenotypic characteristics of drug-resistant strains directly isolated from patient serum, and achieve high application value.

Inactive Publication Date: 2014-12-03
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since these drug-resistant mutants are artificially produced by site-directed mutation technology, only non-naturally occurring mutants are obtained. Artificial site-directed mutation will modify some natural genetic characteristics of the virus, and it is difficult to fully reflect the direct isolation of drug-resistant strains from patient serum. The phenotypic characteristics of
[0006] There is no report about the stable replication of HBV cell lines with C genotype adefovir dipivoxil drug-resistant mutation isolated clinically in my country

Method used

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  • C genotype adefovir dipivoxil drug-resistant HBV (Hepatitis B Virus) stable replication and expression cell line
  • C genotype adefovir dipivoxil drug-resistant HBV (Hepatitis B Virus) stable replication and expression cell line
  • C genotype adefovir dipivoxil drug-resistant HBV (Hepatitis B Virus) stable replication and expression cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of 1.1 times longer HBV expression vector

[0058] The patient has been taking adefovir dipivoxil antiviral treatment for a long time, the viral load has been negative (figure 1 ). Five primers were synthesized inside the HBV sequence for full-length sequencing identification, and the results were completely consistent with the original sequence. The reverse transcriptase region contained a typical adefovir dipivoxil resistance mutation site rtA181V+rtN236T.

Embodiment 2

[0059] Example 2 Stable cell line screening

[0060] (1) Plasmid DNA transfection and cell clone screening Extract the transfection-grade recombinant plasmid pTriEx-1.1-HBV B111, and quantify the concentration with an ultraviolet spectrophotometer. At 37°C, containing 5% CO 2 HepG2 cells were cultured in complete DMEM medium with 10% FBS. Before transfection, the cells were inoculated into 24-well plates and cultured overnight. When the cell fusion reached 80%, the DMEM without serum and antibiotics was replaced, and the DNA was introduced with FuGENE HD liposomes. The specific method was carried out according to the operation manual. 5h after transfection, add FBS to 10%. Set untransfected wells as negative controls. Add G418 (final concentration 600 μg / ml) to the cell culture dish for 48 hours for screening, change the medium every 2 days, and replace it with G418 (300 μg / ml) at a maintenance concentration after the cells in the control group are completely dead to contin...

Embodiment 3

[0062] Example 3 Construction of the relevant detection of the resulting cell line

[0063] 1. Expression of major HBV antigens

[0064] (1) ELISA detection Collect the cell culture supernatant of the HepG2.B111 cell line at passages 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and 60 for ELISA (double antibody sandwich method) detection For the level of HBsAg and HBeAg, the wavelength is set to 450nm. With the increase of cell passage number, the expression of antigen gradually increased and tended to be stable, the average OD value of HBsAg was 2.64, HBeAg was 1.25 ( image 3 ), the corresponding HBsAg and HBeAg of HepG2.2.15 cells were 0.93 and 3.01 ( Figure 4 ).

[0065] (2) Immunohistochemical polylysine-treated slides were placed in a 6-well plate, and 5×10 HepG2.B111 cells were added 5 Each well was allowed to slide cells for 72 hours. After the cells were fixed, two-step method was used for immunohistochemical staining. The primary antibody was rabbit anti-HBs / anti-H...

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Abstract

The invention relates to an adefovir dipivoxil HBV (Hepatitis B Virus) resistant stable replication and expression cell line. The virus is derived from an adefovir dipivoxil drug-resistant chronic hepatitis B patient in China and is a national popular C genotype HBV strain, and the DNA (Deoxyribose Nucleic Acid) polymerase / reverse transcriptase region of the C genotype HBV strain contains a nucleotide analog adefovir dipivoxil drug-resistant mutation site. According to the cell line, virus genomes are integrated to the HepG2 cell chromosome of a human hepatic carcinoma cell line through a screening method, and the autonomous replication and completed life cycle circulation of the virus are generated; the virus is stably secreted, the DNA of the virus is violently replicated, virus particles can be detected in a culture supernatant, and the HBV covalent closed loop-shaped DNA (cccDNA) at a stable level is in a cell nucleus; the HBV replication of the cell line is resistant in the traditional nucleoside (nucleotide) drug adefovir dipivoxil and sensitive in lamivudine, entecavir and tenofovir disoproxil fumarate. The cell line disclosed by the invention can provide a stable and reliable cell model for the evaluation of a drug-resistant HBV drug and the research and development of a new drug.

Description

technical field [0001] The invention belongs to the field of microbial animal cell lines, and relates to a stable replication and expression cell line of hepatitis B virus (HBV). In particular, the contained virus is derived from a Chinese patient's C genotype HBV strain, and its DNA polymerase / reverse transcriptase ( RT) region contains a nucleotide analog adefovir dipivoxil resistance mutation site of the virus stable replication expression cell line. Background technique [0002] The establishment of HBV stable replication cell model, which has a complete virus life cycle and can continuously secrete virus particles for a long time, is an essential tool for standardized screening of anti-HBV drugs in vitro and research on antiviral mechanisms. Due to the compact structure of the HBV genome, different reading frames and highly overlapping regulatory sequences, it is necessary to construct a recombinant vector carrying more than a complete 3.2kb HBV genome sequence in order...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12Q1/70C12Q1/02C12R1/91
Inventor 刘妍徐东平李奇刘文李晓东王琳许智慧
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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